CN104726400A - Animal-source-free component culture method for differentiation from human pluripotent stem cells to germ cells - Google Patents
Animal-source-free component culture method for differentiation from human pluripotent stem cells to germ cells Download PDFInfo
- Publication number
- CN104726400A CN104726400A CN201510094574.4A CN201510094574A CN104726400A CN 104726400 A CN104726400 A CN 104726400A CN 201510094574 A CN201510094574 A CN 201510094574A CN 104726400 A CN104726400 A CN 104726400A
- Authority
- CN
- China
- Prior art keywords
- serum
- free
- nutrient solution
- animal derived
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an animal-source-free component culture method for differentiation from male source pluripotent stem cells to germ cells. Under the conditions of a serum-free animal-source-protein-free culture solution, the male source pluripotent stem cells generate spermatogonia-like stem cells and haploid cells in the in-vitro differentiation process. The invention also discloses a serum-free animal-source-protein-free culture solution system. The serum-free animal-source-protein-free culture method disclosed by the invention is simple to operate, has high repeatability, has wide clinical application value, and provides a basic technical platform for the research of germ cell generation mechanisms and the diagnosis and treatment of male non-obstructive azoospermia.
Description
Technical field
The present invention relates to the biotechnology of human pluripotent stem cells, be specifically related to the non-animal derived component cultural method of a kind of human pluripotent stem cells to germline.
Background technology
The current whole world about has the couple at child-bearing age of 1/6th sterile, and wherein male factor accounts for 50% (Hirsh 2003).Research shows, Europe 20% male sterility patient belong to azoospermia (Bhasin, de Kretser et al.1994), and this ratio in Chinese male infertile patients up to 25% (Wu, Lu et al.2007); Meanwhile, the azoospermia patients of 49% to 93% belongs to the dysfunction of testis own, i.e. primary non-obstructivity azoospermia (Non-obstructive azoospermia, NOA) (Hayashi, Saitou et al.2012).NOA disease patients with clinical manifestations is scarcity and the disappearance of androgone, there is no active drug treatment plan, and adopting the smart person's seminal fluid of tax to carry out artificial insemination is one of current NOA disease " treatment " approach.
In recent years, large quantity research shows, human embryo stem cell (Human Embryonic Stem Cells, hES), people's induced multi-potent stem cells (Human Induced Pluripotent Stem Cells, hiPSC) (be referred to as " human pluripotent stem cells "), possesses ability (Tilgner, the Atkinson et al.2008 that vitro differentiation produces sexual cell, Bucay, Yebra et al.2009, Kee, Angeles et al.2009, Park, Galic et al.2009, Tilgner, Atkinson et al.2010, Eguizabal, Montserrat et al.2011, Medrano, Ramathal et al.2011, Easley, Phillips et al.2012, Gkountela, Li et al.2013, Hayashi and Saitou 2013), especially with the appearance (Takahashi of iPS technology, Tanabe et al.2007), people obtain NOA disease hypotype " the micro-deleted disease of Y chromosome " pathology iPS cell line successively, research shows that Y chromosome disappearance disease iPS cell line is in mouse testis differentiation in vivo process, sexual cell ratio is significantly lower than control group, show the similarity (Ramathal surprising with NOA disease pathological characters, Durruthy-Durruthy et al.2014), correlative study is the research of NOA disease mechanism of causing a disease, pathological diagnosis, clinical treatment provides brand-new technique means.Unfortunately, the multipotential stem cell reported at present is to Germ Cells In vitro, differentiation in vivo system, all there is inefficiency, cultivate composition not clear (serum), exist (mouse embryo fibroblasts Dual culture, nude mice differentiation in vivo) such as a large amount of animal source " pollution ", the pathomechanism research of serious restriction NOA disease and clinical treatment propagation and employment.
Summary of the invention
The present invention is as point of penetration, overcome the problems referred to above of prior art, in conjunction with the current research of multipotential stem cell to germline, groped by lot of experiments combination, construct a kind of brand-new male sex and originate multipotential stem cell to the serum-free of germline, non-animal derived albumen culture system and cultural method, there is science, practicality and clinical value widely.
An object of the present invention is, there is provided a kind of to originate the serum-free of multipotential stem cell to germline, the cultural method of non-animal derived property albumen for the male sex, under the condition adopting serum-free, non-animal derived property albumen nutrient solution, the male sex is originated in multipotential stem cell vitro differentiation process and produces class stem spermatogonium, haploid cell, thus expanded the research field that Mammals multipotential stem cell Differentiation Induction in vitro produces sexual cell.
In the present invention, male sex's multipotential stem cell of originating comprises any one nutrient solution to the serum-free of germline, non-animal derived albumen culture system, i.e. serum-free, non-animal derived property albumen nutrient solution I and serum-free, non-animal derived property albumen nutrient solution II.Wherein, described serum-free, non-animal derived property albumen nutrient solution I comprise: MEM substratum; The dual anti-solution 1% of penicillin/streptomycin; L-glutaminate 2mM; 4-hydroxyethyl piperazine ethanesulfonic acid 10mM; Insulin-Transferrin-selenium-X additive 1%; Without the serum substitute 0.2-3% of xenogeneic components; Prostatropin 1ng/mL; Recombinant human nerve colloid derived neurotrophic factor 10-40ng/mL; Alpha-mercapto ethanol 0.1mM; Lipid enriched material 0.1-0.2%.Described serum-free, non-animal derived property albumen nutrient solution II comprise: MEM substratum; The dual anti-solution 1% of penicillin/streptomycin; L-glutaminate 2mM; 4-hydroxyethyl piperazine ethanesulfonic acid 10mM; Insulin-Transferrin-selenium-X additive 1%; Without the serum substitute 0.2-3% of xenogeneic components; Prostatropin 1ng/mL; Recombinant human nerve colloid derived neurotrophic factor 10-40ng/mL; Vitamins C 50-200ug/mL; Lipid enriched material 0.1-0.2%.
In the present invention, MEM substratum refers to a kind of minimum required nutrient solution being widely used in mammaliancellculture, and be purchased from Minimum Essential Medium (MEM) α of Invitrogen company, its trade mark is numbered 12561-056.
In the present invention, Insulin-Transferrin-selenium-X additive refers to the mixing addO-on therapy of Regular Insulin, Transferrins,iron complexes, selenium, thanomin, be widely used in Mammals serum-free cell culture, to cellular energy metabolism, anti-oxidant, iron ion transports, oxygen partial pressure regulates, organoid composition has a very important role.Insulin-Transferrin-selenium-X is purchased from the Insulin-Transferrin-Selenium-X of Invitrogen company, and trade mark is numbered 51500, and the concrete component that its product description provides comprises as follows: Sodium Selenite (anhydrous) 0.00067g/L; Regular Insulin 1.00g/L; Transferrins,iron complexes 0.55g/L; Thanomin 0.20g/L.
In the present invention, the described serum substitute without xenogeneic components, refers to a kind of complete synthetic, non-animal derived protein contamination, is suitable for the human pluripotent stem cells of clinical study cultivation component.Serum substitute without xenogeneic components is purchased from the KnockOut of Invitrogen company
tMsR XenoFree CTS
tM, trade mark is numbered A10992-01.
In the present invention, described lipid enriched material refers to that the lipid that a kind of chemical composition is determined concentrates mixed solution, refer to a kind of grease added ingredients cultivated for vertebrate cells, spinal animal cell non-serum, it has vital role to cellularstructure, Power supply.The lipid enriched material that described lipid enriched material and chemical composition are determined is purchased from the ChemicallyDefined Lipid Concentrate of Invitrogen company, trade mark is numbered 11905031, and the concrete component that its product description provides comprises as follows: arachidonic acid 2.0mg/L; Cholesterol 220.0mg/L; Vitamin-E-acetic ester 70.0mg/L; Ethanol 100%; Linolic acid 10.0mg/L; Linolenic acid 10.0mg/L; Tetradecanoic acid 10.0mg/L; Oleic acid 10.0mg/L; Palmitinic acid 10.0mg/L; Zoomeric acid 10.0mg/L; Poloxamer 90000.0mg/L; Stearic acid 10.0mg/L; Tween-80 2200.0mg/L.
In the present invention, in described serum-free, non-animal derived property albumen nutrient solution, the raw materials such as the dual anti-solution of penicillin/streptomycin, L-glutaminate, Prostatropin, alpha-mercapto ethanol all can be purchased from Invitrogen company.4-hydroxyethyl piperazine ethanesulfonic acid can be purchased from Amresco company.Recombinant human nerve colloid derived neurotrophic factor is purchased from Sino Biological Inc. company.
Preferably, described serum-free, non-animal derived property albumen nutrient solution I comprise: MEM substratum; The dual anti-solution of 1% penicillin/streptomycin; 2mM L-glutaminate; 10mM 4-hydroxyethyl piperazine ethanesulfonic acid; 1% Insulin-Transferrin-selenium-X additive; 0.2% without the serum substitute of xenogeneic components; 1ng/mL Prostatropin; 20ng/mL recombinant human nerve colloid derived neurotrophic factor; 0.1mM alpha-mercapto ethanol; 0.2% lipid enriched material.
In the present invention, the nutrient solution II of described serum-free, non-animal derived property albumen can comprise: MEM substratum; The dual anti-solution 1% of penicillin/streptomycin; L-glutaminate 2mM; 4-hydroxyethyl piperazine ethanesulfonic acid 10mM; Insulin-Transferrin-selenium-X additive 1%; Without the serum substitute 0.2-3% of xenogeneic components; Prostatropin 1ng/mL; Recombinant human nerve colloid derived neurotrophic factor 10-40ng/mL; Vitamins C 50-200ug/mL; Lipid enriched material 0.1-0.2%.
Preferably, the nutrient solution II of described serum-free, non-animal derived property albumen is: MEM substratum; The dual anti-solution of 1% penicillin/streptomycin; 2mM L-glutaminate; 10mM 4-hydroxyethyl piperazine ethanesulfonic acid; 1% Insulin-Transferrin-selenium-X additive; 1% without the serum substitute of xenogeneic components; 1ng/mL Prostatropin (Invitrogen); 20ng/mL recombinant human nerve colloid derived neurotrophic factor; Vitamins C 200ug/mL; The lipid enriched material that 0.2% chemical composition is determined.
The male sex that the present invention proposes originates multipotential stem cell in the serum-free, non-animal derived albumen cultural method of germline, when the male sex originate multipotential stem cell growth reach culture dish growth area 90% converging state time, change use described serum-free, non-animal derived property albumen nutrient solution.The present invention studies and finds and propose the impact of multipotential stem cell density on class stem spermatogonium, haploid cell pick-up rate first, and research finds to cause class stem spermatogonium, one of the main reasons that haploid cell pick-up rate is lower is because cell density is too low.The present invention proposes the cell density that culture system is suitable, e.g., it is good for originating that multipotential stem cell growth reaches when culture dish growth area is not less than 90% converging state as the male sex.
Use the nutrient solution of serum-free of the present invention, non-animal derived property albumen can obtain class stem spermatogonium, haploid cell from male sex's multipotential stem cell of originating in the inventive method, serum-free, non-animal derived property albumen nutrient solution I is used in atomization in vitro at male sex's multipotential stem cell of originating, or choice for use serum-free, non-animal derived property albumen nutrient solution II, improve cytodifferentiation effect further.
In the present invention, the male sex originate multipotential stem cell grow to 90% converging state before the nutrient solution that adopts be people's multipotent stem cells nutrient solution that prior art is general.
In the present invention, at 37 DEG C, 5%CO
2cultivate under saturated humidity condition.
In the present invention, the described male sex originate multipotential stem cell cultivate within 8 ~ 25 days, class stem spermatogonium, haploid cell can be obtained.Preferably, cultivate 10 ~ 15 days.Preferably, cultivate 12 days.
In the present invention, cultivating the class stem spermatogonium obtained is that VASA is positive, PLZF is positive, GPR125 is positive, CD90 is positive, and the class stem spermatogonium of H19 imprinted gene upstream region high methylation.Cultivating the haploid cell obtained is that Acrosin is positive, Prm1 is positive, and the haploid cell of H19 imprinted gene upstream region high methylation.Wherein, in cultured products, the ratio of class stem spermatogonium and haploid cell can be arbitrary proportion, according to experiment particular case.Preferably, the ratio of class stem spermatogonium is 10 ~ 85%, haploid cell ratio 2 ~ 8%.
In a specific embodiment, the present invention is used for male sex's multipotential stem cell of originating, until the male sex originate multipotential stem cell grow to 90% converging state time, general people's multipotent stem cells nutrient solution is replaced by described serum-free, non-animal derived property albumen nutrient solution, is placed in 37 DEG C, 5%CO
2cultivate under saturated humidity condition, between incubation period, every 48h full dose changes nutrient solution, pH is 6.8-7.2, cultivate 12 days, then can obtain the VASA positive in system, PLZF is positive, GPR125 is positive, CD90 is positive, and the class stem spermatogonium of H19 imprinted gene upstream region high methylation, and Acrosin is positive, Prm1 is positive, the haploid cell of H19 imprinted gene upstream region high methylation.
In cultural method of the present invention, every 48h changes described serum-free, non-animal derived property albumen nutrient solution.Before changing liquid, with without the washing of calcium magnesium phosphate buffered saline buffer once, remove dead cell to the impact of follow-up differentiation.First 8 days of differentiation culture, necrocytosis is more, and dead cell is residual to have an impact to late-stage differentiation.
Cultural method of the present invention, adopt the culture technique system of described serum-free, non-animal derived property albumen, the male sex can be made to originate, and pluripotent stem cell differentiation produces the VASA positive, PLZF is positive, GPR125 is positive, CD90 is positive, the class stem spermatogonium of H19 imprinted gene upstream region high methylation, and Acrosin is positive, Prm1 is positive, the haploid cell of H19 imprinted gene upstream region high methylation.
The present invention also proposes a kind of serum-free, non-animal derived property albumen nutrient solution, for serum-free, non-animal derived property albumen nutrient solution I, originate multipotential stem cell in the cultural method of the serum-free of germline, non-animal derived property albumen for the male sex, and it comprises: MEM substratum; The dual anti-solution 1% of penicillin/streptomycin; L-glutaminate 2mM; 4-hydroxyethyl piperazine ethanesulfonic acid 10mM; Insulin-Transferrin-selenium-X additive 1%; Without the serum substitute 0.2-3% of xenogeneic components; Prostatropin 1ng/mL; Recombinant human nerve colloid derived neurotrophic factor 10-40ng/mL; Alpha-mercapto ethanol 0.1mM; Lipid enriched material 0.1-0.2%.
Preferably, described serum-free, non-animal derived property albumen nutrient solution I comprise: MEM substratum; The dual anti-solution of 1% penicillin/streptomycin; 2mM L-glutaminate; 10mM 4-hydroxyethyl piperazine ethanesulfonic acid; 1% Insulin-Transferrin-selenium-X additive; 0.2% without the serum substitute of xenogeneic components; 1ng/mL Prostatropin; 20ng/mL recombinant human nerve colloid derived neurotrophic factor; 0.1mM alpha-mercapto ethanol; 0.2% lipid enriched material.
The invention allows for another kind of serum-free, non-animal derived property albumen nutrient solution II, originate multipotential stem cell in the cultural method of the serum-free of germline, non-animal derived property albumen for the male sex, it comprises: MEM substratum; The dual anti-solution 1% of penicillin/streptomycin; L-glutaminate 2mM; 4-hydroxyethyl piperazine ethanesulfonic acid 10mM; Insulin-Transferrin-selenium-X additive 1%; Without the serum substitute 0.2-3% of xenogeneic components; Prostatropin 1ng/mL; Recombinant human nerve colloid derived neurotrophic factor 10-40ng/mL; Vitamins C 50-200ug/mL; Lipid enriched material 0.1-0.2%.
Preferably, the nutrient solution II of described serum-free, non-animal derived property albumen is: MEM substratum; The dual anti-solution of 1% penicillin/streptomycin; 2mM L-glutaminate; 10mM 4-hydroxyethyl piperazine ethanesulfonic acid; 1% Insulin-Transferrin-selenium-X additive; 1% without the serum substitute of xenogeneic components; 1ng/mL Prostatropin; 20ng/mL recombinant human nerve colloid derived neurotrophic factor; Vitamins C 200ug/mL; 0.2% lipid enriched material.
Wherein, described serum-free, non-animal derived property nutrient solution are used for human pluripotent stem cells in the culture system of germline.
Wherein, described serum-free, non-animal derived property albumen nutrient solution do not contain serum, not containing animal-based protein, definite ingredients, repeatability is strong, is applicable to clinical medical.Described nutrient solution answers lucifuge, 4 DEG C of preservations.Serum-free of the present invention, non-animal derived property albumen cultural method, the addO-on therapy not containing any animal-origin.In cultural method of the present invention and nutrient solution, institute's with medicament is commercially available prod.
The factor playing important influence in culture system of the present invention comprises: the male sex originates multipotential stem cell inoculum density; Serum-free, non-animal derived property albumen nutrient solution:
The male sex originates multipotential stem cell inoculum density: cell paracrine has material impact to stem cells hyperplasia, differentiation.Through great many of experiments room, checking finds in the present invention, and under the culture condition of described serum-free, non-animal derived property albumen nutrient solution, male sex's multipotential stem cell density of originating has remarkably influenced to later stage class stem spermatogonium rate of formation.In a specific embodiment, when the male sex originate multipotential stem cell inoculum density from 30% converge be promoted to 90% converge time, adopt serum-free of the present invention, non-animal derived property albumen nutrient solution cultivates 12 days, obtain PLZF positive class stem spermatogonium ratio and be increased to 63.9% by 34.6%, see Fig. 1.
Serum-free, non-animal derived property albumen nutrient solution: the present inventor is with reference to human pluripotent stem cells serum free culture system of the prior art, adopt described serum-free, non-animal derived property albumen nutrient solution and cultural method, from affecting proliferation of pluripotent stem cells, apoptosis, promote multiple angles such as germline, carry out a large amount of optimum combination test.Research finds, under the culture condition of described serum-free, non-animal derived property albumen nutrient solution, alpha-mercapto ethanol, vitamins C on cell proliferation all have remarkably influenced.In specific embodiment, when removing alpha-mercapto ethanol in nutrient solution, cell survival rate significantly increases, and sees Fig. 2.In nutrient solution, add vitamins C then significantly promote cell proliferation, see Fig. 2.In nutrient solution, remove alpha-mercapto ethanol simultaneously and add vitamins C, male sex's multipotential stem cell state of originating obviously is improved, and cell proliferation is obvious, sees Fig. 2.
The present invention studies discovery further, in culture system, remove alpha-mercapto ethanol, add vitamins C, the male sex originates, and multipotential stem cell adopts described serum-free, non-animal derived property albumen nutrient solution cultivates 12 days, PLZF positive class stem spermatogonium ratio can improve further, be increased to 77.7% by 52.8%, see Fig. 3.Immunoblotting detects that PLZF expressing quantity improves 1.5 times, sees Fig. 3.
In prior art, bovine serum albumin (BSA) is widely used in technical field of cell culture, but because of its batch of unstable, become grading factors containing animal source, relevant cell culture technique is extremely restricted at clinical expansion and application.
The present invention studies discovery further, in described culture system, add 0.2% bovine serum albumin (BSA), 0.2% respectively without the serum substitute (KnockOut SR XenoFree), 1% of xenogeneic components without the serum substitute (KnockOutSR XenoFree) of xenogeneic components, male sex's multipotential stem cell of originating cultivates 12 days, PLZF positive class stem spermatogonium ratio is respectively 49.1%, 36.0%, 51.0%, sees Fig. 4.Result shows, 1% is adopted to replace the BSA in regular growth cultivation without the serum substitute (KnockOut SR XenoFree) of xenogeneic components, PLZF positive class stem spermatogonium ratio is not affected, and its batch of stable, non-animal derived component, the correlation technique system formed, can be applicable in clinical treatment.
In prior art, feeder layer Dual culture mode has been widely used in multipotential stem cell to germline technical field.The present invention studies discovery, in culture system of the present invention, remove feeder layer cells, cultivates 12 days, and obtaining PLZF positive class stem spermatogonium ratio significantly increases, and is increased to 55.4%, sees Fig. 5 by 7.93%.
Feature of the present invention and beneficial effect comprise:
The lipid enriched material (ChemicallyDefined Lipid Concentrate), vitamins C etc. determined containing chemical composition in serum-free of the present invention, non-animal derived property albumen nutrient solution are easily shown in photoxidation decomposition, unstable component, answer lucifuge, 4 DEG C of preservations.
In cultural method of the present invention, when the male sex originate multipotential stem cell enter described serum-free, non-animal derived property albumen culture system time, cell concentration should reach culture dish 90% and converge.Instant invention overcomes the problem causing later stage class stem spermatogonium, haploid cell production rate to decline because cell density is too low.
In cultural method of the present invention, when entering serum-free, non-animal derived property albumen culture system, 1-8 days male sex multipotential stem cell of originating is the adaptive phase, necrocytosis amount is larger, before within every 48 hours, changing liquid, should use without the washing of calcium magnesium phosphate buffered saline buffer once, remove dead cell, avoid residual dead cell to have an impact to later stage class stem spermatogonium, haploid cell.
In cultural method of the present invention, between the incubation period of serum-free, non-animal derived property albumen nutrient solution, every 48 hours when changing nutrient solution.Manipulation in vitro overlong time causes the principal element that cellular layer entirety is curling, come off.
The present invention from affecting proliferation of pluripotent stem cells, apoptosis factor is started with, in conjunction with germline correlative study achievement, put into practice with method by well-designed, adopt the culture technique of serum-free, non-animal derived property albumen nutrient solution, the male sex is originated in multipotential stem cell vitro differentiation process and produces class stem spermatogonium, haploid cell.The present invention adopts serum-free, non-animal derived property albumen culture scheme, not containing animal derived protein and not containing other animal ingredients such as feeder layer cells in cultural method in nutrient solution, nutrient solution of the present invention, cultural method all realize " non-animal derived component " to adapt to clinical expansion, for Non-obstructive Azoospermia pathogenesis (NOA disease pathology) Mechanism Study and clinical treatment thereof provide solid strong technology platform, expand Mammals multipotential stem cell Differentiation Induction in vitro and produced sexual cell research field, show technical progress.
Accompanying drawing explanation
Fig. 1 represents that the male sex originates the impact of multipotential stem cell inoculum density on later stage class stem spermatogonium rate of formation.Wherein, Figure 1A illustrates that, after 30% cell confluency, Figure 1B illustrates after 90% cell confluency.
Fig. 2 represents that alpha-mercapto ethanol, vitamins C are originated on the male sex between serum-free, non-animal derived property albumen nutrient solution incubation period the impact of proliferation of pluripotent stem cells.Wherein, Fig. 2 A is experimental result photo, and Fig. 2 B is MTT analytical method experimental result.
Fig. 3 represents the impact on the positive class stem spermatogonium of PLZF during cultivation 12 days rate of formation of alpha-mercapto ethanol, vitamins C.Wherein, Fig. 3 A is flow cytometry analysis result, and Fig. 3 B is results of immunoblot analysis.
Fig. 4 indicates that serum substitute ratio without xenogeneic components is on the impact of the positive class stem spermatogonium of PLZF during cultivation 12 days rate of formation.Wherein, Fig. 4 A, 4B, 4C respectively shown in, add 0.2% bovine serum albumin (BSA), 0.2% without the serum substitute (KnockOut SR XenoFree), 1% of xenogeneic components without the serum substitute (KnockOut SR XenoFree) of xenogeneic components.
Fig. 5 represents under serum-free, non-animal derived property albumen nutrient solution culture condition, and feeder layer cells is on the impact of cultivation 12 days PLZF positive class stem spermatogonium positive rates.Wherein, Fig. 5 A represents that atomization is without feeder layer cells, and Fig. 5 B represents that atomization contains feeder layer cells.
Fig. 6 A cultivates 12 days under representing serum-free, non-animal derived property albumen nutrient solution culture condition, and flow cytometry analysis detects the PLZF positive class stem spermatogonium ratio in H1 ES cell differentiation system; Fig. 6 B represents the PLZF positive class stem spermatogonium ratio that the P2618 male sex originates in NOA disease induced multi-potent stem cells differentiated system.
Fig. 7 represents under serum-free, non-animal derived property albumen nutrient solution culture condition, H1 male sex's human embryo stem cell, P2618 male sex NOA disease induced multi-potent stem cells of originating of originating cultivates 12 days, the class stem spermatogonium of VASA, PLZF, GPR125, CD90 stem spermatogonium molecule marker positive in producing in system.
Fig. 8 A represents that control group H1 human embryo stem cell H19 imprinted gene upstream region methylates distribution.Fig. 8 B represents under serum-free, non-animal derived property albumen nutrient solution culture condition, H1 male sex's human embryo stem cell, P2618 male sex NOA disease induced multi-potent stem cells of originating of originating cultivates 12 days, the H19 imprinted gene upstream region of the PLZF positive cell produced, presents high methylation.
Fig. 9 A cultivates 12 days under representing serum-free, non-animal derived property albumen nutrient solution culture condition, and flow cytometry analysis detects haploid cell (1N) ratio in H1 ES cell differentiation system; Fig. 9 B represents haploid cell (1N) ratio that the P2618 male sex originates in NOA disease induced multi-potent stem cells differentiated system.
Figure 10 is under serum-free, non-animal derived property albumen nutrient solution culture condition, H1 male sex's human embryo stem cell, P2618 male sex NOA disease induced multi-potent stem cells of originating of originating cultivates 12 days, adopt haploid cell (1N) in selected by flow cytometry apoptosis, enrichment system, and utilize cellular immunofluorescence to detect Acrosin, Prm1 monoploid molecule marker.
Figure 11 is under serum-free, non-animal derived property albumen nutrient solution culture condition, H1 male sex's human embryo stem cell, P2618 male sex NOA disease induced multi-potent stem cells of originating of originating cultivates 12 days, adopt haploid cell (1N) in selected by flow cytometry apoptosis, enrichment system, and H19 imprinted gene upstream region is high methylation to utilize genome bisulfite sequence measurement to detect.
Embodiment
In conjunction with following specific embodiments and the drawings, the present invention is described in further detail, and protection content of the present invention is not limited to following examples.Under the spirit and scope not deviating from inventive concept, the change that those skilled in the art can expect and advantage are all included in the present invention, and are protection domain with appending claims.Implement process of the present invention, condition, reagent, experimental technique etc., except the following content mentioned specially, be universal knowledege and the common practise of this area, the present invention is not particularly limited content.
Embodiment 1 ~ 4:H1 male sex derived embryo stem cell, P2618 male sex NOA disease induced multi-potent stem cells differentiation of originating produces class stem spermatogonium, haploid cell
Inoculation culture method steps:
Human embryo stem cell, P2618 male sex NOA disease induced multi-potent stem cells of originating of the H1 male sex being originated is inoculated in culture dish, when Growth of Cells reaches culture dish 90% converging state, with without calcium magnesium phosphate buffer wash cell once, be replaced by serum-free, non-animal derived property albumen nutrient solution, be placed in 37 DEG C, 5%CO
2cultivate under saturated humidity condition, within every 48 hours, change equal-volume fresh medium, cultivate 12 days, adopt flow cytometry analysis PLZF positive class stem spermatogonium ratio, and cellular immunofluorescence detects VASA, PLZF, GPR125, CD90 stem spermatogonium molecule marker; Enrichment PLZF positive cell, extracts genome, and sulphite is modified, nested PCR amplification H19 imprinted gene upstream region, connects carrier T order-checking, and software analysis statistics amplification section methylates modification situation.Comprehensive above-mentioned molecular biology, cytobiology detected result, pass judgment in culturing process whether occur class stem spermatogonium.Simultaneously, adopt haploid cell ratio in flow cytometry analysis system, flow cytometer enrichment haploid cell, Immunofluorescence test Acrosin, Prm1 haploid cell molecule marker, bisulfite sequencing detects H19 imprinted gene upstream region methylation status, comprehensive above-mentioned molecular biology, cytobiology detected result, pass judgment in culture system whether occur haploid cell.
The formula of serum-free used in each embodiment, non-animal derived property albumen nutrient solution, in table 1.In other embodiments, the recombinant human nerve colloid derived neurotrophic factor of arbitrary numerical value within the scope of 10ng/mL, 40ng/mL, 10 ~ 40ng/mL can be used in nutrient solution; Can with the lipid enriched material of arbitrary numerical value in 0.1%, 0.2%, 0.1 ~ 0.2% scope; The vitamins C of arbitrary numerical value within the scope of 50ug/mL, 200ug/mL, 50-200ug/mL can be used.
Experimental result: the H1 male sex originates human embryo stem cell, the P2618 male sex originates NOA disease induced multi-potent stem cells at serum-free, under non-animal derived property albumen nutrient solution (as shown in table 1) condition, cultivate 12 days, the product that cultivation obtains is through cytobiology, molecular Biological Detection, in system, visible VASA is positive, PLZF is positive, GPR125 is positive, CD90 positive cell, and H19 imprinted gene upstream region presents the class stem spermatogonium of high methylation, visible Acrosin, Prm1 is positive, H19 imprinted gene upstream region presents the haploid cell of high methylation.
Table 1
Flow cytometry analysis, in H1, P2618 clone culture system, PLZF positive class stem spermatogonium ratio is respectively 77.7% and 84.5%, sees Fig. 6; The all visible VASA of cellular immunofluorescence analyzing and testing two strain clone is positive, PLZF is positive, GPR125 is positive, CD90 positive cell, sees Fig. 7; Pcr amplification product sequencing result shows, and PLZF positive cell H19 imprinted gene upstream region presents high methylation, sees Fig. 8.Comprehensive above-mentioned molecular biology, cytobiology detected result, the cell breaking up generation in culture system meets class stem spermatogonium biological characteristics.
Further flow cytometry analysis, in H1, P2618 clone culture system, haploid cell (1N) ratio difference 4.19% and 3.61%, is shown in Fig. 9; Flow cytometer enrichment haploid cell, cellular immunofluorescence detects, and the haploid cell that all visible Acrosin of two strain clones is positive, Prm1 is positive, is shown in Figure 10; Genome bisulfite sequence measurement detects, and the haploid cell of all visible H19 imprinted gene upstream region high methylation of two strain clones, is shown in Figure 11.Comprehensive above-mentioned molecular biology, cytobiology detected result, the cell breaking up generation in culture system meets haploid cell biological characteristics.
As shown in Figure 1, the male sex originate multipotential stem cell inoculum density from 30% converge be promoted to 90% converge time, 12 days are cultivated with serum-free, non-animal derived property albumen nutrient solution, PLZF positive class stem spermatogonium ratio is increased to 63.9% by 34.6%, the impact showing to originate the male sex multipotential stem cell inoculum density produces later stage class stem spermatogonium rate of formation.Wherein, Figure 1A starts with the PLZF positive class stem spermatogonium ratio that nutrient solution of the present invention is cultivated 12 days after representing 30% cell confluency be the PLZF positive class stem spermatogonium ratio started after 34.6%, Figure 1B represents 90% cell confluency with nutrient solution of the present invention is cultivated 12 days is 63.9%.
As shown in Figure 2, between the incubation period of serum-free, non-animal derived property albumen nutrient solution, alpha-mercapto ethanol, vitamins C have an impact to male sex's proliferation of pluripotent stem cells of originating.Wherein, as shown in Figure 2 A, violet staining when upper strata is cell cultures 12 days, cell quantity is large, density is high, in clone's shape time violet staining be dark color, in contrast, then painted more shallow; Lower floor is morphological observation when cultivating 12 days under equal conditions.Result shown in Fig. 2 A shows, time in culture system only containing vitamins C (without alpha-mercapto ethanol), cultivates that 12 days cell quantities are maximum, density is the highest, is to clone shape.MTT analytical method experimental result as shown in Figure 2 B, detect alpha-mercapto ethanol, the impact of vitamins C interpolation on cell viability, then cell quantity is higher for OD490 optical density value height, otherwise cell quantity is less.Wherein, Vc represents vitamins C; 2ME represents alpha-mercapto ethanol.Result shows, cultivates 12 days, and time in culture system only containing vitamins C (without alpha-mercapto ethanol), cell quantity reaches maximum.Only containing vitamins C (without alpha-mercapto ethanol) in culture system, male sex's multipotential stem cell state of originating obviously is improved, and obviously, in nutrient solution of the present invention, vitamin C component on cell proliferation has remarkable facilitation effect in cell proliferation.
As shown in Figure 3, alpha-mercapto ethanol, vitamins C are on the impact of the positive class stem spermatogonium of PLZF during cultivation 12 days rate of formation.Wherein, Fig. 3 A is flow cytometry analysis result, removes alpha-mercapto ethanol and adds vitamins C, and when cultivating 12 days, PLZF positive class stem spermatogonium rate of formation is increased to 77.7% by 52.8%.Fig. 3 B is results of immunoblot analysis, shows to add vitamins C, and when cultivating 12 days, PLZF expressing quantity improves 1.5 times.In figure, Vc represents vitamins C; 2ME represents alpha-mercapto ethanol.
As shown in Figure 4, under the culture condition using serum-free, non-animal derived property albumen nutrient solution II, without the serum substitute ratio of xenogeneic components on the impact of PLZF positive class stem spermatogonium rate of formation during cultivation 12 days.As shown in Fig. 4 A, 4B, 4C, add 0.2% bovine serum albumin (BSA), 0.2% respectively without the serum substitute (KnockOut SR XenoFree), 1% of xenogeneic components without the serum substitute (KnockOut SR XenoFree) of xenogeneic components, male sex's multipotential stem cell of originating cultivates 12 days, and PLZF positive class stem spermatogonium ratio is respectively 49.1%, 36.0%, 51.0%.Adopt 1% without the serum substitute (KnockOut SR XenoFree) of xenogeneic components replace regular growth cultivate in BSA, under described culture system, PLZF positive class stem spermatogonium ratio is not affected.
As shown in Figure 5, under the culture condition using serum-free, non-animal derived property albumen nutrient solution II, feeder layer cells is on the impact of cultivation 12 days PLZF positive class stem spermatogonium positive rates.Wherein, Fig. 5 A indicates without feeder layer cells, PLZF positive class stem spermatogonium ratio that to cultivate with nutrient solution 12 days is that 55.4%, Fig. 5 B represents that containing feeder layer cells, the PLZF positive class stem spermatogonium ratio cultivated 12 days with nutrient solution be 7.93%.Result shows, and the present invention cultivates under without feeder layer cells condition, and PLZF positive class stem spermatogonium ratio is increased to 55.4% by 7.93%.
As shown in Figure 6, under the culture condition using serum-free, non-animal derived property albumen nutrient solution II, cultivate the ratio of the positive class stem spermatogonium of PLZF of 12 days.As shown in Figure 6A, in H1 ES cell differentiation system, PLZF positive class stem spermatogonium ratio 77.7%.As shown in Figure 6B, the P2618 male sex originates in NOA disease induced multi-potent stem cells differentiated system, PLZF positive class stem spermatogonium ratio 84.5%.
As shown in Figure 7, under the culture condition using serum-free, non-animal derived property albumen nutrient solution I or nutrient solution II, H1 male sex's human embryo stem cell, P2618 male sex NOA disease induced multi-potent stem cells of originating of originating cultivates 12 days, the class stem spermatogonium of VASA, PLZF, GPR125, CD90 stem spermatogonium molecule marker positive in producing in system.
As shown in Figure 8 A, control group H1 human embryo stem cell H19 imprinted gene upstream region methylates distribution.As shown in Figure 8 B, under the culture condition using serum-free, non-animal derived property albumen nutrient solution I or nutrient solution II, H1 male sex's human embryo stem cell, P2618 male sex NOA disease induced multi-potent stem cells of originating of originating cultivates 12 days, the H19 imprinted gene upstream region of the PLZF positive cell produced, present high methylation, meet stem spermatogonium biological characteristics.Wherein, solid rim represents generation DNA methylation, and open circle represents DNA methylation does not occur.
As shown in Figure 9, under the culture condition using serum-free, non-animal derived property albumen nutrient solution II, cultivate 12 days, flow cytometry analysis detects in H1 ES cell differentiation system, haploid cell (1N) ratio 4.19%, as shown in Figure 9 A; The P2618 male sex originates in NOA disease induced multi-potent stem cells differentiated system, haploid cell (1N) ratio 3.61%, as shown in Figure 9 B; 1N represents haploid cell.
As shown in Figure 10, under use serum-free, non-animal derived property albumen nutrient solution culture condition I or nutrient solution II, H1 male sex's human embryo stem cell, P2618 male sex NOA disease induced multi-potent stem cells of originating of originating cultivates 12 days, adopt haploid cell (1N) in selected by flow cytometry apoptosis, enrichment system, and utilize cellular immunofluorescence to detect Acrosin, Prm1 monoploid molecule marker.
As shown in figure 11, under use serum-free, non-animal derived property albumen nutrient solution culture condition I or nutrient solution II, H1 male sex's human embryo stem cell, P2618 male sex NOA disease induced multi-potent stem cells of originating of originating cultivates 12 days, adopt haploid cell (1N) in selected by flow cytometry apoptosis, enrichment system, and utilize genome bisulfite sequencing, detect H19 imprinted gene upstream region and present high methylation, meet haploid cell (1N) biological characteristics.Wherein, solid rim represents generation DNA methylation, and open circle represents DNA methylation does not occur.
Experimental result shows, uses the nutrient solution I of serum-free of the present invention, non-animal derived property albumen or serum-free, non-animal derived property albumen nutrient solution II all can realize obtaining class stem spermatogonium, haploid cell from male sex's multipotential stem cell of originating to germline.According to cytobiology detected result, nutrient solution II has more excellent cytodifferentiation efficiency.
Claims (9)
1. a human pluripotent stem cells is to the non-animal derived component cultural method of germline, it is characterized in that, under the condition of serum-free, non-animal derived property albumen nutrient solution, male sex's multipotential stem cell of originating is made to produce class stem spermatogonium, haploid cell in atomization in vitro; Wherein, described serum-free, non-animal derived property albumen nutrient solution comprise serum-free, non-animal derived property albumen nutrient solution I, serum-free, non-animal derived property albumen nutrient solution II:
Described serum-free, non-animal derived property albumen nutrient solution I comprise:
MEM substratum;
The dual anti-solution 1% of penicillin/streptomycin;
L-glutaminate 2mM;
4-hydroxyethyl piperazine ethanesulfonic acid 10mM;
Insulin-Transferrin-selenium-X additive 1%;
Without the serum substitute 0.2-3% of xenogeneic components;
Prostatropin 1ng/mL;
Recombinant human nerve colloid derived neurotrophic factor 10-40ng/mL;
Alpha-mercapto ethanol 0.1mM;
Lipid enriched material 0.1-0.2%;
The nutrient solution II of described serum-free, non-animal derived property albumen comprises:
MEM substratum;
The dual anti-solution 1% of penicillin/streptomycin;
L-glutaminate 2mM;
4-hydroxyethyl piperazine ethanesulfonic acid 10mM;
Insulin-Transferrin-selenium-X additive 1%;
Without the serum substitute 0.2-3% of xenogeneic components;
Prostatropin 1ng/mL;
Recombinant human nerve colloid derived neurotrophic factor 10-40ng/mL;
Vitamins C 50-200ug/mL;
Lipid enriched material 0.1-0.2%.
2. human pluripotent stem cells as a kind of in claim is to the non-animal derived component cultural method of germline, it is characterized in that, use serum-free, non-animal derived property albumen nutrient solution I or serum-free, non-animal derived property albumen nutrient solution II in atomization in vitro at male sex's multipotential stem cell of originating.
3. cultural method as claimed in claim 1, is characterized in that, when the male sex originate multipotential stem cell growth reach culture dish growth area 90% converging state time, use the nutrient solution of described serum-free, non-animal derived property albumen.
4. cultural method as claimed in claim 1, it is characterized in that, described cultural method is at 37 DEG C, 5%CO
2carry out under saturated humidity condition.
5. cultural method as claimed in claim 1, is characterized in that, in described cultural method, male sex's multipotential stem cell of originating is cultivated and can be obtained class stem spermatogonium, haploid cell in 8 ~ 25 days.
6. cultural method as claimed in claim 1, is characterized in that, described class stem spermatogonium is that VASA is positive, PLZF is positive, GPR125 is positive, CD90 is positive, and the stem spermatogonium of H19 imprinted gene upstream region high methylation class; Described haploid cell is that Acrosin is positive, Prm1 is positive, and the haploid cell of H19 imprinted gene upstream region high methylation.
7. cultural method as claimed in claim 1, is characterized in that, described method in the male sex originates pluripotent stem cell differentiation process without feeder layer cells.
8. serum-free, a non-animal derived property albumen nutrient solution, is characterized in that, be serum-free, non-animal derived property albumen nutrient solution I, it comprises:
MEM substratum;
The dual anti-solution 1% of penicillin/streptomycin;
L-glutaminate 2mM;
4-hydroxyethyl piperazine ethanesulfonic acid 10mM;
Insulin-Transferrin-selenium-X additive 1%;
Without the serum substitute 0.2-3% of xenogeneic components;
Prostatropin 1ng/mL;
Recombinant human nerve colloid derived neurotrophic factor 10-40ng/mL;
Alpha-mercapto ethanol 0.1mM;
Lipid enriched material 0.1-0.2%.
9. serum-free, a non-animal derived property albumen nutrient solution, is characterized in that, be serum-free, non-animal derived property albumen nutrient solution II, it comprises:
MEM substratum;
The dual anti-solution 1% of penicillin/streptomycin;
L-glutaminate 2mM;
4-hydroxyethyl piperazine ethanesulfonic acid 10mM;
Insulin-Transferrin-selenium-X additive 1%;
Without the serum substitute 0.2-3% of xenogeneic components;
Prostatropin 1ng/mL;
Recombinant human nerve colloid derived neurotrophic factor 10-40ng/mL;
Vitamins C 50-200ug/mL;
Lipid enriched material 0.1-0.2%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510094574.4A CN104726400B (en) | 2015-03-03 | 2015-03-03 | Non-animal derived component cultural method of the human pluripotent stem cells to germline |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510094574.4A CN104726400B (en) | 2015-03-03 | 2015-03-03 | Non-animal derived component cultural method of the human pluripotent stem cells to germline |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104726400A true CN104726400A (en) | 2015-06-24 |
| CN104726400B CN104726400B (en) | 2018-02-23 |
Family
ID=53450818
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510094574.4A Active CN104726400B (en) | 2015-03-03 | 2015-03-03 | Non-animal derived component cultural method of the human pluripotent stem cells to germline |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104726400B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112446A (en) * | 2015-06-25 | 2015-12-02 | 中国医学科学院基础医学研究所 | Method for high-efficiency establishment of genetically modified animal model through haploid stem cells |
| CN107034178A (en) * | 2017-03-07 | 2017-08-11 | 上海交通大学医学院附属仁济医院 | The pluripotent stem cell differentiation of inducing in vitro human is the method for stem spermatogonium |
| CN109055306A (en) * | 2018-06-25 | 2018-12-21 | 佛山科学技术学院 | A kind of system and method for no feeder layer free serum culture spermatogonial stem cells into mouse |
-
2015
- 2015-03-03 CN CN201510094574.4A patent/CN104726400B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| CHARLES A. EASLEY等: "Direct Differentiation of Human Pluripotent Stem Cells into Haploid Spermatogenic Cells", 《CELL REPORTS》 * |
| 冯年花等: "人诱导性多能干细胞向神经干细胞分化的方法探讨", 《中国病理生理杂志》 * |
| 杜军慧等: "诱导多能干细胞体外定向分化为雄性生殖细胞研究进展", 《中国农业科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112446A (en) * | 2015-06-25 | 2015-12-02 | 中国医学科学院基础医学研究所 | Method for high-efficiency establishment of genetically modified animal model through haploid stem cells |
| CN107034178A (en) * | 2017-03-07 | 2017-08-11 | 上海交通大学医学院附属仁济医院 | The pluripotent stem cell differentiation of inducing in vitro human is the method for stem spermatogonium |
| CN109055306A (en) * | 2018-06-25 | 2018-12-21 | 佛山科学技术学院 | A kind of system and method for no feeder layer free serum culture spermatogonial stem cells into mouse |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104726400B (en) | 2018-02-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103243070B (en) | Stem cell medium and application thereof | |
| CN101864393B (en) | Serum-free culture medium without animal origin components for culturing Vero cell micro-carrier | |
| CN106754668B (en) | Stem cell culture solution and injection | |
| CN102634482B (en) | Serum-free complete medium for mesenchymal stem cell | |
| US11339372B2 (en) | Serum-free medium inducing differentiation of umbilical cord mesenchymal stem cell into insulin-secretion-like cell and preparation method and use thereof | |
| CN103881971B (en) | Culture medium for culturing and/or amplifying mesenchymal stem cells and culture method thereof | |
| CN102344906B (en) | Hair follicle stem cell separation culture method | |
| CN104164405A (en) | Serum-free culture system for efficiently culturing human umbilical cord mesenchymal stem cells in vitro | |
| CN104805054A (en) | Serum-free medium of stem cell | |
| CN104762260A (en) | Preparation method and application of adipose-derived mesenchymal stem cells and preparation thereof | |
| CN105670986A (en) | Culture medium for inducing human umbilical cord mesenchymal stem cells to differentiate into islet-like cells and induction method therefor | |
| CN105112362A (en) | Serum-free medium for placenta-derived mesenchymal stem cells and preparation method thereof | |
| CN104232574A (en) | Method for in-vitro directional differentiation inducing of mesenchymal stem cell towards melanocyte | |
| CN104694470A (en) | Serum-free medium for stem cells | |
| CN105112363A (en) | Serum-free medium for human adipose-derived stem cells and preparation method thereof | |
| CN109943526B (en) | A kind of serum-free peptide composition promoting mescenchymal stem cell proliferation | |
| CN106929470A (en) | It is a kind of for derived mesenchymal stem cells in vitro culture and amplification serum free medium | |
| CN103087977A (en) | Culture solution for in vitro efficient amplification of animal cells and application of culture solution | |
| CN101831403B (en) | Method for amplifying mesenchymal stem cells of human umbilical cord and placenta in vitro | |
| CN109988746A (en) | A kind of mescenchymal stem cell adipogenic induction differentiation method | |
| CN104726400A (en) | Animal-source-free component culture method for differentiation from human pluripotent stem cells to germ cells | |
| CN102146359A (en) | Method for extracting original mesenchymal stem cells from placenta and serum-free amplification | |
| CN103421740B (en) | In-vitro culture and proliferation method for human mesenchymal stem cells | |
| CN100540659C (en) | A kind of method and special culture media thereof of cultivating mouse embryo stem cell | |
| CN104164404A (en) | Application of serum-free culture system for efficiently culturing human umbilical cord mesenchymal stem cells in vitro |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |