WO2017088830A1 - 人诱导多能干细胞向睾丸间质细胞的诱导分化方法及其用途 - Google Patents

人诱导多能干细胞向睾丸间质细胞的诱导分化方法及其用途 Download PDF

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WO2017088830A1
WO2017088830A1 PCT/CN2016/107361 CN2016107361W WO2017088830A1 WO 2017088830 A1 WO2017088830 A1 WO 2017088830A1 CN 2016107361 W CN2016107361 W CN 2016107361W WO 2017088830 A1 WO2017088830 A1 WO 2017088830A1
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cells
hips
hncscs
differentiation
lcs
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项鹏
姜美花
李伟强
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Sun Yat Sen University
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Sun Yat Sen University
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Priority to EP16868053.6A priority patent/EP3369809B1/en
Publication of WO2017088830A1 publication Critical patent/WO2017088830A1/zh
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Definitions

  • the invention relates to the technical field of stem cells and tissue engineering, in particular to a method for inducing differentiation of human induced pluripotent stem cells to Leydig cells (LCs) and uses thereof.
  • LCs Leydig cells
  • Leydig cells are distributed in the loose connective tissue between the testicular sperm cells, and the testosterone secreted is the main source of testosterone in adult males.
  • the main treatment for LOH is exogenous androgen replacement therapy, but the therapy has a difficult dose to grasp, can not simulate the secretion characteristics of physiological testosterone, requires long-term medication and is accompanied by Many shortcomings such as complications and various side effects. Due to a series of defects in exogenous androgen drugs, only a small number of patients with androgenetic diseases are currently treated with exogenous androgen drugs. Therefore, it is urgent to explore a treatment for androgenetic diseases. new method.
  • LCs transplantation is the best way to treat testosterone deficiency-related diseases such as LOH. It can better simulate the physiological characteristics of human testosterone secretion, so that serum testosterone concentration and testicular testosterone concentration can be effectively improved, and better treatment can be obtained. At the same time, try to avoid the adverse consequences of the treatment process.
  • the commonly used separation method for Leydig cells is density gradient centrifugation (Ge RS, Dong Q, Sottas CM, Papadopoulos V, Zirkin BR, Hardy MP.
  • iPS cells Induced pluripotent stem cells
  • KLF4, SOX2, OCT4 and c-MYC transcription factors
  • LCs Possible sources include adrenal-gonadal primordium, neural crest, mesonephros or coelomic epithelium [Barsoum, IBand HHYao, Fetal Leydig cells: progenitor cell Maintenance and differentiation. J Androl, 2010.31(1): p.11-5.]. Davidoff's study found that testicular mesenchymal progenitor cells express the neural stem cell marker Nestin and the pericyte marker NG2, further confirming that LC cells may be neural crest origin [Davidoff, MS, Middendorff, R., Enikolopov, G., Riethhamcher, D Holstein, AF, Müller, D., Progenitor cells of the testosterone-producing Leydig cells revealed. J Cell Biol, 2004. 167(5): p.935-44.
  • iPS induced pluripotent stem cells
  • LCs mature Leydig cells
  • NCSCs neural crest stem cells
  • the object of the present invention is to provide a method for inducing differentiation of human induced pluripotent stem cells (hiPS) differentiated into testicular interstitial cells (LCs) by neural crest stem cells (NCSCs) in vitro, and using an animal model to confirm the origin of hiPS cells.
  • LCs have regenerative senescence or damage
  • the ability to injure LCs provides a new regimen for testosterone supplementation in patients with hypogonadism, especially in patients with LOH.
  • the invention provides a method for inducing differentiation of human pluripotent stem cells (hiPS) into Leydig cells (hiPS-hNCSCs-LCs).
  • the above method is mainly based on human induced pluripotent stem cells (hiPS) as "seed” cells, which first induce differentiation of hiPS cells into specific tissue cells, which can be further targeted in vitro.
  • hiPS human induced pluripotent stem cells
  • the specific tissue cells are preferably human neural crest stem cells.
  • a method for inducing differentiation of human induced pluripotent stem cells (hiPS) into Leydig cells comprises the following steps:
  • hiPS human induced pluripotent stem cells
  • hiPS-hNCSCs human neural crest stem cells
  • hiPS-hNCSCs human neural crest stem cells obtained in the step (1) were induced to differentiate into testicular interstitial cells (hiPS-hNCSCs-LCs).
  • the step (1) comprises culturing human induced pluripotent stem cells (hiPS) in a low adhesion culture dish, preferably a Petri dish.
  • hiPS human induced pluripotent stem cells
  • the step (1) comprises inducing differentiation of human induced pluripotent stem cells (hiPS) into neural neural stem cells (hiPS-hNCSCs) in a neural differentiation culture.
  • hiPS human induced pluripotent stem cells
  • hiPS-hNCSCs neural neural stem cells
  • the step (1) comprises culturing a human induced pluripotent stem cell (hiPS) in a neural differentiation culture medium to form an embryo body, and then adhering the culture to a neural crest stem cell culture solution.
  • hiPS human induced pluripotent stem cell
  • the step (1) of the method for inducing differentiation of human induced pluripotent stem cells (hiPS) into Leydig cells comprises human induced pluripotent stem cells (hiPS)
  • the suspension cells are inoculated into a low-adherence culture dish, preferably a Petri dish, and subjected to suspension culture using a neural differentiation culture medium to form an embryo body, and then the embryo body is inoculated to coat with fibronectin.
  • human induced pluripotent stem cells are resuspended using mTeSR culture medium containing a ROCK inhibitor.
  • a neural differentiation culture solution known in the art can be used.
  • the neural differentiation medium containing 50-80% (V / V) Knockout TM DMEM, 5-20% (V / V) Knockout TM SR, 0.5-5% (V / V) chain Green neomycin mixture, 0.5-5 mM L-glutamine, the ⁇ - mercaptoethanol 0.05-0.5 mm; in a further preferred embodiment, the neural differentiation medium containing 80% (V / V) Knockout TM DMEM, 18% Knockout TM SR, 1% (V / V) mixture streptomycin, 1mM L- glutamine, 0.1mM of ⁇ - mercaptoethanol.
  • the neural crest stem cell culture medium is a mixture of DMEM-F12 medium and Neurobasal medium in a ratio of 1:0.1-1. In a further preferred embodiment, the neural crest stem cell culture medium is a mixture of DMEM-F12 medium and Neurobasal medium in a 1:1 ratio.
  • the neural crest stem cell culture solution contains 0.1-5% (v/v) N2, 0.5-10% (v/v) B27, 0.5-5% (V/V) cyan chain Mixture of oxytetracycline, 0.5-5 mM L-glutamine, 0.05-0.5 mM ⁇ -mercaptoethanol, and then add 1-100 ng/mL of basic fibroblast growth factor (bFGF) and 1-100 ng/mL of epidermis Cell growth factor (EGF).
  • bFGF basic fibroblast growth factor
  • EGF epidermis Cell growth factor
  • the neural crest stem cell culture solution contains 1% (v/v) N2, 2% (V/V) B27, 1% (V/V) streptomycin mixture, 1 mM L-Valley Aminoamide, 0.1 mM ⁇ -mercaptoethanol, plus 10 ng/mL of basic fibroblast growth factor (bFGF) and 10 ng/mL of epidermal growth factor (EGF).
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • the neural crest stem cell culture solution is to mix DMEM-F12 medium with Neurobasal medium in a ratio of 1:0.1-1, and add 0.1-5% (V/V) of N2, 0.5. -10% (V/V) B27, 0.5-5% (V/V) streptomycin mixture, 0.5-5 mM L-glutamine, 0.05-0.5 mM ⁇ -mercaptoethanol, plus 1 -100 ng/mL of basic fibroblast growth factor (bFGF) and 1-100 ng/mL of epidermal growth factor (EGF).
  • V/V 0.1-5%
  • N2 0.5. -10% (V/V) B27
  • V/V streptomycin mixture
  • streptomycin mixture 0.5-5 mM L-glutamine
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • the neural crest stem cell culture medium is a mixture of DMEM-F12 medium and Neurobasal medium in a ratio of 1:1, and 1% (V/V) N2, 2% (V/V) B27 is added. , 1% (V/V) streptomycin mixture, 1 mM L-glutamine and 0.1 mM ⁇ -mercaptoethanol, plus 10 ng/mL of basic fibroblast growth factor (bFGF) and 10 ng/mL Epidermal growth factor (EGF).
  • bFGF basic fibroblast growth factor
  • EGF Epidermal growth factor
  • the embryo body is inoculated into a culture plate coated with fibronectin after 5 days of formation.
  • the fibronectin coated plate is coated with polylysine/gelatin/fibronectin.
  • adherently cultured cells are sorted by flow cytometry 5-7 days after adherence.
  • the step (2) of the method for inducing differentiation of human induced pluripotent stem cells (hiPS) into Leydig cells (hiPS-hNCSCs-LCs) of the present invention comprises the person obtained by the amplification step (1) Neural crest stem cells (hiPS-hNCSCs) were subsequently replaced with testicular stromal cells (LCs) to differentiate into culture medium to obtain testicular stromal cells (hiPS-hNCSCs-LCs).
  • human neural crest stem cells hiPS-hNCSCs
  • LC differentiation medium when they are brought to a density of 60%.
  • the testicular interstitial cells (LCs) LCs differentiation culture medium is added to the DMEM-F12 medium in a volume percentage of 0.1%-20% (V/V) of calf serum (FCS), 0.1- 10nM triiodothyronine (T3), 0.1-20ng/ml luteinizing hormone (LH), 5-100ng/ml insulin-like growth factor (IGF-I), 1-50ng/ml platelet-derived growth factor BB (PDGFBB).
  • V/V calf serum
  • T3 triiodothyronine
  • LH 0.1-20ng/ml luteinizing hormone
  • IGF-I insulin-like growth factor
  • PDGFBB platelet-derived growth factor BB
  • the Leydig cells (LCs) differentiation culture medium is supplemented with 2% (v/v) calf serum (FCS), 1 nM triiodothyronine in DMEM-F12 medium ( T3), 1 ng/ml luteinizing hormone (LH), 70 ng/ml insulin-like growth factor (IGF-I), 10 ng/ml platelet-derived growth factor BB (PDGF-BB).
  • FCS calf serum
  • T3 1 nM triiodothyronine in DMEM-F12 medium
  • LH 1 ng/ml luteinizing hormone
  • IGF-I insulin-like growth factor
  • PDGF-BB platelet-derived growth factor BB
  • the induction time in the above step (2) is 14 days.
  • a further aspect of the invention provides the use of the testicular interstitial cells (hiPS-hNCSCs-LCs) of the invention in the preparation of a medicament for increasing testosterone levels, and the correlation caused by the preparation of a low therapeutic testosterone level The application of drugs for diseases.
  • hiPS-hNCSCs-LCs testicular interstitial cells
  • the Leydig cells (hiPS-hNCSCs-LCs) of the invention increase serum testosterone levels.
  • hNCSCs or “hiPS-hNCSCs” as used in the present invention refers to human neural crest stem cells (hNCSCs) obtained by human induced pluripotent stem cell (hiPS)-induced differentiation.
  • hNCSCs human neural crest stem cells obtained by human induced pluripotent stem cell (hiPS)-induced differentiation.
  • hiPS-hNCSCs-LCs or “hNCSCs-LCs” as used in the present invention refers to Leydig cells (LCs) obtained by differentiation of human neural crest stem cells (hNCSCs) induced by human induced pluripotent stem cells (hiPS). ).
  • the protocol of the present invention is to induce differentiation of human iPS cell lines into neural crest stem cells (hNCSCs), followed by further induction of differentiation into Leydig cells (LCs).
  • hNCSCs neural crest stem cells
  • LCs Leydig cells
  • hiPSCs Human iPS cell lines
  • the cells can be resuspended in mTeSR culture medium containing a ROCK inhibitor.
  • the suspension cells were seeded in a low-adhesion culture dish and cultured using a neural differentiation culture medium to form an embryo body structure.
  • the neural differentiation medium can be, for example, containing 80% Knockout TM DMEM, 18% Knockout TM SR, 1% anti-dual, 1mM L- glutamic acid, and the 0.1mM ⁇ - mercaptoethanol.
  • the formed embryo body was inoculated into a fibronectin-coated culture plate for adherent culture.
  • the inoculation time can be 5 days after the formation of the embryo body.
  • the plates can be coated with polylysine/gelatin/fibronectin.
  • the neural crest stem cell culture solution may be added to the culture plate, and the neural crest stem cell culture solution may be mixed with the Neurosal medium in a ratio of 1:1 in a ratio of 1% (V/V) N2, 2%.
  • the neural crest stem cell culture solution may be a mixture of DMEM/F12 medium and Neurobasal medium in a ratio of 1:1, and added 1% (V/V) N2, 2% (V/V) B27, 1% (V). /V) Streptomycin mixture, 1 mM L-glutamic acid and 0.1 mM ⁇ -mercaptoethanol, followed by 10 ng/mL bFGF and 10 ng/mL EGF.
  • the neural crest stem cells (hNCSCs or hiPS-hNCSCs) obtained in the step (1) were induced to differentiate into Leydig cells (hiPS-hNCSCs-LCs or hNCSCs-LCs).
  • the step (2) may be the neural crest stem cells (hNCSCs) obtained in the amplification step (1), and induced to differentiate in the testicular stromal cells (LCs) differentiation culture medium for 14 days to obtain Leydig cells (hNCSCs-LCs).
  • hNCSCs neural crest stem cells
  • LCs testicular stromal cells
  • the obtained neural crest stem cells can be amplified in the neural crest stem cell culture solution. Further, the LCs differentiation culture solution can be replaced when the hNCSCs are expanded to a density of 60%.
  • the LCs differentiation culture solution may be a volume percentage of 0.1%-20% of calf serum (FCS), 0.1-10 nM of triiodothyronine (T3), 0.1-20 ng/ml in DMEM-F12 medium. Luteinizing hormone (LH), 5-100 ng/ml insulin-like growth factor (IGF-I), 1-50 ng/ml platelet-derived growth factor BB (PDGFBB).
  • the obtained hNCSCs-LCs expressed 3 ⁇ -HSD, P450C17, StAR, SF-1, and secreted testosterone.
  • the testicular interstitial cells (hiPS-hNCSCs-LCs) obtained by the method of the present invention were transplanted into a rat animal model for removing Leydig cells (the specific apoptosis inducer for the detection of rat Leydig cells).
  • the apoptotic model of the Leydig cells established by the alkane (EDS), the EDS model evaluates the role of the cells in the testicular microenvironment.
  • EDS alkane
  • human iPS cells are induced to differentiate and sorted to obtain neural crest stem cells, and induced to differentiate into neural crest stem cells in an in vitro induction medium to obtain testicular stromal cells and secrete testosterone.
  • V/V refers to the percentage by volume of each component in the culture or culture medium.
  • the researchers of the present invention have conducted intensive research for a long time to provide treatment for testosterone deficiency diseases such as LOH.
  • a new idea is to use human induced pluripotent stem cells (hiPS) as a source of Leydig cells (LCs), and experimentally confirmed that LC derived from hiPS cells has the ability to regenerate senescence or damage LC cells, and It can better simulate the physiological characteristics of human testosterone secretion, so that serum testosterone concentration and testicular testosterone concentration can be effectively improved, and better therapeutic effect can be obtained, while avoiding the adverse consequences in the treatment process.
  • hiPS human induced pluripotent stem cells
  • LCs Leydig cells
  • Figure 1 is an immunofluorescence staining of white light photographs and differentiated NCSCs markers after formation of embryoid bodies and suspension cultures during induction of differentiation of hiPSCs into hNCSCs.
  • AC is a white light photograph
  • A amplified cultured hiPSCs
  • B suspension cultured embryo body
  • C adherent cultured embryo body, which forms a neuron-ring structure and migrates outward
  • D is a specific cell of NCSCs after differentiation Expression of sex markers (immunofluorescence staining).
  • Scale bar 100 ⁇ m.
  • Figure 2 is a photograph of white light and fluorescent staining of biological characteristics of hNCSCs.
  • Figure 3 is a photograph of white light and immunofluorescence staining of hNCSCs differentiated into peripheral neurons and Schwann cells.
  • A The morphology of hNCSCs before and after differentiation into peripheral neurons was observed under light microscope.
  • B-D is the result of immunofluorescence staining.
  • B.hNCSCs differentiated into peripherin+/Tuj+ peripheral neurons;
  • C.hNCSCs differentiated into TH+/Tuj+ peripheral sympathetic neurons;
  • Scale bar 100 ⁇ m.
  • Figure 4 is a photograph of white light and histochemical staining of hNCSCs induced differentiation into hNCSCs-MSCs.
  • A. The morphology of hNCSCs induced to differentiate into hNCSCs-MSCs before and after white light microscopy;
  • B. The chemical staining method was used to culture hNCSCs-MSC in a suitable induced differentiation solution for a period of time after the Alizarin Red S staining, Tapprodine Blue staining, and Oil Red O staining;
  • C hNCSCs-MSC in the appropriate induced differentiation solution
  • the ⁇ SMA staining map after a period of culture. Scale bar 100 ⁇ m.
  • Figure 5 is a graph of LC marker expression in hNCSCs-LCs.
  • Figure 6 is a graph showing secretion of testosterone in vitro culture conditions of hNCSCs-LCs.
  • Figure 7 is a graph showing the effect of hNCSCs-LCs transplantation on serum testosterone levels in EDS model rats.
  • hiPSCs cells were grown in a flat clone-like manner when expanded on Matrigel. The cells are arranged closely, as shown in Figure 1A.
  • the hiPSCs cells were digested into small pieces with 0.5 mmol/L EDTA, and the cells were resuspended in mTeSR medium containing ROCK inhibitor.
  • the ROCK inhibitor used therein was Y27632 (Calbiochem, San Diego, CA).
  • the neural differentiation culture solution described in the present embodiment is not limited to the above formula, the neural differentiation culture solution of the present invention is a neural differentiation medium containing a volume percentage of 50-80% Knockout DMEM, the volume percentage of 5-20% Knockout TM SR, the volume percentage of 0.5 to 5% penicillin-streptomycin mixture, 0.5-5mM
  • Knockout TM DMEM and Knockout TM SR were purchased from Invitrogen, Carlsbad, CA.
  • Adherent culture After 5 days of suspension culture to form embryoid bodies, the spheroidal embryo bodies are inoculated into a culture plate coated with polylysine/gelatin/fibronectin for adherent culture, and neural crest stem cell culture solution is used ( DMEM-F12 medium was mixed with Neurobasal medium in a ratio of 1:1, and 1% (V/V) N2, 2% (V/V) B27, 1% (V/V) streptomycin mixture was added.
  • DMEM-F12 medium was mixed with Neurobasal medium in a ratio of 1:1, and 1% (V/V) N2, 2% (V/V) B27, 1% (V/V) streptomycin mixture was added.
  • the nerves in this example The sputum stem cell culture solution is not limited to the above formula, and the neural crest stem cell culture solution is prepared by mixing the DMEM-F12 medium and the Neurobasal medium in a ratio of 1:0.1-1 using the neural crest stem cell culture solution of the present invention.
  • the volume percentage is 0.1-5% of N2, the volume percentage is 0.5-10% of B27, and the volume percentage is 0.5-5% of blue.
  • bFGF basic fibroblast growth factor
  • Fig. 1D Immunofluorescence staining of adherent cultured cells revealed that, as shown in Fig. 1D, markers such as Pax6 and Sox2 were mainly expressed in cells at the central part of the nerve wreath, and neural stem stem cell-specific markers AP2 ⁇ , Sox10, P75, HNK1, etc. Mainly expressed in cells that migrate outward, indicating After the adherent culture in the embryo culture stage, hiPSCs can be induced to differentiate into hNCSCs.
  • the cells were sieved by a nylon sieve, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, 1 mL of PBS was added, and the mixture was mixed, and 20 ⁇ L of the cell suspension was aspirated for cell counting.
  • the remaining cells were divided into four groups for antibody labeling: IgG negative control group, P75 antibody single labeling group, HNK1 antibody single labeling group, and P75+/HNK1+ antibody sample group, and 20 ⁇ L of antibody label was added per 10 6 cells.
  • the IgG negative control cell suspension was flow-loaded using a flow cytometer (BD influx cell sorter), and a negative fluorescent signal region was selected as a negative control, and cells having a fluorescence intensity higher than 10 times higher than that of the negative control were collected.
  • Flow cytometry analysis showed that about 80-90% of the hiPSCs cells expressed NCSC-specific markers HNK1 and P75 (Fig. 2A) after induction of differentiation, indicating that most cells have differentiated into hNCSCs after induction.
  • hNCSCs Purified hNCSCs were obtained by flow sorting, and adherent culture was carried out at 5 ⁇ 10 4 -1 ⁇ 10 5 cells/cm 2 . As shown in Fig. 2B, the cell morphology of the adherent culture was relatively uniform. The hNCSCs were digested and subcultured in a low-adhesion culture plate. As shown in Fig. 2C, the cells formed neurospheres of relatively uniform size. Immunofluorescence detection was performed on the sorted hNCSCs cells. As shown in Fig. 2D, the cells were maintained to express the markers specific for NCSCs, such as P75 and Sox10. This indicates that the cells are capable of maintaining the characteristics of NCSCs under the existing culture amplification conditions.
  • markers specific for NCSCs such as P75 and Sox10. This indicates that the cells are capable of maintaining the characteristics of NCSCs under the existing culture amplification conditions.
  • hNCSCs Biological characteristics of hNCSCs were identified. After inoculation of hNCSCs into a multi-lysine/gelatin/fibronectin-coated multi-well plate, the induction medium was replaced with the corresponding cells to induce differentiation. As shown in Fig. 3A, cell morphology was significantly changed after 2 weeks of differentiation of peripheral neurons, and the cell bodies became round and elongated filamentous protrusions appeared. After 3-4 weeks, the cells were stained and identified. As shown in Figures 3B and 3C, sympathetic neurons with peripherin+/Tuj+ peripheral neurons and TH+/Tuj+ were observed. The hNCSCs were induced to differentiate with Schwann cell-inducing solution, and stained 4 weeks later. As shown in Fig. 3D, Schwann cells with GFAP+/S100b+ were observed.
  • hNCSCs Differentiation of hNCSCs in MSC cell culture medium (low glucose DMEM, 10% FBS) for 7 days Then, it differentiated into MSC (mesenchymal stem cells) cells. As shown in Fig. 4A, the cell morphology was changed into a fusiform shape and grew in a spiral shape. The MSCs (hNCSCs-MSC) induced by hNCSCs were further characterized for multi-directional differentiation.
  • Example 2 induced differentiation from hNCSCs into Leydig cells (hiPS-hNCSCs-LCs or hNCSCs-LCs)
  • hNCSCs were expanded to a testicular stromal cell (LCs) differentiation medium at a density of 60% (DMEM-F12 medium (Hyclone, SH30023.018) was added with 2% by volume of calf serum ( FCS), 1 nM triiodothyronine (T3) (Sigma, T2877), 1 ng/ml luteinizing hormone (LH) (Sigma, L6420), 70 ng/ml insulin-like growth factor (IGF-I) (PeproTech , 100-11), 10 ng/ml platelet-derived growth factor BB (PDGF-BB) (PeproTech, 500-P47), induced for 14 days, cell differentiation, cell supernatant collection, fixed cells.
  • FCS calf serum
  • T3 triiodothyronine
  • LH luteinizing hormone
  • IGF-I insulin-like growth factor
  • PDGF-BB platelet-derived growth factor BB
  • LCs markers 3 ⁇ -HSD, P450c17, steroidogenic acute regulatory protein (StAR), and steroidogenic factor 1 (SF-1) immunofluorescence Marker expression
  • immunostaining analysis indicated that hNCSCs induced differentiated cells (hNCSCs) -LCs) expressed 3 ⁇ -HSD, P450C17, StAR, SF-1.
  • Testosterone levels in culture medium were determined by testosterone ELISA.
  • Figure 6 it was found that hNCSCs-LCs differentiated in vitro increased testosterone secretion, indicating hNCSCs. Can differentiate into mature Leydig cells.
  • testicular stromal cells (LCs) differentiation culture solution is not limited to the above formula, and the testicular stromal cells (LCs) differentiation culture liquid uses the testicular stromal cells (LCs) differentiation culture solution (DMEM-F12 medium) of the present invention.
  • LCs testicular stromal cells
  • DEM-F12 medium testicular stromal cells differentiation culture solution
  • LCs differentiation culture solution
  • FCS calf serum
  • T3 triiodothyronine
  • LH 5-100ng/ml insulin-like growth factor
  • IGF-I insulin-like growth factor
  • platelet-derived growth factor BB each of the specific combinations of PDGFBB can achieve the effects as shown in Figs. 5 and 6.
  • Example 3 The role of hiPS-hNCSCs-LCs (or hNCSCs-LCs) in vivo
  • EDS apoptosis inducer dimethoate
  • EDS-control rats were intraperitoneally injected with EDS (75 mg/kg body weight) on day 0, and on the fourth day, 20 ⁇ l of physiological saline (10 ⁇ l/unilateral testes) were injected into the testes.
  • Rats in the cell group were intraperitoneally injected with EDS (75 mg/kg body weight) on day 0, and on day 4, hNCSCs-LCs were cultured for 5-7 days in LC medium (1.5 ⁇ 10 6 resuspended in 10 ⁇ l PBS). / Unilateral testis) transplanted into the rat testis. Serum testosterone concentrations were measured on day 10 post-transplantation. Results As shown in Figure 7, transplantation of hNCSCs-LCs increased serum testosterone levels.

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Abstract

提供一种诱导人多能干细胞（hiPS）经神经嵴干细胞（NCSCs）分化为睾丸间质细胞（LCs）的体外定向诱导分化方法。

Description

人诱导多能干细胞向睾丸间质细胞的诱导分化方法及其用途
本申请要求申请日为2015年11月27日的中国专利申请CN201510845021.8的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及干细胞与组织工程技术领域，具体涉及人诱导多能干细胞向睾丸间质细胞(LCs)的诱导分化方法及其用途。
背景技术
睾丸间质细胞(Leydig cells,LCs)分布于睾丸生精细胞管之间的疏松结缔组织，其分泌的睾酮是成年男性体内睾酮的主要来源。目前,LOH(迟发性性腺功能减退症)的主要治疗方法是外源性雄激素药物替代疗法，但是该疗法存在补充剂量难以掌握、无法模拟生理性睾酮的分泌特点、需长期用药并伴随着多种并发症及各种副作用等缺点。由于外源性雄激素药物存在一系列的缺陷，导致目前仅有很少一部分雄激素低下性疾病患者在接受外源性雄激素药物的治疗，因此迫切需要探索一种治疗雄激素低下性疾病的新方法。LCs移植是治疗LOH等睾酮缺乏相关疾病的最佳手段，它能够更好的模拟人体睾酮分泌的生理特点，使血清睾酮浓度和睾丸局部睾酮浓度都能得到有效的提高，可以获得更好的治疗效果的同时，尽量避免治疗过程中产生的不良后果。然而，由于LC发育起源不明，而且存在取材困难、数量稀少、扩增困难等缺陷明显制约了其临床应用前景。目前睾丸间质细胞常用的分离方法为密度梯度离心法(Ge RS,Dong Q,Sottas CM,Papadopoulos V,Zirkin BR,Hardy MP.In search of rat stem Leydig cells:identification,isolation,and lineage-specific development.Proc Natl Acad Sci U S A 2006；103:2719-2724； Stanley E,Lin CY,Jin S,Liu J,Sottas CM,Ge R,et al.Identification,proliferation,and differentiation of adult Leydig stem cells.Endocrinology 2012；153:5002-5010.)。
诱导多能干细胞(induced pluripotent stem cell，iPS细胞)具备了与胚胎干细胞相似的自我更新和多向分化潜能，同时因其可来源于自身成体细胞，克服了胚胎干细胞存在的伦理与免疫源性等诸多问题，成为研究人类疾病发病机制、开展组织细胞替代治疗的重要细胞来源。个体来源的成体细胞经过转录因子(KLF4,SOX2,OCT4和c-MYC)导入重编程为多能干细胞，从而使自体移植成为可能。以iPS细胞作为“种子”细胞，可以将其在体外大量扩增并诱导分化为特定的组织细胞。
LCs的可能来源包括肾上腺-性腺原基(adrenal-gonadal primordium)、神经嵴(neural crest)、中肾(mesonephros)或者体腔上皮(coelomic epithelium)[Barsoum,I.B.and H.H.Yao,Fetal Leydig cells:progenitor cell maintenance and differentiation.J Androl,2010.31(1):p.11-5.]。Davidoff的研究发现，睾丸间质祖细胞表达神经干细胞标志Nestin和周细胞标志NG2，进一步证实了LC细胞有可能是神经嵴起源[Davidoff,M.S.,Middendorff,R.,Enikolopov,G.,Riethmacher,D.,Holstein,A.F.,Müller,D.,Progenitor cells of the testosterone-producing Leydig cells revealed.J Cell Biol,2004.167(5):p.935-44.]。
发明内容
本发明人发现，诱导多能干细胞(induced pluripotent stem cell，iPS)在体外经神经嵴干细胞(neural crest stem cells，NCSCs)，可以分化为成熟睾丸间质细胞(Leydig cells,LCs)。本发明的目的是提供了一种人诱导多能干细胞(hiPS)经神经嵴干细胞(NCSCs)分化为睾丸间质细胞(LCs)的体外定向诱导分化方法，并利用动物模型证实来源于hiPS细胞的LCs具备再生衰老或损 伤LCs的能力，为性腺功能低下的病人尤其是LOH病人提供新的补充睾酮的方案。
本发明采用以下技术方案予以实现：
本发明一方面提供了一种经人诱导多能干细胞(hiPS)向睾丸间质细胞(hiPS-hNCSCs-LCs)进行诱导分化的方法。
在进一步的的实施方案中，上述方法主要是以人诱导多能干细胞(hiPS)作为“种子”细胞，先将hiPS细胞诱导分化为特定的组织细胞，所述特定的组织细胞可在体外进一步定向分化为睾丸间质细胞(Leydig cells,LCs)，优选地，所述特定的组织细胞优选人神经嵴干细胞。
在进一步的的实施方案中，一种人诱导多能干细胞(hiPS)向睾丸间质细胞(hiPS-hNCSCs-LCs)的诱导分化方法，具体包括如下步骤：
(1)使人诱导多能干细胞(hiPS)诱导分化为人神经嵴干细胞(hiPS-hNCSCs)；
(2)使步骤(1)中获得的人神经嵴干细胞(hiPS-hNCSCs)诱导分化为睾丸间质细胞(hiPS-hNCSCs-LCs)。
在进一步的的实施方案中，所述步骤(1)包括将人诱导多能干细胞(hiPS)接种在低粘附性的培养皿中进行培养，优选Petri培养皿。
在进一步的的实施方案中，所述步骤(1)包括使人诱导多能干细胞(hiPS)在神经分化培养液中诱导分化为人神经嵴干细胞(hiPS-hNCSCs)。
在进一步的的实施方案中，所述步骤(1)包括使人诱导多能干细胞(hiPS)在神经分化培养液中进行培养，形成胚体后，用神经嵴干细胞培养液进行贴壁培养。
在一个具体的实施方案中，本发明的人诱导多能干细胞(hiPS)向睾丸间质细胞(hiPS-hNCSCs-LCs)的诱导分化方法的步骤(1)包括将人诱导多能干细胞(hiPS)消化后重悬，将悬浮细胞接种在低粘附性的培养皿，优选Petri培养皿中，使用神经分化培养液进行悬浮培养，形成胚体，随后将胚体接种到用纤连蛋白包被的培养板中用神经嵴干细胞培养液进行贴壁培养，将贴壁培养 的细胞通过流式细胞仪分选出P75+/HNK1+双阳细胞，即为人神经嵴干细胞(hiPS-hNCSCs)。
在一个具体的实施方案中，使用含有ROCK抑制剂的mTeSR培养液重悬人诱导多能干细胞(hiPS)。
可以使用本领域已知的神经分化培养液。在一个优选的实施方案中，神经分化培养液含有50-80％(V/V)KnockoutTMDMEM、5-20％(V/V)KnockoutTMSR、0.5-5％(V/V)青链霉素混合液、0.5-5mM L-谷氨酰胺、0.05-0.5mM的β-巯基乙醇；在进一步优选的实施方案中，神经分化培养液含有80％(V/V)KnockoutTMDMEM、18％KnockoutTMSR、1％(V/V)青链霉素混合液、1mM L-谷氨酰胺、0.1mM的β-巯基乙醇。
可以使用本领域已知的神经嵴干细胞培养液。在一个优选的实施方案中，神经嵴干细胞培养液为按1:0.1-1比例将DMEM-F12培养基与Neurobasal培养基混合。在进一步优选的实施方案中，神经嵴干细胞培养液为按1:1比例将DMEM-F12培养基与Neurobasal培养基混合。
在进一步优选的实施方案中，神经嵴干细胞培养液含有0.1-5％(V/V)的N2、0.5-10％(V/V)的B27、0.5-5％(V/V)的青链霉素混合液、0.5-5mM L-谷氨酰胺，0.05-0.5mM的β-巯基乙醇，再加入1-100ng/mL的碱性成纤维细胞生长因子(bFGF)和1-100ng/mL的表皮细胞生长因子(EGF)。优选地，所述神经嵴干细胞培养液含有1％(V/V)的N2、2％(V/V)的B27、1％(V/V)的青链霉素混合液、1mM L-谷氨酰胺，0.1mM的β-巯基乙醇，再加入10ng/mL的碱性成纤维细胞生长因子(bFGF)和10ng/mL的表皮细胞生长因子(EGF)。
在进一步优选的实施方案中，所述神经嵴干细胞培养液为按1:0.1-1比例将DMEM-F12培养基与Neurobasal培养基混合，并添加0.1-5％(V/V)的N2、0.5-10％(V/V)的B27、0.5-5％(V/V)的青链霉素混合液、0.5-5mM L-谷氨酰胺，0.05-0.5mM的β-巯基乙醇，再加入1-100ng/mL的碱性成纤维细胞生长因子(bFGF)和1-100ng/mL的表皮生长因子(EGF)。
在一个具体的实施方案中，神经嵴干细胞培养液为按1:1比例将DMEM-F12培养基与Neurobasal培养基混合，并添加1％(V/V)N2、2％(V/V)B27、1％(V/V)青链霉素混合液、1mM L-谷氨酰胺和0.1mM的β-巯基乙醇，再加入10ng/mL的碱性成纤维细胞生长因子(bFGF)和10ng/mL的表皮细胞生长因子(EGF)。
在一个具体的实施方案中，胚体形成5天后接种到用纤连蛋白包被的培养板中。在一个优选的实施方案中，所述用纤连蛋白包被的培养板是用多聚赖氨酸/明胶/纤连蛋白包被的。
在一个具体的实施方案中，贴壁培养的细胞在贴壁5-7天后用流式细胞仪进行分选。
在一个具体的实施方案中，本发明的人诱导多能干细胞(hiPS)向睾丸间质细胞(hiPS-hNCSCs-LCs)的诱导分化方法的步骤(2)包括扩增步骤(1)获得的人神经嵴干细胞(hiPS-hNCSCs)，随后更换为睾丸间质细胞(LCs)分化培养液诱导分化，获得睾丸间质细胞(hiPS-hNCSCs-LCs)。
在一个具体的实施方案中，在上述步骤(2)中，扩增人神经嵴干细胞(hiPS-hNCSCs)使之达到60％的密度时更换为LC分化培养液。
在优选的实施方案中，睾丸间质细胞(LCs)LCs分化培养液为在DMEM-F12培养基中加入体积百分比为0.1％-20％(V/V)的小牛血清(FCS)、0.1-10nM三碘甲腺原氨酸(T3)、0.1-20ng/ml促黄体生成素(LH)、5-100ng/ml类胰岛素生长因子(IGF-I)、1-50ng/ml血小板来源生长因子BB(PDGFBB)。
在优选的实施方案中，睾丸间质细胞(LCs)分化培养液为在DMEM-F12培养基中加入2％(V/V)的小牛血清(FCS)、1nM三碘甲腺原氨酸(T3)、1ng/ml促黄体生成素(LH)、70ng/ml类胰岛素生长因子(IGF-I)、10ng/ml血小板来源生长因子BB(PDGF-BB)。
在一个具体的实施方案中，上述步骤(2)中的诱导时间为14天。
本发明的又一方面提供本发明的睾丸间质细胞(hiPS-hNCSCs-LCs)在制备提高睾酮水平的药物中的应用，以及在制备治疗睾酮水平低下导致的相关 疾病的药物中的应用。
在一个具体的实施方案中，本发明的睾丸间质细胞(hiPS-hNCSCs-LCs)可以提高血清睾酮水平。
本发明中所使用的“hNCSCs”或“hiPS-hNCSCs”是指由人诱导多能干细胞(hiPS)诱导分化获得的人神经嵴干细胞(hNCSCs)。
本发明中所使用的“hiPS-hNCSCs-LCs”或“hNCSCs-LCs”是指由人诱导多能干细胞(hiPS)诱导分化的人神经嵴干细胞(hNCSCs)诱导分化获得的睾丸间质细胞(LCs)。
本发明的方案是将人iPS细胞系诱导分化为神经嵴干细胞(hNCSCs)，随后进一步诱导分化为睾丸间质细胞(LCs)。其具体步骤如下：
(1)人iPS细胞系(hiPSCs)向神经嵴干细胞(hNCSCs或称hiPS-hNCSCs)的诱导分化：
①制备细胞悬液。将人iPS细胞系(hiPSCs)消化成小团块状后重悬。
其中可使用含有ROCK抑制剂的mTeSR培养液重悬细胞。
②诱导分化。将悬浮细胞接种在低粘附性的培养皿中，使用神经分化培养液进行培养形成胚体结构。
其中神经分化培养液可以，例如，含有80％KnockoutTMDMEM、18％KnockoutTMSR、1％双抗、1mM L-谷氨酸、和0.1mM的β-巯基乙醇。
③贴壁培养。将形成的胚体接种于纤连蛋白包被的培养板中进行贴壁培养。
其中，接种时间可为胚体形成5天后。培养板可用多聚赖氨酸/明胶/纤连蛋白包被。培养板中可加入神经嵴干细胞培养液，所述神经嵴干细胞培养液可以是按1:1比例将DMEM/F12培养基与Neurobasal培养基混合，并添加1％(V/V)N2、2％(V/V)B27、1％(V/V)青链霉素混合液、1mM L-谷氨酸和0.1mM的β-巯基乙醇，再加入10ng/mL的bFGF和10ng/mL的EGF。
④流式分选。使用流式细胞仪分选出P75+/HNK1+的细胞，即神经嵴干细胞(hNCSCs或称hiPS-hNCSCs)。
所述神经嵴干细胞培养液可以是按1:1比例将DMEM/F12培养基与Neurobasal培养基混合，并添加1％(V/V)N2、2％(V/V)B27、1％(V/V)青链霉素混合液、1mM L-谷氨酸和0.1mM的β-巯基乙醇，再加入10ng/mL的bFGF和10ng/mL的EGF。
(2)使步骤(1)获得的神经嵴干细胞(hNCSCs或称hiPS-hNCSCs)向睾丸间质细胞(hiPS-hNCSCs-LCs或称hNCSCs-LCs)诱导分化。
步骤(2)可以是扩增步骤(1)获得的神经嵴干细胞(hNCSCs)，并在睾丸间质细胞(LCs)分化培养液中诱导分化14天，获得睾丸间质细胞(hNCSCs-LCs)。
其中，可以在神经嵴干细胞培养液中扩增获得的神经嵴干细胞(hNCSCs)。进一步，可以在hNCSCs扩增达到60％的密度时换成LCs分化培养液。LCs分化培养液可以是在DMEM-F12培养基中加入体积百分比为0.1％-20％的小牛血清(FCS)、0.1-10nM三碘甲腺原氨酸(T3)、0.1-20ng/ml促黄体生成素(LH)、5-100ng/ml类胰岛素生长因子(IGF-I)、1-50ng/ml血小板来源生长因子BB(PDGFBB)。所获得的hNCSCs-LCs表达3β-HSD，P450C17，StAR，SF-1，并分泌睾酮。
将通过本发明方法获得的睾丸间质细胞(hiPS-hNCSCs-LCs)移植到去除睾丸间质细胞的大鼠动物模型(应用大鼠睾丸间质细胞的特异性凋亡诱导剂二甲磺酸乙烷(EDS)建立的睾丸间质细胞的凋亡模型，即EDS模型)，评价该细胞在睾丸微环境中的作用。结果发现，该细胞移植后可以提高血清睾酮的浓度，因此本发明的方法得到的睾丸间质细胞(hiPS-hNCSCs-LCs)可以用于制备治疗睾酮水平低下相关疾病的药物。
本发明将人iPS细胞经诱导分化及细胞分选获得神经嵴干细胞，并在体外诱导培养基中诱导分化神经嵴干细胞，获得睾丸间质细胞并分泌睾酮。将本发明的睾丸间质细胞(hiPS-hNCSCs-LCs)移植到用EDS消除了睾丸间质细胞的大鼠模型中，可提高模型动物的血清睾酮水平。
本文中出现的“V/V”均指各成分在培养液或培养基中所占的体积百分比。
本发明的研究人员经过长期深入研究，为LOH等睾酮缺乏疾病的治疗提 供新的思路，即以人诱导多能干细胞(hiPS)作为睾丸间质细胞(Leydig cells,LCs)的来源，并且通过实验证实来源于hiPS细胞的LC具有再生衰老或损伤LC细胞的能力，以及能够更好的模拟人体睾酮分泌的生理特点，使血清睾酮浓度和睾丸局部睾酮浓度都能得到有效的提高，可以获得更好的治疗效果的同时，尽量避免治疗过程中产生的不良后果。从而一方面克服了传统外源性的雄激素药物疗法无法模拟机体睾酮的生理情况，存在补充剂量难以掌握、需长期用药并伴随着多种并发症及各种副作用等缺点，另一方面还解决了由于LC发育起源不明，存在取材困难、数量稀少、扩增困难等缺陷。
附图说明
图1是hiPSCs向hNCSCs的诱导分化过程中形成胚体和悬浮培养后的白光照片和分化后的NCSCs标志物的免疫荧光染色图。其中A-C为白光照片，A.扩增培养的hiPSCs；B.悬浮培养的胚体；C.贴壁培养的胚体，其形成神经花环结构，并向外迁移；D是分化后细胞中NCSCs特异性标志物的表达(免疫荧光染色)。其中Scale bar＝100μm。
图2是对hNCSCs进行生物学特性分析的白光和荧光染色照片。A.用流式细胞仪检测NCSCs的标志物HNK1和P75在hiPSCs中的表达的图；B.流式细胞分选后贴壁培养的hNCSCs的白光照片；C.悬浮培养后的hNCSCs的白光照片；D.扩增培养的hNCSCs中NCSCs特异性标志物P75和Sox10的表达图。Scale bar＝100μm。
图3是hNCSCs向外周神经元和施旺细胞分化的白光和免疫荧光染色照片。A.白光光镜下观察hNCSCs向外周神经元分化前后的细胞形态。B-D为免疫荧光染色结果。B.hNCSCs分化形成peripherin+/Tuj+外周神经元；C.hNCSCs分化为TH+/Tuj+外周交感神经元；D.hNCSCs分化形成GFAP+/S100B+施旺细胞。Scale bar＝100μm。
图4是hNCSCs诱导分化为hNCSCs-MSC的白光和组织化学染色照片。A.白光光镜下观察hNCSCs诱导分化为hNCSCs-MSC前后的细胞形态；B. 利用化学染色方法将hNCSCs-MSC在合适的诱导分化液中培养一段时间以后的Alizarin Red S染色图、Tuluidine Blue染色图、和Oil Red O染色图；C，将hNCSCs-MSC在合适的诱导分化液中培养一段时间以后的αSMA染色图。Scale bar＝100μm。
图5是hNCSCs-LCs中LC标志物表达的图。
图6是hNCSCs-LCs体外培养条件下分泌睾酮的图。
图7是说明hNCSCs-LCs移植对EDS模型大鼠的血清睾酮水平的影响的图。
具体实施方式
可以理解的是，在此描述的特定实施方式通过举例的方式来表示，其并不作为对本发明的限制。在不偏离于本发明范围的情况下，本发明的主要特征可以用于各种实施方式。本领域的技术人员将会意识到或能够确认，仅仅使用常规实验，许多等同物都能应用于本文所描述的特定步骤中。这些等同物被认为处在本发明的范围之内，并且被权利要求所覆盖。
为使本发明的目的、技术方案和优点更加清楚明了，下面结合具体实施方式并参照附图，对本发明进一步详细说明。应该理解，这些描述只是示例性的，而并非要限制本发明的范围。此外，在以下说明中，省略了对公知结构和技术的描述，以避免不必要地混淆本发明的概念。
实施例1从hiPS细胞系向hNCSCs分化
1)制备细胞悬液：使用已建立的人iPS细胞系(hiPSCs，Hum Mol Genet.2013,22(11):2221-33)，hiPSCs细胞在Matrigel上扩增培养时呈扁平的克隆样生长，细胞排列紧密，如图1A所示。将hiPSCs细胞用0.5mmol/L的EDTA消化成小块状，用含有ROCK抑制剂的mTeSR培养液重悬细胞。其中所使用的ROCK抑制剂为Y27632(Calbiochem,San Diego,CA)。
2)诱导分化：收集悬浮细胞，接种在Petri培养皿中，使用神经分化培养 液(80％KnockoutTMDMEM、18％KnockoutTMSR、1％(V/V)青链霉素混合液、1mM L-谷氨酸、0.1mM的β-巯基乙醇)悬浮培养，形成透亮球状的胚体，如图1B所示(本实施例中所述神经分化培养液不限于采用如上配方，所述神经分化培养液采用本发明所述的神经分化培养液含有体积百分比为50-80％Knockout DMEM、体积百分比为5-20％KnockoutTMSR、体积百分比为0.5-5％青链霉素混合液、0.5-5mM L-谷氨酰胺、0.05-0.5mM的β-巯基乙醇中的任意一种具体组合时，均可达到如附图1B所示的效果)。其中KnockoutTMDMEM和KnockoutTMSR均购自Invitrogen,Carlsbad，CA。
3)贴壁培养：悬浮培养形成胚体5天后，将球状胚体接种到用多聚赖氨酸/明胶/纤连蛋白包被的培养板中进行贴壁培养，使用神经嵴干细胞培养液(按1:1比例将DMEM-F12培养基与Neurobasal培养基混合，并添加1％(V/V)N2、2％(V/V)B27、1％(V/V)青链霉素混合液、1mM L-谷氨酸和0.1mM的β-巯基乙醇，再加入10ng/mL的碱性成纤维细胞生长因子bFGF(Invitrogen，13256029)和10ng/mL的表皮细胞生长因子EGF(PeproTech，NO.62253-63-8)，隔天换液。贴壁2天后即可见细胞团中央部位出现明显的神经花环结构，并见细胞向外迁移，如图1C所示。(本实施例中所述神经嵴干细胞培养液不限于采用如上配方，所述神经嵴干细胞培养液采用本发明所述的神经嵴干细胞培养液为按1:0.1-1比例将DMEM-F12培养基与Neurobasal培养基混合，并添加体积百分比为0.1-5％的N2、体积百分比为0.5-10％的B27、体积百分比为0.5-5％的青链霉素混合液、0.5-5mML-谷氨酰胺，0.05-0.5mM的β-巯基乙醇，再加入1-100ng/mL的碱性成纤维细胞生长因子(bFGF)和1-100ng/mL的表皮生长因子(EGF)中的任意一种具体组合时，均可达到如附图1C所示的效果。)
对贴壁培养的细胞进行免疫荧光染色检测发现，如图1D所示，Pax6、Sox2等标志物主要表达在神经花环中央部位的细胞，神经嵴干细胞特异性标志物AP2α、Sox10、P75、HNK1等主要表达在向外迁移的细胞中，表明经 过胚体培养阶段再贴壁培养后，可以将hiPSCs诱导分化成hNCSCs。
4)流式分选：贴壁5天后，将胚体消化成单个细胞，用抗P75和HNK1的流式抗体标记细胞后，进行P75+/HNK1+双阳细胞的流式分选。分选时吸管吸去培养液，用PBS洗2遍，加入Accutase消化已分化的hiPSCs，37℃作用3-5分钟后观察细胞变圆变透亮，加入培养基终止消化并用枪头把细胞吹散后，利用尼龙筛，过筛细胞并1500rmp离心5min、弃去上清，加入1mL PBS后混匀，吸取20μL细胞悬液进行细胞计数。余下细胞分为四组进行抗体标记:IgG阴性对照组、P75抗体单标组、HNK1抗体单标组和P75+/HNK1+抗体样本组，每106细胞量加入20μL抗体标记。使用流式细胞仪(BD influx cell sorter)先对IgG阴性对照组细胞悬液进行流式上样，选出阴性荧光信号区域作为阴性对照，收集荧光强度高于阴性对照10倍以上的细胞。流式检测分析可见诱导分化后约80-90％的hiPSCs细胞表达NCSC特异性标志物HNK1和P75(图2A)，说明经过诱导后多数细胞已分化为hNCSCs。
通过流式分选即可获得纯化的hNCSCs，按5×104-1×105细胞/cm2进行贴壁培养，如图2B所示，贴壁培养的细胞形态较为均一。将hNCSCs消化传代后接种在低粘附性培养板中，如图2C所示，细胞形成大小较为均一的神经球体。对分选获得的hNCSCs细胞进行免疫荧光检测，如图2D所示，可见细胞维持表达NCSCs特异性的标志物P75、Sox10等。这表明在现有的培养扩增条件下，细胞能够维持NCSCs的特性。
对hNCSCs进行生物学特性鉴定。将hNCSCs接种至用多聚赖氨酸/明胶/纤连蛋白包被的多孔板中后，换成相应细胞的诱导培养基进行诱导分化。如图3A所示，外周神经元诱导分化2周后可见细胞形态发生明显改变，细胞胞体变圆并有细长的丝状突起出现。3-4周后对细胞进行染色鉴定，如图3B和3C所示，可见有peripherin+/Tuj+外周神经元、TH+/Tuj+的交感神经元出现。对hNCSCs用施旺细胞诱导液进行诱导分化，4周后进行染色，如图3D所示，可以见有GFAP+/S100b+的施旺细胞出现。
将hNCSCs在MSC细胞培养液(低糖DMEM，10％FBS)中诱导分化7天 后，分化为MSC(mesenchymal stem cells,间充质干细胞)细胞，如图4A所示，可见细胞形态变为梭形，并呈漩涡状生长。对hNCSCs诱导而来的MSC(hNCSCs-MSC)进一步进行多向分化能力鉴定。在合适的诱导分化液中培养一定时间后，如图4B所示，Alizarin Red S染色钙结节形成证实hNCSCs-MSC分化为成骨细胞，Tuluidine Blue染色证实hNCSCs-MSC已向软骨细胞分化，Oil Red O染色脂滴形成表明hNCSCs-MSC已向脂肪细胞分化。在合适的诱导分化液中培养一定时间后，如图4C所示，αSMA染色显示hNCSCs-MSC已分化为平滑肌细胞。说明培养的hNCSCs具有多向分化能力。
实施例2从hNCSCs诱导分化为睾丸间质细胞(hiPS-hNCSCs-LCs或称hNCSCs-LCs)
扩增获得的hNCSCs细胞，达到60％的密度时换成睾丸间质细胞(LCs)分化培养液(DMEM-F12培养基(Hyclone，SH30023.018)中加入体积百分比为2％的小牛血清(FCS)、1nM三碘甲腺原氨酸(T3)(Sigma，T2877)、1ng/ml促黄体生成素(LH)(Sigma，L6420)、70ng/ml类胰岛素生长因子(IGF-I)(PeproTech，100-11)、10ng/ml血小板来源生长因子BB(PDGF-BB)(PeproTech，500-P47)，诱导14天，进行细胞分化，收集细胞上清，固定细胞。通过免疫荧光检测成熟LCs相关标志物的表达(LCs标志物3β-HSD、P450c17、steroidogenic acute regulatory protein(StAR)、以及steroidogenic factor 1(SF-1))，如图5所示，免疫染色分析表明hNCSCs诱导分化的细胞(hNCSCs-LCs)表达3β-HSD，P450C17，StAR，SF-1。通过睾酮ELISA检测来测定培养液中睾酮水平，如图6所示,发现在体外分化后的hNCSCs-LCs逐渐增加睾酮分泌，表明hNCSCs可以分化为成熟的睾丸间质细胞。(本实施例中所述睾丸间质细胞(LCs)分化培养液不限于采用如上配方，所述睾丸间质细胞(LCs)分化培养液采用本发明所述的睾丸间质细胞(LCs)分化培养液(DMEM-F12培养基中加入体积百分比为0.1％-20％的小牛血清(FCS)、0.1- 10nM三碘甲腺原氨酸(T3)、0.1-20ng/ml促黄体生成素(LH)、5-100ng/ml类胰岛素生长因子(IGF-I)、1-50ng/ml血小板来源生长因子BB(PDGFBB中的任意一种具体组合时，均可达到如附图5和图6所示的效果。)
实施例3hiPS-hNCSCs-LCs(或称hNCSCs-LCs)在体内的作用
先前的研究表明用大鼠睾丸间质细胞的特异性凋亡诱导剂二甲磺酸乙烷(EDS)处理4天能够耗尽睾丸间质细胞，因此向大鼠腹腔注射EDS建立EDS模型,。选择三组成年大鼠，分别为正常对照组、EDS-对照组、细胞移植组。正常对照组在第0天和第4天，分别腹腔注射相同体积的生理盐水。EDS-对照组大鼠在第0天向腹腔注射EDS(75mg/kg体重)，在第4天向睾丸内注射20μl生理盐水(10μl/单侧睾丸)。细胞组的大鼠在第0天向腹腔内注射EDS(75mg/kg体重)，在第4天,将LC培养液中培养了5-7天的hNCSCs-LCs(1.5x106重悬于10μl PBS/单侧睾丸)移植到大鼠睾丸内。移植后第10天时测量血清睾酮浓度。结果如图7所示，移植hNCSCs-LCs能够提高血清睾酮的水平。
应当理解的是，本发明的上述具体实施方式仅仅用于示例性说明或解释本发明的原理，而不构成对本发明的限制。因此，在不偏离本发明的精神和范围的情况下所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。此外，本发明所附权利要求旨在涵盖落入所附权利要求范围和边界、或者这种范围和边界的等同形式内的全部变化和修改例。

Claims (19)

  1. 一种经人诱导多能干细胞(hiPS)向睾丸间质细胞(hiPS-hNCSCs-LCs)进行诱导分化的方法。
  2. 根据权利要求1所述人诱导多能干细胞(hiPS)向睾丸间质细胞(hiPS-hNCSCs-LCs)的诱导分化方法，其特征在于：
    (1)使人诱导多能干细胞(hiPS)诱导分化为人神经嵴干细胞(hiPS-hNCSCs)；
    (2)使步骤(1)中获得的人神经嵴干细胞(hiPS-hNCSCs)诱导分化为睾丸间质细胞(hiPS-hNCSCs-LCs)。
  3. 根据权利要求2所述的方法，其特征在于，所述步骤(1)包括将人诱导多能干细胞(hiPS)接种在低粘附性的培养皿中进行培养。
  4. 根据权利要求2所述的方法，其特征在于，所述步骤(1)包括使人诱导多能干细胞(hiPS)在神经分化培养液中诱导分化为人神经嵴干细胞(hiPS-hNCSCs)。
  5. 根据权利要求2所述的方法，其特征在于，所述步骤(1)包括使人诱导多能干细胞(hiPS)在神经分化培养液中进行培养，形成胚体后，用神经嵴干细胞培养液进行贴壁培养。
  6. 根据权利要求2所述的方法，其特征在于，所述步骤(1)包括将人诱导多能干细胞(hiPS)消化后重悬，将悬浮细胞接种在低粘附性的培养皿，使用神经分化培养液进行悬浮培养，形成胚体，随后将胚体接种到用纤连蛋白包被的培养板中，用神经嵴干细胞培养液进行贴壁培养，将贴壁培养的细胞通过流式细胞仪分选出P75+/HNK1+双阳细胞，即为人神经嵴干细胞(hiPS-hNCSCs)。
  7. 根据权利要求3或6所述的方法，其特征在于，所述低粘附性的培养皿为Petri培养皿。
  8. 根据权利要求4-7任一项所述的方法，其特征在于，所述神经分化 培养液含有体积百分比为50-80％ Knockout DMEM、体积百分比为5-20％KnockoutTMSR、体积百分比为0.5-5％青链霉素混合液、0.5-5mM L-谷氨酰胺、0.05-0.5mM的β-巯基乙醇。
  9. 根据权利要求4-7任一项所述的方法，其特征在于，所述神经分化培养液含有体积百分比为80％ KnockoutTMDMEM、体积百分比为18％KnockoutTMSR、体积百分比为1％青链霉素混合液、1mM L-谷氨酰胺、0.1mM的β-巯基乙醇。
  10. 根据权利要求5-6任一项所述的方法，其特征在于，所述神经嵴干细胞培养液含有体积百分比为0.1-5％的N2、体积百分比为0.5-10％的B27、体积百分比为0.5-5％的青链霉素混合液、0.5-5mM L-谷氨酰胺，0.05-0.5mM的β-巯基乙醇，再加入1-100ng/mL的碱性成纤维细胞生长因子(bFGF)和1-100ng/mL的表皮生长因子(EGF)。
  11. 根据权利要求5-6任一项所述的方法，其特征在于，所述神经嵴干细胞培养液含有体积百分比为1％的N2、体积百分比为2％的B27、体积百分比为1％的青链霉素混合液、1mM L-谷氨酰胺，0.1mM的β-巯基乙醇，再加入10ng/mL的碱性成纤维细胞生长因子(bFGF)和10ng/mL的表皮生长因子(EGF)。
  12. 根据权利要求5-6任一项所述的方法，其特征在于，所述神经嵴干细胞培养液为按1:0.1-1比例将DMEM-F12培养基与Neurobasal培养基混合，并添加体积百分比为0.1-5％的N2、体积百分比为0.5-10％的B27、体积百分比为0.5-5％的青链霉素混合液、0.5-5mM L-谷氨酰胺，0.05-0.5mM的β-巯基乙醇，再加入1-100ng/mL的碱性成纤维细胞生长因子(bFGF)和1-100ng/mL的表皮生长因子(EGF)。
  13. 根据权利要求5-6任一项所述的方法，其特征在于，所述神经嵴干细胞培养液为按1:1比例将DMEM-F12培养基与Neurobasal培养基混合，并添加体积百分比为1％的N2、体积百分比为2％的B27、体积百分比为1％的青链霉素混合液、1mM L-谷氨酰胺，0.1mM的β-巯基乙醇， 再加入10ng/mL的碱性成纤维细胞生长因子(bFGF)和10ng/mL的表皮生长因子(EGF)。
  14. 根据权利要求2所述的方法，其特征在于，所述步骤(2)包括扩增步骤(1)获得的hiPS-hNCSCs，随后更换为睾丸间质细胞(LCs)分化培养液诱导分化，获得睾丸间质细胞(hiPS-hNCSCs-LCs)。
  15. 根据权利要求14所述的方法，其特征在于，所述睾丸间质细胞(LCs)分化培养液为在DMEM-F12培养基中加入体积百分比为0.1％-20％的小牛血清(FCS)、0.1-10nM三碘甲腺原氨酸(T3)、0.1-20ng/ml促黄体生成素(LH)、5-100ng/ml类胰岛素生长因子(IGF-I)、1-50ng血小板来源生长因子BB(PDGF-BB)。
  16. 根据权利要求14所述的方法，其特征在于，所述睾丸间质细胞(LCs)分化培养液为在DMEM-F12培养基中加入体积百分比为2％的小牛血清(FCS)、1nM三碘甲腺原氨酸(T3)、1ng/ml促黄体生成素(LH)、70ng/ml类胰岛素生长因子(IGF-I)、10ng/ml血小板来源生长因子BB(PDGF-BB)。
  17. 根据权利要求1-16任一项的方法获得的睾丸间质细胞(hiPS-hNCSCs-LCs)在制备提高睾酮水平的药物中的应用。
  18. 根据权利要求1-16任一项的方法获得的睾丸间质细胞(hiPS-hNCSCs-LCs)在制备治疗睾酮水平低下导致的相关疾病的药物中的应用。
  19. 根据权利要求15或16的方法，其中睾酮水平为血清睾酮水平。
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