WO2025199576A1 - Cellules souches/progénitrices hématopoïétiques et leurs procédés de production - Google Patents

Cellules souches/progénitrices hématopoïétiques et leurs procédés de production

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WO2025199576A1
WO2025199576A1 PCT/AU2025/050292 AU2025050292W WO2025199576A1 WO 2025199576 A1 WO2025199576 A1 WO 2025199576A1 AU 2025050292 W AU2025050292 W AU 2025050292W WO 2025199576 A1 WO2025199576 A1 WO 2025199576A1
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cells
population
medium
concentration
cell
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Elizabeth Ng
Edouard Stanley
Andrew Elefanty
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Murdoch Childrens Research Institute
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Murdoch Childrens Research Institute
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Priority claimed from AU2024900798A external-priority patent/AU2024900798A0/en
Application filed by Murdoch Childrens Research Institute filed Critical Murdoch Childrens Research Institute
Publication of WO2025199576A1 publication Critical patent/WO2025199576A1/fr
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Definitions

  • the invention relates to differentiating a pluripotent stem cell (PSC) into a definitive haematopoietic stem/progenitor cell which is capable of multilineage engraftment, and to therapeutic uses of such haematopoietic stem/progenitor cells.
  • PSC pluripotent stem cell
  • HSC Haematopoietic stem cell transplantation permits the reconstitution of the blood cell compartment, for example, in patients with haematopoietic disorders or receiving myeloablative therapy.
  • the provision of HSCs from differentiated induced pluripotent stem cells (iPSCs) or embryonic stem cells would be beneficial.
  • iPSCs differentiated induced pluripotent stem cells
  • HSCs derived from patient induced PSCs would circumvent the donor-host mismatch that leads to graft- versus-host-disease, a major source of morbidity and mortality in recipients of imperfectly matched allogenic transplants.
  • transplantable HSCs via in vitro haematopoietic differentiation has proved challenging, in part, due to difficulties distinguishing between cells resembling derivatives of the yolk-sac (and lacking re-populating activity), and those representing aorta-gonad-mesonephros (AGM)-like haematopoiesis (which possess repopulating activity).
  • AGM aorta-gonad-mesonephros
  • HSPCs hematopoietic stem / progenitor cells
  • a first aspect provides a method for generating a population of definitive haematopoietic stem/progenitor cells (HSPCs), the method comprising: a) culturing a population of mesoderm cells obtained from a population of PSCs in a medium comprising a WNT agonist, an ACTIVIN antagonist, a FGF, vascular endothelial growth factor (VEGF), and a retinoic acid signalling agent; b) culturing the population of cells from step a) in a medium comprising a FGF, VEGF, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), and a retinoic acid signalling agent, wherein the concentration of the retinoic acid signalling agent is 10 to 50 fold greater than the concentration of the retinoic acid signalling agent in the medium of step a), and wherein the concentration of VEGF is 2 to 10 fold greater than the concentration of VEGF
  • the method step a) comprises culturing the population of cells for a time sufficient for the generation of a population of cells expressing a pattern of HOXA genes including one or more of H0XA1, H0XA2, H0XA3, H0XA4, H0XA5, H0XA6, H0XA7, H0XA9, HOXA10, preferably one or more of H0XA5, H0XA7, H0XA9, and HOXA10, even more preferably H0XA5, H0XA7, H0XA9, and HOXA10.
  • the method step a) comprises culturing the population of cells for a period of about 2 days.
  • the present invention provides a method for differentiating a population of pluripotent stem cells (PSCs) into a population of definitive haematopoietic stem/progenitor cells (HSPCs), the method comprising: i) culturing the population of PSCs in a basal medium comprising a WNT agonist, a fibroblast growth factor (FGF), and Activin A; ii) culturing the population of cells comprising mesoderm cells from step i) in a medium comprising a WNT agonist, an ACTIVIN antagonist, a FGF, vascular endothelial growth factor (VEGF), and a retinoic acid signalling agent; iii) culturing the population of cells from step ii) in a medium comprising a FGF, VEGF, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), and a retinoic acid signalling agent, wherein
  • the method step i) comprises culturing the population of cells for a time sufficient for the generation of a population of mesoderm cells expressing CD 13 and CD90 on the cell surface. In a further embodiment, the method step i) comprises culturing the population of cells for a period of about 1 day.
  • the method step ii) comprises culturing the population of cells for a time sufficient for the generation of a population of cells expressing a pattern of HOXA genes including one or more of H0XA1, H0XA2, H0XA3, H0XA4, H0XA5, H0XA6, H0XA7, H0XA9, HOXA10, preferably one or more of HOXA5, HOXA7, HOXA9, and HOXA10, even more preferably HOXA5, HOXA7, HOXA9, and HOXA10.
  • the method step ii) comprises culturing the population of cells for a period of about 2 days.
  • the method step iii) comprises culturing the population of cells for a time sufficient for generation of a population of cells expressing CD34 on the cell surface. In a further embodiment, the method step iii) comprises culturing the population of cells for a period of about 2 days.
  • the method step iv) comprises culturing the population of cells for a time sufficient for generation of a population of cells co-expressing CD34 and CXCR4 on the cell surface. In a further embodiment, the method step iv) comprises culturing the population of cells for a period of about 2 days.
  • the method step v) comprises culturing the population of cells for a time sufficient for generation of a population of cells expressing CD34 without CXCR4 on the cell surface. In a further embodiment, the method step v) comprises culturing the population of cells for a period of about 4 days.
  • the method step vi) comprises culturing the population of cells for a time sufficient for generation of a population of definitive HSPCs. In a further embodiment, the method vi) comprises culturing the population of cells for a period of about 3 days. In one embodiment, the medium in step vi) further comprises, a TGF-beta pathway activator, a BMP, or a combination of one or more thereof.
  • the WNT agonist is CHIR99021.
  • the CHIR99021 is present in said medium at a concentration of about 4 pM.
  • the concentration of ACTIVIN A in the medium of step i) is about 5 ng/ml to 50 ng/mL. In a further embodiment, the concentration of ACTIVIN A in the medium of step i) is about 30 ng/mL.
  • the medium of step d) or step vi) comprises StemRegenninl (SRI), FLT3 receptor ligand (FLT3L), interleukin 3 (IL-3), and/or erythropoietin (EPO).
  • SRI StemRegenninl
  • FLT3L FLT3 receptor ligand
  • IL-3 interleukin 3
  • EPO erythropoietin
  • the FGF is FGF2, and/or wherein IGF comprises IGF1 and/or IGF2, optionally in equal concentrations when IGF1 and IGF2 are both present.
  • the method further comprises dissociating EBs in suspension following step d) or step vi) and harvesting cells dissociated from the EBs.
  • Figure 2 shows Multilineage engraftment depends on CHIR and retinoids during iPSC differentiation,
  • Numbers indicate concentration of CHIR (CH) in pM, and concentrations of BMP4 (B) and ACTIVIN A (A) in ng/ml.
  • ROL or RETA retinoid
  • BM cells comprised CD19 + B cells, CD33 + and CD13 + myeloid cells, and CD45 + CD34 + CD38 lo/_ HSC-like cells (HSC) (boxed in red),
  • HSC HSC-like cells
  • the spleen contained CD45 + CD19 + sIGM + B cells
  • the thymus contained immature CD45 + CD3“ thymocytes including CD4 CD8’ cells, transitioning through immature CD4 + to CD4 + CD8 + double positive cell states, whilst CD45 + CD3 + thymocytes included CD4 + CD8 + double positive and CD4 + and CD8 + single positive cells.
  • Figure 5 shows robust hematopoietic engraftment with cells differentiated using protocol #3.
  • BM bone marrow
  • S spleen
  • a Engraftment of bone marrow
  • b PB1.1 BFP
  • c PB5.1
  • PB 10.5 cells showing the phenotype of engrafting cells and the level of engraftment. Error bars, mean+SEM.
  • e - h Tissue distribution of engrafting cells in MLE recipients of RM TOM (e).
  • Each circle represents one animal, color coded to represent myeloid (M), myelo- lymphoid (ML), myelo-erythroid (ME) and erythro-myelo-lymphoid multilineage (MLE) patterns of engraftment. Error bars, mean+SEM.
  • Figure 7 shows schematic outline of the growth factors used for iPSC differentiation in screening protocols #1 and #2 and in protocol #3. Cohorts of mice transplanted with each protocol are indicated. Concentrations of growth factors used are provided below.
  • Figure 8 shows flow cytometry live cell gating strategy and negative control samples. Data collected in the indicated fluorochrome channels for unstained suspension hematopoietic cells and disaggregated swirling embryoid body (EB) cells. See also Figure Id and le. The same strategy was used for evaluation of iPSC engraftment in mouse hematopoietic tissues shown in Figures 4d- g, 5i-j, 6c and Figures 15b, c and 18a - d.
  • Each circle represents one animal, color coded to indicate myeloid (M), myelo-lymphoid (ML), lympho- myeloid (LM) and erythro-myelo-lymphoid multilineage (MLE) patterns of engraftment.
  • M myeloid
  • ML myelo-lymphoid
  • LM lympho- myeloid
  • MLE erythro-myelo-lymphoid multilineage
  • Figure 12 shows effects of retinoids on expression of stem cell genes during the endothelial to hematopoietic transition in vitro and expression of retinoid dependent genes in iPSC -differentiated cells and CS10 - CS17 human embryos,
  • RAA retinyl acetate
  • Figure 17 shows removal of VEGF at day 7 of differentiation accelerates the loss of arterial endothelial markers,
  • (b) CD34 expression in RM TOM cells by flow cytometry correlated with VEGF concentration. Error bars, mean+SEM, n 3. One-way ANOVA test for CD34 linear trend, P ⁇ 0.0001.
  • (c) Percentage of RM TOM CXCR4 + CD73 lo/+ arterial cells, subsetted from CD34 + cells, by flow cytometry correlated with VEGF concentration. Error bars, mean+SEM, n 3.
  • this workflow recapitulates clinical hematopoietic stem cell transplantation, in which donor hematopoietic cells harvested from the bone marrow or peripheral blood are cryopreserved prior to intravenous transplantation into the recipient.
  • the invention generally relates to methods and compositions for differentiating stem cells toward a definitive hematopoietic cell fate. More particularly, the invention provides a multi-stage, fully defined culture system wherein PSCs (e.g. iPSCs) or PSC-derived cells at various stages of development can be induced to assume a definitive hematopoietic phenotype, including definitive hemogenic endothelium, to hematopoietic stem/progenitor cells (HSPCs) and their progeny, wherein the HSPCs have multiple lineage engraftment capacity. That is, the invention provides methods, and cells obtained by such methods, involving the coordinated temporal provision of cell culture components (e.g.
  • WNT- agonists that permits a definitive HSPC fate to be assumed (e.g. a CD34 + definitive hematopoietic population) by PSC- derived cells wherein the HSPCs are capable of multilineage engraftment.
  • a cell includes one cell, one or more cells and a plurality of cells.
  • the term “about” as used herein contemplates a range of values for a given number of ⁇ 25% the magnitude of that number. In other embodiments, the term “about” contemplates a range of values for a given number of ⁇ 30%, ⁇ 20%, ⁇ 15%, ⁇ 10%, or ⁇ 5% the magnitude of that number. For example, in one embodiment, “about 3 pM” indicates a value of 2.7 to 3.3 pM (i.e. 3 pM ⁇ 10%), and the like.
  • differentiation processes include ordered, sequential events
  • the timing of the events may be varied by at least 25%.
  • a particular step may be disclosed in one embodiment as lasting one day, the event may last for more or less than one day.
  • “one day” may include a period of about 18 to about 30 hours.
  • periods of time may vary by ⁇ 20%, ⁇ 15%, ⁇ 10%, or ⁇ 5% of that period of time.
  • Periods of time indicated that are multiple day periods may be multiples of “one day,” such as, for example, about two days may span a period of about 36 to about 60 hours, and the like.
  • time variation may be lessened, for example, where 1 day is 24 ⁇ 3 hours; 3 days is 72 ⁇ 3 hours; 4 days is 96 ⁇ 3 hours; 5 days is 120 ⁇ 3 hours; 6 days is 144 ⁇ 3 hours; 7 days is 168 ⁇ 3 hours; 11 days is 264 ⁇ 3.
  • about 3 days may be 2.5, 3 or 3.5 days, about 4 days may be 3.5, 4 or 4.5 days, about 5 days may be 4.5, 5 or 5.5 days, about 6 days may be 5.5, 6 or 6.5 days, about 7 days may be 6.5, 7 or 7.5 days, about 11 days may be 10, 10.5, 11, 11.5, or 12 days, about 21 days may be 20, 20.5, 21, 21.5, or 22 days.
  • pluripotent stem cell and “PSC” refer to cells that display pluripotency.
  • human pluripotent stem cell and “hPSC” refer to cells derived, obtainable or originating from human tissue that display pluripotency.
  • the hPSC may be a human embryonic stem cell or a human induced pluripotent stem cell (iPSC).
  • Human pluripotent stem cells may be derived from inner cell mass or reprogrammed using Yamanaka factors from many foetal or adult somatic cell types.
  • the generation of hPSCs may be possible using somatic cell nuclear transfer.
  • induced pluripotent stem cell and “iPSC” and “hiPSC” (human iPSC) refer to cells derivable, obtainable or originating from adult somatic cells of any type reprogrammed to a pluripotent state through the expression of exogenous genes, such as transcription factors, including but not limited to a preferred combination of OCT4, SOX2, KLF4 and c-MYC.
  • hiPSC show levels of pluripotency equivalent to hESC but can be derived from an individual for autologous therapy with or without concurrent gene correction prior to differentiation and cell delivery.
  • the method disclosed herein could be applied to any pluripotent stem cell derived from any individual or a hPSC subsequently modified to generate a mutant model using gene-editing or a mutant hPSC corrected using gene-editing.
  • Gene-editing could be by way of CRISPR, TALEN or ZF nuclease technologies.
  • the term “mesoderm” refers to one of the three germinal layers that appears during early embryogenesis and which gives rise to various specialized cell types including blood cells of the circulatory system, muscles, the heart, the dermis, skeleton, and other supportive and connective tissues.
  • a "progenitor cell” is a cell which is capable of differentiating along one or a plurality of developmental pathways, with or without self -renewal. Typically, progenitor cells are unipotent or oligopotent and are capable of at least limited self- renewal.
  • differentiate relate to progression of a cell from an earlier or initial stage of a developmental pathway to a later or more mature stage of the developmental pathway.
  • undifferentiated in this context, relate to a cell from an earlier or initial stage of a developmental pathway or a cell that has not yet developed into a specialized cell type. It will be appreciated that in this context “differentiated' does not mean or imply that the cell is fully differentiated and has lost pluripotency or capacity to further progress along the developmental pathway or along other developmental pathways. Differentiation may be accompanied by cell division.
  • the stage or state of differentiation of a cell may be characterized by the expression and/or non-expression of one or more specific markers.
  • the expression of “signature” or “milestone” markers may be used in determining or defining the stage or state of differentiation instead of using the period of time defined in days and/or hours.
  • markers is meant nucleic acids or proteins that are encoded by the genome of a cell, cell population, lineage, compartment or subset, whose expression or pattern of expression changes throughout development. Nucleic acid marker expression may be detected or measured by any technique known in the art including nucleic acid sequence amplification (e.g. polymerase chain reaction) and nucleic acid hybridization (e.g.
  • Protein marker expression may be detected or measured by any technique known in the art including flow cytometry, immunohistochemistry, immunoblotting, protein arrays, protein profiling (e.g. 2D gel electrophoresis), although without limitation thereto.
  • Such terms are commonplace and well-understood by the skilled person when characterizing cell phenotypes.
  • a skilled person would conclude the presence or evidence of a distinct signal for the marker when carrying out a measurement capable of detecting or quantifying the marker in or on the cell.
  • the presence or evidence of the distinct signal for the marker would be concluded based on a comparison of the measurement result obtained for the cell to a result of the same measurement carried out for a negative control (for example, a cell known to not express the marker) and/or a positive control (for example, a cell known to express the marker).
  • a positive cell may generate a signal for the marker that is at least 1.5 -fold higher than a signal generated for the marker by a reference cell (e.g. negative control cell) or than an average signal generated for the marker by a population of reference or negative control cells, e.g., at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold higher, at least 100-fold higher, or even higher.
  • a reference cell e.g. negative control cell
  • an average signal generated for the marker by a population of reference or negative control cells e.g., at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold higher, at least 100-fold higher, or even higher.
  • a positive cell may generate a signal for the marker that is 3.0 or more standard deviations, e.g., 3.5 or more, 4.0 or more, 4.5 or more, or 5.0 or more standard deviations, higher than an average signal generated for the marker by a population of reference or negative control cells.
  • the terms “culture medium”, “cell culture medium”, “defined medium”, and the like refer to media that are suitable to support the growth of cells in vitro (i.e., cell cultures, cell lines, etc.). It is not intended that the term be limited to any particular culture medium. For example, it is intended that the definition encompass maintenance media as well as other media for the differentiation or specialization of cells. Indeed, it is intended that the term encompass any culture medium suitable for the growth of the cell cultures and cells of interest.
  • the cell culture medium used in various steps includes a basal medium which is supplemented. In some embodiments, the basal medium is SPELS medium.
  • Enriched as in an enriched population of cells, can be defined phenotypically based upon the increased number of a specific subset or subtype of cells having a particular marker, or combination of markers, or having one or more markers and lacking one or more other markers, in a fractionated, or expanded, set of cells as compared with the number of cells having the marker, combination of markers, or having one or more markers and lacking one or more other markers, in the unfractionated or unexpanded set of cells.
  • tissue means an aggregate of cells.
  • the cells in the tissue are cohered or fused.
  • a definitive haematopoietic stem/progenitor cell described herein or produced by the methods described herein may be further characterized by gene and protein expression as detailed in the examples and figures.
  • embryoid body and “EB” refers to a three-dimensional aggregate of PSCs.
  • EBs in may be cultured in suspension, thus making EB cultures scalable for clinical applications.
  • the three-dimensional structure of EBs including the establishment of complex cell adhesions and paracrine signaling within the EB microenvironment, enables differentiation and morphogenesis which yields microtissues that are similar to native tissue structures, for example the AGM as disclosed herein.
  • Methods for culturing EBs are known in the art.
  • a specific example of a spin or swirling EB culture method that may be used in the present invention is described in Ng et al. Nature Protocols 3, 768-776 (2008).
  • “reduced,” “reduction,” “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more.
  • “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • agonist or “activator” may be used interchangeably and as used herein means an activator, for example, of a pathway or signalling molecule.
  • An agonist of a molecule can retain substantially the same, or a subset, of the biological activities of the molecule (e.g. FGF).
  • FGF biological activity of the molecule
  • an FGF agonist or FGF activator means a molecule that selectively activates FGF signalling.
  • a selective inhibitor for example of a pathway or signalling molecule.
  • An inhibitor or antagonist of a molecule e.g. BMP4 inhibitor
  • BMP4 inhibitor can inhibit one or more of the activities of the naturally occurring form of the molecule.
  • a BMP4 inhibitor is a molecule that selectively inhibits BMP signalling mediated by BMP4.
  • the term “therapeutic composition” refers to a composition comprising a haematopoietic stem/progenitor cell of or produced according to the invention that has been formulated for administration to a subject.
  • the therapeutic composition is sterile.
  • the therapeutic composition is pyrogen-free.
  • terapéuticaally effective amount refers to an amount of the haematopoietic stem/progenitor cell of or produced according to the invention effective to treat a condition, disease or disorder in a subject.
  • treat refers to both therapeutic treatment and prophylactic or preventative measures, wherein the aim is to prevent or ameliorate a condition, disease or disorder in a subject or slow down (lessen) progression of a condition, disease or disorder in a subject.
  • Subjects in need of treatment include those already with the condition, disease or disorder as well as those in which the condition, disease or disorder is to be prevented.
  • preventing refers to keeping from occurring, or to hinder, defend from, or protect from the occurrence of a condition, a disease or disorder, including an abnormality or symptom.
  • a subject in need of prevention may be prone to develop the condition, disease or disorder.
  • ameliorate or “amelioration” refers to a decrease, reduction or elimination of a condition, a disease or disorder, including an abnormality or symptom.
  • a subject in need of treatment may already have the condition, disease or disorder, or may be prone to have the condition, disease or disorder, or may be in whom the condition, disease or disorder is to be prevented.
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • the methods of the present invention comprise a WNT pathway activator or a WNT agonist.
  • the WNT pathway activator or WNT agonist is selected from the group consisting of CHIR99021 (6-[[2-[[4-(2,4-Dichlorophenyl)5-(5-methyl- lH-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), Wntl, Wnt-2, Wnt- 2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a/b, Wnt-7b, Wnt48 8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-lOa, Wnt-lOb, Wnt-11, Wnt-16b, RSPO co-agonist
  • the Wnt pathway activator is CHIR99021.
  • the WNT agonist in the cell culture media is CHIR99021 ((CHIR) CAS 252917-06-9).
  • the concentration of CHIR99021 in the medium is about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about
  • the methods of the present invention comprise a TGF-beta pathway activator, for example, Activin A.
  • the TGF-beta pathway activator is selected from the group consisting of Activin A, TGF-betal, TGF-beta2, TGF-beta3, IDE1/2 (IDE1 (l-[2-[(2Carboxyphenyl)methylene]hydrazide]heptanoic acid), IDE2 (Heptanedioic acid- 1 -(249 cyclopentylidenehydrazide)), and Nodal.
  • the TGF-beta pathway activator is Activin A.
  • the concentration of Activin A in the medium is about 1 ng/mE, about 2 ng/mL, about 3 ng/mL about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24 ng/mL, about 25 ng/mL, about 26 ng/mL, about 27 ng/mL, about 28 ng/mL, about 29 ng/mL, about 30 ng/mL,
  • the TGF-beta pathway activator is TGF-beta 1.
  • the concentration of TGF-beta in the medium used in the methods of the invention is about 0.1 ng/ml, about 1 ng/mL, about 2 ng/mL, about 3 ng/mL about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, or about 10 ng/mL.
  • the methods of the present invention comprise FGF.
  • the FGF is selected from the group consisting of FGF2, FGF4, FGF9, FGF19, FGF21, FGF3, FGF5, FGF6, FGF8a, FGF16, FGF17, FGF18, FGF20 and FGF23.
  • the FGF is FGF2.
  • the concentration of FGF2, also known as basic fibroblast growth factor (bFGF), in the medium is about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24 ng/mL, about 25 ng/mL, about 26 ng/mL, about 27 ng/mL, about 28 ng/mL, about 29
  • the methods of the present invention comprise a TGF-beta pathway inhibitor or ACTIVIN antagonist.
  • the TGF-beta pathway inhibitor/ACTIVIN antagonist is selected from the group consisting of A-83-01 (3-(6-Methyl-2- pyridinyl)-N-phenyl-4-(4-quinolinyl)- 1 H-pyrazole- 1 carbothioamide) , D4476 (4- [4-(2,3 -Dihydro- l,4-benzodioxin-6-yl)-5-(2-pyridinyl)-lHimidazol-2-yl]benzamide), GW 788388 (4-[4-[3-(2- Pyridinyl)- lH-pyrazol-4-yl]-2-pyridinyl]-N(tetrahydro-2H-pyran-4-yl)-benzamide), LY 364947 (4-[3-(2-Pyridin
  • the ACTIVIN antagonist is SB431542.
  • the concentration of SB431542 in the medium is about 0.1 pM, about 0.5 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, or about 10 pM.
  • the methods of the present invention comprise a Rho kinase inhibitor (ROCKi).
  • the ROCK inhibitor is thiazovivin, Y27632, or pyrintegrin.
  • the ROCK inhibitor is thiazovivin.
  • the concentration of Y-27263 in the medium is about 1 pM, about 2 pM, about 5 pM, about 8 pM, about 8.2 pM, about 8.4 pM, about 8.6 pM, about 8.8 pM, about 9 pM, about 9.2 pM, about 9.4 pM, about 9.6 pM, about 9.8 pM, about 10 pM, about 10.2 pM, about 10.4 pM, about 10.6 pM, about 10.8 pM, about 11 pM, about 11.2 pM, about 11.4 pM, about 11.6 pM, about 11.8 pM, about 12 pM, about 15 pM, about 20 pM, about 25 pM or about 50 pM.
  • the concentration of thiazovivin in the medium is about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1 pM, about 1.1 pM, 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.2 pM, about 2.4 pM, about 2.6 pM, about 2.8 pM, about 3 pM, about 3.2 pM, about 3.4 pM, about 3.6 pM, about 3.8 pM, about 4 pM, about 4.2 pM, about 4.6 pM, about 4.8 pM or about 5 pM.
  • the methods of the present invention comprise vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • the VEGF includes human VEGF family members such as VEGFA as well as non-human VEGF.
  • the concentration of VEGF in the medium is about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, about 300 ng/mL, about 350 ng/mL, about 400 ng/mL, about 450
  • the methods of the present invention comprise a retinoic acid signalling agent.
  • the retinoic acid signalling agent is a retinoid is selected from retinol or retinyl acetate (RETA).
  • the retinoid is RETA.
  • the concentration of RETA in the medium is about 0.01 pM, 0.02 pM, 0.03 pM, 0.04 pM, 0.05 pM, 0.06 pM, 0.07 pM, 0.08 pM, 0.09 pM, 0.1 pM, 0.15 pM, 0.16 pM, 0.17 pM, 0.18 pM, 0.19 pM, 0.2 pM, 0.25 pM, 0.30 pM, 0.35 pM, 0.4 pM, 0.45 pM, 0.5 pM, 0.55 pM, 0.6 pM, 0.65 pM, 0.7 pM, 0.75 pM, 0.85 pM, 0.9 pM, 0.95 pM, 1 pM, 1.1 pM, 1.2 pM, 1.3 pM, 1.4 pM, 1.5 pM, 1.6 pM, 1.7 pM, 1.8 pM, 1.9 pM, 2
  • the methods of the present invention comprise a BMP.
  • the BMP is selected from the group consisting of BMP4, BMP2 and BMP7.
  • the BMP is BMP4.
  • the concentration of bone morphogenetic protein 4 (BMP4) in the medium used is about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70
  • the methods of the present invention comprise an IGF.
  • the IGF is selected from the group consisting of IGF1 and IGF2.
  • the medium comprises both IGF1 and IGF2.
  • the concentration of IGF and/or IGF2, in the medium used is about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, about 20 ng/mL, about 21 ng/mL, about 22 ng/mL
  • the methods of the present invention comprise stem cell factor (SCF).
  • SCF stem cell factor
  • the concentration of SCF in the medium is about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24 ng/mL, about 25
  • the methods of the present invention comprise thrombopoietin (TPO).
  • TPO thrombopoietin
  • the concentration of TPO in the medium is about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24 ng/mL, about 25 ng/mL, about 26 ng/mL, about 27
  • HSPCs definitive haematopoietic stem/progenitor cells
  • the invention provides a method of using a multistage process to generate and expand definitive HSPCs.
  • the method begins with a stage wherein a pluripotent stem cell is differentiated to a mesodermal cell, next the mesodermal cells are patterned to express HOXA genes, in a next stage, the patterned mesodermal cells are differentiated (and expanded) to hemogenic endothelium (HE).
  • HE hemogenic endothelium
  • the HE cells are differentiated to undergo endothelial to haematopoietic transition into definitive HSPCs while being expanded at same time.
  • the invention also provides a method of generating and expand definitive HSPCs that comprises differentiating pluripotent stem cell-derived mesodermal cells to give rise to HE, and differentiating the HE to give rise to HSPC.
  • the present invention provides a method for generating a population of definitive haematopoietic stem/progenitor cells (HSPCs), the method comprising: a) culturing a population of mesoderm cells obtained from a population of PSCs in a medium comprising a WNT agonist, an ACTIVIN antagonist, a FGF, vascular endothelial growth factor (VEGF), and a retinoic acid signalling agent; b) culturing the population of cells from step a) in a medium comprising a FGF, VEGF, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), and a retinoic acid signalling agent, wherein the concentration of the retinoic acid signalling agent is 10 to 50 fold greater than the concentration of the retinoic acid signalling agent in the medium of step a), and wherein the concentration of VEGF is 2 to 10 fold greater than the concentration of VEGF in the medium of step
  • the culturing of the population of mesoderm cells in step a) is performed for a time sufficient for the generation of a population of cells expressing a pattern of HOXA genes including one or more of H0XA1, H0XA2, H0XA3, H0XA4, H0XA5, H0XA6, H0XA7, H0XA9, HOXA10.
  • the culturing of the population of mesoderm cells in step a) is performed for a time sufficient for the generation of a population of cells expressing one or more of H0XA5, H0XA7, H0XA9, and HOXA10.
  • the culturing in step a) is performed for about 2 to about 72 hours, In one embodiment, the culturing in step a) is performed for about 2 days. Preferably, the culturing in step a) is performed for about 48 hours.
  • the WNT agonist is CHIR99021, optionally, wherein the medium comprises about 2 pM, to about 5 pM CHIR99021, preferably 4 pM CHIR99021.
  • the ACTIVIN antagonist is SB431542, optionally, wherein the medium comprises about 2 pM, to about 5 pM SB431542, preferably about 3 or 4 pM SB431542.
  • the medium comprises about 5 to about 50 ng/mL FGF, preferably about 20 ng/mL FGF. In one embodiment, the medium comprises about 5 to about 50 ng/mL VEGF, preferably about 25 ng/mL VEGF. In another embodiment, the retinoic acid signalling agent is RETA, optionally, wherein the medium comprises about 50 nM to about 100 nM RETA, preferably about 50 nM RETA.
  • the culturing of the population of cells from step a) in step b) is performed for a time sufficient for generation of a population of cells expressing CD34 on the cell surface.
  • the culturing in step b) is performed for about 2 to about 72 hours, In one embodiment, the culturing in step b) is performed for about 2 days. Preferably, the culturing in step b) is performed for about 48 hours.
  • the medium comprises about 5 ng/mL to about 50 ng/mL FGF, preferably about 20 ng/mL FGF.
  • the concentration of VEGF in the medium is about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about 10 fold greater than the concentration of VEGF in the medium of step a). In one embodiment, the concentration of VEGF in the medium is about 5 fold greater than the concentration of VEGF in the medium of step a). In one embodiment, the medium comprises about 50 to about 200 ng/mL VEGF, preferably about 150 ng/mL VEGF.
  • the concentration of the retinoic acid signalling agent in the medium is about 10 fold, about 15 fold, about 20 fold, about 25 fold, about 30 fold, about 35 fold, about 40 fold, about 45 fold, or about 50 fold greater than the concentration of the retinoic acid signalling agent in the medium of step a). In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 20 fold greater than the concentration of the retinoic acid signalling agent in the medium of step a). In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 40 fold greater than the concentration of the retinoic acid signalling agent in the medium of step a). In another embodiment, the retinoic acid signalling agent is RETA.
  • the medium comprises about 0.5 pM, to about 4 pM, RETA, preferably about 2 pM RETA. In one embodiment, the medium comprises about 5 ng/mL to about 50 ng/mL of an IGF, preferably about 10 ng/mL IGF1 and/or IGF2, even more preferably 10 ng/mL of each of IGF1 and IGF2. In one embodiment, the medium comprises about 5 ng/mL to about 50 ng/mL BMP4, preferably about 20 ng/mL BMP4.
  • the culturing of the population of cells from step b) in step c) is performed for a time sufficient for generation of a population of cells co-expressing CD34 and CXCR4 on the cell surface.
  • the culturing in step c) is performed for at least about 2 days and up to about 6 days.
  • the culturing in step c) is performed for about 6 days.
  • the culturing in step c) is performed for about 48 hours.
  • the medium comprises about 5 to about 50 ng/mL FGF, preferably about 20 ng/mL FGF.
  • the medium comprises about 50 ng/mL to about 200 ng/mL VEGF, preferably about 150 ng/mL VEGF.
  • the concentration of the retinoic acid signalling agent in the medium is about 10 fold, about 15 fold, about 20 fold, about 25 fold, about 30 fold, about 35 fold, about 40 fold, about 45 fold, or about 50 fold lower than the concentration of the retinoic acid signalling agent in the medium of step b). In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 20 fold lower than the concentration of the retinoic acid signalling agent in the medium of step b).
  • the concentration of the retinoic acid signalling agent in the medium is about 40 fold lower than the concentration of the retinoic acid signalling agent in the medium of step b).
  • the retinoic acid signalling agent is RETA.
  • the medium comprises about 50 nM to about 100 nM RETA, preferably about 100 nM RETA.
  • the medium comprises about 5 ng/mL to about 50 ng/mL of an IGF, preferably about 10 ng/mL IGF1 and/or IGF2, even more preferably 10 ng/mL of each of IGF1 and IGF2.
  • the concentration of the BMP4 in the medium is the same as that in the medium in step b).
  • the concentration of the BMP4 in the medium is about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 11 fold, about 12 fold, about 13 fold, about 14 fold, about 15 fold, about 16 fold, about 17 fold, about 18 fold, about 19 fold, or about 20 fold lower than the concentration of BMP4 in the medium of step b).
  • the concentration of BMP4 in the medium is about 10 fold lower than the concentration of BMP4 in the medium of step b).
  • the medium comprises about 0.5 to about 5 ng/mL BMP4, preferably about 2 ng/mL BMP4.
  • the culturing of the population of cells from step c) in step d) is performed for a time sufficient for generation of a population of cells co-expressing CD34, CD90 and CD45 on the cell surface.
  • the culturing in step d) is performed for about 1 day up to about 5 days.
  • the culturing in step d) is performed for about 2 days.
  • the culturing in step d) is performed for about 48 hours.
  • the medium comprises about 5 ng/mL to about 50 ng/mL FGF, preferably about 10 ng/mL FGF.
  • the medium does not comprise VEGF.
  • the retinoic acid signalling agent is RETA, optionally wherein the medium comprises about 50 nM to about 100 nM RETA, preferably about 50 nM RETA.
  • the medium comprises about 5 ng/mL to about 50 ng/mL of an IGF, preferably about 10 ng/mL IGF1 and/or IGF2, even more preferably 10 ng/mL of each of IGF1 and IGF2.
  • the medium comprises about 5 to about 50 ng/mL TPO, preferably about 10 ng/mL TPO.
  • the medium comprises about 5 to about 50 ng/mL SCF, preferably about 10 ng/mL SCF.
  • the medium does not comprise BMP4.
  • the medium in step d) further comprises, a TGF-beta pathway activator, a BMP pathway activator, or a combination of one or more thereof.
  • step a) occurs from day 0 to day 2.
  • step b) occurs from day 2 to day 4.
  • step c) occurs from day 4 to day 10.
  • step d) occurs from day 10 to day 12, day 13 or day 14, or day 15.
  • step a) occurs from day 0 to day 2
  • step b) occurs from day 2 to day 4
  • step c) occurs from day 4 to day 10
  • step d) occurs from day 10 to day 12, day 13 or day 14, or day 15.
  • the population of mesoderm cells are in embryoid bodies (EBs).
  • EBs embryoid bodies
  • the present invention provides a method for differentiating a population of pluripotent stem cells (PSCs) into a population of definitive haematopoietic stem/progenitor cells (HSPCs), the method comprising: i) culturing the population of PSCs in a basal medium comprising a WNT agonist, a fibroblast growth factor (FGF), and Activin A; ii) culturing the population of cells comprising mesoderm cells from step i) in a medium comprising a WNT agonist, an ACTIVIN antagonist, a FGF, vascular endothelial growth factor (VEGF), and a retinoic acid signalling agent; iii) culturing the population of cells from step ii) in a medium comprising a FGF, VEGF, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), and a retinoic acid signalling agent, wherein the
  • the culturing of the population of cells in step i) is performed for a time sufficient to generate a population of mesoderm cells expressing CD13 and CD90 on the cell surface.
  • the culturing in step ii) is performed for about 2 to about 48 hours.
  • the culturing in step i) is performed for about 1 day.
  • the culturing in step i) is performed for about 24 hours.
  • the WNT agonist is CHIR99021, optionally wherein the medium comprises about 2 pM, to about 5 pM CHIR99021, preferably 4 pM.
  • the medium comprises about 5 ng/mL to about 50 ng/mL FGF, preferably about 20 ng/mL FGF.
  • the concentration of ACTIVIN A in the medium of step i) is about 5 ng/ml to 50 ng/mL, preferably wherein the concentration is about 30 ng/mL.
  • the medium further comprises a BMP, preferably BMP4.
  • the concentration of ACTIVIN A in the medium of step i) is about 5 ng/ml and the medium further comprises a BMP, preferably BMP4, at a concentration of about 3 ng/mL.
  • the medium also comprises a ROCK inhibitor.
  • the ROCK inhibitor is thiazovivin.
  • the medium further comprises about 0.5 pM to about 5 pM, preferably about 1 pM ROCK inhibitor.
  • the culturing of the population of cells from step i) in step ii) is performed for a time sufficient for the generation of a population of cells expressing a pattern of HOXA genes including one or more of H0XA1, H0XA2, H0XA3, H0XA4, H0XA5, H0XA6, H0XA7, H0XA9, and HOXA10.
  • the culturing of the population of mesoderm cells in step ii) is performed for a time sufficient to generate a population of cells expressing one or more of H0XA5, H0XA7, H0XA9, and HOXA10.
  • the culturing in step ii) is performed for about 2 to about 72 hours. In one embodiment, the culturing in step ii) is performed for about 2 days. Preferably, the culturing in step ii) is performed for about 48 hours.
  • the WNT agonist is CHIR99021, optionally wherein the medium comprises about 2 pM, to about 5 pM CHIR99021, preferably 4 pM CHIR9902L
  • the ACTIVIN antagonist is SB431542, optionally wherein the medium comprises about 2 pM, to about 5 pM SB431542, preferably about 3 or 4 pM SB431542.
  • the culturing in step iii) is performed for about 2 to about 72 hours. In one embodiment, the culturing in step iii) is performed for about 2 days. Preferably, the culturing in step iii) is performed for about 48 hours.
  • the medium comprises about 5 ng/mL to about 50 ng/mL FGF, preferably about 20n g/mL FGF. In one embodiment, the concentration of VEGF in the medium is about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about 10 fold greater than the concentration of VEGF in the medium of step ii).
  • the concentration of VEGF in the medium is about 5 fold greater than the concentration of VEGF in the medium of step ii).
  • the medium comprises about 50 ng/mL to about 200 ng/mL VEGF, preferably about 150 ng/mL VEGF.
  • the concentration of the retinoic acid signalling agent in the medium is about 10 fold, about 15 fold, about 20 fold, about 25 fold, about 30 fold, about 35 fold, about 40 fold, about 45 fold, or about 50 fold greater than the concentration of the retinoic acid signalling agent in the medium of step ii).
  • the concentration of the retinoic acid signalling agent in the medium is about 20 fold greater than the concentration of the retinoic acid signalling agent in the medium of step ii). In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 40 fold greater than the concentration of the retinoic acid signalling agent in the medium of step ii). In another embodiment, the retinoic acid signalling agent is RETA. In one embodiment, the medium comprises about 0.5 pM, to about 4 pM, RETA, preferably about 2 pM RETA.
  • the medium comprises about 5 ng/mL to about 50 ng/mL of an IGF, preferably about 10 ng/mL IGF1 and/or IGF2, even more preferably 10 ng/mL of each of IGF1 and IGF2. In one embodiment, the medium comprises about 5 ng/mL to about 50 ng/mL BMP4, preferably about 20n g/mL BMP4.
  • the culturing of the population of cells from step iii) in step iv) is performed for a time sufficient to generate a population of cells co-expressing CD34 and CXCR4 on the cell surface.
  • the culturing in step iv) is performed for at least about 2 days and up to about 6 days.
  • the culturing in step iv) is performed for about 2 days.
  • the culturing in step iv) is performed for about 4 days.
  • the medium comprises about 5 ng/mL to about 50 ng/mL FGF, preferably about 20 ng/mL FGF.
  • the medium comprises about 50 ng/mL to about 200 ng/mL VEGF, preferably about 150 ng/mL VEGF.
  • the concentration of the retinoic acid signalling agent in the medium is about 10 fold, about 15 fold, about 20 fold, about 25 fold, about 30 fold, about 35 fold, about 40 fold, about 45 fold, or about 50 fold lower than the concentration of the retinoic acid signalling agent in the medium of step iii). In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 20 fold lower than the concentration of the retinoic acid signalling agent in the medium of step iii).
  • the concentration of the retinoic acid signalling agent in the medium is about 40 fold lower than the concentration of the retinoic acid signalling agent in the medium of step iii).
  • the retinoic acid signalling agent is RETA.
  • the medium comprises about 50 nM to about 100 nM RETA, preferably about 100 nM RETA.
  • the medium comprises about 5 ng/mL to about 50 ng/mL of an IGF, preferably about 10 ng/mL IGF1 and/or IGF2, even more preferably 10 ng/mL of each of IGF1 and IGF2.
  • the concentration of the BMP4 in the medium is about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 11 fold, about 12 fold, about 13 fold, about 14 fold, about 15 fold, about 16 fold, about 17 fold, about 18 fold, about 19 fold, or about 20 fold lower than the concentration of BMP4 in the medium of step iii). In one embodiment, the concentration of BMP4 in the medium is about 10 fold lower than the concentration of BMP4 in the medium of step iii). In another embodiment, the medium comprises about 0.5 ng/mL to about 5 ng/mL BMP4, preferably about 2 ng/mL BMP4.
  • the culturing of the population of cells from step iv) in step v) is performed for a time sufficient to generate a population of cells expressing CD34 without CXCR4 on the cell surface.
  • the culturing in step v) is performed for about 1 day up to about 5 days.
  • the culturing in step v) is performed for about 2 days.
  • the culturing in step v) is performed for about 48 hours.
  • the medium comprises about 5 to about 50 ng/mL FGF, preferably about 10 ng/mL FGF.
  • the retinoic acid signalling agent is RETA, optionally wherein the medium comprises about 50 nM to about 100 nM RETA, preferably about 50 nM RETA.
  • the medium comprises about 5 ng/mL to about 50 ng/mL of an IGF, preferably about 10 ng/mL IGF1 and/or IGF2, even more preferably 10 ng/mL of each of IGF1 and IGF2.
  • the medium comprises about 0.5 ng/mL to about 5 ng/mL BMP4, preferably about 2 ng/mL BMP4.
  • the medium in step v) is identical to that employed for step iv) except that VEGF is excluded.
  • the method further comprises including in the culture medium in step v) about 5 ng/mL to about 50 ng/mL SCF, preferably about 10 ng/mL SCF after about 24 hours, or after about 48h when the culturing in step iv) occurs for greater than 48 hours.
  • the method further comprises including in the culture medium in step v) after 48 hours a TGF-beta pathway activator, a BMP pathway activator, or a combination of one or more thereof.
  • the culturing of the population of cells from step v) in step vi) is performed for a time sufficient for to generate a population of definitive HSPCs.
  • the culturing in step vi) is performed from about 1 day up to about 10 days.
  • the culturing in step vi) is performed from about 1 day up to about 5 days.
  • the culturing in step vi) is performed for about 3 days.
  • the culturing in step vi) is performed for about 48 - 72 hours.
  • the medium comprises about 5 ng/mL to about 50 ng/mL FGF, preferably about 10 ng/mL FGF.
  • the medium does not comprise VEGF.
  • the retinoic acid signalling agent is RETA, optionally wherein the medium comprises about 50 nM to about 100 nM RETA, preferably about 50 nM RETA.
  • the medium comprises about 5 ng/mL to about 50 ng/mL of IGF, preferably about 10 ng/mL IGF1 and/or IGF2, even more preferably 10 ng/mL of each of IGF1 and IGF2.
  • the medium comprises about 5 ng/mL to about 50 ng/mL TPO, preferably about 10 ng/mL TPO.
  • the medium comprises about 5 ng/mL to about 50 ng/mL SCF, preferably about 10 ng/mL SCF.
  • the medium does not comprise BMP4.
  • the medium further comprises StemRegenin 1 (SRI) (4-[2-[[2-benzo[b]thien-3-yl-9-(l-methylethyl)-9H-purin-6-yl]amino]ethyl]-phenol).
  • SRI StemRegenin 1
  • the medium comprises about 2 nM to about 500 nM, preferably about 100 nM StemRegenin.
  • the method further comprises including in the culture medium in step vi) a TGF-beta pathway activator, a BMP pathway activator, or a combination of one or more thereof.
  • step i) occurs from day 0 to day 1
  • step ii) occurs from day 1 to day 3
  • step iii) occurs from day 3 to day 5
  • step iv) occurs from day 5 to day 7
  • step v) occurs from day 7 to day 11
  • step vi) occurs from day 11 to day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21 or for a time sufficient for generation of a population of definitive HSPCs.
  • the population of definitive HSPCs express one or more of HLF, SPINK2, HOXA. RUNX1, MECOM, MEET3, HLF, HOXA9.
  • the population of definitive HSPCs are HLF+SP1NK2+.
  • the definitive the population of definitive HSPCs are CD34+.
  • the population of definitive HSPCs express CD34, CD45, and one or more of CD90, CD44, KIT, CD201, ITGA3 and ITGA6 on the cell surface.
  • the above methods comprise culturing the population of cells using an embryoid body protocol.
  • the cells are cultured with swirling.
  • the cells are cultured according to a swirling Embryoid Body (SwB) protocol as described in Motazedian, A. et al. Nature cell biology 22, 60-73 (2020).
  • about 2.0 x 10 6 - about 2.0 x 10 7 iPSCs are differentiated in 60 mm dishes.
  • dissociated iPSCs are transferred to a nontissue culture treated 60 mm dish and cultured in 5 ml of SPELS medium.
  • the cell culture dishes are placed on an orbital shaker.
  • the methods yield approximately 1.4 x 10 8 CD34 + hematopoietic cells by day 14, a yield of about 7 CD34 + cells for each input iPSC.
  • the method further comprises harvesting single cells, cell aggregates or clusters and/or EBs in suspension following step d) or step vi). In another embodiment, the method further comprises dissociating or disaggregating cell aggregates or EBs in suspension following step d) or step vi) and harvesting cells dissociated or disaggregated from the EBs or cell aggregates or clusters. In another embodiment of the above methods, the method further comprising enriching, or sorting the harvested cells, such as for CD34 + cells. In one embodiment, the cells are enriched or sorted using magnetic bead separation. In another embodiment, the cells are enriched using cell sorting. In one embodiment, the cells are enriched for CD34 + cells.
  • the above methods further comprise sorting the obtained cells, such as following step d) or step vi) or following any harvesting or dissociation or disaggregation, using CD34, CD44, CD45, CD73, CD90, KIT, CD201, ITGA3, ITGA6 and/or CXCR4.
  • the above method further comprises sorting using CD34 positive.
  • the sorting uses CD34 positive and CD44 positive.
  • the sorting uses CD34 positive and CD45 positive.
  • the sorting uses CD34 positive, CD44 positive and CD45 positive.
  • the sorting uses CD34 positive, CD44 positive, CD45 positive and CD90 positive.
  • the sorting uses CD34 positive, CD44 positive, CD45 positive, CD90 positive and KIT positive. In some embodiments, the sorting uses CD34 positive and CD73 negative. In some embodiments, the sorting uses CD34 positive, CD73 negative, and CXCR4 lo/negative. In some embodiments, the sorting uses CD34 positive and/or CD201 positive, ITGA3 positive, or ITGA6 positive.
  • the population of cells is cultured under hypoxic conditions, or low oxygen tension, preferably between about 2% and about 10% oxygen.
  • the above methods comprise a step of cry opreserving the harvested, enriched and/or sorted cells. That is, the population of definitive HSPCs may be optionally cryopreserved and subsequently thawed and further cultured or administered to a subject.
  • the method further comprises cryopreserving the population of HSPCs following any of the aforementioned methods.
  • a cryopreserved population of HSPCs obtained according to the methods described herein.
  • a composition comprising the cryopreserved population of HSPCs.
  • the present invention provides culture methods that yield definitive HSPCs capable of in vitro differentiation, ex vivo modulation, and the capacity to engraft multiple lineages long term.
  • any pluripotent stem cell population including a human embryonic stem cell population (hESC) or a human induced pluripotent stem cell population (iPSCs), can be used as the starting material to derive HSPCs using the methods described herein.
  • the population of pluripotent progenitor cells is a human iPSC population.
  • the iPSC cells grown in feeder-free conditions.
  • cells obtained from a subject can be subjected to methods to generate patient specific iPSCs which can then be differentiated using the methods described herein.
  • the cells undergoing haematopoietic differentiation are cultured in SPELS medium, comprising IMDM/F12 media supplemented with 0.1% (or 0.05%) poly vinyl alcohol (PVA); linoleic and linolenic acid (125 ng/mL each), soybean oil (1 pg/mL), alpha-tocopherol (50 nM) , L-ascorbic acid-2-phosphate (50 pg/mL), L-ascorbic acid (50
  • SPELS medium comprising IMDM/F12 media supplemented with 0.1% (or 0.05%) poly vinyl alcohol (PVA); linoleic and linolenic acid (125 ng/mL each), soybean oil (1 pg/mL), alpha-tocopherol (50 nM
  • a haematopoietic stem/progenitor cell produced by or obtained according to the methods described herein or a therapeutic composition comprising such a cell may be used for treating a condition, disease or disorder requiring HSC transplantation following myeloablative therapy in a subject.
  • a haematopoietic stem/progenitor cell produced according to the methods described herein or a therapeutic composition comprising such a cell may be used for treating a condition, disease or disorder requiring HSC transplantation that does not require myeloablative therapy in a subject.
  • the present invention provides a method for treating a condition, disease or disorder requiring HSC transplantation, the method comprising administering to a subject the cell or population of cells or a therapeutic composition comprising the cell or population of cells, wherein the cell or population of cells is produced according to the methods described herein.
  • the present invention provides a use of a cell or population of cells, or a therapeutic composition comprising the cell or population of cells, in the manufacture of a medicament for treating a condition, disease or disorder requiring HSC transplantation, wherein the cell or population of cells is produced according to the methods described herein.
  • the present invention provides a cell or population of cells for use in treating a condition, disease or disorder requiring HSC transplantation, wherein the cell or population of cells is produced according to the methods described herein.
  • the haematopoietic stem/progenitor cell produced according to the invention will be formulated, dosed, and administered in a fashion consistent with good medical practice.
  • Factors for consideration in this context include the particular type of condition, disease or disorder being treated, the particular subject being treated, the clinical condition of the subject, the site of administration, the method of administration, the scheduling of administration, possible sideeffects and other factors known to medical practitioners.
  • the therapeutically effective amount of the haematopoietic stem/progenitor cell produced according to the invention to be administered will be governed by such considerations.
  • the haematopoietic stem/progenitor cell may be administered to a subject by any suitable method including intravenous (IV), intra-arterial, intramuscular, intraperitoneal, intracerobrospinal, subcutaneous (SC), intra- articular, intrasynovial, intrathecal, intracoronary, transendocardial, surgical implantation, topical and inhalation (e.g. intrapulmonary) routes.
  • IV intravenous
  • intra-arterial intramuscular
  • intraperitoneal intracerobrospinal
  • SC subcutaneous
  • intra- articular intrasynovial
  • intrathecal intracoronary
  • transendocardial surgical implantation
  • topical and inhalation e.g. intrapulmonary
  • kits comprising one or more of a cell or population of cells produced according to a method described herein, a product or composition comprising a cell or population of cells produced, optionally comprising a further therapeutic agent, or optionally wherein the cell comprises a reporter system or other modification, according to a method described herein.
  • the kits may further comprise one or more of components selected from an agonist, inhibitor, medium, apparatus or other component that can be used in a method described herein, instructions for use, for example instructions on how to generate the cells, perform an assay, harvest, isolate, or administer the cell or population of cells, composition, or product, and a vial or other container for housing one of these aforementioned cells, compositions, products, agonists, inhibitors, media etc.
  • kits for use in generating a population of definitive haematopoietic stem/progenitor cells comprising one or more components selected from the group consisting of: a WNT agonist, a fibroblast growth factor (FGF), Activin A, an ACTIVIN antagonist, vascular endothelial growth factor (VEGF), a retinoic acid signalling agent, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), a stem cell factor (SCF), thrombopoietin (TPO), StemRegenninl (SRI), FLT3 receptor ligand (FLT3L), interleukin 3 (IL-3), erythropoietin (EPO), and a ROCK inhibitor.
  • the kit components are provided in amounts as described in the paragraphs above referring to “cell culture components”.
  • the kit further comprises one or more basal media.
  • kits when used for generating a population of definitive haematopoietic stem/progenitor cells (HSPCs), wherein said kit comprising one or more components selected from the group consisting of: a WNT agonist, a fibroblast growth factor (FGF), Activin A, an ACTIVIN antagonist, vascular endothelial growth factor (VEGF), a retinoic acid signalling agent, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), a stem cell factor (SCF), thrombopoietin (TPO), StemRegenninl (SRI), FLT3 receptor ligand (FLT3L), interleukin 3 (IL-3), erythropoietin (EPO), and a ROCK inhibitor.
  • the kit components are provided in amounts as described in the paragraphs above referring to “cell culture components”.
  • the kit comprises one or more basal media.
  • the kit of the foregoing embodiments comprises SPELS medium or components to provide SPELS medium, wherein said medium comprises IMDM/F12 media supplemented with 0.1% (or 0.05%) poly vinyl alcohol (PVA); linoleic and linolenic acid (125 ng/mL each), soybean oil (1 pg/mL), alpha-tocopherol (50 nM) , L-ascorbic acid-2-phosphate (50 pg/mL), L-ascorbic acid (50 pg/mL, IxGlutaMAX, ITSE AF blood-free cell culture media supplement (50 pg/mL), and lx Non Essential Amino Acids (MEM).
  • PVA poly vinyl alcohol
  • linoleic and linolenic acid 125 ng/mL each
  • soybean oil (1 pg/mL
  • alpha-tocopherol 50 nM
  • L-ascorbic acid-2-phosphate 50 pg/m
  • iPSC Induced pluripotent stem cell
  • RM TOM iPSCs constitutively expressing a tdTOMATO transgene from the GAPDH locus, were derived from human foreskin fibroblasts purchased from ATCC and reprogrammed using the hSTEMCCAloxP four-factor lentiviral vector, and integrated vector sequences were removed using Cre recombinase.
  • PB1.1 (male), PB 10.5 (male) and PB5.1 (female) iPSCs were reprogrammed from the peripheral blood of a healthy volunteers with Sendai virus carrying the reprogramming factors POU5F1, SOX2, KLF4 and MYC.
  • PB1.1 was engineered to express mTagBFP2 from the GAPDH locus. Following vector integration, Cre recombinase was used to excise the antibiotic selectable marker from this version of the targeting vector.
  • Human iPSC lines were maintained by coculture with mouse embryo fibroblasts in KOSR medium (Thermofisher), or adapted to culture on Matrigel (Coming) in Essential 8 medium (Thermofisher).
  • KOSR medium Thermofisher
  • Matrigel Coming
  • Essential 8 medium Thermofisher
  • SPELS medium includes non-essential amino acids, but not albumin or Protein Free Hybridoma Medium. SPELS medium was supplemented during differentiation with various cell culture components as described in detail herein.
  • rh fibroblast growth factor FGF2 (PeproTech) and 1 pM Thiazovivin (Selleck Chem). Starting at day 1, medium changes occurred every 2 days during the differentiation. From day 1 - day 3, mesoderm was patterned to HOXA expression with 3 pM CHIR99021 (Tocris Biosciences) and 4 pM SB431542 (Cayman Chemicals or Selleck Chemical), 25 ng/ml rh vascular endothelial growth factor (VEGF, PeproTech), 25 ng/ml rh stem cell factor (SCF, PeproTech) and 20 ng/ml rh FGF2.
  • VEGF vascular endothelial growth factor
  • SCF Ste cell factor
  • the medium was supplemented with 20 ng/ml rh BMP4, 50 ng/ml rh VEGF, 20 ng/ml rh FGF2, 50 ng/ml rh SCF and 10 ng/ml rh insulin-like growth factor 2 (IGF2, PeproTech).
  • IGF2 insulin-like growth factor 2
  • PeproTech In selected transplantation experiments, cultures were supplemented at day 3 of differentiation with 2 pM retinol (ROL) or 2 pM retinyl acetate (RETA), which was removed at the day 5 medium change.
  • ROL 2 pM retinol
  • FDA 2 pM retinyl acetate
  • 10 ng/ml APELIN peptide was included from day 5 to day 9. From day 11 of differentiation, growth factors were modified to include 50 ng/ml rh VEGF, 50 ng/ml rh SCF, 50 ng/ml rh thrombopoietin (TPO, PeproTech), 10 ng/ml rh FGF2 and 20 nM Stemregeninl (SRI, Selleck Chemical). Early experiments also included 10 ng/ml rh FLT3 receptor ligand (FLT3L, PeproTech) and 10 ng/ml rh IL3 (PeproTech). Blood cells were shed into the medium after 10-12 days of differentiation.
  • FLT3L FLT3 receptor ligand
  • PeproTech 10 ng/ml rh IL3
  • CD34 antibody-conjugated magnetic beads (Miltenyi Biotec) were used according to the manufacturer's instructions, to enrich CD34 + cells from disaggregated embryoid bodies and deplete cultures of stromal cells prior to cryopreservation.
  • HOXA expression was induced as above (3 pM CHIR99021, 4 pM SB431542, 25 ng/ml rh VEGF, 25 ng/ml rh SCF, and 20 ng/ml rh FGF2).
  • the medium was supplemented with 20 ng/ml rh BMP4, 50 ng/ml rh VEGF, 20 ng/ml rh FGF2, 50 ng/ml rh SCF 10 ng/ml rh IGF2, and 2 pM RETA.
  • the retinoid was removed at the day 5 medium change (control), or RETA supplementation was repeated at 2 day intervals during the differentiation as shown in Figure 4a at concentrations between 100 nM and 2 pM.
  • the medium was supplemented with 2 ng/ml rh BMP4, 50 ng/ml rh VEGF, 20 ng/ml rh FGF2, 50 ng/ml rh SCF and 10 ng/ml rh IGF2 with or without RETA at 2 pM or 100 nM.
  • growth factors included were 50 ng/ml rh VEGF, 50 ng/ml rh SCF, 50 ng/ml rh thrombopoietin (TPO, PeproTech), 10 ng/ml rh FGF2 and 20 nM SRI, with and without RETA at 2 pM or 100 nM.
  • TPO PeproTech
  • 10 ng/ml rh FGF2 and 20 nM SRI with and without RETA at 2 pM or 100 nM.
  • suspension haematopoietic cells were pooled in some experiments with MACS-enriched CD34 + cells from disaggregated SwBs, analysed by FACS, and cryopreserved for transplantation.
  • the medium was supplemented with 20 ng/ml rh BMP4, 2 pM RETA, 150 ng/ml rh VEGF, 20 ng/ml rh FGF2, 10 ng/ml rh IGF2 and 10 ng/ml rh insulin-like growth factor 1 (IGF1, PeproTech).
  • rh BMP4 and RETA were reduced to 2 ng/ml and 100 nM respectively and all other cytokines were as for day 3.
  • rh VEGF was removed, rh BMP4 and RETA were retained at 2 ng/ml 100 nM respectively, while rh FGF2, rh IGF1 and rh IGF2 were supplemented at 10 ng/ml.
  • rh SCF was included at 10 ng/ml, and all other cytokines were as for day 7. From day 11 onwards, rh BMP was removed.
  • SwBs were cultured in 10 ng/ml rh SCF, rh thrombopoietin (TPO PeproTech), rh FGF2, rh IGF1, rh IGF2, and 100 nM RETA, and 20 nM Stemregenin 1 (SRI, Selleck Chem). From day 14 to 16, suspension haematopoietic cells were analysed by FACS before being cryopreserved for transplantation.
  • TPO PeproTech rh thrombopoietin
  • SRI Stemregenin 1
  • Red cell lysis of peripheral blood samples was performed by incubating 100 pF of blood with 10 mF of Ammonium chloride lysis buffer (155 mM NH4CI/ 12 mM NaHCCh/ 0.1 mM EDTA) at 37°C for 15 min. Cells were pelleted and washed with phosphate buffered saline. For analysis, all samples were resuspended in phosphate buffered saline supplemented with 2% fetal calf serum (PBS/2%FCS). Directly conjugated antibodies directed against cell surface antigens, were used to identify dissociated cells by flow cytometric analysis during differentiation and in single cell suspensions from hematopoietic tissues and peripheral blood samples from transplanted mice.
  • Ammonium chloride lysis buffer 155 mM NH4CI/ 12 mM NaHCCh/ 0.1 mM EDTA
  • mice were sourced from JAX Mice and Services (stock number 0266220) at The Jackson Eaboratory (Maine, USA) and a colony was established at the Murdoch Children's Research Institute.
  • the Murdoch Children's Research Institute animal ethics committee approved all animal protocols (reference A885 and A954), and experiments were carried out under its guidelines for the care and use of laboratory animals.
  • CB mononuclear cells from four independent cords (0.7% - 2.7% CD34 + ) were thawed and mononuclear cells estimated to contain 3.5 x 10 2 - 2.7 x 10 4 CD34 + cells were transplanted in a similar manner.
  • Tissues were harvested for analysis from most recipients at least 16 weeks, and up to 24 weeks, post engraftment.
  • Single cell suspensions were generated from peripheral blood, bone marrow (femurs and tibiae), spleen and, where visible, thymic tissue. Cells were analyzed by flow cytometry for surface antigens indicative of erythroid, myeloid, B cell, T cell and stem cell compartments. Residual bone marrow and spleen samples from repopulated mice were cryopreserved for further analyses including secondary transplantation.
  • Cryopreserved bone marrow samples from selected multilineage engrafted mice were transplanted (3 x 10 5 - 2 x 10 6 total bone marrow cells per mouse) into NBSGW recipients by tail vein injection, and bone marrow and spleen analyzed after 13 - 20 weeks.
  • 'FindAllMarkers' generated a list of cluster specific genes for each cluster. These genes were then compared with known markers of a cell type to assign cluster identities. Differential gene analysis between clusters was completed with the 'FindMarkers' function, whilst differential genes expressed between samples utilized a pseudobulk method based on average counts.
  • each single cell within a preselected cell cluster acted as a replicate thus allowing for the RNA expression level across the cluster to be treated as a bulk RNA sample. Differing conditions of the same cluster could therefore utilize the same analysis strategies as used in bulk RNA sequencing.
  • the Voom/limma method on the Degust web portal was used to identify differentially expressed genes in the 'Artl', 'HE' and 'HSPC1' clusters identified in Figure 11. Cells from these three clusters were also pooled and reclustered with a higher resolution to investigate the endothelial to hematopoietic transition.
  • ACTINN version 2 was used as an unsupervised neural network based method to identify subsets of the haematopoietic ally differentiated iPSC population based on a comparison to a reference dataset of human embryonic and CB derived endothelial and hematopoietic cell populations (Calvanese, V. et al. Nature 604, 534-540 (2022)).
  • the ACTINN data sets used in Figure 3h comprised 27 samples of hematovascular cells from gestational day 22-24 (CS 10-11) embryo and yolk sac, day 29-36 (CS 14-15) AGM, yolk sac, embryonic liver and placenta, week 6, 8, 11 and 15 embryonic and fetal liver HSPCs, and CB HSCs and progenitor cells.
  • the expression matrix for the reference training data and the cell type annotation of the cells are accessible in GitHub (https://github.com/mikkolalab/Human-HSC-Ontogeny). Cells expressing HLF and SPINK2 from the differentiated iPSCs were matched to 19 of the 27 reference samples. Results from these 19 data sets that are shown in Figure 3h.
  • Example 1 Differentiation of iPSCs to CD34 expressing hematopoietic cells
  • Induced pluripotent stem cells were differentiated to hematopoietic cells using a swirling embryoid body (EB) protocol, in which iPSCs were seeded into dishes that were incubated on a rotating platform (Calvanese, V. et al. Nature 604, 534-540 (2022) and Motazedian, A. et al. Nature cell biology 22, 60-73 (2020)) (see Figure 7 for protocol details, Figure la, b).
  • Mesoderm was induced in albumin-free SPELS medium (made in house, see Methods) using a combination of the WNT-agonist CHIR99021 (CHIR), FGF2, BMP4 and/or ACTIVIN A for 24 hours ( Figure la).
  • HOXA genes were achieved by culture for 48 hours with CHIR and the ALK-kinase inhibitor SB431542 (SB). From day 3, mesoderm was further differentiated to hemogenic endothelium, with or without a 2 -day pulse of a retinoic acid precursor, retinyl acetate (RETA) or retinol (ROL). From day 7, cells undergoing an endothelial to hematopoietic transition were visible as protrusions on the surface of the embryoid bodies, reminiscent of intra-arterial hematopoietic clusters of blood cells that are seen emerging from the aorta in the embryonic AGM ( Figure 1c).
  • RETA retinyl acetate
  • ROL retinol
  • the endothelial populations present at this time included CXCR4 + CD73 + arterial cells, CXCR4 CD73 hl venous cells and CXCR4 CD73’ hemogenic cells. From day 14 - 16, the suspension hematopoietic cells were cryopreserved for further analyses ( Figure Id). In some experiments, CD34 + cells enriched from embryoid bodies by magnetic bead separation ( Figure le) were also cryopreserved.
  • Example 2 Multilineage engrafting cells require retinoids during iPSC differentiation
  • the inventors assessed combinations of CHIR, ACTIVIN A, BMP4 and a retinoid during the mesoderm induction and patterning stages (screening protocol #1, see Figure 7 and Figure 9a), to determine whether any supported the generation of engraftable human hematopoietic cells.
  • the CD34 + hematopoietic cells generated from an iPSC line constitutively expressing a tandem TOMATO fluorescent protein (RM TOM) ( Figure 1c) were cryopreserved prior to thawing and injection into the tail vein of NOD,B6.Prk ⁇ 7c scld t w4i/W4i (NBSGW) mice, mimicking the workflow in clinical HSC transplantation.
  • R TOM tandem TOMATO fluorescent protein
  • mice (totaling 134, denoted cohort #1) were injected with cells differentiated under one of 12 mesoderm induction and patterning protocols in screening protocol #1.
  • Groups of mice (totaling 134, denoted cohort #1) were injected with RM TOM cells differentiated under one of 12 mesoderm induction and patterning protocols (Figure 9a).
  • Human cell contributions in the bone marrow and spleen were determined post transplantation based on coincident expression of the TOMATO reporter with human lympho-myeloid (CD45 and/or CD43) and erythroid (GYPA) cell surface markers by flow cytometry.
  • mice most frequent were myeloid restricted stem cells that gave low-level bone marrow engraftment (human cells, 0.7+0.1%) in 58/134 transplant recipients, or myelo- lymphoid stem cells that led to mixture of bone marrow myeloid cells (human cells, 0.5+0.1%) accompanied by B lymphocytes in the spleen (human cells, 0.02+0.003%) in 30/134 mice (Figure 9c).
  • mice (12/134) were engrafted by stem cells displaying multilineage differentiation resulting in erythroid, myeloid and lymphoid reconstitution (denoted multilineage engraftment, MLE).
  • the mouse groups in which MLE engraftment occurred were predominantly those receiving cells in which mesoderm was induced with 4 pM CHIR on day 0, and a pulse of a retinoic acid precursor (ROL or RETA) was included from day 3 - day 5 of differentiation ( Figure 2a-d). Indeed, 17.6% (9/51) mice transplanted with cells treated with the combination of 4 pM CHIR and retinoid showed erythroid, myeloid and lymphoid cell engraftment ( Figure 2e).
  • Example 3 Transcriptional similarity of in vitro differentiated iPSCs and human AGM
  • scRNA seq Single cell RNA sequencing (scRNA seq) of differentiated iPSCs was performed to search for transcriptional signatures accounting for functional differences between cells differentiated with and without retinoid and to allow comparisons with published data sets of human AGM (e.g. Calvanese, V. et al. Nature 604, 534-540 (2022)).
  • Two iPSC lines were profiled, the RM TOM line described above and an independent line in which the mTagBFP2 fluorescent protein was expressed from the GAPDH locus of PB 1.1 iPSCs (denoted PB1.1 BFP).
  • UMAP Uniform manifold approximation projection
  • retinoids minimally impacted stem cell gene expression ( Figure 12a), but most notably influenced genes associated with retinoic acid metabolism such as CYP26B1, DHRS3, CRABP2, RARB and RARG, modulators of WNT and FGF signalling such as SHISA3, DKK1, RSPO1 and WNT4, as well as genes associated with vascular and hematopoietic development such as FOXC2 and CD38 ( Figures 11 and 12).
  • Retinoid metabolism genes were similarly up-regulated in the HE cluster, as were genes related to Tube Development (gene ontology term G0:0035295, FDR 3.8 x 10-6) ( Figure Ilf).
  • Genes enriched in the HSPC1 cluster included those associated with Positive Regulation of Multicellular Organismal Process (gene ontology term G0:0051240, FDR 1.04 x 10-16), and Genes Upregulated in HL-60 Myeloid Cells in Response to Retinoic Acid (FDR 1.03 x 10-23) (Figure Ilf). Interestingly, expression of many retinoid responsive genes was only induced if the retinoids were included until at least day 11 of differentiation ( Figure 12b).
  • the inventors compared the transcriptional profiles of the iPSC-derived HLF*SPINK2 + HSPC cells to similar HLF + SP1NK2 + stem cell-like populations from human embryos at CS 14 and 15, examining the expression of a selected range of relevant genes (Figure 3g).
  • Figure 3g For a more extensive comparison between in vitro and human embryo derived samples, a suite of scorecards developed in profiling studies of hematopoietic development in human embryos (Calvanese, V. et al. Nature 604, 534-540 (2022)) (Example 4 and Figure 13 and 14).
  • These studies benchmarked the HLP + SP1NK2 + HSPCs generated using the methods of the present invention against CS14 and CS 15 human embryo cells, demonstrating a high level of concordance between the transcriptional profiles across the 9 scorecards of genes examined.
  • a machine learning algorithm (Ma, F. & Pellegrini, M. Bioinformatics 36, 533-538 (2020)), was employed to compare the expression profiles of day 14 differentiated iPSCs to a human reference data set comprising hematovascular cells from gestational day 22-24 (CS 10- 11) embryo and yolk sac, day 29-36 (CS 14-15) AGM, yolk sac, embryonic liver and placenta, week 6, 8, 11 and 15 embryonic and fetal liver HSPCs, and cord blood (CB) stem and progenitor cells (Calvanese, V. et al. Nature 604, 534-540 (2022)).
  • HLB + SP1NK2 + HSPCs were most closely related to cells categorized as hematopoietic stem and progenitor cells (HSPCs) in CS 14-15 AGM, placenta and yolk sac (Figure 3h).
  • HSPCs hematopoietic stem and progenitor cells
  • Figure 3h Dissecting the allocation of cells to these categories from the two cell lines and the different durations of retinoid treatment, revealed that the RM TOM line mapped predominantly to the CS 14/15 AGM HSPCs whilst the PB 1.1 BFP cells were more similar to CS 14/15 YS and placental HSPCs.
  • the longer duration of retinoid increased the proportion of CS 14/15 AGM HSPCs and decreased the CS 14/15 YS and placental HSPCs ( Figure 3h).
  • Example 4 Transcriptional profiles of iPSC-derived hematopoietic cells resemble those of hematopoietic cells from the AGM.
  • HSC-derived cells Although the proportion of cells expressing H0XA9, MEETS and MECOM were a little lower than in the CS14 and CS15 embryo reference samples. Similarly, there were some genes enriched in HSCs or shared with endothelium that were expressed in a higher percentage of cells in embryo samples, such as STAT5A, GATA2, SEEP. AEDH1A1, PROCR and EMCN ( Figure 13).
  • the 'Signaling' scorecard showed broad concordance between embryo and iPSC-derived samples with the notable exceptions of the retinoid metabolising enzyme ALDH1A1, the BMP-responsive transcription factor ID3, and some differences in the balance of JAK-STAT signaling genes ( Figure 14).
  • the transcriptional regulators and the hematopoietic lineage genes in the 'Endothelial to hematopoietic transition' scorecards were concordantly expressed in iPSC-derived and embryo samples.
  • iPSC-derived arterial cells ( Figure 14f) expressed very low levels of the aortic (pre- hemogenic endothelium) genes.
  • Example 5 Multilineage engrafting cells are generated from cultures treated with retinoids throughout differentiation
  • Multi-lineage engraftment was seen in 6/25 (24%) mice transplanted with cells treated with RETA from day 3 - day 5, the duration of RETA that was successful for engraftment in cohort#l, and in 7/19 (36.8%) mice receiving cells exposed to RETA treatment from day 3 - day 13, although the difference did not achieve statistical significance. All mice with MLE were analysed >16 weeks post transplantation, with one exception (15.7 w).
  • Example 7 Modulating VEGF signaling enhances multilineage engraftment in recipients from multiple independent iPSC lines
  • VEGF acts in a dose-dependent manner to drive endothelium generation and arterialization in differentiating pluripotent stem cells.
  • published data showed that VEGF suppressed hematopoietic progenitor development from endothelium by blocking the upregulation of RUNX1 expression, a critical marker of hemogenic endothelium and HSCs in the human embryo.
  • a range of VEGF concentrations were trialed from day 3 - day 7, during endothelial generation, followed by continuing or removing VEGF to determine which best enhanced the endothelial to hematopoietic transition.
  • protocol#3 generated more robustly engrafting cells.
  • Limit dilution transplantation experiments with this protocol were not performed, but analysis of the overall engraftment results given above suggests that the frequency of multilineage engrafting cells is still low, at 1/3.0 x 10 6 for the PB TOM, 1/3.1 x 10 6 for the PB 1.1 BFP, 1/6.2 x 10 6 for the PB5.1 and 1/4.3 x 10 6 for the PB 10.5.
  • mice #1-7 multilineage engrafted mice were seen in 19.6% of recipients transplanted with CD34 + suspension blood cells, 23.8% of those receiving CD34 enriched cells from the embryoid bodies, and 24.5% of mice that received both suspension blood cells and CD34- enriched cells from the embryoid bodies. These results demonstrated that similar proportions of stem cells were present in CD34 + cells from both sources.
  • Example 9 Umbilical cord blood mononuclear cells display dose dependent engraftment
  • mice were transplanted with 5 x 10 4 - 2.5 x 10 6 CB mononuclear cells isolated from four separate cords that comprised 0.7 - 2.7% CD34 + cells, resulting in transplantation of 3.5 x 10 2 - 2.7 x 10 4 CD34 + cells.
  • Multilineage engraftment was observed in most recipients (14/15) of mononuclear cells estimated to contain > 6.0 x 10 3 CD34 + cells ( Figure 6b), and the estimated frequency of repopulating CB stem cells was 1/6.3 x 10 3 CD34 + cells by limit dilution assay ( Figure 21b), consistent with reports in the literature. Similar to the findings observed with iPSC derived iHSC transplants, higher levels of engraftment were observed in female mice engrafted with CB cells ( Figure 21b, c).
  • mice receiving fewer than 6.0 x 10 3 CB CD34 + cells showed lower total proportions of human cells in the bone marrow and frequently displayed restricted lineage engraftment with myeloid or myeloid and lymphoid lineages (Figure 6b).
  • This positive correlation between engraftment level and lineage complexity in recipients of CB stem cells mirrored the similar correlation observed in the iPSC-derived blood cell transplants.
  • Multilineage engrafting cells with high proliferative capacity are less abundant than myeloid or myeloid/lymphoid restricted stem cells with low proliferative capacity.
  • the profile of engrafted lineages was similar between CB and RM TOM, PB5.1 and PB 10.5 recipients, although T cell engraftment was greater in the RM TOM mice and the stem cell compartment was smaller (compare Figure 6c-g with Figure 19a-d).
  • PB 1.1 BFP recipients displayed prominent erythroid engraftment in the BM and SPL, with commensurately lower levels of lymphoid and myeloid engraftment, although their stem cell compartment was maintained (Figure 19c-d).
  • Heterogeneity in the distribution of lineages in individual multilineage engrafted CB mice can be appreciated in the bar graphs in Figure 6h, where the most abundant lineages were B and myeloid cells, with few mice displaying large erythroid populations and few cases of T cell engraftment.
  • Haematopoietic stem/progenitor cells produced according to the present disclosure will be administered to a subject diagnosed with acute myeloid leukaemia (AML) at a dose of 3 x 10 6 cells/kg who has undergone myeloablative therapy according to standard protocols prior to allogeneic HSC transplantation. Administration of the haematopoietic stem/ progenitor cells will result in an equivalent or improved response in the subject.
  • AML acute myeloid leukaemia
  • Haematopoietic stem/progenitor cells produced according to the present disclosure will be administered to a subject diagnosed with acute lymphocytic leukaemia (ALL) at a dose of 3 x 10 6 cells/kg who has undergone myeloablative therapy according to standard protocols prior to allogeneic HSC transplantation. Administration of the haematopoietic stem/ progenitor cells will result in an equivalent or improved response in the subject.
  • ALL acute lymphocytic leukaemia

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Abstract

L'invention concerne la différenciation d'une cellule souche pluripotente (PSC) en une cellule souche/progénitrice hématopoïétique définitive qui est capable de prise de greffe multiligneuse, et des utilisations thérapeutiques de telles cellules souches/progénitrices hématopoïétiques.
PCT/AU2025/050292 2024-03-25 2025-03-25 Cellules souches/progénitrices hématopoïétiques et leurs procédés de production Pending WO2025199576A1 (fr)

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US20190144828A1 (en) * 2016-05-13 2019-05-16 Murdoch Childrens Research Institute Haematopoietic stem/progenitor cells
WO2020154412A1 (fr) * 2019-01-22 2020-07-30 Washington University Compositions et procédés de génération de cellules souches hématopoïétiques (csh)

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US20190144828A1 (en) * 2016-05-13 2019-05-16 Murdoch Childrens Research Institute Haematopoietic stem/progenitor cells
WO2020154412A1 (fr) * 2019-01-22 2020-07-30 Washington University Compositions et procédés de génération de cellules souches hématopoïétiques (csh)

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BROWN GEOFFREY: "Retinoic acid receptor regulation of decision-making for cell differentiation", FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY, vol. 11, 4 April 2023 (2023-04-04), XP093278442, ISSN: 2296-634X, DOI: 10.3389/fcell.2023.1182204 *
LUFF STEPHANIE A., CREAMER J. PHILIP, VALSONI SARA, DEGE CARISSA, SCARFÒ REBECCA, DACUNTO ANALISA, CASCIONE SARA, RANDOLPH LAUREN : "Identification of a retinoic acid-dependent haemogenic endothelial progenitor from human pluripotent stem cells", NATURE CELL BIOLOGY, vol. 24, no. 5, 1 May 2022 (2022-05-01), London, pages 616 - 624, XP093361860, ISSN: 1465-7392, DOI: 10.1038/s41556-022-00898-9 *

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