WO2016185457A1 - Méthodes pour favoriser la lymphagiogenèse - Google Patents

Méthodes pour favoriser la lymphagiogenèse Download PDF

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WO2016185457A1
WO2016185457A1 PCT/IL2015/051030 IL2015051030W WO2016185457A1 WO 2016185457 A1 WO2016185457 A1 WO 2016185457A1 IL 2015051030 W IL2015051030 W IL 2015051030W WO 2016185457 A1 WO2016185457 A1 WO 2016185457A1
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cells
wnt
lec
stem cells
pathway
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Karina YANIV
Yogev SELA
Julian NICENBOIM
Lihee ASAF
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Yeda Research and Development Co Ltd
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C12N5/0692—Stem cells; Progenitor cells; Precursor cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5064—Endothelial cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled

Definitions

  • the present invention in some embodiments thereof, relates to methods of promoting lymphangiogenesis and, more particularly, but not exclusively, to use of the Wnt pathway therefore.
  • the lymphatic system plays a crucial role in normal and pathological conditions. It is essential for maintaining fluid homeostasis, for immune responses and for dietary lipid absorption, and is exploited by tumors to metastasize. Close to a century ago, two models describing the origins of the lymphatic system were proposed. While Florence Sabin [Sabin F.R., American Journal of Anatomy (1902) 1: 367-389] suggested a venous origin for the lymphatic endothelium, the second model, put forward by Huntington and McClure [Huntington G. and McClure C, Am. J. Anat.
  • lymphatic vessels form by concrescence of discontinuous and independent lymph vesicles, and that mesenchymal-derived cells constitute the walls of the lymphatic vessels.
  • Studies performed during the last decade, involving in vivo imaging in zebrafish, and lineage tracing in mice have extensively confirmed Sabin' s hypothesis.
  • mesenchymal lymphangioblast-derived lymphatic vessels has been described in Xenopus, and chick embryos. At present, the embryonic origins of the lymphatic endothelium still remain controversial.
  • Assembly of the lymphatic vascular network is considered a stepwise process, which begins at approximately gestational week E9.5 when the expression of Proxl, a master regulator of lymphatic differentiation and maintenance is first detected in a subpopulation of endothelial cells (ECs) within the cardinal vein (CV).
  • ECs endothelial cells
  • CV cardinal vein
  • VEGF-C Vascular endothelial growth factor C
  • zebrafish was recently shown to possess a lymphatic system that shares many similarities with lymphatic vessels found in higher vertebrates.
  • dpf days-post-fertilization
  • PACs parachordal cells
  • PCV posterior cardinal vein
  • Wnt (wingless-type MMTV integration site family) protein family is a diverse family of 19 secreted cysteine -rich glycoproteins that operate via three pathways: the canonical Wnt pathway, the non-canonical planar cell polarity (PCP) pathway, and the non-canonical Wnt/calcium pathway. These pathways control cell differentiation, proliferation and motility.
  • Wnt proteins are not intrinsically canonical or non-canonical, pathway decisions are determined by distinct sets of receptors and co-receptors. Thus, pathway decisions may be cell type- specific and may depend on receptor and co-receptor availability. Dysregulation of WNT signaling pathways has been observed under pathological conditions, e.g., in cancer, rheumatoid arthritis and Alzheimer's disease.
  • Buttler et al. [Buttler K. et al. Developmental Biology (2013) 381(2): 365-376] reported a link between Wnt5a, acting via a non-canonical pathway, and the morphogenesis of dermal lymphatic capillaries. Specifically, Buttler et al. teach that Wnt5a as well as other non-canonical pathway components are expressed in skin-derived lymphatic endothelial cells (LECs) and play important roles in the morphogenesis of dermal lymphatic capillaries (i.e. Wnt5a deficiency results in a significantly lower number of dermal lymphatic capillaries, while the mean size of individual lymphatics and the LEC number per vessel are greater). The study was performed in Wnt5a knockout mice. No ectopic effect of Wnt5a on stem cells was studied.
  • LECs skin-derived lymphatic endothelial cells
  • Wnt-1 polynucleotides and/or Wnt-1 polypeptides reduce VEGF-C expression, lymphangiogenesis and cancer metastasis.
  • lymphatic endothelial cell (LEC) specification comprising upregulating the activity of Wnt canonical pathway in stem cells and/or vascular progenitor cells and/or angioblasts, thereby promoting LEC specification.
  • a method of inducing lymphangiogenesis comprising: (a) promoting LEC specification in stem cells and/or vascular progenitor cells and/or angioblasts so as to obtain a LEC committed cell according to the method of some embodiments of the invention; and (b) subjecting the LEC committed cell to a signal selected from the group consisting of a LEC morphogenesis signal, a LEC proliferation signal and a LEC survival signal, thereby inducing lymphangiogenesis.
  • an isolated population of cells generated according to the method of some embodiments of the invention.
  • a pharmaceutical composition comprising the isolated population of cells of some embodiments of the invention and a pharmaceutically acceptable carrier or diluent.
  • an agent capable of upregulating the activity of Wnt canonical pathway for the manufacture of a medicament identified for treating or preventing a disease or disorder related to lymphangiogenesis.
  • a method of treating or preventing a disease or disorder related to lymphangiogenesis in a subject in need thereof comprising administering to the subject the isolated population of cells of some embodiments of the invention, thereby treating or preventing the disease or disorder related to lymphangiogenesis.
  • a method of treating or preventing a disease or disorder related to lymphangiogenesis in a subject in need thereof comprising administering to the subject an agent capable of upregulating the activity of Wnt canonical pathway, thereby treating or preventing the disease or disorder related to lymphangiogenesis.
  • LECs lymphatic endothelial cells
  • a method of screening for an agent capable of upregulating the activity of Wnt canonical pathway for the formation of LECs comprising: (a) contacting the agent with stem cells and/or vascular progenitor cells and/or angioblasts; (b) analyzing an expression of a lymphatic marker on the stem cells and/or vascular progenitor cells and/or angioblasts; and (c) comparing the expression of the marker following the contacting to prior to the contacting, wherein an expression of the marker is indicative of an agent suitable for formation of LECs.
  • the method further comprises differentiating the stem cells into endothelial precursor cells or angioblasts prior to the promoting the LEC specification.
  • the method further comprises the step of selecting LECs from the stem cells and/or vascular progenitor cells.
  • selecting is effected by analyzing expression of a marker selected from the group consisting of proxl, lymphatic vessel endothelial hyaluronan receptor-1 (lyvel), flt4, soxl8, nr2f2, podoplanin (Pdpn), CD31 and GP38.
  • a marker selected from the group consisting of proxl, lymphatic vessel endothelial hyaluronan receptor-1 (lyvel), flt4, soxl8, nr2f2, podoplanin (Pdpn), CD31 and GP38.
  • the LEC morphogenesis signal comprises upregulating the activity of Wnt non-canonical pathway in the LEC.
  • the LEC morphogenesis signal comprises an endothelial growth factor.
  • the method is effected ex-vivo.
  • the upregulating the activity of Wnt canonical pathway is effected by contacting the stem cells and/or vascular progenitor cells and/or angioblasts with an inhibitor of a Wnt canonical pathway member selected from the group consisting of an Axin, a GSK3, an adenomatosis polyposis coli (APC) and a sFRP.
  • a Wnt canonical pathway member selected from the group consisting of an Axin, a GSK3, an adenomatosis polyposis coli (APC) and a sFRP.
  • the inhibitor of a Wnt canonical pathway factor is a small molecule.
  • Wnt canonical pathway is effected by contacting the stem cells and/or vascular progenitor cells and/or angioblasts with an activator of a Wnt canonical pathway member selected from the group consisting of a Wnt5b, a Wnt3a, a Wnt7a, a Wnt7b, a LRP5/6, a ⁇ -catenin,
  • Dishevelled (Dsh), PP2A, ARFGAP1, and TCF.
  • the Wnt5b is selected from the group consisting of a Wnt5b polypeptide, a Wnt5b analogue and a Wnt5b polynucleotide.
  • Wnt non-canonical pathway is effected by contacting the LEC with an activator of a Wnt non-canonical pathway member selected from the group consisting of a Wnt5a, Wnt4,
  • the stem cells and/or vascular progenitor cells are derived from a source selected from the group consisting of hematopoietic stem cells, mesodermal progenitor cells, endothelial progenitor cells, angioblasts, induced pluripotent stem cells (iPS) and human embryonic stem cells
  • the use further comprises an agent capable of upregulating the activity of Wnt non-canonical pathway.
  • the method further comprises a administering to the subject an agent capable of upregulating the activity of Wnt non- canonical pathway.
  • the disease or disorder related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor, graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema, chronic wound, lymphedema, lymphatic fistula, lymphorrea and lymphocele/seroma formation.
  • the LECs are syngeneic or allogeneic with respect to the subject.
  • the subject is a human subject.
  • the lymphatic marker is selected from the group consisting proxl, lyvel, flt4, soxl8, nr2f2, Pdpn, CD31 and GP38.
  • FIGs. 1A-G show that lymphatic progenitors originate in the vPCV.
  • Figures 1E-G illustrate photoswitching of ventral ( Figure IE) and dorsal (Figure IF) PCV in Tgiflil :gal4;uasKaede) embryos at 24 hpf (light-blue arrowheads), and
  • FIGs. 1H-T show that lymphatic progenitors originate in the vPCV.
  • Figures 1H-K are snapshots from a time-lapse sequence of a Tg (flil:nEGFP) y? zebrafish embryo, showing the origin of a PAC cell (yellow) in the vPCV;
  • Figures 1L-M are snapshots of dPCV (1M)-, and vPCV (1L)- Kaede photoconverted cells at 48 hpf;
  • Figures lN-0 are snapshots of Kaede-pho to switched "medial" (IN)-, and "early lateral" (10)- angioblasts;
  • Figure IT is a quantification of symmetric and asymmetric division events in the vPCV
  • FIGs. 2A-Q show that vPCV cells are specialized angioblasts.
  • Figures 2A-D are snapshots from a time-lapse movie of a Tg(flil :gal4;uasKaede) embryo showing photoswitched-vPCV cell (light-blue arrowhead), which generates PACs (white arrowhead) through asymmetric division;
  • Figures 2E-G show a single photoswitched- vPCV cell in Tg(flil :gal4;uas Kaede) embryo at 24 hpf (light-blue arrowhead), whose progeny populates the SIA (white arrows) and SIV (yellow arrows) at 56 hpf;
  • Figures 2H- I show the location of vPCV-, and dPCV- progeny at 56 hpf, following photo switching at different stages (n pho to S witched-vPCv n pho to S witched-dPC
  • FIGs. 3A-K show cell proliferation in the dPCV, vPCV and DA.
  • Figures 3A-H show a phospho-histone H3 staining illustrating no difference in the number of proliferative ECs among the DA, dPCV and vPCV
  • FIGs. 4A-H show additional fates of vPCV cells.
  • Figure 4 A is a schematic representation of the Subintestinal plexus at 72 hpf.
  • Subintestinal Vein (SIV, green) interconnecting SI vessels (purple), Supraintestinal Artery (SIA, pink), Posterior Cardinal Vein (PCV, blue), Dorsal Aorta (DA, red);
  • Figures 4B-C show quantification of the number of Intersegmental Arteries (ISA) and Intersegmental Veins (ISV) in the first four segments of Tg(fltl_9a_cFos:GFP; lyvel:dsRed2) double transgenic embryos IS# denotes the position of Intersegmental Vessel;
  • Figures 4D-E are confocal images of Tg(lyvel:dsRed2) (4D) and Tg(fltl _9a_cFos:GFP ; lyvel:dsRed
  • FIGs. 5A-D show global expression profiling via RNASeq.
  • Figure 5 A is a schematic illustration of an experimental setup used for RNASeq analysis of FACS isolated vPCV and dPCV cells;
  • Figure 5B shows FACS isolation of green versus red (photoconverted) ECs from Tg(flil :gal4;uasKaede) embryos following photoswitching of dorsal or ventral PCV
  • Figure 5D shows Gene Ontology enrichment in vPCV versus dPCV cells (results represent 2 out of 4 independent biological repeats). Error bars, geometrical mean + s.e.m.
  • FIGs. 6A-H show that LEC specification is induced in the vPCV angioblasts.
  • Figures 6A-E show that a (proxla:TagRFP-flil:EGFP) + cell in the vPCV (yellow) generates a daughter cell that translocates dorsally, buds from the PCV and forms PACs (white arrowheads);
  • Figure 6G shows Proxl immuno staining at 24 hpf illustrating expression in vPCV cell (light-blue arrowheads);
  • Figure 6H shows a quantification of (fltl_9a:GFP;proxla:TagRFP) + cells
  • FIGs. 7A-L show that Wnt5b is necessary and sufficient for LEC specification.
  • Figures 7A-B are histological sections at 22 and 24 hpf, depicting the position of the vPCV angioblasts (light-blue arrows) and the endoderm (depicted by the letter 'e');
  • Figure 7C is a schematic illustration of the Posterior Cardinal Vein (PCV), the ventral side of the PCV (vPCV) and the endoderm (depicted by the letter 'e');
  • Figure 7E shows an in situ hybridization at 18 hpf illustrating expression of wnt5b mRNA in the endoderm
  • FIGS. 7G-I show that Wnt5b overexpression in Tg(hsp70l:wnt5b-GFP;lyvel:dsRed2) embryos induces ectopic lymphangiogenesis
  • FIGs. 8A-M show that Wnt5b is required during lymphatic development.
  • Figures 8A-B show PAC-containing segments in WT (arrowhead) and cas mutants (asterisks);
  • Figures 8C-D show in situ hybridization at 20 hpf illustrating expression of wnt5b mRNA (blue arrowhead) in the endoderm of WT embryos;
  • Figures 8E-F show PAC-containing segments in UI (arrowhead) and wnt5b MO-injected embryos (asterisks);
  • Figure 8G is a graph illustrating ppt mutants injected with wnt5b MO (subdose) which display significant reduction in PAC number ppti wnt 5b MO
  • Figures 8H-J show wnt5b morphants which exhibit marked reduction in the number of TD-containing segments (asterisks) as compared to uninjected
  • FIGs. 9A-H show specificity of Wnt5b role in lymphatic specification.
  • Figures 9D-E and 9G-H 60 ⁇ . Error bars mean + s.e.m.
  • FIGs. 10A-U show that loss of Wnt5b specifically affects lymphatic specification.
  • Figures 10A-D show selected frames from a time-lapse sequence of a Tgiflil :gal4;uasKaede) embryo injected with wnt5b MO.
  • Photoconverted vPCV cell (white arrow) divides normally (arrows at 48 hpf point to 2 daughter cells), but does not engage in dorsal migration to generate PACs;
  • Figures 10E-L show in situ hybridization of Ctrl MO-, and wnt5b MO-injected zebrafish at 30 hpf, with lyvel, soxl8, nr2f2 and cdh5 probes, showing specific decrease in lymphatic marker expression in the floor of the PCV (white arrowheads) of wnt5b morphants.
  • pan-endothelial marker- cdh5- as well as the arterial expression of soxl8, remain unchanged in wnt5b morphants;
  • Figures 10M-R show that vegfc and ccbel mRNA levels remain unaltered in sox32-, and wnt5b- morphants;
  • Figures 10S-T show an immuno staining of Proxl illustrating significant increase in protein levels following ectopic activation of Wnt5b in Tg(hsp70l:wnt5b; flil.EGFP) embryos (co-localization channel is shown in yellow, white arrowheads);
  • FIGs. 11A-H show that Wnt5b induces lymphatic specification in zebrafish and hESCs.
  • Figure HE shows proxla mRNA levels in 24 hpf Tg(flil:EGFP;proxla:KalT4-UAS:uncTagRFP;hsp70l:wnt5b-GFP) embryos following heat-shock at 21 hpf
  • Figures 11F-G show fraction of LYVE1 + cells; and
  • Figure 11H shows PROXl mRNA levels in hESC-derived angioblasts treated with WNT5B Scale bar 60 ⁇ .
  • FIGs. 12A-H show that Wnt5b controls lymphatic specification through activation of the canonical pathway.
  • FIGs. 13 A- J show that Wnt5b controls lymphatic specification through activation of the canonical pathway.
  • Figures 13B-F show quantification of PAC-containing segments in the trunk of UI, tcf7, lefl and tcf4 MO-injected embryos 3, niefi-MO and
  • Figures 13G-J show photoswitching of vPCV cells in tcf7 MO-injected Tg(flil :gal4;uasKaede) embryos (white arrowheads) at 24 hpf.
  • At 48 hpf photoconverted-, red vPCV cells (arrowheads) remain in the PCV and do not generate PACs.
  • Scale bar 30 ⁇ . Error bars, mean + s.e
  • FIGs. 14A-G show that Wnt5b induces LEC specification through activation of the canonical pathway.
  • Figures 14A-B show Tg(7xTCF-Xla.Siam:nlsmCherryflil:EGFP) embryo illustrating TCF activity in vPCV cells at 24 hpf (light-blue arrowheads), and PACs at 48 hpf (white arrows);
  • Figure 14C shows Tg(7xTCF- Xla.Siam:nlsmCherryfltl_9a_cFos:GFP) embryo illustrating TCF activity in fltl_9a + vPCV angioblasts at 24 hpf (light-blue arrowheads);
  • Figure 14D shows Kalt4 mRNA levels in Tg(proxla:KalT4-UAS:uncTagRFP;hsp70l:wnt5b-GFP) 24 hpf embryos, following
  • FIGs. 15A-H show specific activation of TCF in lymphatic progenitors.
  • FIGs. 16A-C are schematical illustrations of the Wnt canonical and non-canonical pathways.
  • Canonical pathway Figure 16A
  • non-canonical PCP pathway Figure 16B
  • non-canonical Wnt/calcium pathway Figure 16C
  • the present invention in some embodiments thereof, relates to methods of promoting lymphangiogenesis and, more particularly, but not exclusively, to use of the Wnt pathway therefore.
  • a fundamental question in developmental and regenerative biology is how cells acquire their fate. Multipotent progenitors undergo cell-fate restriction in response to cues from the microenvironment, the nature of which is poorly understood. In the case of the lymphatic system, venous cells from the cardinal vein (CV) are thought to generate lymphatic vessels, through trans-differentiation.
  • CV cardinal vein
  • the present inventors While reducing the present invention to practice, the present inventors have uncovered that the canonical and non-canonical Wnt pathways are involved in different stages of lymphangiogenesis. Specifically, the present inventors have uncovered that the Wnt canonical pathway is involved in lymphatic endothelial cell (LEC) specification while the non-canonical Wnt pathway is involved in additional steps of lymphatic morphogenesis and remodeling. This elucidation paves the way towards the generation of lymphatic vessels in vitro and for the treatment of lymphatic disorders or diseases in vivo.
  • LEC lymphatic endothelial cell
  • lymphatic progenitors arise from a previously uncharacterized niche of specialized angioblasts within the floor of the Posterior Cardinal Vein (PCV), which bears the potential to also generate arterial and venous fates (see results in zebrafish embryos, Examples 1 and 2 of the Examples section which follows).
  • PCV Posterior Cardinal Vein
  • these cells develop in close proximity to the endoderm, which serves as source of Wnt5b - a novel inductive signal promoting the angioblast-to-lymphatic transition (see Example 3 of the Examples section which follows).
  • Wnt5b as a lymphatic inductive signal that promotes the "angioblast-to-lymphatic" transition in human embryonic stem cells (see Example 3 of the Examples section which follows).
  • Wnt5b induces LEC specification through activation of Wnt canonical pathway (see Example 4 of the Examples section which follows). Taken together, these results substantiate the value of upregulating the Wnt signaling pathways for promoting lymphangiogenesis.
  • lymphatic endothelial cell (LEC) specification comprising upregulating the activity of Wnt canonical pathway in stem cells and/or vascular progenitor cells and/or angioblasts, thereby promoting LEC specification.
  • LEC specification as used herein is interchangeably used with the terms “LEC differentiation” or “determination” and refers to the process by which stem cells and/or vascular progenitor cells and/or angioblasts change into lymphatic endothelial cells (LECs). Change into LECs may be indicated, for example, by changes in marker expression, gene expression, biochemistry, structure and/or function of the cells, e.g. LECs typically express remarkably high levels of specific transcription factors and receptors, such as, but not limited to, proxl, lyvel, podoplanin and vegfr3. Markers of LECs are described in detail below.
  • lymphatic endothelial cell refers to a cell which makes up the lymphatic endothelium (LE) that lines the lymphatic vascular system, and expresses typical LEC markers including, but not limited to, the intracellular expression of Proxl, lymphatic vessel endothelial hyaluronan receptor-1 (lyvel), VEGFR- 3, flt4, soxl8, nr2f2, neuropilin 2 (Nrp2), forkhead box C2 (Foxc2), podoplanin (Pdpn), angiopoietin 2 (Ang2), ephrin B2 (Efnb2), cxcr4, activin receptor-like kinase 1 (ALK-1), activin receptor-like kinase 2 (ALK-2), protein phosphatase 1 regulatory (inhibitor) subunit 13B (Asppl) and/or core 1 synthase glycoprotein-N-ace
  • LEC lymphatic endothelium
  • LECs typically also produce chemokines, including but not limited to, CCL21.
  • LECs express lyvel, proxl, Pdpn and/or VEGFR-3. Determination of LECs may be carried out using any method known in the art, e.g. by FACS analysis, by PCR or by ELISA, selecting cells expressing the LEC markers as described in detail above.
  • a "LEC committed cell” is a cell, which has been specified particularly into a LEC fate.
  • stem cells refers to cells which are capable of remaining in an undifferentiated state (e.g., totipotent, pluripotent or multipotent stem cells) for extended periods of time in culture until induced to differentiate into other cell types having a particular, specialized function (e.g., fully differentiated cells).
  • Totipotent cells such as embryonic cells within the first couple of cell divisions after fertilization are the only cells that can differentiate into embryonic and extra-embryonic cells and are able to develop into a viable human being.
  • pluripotent or multipotent stem cells refers to cells which can differentiate into all three embryonic germ layers, i.e.
  • the pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS).
  • the multipotent stem cells include adult stem cells and hematopoietic stem cells.
  • embryonic stem cells refers to embryonic cells which are capable of differentiating into cells of all three embryonic germ layers (i.e. , endoderm, ectoderm and mesoderm), or remaining in an undifferentiated state.
  • embryonic stem cells may comprise cells which are obtained from the embryonic tissue formed after gestation (e.g., blastocyst) before implantation of the embryo (i.e., a pre-implantation blastocyst), extended blastocyst cells (EBCs) which are obtained from a post-implantation/pre- gastrulation stage blastocyst (see WO2006/040763), embryonic germ (EG) cells which are obtained from the genital tissue of a fetus any time during gestation, preferably before 10 weeks of gestation, and cells originating from an unfertilized ova which are stimulated by parthenogenesis (parthenotes).
  • gestation e.g., blastocyst
  • EBCs extended blastocyst cells
  • EG embryonic germ
  • Induced pluripotent stem cells are cells obtained by de-differentiation of adult somatic cells which are endowed with pluripotency (i.e., being capable of differentiating into the three embryonic germ cell layers, i.e., endoderm, ectoderm and mesoderm).
  • pluripotency i.e., being capable of differentiating into the three embryonic germ cell layers, i.e., endoderm, ectoderm and mesoderm.
  • such cells are obtained from a differentiated tissue (e.g., a somatic tissue such as skin) and undergo de-differentiation by genetic manipulation, which re-program the cell to acquire embryonic stem cells characteristics.
  • the induced pluripotent stem cells are formed by inducing the expression of Oct-4, Sox2, Kfl4 and c-Myc in a somatic stem cell.
  • adult stem cells also called “tissue stem cells” or a stem cell from a somatic tissue refers to any stem cell derived from a somatic tissue [of either a postnatal or prenatal animal (especially the human)] .
  • the adult stem cell is generally thought to be a multipotent stem cell, capable of differentiation into multiple cell types.
  • Adult stem cells can be derived from any adult, neonatal or fetal tissue such as adipose tissue, skin, kidney, liver, prostate, pancreas, intestine, bone marrow and placenta.
  • Hematopoietic stem cells which may also referred to as adult tissue stem cells, include stem cells obtained from blood or bone marrow tissue of an individual at any age or from cord blood of a newborn individual.
  • Preferred stem cells according to this aspect of some embodiments of the invention are embryonic stem cells, preferably of a human or primate (e.g., monkey) origin.
  • Human hematopoietic stem cells (HSCs) are typically positive for the expression of the markers CD34, CD59 and/or Thyl/CD90 and may be positive for expression of CD38 and/or C-kit/CDl 17.
  • Placental and cord blood stem cells may also be referred to as "young stem cells”.
  • the embryonic stem cells of some embodiments of the invention can be obtained using well-known cell-culture methods.
  • human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or from in vitro fertilized (IVF) embryos. Alternatively, a single cell human embryo can be expanded to the blastocyst stage.
  • the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by immunosurgery, in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting.
  • ICM inner cell mass
  • the ICM is then plated in a tissue culture flask containing the appropriate medium which enables its outgrowth. Following 9 to 15 days, the ICM derived outgrowth is dissociated into clumps either by a mechanical dissociation or by an enzymatic degradation and the cells are then re-plated on a fresh tissue culture medium. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and re-plated. Resulting ES cells are then routinely split every 4-7 days. For further details on methods of preparation human ES cells see Thomson et al., [U.S. Pat. No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol.
  • ES cells can also be used according to some embodiments of the invention.
  • Human ES cells can be purchased from the NIH human embryonic stem cells registry [Hypertext Transfer Protocol://grants (dot) nih (dot) gov/stem_cells/registry/current (dot) htm].
  • Non-limiting examples of commercially available embryonic stem cell lines are BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, HUES 1, HUES 2, HUES 3, HUES 4, HUES 5, HUES 6, HUES 7, HUES 8, HUES 9, HUES 10, HUES 11, HUES 12, HUES 13, HUES 14, HUES 15, HUES 16, HUES 17, HUES 18, HUES 19, HUES 20, HUES 21, HUES 22, HUES 23, HUES 24, HUES 25, HUES 26, HUES 27, HUES 28, CyT49, RUES 3, WA01, UCSF4, NYUES 1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, UCLA 1, UCLA 2, UCLA 3, WA077 (H7)
  • ES cells can be obtained from other species as well, including mouse
  • EBCs Extended blastocyst cells
  • EBCs can be obtained from a blastocyst of at least nine days post fertilization at a stage prior to gastrulation.
  • the zona pellucida Prior to culturing the blastocyst, the zona pellucida is digested [for example by Tyrode' s acidic solution (Sigma Aldrich, St Louis, MO, USA)] so as to expose the inner cell mass.
  • the blastocysts are then cultured as whole embryos for at least nine and no more than fourteen days post fertilization (i.e., prior to the gastrulation event) in vitro using standard embryonic stem cell culturing methods.
  • EG cells are prepared from the primordial germ cells obtained from fetuses of about 8-11 weeks of gestation (in the case of a human fetus) using laboratory techniques known to anyone skilled in the arts.
  • the genital ridges are dissociated and cut into small chunks which are thereafter disaggregated into cells by mechanical dissociation.
  • the EG cells are then grown in tissue culture flasks with the appropriate medium.
  • the cells are cultured with daily replacement of medium until a cell morphology consistent with EG cells is observed, typically after 7-30 days or 1-4 passages.
  • Shamblott et al. [Proc. Natl. Acad. Sci. USA 95: 13726, 1998] and U.S. Pat. No. 6,090,622.
  • Embryonic stem cells e.g., human ESCs
  • parthenogenesis e.g., Zhenyu Lu et al., 2010. J. Assist Reprod. Genet. 27:285-291; "Derivation and long-term culture of human parthenogenetic embryonic stem cells using human foreskin feeders", which is fully incorporated herein by reference).
  • Parthenogenesis refers to the initiation of cell division by activation of ova in the absence of sperm cells, for example using electrical or chemical stimulation.
  • the activated ovum (parthenote) is capable of developing into a primitive embryonic structure (called a blastocyst) but cannot develop to term as the cells are pluripotent, meaning that they cannot develop the necessary extra-embryonic tissues (such as amniotic fluid) needed for a viable human fetus.
  • iPS Induced pluripotent stem cells
  • somatic cells can be generated from somatic cells by genetic manipulation of somatic cells, e.g., by retroviral transduction of somatic cells such as fibroblasts, hepatocytes, gastric epithelial cells with transcription factors such as Oct-3/4, Sox2, c-Myc, and KLF4 [Yamanaka S, Cell Stem Cell. 2007, l(l):39-49; Aoi T, et al., Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells. Science. 2008 Feb 14. (Epub ahead of print); IH Park, Zhao R, West JA, et al.
  • Fetal stem cells can be isolated using various methods known in the art such as those disclosed by Eventov-Friedman S, et al., PLoS Med. 2006, 3: e215; Eventov-Friedman S, et al., Proc Natl Acad Sci U S A. 2005, 102: 2928-33; Dekel B, et al., 2003, Nat Med. 9: 53-60; and Dekel B, et al., 2002, J. Am. Soc. Nephrol. 13: 977-90.
  • Hematopoietic stem cells can be isolated using various methods known in the arts such as those disclosed by "Handbook of Stem Cells” edit by Robert Lanze, Elsevier Academic Press, 2004, Chapter 54, pp609-614, isolation and characterization of hematopoietic stem cells, by Gerald J Spangrude and William B Stayton.
  • adult tissue stem cells are based on the discrete location (or niche) of each cell type included in the adult tissue, i.e., the stem cells, the transit amplifying cells and the terminally differentiated cells [Potten, C. S. and Morris, R. J. (1988). Epithelial stem cells in vivo. J. Cell Sci. Suppl. 10, 45-62].
  • an adult tissue such as, for example, prostate tissue is digested with Collagenase and subjected to repeated unit gravity centrifugation to separate the epithelial structures of the prostate (e.g., organoids, acini and ducts) from the stromal cells.
  • Organoids are then disaggregated into single cell suspensions by incubation with Trypsin/EDTA (Life Technologies, Paisley, UK) and the basal, CD44-positive, stem cells are isolated from the luminal, CD57-positive, terminally differentiated secretory cells, using anti-human CD44 antibody (clone G44-26; Pharmingen, Becton Dickinson, Oxford, UK) labeling and incubation with MACS (Miltenyi Biotec Ltd, Surrey, UK) goat anti-mouse IgG microbeads.
  • MACS Miltenyi Biotec Ltd, Surrey, UK
  • the cell suspension is then applied to a MACS column and the basal cells are eluted and re- suspended in WAJC 404 complete medium [Robinson, E.J. et al. (1998). Basal cells are progenitors of luminal cells in primary cultures of differentiating human prostatic epithelium Prostate 37, 149-160].
  • basal stem cells can adhere to basement membrane proteins more rapidly than other basal cells [Jones, P.H. et al. (1995). Stem cell patterning and fate in human epidermis. Cell 60, 83-93; Shinohara, T., et al. (1999). ⁇ - and a6-integrin are surface markers on mouse spermatogonial stem cells. Proc. Natl. Acad. Sci.
  • the CD44 positive basal cells are plated onto tissue culture dishes coated with either type I collagen (52 ⁇ g/ml), type IV collagen (88 ⁇ g/ml) or laminin 1 (100 ⁇ g/ml; Biocoat®, Becton Dickinson) previously blocked with 0.3 % bovine serum albumin (fraction V, Sigma-Aldrich, Poole, UK) in Dulbecco's phosphate buffered saline (PBS; Oxoid Ltd, Basingstoke, UK). Following 5 minutes, the tissue culture dishes are washed with PBS and adherent cells, containing the prostate tissue basal stem cells are harvested with trypsin-EDTA.
  • type I collagen 52 ⁇ g/ml
  • type IV collagen 88 ⁇ g/ml
  • laminin 1 100 ⁇ g/ml
  • Biocoat® Becton Dickinson
  • BM-derived stem cell BM-derived stem cell, mesenchymal stem cells
  • the stem cells utilized by some embodiments of the invention are BM-derived stem cells including hematopoietic, stromal or mesenchymal stem cells (Dominici, M et al., 2001. Bone marrow mesenchymal cells: biological properties and clinical applications. J. Biol. Regul. Homeost. Agents. 15: 28-37).
  • BM- derived stem cells may be obtained from iliac crest, femora, tibiae, spine, rib or other medullar spaces.
  • mesenchymal stem cells are the formative pluripotent blast cells, and as such are preferred for use with some embodiments of the invention.
  • Mesenchymal stem cells give rise to one or more mesenchymal tissues (e.g., adipose, osseous, cartilaginous, elastic and fibrous connective tissues, myoblasts) as well as to tissues other than those originating in the embryonic mesoderm (e.g., neural cells) depending upon various influences from bioactive factors such as cytokines.
  • MSCs mesenchymal stem cells
  • mesenchymal stem cell cultures are generated by diluting BM aspirates (usually 20 ml) with equal volumes of Hank's balanced salt solution (HBSS; GIBCO Laboratories, Grand Island, NY, USA) and layering the diluted cells over about 10 ml of a Ficoll column (Ficoll-Paque; Pharmacia, Piscataway, NJ, USA). Following 30 minutes of centrifugation at 2,500 x g, the mononuclear cell layer is removed from the interface and suspended in HBSS.
  • HBSS Hank's balanced salt solution
  • MSC complete medium
  • FCS fetal calf serum
  • Resuspended cells are plated in about 25 ml of medium in a 10 cm culture dish (Corning Glass Works, Corning, NY) and incubated at 37 °C with 5 % humidified C0 2 . Following 24 hours in culture, nonadherent cells are discarded, and the adherent cells are thoroughly washed twice with phosphate buffered saline (PBS). The medium is replaced with a fresh complete medium every 3 or 4 days for about 14 days. Adherent cells are then harvested with 0.25 % trypsin and 1 mM EDTA (Trypsin/EDTA, GIBCO) for 5 min at 37 °C, replated in a 6-cm plate and cultured for another 14 days.
  • Trypsin/EDTA GIBCO
  • Cells are then trypsinized and counted using a cell counting device such as for example, a hemocytometer (Hausser Scientific, Horsham, PA). Cultured cells are recovered by centrifugation and resuspended with 5 % DMSO and 30 % FCS at a concentration of 1 to 2 X 10 6 cells per ml. Aliquots of about 1 ml each are slowly frozen and stored in liquid nitrogen.
  • a cell counting device such as for example, a hemocytometer (Hausser Scientific, Horsham, PA).
  • mesenchymal stem cell fraction frozen cells are thawed at 37 °C, diluted with a complete medium and recovered by centrifugation to remove the DMSO. Cells are resuspended in a complete medium and plated at a concentration of about 5,000 cells/cm . Following 24 hours in culture, nonadherent cells are removed and the adherent cells are harvested using Trypsin/EDTA, dissociated by passage through a narrowed Pasteur pipette, and preferably replated at a density of about 1.5 to about 3.0 cells/cm . Under these conditions, MSC cultures can grow for about 50 population doublings and be expanded for about 2000 fold [Colter DC, et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA. 97: 3213-3218, 2000].
  • MSC cultures utilized by some embodiments of the invention preferably include three groups of cells which are defined by their morphological features: small and agranular cells (referred to as RS-1, hereinbelow), small and granular cells (referred to as RS-2, hereinbelow) and large and moderately granular cells (referred to as mature MSCs, hereinbelow).
  • RS-1 small and agranular cells
  • RS-2 small and granular cells
  • mature MSCs large and moderately granular cells
  • MSCs When MSCs are cultured under the culturing conditions of some embodiments of the invention they exhibit negative staining for the hematopoietic stem cell markers CD34, CDl lB, CD43 and CD45. A small fraction of cells (less than 10 %) are dimly positive for CD31 and/or CD38 markers. In addition, mature MSCs are dimly positive for the hematopoietic stem cell marker, CD 117 (c-Kit), moderately positive for the osteogenic MSCs marker, Stro-1 [Simmons, P. J. & Torok-Storb, B. (1991). Blood 78, 5562] and positive for the thymocytes and peripheral T lymphocytes marker, CD90 (Thy-1). On the other hand, the RS-1 cells are negative for the CD 117 and Strol markers and are dimly positive for the CD90 marker, and the RS-2 cells are negative for all of these markers.
  • undifferentiated stem cells are of a distinct morphology, which is clearly distinguishable from differentiated cells of embryo or adult origin by the skilled in the art. Typically, undifferentiated stem cells have high nuclear/cytoplasmic ratios, prominent nucleoli and compact colony formation with poorly discemable cell junctions. Additional features of undifferentiated stem cells are further described hereinunder.
  • mesodermal progenitor cells refers to the population of cells that exhibit both mesenchymal and endothelial differentiation potential. According to one embodiment, mesodermal progenitor cells are capable of differentiating into mesodermal angioblasts and endothelial cells (EC) under specific conditions.
  • Mesodermal progenitor cells can be selected by the phenotype CD105 + SSEA-4 + CD90 neg and typically lack the markers, MSCA-1, CD166, CD271, W5B5, and CD146. MPCs typically express the pluripotency-associated marker SSEA-4 and the nuclear factors Oct-3/4 and Nanog.
  • Mesodermal progenitor cells may be obtained, generated and/or selected by any method known to one of skill in the art, for example, those disclosed in U.S. Patent Application No. 20100150887, incorporated herein by reference.
  • Another protocol to harvest mesodermal progenitor cells (MPCs) e.g. from human bone marrow mono-nucleate cells (BMMNCs) supplemented with autologous serum can be found in Trombi L. et al. [Trombi L. et al. (2009) Selective culture of mesodermal progenitor cells. Stem Cells Dev 18(8): 1227-1234 and Petrini M. et al., Stem Cells and Development (2009) 18(6): 857- 866, both incorporated herein by reference].
  • vascular progenitor cells refers to adult stem cells which are capable of differentiating into endothelial lineage.
  • Vascular progenitor cells can be induced to differentiate into endothelial progenitor cells and endothelial cells (EC) under specific conditions.
  • Vascular progenitor cells can be selected by expression of characteristic markers, such as but not limited to, CD34, CD31 (PECAM-1), CD144, CD 146, CD 105, PDGFR-beta, KDR and/or ETV2.
  • characteristic markers such as but not limited to, CD34, CD31 (PECAM-1), CD144, CD 146, CD 105, PDGFR-beta, KDR and/or ETV2.
  • vascular progenitor cells do not express CD133.
  • endothelial progenitor cells refer to cells of the endothelial lineage capable of differentiating into mature endothelial cells, for example, into blood vessel endothelial cells.
  • EPCs include monocytic EPCs and hemangioblastic EPCs.
  • Endothelial progenitor cells may be obtained by any method known to one of skill in the art, for example, those disclosed in U.S. Patent Application No. 20130224116, incorporated herein by reference.
  • EPCs may be selected for expression of any of sphingosine kinase 1 (SK-1), CD34, CD14, CD133, CD45, CD31 and/or VEGFR2.
  • EPCs may not express significant or above background levels of CD 144 and/or vWF and/or eNOS and/or Tie2.
  • EPCs may produce pro-angiogenic factors, e.g., hepatocyte growth factor and/or insulin-like growth factor- 1 and/or basic fibroblast growth factor and/or VEGF.
  • EPCs do not adhere to tissue culture plastic-ware, optionally plastic-ware coated with extracellular matrix or a component thereof (e.g., fibronectin).
  • EPCs may be isolated from 4-7 day cultured non-adherent CD133 expressing mononuclear cells.
  • angioblasts refers to cells of the embryonic mesenchymal tissue committed to endothelial lineage differentiation.
  • Angioblasts are capable of differentiating into endothelial progenitor cells and endothelial cells (EC) under specific conditions.
  • angioblasts can be selected by expression of VEGFR2, CD31, CD133/1 and/or CD117. They may also express the transcription factor GATA-2 and/or ETV2.
  • angioblasts refers to multipotent angioblasts (e.g. meso-angioblasts or mesodermal angioblasts, i.e. multipotent, self- renewing cells that differentiate into most mesodermal tissues). Such cells can be identified by expression of the marker Flk-1, and optionally ETV2.
  • multipotent angioblasts e.g. meso-angioblasts or mesodermal angioblasts, i.e. multipotent, self- renewing cells that differentiate into most mesodermal tissues.
  • Such cells can be identified by expression of the marker Flk-1, and optionally ETV2.
  • Angioblasts can be obtained by any method known in the art, for example, angioblasts may be obtained from stem cells in the bone marrow. According to one embodiment, the angioblasts are bone marrow derived angioblasts, as described by Kocher A et al., Nat Med. (2001) 7(4):430-6.
  • the angioblasts are angioblast-like progenitor cells derived from fibroblasts as taught by Kurian L. et al. Nature Methods 10, 77-83 (2013), incorporated herein by reference. Other methods of obtaining angioblasts are described in Yamashita, J. et al. Nature 408: 92-96 (2000) and in Kusuma, S. et al., Proceedings of the National Academy of Sciences of the United States of America 110: 12601-12606 (2013), both incorporated herein by reference.
  • promoting LEC specification in stem cells or vascular progenitor cells and/or angioblasts so as to obtain a LEC committed cell is effected by upregulating the activity of Wnt canonical pathway in the stem cells or vascular progenitor cells and/or angioblasts.
  • Wnt canonical pathway refers to the Wnt signaling pathway which activates transcription of a TCF regulated gene (i.e., a gene comprising a Tcf/LEF consensus binding site in its promoter), including TCF1, TCF2, TCF3, LEF1 and LEF2.
  • a TCF regulated gene i.e., a gene comprising a Tcf/LEF consensus binding site in its promoter
  • TCF1, TCF2, TCF3, LEF1 and LEF2 i.e., Tcf/LEF consensus binding site in its promoter
  • activation of the Wnt signaling pathway results in the activation of transcription of a Wnt target gene.
  • Wnt target genes are described in further detail below.
  • the pathway involves signaling from Wnt receptors (e.g.
  • Frizzleds to ⁇ -catenin.
  • the activation of the Wnt canonical signaling pathway results in an increase in activity of ⁇ -catenin, such as an accumulation of active ⁇ -catenin in the cytoplasm, such as an increase in the amount of phosphorylated ⁇ -catenin in the cytoplasm.
  • the canonical pathway functions through the activation of the Wnt receptor to deactivate the destruction complex-GSK3, Axin, APC and CKla and to stabilize ⁇ -catenin so as to mediate its function in transcription.
  • Wnt signaling begins when one of the Wnt proteins binds to a Frizzled (Fz) family receptor, a G-protein coupled receptors (GPCRs).
  • Fz Frizzled
  • GPCRs G-protein coupled receptors
  • co-receptors may also be required alongside the interaction between the Wnt protein and Fz receptor.
  • co-receptors include, for example, the lipoprotein receptor-related protein (LRP)-5/6, the receptor tyrosine kinase (Ryk), or ROR2.
  • LRP lipoprotein receptor-related protein
  • Ryk receptor tyrosine kinase
  • ROR2 phosphoprotein Dishevelled
  • Dsh then becomes phosphorylation and activated and inhibits the activity of GSK3 of the destruction complex (described in detail below).
  • This allows ⁇ -catenin to accumulate and localize to the nucleus and subsequently induce a cellular response via gene transduction alongside the TCF/LEF (T-cell factor/lymphoid enhancing factor) transcription factors. Without Wnt signaling, ⁇ -catenin does not accumulate in the cytoplasm since the destruction complex degrades it.
  • This destruction complex includes the following proteins: Axin, adenomatosis polyposis coli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3, e.g.
  • GSK ⁇ GSK ⁇
  • CKla casein kinase la
  • composition which acts to upregulate activity or expression of ⁇ - catenin is contemplated herein.
  • the activity of Wnt canonical signaling in the stem cells or vascular progenitor cells or angioblasts may be increased by 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 % or 100 % or more, as compared to stem cells or vascular progenitor cells or angioblasts not treated by a composition which acts to upregulate Wnt canonical signaling activity, to effect differentiation of the stem cells or vascular progenitor cells or angioblasts towards LEC.
  • the activity or activation of the Wnt canonical signaling pathway may be assayed using any method known to one of skill in the art, such as described below.
  • upregulating the activity of Wnt canonical pathway is effected by contacting the stem cells and/or vascular progenitor cells and/or angioblasts with an activator of a Wnt canonical pathway member, including but not limited to, Wnt5b, a Wnt3a, a Wnt7a, a Wnt7b, a LRP5/6, a ⁇ -catenin, Dishevelled (Dsh), PP2A, ARFGAP1, and TCF.
  • a Wnt canonical pathway member including but not limited to, Wnt5b, a Wnt3a, a Wnt7a, a Wnt7b, a LRP5/6, a ⁇ -catenin, Dishevelled (Dsh), PP2A, ARFGAP1, and TCF.
  • Upregulation of the Wnt canonical pathway can be effected at the protein or nucleic acid level, as summarized infra.
  • upregulating the activity of Wnt canonical pathway is effected by down-regulation of any antagonist or negative regulator or component of the Wnt canonical pathway.
  • exemplary antagonist or negative regulator or component of the Wnt pathway include, but are not limited to, Axin, glycogen synthase kinase 3 (GSK3, e.g. GSK3p), APC and sFRP (Secreted frizzled-related proteins).
  • Down-regulation can be effected on the genomic and/or the transcript level as summarized in further detail hereinbelow.
  • Wnt non-canonical pathway refers to the ⁇ -catenin- independent signaling pathway. Typically this pathway can be divided into two distinct branches, the Planar Cell Polarity pathway (or PCP pathway) and the Wnt/Ca 2+ pathway.
  • the Wnt non-canonical pathway is also illustrated in Figures 16B-C (PCP and Wnt/calcium pathways, respectively).
  • PCP pathway Planar Cell Polarity pathway
  • Wnt/calcium pathways respectively.
  • signaling is transduced through Fzd independent of LRP5/6 leading to the activation of Dsh.
  • Dsh through Daaml mediates activation of Rho which in turn activates Rho kinase (ROCK). Daaml also mediates actin polymerization through the actin binding protein Profilin. Dsh also mediates activation of Rac, which in turn activates JNK.
  • ROCK Rho kinase
  • Any means for increasing Wnt non-canonical signaling activity may be used, including both direct and indirect modulation. These may include for example, modulating the expression of any endogenous gene for any member of the Wnt non-canonical signaling pathway (described in further detail below) at the transcriptional, translational or post-translational level, such as modulating the persistence or breakdown of messenger RNA for the member of the Wnt non-canonical signaling pathway, modulating the persistence or breakdown of protein, etc. They may also include modulation of the activity of a member of the Wnt non-canonical signaling pathway, such as by use of agonists thereof. Furthermore, the expression and/or activity of activators of any member of the Wnt non-canonical signaling pathway may be modulated to upregulate Wnt non-canonical signaling activity. These are described in further detail below.
  • the activity of Wnt non-canonical signaling in LEC committed cells may be increased by 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 % or 100 % or more, as compared to LEC committed cells not effected by a LEC morphogenesis signal, to effect morphogenesis of the LEC committed cell.
  • the activity or activation of the Wnt non-canonical signaling pathway may be assayed using any method known to one of skill in the art, such as described below.
  • upregulating the activity of Wnt non-canonical pathway is effected by contacting a LEC with an activator of a Wnt non-canonical pathway member, including but not limited to, Wnt5a, Wnt4, Wntl l, Ror2, Ryk, G protein, Dishevelled (Dsh) and Dishevelled-associated activator of morphogenesis 1 (DAAM1).
  • a Wnt non-canonical pathway member including but not limited to, Wnt5a, Wnt4, Wntl l, Ror2, Ryk, G protein, Dishevelled (Dsh) and Dishevelled-associated activator of morphogenesis 1 (DAAM1).
  • Upregulation of the Wnt non-canonical pathway can be effected at the protein or nucleic acid level, as summarized infra.
  • Upregulation of the activity of Wnt canonical or non-canonical pathways can be effected at the genomic level (e.g., polynucleotide sequences encoding the expression product of interest and/or activation of transcription via promoters, enhancers, regulatory elements), at the transcript level (i.e., correct splicing, polyadenylation, activation of translation, cDNA introduction) or at the protein level e.g., administering the protein of interest (e.g., soluble protein, e.g., secreted).
  • genomic level e.g., polynucleotide sequences encoding the expression product of interest and/or activation of transcription via promoters, enhancers, regulatory elements
  • the transcript level i.e., correct splicing, polyadenylation, activation of translation, cDNA introduction
  • protein level e.g., administering the protein of interest (e.g., soluble protein, e.g., secreted).
  • An agent capable of upregulating expression of a Wnt canonical or non-canonical pathway component may be an exogenous polynucleotide sequence designed and constructed to express at least a functional portion of a Wnt canonical or non-canonical pathway component [e.g. for Wnt canonical pathway: a Frizzled receptor, an LRP co- receptor, a Wnt ligand, a Dishevelled (Dsh), PP2A, ARFGAPl, dominant stable mutated ⁇ -catenin (e.g.
  • the exogenous polynucleotide sequence may be a DNA or RNA sequence encoding a molecule capable of upregulating the Wnt canonical or non-canonical pathway component (e.g. for Wnt canonical pathway: a Frizzled receptor, a Wnt ligand, a Dishevelled (Dsh), PP2A or ARFGAPl; e.g. for Wnt non-canonical pathway: Dsh, DAAM1, Wnt4, Wnt5a or Wntl l).
  • Wnt canonical pathway e.g. for Wnt canonical pathway: a Frizzled receptor, a Wnt ligand, a Dishevelled (Dsh), PP2A or ARFGAPl
  • Wnt non-canonical pathway Dsh, DAAM1, Wnt4, Wnt5a or Wntl l
  • a Wnt canonical or non-canonical pathway component e.g. for Wnt canonical pathway: a Frizzled receptor, a Wnt ligand, a Dishevelled (Dsh), PP2A or ARFGAPl; e.g. for Wnt non-canonical pathway: Dsh, DAAM1, Wnt4, Wnt5a or Wntl l
  • a Wnt canonical or non-canonical pathway component e.g. for Wnt canonical pathway: a Frizzled receptor, a Wnt ligand, a Dishevelled (Dsh), PP2A or ARFGAPl
  • Dsh Dishevelled
  • PP2A PP2A
  • ARFGAPl e.g. for Wnt non-canonical pathway: Dsh, DAAM1, Wnt4, Wnt5a or Wntl l
  • Wnt canonical or non-canonical pathway components e.g. for Wnt canonical pathway: a Frizzled receptor, a Wnt ligand, a Dishevelled (Dsh), PP2A, ARFGAPl, etc.; e.g. for Wnt non-canonical pathway: Dsh, DAAM1, Wnt4, Wnt5a or Wntl l
  • Wnt canonical or non-canonical pathway component e.g.
  • Wnt canonical pathway a Frizzled receptor, a Wnt ligand, a Dishevelled (Dsh), PP2A or ARFGAPl; e.g. for Wnt non-canonical pathway: Dsh, DAAMl, Wnt4, Wnt5a or Wntl l) is available from several databases including the GenBank database available at the National Center for Biotechnology Information at www(dot)ncbi(dot)nlm(dot)nih(dot)gov/.
  • Wnt canonical or non-canonical pathway component e.g. for Wnt canonical pathway: a Frizzled receptor, a Wnt ligand, a Dishevelled (Dsh), PP2A or ARFGAPl; e.g. for Wnt non-canonical pathway: Dsh, DAAMl, Wnt4, Wnt5a or Wntl l
  • a polynucleotide sequence encoding the Wnt canonical or non-canonical pathway component e.g.
  • a Frizzled receptor a Wnt ligand, a Dishevelled (Dsh), PP2A, ARFGAPl, DAAMl, exemplary GenBank Accession numbers are provided hereinbelow
  • a nucleic acid construct suitable for mammalian cell expression.
  • Such a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.
  • nucleic acid construct of some embodiments of the invention can also utilize homologues which exhibit the desired activity (i.e., promote LEC specification).
  • nucleic acid construct of some embodiments of the invention can also utilize homologues which exhibit the desired activity (i.e., promote LEC specification).
  • Such homologues can be, for example, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical to the desired sequences, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals -9.
  • Constitutive promoters suitable for use with some embodiments of the invention are promoter sequences which are active under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV).
  • Inducible promoters suitable for use with some embodiments of the invention include for example the tetracycline-inducible promoter (Zabala M, et al., Cancer Res. 2004, 64(8): 2799-804).
  • the nucleic acid construct (also referred to herein as an "expression vector") of some embodiments of the invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).
  • typical cloning vectors may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal.
  • such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
  • the nucleic acid construct of some embodiments of the invention typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of some embodiments of the invention.
  • Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements.
  • the TATA box located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis.
  • the other upstream promoter elements determine the rate at which transcription is initiated.
  • the promoter utilized by the nucleic acid construct of some embodiments of the invention is active in the specific cell population transformed.
  • cell type-specific and/or tissue-specific promoters include promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al.
  • neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473- 5477], pancreas- specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland- specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166).
  • Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for some embodiments of the invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by reference.
  • CMV cytomegalovirus
  • the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of mRNA translation.
  • Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream.
  • Termination and polyadenylation signals that are suitable for some embodiments of the invention include those derived from SV40.
  • the expression vector of some embodiments of the invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA.
  • a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.
  • the vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.
  • the expression vector of some embodiments of the invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
  • IRS internal ribosome entry site
  • the individual elements comprised in the expression vector can be arranged in a variety of configurations. For example, enhancer elements, promoters and the like, and even the polynucleotide sequence(s) encoding a Wnt canonical or non-canonical pathway component (e.g.
  • a Frizzled receptor a Wnt ligand, a Dishevelled (Dsh), PP2A, ARFGAP1 or DAAM1
  • a Frizzled receptor a Wnt ligand, a Dishevelled (Dsh), PP2A, ARFGAP1 or DAAM1
  • Dsh Dishevelled
  • PP2A a Dishevelled
  • ARFGAP1 or DAAM1 Dishevelled
  • mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used.
  • SV40 vectors include pSVT7 and pMT2.
  • Vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205.
  • exemplary vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms.
  • viruses infect and propagate in specific cell types.
  • the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
  • the type of vector used by some embodiments of the invention will depend on the cell type transformed. The ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein.
  • bone marrow cells can be targeted using the human T cell leukemia virus type I (HTLV-I) and kidney cells may be targeted using the heterologous promoter present in the baculovirus Autographa californica nucleopolyhedro virus (AcMNPV) as described in Liang CY et al., 2004 (Arch Virol. 149: 51-60).
  • HTLV-I human T cell leukemia virus type I
  • AcMNPV Autographa californica nucleopolyhedro virus
  • Recombinant viral vectors are useful for in vivo expression of a Wnt canonical or non-canonical pathway component (e.g. a Frizzled receptor, a Wnt ligand, a Dishevelled (Dsh), PP2A, ARFGAP1, or DAAM1) since they offer advantages such as lateral infection and targeting specificity.
  • a Wnt canonical or non-canonical pathway component e.g. a Frizzled receptor, a Wnt ligand, a Dishevelled (Dsh), PP2A, ARFGAP1, or DAAM1
  • Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles.
  • Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.
  • preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].
  • the most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses.
  • a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus -defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post- translational modification of messenger.
  • Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct.
  • LTRs long terminal repeats
  • such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of some embodiments of the invention.
  • the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
  • such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
  • Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
  • Administration of the a Wnt canonical or non-canonical pathway component expressing cells of some embodiments of the invention can be effected using any suitable route such as intravenous, intra peritoneal, intra kidney, intra gastrointestinal track, subcutaneous, transcutaneous, intramuscular, intracutaneous, intrathecal, epidural and rectal.
  • the Wnt canonical or non- canonical pathway component expressing cells of some embodiments of the invention are introduced to the individual using intravenous, intra kidney, intra gastrointestinal track and/or intra peritoneal administrations.
  • Wnt canonical or non-canonical pathway component e.g. a Frizzled receptor, a
  • Wnt ligand, a Dishevelled (Dsh), PP2A, ARFGAPl, DAAMl) expressing cells of some embodiments of the invention can be derived from either autologous sources such as self bone marrow cells or from allogeneic sources such as bone marrow or other cells derived from non- autologous sources. Since non- autologous cells are likely to induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non- autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells or tissues in immunoisolating, semipermeable membranes before transplantation.
  • Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat-sheet and hollow- fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).
  • microcapsules Methods of preparing microcapsules are known in the arts and include for example those disclosed by Lu MZ, et al., Cell encapsulation with alginate and alpha- phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479-83, Chang TM and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu MZ, et al., A novel cell encapsulation method using photosensitive poly(allylamine alpha- cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.
  • microcapsules are prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 ⁇ .
  • HEMA 2-hydroxyethyl methylacrylate
  • MAA methacrylic acid
  • MMA methyl methacrylate
  • Such microcapsules can be further encapsulated with additional 2-5 ⁇ ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S.M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56).
  • microcapsules are based on alginate, a marine polysaccharide (Sambanis, A.
  • microcapsules in diabetes treatment. Diabetes Thechnol. Ther. 2003, 5: 665-8) or its derivatives.
  • microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.
  • An agent capable of upregulating a Wnt canonical or non-canonical pathway component may also be any compound which is capable of increasing the transcription and/or translation of an endogenous DNA or mRNA encoding the Wnt canonical or non- canonical pathway component and thus increasing endogenous Wnt pathway activity.
  • An agent capable of upregulating a Wnt canonical or non-canonical pathway component may also be an exogenous polypeptide including at least a functional portion (as described hereinabove) of the a Wnt canonical or non-canonical pathway component (e.g. a Frizzled receptor, a Wnt ligand, a Dishevelled (Dsh), PP2A, ARFGAP1 or DAAMl).
  • a Frizzled receptor, a Wnt ligand, a Dishevelled (Dsh), PP2A, ARFGAP1 or DAAMl may also be an exogenous polypeptide including at least a functional portion (as described hereinabove) of the a Wnt canonical or non-canonical pathway component (e.g. a Frizzled receptor, a Wnt ligand, a Dishevelled (Dsh), PP2A, ARFGAP1 or DAAMl).
  • Wnt canonical or non-canonical pathway component e.g. a Wnt canonical or non-canonical pathway component
  • Frizzled receptor a Wnt ligand, a Dishevelled (Dsh), PP2A, ARFGAP1 or DAAMl
  • a Wnt canonical or non-canonical pathway component can be also achieved by introducing at least one substrate of a Wnt canonical or non-canonical pathway component.
  • any means for increasing Wnt canonical or non-canonical signaling activity may be used, including both direct and indirect modulation.
  • These may include for example, modulating the expression of any endogenous gene for any member of the Wnt canonical or non-canonical signaling pathway (described in further detail below) at the transcriptional, translational or post-translational level, such as degradation of messenger RNA for the member of the Wnt canonical or non-canonical signaling pathway, modulating the persistence or degradation of protein, etc.
  • They may also include modulation of the activity of a member of the Wnt canonical or non-canonical signaling pathway, such as by use of agonists thereof.
  • the expression and/or activity of activators of any member of the Wnt canonical or non-canonical signaling pathway may be modulated to upregulate Wnt canonical or non-canonical signaling activity.
  • Wnt canonical signaling pathway is activated by activating any of the receptors for Wnt canonical signaling, i.e., a Wnt receptor.
  • any of the Frizzled receptors may be up-regulated to activate the Wnt canonical signaling pathway.
  • a constitutively active receptor may be used (as further detailed hereinbelow).
  • Exemplary Frizzled receptors include, but are not limited to, FZD1 (frizzled- 1, accession nos. NM_003505.1, NP_003496.1), FZD2 (frizzled-2, accession nos.
  • FZD3 (frizzled-3, accession nos. NM_017412.3 and NP_059108.1)
  • FZD4 (frizzled-4, accession nos. NM_012193.3 and NP_036325.2)
  • FZD5 (frizzled-5, accession nos. NM_003468.3 and NP_003459.2)
  • FZD6 (frizzled-6, accession nos. NM_001164615.1 and NP_001158087.1)
  • FZD7 (frizzled-7, accession nos. NM_003507.1 and NP_003498.1)
  • FZD8 (frizzled-8, accession nos.
  • Receptor activation may be achieved in a number of ways, for example, by upregulating the expression of the receptor. This may be achieved, for example, by ligating a polynucleotide sequence encoding the receptor into a suitable expression vector and introducing same into the stem cell or vascular progenitor cell or angioblast. Furthermore, receptor activation may be achieved by introduction of a constitutively active Frizzled receptor to the stem cell or vascular progenitor cell or angioblast, for example by ligating a polynucleotide sequence encoding the constitutively active receptor into a suitable expression vector and introducing same into the stem cell or vascular progenitor cell or angioblast.
  • An exemplary constitutively active Frizzled receptor is described in Holmen et al., Journal of Biological Chemistry (2002) 277(38): 34727- 34735, incorporated herein by reference.
  • the Norrin ligand which binds to Frizzled with high affinity may be used to activate the Wnt canonical signaling pathway.
  • the R-spondin2 protein which also binds to the Frizzled receptors may also be used in accordance with the present teachings.
  • Receptors for Wnt may also be activated by binding of Wnt ligand.
  • the Wnt canonical signaling pathway may be activated by increasing the activity or expression of Wnt ligand, or by decreasing the activity or expression of antagonists of Wnt or Frizzled.
  • Exemplary Wnt ligands include, but are not limited to, Wntl (wingless-type MMTV integration site family, member 1, accession nos. NM_005430.3 and NP_005421.1), Wnt2 (wingless-type MMTV integration site family, member 2, accession nos. NMJ303391.2 and NP_003382.1), Wnt2b (wingless-type MMTV integration site family, member 2b, accession nos. NM_001291880.1 and NP_001278809.1), Wnt3 (wingless-type MMTV integration site family, member 3, accession nos.
  • Wnt3a wingless-type MMTV integration site family, member 3a, accession nos. NMJ333131.3 and NP_149122.1
  • Wnt5b wingless-type MMTV integration site family, member 5b, accession nos. NM_030775.2, NM_032642.2, NP_110402.2 and NP_116031.1
  • Wnt6 wingless-type MMTV integration site family, member 6, accession nos. NM_006522.3 and NP_006513.1
  • Wnt7a wingless-type MMTV integration site family, member 7a, accession nos.
  • NM_004625.3 and NP_004616.2 Wnt7b (wingless-type MMTV integration site family, member 7b, accession nos. NM_058238.2 and NP_478679.1)
  • Wnt8a wingless-type MMTV integration site family, member 8a, accession nos. NM_001300938.1, NM_001300939.1, NM_058244.3, NP_001287867.1, NP_001287868.1 and NP_490645.1)
  • Wnt8b wingless- type MMTV integration site family, member 8b, accession nos.
  • Wnt9a wingless-type MMTV integration site family, member 9a, accession nos. NM_003395.2 and NP_003386.1
  • Wnt9b wingless-type MMTV integration site family, member 9b, accession nos. NM_003396.1 and NP_003387.1
  • WntlOa wingless-type MMTV integration site family, member 10a, accession nos. NM_025216.2 and NP_079492.2
  • WntlOb wingless-type MMTV integration site family, member 10b, accession nos.
  • NM_003394.3 and NP_003385.2 wingless- type MMTV integration site family, member 16, accession nos. NM_016087.2, NM_057168.1, NP_057171.2 and NP_476509.1).
  • the Wnt ligand comprises Wnt5b.
  • the invention contemplates using a Wnt that signals through the non-canonical Wnt signaling pathway.
  • Wnts that signal through the non-canonical pathway include, but are not limited to, Wnt4 (wingless-type MMTV integration site family, member 4, accession nos. NM_030761.4 and NP_110388.2), Wnt5a (wingless-type MMTV integration site family, member 5a, accession nos.
  • the Wnt comprises a polypeptide, an analogue or a polynucleotide (as discussed in detail above).
  • the LEC committed cell of some embodiments of the invention may be exposed to any of the known Wnt non-canonical pathway ligands, additionally or alternatively, upregulating the activity of Wnt non-canonical pathway may be effected by contacting the LEC with an activator of any Wnt non-canonical pathway member.
  • Wnt non-canonical pathway members include, but are not limited to, Ror2 (receptor tyrosine kinase-like orphan receptor 2, accession nos. NM_004560.3 and NP_004551.2), Ryk (receptor-like tyrosine kinase, accession nos.
  • NM_001005861.2, NM_002958.3, NP_001005861.1 and NP_002949.2) G protein (homo sapiens guanine nucleotide binding protein (G protein), alpha 11 (Gq class) (GNA11), accession nos. NM_002067.4 and NP_002058.2), Dishevelled e.g. Dvll (dishevelled segment polarity protein 1, accession nos. NMJ304421.2 and NP_004412.2), Dvl2 (dishevelled segment polarity protein 2, accession nos. NM_004422.2 and NP_004413.1) or Dvl3 (dishevelled segment polarity protein 3, accession nos.
  • DAAM1 (dishevelled associated activator of morphogenesis 1, accession nos. NM_001270520.1, NM_014992.2, NP_001257449.1 and NP_055807.1) or DAAM2 (dishevelled associated activator of morphogenesis 2, accession nos. NM_001201427.1, NM_015345.3, NP_001188356.1 and NP_056160.2).
  • any of the proteinaceous members of the signaling pathways described herein may be provided in the form of the polynucleotide (especially in the case of transmembrane proteins), polypeptides or analogues as further defined hereinbelow.
  • the Wnt ligand comprises a polypeptide, an analogue or a polynucleotide.
  • polypeptide encompasses a naturally occurring polypeptide which is comprised solely of natural amino acid residues or synthetically prepared polypeptides, comprised of a mixture of natural and modified (non-natural) amino acid residues.
  • analogue is used herein to extend to any amino acid derivative of the polypeptides described herein, as long as their function e.g., promoting LEC specification, is maintained.
  • polynucleotide refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
  • the stem cell and/or vascular progenitor cell and/or angioblast may be exposed to any of the known Wnt ligands, such as purified polypeptides.
  • Wnt ligands are available commercially from R&D Systems (Minnesota, USA), from PeproTech, Inc (New Jersey, USA) and from Calbiochem, and may also be made by recombinant means, such as by expression of an expression vector comprising a Wnt nucleic acid in a suitable host cell.
  • the Wnt canonical or non-canonical signaling pathway is activated by increasing the expression of Wnt ligands.
  • activation of the Wnt signaling pathway by Wnt over-expression is described in detail in U.S. application no. 2004/0014209, incorporated herein by reference.
  • the teachings of U.S. 2004/0014209 may be used to prepare mRNAs from expression constructs and plasmids comprising Wnt sequences, for injection into stem cells or vascular progenitor cells or angioblasts to promote LEC specification.
  • expression vectors may be transiently or permanently transfected into stem cells or vascular progenitor cells or angioblasts to achieve the same purpose (as described in detail hereinabove).
  • the Wnt canonical or non-canonical signaling pathway may be activated ex vivo by exposing the stem cell or vascular progenitor cell or angioblast to medium containing the Wnt ligand.
  • a medium is a medium in which Wnt secreting cells, such as cells transfected with Wnt expression vectors, is growing.
  • the presence of the appropriate Wnt ligand in the conditioned medium may be established through known means, such as by Western blots.
  • Cells producing Active Wnt include, but are not limited to, Mouse Wnt3A (ATCC CRL-2647).
  • the Wnt canonical signaling pathway may be activated by upregulating the activity or Dishevelled (Dsh) (e.g. DVL1, DVL2, DVL3, and DvllLl), ⁇ -catenin, LDL-receptor related proteins LRP5 or LRP6 (LRP5/6), PP2A, ADP- ribosylation factor 1 (ARFGAPl) and/or T-cell factor (TCF)/Lymphoid enhancing factor (LEF) family of proteins.
  • Dsh Dishevelled
  • LRP5 or LRP6 LRP5/6
  • PP2A LDL-receptor related proteins
  • ADP- ribosylation factor 1 ARFGAPl
  • TCF T-cell factor
  • LMF Lymphoid enhancing factor
  • serine/threonine phosphatase PP2A activity may be upregulated using the small molecule IQ-1, commercially available e.g. from Tocris Bioscience or from Santa Cruz Biotech.
  • ARFGAPl activity may be upregulated using the small molecule QS 11, commercially available e.g. from Tocris Bioscience or from Abeam.
  • ⁇ -catenin activity may be upregulated using the small molecule deoxycholic acid (DC A).
  • the Wnt canonical signaling pathway may be activated by decreasing the activity or expression of antagonists of the Wnt pathway (e.g. of Wnt or Frizzled).
  • activation of a Wnt canonical signaling pathway is achieved by down-regulation of any antagonist or negative regulator or component of the Wnt canonical pathway.
  • exemplary antagonist or negative regulator or component of the Wnt pathway include, but are not limited to, Axin, glycogen synthase kinase 3 (GSK3, e.g. GSK3p), APC and sFRP (Secreted frizzled-related proteins).
  • Down-regulation can be effected on the genomic and/or the transcript level using a variety of molecules which interfere with transcription and/or translation [e.g., RNA silencing agents (e.g., antisense, siRNA, shRNA, micro-RNA), Ribozyme DNAzyme and a CRISPR system (e.g. CRISPR/Cas)], or on the protein level using e.g., and small molecules, antagonists, antibodies, enzymes that cleave the polypeptide or inhibit functionality of the peptide and the like.
  • RNA silencing agents e.g., antisense, siRNA, shRNA, micro-RNA
  • Ribozyme DNAzyme and a CRISPR system e.g. CRISPR/Cas
  • CRISPR/Cas CRISPR/Cas
  • agents capable of downregulating expression level and/or activity of an antagonist or negative regulator or component of the Wnt pathway e.g. Axin, GSK3, APC and sFRP.
  • an agent capable of downregulating an antagonist or negative regulator or component of the Wnt pathway e.g. Axin, GSK3, APC and sFRP
  • an antibody or antibody fragment capable of specifically binding to the antagonist or negative regulator or component of the Wnt pathway e.g. Axin, GSK3, APC and sFRP
  • the antibody specifically binds at least one epitope of the antagonist or negative regulator or component of the Wnt pathway.
  • epitope of the antagonist or negative regulator or component of the Wnt pathway e.g. the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • antibody as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages.
  • These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of
  • Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker.
  • sFv single-chain antigen binding proteins
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11: 1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and coworkers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)].
  • human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • RNA silencing refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene.
  • RNA silencing has been observed in many types of organisms, including animals, and fungi.
  • RNA silencing agent refers to an RNA which is capable of specifically inhibiting or “silencing" the expression of a target gene.
  • the RNA silencing agent is capable of preventing complete processing (e.g, the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism.
  • RNA silencing agents include noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated.
  • Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.
  • the RNA silencing agent is capable of inducing RNA interference.
  • the RNA silencing agent is capable of mediating translational repression.
  • the RNA silencing agent is specific to the target RNA (e.g., an antagonist or negative regulator or component of the Wnt pathway e.g. Axin, GSK3, APC and sFRP) and does not cross inhibit or silence a gene or a splice variant which exhibits 99% or less global homology to the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% global homology to the target gene.
  • the target RNA e.g., an antagonist or negative regulator or component of the Wnt pathway e.g. Axin, GSK3, APC and sFRP
  • RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs).
  • siRNAs short interfering RNAs
  • the corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi.
  • the process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla.
  • Such protection from foreign gene expression may have evolved in response to the production of double- stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single- stranded RNA or viral genomic RNA.
  • dsRNAs double- stranded RNAs
  • RNA-induced silencing complex RISC
  • some embodiments of the invention contemplates use of dsRNA to downregulate protein expression from mRNA.
  • the dsRNA is greater than 30 bp.
  • the use of long dsRNAs i.e. dsRNA greater than 30 bp
  • the use of long dsRNAs can provide numerous advantages in that the cell can select the optimal silencing sequence alleviating the need to test numerous siRNAs; long dsRNAs will allow for silencing libraries to have less complexity than would be necessary for siRNAs; and, perhaps most importantly, long dsRNA could prevent viral escape mutations when used as therapeutics.
  • the invention contemplates introduction of long dsRNA (over 30 base transcripts) for gene silencing in cells where the interferon pathway is not activated (e.g. embryonic cells and oocytes) see for example Billy et al., PNAS 2001, Vol 98, pages 14428-14433. and Diallo et al, Oligonucleotides, October 1, 2003, 13(5): 381-392. doi: 10.1089/154545703322617069.
  • long dsRNA over 30 base transcripts
  • the invention also contemplates introduction of long dsRNA specifically designed not to induce the interferon and PKR pathways for down-regulating gene expression.
  • Shinagwa and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP, to express long double-strand RNA from an RNA polymerase II (Pol II) promoter. Because the transcripts from pDECAP lack both the 5'-cap structure and the 3'-poly(A) tail that facilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP does not induce the interferon response.
  • siRNAs small inhibitory RNAs
  • siRNA refers to small inhibitory RNA duplexes (generally between 18- 30 basepairs) that induce the RNA interference (RNAi) pathway.
  • RNAi RNA interference
  • siRNAs are chemically synthesized as 21mers with a central 19 bp duplex region and symmetric 2- base 3 '-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100-fold increase in potency compared with 21mers at the same location.
  • RNA silencing agent of some embodiments of the invention may also be a short hairpin RNA (shRNA).
  • RNA agent refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop.
  • oligonucleotide sequences that can be used to form the loop include 5'-UUCAAGAGA- 3' (Brummelkamp, T. R. et al. (2002) Science 296: 550) and 5'-UUUGUGUAG-3' (Castanotto, D. et al. (2002) RNA 8: 1454). It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem-loop or hairpin structure comprising a double- stranded region capable of interacting with the RNAi machinery.
  • RNA silencing agents suitable for use with some embodiments of the invention can be effected as follows. First, the mRNA sequence of the antagonist or negative regulator or component of the Wnt pathway (e.g. Axin, GSK3, APC and sFRP) is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites.
  • UTRs untranslated regions
  • UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www(dot)ambion(dot)com/techlib/tn/91/912(dot)html).
  • potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server
  • Qualifying target sequences are selected as template for siRNA synthesis.
  • Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %.
  • Several target sites are preferably selected along the length of the target gene for evaluation.
  • a negative control is preferably used in conjunction.
  • Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome.
  • a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
  • a suitable siRNA can be an Axin siRNA commercially available from Santa Cruz Biotechnology or from OriGene, a GSK3 siRNA commercially available from SignalChem, an APC siRNA commercially available from Santa Cruz Biotechnology, and a sFRP siRNA commercially available from OriGene.
  • RNA silencing agent of some embodiments of the invention need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.
  • the RNA silencing agent provided herein can be functionally associated with a cell-penetrating peptide.
  • a "cell-penetrating peptide” is a peptide that comprises a short (about 12-30 residues) amino acid sequence or functional motif that confers the energy-independent (i.e., non-endocytotic) translocation properties associated with transport of the membrane-permeable complex across the plasma and/or nuclear membranes of a cell.
  • the cell-penetrating peptide used in the membrane-permeable complex of some embodiments of the invention preferably comprises at least one non-functional cysteine residue, which is either free or derivatized to form a disulfide link with a double-stranded ribonucleic acid that has been modified for such linkage.
  • Representative amino acid motifs conferring such properties are listed in U.S. Pat. No. 6,348,185, the contents of which are expressly incorporated herein by reference.
  • the cell-penetrating peptides of some embodiments of the invention preferably include, but are not limited to, penetratin, transportan, plsl, TAT(48-60), pVEC, MTS, and MAP.
  • RNA silencing agent may be a miRNA.
  • miRNA refers to a collection of non-coding single-stranded RNA molecules of about 19-28 nucleotides in length, which regulate gene expression. miRNAs are found in a wide range of organisms (viruses. fwdarw. humans) and have been shown to play a role in development, homeostasis, and disease etiology.
  • the pri-miRNA is typically part of a polycistronic RNA comprising multiple pri-miRNAs.
  • the pri-miRNA may form a hairpin with a stem and loop.
  • the stem may comprise mismatched bases.
  • the hairpin structure of the pri-miRNA is recognized by Drosha, which is an RNase III endonuclease. Drosha typically recognizes terminal loops in the pri-miRNA and cleaves approximately two helical turns into the stem to produce a 60-70 nucleotide precursor known as the pre-miRNA.
  • Drosha cleaves the pri-miRNA with a staggered cut typical of RNase III endonucleases yielding a pre-miRNA stem loop with a 5' phosphate and ⁇ 2 nucleotide 3' overhang. It is estimated that approximately one helical turn of stem (-10 nucleotides) extending beyond the Drosha cleavage site is essential for efficient processing.
  • the pre-miRNA is then actively transported from the nucleus to the cytoplasm by Ran-GTP and the export receptor Ex-portin-5.
  • the double-stranded stem of the pre-miRNA is then recognized by Dicer, which is also an RNase III endonuclease. Dicer may also recognize the 5' phosphate and 3' overhang at the base of the stem loop. Dicer then cleaves off the terminal loop two helical turns away from the base of the stem loop leaving an additional 5' phosphate and ⁇ 2 nucleotide 3' overhang.
  • the resulting siRNA-like duplex which may comprise mismatches, comprises the mature miRNA and a similar-sized fragment known as the miRNA*.
  • the miRNA and miRNA* may be derived from opposing arms of the pri- miRNA and pre-miRNA. MiRNA* sequences may be found in libraries of cloned miRNAs but typically at lower frequency than the miRNAs.
  • RISC RNA-induced silencing complex
  • the miRNA strand of the miRNA:miRNA* duplex When the miRNA strand of the miRNA:miRNA* duplex is loaded into the RISC, the miRNA* is removed and degraded.
  • the strand of the miRNA:miRNA* duplex that is loaded into the RISC is the strand whose 5' end is less tightly paired. In cases where both ends of the miRNA:miRNA* have roughly equivalent 5' pairing, both miRNA and miRNA* may have gene silencing activity.
  • the RISC identifies target nucleic acids based on high levels of complementarity between the miRNA and the mRNA, especially by nucleotides 2-7 of the miRNA.
  • a number of studies have looked at the base-pairing requirement between miRNA and its mRNA target for achieving efficient inhibition of translation (reviewed by B artel 2004, Cell 116-281).
  • the first 8 nucleotides of the miRNA may be important (Doench & Sharp 2004 GenesDev 2004-504).
  • other parts of the microRNA may also participate in mRNA binding.
  • sufficient base pairing at the 3' can compensate for insufficient pairing at the 5' (Brennecke et al, 2005 PLoS 3- e85).
  • the target sites in the mRNA may be in the 5' UTR, the 3' UTR or in the coding region.
  • multiple miRNAs may regulate the same mRNA target by recognizing the same or multiple sites.
  • the presence of multiple miRNA binding sites in most genetically identified targets may indicate that the cooperative action of multiple RISCs provides the most efficient translational inhibition.
  • MiRNAs may direct the RISC to downregulate gene expression by either of two mechanisms: mRNA cleavage or translational repression.
  • the miRNA may specify cleavage of the mRNA if the mRNA has a certain degree of complementarity to the miRNA. When a miRNA guides cleavage, the cut is typically between the nucleotides pairing to residues 10 and 11 of the miRNA.
  • the miRNA may repress translation if the miRNA does not have the requisite degree of complementarity to the miRNA. Translational repression may be more prevalent in animals since animals may have a lower degree of complementarity between the miRNA and binding site.
  • miRNA mimic refers to synthetic non-coding RNAs that are capable of entering the RNAi pathway and regulating gene expression.
  • miRNA mimics imitate the function of endogenous microRNAs (miRNAs) and can be designed as mature, double stranded molecules or mimic precursors (e.g., or pre-miRNAs). miRNA mimics can be comprised of modified or unmodified RNA, DNA, RNA-DNA hybrids, or alternative nucleic acid chemistries (e.g., LNAs or 2'-0,4'-C-ethylene-bridged nucleic acids (EN A)). For mature, double stranded miRNA mimics, the length of the duplex region can vary between 13-33, 18-24 or 21-23 nucleotides.
  • the miRNA may also comprise a total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides.
  • the sequence of the miRNA may be the first 13-33 nucleotides of the pre-miRNA.
  • the sequence of the miRNA may also be the last 13-33 nucleotides of the pre-miRNA.
  • a suitable miRNA can be a miRNA targeting Axin commercially available from Genecopoeia, a miRNA targeting GSK3 commercially available from OriGene, a miRNA targeting APC commercially available from OriGene, and a miRNA targeting sFRP commercially available from OriGene.
  • contacting stem cells or vascular progenitor cells or angioblasts with a miRNA may be affected in a number of ways:
  • the pre- miRNA sequence may comprise from 45-90, 60-80 or 60-70 nucleotides.
  • the sequence of the pre-miRNA may comprise a miRNA and a miRNA* as set forth herein.
  • the sequence of the pre-miRNA may also be that of a pri-miRNA excluding from 0-160 nucleotides from the 5' and 3' ends of the pri-miRNA. 4.
  • the pri- miRNA sequence may comprise from 45-30,000, 50-25,000, 100-20,000, 1,000-1,500 or 80-100 nucleotides.
  • the sequence of the pri-miRNA may comprise a pre-miRNA, miRNA and miRNA*, as set forth herein, and variants thereof.
  • Preparation of miRNAs mimics can be effected by chemical synthesis methods or by recombinant methods.
  • Another agent capable of downregulating an antagonist or negative regulator or component of the Wnt pathway is a DNAzyme molecule capable of specifically cleaving a mRNA transcript or DNA sequence of the antagonist or negative regulator or component of the Wnt pathway (e.g. Axin, GSK3, APC and sFRP).
  • DNAzymes are single- stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology 1995;2:655; Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci.
  • DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL.
  • Downregulation of an antagonist or negative regulator or component of the Wnt pathway can also be effected by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the antagonist or negative regulator or component of the Wnt pathway (e.g. Axin, GSK3, APC and sFRP).
  • an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the antagonist or negative regulator or component of the Wnt pathway (e.g. Axin, GSK3, APC and sFRP).
  • Design of antisense molecules which can be used to efficiently downregulate an antagonist or negative regulator or component of the Wnt pathway e.g. Axin, GSK3, APC and sFRP
  • the first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells
  • the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits
  • a suitable antisense oligonucleotides can be an antisense oligonucleotides targeted against GSK3 commercially available from Isis Pharmaceuticals.
  • antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Holmund et al., Curr Opin Mol Ther 1:372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz Curr Opin Mol Ther 1:297-306 (1999)].
  • Another agent capable of downregulating an antagonist or negative regulator or component of the Wnt pathway is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding an antagonist or negative regulator or component of the Wnt pathway (e.g. Axin, GSK3, APC and sFRP).
  • Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)].
  • ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al., Clin Diagn Virol. 10: 163-71 (1998)]. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials.
  • ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models.
  • HEPTAZYME a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated - WEB home page).
  • Another agent capable of downregulating an antagonist or negative regulator or component of the Wnt pathway is a RNA-guided endonuclease technology e.g. CRISPR system.
  • CRISPR system also known as Clustered Regularly Interspaced Short Palindromic Repeats refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated genes, including sequences encoding a Cas gene (e.g. CRISPR-associated endonuclease 9), a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a "direct repeat” and a tracrRNA -processed partial direct repeat) or a guide sequence (also referred to as a "spacer”) including but not limited to a crRNA sequence (i.e. an endogenous bacterial RNA that confers target specificity yet requires tracrRNA to bind to Cas) or a sgRNA sequence (i.e. single guide RNA).
  • a crRNA sequence i.e. an endogenous bacterial RNA that confers target specificity yet requires tracrRNA
  • one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system.
  • one or more elements of a CRISPR system (e.g. Cas) is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes, Neisseria meningitides, Streptococcus thermophilus or Treponema denticola.
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).
  • target sequence refers to a sequence to which a guide sequence (i.e. guide RNA e.g. sgRNA or crRNA) is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. Thus, according to some embodiments, global homology to the target sequence may be of 50 %, 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 % or 99 %.
  • a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • a target sequence is located in the nucleus or cytoplasm of a cell.
  • the CRISPR system comprises two distinct components, a guide RNA (gRNA) that hybridizes with the target sequence, and a nuclease (e.g. Type-II Cas9 protein), wherein the gRNA targets the target sequence and the nuclease (e.g. Cas9 protein) cleaves the target sequence.
  • the guide RNA may comprise a combination of an endogenous bacterial crRNA and tracrRNA, i.e. the gRNA combines the targeting specificity of the crRNA with the scaffolding properties of the tracrRNA (required for Cas9 binding).
  • the guide RNA may be a single guide RNA capable of directly binding Cas.
  • a CRISPR complex comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins
  • formation of a CRISPR complex results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.
  • the tracr sequence which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g.
  • a wild-type tracr sequence may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence.
  • the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of a CRISPR complex. As with the target sequence, a complete complementarity is not needed, provided there is sufficient to be functional. In some embodiments, the tracr sequence has at least 50 %, 60 %, 70 %, 80 %, 90 %, 95 % or 99 % of sequence complementarity along the length of the tracr mate sequence when optimally aligned.
  • Introducing CRISPR/Cas into a cell may be effected using one or more vectors driving expression of one or more elements of a CRISPR system such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites.
  • a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors.
  • two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector.
  • CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream” of) or 3' with respect to ("downstream” of) a second element.
  • the coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction.
  • a single promoter may drive expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron).
  • TFOs triplex forming oligonucleotides
  • the triplex-forming oligonucleotide has the sequence correspondence: oligo 3'-A G G T duplex 5'-A G C T duplex 3'-T C G A
  • the A-AT and G-GC triplets have the greatest triple helical stability (Reither and Jeltsch, BMC Biochem, 2002, Septl2, Epub).
  • the same authors have demonstrated that TFOs designed according to the A-AT and G-GC rule do not form non-specific triplexes, indicating that the triplex formation is indeed sequence specific.
  • triplex forming sequence preferably are at least 15, more preferably 25, still more preferably 30 or more nucleotides in length, up to 50 or 100 bp.
  • Transfection of cells for example, via cationic liposomes
  • TFOs Transfection of cells (for example, via cationic liposomes) with TFOs, and formation of the triple helical structure with the target DNA induces steric and functional changes, blocking transcription initiation and elongation, allowing the introduction of desired sequence changes in the endogenous DNA and resulting in the specific downregulation of gene expression.
  • Examples of such suppression of gene expression in cells treated with TFOs include knockout of episomal supFGl and endogenous HPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res.
  • TFOs designed according to the abovementioned principles can induce directed mutagenesis capable of effecting DNA repair, thus providing both downregulation and upregulation of expression of endogenous genes (Seidman and Glazer, J Clin Invest 2003;112:487-94).
  • Detailed description of the design, synthesis and administration of effective TFOs can be found in U.S. Patent Application Nos. 2003 017068 and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 to Emanuele et al, and U.S. Pat. No. 5,721,138 to Lawn.
  • Another agent capable of downregulating an antagonist or negative regulator or component of the Wnt pathway e.g. Axin, GSK3, APC and sFRP
  • Axin, GSK3, APC and sFRP would be any molecule which binds to and/or cleaves the antagonist or negative regulator or component of the Wnt pathway (e.g. Axin, GSK3, APC and sFRP).
  • Such molecules can be antagonists, or inhibitory peptides.
  • a non-functional analogue of at least a catalytic or binding portion of an antagonist or negative regulator or component of the Wnt pathway can be also used as an agent which downregulates the antagonist or negative regulator or component of the Wnt pathway (e.g. Axin, GSK3, APC and sFRP).
  • Another agent which can be used along with some embodiments of the invention to downregulate an antagonist or negative regulator or component of the Wnt pathway is a molecule which prevents activation or substrate binding.
  • upregulation of the Wnt canonical pathway is achieved by inhibiting GSK3 kinase activity.
  • Inhibiting GSK3 activity may be achieved by inhibiting the enzymatic activity of GSK3, e.g. by use of chemical inhibitors or antagonists, which may be competitive or non-competitive, as described below.
  • Such inhibitors may include kinase inhibitors.
  • GSK3 activity may be down-regulated by down-regulating the expression of GSK3 protein, such as by use of antisense RNA, or RNAi, or siRNA or by inhibiting the conversion of inactive forms of GSK3 to active forms, or by increasing the rate of degradation of GSK-3.
  • the methods and compositions described here may also employ loss of function and dominant negative mutations in GSK-3, described for example in Hedgepeth et al. (1997) Dev Biol. 185(1):82-91.
  • Kinase deficient mutants of GSK3, for example as described in Crowder and Freeman, 2000, J. Biol. Chem. 275 (2000), pp. 34266-34271, incorporated herein by reference, may be transfected into stem cells and/or vascular progenitor cells or angioblasts to achieve LEC specification.
  • Fibroblast growth factor may also be used as an activator of Wnt canonical signaling pathway as exposure to FGF activates Akt and thus inhibits GSK3, described in Hashimoto et al., 2002, J. Biol. Chem. 277 (2002), pp. 32985-32991, incorporated herein by reference.
  • Wnt canonical signaling pathway may also be activated via upregulation, for example, over-expression, of FRAT1, a negative regulator of GSK3, as described in Crowder and Freeman, 2000, J. Biol. Chem. 275 (2000), pp. 34266-34271 and Culbert, et al., 2001, FEBS Lett. 507 (2001), pp. 288-294, incorporated herein by reference.
  • GSK-3 is down-regulated using a chemical inhibitor.
  • a number of chemical inhibitors of GSK3 activity are known in the art, as described in for example U.S. Pat. No. 6,441,053, incorporated herein by reference.
  • the chemical inhibitors of GSK3 comprise lithium and its salts, including lithium chloride (LiCl); indirubins, e.g., Tyrian purple indirubins; 6- bromoindirubin-3'-oxime (BIO); iGSK-3 and its variants, SB-216763 and SB-415286, (i.e. structurally distinct maleimides).
  • Additional GSK3 inhibitors are commercially available, e.g. from Calbiochem (San Diego, USA) and include e.g.
  • upregulation of the Wnt canonical pathway is achieved by inhibiting Axin activity.
  • Inhibiting Axin activity may be achieved using any method known in the art, e.g. by RNAi as described in detail above.
  • upregulation of the Wnt canonical pathway is achieved by inhibiting APC activity.
  • Inhibiting APC activity may be achieved using any method known in the art, e.g. by RNAi as described in detail above.
  • upregulation of the Wnt canonical pathway is achieved by inhibiting sFRP (e.g. sFRP-1) activity.
  • sFRP e.g. sFRP-1
  • Inhibiting sFRP activity may be achieved using any method known in the art, e.g. by RNAi or using the small molecule inhibitor WAY-316606, commercially available e.g. from Tocris Bioscience and R&D systems. Further methods are as described in detail hereinabove.
  • Wnt canonical signaling may be activated by use of the cyclin dependent kinase (CDK) inhibitors described in Leclerc, et al., (2001), J. Biol. Chem. 276 (2001), pp. 251-260, and Knockaert et al., (2002), J. Biol. Chem. 277 (2002), pp. 25493-25501, incorporated herein by reference.
  • CDK cyclin dependent kinase
  • any of the components of the Wnt canonical signaling pathway may be modulated (e.g. upregulated or downregulated dependent on the target) in order to activate the pathway.
  • Activation of the Wnt canonical signaling pathway in stem cells or vascular progenitor cells or angioblasts may be assessed in a number of ways known in the art. In general, such an assay will seek to detect the modulation of the target component, or a component downstream of the component which is the target of activation.
  • an assay for activation of the Wnt canonical signaling pathway may comprise detection of a reduced activity of GSK3.
  • GSK3 activity may be assessed in a number of ways, for example, utilizing GSK3 Kinase Assays. Such an assay may be particularly suitable where GSK3 activity is targeted for inhibition as a means to activate the Wnt canonical signaling pathway.
  • an assay for activation of the Wnt canonical signaling pathway may comprise detecting accumulation of ⁇ -catenin in the cytoplasm, or the nucleus, or both, when an increase in the amount or quantity of ⁇ -catenin in either or both locations may be assessed as an indication of Wnt canonical signaling pathway activation.
  • This may be achieved by making nuclear or cytoplasmic extracts of the cells in question, e.g., stem cells and/or vascular progenitor cells and/or angioblasts, using means known in the art, and detection of ⁇ -catenin protein by antibody Western blots.
  • Particularly useful assays include, for example, those assays which detect active forms of ⁇ -catenin, or non- phosphorylated forms of ⁇ -catenin, using antibodies specific for such forms.
  • phosphorylation of Dishevelled or phosphorylation of the
  • LRP may also be detected as a means of gauging activation of Wnt canonical signaling pathway.
  • activation of the Wnt non-canonical signaling pathway may be assessed using any method known to one of skill in the art.
  • an assay for activation of the Wnt non-canonical signaling pathway may comprise detection of intracellular Ca 2+ release.
  • an assay for activation of the Wnt non-canonical signaling pathway may comprise detecting accumulation of Dsh, Daaml, Rho, Rho kinase (ROCK), Rac and/or JNK in the cytoplasm, or the nucleus, or both, when an increase in the amount or phosphorylation of Dsh, Daaml, Rho, Rho kinase (ROCK), Rac and/or JNK in either or both locations may be assessed as an indication of Wnt non-canonical signaling pathway activation.
  • ROCK Dsh, Daaml, Rho, Rho kinase
  • Particularly useful assays include, for example, those assays which detect active forms of Dsh, Daaml, Rho, Rho kinase (ROCK), Rac and/or JNK, or non-phosphorylated forms thereof, using antibodies specific for such forms.
  • the Wnt canonical signaling pathway in the stem cells or vascular progenitor cells or angioblasts may be activated for 6 hours or more, 12 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, or 14 days or more.
  • the Wnt canonical signaling pathway in the stem cells or vascular progenitor cells or angioblasts is activated for 6 hours to 14 days, for 1 to 14 days, for 2 to 14 days, for 4 to 14 days, for 6 to 14 days, for 8 to 14 days, for 4 to 10 days, for 4 to 8 days, for 6 to 8 days or 6 to 7 days.
  • the Wnt canonical signaling pathway may activated for as long as necessary, depending on the application, for example, the Wnt canonical signaling pathway may be activated for 1 week, 2 weeks, 3 weeks, 4 weeks, etc, as necessary. In such embodiments, the Wnt canonical signaling pathway may be activated continuously during that time.
  • the stem cells or vascular progenitor cells or angioblasts are first induced to differentiate into endothelial cells prior to promoting LEC specification.
  • the stem cells or vascular progenitor cells or angioblasts may be treated with a factor known to induce endothelial cell differentiation, e.g. Collagen (e.g. Collagen-IV), vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • the stem cells or vascular progenitor cells or angioblasts may be further treated with an agent which blocks differentiation into other cell types, e.g. into smooth muscle cells, such an agent may include e.g. SB431542.
  • Isolation and selection of ECs may be carried out using any method known in the art, e.g. by FACS analysis, selecting cells expressing the surface markers KDR, CD31 and/or or CD 144 (VE-cadherin).
  • the stem cells or vascular progenitor cells or angioblasts are induced to differentiate towards the endothelial lineage by first being seeded as single cells on Collagen-IV coated plates at 1-20 x 10 4 cells/cm 2 (e.g. 5 x 10 4 cells/cm 2 ) and cultured with MEM-alpha, 5-20 % FBS (e.g. 10 %) and 0.01-1 mM ⁇ - mercaptoethanol (e.g. 0.1 mM) for 3-10 days (e.g. 6 days). On the last day (e.g. day 6), cells are re-seeded on collagen-IV coated plates at 0.5-5 x 10 4 cells/cm 2 (e.g.
  • the endothelial cells may be subjected to Wnt ligand, e.g. Wnt5b (e.g. 100 ng/ml) starting at day 3-10 (e.g. day 6) every other day for about 3-10 days (e.g. 6 days).
  • Wnt ligand e.g. Wnt5b (e.g. 100 ng/ml) starting at day 3-10 (e.g. day 6) every other day for about 3-10 days (e.g. 6 days).
  • Isolation and selection of LECs may be carried out using any method known in the art, e.g. by FACS analysis, selecting cells expressing the LEC markers as described in detail above, e.g. proxl, lyvel, flt4, soxl8, nr2f2, CD31 and GP38.
  • activation of the Wnt canonical signaling pathway may be used to cause a stem cell or vascular progenitor cell or angioblast which is still pluripotent, but already committed to differentiation along a specific pathway (e.g., an arterial or venous pathway), to enter a lymphatic differentiation pathway.
  • a specific pathway e.g., an arterial or venous pathway
  • stem cells or vascular progenitor cells or angioblasts may be further subjected to additional factors that can enhance or promote LEC specification.
  • expression of the orphan nuclear receptor COUP transcription factor II (COUP-TFII), SRY-related HMG-domain transcription factor Soxl8, ERK and/or homeobox transcription factor Proxl may be upregulated in the stem cells or vascular progenitor cells or angioblasts to promote LEC specification.
  • a method of inducing lymphangiogenesis comprising: (a) promoting LEC specification in stem cells or vascular progenitor cells or angioblasts so as to obtain a LEC committed cell according to the method of some embodiments of the invention; and (b) subjecting the LEC committed cell to a LEC morphogenesis signal, thereby inducing lymphangiogenesis.
  • lymphatic vessel e.g. capillaries, collecting vessels, and ducts.
  • LEC committed cells are subjected to a LEC morphogenesis signal, a LEC proliferation signal or a LEC survival signal.
  • LEC morphogenesis signal refers to any agent which induces lymphangiogenesis in the LEC committed cells.
  • LEC proliferation signal refers to any agent which induces cell division in the LEC committed cells.
  • LEC survival signal refers to any agent which reduced cell death in the LEC committed cells.
  • the LEC morphogenesis, proliferation or survival signal comprises an endothelial growth factor.
  • the endothelial growth factor is a vascular endothelial growth factor (VEGF) or a receptor thereof.
  • VEGF vascular endothelial growth factor
  • the VEGF is VEGF-C or VEGF-D, or their receptors VEGFR-2 or VEGFR- 3.
  • the factor is a collagen and calcium-binding EGF domain-containing protein 1 (ccbel) previously shown to be required for embryonic lymphangiogenesis and venous sprouting through upregulation of Vegfc (Hogan, 2009 or Le Guen, 2009).
  • the LEC committed cell may be exposed to endothelial growth factor (e.g. VEGF) ex vivo in cell medium comprising the endothelial growth factor.
  • endothelial growth factor e.g. VEGF
  • the LEC morphogenesis signal comprises upregulating the activity of a Wnt non-canonical pathway in the LECs (as described in detail hereinabove).
  • the LEC committed cells may be further subjected to additional factors that can enhance or promote lymphangiogenesis.
  • expression of VEGF, SH2 domain containing leukocyte protein of 76 kDa (Slp76), spleen tyrosine kinase (Syk) and phospholipase C, gamma 2 (Plcg2) may be upregulated in the LEC committed cells to promote lymphangiogenesis.
  • Selecting LECs that have undergone lymphangiogenesis can be effected using any method known in the art, e.g. by microscopy, isolating those cells that have formed any of the lymphatic vasculature (lymphatic capillaries, collecting vessels, and ducts) or by phenotypic cell sorting (e.g. FACS) selecting cells expressing LEC markers e.g.
  • Neuropilin 2 neuropilin 2 (Nrp2), forkhead box C2 (Foxc2), podoplanin (Pdpn), angiopoietin 2 (Ang2), ephrin B2 (Efnb2), protein phosphatase 1 regulatory (inhibitor) subunit 13B (Asppl) and/or core 1 synthase glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase 1 (T-synthase).
  • Nrp2 neuropilin 2
  • Foxc2 podoplanin
  • Ang2 angiopoietin 2
  • Efnb2 ephrin B2
  • protein phosphatase 1 regulatory (inhibitor) subunit 13B Asppl
  • stem cells or vascular progenitor cells or angioblasts are preferably treated with the agent of the present invention (as detailed in further detail hereinabove), following which they are administered to the subject in need thereof.
  • a method of treating or preventing a disease or disorder related to lymphangiogenesis in a subject in need thereof comprising administering to the subject the isolated population of cells of some embodiments of the invention, thereby treating or preventing the disease or disorder related to lymphangiogenesis.
  • treating refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology.
  • pathology disease, disorder or condition
  • Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
  • the term "preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.
  • the term "subject” refers to an animal, preferably a mammal, or a human being, of any age or sex, who suffers from or is predisposed to a disease or disorder related to lymphangiogenesis.
  • lymphangiogenesis diseases or disorders related to lymphangiogenesis include any condition in which there is a deviation from or interruption of the normal structure or function of the lymph or lymph vessels. Collectively, such diseases and disorders are referred to herein as “lymphatic disorders or diseases.”
  • Lymphatic disorders or diseases include, but are not limited to, lymphedema e.g. lymphangiomatosis, lymphangioleiomyomatosis, mixed vascular/lymphatic malformation syndromes or conditions, e.g. Turner- Weber and Klippel Trenaunay Syndrome, conditions arising from filariasis, trauma, infection or surgeries e.g. of the breast, prostate, uterus, cervix, abdomen, orthopedic, cosmetic (liposuction), malignant melanoma, conditions arising from treatments used for malignant diseases, e.g. lymphoma (e.g.
  • Hodgkin's and non-Hodgkin's lymphoma radiation therapy, sport injuries, tattooing, diabetes, obesity and any physical insult to the lymphatic pathways, the inability to control infections (e.g. such as that associated with HIV/AIDS), the inability to deliver antibiotic and anti-viral medication to infected tissues and organs, inflammatory and auto-immune diseases (e.g. such as but not limited to, rheumatoid arthritis and systemic lupus erythematosis, scleroderma, Wegener's granulomatosis), lymphatic insufficiency of the internal organs, impairment of lymphatic development in the intestines (e.g. which leads to malabsorption, ascites, underdevelopment from malnutrition, immune malfunction) and pulmonary lymphangiectasia, cystic hygromas and lymphangiomas that may lead to impaired vision, swallowing and breathing.
  • infections e.g. such as that associated with HIV/AIDS
  • the lymphatic disorders or diseases include, but are not limited to, cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor, graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema, chronic wound (e.g. chronic wound not healing because of lymphorrhea or lymph secretions in surgical wounds), lymphedema, lymphatic fistula, lymphorrea and lymphocele/seroma formation.
  • cancer melanoma
  • solid tumor e.g., breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor, graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema, chronic wound (e.g. chronic wound not healing because of lymphorrhea or lymph secretions in surgical wounds), lymphedema, lymphatic fistula, lymphorrea and lympho
  • lymphatic endothelial cells LECs
  • the method comprising generating LECs according to the method of some embodiments of the invention; and transplanting the LECs into the subject, thereby transplanting the LECs.
  • transplanting refers to providing the LECs of the present invention, using any suitable route.
  • ex-vivo treated cells of the present invention can be effected using any suitable route of introduction, such as intravenous, intraperitoneal, intra-kidney, intra-gastrointestinal track, subcutaneous, transcutaneous, intramuscular, intracutaneous, intrathecal, epidural, and rectal.
  • the ex-vivo treated cells of the present invention may be introduced to the individual using an intravenous mode of administration.
  • the stem cells and/or vascular progenitor cells or angioblasts may be obtained from any autologous or non- autologous (i.e., allogeneic or xenogeneic) donor.
  • cells may be isolated from a human cadaver or from a human hematopoietic cell donor.
  • cells may be obtained from any xenogeneic donor (e.g. porcine origin).
  • Stem cells and/or vascular progenitor cells or angioblasts of xenogeneic origin are preferably obtained from a source which is known to be free of zoonoses, such as porcine endogenous retroviruses.
  • human-derived stem cells and/or vascular progenitor cells or angioblasts are preferably obtained from substantially pathogen-free sources.
  • non-autologous cells are likely to induce an immune reaction when administered to the body
  • approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells in immunoisolating, semipermeable membranes before transplantation.
  • cells may be uses which do not express xenogenic surface antigens, such as those developed in transgenic pigs.
  • immunosuppressive agents which may be used in conjunction with the ex-vivo treatment include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE.sup.R), etanercept, TNF. alpha, blockers, a biological agent that targets an inflammatory cytokine, and Non-Steroidal Anti-Inflammatory Drug (NSAIDs).
  • methotrexate cyclophosphamide
  • cyclosporine cyclosporin A
  • chloroquine hydroxychloroquine
  • sulfasalazine sulphasalazopyrine
  • gold salts gold salts
  • NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.
  • treating or preventing a disease or disorder related to lymphangiogenesis is effected by administering to the subject the agent per se capable of upregulating the activity of Wnt canonical pathway.
  • the method may be further effected by administering to the subject an agent capable of inducing LEC morphogenesis signal (e.g. upregulating the activity of Wnt non-canonical pathway).
  • the agent as detailed hereinabove
  • the ex-vivo treated LECs or the agent capable of upregulating the activity of Wnt canonical and/or non-canonical pathway of the present invention can be administered to the individual per se or as part of a pharmaceutical composition which also includes a physiologically acceptable carriers or excipients.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the ex-vivo treated LECs or the agent capable of upregulating the activity of Wnt canonical and/or non-canonical pathway accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
  • molecular manipulation of the agent e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB
  • pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers)
  • the transitory disruption of the integrity of the BBB by hyperosmotic disruption resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
  • each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
  • compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (LECs or agent capable of upregulating the activity of Wnt canonical and/or non-canonical pathway) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., a disease or disorder related to lymphangiogenesis) or prolong the survival of the subject being treated.
  • active ingredients e.g., a disease or disorder related to lymphangiogenesis
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
  • Dosage amount and interval may be adjusted individually to provide ample levels of the active ingredient which are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • a method of screening for an agent capable of upregulating the activity of Wnt canonical pathway for the formation of LECs comprising: contacting the agent with stem cells and/or vascular progenitor cells or angioblasts; analyzing an expression of a lymphatic marker on the stem cells and/or vascular progenitor cells or angioblasts; and comparing the expression of the marker following the contacting to prior to the contacting, wherein an expression of the marker is indicative of an agent suitable for formation of LECs.
  • the lymphatic marker includes, but is not limited to,
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • the Tg(fltl_9a_cFos:GFP) wz2 reporter was generated by cloning the previously identified zebrafish fltl_9a enhancer into pGW_cFosGFP as previously described [Fisher, S. et al., Nat Protoc (2006) 1: 1297- 1305].
  • the Tg(kdrl:Kaede) wz3 was generated by cloning a Kaede fragment in a Tol2- compatible vector containing 2.5 kb from the kdrl promoter using the Gateway methodology [Villefranc J. A. et al., Dev Dyn (2007) 236: 3077-3087].
  • the lyvel (5 ' -AGACGTGGGTGAAATCCAAG-3 ' and 5'- GATGATGTTGCTGCATGTCC-3 ' , SEQ ID NOs: 1 and 2, respectively), wnt5b (5'- ATGGATGTGAGAATGAACC AAGGAC-3 ' and 5'-
  • CTACTTGCAC ACAAACTGGTCTACG-3 ' , SEQ ID NOs: 3 and 4, respectively), and vegfc (5 ' -C ATC AGCACTTCATACATCAGC-3 ' and 5'- GTCC AGTCTTCCCC AGTATG-3 ' , SEQ ID NOs: 5 and 6, respectively) probes were amplified by PCR from 24 hpf cDNA.
  • Tg(hsp70l:wnt5b-GFP) embryos were heat- shocked at 37 °C for 25 minutes and analyzed for parachordal cells (PAC) number at 56 hpf.
  • PAC parachordal cells
  • Tg(hsp70l:wnt5b-GFP) embryos were heat-shocked at 19- 20 hpf, for 25 minutes and fixed as described above at 28 hpf.
  • Tg(proxl :KalT4-UAS:uncTagRFP ;hsp70l:wnt5b-GFP) embryos were heat-shocked at 21 hpf, for 25-30 minutes.
  • IWR1 (Sigma) and TNP-470 (Sigma) were dissolved in Dimethyl sulfoxide (DMSO) as previously described [Cirone, P. et al., Angiogenesis (2008) 11: 347-360]. Embryos were treated with 30 ⁇ IWR1, for 2 days starting at 20 hpf. TNP-470 was added at a concentration of 25 ⁇ ⁇ , for 1 day starting at 23 hpf. PAC formation was assessed at 3 dpf.
  • DMSO Dimethyl sulfoxide
  • sox32 [Griffin, K. J. and Kimelman, D., Nat Cell Biol (2002) 4: 821-825] (1 ng), tcf7 [Nyholm, M. K. et al, Development (Cambridge, England) (2007) 134: 735-746] (8 ng), lefl [Dorsky, R. I. et al., Dev Biol (2002) 241: 229-237] (3 ng), wnt5b [Lele, Z. et al., Genesis (2001) 30: 190-194] (7.5 ng or 4 ng for subdose), vegfc [Yaniv, K. et al.
  • h-Proxl (5 ' -CC ACTGACC AGAC AGAAGCA-3 ' and 5 ' -TGGGCTCTGAAATGGATAGG-3 ' , SEQ ID NOs: 21 and 22, respectively), h-beta-actin (5 ' -TCC ACCTTCC AGC AGATGTG- 3' and 5'- GCATTTGCGGTGGACGAT-3 ' , SEQ ID NOs: 23 and 24, respectively), h- Flt4 (5 ' - AAG AAGTTCC ACC ACC AAAC AT-3 ' and 5'-
  • Tgiflil gal4 ;uasKaede embryos.
  • Photo switching was performed using a 405 nm laser.
  • ECs in 4 segments of Tg(kdrl:Kaede) embryos were photoswitched at 17-18 hpf and 20-21 hpf respectively. 28 hours later, embryos were imaged, and the number of green versus red PACs was counted in 6 segments over the photoswitched area.
  • PAC related phenotypes For quantification of phenotypes, the average number of PACs/TD/cell number in 9-10 segments over the yolk extension was calculated. Embryos with no fluorescence or with gross vascular morphological defects were excluded from quantification. For analysis, embryos that meet all the criteria above were randomly selected. For quantification of PAC related phenotypes in ppt mutants, a subdose of wnt5b MO (4 ng) was injected into ppt embryos to abolish maternal RNA contribution as previously described [Kim, H. J. et al., BMC biology (2005) 3: 23].
  • Confocal imaging was performed using a Zeiss LSM 780 upright confocal microscope (Carl Zeiss, Jena, Germany) with a W-Plan Apochromat x20 objective, NA 1.0. Fluorescent proteins were excited sequentially with single-photon lasers (488 nm, 563 nm). 2Photon imaging of eGFP was carried out at 920 nm. Time-lapse, in-vivo imaging was performed as previously described [Ben Shoham, A. et al., Development (Cambridge, England) (2012) 139: 3859-3869] using a custom-built chamber for perfusion of embryos with temperature-controlled physiological medium, z-stacks were acquired at 2.5-3 ⁇ increments, every 10-12 minutes.
  • Tgiflil :gal4;uasKaede embryos at 24 hpf, 6 embryos per group were used for FACS isolation of Kaede photoconverted (red) ECs.
  • Single-cell suspensions were prepared as previously described [Avraham-Davidi I. et al., Nature medicine (2012) supra] with some modifications (the embryos were not chopped, and no Liberase was used). Sorting was performed at 4 °C in a FACSAria cell sorter using a 70- ⁇ nozzle.
  • RNASeq was performed as previously described [Hashimshony, T. et al., Cell reports (2012) 2: 666-673] with the following modification - a new set of primers was used with a shorter barcode, and a 5 base UMI to enable transcript counting [described in Hashimshony, T. et al., (2012) supra] .
  • CEL-Seq data was normalized by dividing the reads of each gene by the total reads of the sample and multiplying by 10,000 (transcript per 10,000). Genes without any detected expression, or with expression detected only in one sample were filtered out. For identification of significant differentially expressed genes, fold change was calculated and a two-sample t-test was conducted.
  • Tg(flil:EGFP) embryos were fixed in 4 % PFA for 20 minutes at RT, embedded in Gelatin-Bovine albumin medium (0.35 % Gelatin, 21 % Bovine albumin) as previously described [Levin, M. Biochem Biophys Methods (2004) 58: 85-96]. 50-100 ⁇ cross- sections were obtained using a Leica VT 1000s vibratome and stained in 1:200 dilution of TRITC-Phalloidin (Sigma) as previously described [Jin, S. W. et al.,. Development (Cambridge, England) (2005) 132: 5199-5209].
  • hESCs Human embryonic stem cells
  • H9 cells were seeded as single cells on Collagen-IV (Sigma) coated plates at 5 x 10 4 cells/cm 2 and cultured with MEM-alpha (Invitrogen), 10 % FBS (Hyclone) and 0.1 mM ⁇ -mercaptoethanol for 6 days.
  • H9 cells were obtained and handled by the Stem Cells Research Center at the
  • PCV Posterior Cardinal Vein
  • lymphatic endothelial cells within the Posterior Cardinal Vein (PCV) of zebrafish embryos were fate-mapped.
  • Tg(flil:EGFP) yl Figures 1A-D
  • Tg(flil:nEGFPf Figures 1H-K embryos were imaged starting at 22-24 hpf and until 60 hpf, when PACs are fully discernible. Tracking of PAC-LECs back in time and space demonstrated that 81 % of these cells originated in the ventral side of the PCV (vPCV) as compared to 19 % that originated in the dorsal-PC V (dPCV).
  • Tg(flil :gal4 ubsS ;uasKaede rkS ) embryos were used which express the photoconvertible molecule Kaede in ECs.
  • Pan-Kaede photoconversion of vPCV cells at 24 hpf rendered approximately 90 % red PACs, indicating that they originated in the floor of the PCV.
  • less than 10 % red PACs were observed following dPCV photoconversion ( Figures 1E-G and Figures 1L-M).
  • vPCV cells generated PACs also in plcgl mutants ( Figures 1P-S), which lack arterial ISVs as well as blood flow, but develop venous sprouts and PACs, suggesting that the specification of lymphatic progenitors is not affected by nearby arteries or by blood circulation.
  • ventral PCV harbors a niche of specialized angioblasts
  • lymphatic vessels originate from a novel population of PCV angioblasts and not from fully differentiated venous ECs as previously postulated, prompted the present inventors to enquire into the molecular signature of these cells.
  • Global expression profiling via RNASeq revealed significant enrichment of well-established angioblast, lymphatic, and arterial markers in the vPCV versus dPCV cells ( Figure 2Q and Figures 5C-D). It was then asked when these progenitors acquire a lymphatic fate.
  • the expression of the transcription factor Proxl in certain cells of the CV marks the onset of lymphatic specification.
  • TgBAC(proxla:KalT4-UAS:uncTagRFP) mm5 embryos were imaged. It was found that the first cells expressing proxla are already visible at 22-24 hpf in the ventral-PCV ( Figures 6A-E). Later on these cells divide, translocate to the dorsal PCV, and bud from the PCV to generate PACs ( Figures 6A-E).
  • WNT5B induced a marked increase in the fraction of LYVE1 + cells detected in the culture, as well as in the levels of PROX1 (Figure 11H) and FLT4 ( Figure 10U) mRNAs, indicating that the role of Wnt5b as potent inducer of LEC specification is evolutionarily conserved. Furthermore these findings suggest that Wnt5b acts directly on vascular progenitors to promote the "angioblast-to-lymphatic" specification.
  • Wnt5b induces LEC specification through activation of Wnt canonical pathway
  • Wnt5 is mostly referred to as a non- canonical Wnt ligand, which can also repress and/or activate the canonical pathway in different contexts. It is well-established that a key step in the activation of canonical- Wnt pathway is the inhibition of a destruction complex composed of APC, Axin, GSK3P, and other proteins, which results in stabilization and nuclear translocation of cytoplasmic ⁇ - catenin. The present inventors therefore began by analyzing lymphatic development following manipulation of Axin and APC.
  • lymphatic defects derived from Wnt/p-catenin inhibition could be secondary to Wnt5b- signaling depletion. Alternatively, they could reflect a cell- autonomous requirement for Wnt-signaling within prospective LEC progenitors.
  • ⁇ -catenin/TCF activity within vPCV-angioblasts was assessed using Tg(flil :EGFP;7xTCF-Xla.Siam:nlsmCherryf a5 double transgenic embryos. As seen in Figures 14A-B, TCF activity was detected in these cells at 24 hpf, and in PACs at 48 hpf.
  • proxl mRNA levels observed in zebrafish and hESCs could result from either transcriptional regulation, or post-transcriptional modifications that alter RNA stability of the proxl transcript.
  • TgBAC(proxla:KalT4-UAS:uncTagRFP) zebrafish reporter was utilized (Figure HE), in which the KalT4 fragment recapitulates the transcriptional activation of the endogenous proxla promoter, without being subjected to the post- transcriptional modifications of the proxla gene (the KalT4 cassette possess its own 3'UTR).

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Abstract

L'invention concerne un procédé pour favoriser la spécification des cellules endothéliales lymphatiques (LEC). La méthode consiste à réguler à la hausse l'activité de la voie canonique Wnt dans des cellules souches et/ou des cellules progénitrices vasculaires et/ou des angioblastes, ce qui permet de favoriser la spécification de LEC. L'invention concerne également des méthodes pour induire la lymphagiogenèse L'invention concerne en outre des compositions pharmaceutiques et des méthodes de traitement.
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WO2020215091A1 (fr) * 2019-04-19 2020-10-22 The Regents Of The University Of Colorado, A Body Corporate Traitement de maladie vasculaire et lymphatique
CN112326965A (zh) * 2020-10-22 2021-02-05 南京医科大学 Daam1蛋白在制备肾透明细胞癌诊断及预后评估试剂盒中的应用
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