CN105368773A - Method for obtaining haploid germ cells through in-vitro differentiation of human skin stem cells - Google Patents
Method for obtaining haploid germ cells through in-vitro differentiation of human skin stem cells Download PDFInfo
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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Abstract
The invention relates to a method for obtaining haploid germ cells through differentiation of human skin stem cells. The method comprises the following operation steps: after in-vitro separation, in-vitro culture is carried out on human skin derived stem cells: wherein separated human skin derived stem cells form a spherical cloning sample structure during in-vitro suspension culture, subculturing is carried out every four days and human skin derived stem cell cloning spheres are obtained after subculturing; human skin derived stem cells are induced to germ cell sample cells; human skin derived stem cells are induced and differentiated towards haploid germ cells, and after differentiation is carried out for about 18 days, DNA ploidy analysis can detect that 1.79% of haploid sample cells are formed. With the adoption of the method provided by the invention, primordial germ cells, of which the morphology and specific molecular marker expression are similar to those of normal primordial germ cells in the human body are obtained, the human haploid germ cells are obtained in a differentiation process of a proper condition culture medium and a good in-vitro model is provided for research on a generating mechanism of human germ cells.
Description
Technical field:
The present invention relates to the method for a kind of human skin stem cell in vitro differentiation haploid germ cells, particularly relate to one and comprise in-vitro separation human skin Derived Stem Cells, In vitro Suspension is cultivated, and the method for the clonal structure of acquisition being carried out the induction of sexual cell like cell and haploid induction obtains the method for the haploid germ cells of people.Belong to the field of biomedicine technology of reproductive medicine.
Background technology:
Research for stem cell has had history for a long time, and stem cell is exactly utilize differentiation of stem cells to go out reproduction gamete at one of the study hotspot of field of reproduction, to solving the infertile problem of the mankind; On the other hand, up to the present, the generating process of reproduction gamete also has much mechanism undiscovered, sets up the external platform utilizing stem cell to break up to reproduction gamete, contribute to the generating process that we better study reproduction gamete, make us better understand the combination of both sexes gamete.2009, Kee etc. utilized lentiviral vectors, and the embryonic stem cell (ESCs) to the mankind proceeds to three gene Dazl of DAZ family, Daz, Boule, and success obtains monoploid sperm like cell in vitro.And in mouse, Hayashi etc. utilized ESCs and the induced multi-potent stem cells (iPSCs) of mouse in 2011, vitro differentiation obtains archeocyte (PGCs), the PGCs obtained will be broken up afterwards, be expelled to the injection of mouse convoluted seminiferous tubule to grow, finally obtain ripe sperm, and can complete and be fertilized and create offspring; The maturation of these technology, facilitates the research utilizing adult stem cell (ASCs) to break up sexual cell.But, regardless of ESCs cell or iPSCs cell, clinical application all exists respective drawback, and the skin-derived stem cell (hSDSCs) of people comes from patient self, clinical application does not exist the problems such as histocompatibility, tumorigenicity and ethics.Therefore hSDSCs becomes the focus of people's research in recent years gradually.2006, Dyce etc. utilized the skin-derived stem cell (SDSCs) of tire pig to obtain ovocyte like cell; 2011, Dyce etc., by the SDSCs of mouse, have successfully been obtained the ovocyte like cell (OLCs) with obvious zona pellucida structure.These researchs show, skin-derived stem cell has the potential of differentiation sexual cell.
Comprehensive progress recently, multipotent stem cells has great research potential to the differentiation of sexual cell, and important progress has been achieved on ESCs and iPSCs, skin-derived stem cell has multi-lineage potential too, thus also can as a kind of source of human stem cell to germline.
Summary of the invention:
The object of the invention is to the shortcoming overcoming prior art, provide a kind of human skin stem cell in vitro to break up the technological method of haploid germ cells.The inventive method is by being separated human skin Derived Stem Cells, subculture in vitro separately is cultivated, induced synthesis sexual cell like cell, then the skin-derived stem cell of specific conditioned medium cultivator is adopted, flow cytomery after for some time differentiation, the formation of monoploid sample sexual cell can be detected, and fluorescence in situ hybridization (FISH) result also demonstrate that the formation of class haploid cell.
In order to realize foregoing invention object, the inventive method operates in accordance with the following steps:
The first step, the in-vitro separation of human skin Derived Stem Cells: the skin histology of people is removed fatty tissue and blood clot under the microscope, transfer in culture dish, with scissors, skin histology is cut into small pieces, afterwards with DMEM/F12DMEM/F-12 (the cell culture fluid Dulbecco'sModifiedEagleMedium containing 100U/ml penicillin (penicillin) and 100mg/ml Streptomycin sulphate (streptomycin), F-12NutrientMixture, Gibco company) nutrient solution cleans 3 times, nutrient solution is discarded after centrifugal, by 37 DEG C of digestion in the Digestive system of skin histology pancreatin surrogate TrypLEExpress (Gibco company) that shreds, after digestion terminates, add DMEM/F12 nutrient solution and again clean 3 times, after cleaning terminates, with rifle head piping and druming skin histology block, skin-derived stem cell in skin histology block is split away off, afterwards with cell sieve by unicellularly separating of blowing and beating,
Second step, the skin-derived stem cell of vitro culture of human: the skin-derived stem cell medium of the single cell suspension employment of acquisition is carried out subculture in vitro separately cultivation, the human skin Derived Stem Cells of vitro culture can observe the formation of colony morphology after cultivating 2-3 days under inverted microscope, the skin-derived stem cell of people is gone down to posterity once every 3-4 days needs, when going down to posterity, original skin progenitor cell clone and nutrient solution are all transferred in centrifuge tube, centrifugal, discard supernatant afterwards, add fresh nutrient solution, clone ball is blown and beaten the 1/2-1/3 to original volume, continue to cultivate in the culture dish that access one is new afterwards, described human skin Derived Stem Cells nutrient solution comprises DMEM/F12 (Dulbecco'sModifiedEagleMedium, F-12NutrientMixture), 20ng/ml Urogastron (EGF), 2%B-27 serum substitute (serumsupplement), 40ng/ml Prostatropin (bFGF), 100U/ml penicillin (penicillin) and 100mg/ml Streptomycin sulphate (streptomycin),
3rd step, human skin Derived Stem Cells is induced to sexual cell like cell: collect the clone of the human skin Derived Stem Cells after twice of going down to posterity, TrypLEExpress is digested to single cell suspension, with the resuspended single cell suspension of sexual cell inducing culture, cell suspension is received in 24 orifice plates of suspension and continue to cultivate, change liquid every three days once; Described sexual cell inducing culture comprises TCM199,5 μ l/ml Regular Insulin Transferrins,iron complexes Sodium Selenite (ITS), 3mg/ml bovine serum albumin (BSA), 1mg/ml Pp63 glycophosphoproteins (fetuin), 0.23mM Sodium.alpha.-ketopropionate, 1ng/ml Urogastron (EGF), 20ng/ml activin A (ActivinA) and 30ng/ml bone morphogenetic protein 4 (BMP4), 100U/ml penicillin and 100mg/ml Streptomycin sulphate;
4th step, formed with conditioned medium induction monoploid sample sexual cell: the human skin Derived Stem Cells clone TrypLEExpress induced by sexual cell four days digests, by centrifugal for the single cell suspension obtained, after discarding supernatant, use conditioned medium re-suspended cell, receive in 24 adherent orifice plates and continue to cultivate, within in culturing process two days, change liquid once, when changing liquid, lightly from the old nutrient solution of edge sucking-off 150 μ l, add the conditioned medium that 200 μ l are fresh, in differentiation-inducing process, DNA ploidy analyzing and testing finds at about the 18th day that breaks up, the formation of the haploid cell of 1.79% can be detected, described conditioned medium comprises DMEM/F12 (Dulbecco'sModifiedEagleMedium, F-12NutrientMixture), the foetal calf serum (FBS) of 15%, 1500U/ml leukaemia inhibitory factor (LIF), 50mIU follitropin (FSH), 10ng/ml Urogastron (EGF), 2%B-27 serum substitute (serumsupplement), 5 μ l/ml Regular Insulin Transferrins,iron complexes Sodium Selenite (ITS), 100U/ml penicillin and 100mg/ml Streptomycin sulphates.
Described in the inventive method the 4th step, human skin Derived Stem Cells is when haploid induction conditioned medium is differentiation-inducing, by detecting the expression of differentiation different time SCP3 and γ H2AX, the SCP3 staining signals of wire can be detected, thus demonstrate human skin Derived Stem Cells when haploid induction conditioned medium is differentiation-inducing, can reduction division be started; The human skin Derived Stem Cells broken up by fluorescence in situ hybridization detection, can detect that the class haploid cell of single fluorescent signal exists.
The inventive method successfully utilizes external mechanical process from the skin histology of people, isolate the skin-derived stem cell of people, through vitro culture, sexual cell is induced, and the atomization such as conditioned medium induction, obtains the human haploid sexual cell like cell of expressing sexual cell mark.The inventive method is that the mechanism better studying Human germ cells provides a kind of well external model, meanwhile, utilizes human skin Derived Stem Cells to germline, also for infertility patient brings new hope.
Accompanying drawing illustrates:
Fig. 1 is the clone figure that skin-derived Stem cells cultured in vitro is formed.
Fig. 2 behaves the sexual cell like cell figure of the stem cell external evoked formation of skin-derived.
Fig. 3 is the monoploid sample sexual cell figure of flow cytometer detection human skin Derived Stem Cells induced synthesis.
Fig. 4 is the monoploid sample sexual cell figure formed by fluorescence in situ hybridization detection.
Embodiment:
The inventive method to be further elaborated by specific embodiment below in conjunction with accompanying drawing.
Embodiment 1,
1, the tissue positioned of human skin Derived Stem Cells
By the skin histology of people with 4% paraformaldehyde 4 DEG C fixedly spend the night, after fixing, tissue block gauze is wrapped up, to be put in wide-necked bottle and under being placed in water tap, allowing tap water inject bottle slowly, rinse, rinsing 2-3h.Dewater according to following steps after flushing terminates: 50% alcohol (15min)-70% alcohol (15min)-80% alcohol (20min)-90% alcohol (20min)-95% alcohol I (10min)-95% alcohol II (20min)-raw spirit I (30min)-raw spirit II (30min)-alcohol: dimethylbenzene (volume ratio 1:1,20min).Then through two step dimethylbenzene transparent (often walking 15min).Afterwards tissue block is put into wax cup, after twice each 1.5h of waxdip, tissue block is embedded in embedded box.Tissue is cut into the white tiles of 5 μm by embedded tissue in paraffin slicing machine.For HE staining, the first will be chopped white sheet into 60 degrees hot baked pieces of 1-2 h, after dyeing according to the following steps: I (5-10 min) - dimethylbenzene xylene II (5-10 min), anhydrous alcohol I (5 min) - anhydrous alcohol II (3-5 min), 95% alcohol I II (2-3 min), 95% alcohol (2-3 min) - 80% min alcohol (2 min) - 70% alcohol (2 min), 50% alcohol (2 min), distilled water (2 min), hematoxylin (7 min), water (2 min), distilled water (2 min) - 1% hydrochloric acid alcohol (3-5 s) - tap water (5 min) - distilled water (2 min) - 50% alcohol (2 min) - 70% alcohol (2 min) - 80% min alcohol (2 min) - eosin (30 s) - 95% alcohol I II (2-5 min), 95% alcohol (2-5 min), anhydrous alcohol I (5 min) - anhydrous alcohol II (3-5 min) - xylene I (5 min) - xylene II (5-10 min).On sample, drip neutral gum after dyeing terminates and cover cover glass, to preserve for a long time.Can see that the cell having some structures very fine and close in subcutaneous hair follicle structure exists by the HE dyeing of human skin.For histochemical staining, first the white tiles cut is put into 60 degree of thermal station and bakes sheet 2h, often walk 15min through twice dimethylbenzene and the paraffin in section is purified.Then rehydration is carried out according to following table.
Afterwards, citric acid antigen repair liquid is put in the section of rehydration, 10min at 96 DEG C.At room temperature be cooled to room temperature afterwards.After repair liquid is cooled to room temperature, 5min washed by the Tris damping fluid (be called for short TBS) slice, thin piece being put into pre-warm, then proceed to pre-warm 0.1%Tween-20 Tris damping fluid (being called for short TBST) in wash 5min.Slice, thin piece after antigen retrieval, add that confining liquid BDT (30mgBSA and 100ul lowlenthal serum is dissolved in 900ulTBS) closes, about consumption is 50 μ l, and room temperature closes 30min.Close after terminating, add Beta1-integrin (Abcam company) primary antibodie, be placed on 4 DEG C of overnight incubation in wet box.After primary antibodie terminates, add two anti-(the green skies companies) of having diluted, after anti-with ParafilmTM two, put into wet box, seal with sealed bag, hatch 30min for 37 degree.After hatching end, with Hoechst33342 (green skies company), dye 5min, then uses Vectashield (Vector company) mounting.Experimental result shows, and the skin-derived stem cell of Isolation and culture is mainly derived from hair follicle structure, and is mainly positioned the knuckle district of hair follicle structure.Immunohistochemistry result shows the cell mass that there is the Beta1-integrin positive clearly in hair follicle structure.
2, the in-vitro separation of human skin Derived Stem Cells
The skin histology of people is removed fatty tissue and blood clot under the microscope, transfers in the culture dish of 6cm, with scissors, skin histology is cut into 1mm
3the fritter of left and right, 3 times are cleaned afterwards with DMEM/F12 (Gibco company) nutrient solution containing 100U/ml penicillin and 100mg/ml Streptomycin sulphate (Hyclone company), nutrient solution is discarded after centrifugal, by 37 DEG C of digestion 15-20min in the Digestive system of the TrypLEExpress (Gibco company) of skin histology 2ml that shreds, after digestion terminates, add DMEM/F12 and again clean 3 times, after cleaning terminates, with the rifle head piping and druming skin histology block of 1ml, skin-derived stem cell in skin histology block is split away off, afterwards with 400 object cells sieves by unicellularly separating of blowing and beating.
3, the vitro culture of human skin Derived Stem Cells
The human skin Derived Stem Cells of in-vitro separation, employment skin-derived stem cell medium comprises DMEM/F12, 2%B-27 serum substitute (Gibco company), 20ng/ml Urogastron (R & D company), 40ng/ml Prostatropin (Peprotech company), 100U/ml penicillin and 100mg/ml Streptomycin sulphate (Hyclone company) suspension culture, just cultured cells is designated as p0 generation, 1 postscript that goes down to posterity is p1 generation, when going down to posterity, original skin progenitor cell clone and nutrient solution are all transferred in the centrifuge tube of 15ml, the centrifugal 3min of 1500rpm, discard supernatant afterwards, add the nutrient solution that 1ml is fresh, clone ball is blown and beaten the 1/2-1/3 to original volume, continue to cultivate in the culture dish that access one is new afterwards, Fig. 1 is the human skin Derived Stem Cells clonal structure of p2 for formation, and wherein, left figure is the clonal structure of p2 for formation when 2 days, and right figure is the clonal structure of p2 for formation when 4 days, and along with the prolongation of incubation time, clonal structure is more and more finer and close, and edge is more and more clear.
3, human skin Derived Stem Cells is induced to sexual cell like cell
Collect the human skin Derived Stem Cells in 4 days generations of p2, centrifugal rear supernatant discarded, add TrypLEExpress and digest about 3min, after stopping digestion, clean a human skin Derived Stem Cells with nutrient solution, fully to remove remaining TrypLEExpress, by centrifugal for single cell suspension 1500rpm 5min, with the resuspended single cell suspension of sexual cell inducing culture, cell suspension is received in 24 orifice plates of suspension and cultivate, change liquid every three days once, sexual cell inducing culture comprises TCM199 (Hyclone company), 3mg/ml bovine serum albumin (is called for short BSA, Solarbio company), 1mg/ml Pp63 glycophosphoproteins (fetuin, Calbiochem company), 5 μ l/ml Regular Insulin Transferrins,iron complexes Sodium Selenites (are called for short ITS, Gibco company), 0.23mM Sodium.alpha.-ketopropionate (Hyclone company), 1ng/ml Urogastron (EGF), 20ng/ml activin A (is called for short ActivinA, Sigma company), (BMP4 is called for short with 30ng/ml bone morphogenetic protein 4, R & D company), 100U/ml penicillin and 100mg/ml Streptomycin sulphate (Hyclone company).Fig. 2 is the result of embryoid body induction, and wherein, left figure is embryoid body cellular prion protein when inducing 0 day, and right figure is that embryoid body induces the embryoid body spline structure formed when 3 days.Can the embryonic stem cell of the similar people of skin-derived stem cell of finder and inducing pluripotent stem cells, comparison green structure can be formed in the process of embryoid body induction.
4, synaptonemal complex fluorescent dye detects human skin Derived Stem Cells and enters reduction division
Collector's skin-derived stem cell, single cell suspension is digested to TrypLEExpress, then the hypotonic 30min of citric acid hypotonic medium is used, afterwards by cell suspension with 1% paraformaldehyde fix room temperature overnight incubation, the photoflo of 0.04% (Kodak company) is used to hatch 4min afterwards, then the lowlenthal serum of 1% (Boster company) 37 DEG C of closed 30min are used, close after terminating, add humanSCP3 (SynaptonemalComplexProtein3, rabbitpolyclonal, 1:200, NovusBiologicals company) and γ H2AX (PhosphorylatedHistoneH2AX, mousepolyclonal, 1:200, Abcam company) primary antibodie, 6-8h is hatched for 37 DEG C after adding primary antibodie.After hatching end, two anti-goatanti-rabbitIg-CY3/FITC-conjugated (1:100, Sigma company) two anti-37 DEG C hatch 2h, then dye 5min to use Hoechst33342 (green skies company), and with Vectashield (Vector company) mounting.Synaptonemal complex fluorescent dye result shows, the human skin Derived Stem Cells of differentiation can detect the SCP3 of wire signal and the γ H2AX coloration result of dot signal after differentiation-inducing, when the skin-derived stem cell showing people is differentiation-inducing in vitro, reduction division can be entered.
5, human skin Derived Stem Cells is induced to haploid germ cells
Collect embryoid body spline structure, add TrypLEExpress and digest about 3min, with pipettor, clonal structure piping and druming is become single cell suspension, fresh medium re-suspended cell is added again after stopping digestion, the centrifugal 5min of 1500rpm, abandons supernatant, fully to discard Digestive system, by resuspended for the single cell suspension conditioned medium collected, receive in 24 adherent orifice plates and continue to cultivate, change liquid every three days once, when changing liquid, the old nutrient solution of sucking-off 150 μ l, adds the nutrient solution that 200 μ l are new; In culturing process, collecting cell carries out DNA ploidy analysis, in the formation that the monoploid sample sexual cell about having 1.79% ratio can be detected on the 18th day of differentiation.Described conditioned medium comprises DMEM/F12 (Dulbecco'sModifiedEagleMedium, F-12NutrientMixture), the foetal calf serum (FBS) of 15%, 1500U/ml leukaemia inhibitory factor (LIF), 50mIU follitropin (FSH), 10ng/ml Urogastron (EGF), 2%B-27 serum substitute (serumsupplement), 5 μ l/ml Regular Insulin Transferrins,iron complexes Sodium Selenite (ITS), 100U/ml penicillin and 100mg/ml Streptomycin sulphates.Fig. 3 is the result of the monoploid sample sexual cell that flow cytometer detection human skin Derived Stem Cells is formed.Result shows, and after human skin Derived Stem Cells is differentiation-inducing in vitro, can form class haploid cell, and the 25th day of differentiation, monoploid formation efficiency was the highest.
6, fluorescence in situ hybridization detection human skin Derived Stem Cells breaks up the monoploid sample sexual cell obtained
Collect the human skin Derived Stem Cells single cell suspension after differentiation, after resuspended with the PBS of 50 μ l, drip to that APES (middle mountain gold bridge company) processes on slide glass, be placed in thermal station oven dry, then fix 5min by Carnoy stationary liquid room temperature.With 2xSSC incubated at room 10min after fixing end, then use the paraformaldehyde of 1% (Solarbio company) room temperature to fix 10min, slice, thin piece is transferred to incubated at room 10min in the SSCbuffer of 0.1%NP-40 (Sheng Gong company).Then slice, thin piece is dewatered according to 80% alcohol (2min)-90% alcohol (2min)-100 alcohol (2min).Deng slice, thin piece air-dry after, add hybridization buffer, be specifically formulated as follows:
After adding hybridization buffer, slice, thin piece rubbercement (Elmer ' s company) is sealed, to be then placed in wet box 37 DEG C and to hatch 48h, after hatching end, remove rubbercement, slice, thin piece to be placed in 2xSSC 45 DEG C and to hatch 30min.After hatching end, contaminate core 5min with Hoechst33342, and use Vectashield mounting.Fig. 4 behaves the fluorescence in situ hybridization result of the monoploid sample sexual cell obtained after skin-derived differentiation of stem cells, and relative to normal diploid somatocyte, the monoploid sample sexual cell of formation has single fluorescent signal.
The inventive method obtains expresses similar archeocyte with normal archeocyte form in human body and specific molecular marker, and in the atomization of suitable conditioned medium, can obtain the haploid germ cells of the mankind.
Claims (2)
1. the method for a human skin stem cell in vitro differentiation haploid germ cells, it is characterized in that operating in accordance with the following steps: the first step, the in-vitro separation of human skin Derived Stem Cells: the skin histology of people is removed fatty tissue and blood clot under the microscope, transfer in culture dish, with scissors, skin histology is cut into small pieces, 3 times are cleaned afterwards with the DMEM/F12 nutrient solution containing 100U/ml penicillin and 100mg/ml Streptomycin sulphate, nutrient solution is discarded after centrifugal, by 37 DEG C of digestion in the Digestive system of skin histology pancreatin surrogate TrypLEExpress that shreds, after digestion terminates, add DMEM/F12 nutrient solution and again clean 3 times, after cleaning terminates, with rifle head piping and druming skin histology block, skin-derived stem cell in skin histology block is split away off, afterwards with cell sieve by unicellularly separating of blowing and beating, second step, the skin-derived stem cell of vitro culture of human: the skin-derived stem cell medium of the single cell suspension employment of acquisition is carried out subculture in vitro separately cultivation, the human skin Derived Stem Cells of vitro culture can observe the formation of colony morphology after cultivating 2-3 days under inverted microscope, the skin-derived stem cell of people was gone down to posterity once every 3-4 days, when going down to posterity, original skin progenitor cell clone and nutrient solution are all transferred in centrifuge tube, centrifugal, discard supernatant afterwards, add fresh nutrient solution, clone ball is blown and beaten the 1/2-1/3 to original volume, continue to cultivate in the culture dish that access one is new afterwards, described human skin Derived Stem Cells nutrient solution comprises DMEM/F12,20ng/ml Urogastron, 2%B-27 serum substitute, 40ng/ml Prostatropin, 100U/ml penicillin and 100mg/ml Streptomycin sulphate, 3rd step, human skin Derived Stem Cells is induced to sexual cell like cell: collect the clone of the human skin Derived Stem Cells after twice of going down to posterity, TrypLEExpress is digested to single cell suspension, with the resuspended single cell suspension of sexual cell inducing culture, cell suspension is received in 24 orifice plates of suspension and continue to cultivate, change liquid every three days once, described sexual cell inducing culture comprises TCM199,5 μ l/ml Regular Insulin Transferrins,iron complexes Sodium Selenites, 3mg/ml bovine serum albumin, 1mg/ml Pp63 glycophosphoproteins, 0.23mM Sodium.alpha.-ketopropionate, 1ng/ml Urogastron, 20ng/ml activin A and 30ng/ml bone morphogenetic protein 4,100U/ml penicillin and 100mg/ml Streptomycin sulphate, 4th step, formed with conditioned medium induction monoploid sample sexual cell: the human skin Derived Stem Cells clone TrypLEExpress induced by sexual cell four days digests, by centrifugal for the single cell suspension obtained, after discarding supernatant, use conditioned medium re-suspended cell, receive in 24 adherent orifice plates and continue to cultivate, within in culturing process two days, change liquid once, when changing liquid, lightly from the old nutrient solution of edge sucking-off 150 μ l, add the conditioned medium that 200 μ l are fresh, in differentiation-inducing process, DNA ploidy analyzing and testing finds at the 18th day that breaks up, the formation of the haploid cell of 1.79% can be detected, described conditioned medium comprises DMEM/F12, the foetal calf serum of 15%, 1500U/ml leukaemia inhibitory factor, 50mIU follitropin, 10ng/ml Urogastron, 2%B-27 serum substitute, 5 μ l/ml Regular Insulin Transferrins,iron complexes Sodium Selenite, 100U/ml penicillin and 100mg/ml Streptomycin sulphates.
2. the method for a kind of human skin stem cell in vitro differentiation haploid germ cells according to claim 1, it is characterized in that described in the 4th step, human skin Derived Stem Cells is when haploid induction conditioned medium is differentiation-inducing, by detecting the expression of differentiation different time SCP3 and γ H2AX, the SCP3 staining signals of wire can be detected, human skin Derived Stem Cells, when haploid induction conditioned medium is differentiation-inducing, can start reduction division; The human skin Derived Stem Cells broken up by fluorescence in situ hybridization detection, can detect that the class haploid cell of single fluorescent signal exists.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106754661A (en) * | 2016-12-11 | 2017-05-31 | 青岛农业大学 | A kind of skin progenitor cell is divided into the technical method of sperm through ectopic development |
| CN108611316A (en) * | 2018-05-04 | 2018-10-02 | 江南大学附属医院 | A method of induction human fibroblasts transdifferentiation is human germ-line |
| CN113957038A (en) * | 2021-10-22 | 2022-01-21 | 青岛农业大学 | In-vitro separation method of human skin stem cells |
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| CN103146644A (en) * | 2013-02-27 | 2013-06-12 | 青岛农业大学 | Technical method for differentiating mouse hair follicle stem cells to primordial germ cells in vitro |
| CN103275928A (en) * | 2013-06-18 | 2013-09-04 | 青岛农业大学 | Technical method for differentiating human skin stem cells in vitro into primordial germ cells |
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| CN103146644A (en) * | 2013-02-27 | 2013-06-12 | 青岛农业大学 | Technical method for differentiating mouse hair follicle stem cells to primordial germ cells in vitro |
| CN103275928A (en) * | 2013-06-18 | 2013-09-04 | 青岛农业大学 | Technical method for differentiating human skin stem cells in vitro into primordial germ cells |
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| GE W ET AL.: "Differentiation of early germ cells from human skin-derived stem cells without exogenous gene integration", 《SCI REP》 * |
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| 陈春雷等: "不同类型干细胞向生殖细胞分化及应用潜能的比较与评价", 《青岛农业大学学报（自然科学版）》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754661A (en) * | 2016-12-11 | 2017-05-31 | 青岛农业大学 | A kind of skin progenitor cell is divided into the technical method of sperm through ectopic development |
| CN106754661B (en) * | 2016-12-11 | 2020-04-07 | 青岛农业大学 | Technical method for differentiating skin stem cells into sperms through ectopic development |
| CN108611316A (en) * | 2018-05-04 | 2018-10-02 | 江南大学附属医院 | A method of induction human fibroblasts transdifferentiation is human germ-line |
| CN113957038A (en) * | 2021-10-22 | 2022-01-21 | 青岛农业大学 | In-vitro separation method of human skin stem cells |
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