CN117721072A - Method for obtaining human spermatogonial stem cells in vitro and human spermatogonial stem cell culture capable of being stably cultured in vitro for long term - Google Patents
Method for obtaining human spermatogonial stem cells in vitro and human spermatogonial stem cell culture capable of being stably cultured in vitro for long term Download PDFInfo
- Publication number
- CN117721072A CN117721072A CN202311191974.8A CN202311191974A CN117721072A CN 117721072 A CN117721072 A CN 117721072A CN 202311191974 A CN202311191974 A CN 202311191974A CN 117721072 A CN117721072 A CN 117721072A
- Authority
- CN
- China
- Prior art keywords
- cells
- cell
- spermatogonial stem
- human
- ssc
- 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.)
- Pending
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 134
- 241000282414 Homo sapiens Species 0.000 title claims abstract description 126
- 238000000034 method Methods 0.000 title claims abstract description 51
- 238000000338 in vitro Methods 0.000 title claims abstract description 35
- 238000004113 cell culture Methods 0.000 title claims abstract description 17
- 230000007774 longterm Effects 0.000 title claims description 15
- 210000004027 cell Anatomy 0.000 claims abstract description 228
- 239000001963 growth medium Substances 0.000 claims abstract description 10
- 230000022131 cell cycle Effects 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 239000002609 medium Substances 0.000 claims description 27
- 210000001550 testis Anatomy 0.000 claims description 27
- 230000000920 spermatogeneic effect Effects 0.000 claims description 21
- 210000001519 tissue Anatomy 0.000 claims description 19
- 238000012163 sequencing technique Methods 0.000 claims description 16
- 108010085895 Laminin Proteins 0.000 claims description 9
- 230000029087 digestion Effects 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 7
- 108010082117 matrigel Proteins 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- 102100027766 Atlastin-1 Human genes 0.000 claims description 6
- 101000825172 Dictyostelium discoideum Spore germination protein 3 Proteins 0.000 claims description 6
- 101000936983 Homo sapiens Atlastin-1 Proteins 0.000 claims description 6
- 102000004142 Trypsin Human genes 0.000 claims description 6
- 108090000631 Trypsin Proteins 0.000 claims description 6
- 239000011324 bead Substances 0.000 claims description 6
- 239000006285 cell suspension Substances 0.000 claims description 6
- 239000012091 fetal bovine serum Substances 0.000 claims description 6
- 239000011159 matrix material Substances 0.000 claims description 6
- 239000012588 trypsin Substances 0.000 claims description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 238000010186 staining Methods 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 230000000284 resting effect Effects 0.000 claims description 4
- 102000029816 Collagenase Human genes 0.000 claims description 3
- 108060005980 Collagenase Proteins 0.000 claims description 3
- 101000994365 Homo sapiens Integrin alpha-6 Proteins 0.000 claims description 3
- 102100032816 Integrin alpha-6 Human genes 0.000 claims description 3
- 210000002469 basement membrane Anatomy 0.000 claims description 3
- 229960002424 collagenase Drugs 0.000 claims description 3
- 238000001976 enzyme digestion Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
- 238000010257 thawing Methods 0.000 claims description 3
- 210000004881 tumor cell Anatomy 0.000 claims description 3
- 101100203780 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) SPG5 gene Proteins 0.000 claims description 2
- 239000002771 cell marker Substances 0.000 claims description 2
- 230000006862 enzymatic digestion Effects 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 210000004379 membrane Anatomy 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 230000004083 survival effect Effects 0.000 claims description 2
- 230000001413 cellular effect Effects 0.000 claims 2
- 230000004663 cell proliferation Effects 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 description 9
- 230000004069 differentiation Effects 0.000 description 9
- 230000035755 proliferation Effects 0.000 description 8
- 238000010586 diagram Methods 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000002073 fluorescence micrograph Methods 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 3
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 3
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 206010003883 azoospermia Diseases 0.000 description 3
- 238000012650 click reaction Methods 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 210000002863 seminiferous tubule Anatomy 0.000 description 3
- 230000021595 spermatogenesis Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 2
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 230000006820 DNA synthesis Effects 0.000 description 2
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 2
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 description 2
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 201000010208 Seminoma Diseases 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000010362 genome editing Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 238000003365 immunocytochemistry Methods 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 208000000509 infertility Diseases 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 231100000535 infertility Toxicity 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000003147 molecular marker Substances 0.000 description 2
- 238000011580 nude mouse model Methods 0.000 description 2
- 230000000414 obstructive effect Effects 0.000 description 2
- 208000008634 oligospermia Diseases 0.000 description 2
- 230000036616 oligospermia Effects 0.000 description 2
- 231100000528 oligospermia Toxicity 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- -1 small molecule Azide Chemical class 0.000 description 2
- 238000011476 stem cell transplantation Methods 0.000 description 2
- 229950003937 tolonium Drugs 0.000 description 2
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- 238000012605 2D cell culture Methods 0.000 description 1
- 102100024438 Adhesion G protein-coupled receptor A3 Human genes 0.000 description 1
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- COVZYZSDYWQREU-UHFFFAOYSA-N Busulfan Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 101000833357 Homo sapiens Adhesion G protein-coupled receptor A3 Proteins 0.000 description 1
- 101100281008 Homo sapiens FGF2 gene Proteins 0.000 description 1
- 101000830411 Homo sapiens Probable ATP-dependent RNA helicase DDX4 Proteins 0.000 description 1
- 101710128836 Large T antigen Proteins 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- 208000007466 Male Infertility Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102100024770 Probable ATP-dependent RNA helicase DDX4 Human genes 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 210000001766 X chromosome Anatomy 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229960002092 busulfan Drugs 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 229940068840 d-biotin Drugs 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 229960005309 estradiol Drugs 0.000 description 1
- 125000002534 ethynyl group Chemical group [H]C#C* 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 210000001368 germline stem cell Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000009027 insemination Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 229960000448 lactic acid Drugs 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000003794 male germ cell Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 238000001531 micro-dissection Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000513 principal component analysis Methods 0.000 description 1
- 230000002206 pro-fibrotic effect Effects 0.000 description 1
- 229960003387 progesterone Drugs 0.000 description 1
- 239000000186 progesterone Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 230000007651 self-proliferation Effects 0.000 description 1
- 238000012174 single-cell RNA sequencing Methods 0.000 description 1
- 229960001471 sodium selenite Drugs 0.000 description 1
- 239000011781 sodium selenite Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 125000001425 triazolyl group Chemical group 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to a method for obtaining human spermatogonial stem cells in vitro, a cell population of the isolated human spermatogonial stem cells obtained by the method, and a human spermatogonial stem cell culture which can be stably cultured in vitro for a long time. The invention provides a method and a culture medium capable of stably culturing human spermatogonial stem cells in vitro for a long time, wherein the culture medium can keep cell proliferation and cell stem property. The invention also establishes a cell line ApLCs (human spermatogonial stem cell line Apale-like cells) of the personalized human spermatogonial stem cells from a patient. The invention comprehensively identifies the obtained human spermatogonial stem cell culture at the cell morphology, phenotype, molecular level and functional level, and proves that the human spermatogonial stem cell culture is the human spermatogonial stem cell with active cell cycle.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for obtaining human spermatogonial stem cells in vitro, a cell population of the isolated human spermatogonial stem cells obtained by the method, and a human spermatogonial stem cell culture which can be stably cultured in vitro for a long time.
Background
National census data published by the national statistical office show that the population growth of China slows down, showing the aggravation of the aging problem. In addition to the reduction of subjective fertility will of young people caused by various factors, the trend of infertility to younger age is also one of the important reasons behind the aggravation of population aging. In recent 10 years, the number of people with infertility in China is rapidly increased, and the occurrence rate of male infertility is gradually increased, wherein patients with insemination, oligospermia and oligospermia caused by unknown reasons are increased year by year. Therefore, research on the spermatogenesis mechanism and its influencing factors has become a research hotspot.
Spermatogenesis is at the earliest initiated by self-proliferation and differentiation of spermatogonial stem cells (spermatogonial stem cells, SSCs), the only adult stem cells that a male or human transmits genetic material to offspring, the basis for the male or human spermatogenic process, which involves the complex process of self-renewal and differentiation of spermatogonial stem cells into mature sperm. Because of the complex influence of the structure and function of the whole body and testes, it is difficult to study SSCs in vivo, so that the isolated cultured human SSCs establish a stable SSCs line in vitro, and a cell model is provided for researching the proliferation and differentiation of SSCs.
The following list of several prior art literature techniques for in vitro obtaining spermatogenic stem cells is presented to reveal recent research and development concerning proliferation and differentiation of SSCs.
In 2009, hooman Sadri-Ardekani et al (Sadri-Ardekani H, mizrak SC, van Daalen SK, korver CM, roepers-Gajadien HL, koruji M, hovingh S, de Reijke TM, de la Rosette JJ, van der Veen F, de Rooij DG, repping S, van Pelt AM. Propang of human spermatogonial stem cells in vitro. JAMA.2009Nov 18;302 (19): 2127-34.) human testis tissue was used to isolate single cells, enriched by differential attachment, and 20ng/mL EGF,10ng/mL GDNF,10ng/mL LIF,10ng/mL bFGF were added to the culture SSC, stemPro-34 medium in Human placental laminin coated dishes. Functional identification is carried out by using a human-mouse spermatogonial stem cell transplantation experiment, and immunocytochemistry and RT-PCR are used for detecting key factor expression, wherein the maximum culture time is 196 days.
In 2010, lim JJ et al (Lim JJ, sung SY, kim HJ, song SH, hong JY, yoon TK, kim JK, kim KS, lee DR.Long-term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non-obstructive azoospermia under exogenous feeder-free culture conditions.cell Prolif.2010Aug;43 (4): 405-17.doi:10.1111/j.1365-2184.2010.00691.X.PMID:20590666; PMCID: PMC 6495878.) enriched SSC from human testis single cells using CD9 magnetic beads, differential adherence followed by culture in matrigel coated dishes, stemPro-34 medium supplemented with 10ng/mLGDNF,10ng/mL bFGF,20ng/mL EGF,10,000U/mL LIF, key apoptosis detected using immunocytochemistry, RT-PCR, and culture time was greater than 6 months.
In 2011, kokkinaki M et al (Kokkinaki M, djourabtche A, golestaneh N.Long-term Culture of Human SSEA-4Positive Spermatogonial Stem Cells (SSCs). J Stem Cell Res Ther.20112Nov11; 2 (2): 2488.doi:10.4172/2157-7633.S2-003.PMID:24466499; PMCID: PMC 3898576) enriched SSEA4 magnetic beads from human testis single cells, cultured in a laminin-coated dish after differential adherence, stempro-34 medium supplemented with 10ng/mLGDNF,10ng/mL bFGF,20ng/mL EGF,10,000U/mL LIF. Cell morphology was observed using an optical microscope and cell clone counts were performed and RT-PCR was performed to detect key factor expression. The maximum incubation time was 5 months.
In 2015, hou J et al (Hou J, niu M, liu L, zhu Z, wang X, sun M, yuan Q, yang S, zeng W, liu Y, li Z, he Z. Estabishi strain and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor formation. Sci Rep.2015Nov 20; 5:16922.) used human testis tissue to isolate single cells, after differential wall enrichment, SV40 was transfected with lentivirus to immortalize cells, SSC was sorted with GPR125 beads, and then cultured in DMEM/F12 medium containing 10% FBS and 1% antibiotics.
However, according to the above-mentioned literature report, there are also the following unsolved technical problems:
(1) There is no currently internationally recognized effective in vitro medium for human spermatogonial stem cells, and the stem properties of spermatogonial stem cells are difficult to maintain for a long period of time, but only remain viable for a short period of time, under inappropriate medium/culture conditions.
(2) There is currently no internationally unedited cell line derived from human primary spermatogenic stem cells. Part of the literature discloses gene editing of human spermatogonial stem cells, such as integration of SV40 (simian virus 40) large T antigen fragments into the genome of the cells for immortalization purposes to achieve immortalization of the cells. The problem with this technique is that the genome of human spermatogonial stem cells is artificially altered to have the characteristics of tumor cells, and therefore cannot be returned to the patient, and clinical transformation is limited.
(3) Part of the literature for long-term culture of human spermatogonial stem cells is not comprehensive in identification of cultures and cannot be convinced. For example, molecular level identification uses only RT-PCR rather than comprehensive high throughput transcriptome sequencing, lack of morphological identification at the most accurate electron microscopy level, lack of functional identification of gold standard for human-murine spermatogonial stem cell transplantation, and the like.
According to the existing literature report, if a spermatogonial stem cell culture system is to be established, a sufficient amount of human testis tissue must be obtained, and enough human primary spermatogonial stem cells must be isolated to search for proper culture conditions, but the process takes a lot of time and economic cost, and the operation of normal testis is very rare, clinical samples are difficult to obtain, so that few people are specialized in this work. At present, no effective human spermatogonial stem cell in-vitro culture medium is known internationally, and no universal cell line which is not subjected to gene editing and is derived from human primary spermatogonial stem cells is known, so that the method becomes an obstacle for obtaining the spermatogonial stem cells in vitro and obtaining the spermatogonial stem cells which can be stably cultured in vitro for a long time.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a method for obtaining human spermatogonial stem cells in vitro, and a cell population of isolated human spermatogonial stem cells obtained by the method, a human spermatogonial stem cell culture which can be stably cultured in vitro for a long period of time.
In order to achieve the above object, the solution adopted by the present invention is as follows:
in a first aspect, the present invention provides a method for obtaining human spermatogonial stem cells in vitro, comprising the steps of:
isolating human Spermatogonial Stem Cells (SSC) from human testis tissue;
the isolated human spermatogonial stem cells were placed in SSC medium for cell subculture.
Preferably, the step of isolating human Spermatogonial Stem Cells (SSC) from human testis tissue comprises: cutting testis under aseptic environment, removing white film in Hank's liquid, mechanically cutting testis tissue, treating with two-step enzyme digestion method, counting dead cells under 5% by trypan blue staining, filtering with 100 μm and 40 μm cell filter screen sequentially to remove impurities, centrifuging at 4deg.C for 5min, and sorting with ITGA6 magnetic beads.
Preferably, the two-step enzymatic digestion process comprises the steps of: digestion was performed with 1mg/mL collagenase IV at 37℃for 15min, 0.25% trypsin/1. Mu.M EDTA, and after 15min, digestion was stopped with DMEM containing 10% fetal bovine serum.
Preferably, the step of subjecting the isolated human spermatogonial stem cells to cell subculture in SSC medium comprises: placing 5% suspension-grown single cells of human spermatogonial stem cells isolated from human testis tissue in SSC culture medium to obtain cell solution containing SSC, placing the cell solution in orifice plate, placing at 37deg.C and 5% O 2 7% CO 2 2D cell subculturing was performed in an incubator.
Preferably, the components of the SSC medium and the working concentrations of the components are as follows:
preferably, the cell concentration of the SSC-containing cell fluid is (0.1-10) x 10 6 SSC cells/2 ml SSC medium, preferably 1X 10 6 SSC cells/2 ml SSC medium.
Preferably, the well plate is coated with laminin in advance, and the SSC culture medium is preheated at room temperature in advance; after 2D cell subculture, the method further comprises the steps of: 1ml of SSC medium preheated at room temperature was supplemented on day 3 after passage and at 37℃with 5% O 2 7% CO 2 Continuously culturing in an incubator; on day 5 after passaging, cell passaging was performed again.
Preferably, the method further comprises the step of 3D cell clone formation, comprising: thawing corning basement membrane matrix, digesting with 0.25% trypsin/1 μm EDTA, centrifuging at 1000rpm, discarding supernatant, re-suspending cells with 1mL SSC medium, counting with a cell counter, and adjusting the concentration of cell suspension to (1-10) x 10 4 cells/mL, preferably 5 x 10 4 cells/mL; taking 10 mu L of cell suspension in an EP tube, centrifuging at 1000rpm, discarding supernatant, re-suspending 500 SSC cells with pre-cooled substrate membrane matrix of re-suspended cell Corning, dripping the obtained mixed solution of SSC cells and matrigel into an orifice plate, adding the SSC culture medium into the orifice plate, and then adding the orifice plate at 37deg.C and 5% O 2 7% CO 2 Culturing was continued in the incubator, and at D0, D4, D7, D10 and D14 days, photographing was performed with Olympus IX83 microscope, and the colony formation state was observed.
In a second aspect, the present invention also provides an isolated population of human spermatogonial stem cells obtained by a method of obtaining human spermatogonial stem cells in vitro as described above, said isolated population of human spermatogonial stem cells expressing at least one spermatogonial stem cell marker ApLCs (human spermatogonial stem cell line aple-like cells); the spermatogonial stem cell markers ApLCs are defined as follows: more than 90% of the isolated human spermatogonial stem cells are in cell morphology consistent with undifferentiated spermatogonial cells Apale with self-renewal capacity in human primary spermatogonial stem cells.
Preferably, the spermatogonial stem cell markers ApLCs (human spermatogonial stem cell line aple-like cells) are capable of long-term survival in vitro and are capable of differentiating into spermatogenic cells without forming tumor cells.
Preferably, the spermatogonial stem cell markers ApLCs (human spermatogonial stem cell line aple-like cells) are subjected to single cell sequencing, and unsupervised cluster analysis reveals that the cells are divided into 5 sub-populations C1, C2, C3, C4, C5 in different states of the cell cycle.
Preferably, the human primary spermatogenic stem cells are divided into 6 sub-populations of SPG1, SPG2, SPG3, SPG4, SPG5, SPG6 after single cell sequencing; wherein, the molecular characteristics of resting spermatogenic cells Adark in the human primary spermatogenic stem cells correspond to SPG2 and SPG3, and the molecular characteristics of spermatogenic cells Apale with self-renewal capacity in the human primary spermatogenic stem cells correspond to SPG3, SPG4 and SPG 5; the molecular characteristics of 5 subpopulations C1, C2, C3, C4, C5 obtained after single cell sequencing of the spermatogonial stem cell markers ApLCs (human spermatogonial stem cell line aple-like cells) were all annotated as SPG4.
In a third aspect, the present invention also provides a human spermatogonial stem cell culture obtainable by the method for obtaining human spermatogonial stem cells in vitro as described above, which culture is stable in vitro over a long period of time.
Preferably, the human spermatogonial stem cell culture capable of long term stable in vitro culture comprises human spermatogonial stem cell line aple-like cells (ApLCs), more than 90% of the human spermatogonial stem cells obtained by the method for obtaining human spermatogonial stem cells in vitro as described above have a cell morphology consistent with the undifferentiated spermatogonial cells aple with self-renewal capacity in human primary spermatogonial stem cells.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention provides a method and a culture medium capable of stably culturing human spermatogonial stem cells in vitro for a long time, wherein the culture medium can keep cell proliferation and cell stem property.
(2) The invention establishes a cell line ApLCs (human spermatogonial stem cell line Apale-like cells) of personalized human spermatogonial stem cells from a patient.
(3) The invention comprehensively identifies the obtained human spermatogonial stem cell culture at the cell morphology, phenotype, molecular level and functional level, and proves that the human spermatogonial stem cell culture is the human spermatogonial stem cell with active cell cycle.
Drawings
FIG. 1 is a diagram of cells grown in 2D culture in semi-suspension from SSC cells isolated and cultured in accordance with an embodiment of the present invention.
FIG. 2 is a graph showing the dynamic cell proliferation of SSC cells isolated and cultured in 2D culture according to the example of the present invention.
FIG. 3 shows the formation of clones from isolated SSC cells cultured in the examples of the present invention when cultured in 3D, mCherry was transfected into SSC, flow cytometry sorted, cultured in matrigel, and visible red clones were observed under a fluorescence microscope.
FIG. 4 is a graph showing the proliferation of clones produced by SSC cells isolated and cultured in 3D culture according to the examples of the present invention.
FIG. 5 is a fluorescence microscope image of isolated cultured SSC cells labeled with EdU in accordance with an embodiment of the present invention.
FIG. 6 is a fluorescence microscope image of Eu-labeled nascent RNA isolated from cultured SSC cells in an embodiment of the present invention.
FIG. 7 is a fluorescence microscope image of the detection of SSC cells isolated and cultured by immunofluorescence labeled Ki67 in the examples of the present invention.
FIG. 8 is a graph showing SSC proliferation curves of isolated and cultured SSC cells using real-time imaging techniques for monitoring different culture algebra, where P is the number of cell algebra, in accordance with an embodiment of the present invention.
FIG. 9 is a graph showing cell multiplication times at different generations of SSC cells isolated and cultured according to the present invention, wherein P is the number of cell generations.
FIG. 10 shows cell cycle profiles of different passage numbers of isolated and cultured SSC cells according to an embodiment of the present invention, where P is the passage number.
FIG. 11 is a diagram showing the ratio of apoptosis of different generations of SSC cells isolated and cultured by flow cytometry according to an embodiment of the present invention, wherein P is the number of cells.
FIG. 12 is a fluorescence microscope image of the identification of cell morphology by toluidine blue staining of SSC cells isolated and cultured in the examples of the present invention.
FIG. 13 is a scanning electron microscope image of the isolated and cultured SSC cells used to identify cell morphology in an embodiment of the present invention.
FIG. 14 is a diagram showing the primary source analysis of cell line ApLCs from other human primary spermatogenic stem cell components isolated from cultured SSC cells in accordance with an embodiment of the present invention.
FIG. 15 is a graph showing unsupervised clustering and dryness scoring analysis of single cell sequencing of cell line ApLCs isolated from cultured SSC cells in the examples of the present invention.
FIG. 16 is a diagram of a gene annotation analysis of single cell sequencing of cell line ApLCs isolated from cultured SSC cells in the examples of the present invention.
FIG. 17 is a fluorescence microscope image of the expression of essential proteins of spermatogonial stem cells by immunofluorescence test cell line ApLCs isolated from cultured SSC cells according to an embodiment of the present invention.
FIG. 18 is a diagram showing that SSC cells isolated and cultured in the examples of the present invention demonstrate that the cell line ApLCs has high stem property by a xenograft experiment.
Detailed Description
Definition of terms:
culturing: "culture" as used in this example refers to the propagation of cells under controlled conditions such that cell division and an increase in the number of cells occur.
Differentiation: "differentiation" as used in this example refers to the modification of a cell to a particular form or function. In cells, differentiation results in more committed cells.
Long-term culture: "Long term culture" as used in this example means that the cells are propagated under controlled conditions longer than at least two months or more than 10 passages. Preferably, the long-term culture is cultured for more than 4 months, more than 6 months or more than 1 year. Preferably, the long-term culture is passaged for more than 15 passages, more than 18 passages or more than 20 passages. The duration of long-term cultures is highly dependent on the individual cells, and variability can exist between cell lines.
Stem cells: "Stem cells" as used in this example refers to cells that are capable of self-renewal (ability to undergo numerous cell division cycles while maintaining an undifferentiated state) and at least multipotent (ability to differentiate into more than one specialized cell type).
The technical scheme of the present invention will be further described with reference to specific examples, but the scope of the present invention is not limited to these examples. All changes and equivalents that do not depart from the gist of the invention are intended to be within the scope of the invention.
Examples:
< isolation, sorting and culture of human spermatogonial Stem cells >
The invention provides a method for obtaining human spermatogonial stem cells in vitro, which comprises the following steps:
step one, cell separation and sorting:
cutting testis under aseptic condition, placing in Hank's solution to strip white film, mechanically cutting testis tissue, and treating with two-step enzyme digestion method. Digestion with 1mg/mL collagenase IV (Gibco Co.) was carried out at 37℃for 15min with shaking every 5 min. Digestion was then performed with 0.25% trypsin/1. Mu.M EDTA (Sigma Co.) and the samples were blown with sterile pipettes every 5 min. Digestion was stopped after 15min with DMEM containing 10% fetal bovine serum. The number of dead cells counted by trypan blue staining was below 5%. The mixture was filtered through a 100 μm and 40 μm cell strainer (BD Co.) to remove impurities, and 300g of the cell isolate was centrifuged at 4℃for 5min, followed by separation with ITGA6 beads (Miltenyi Co.).
Step two, 2D cell culture:
1. laminin (Laminin, sigma) coated well plates: surface coverage 1. Mu.g/cm 2 Laminin was coated on the surface of the well plate at 37℃overnight, the liquid in the dish was removed, and about 1mL of 1 XPBS solution was added to the dish and gently washed 2 times. Then, about 1mL of DPBS was added to the dish, and the coated well plate was ready for use.
2. Cell passage: in cell 2D culture, 95% of cells were clonally grown in half-suspension and 5% of cells were single cells grown in suspension. Old cultures in transfer dishes were based on sterile 15ml centrifuge tubes, with 0.5ml of 0.25% trypsin per well of six well plates, and digestion was stopped with DMEM containing 10% fetal bovine serum after 2min at 37 ℃. Centrifugation at 1000rpm at normal temperature3min. And (5) discarding the waste liquid. SSC medium (see table 1) was pre-warmed at room temperature, resuspended, trypan blue stained counted and cell concentration adjusted to 1 x 106SSC cells/2 ml SSC medium. Discarding DPBS in laminin coated pore plate, taking 1×10 6 SSC cells were in laminin-coated six-well wells. Placed at 37 ℃,5% O 2 7% CO 2 Culturing in an incubator.
3. Cell replacement fluid: 1ml of SSC medium pre-warmed at room temperature was added on day 3 after passage. 37 ℃,5% O 2 7% CO 2 Culturing is continued in the incubator. On day 5 after passage, cell passages or other functional experiments can be performed.
TABLE 1SSC Medium composition
| The components | Branding | Working concentration |
| D-(+)-glucose | Sigma | 6mg/ml |
| Stempro-34supplement | ThermoFish | 20μl/ml |
| Insulin-Transferrin-Sodium selenite | Gibco | 10μl/ml |
| FBS | Gibco | 10μl/ml |
| Pyruvic acid | Gibco | 10μl/ml |
| Anti-anti | Gibco | 10μl/ml |
| Non-Essential Amino Acids | Gibco | 10μl/ml |
| GlutaMAX TM | Gibco | 10μl/ml |
| MEM Vitamin Solution | Gibco | 10μl/ml |
| 2-Mercaptoethanol | Gibco | 0.1mM |
| Ascorbic Acid | Sigma | 10 -4 M |
| DL-Lactic Acid | Sigma | 1μl/ml |
| d-Biotin | MERCK | 10μg/ml |
| Human FGF2 | PeproTech | 10ng/ml |
| β-Estradiol | Sigma | 30ng/ml |
| Progesterone | Sigma | 60ng/ml |
| EGF | PeproTech | 20ng/ml |
| ACTA | Invitrogen | 10ng/ml |
| GDNF | R&D SYSTEM | 15ng/ml |
| LIF | MCE | 10 3 U/ml |
| AKT i | MCE | 100nM |
| Plasmocin TM prophylactic | InvivoGen | 2.5μg/ml |
| Rock i | MCE | 10μg/ml |
| Putrecine | Sigma | 60μM |
Step three, 3D cell clone formation:
1. thawing the corning basement membrane matrix according to conventional methods: the day before the experiment, conning Matrigel @ The substrate (356321) was thawed overnight in a refrigerator at 4 ℃ and the vials were rotated to ensure material dispersion.
2. Pipettes, tips and 1.5mL EP tubes require pre-cooling.
3. The cells were resuspended in 1mL of SSC medium as shown in Table 1 by trypsinization, centrifugation at 1000rpm for 3 minutes, and the cell counter was used. Adjusting the cell suspension concentration to 5 x 10 4 cells/mL。
4. 10. Mu.L of the cell suspension was centrifuged at 1000rpm for 3 minutes in a 1.5mL EP tube and the supernatant was discarded and 50. Mu.L of pre-chilled re-suspended cell Conning Matrigel was used @ The matrix resuspended 500 SSC cells. The obtained SSC cell and matrigel mixture was dropped into each well (Perkin Elmer) in a 24-well plate.
5. The matrigel was allowed to set by placing the 24-well plate at 37℃for 10 minutes.
6. 1mL of SSC medium was gently added to each well. Placed at 37 ℃ and 5% CO 2 The culture was carried out in the incubator for 14 days, and half of the medium was replaced every 3 days.
7. At D0, D4, D7, D10 and D14 days, the clone formation status was observed by photographing with Olympus IX83 microscope.
< identification of biological Properties of human spermatogonial Stem cells >
1. In 2D culture, the cultured SSC cells grew in semi-suspension without adherence (fig. 1). Cell growth status was monitored dynamically in real time, and cell number doubling times were seen to be 36-48 hours (FIG. 2). Cells formed clearly visible clones when cultured for 6-7 days in 3D, with a diameter doubling time of 2.5 days on average (fig. 3, 4).
2. The cultured SSC cells were labeled with EdU (5-methyl-2 '-deoxyuretine) and fluorescent microscopy revealed that the cell's nascent DNA synthesis was vigorous, indicating that the cell had active DNA replication (FIG. 5).
EdU (5-methyl-2' -deoxyuridine) is a thymidine analog that can be incorporated into newly synthesized DNA in place of thymidine during DNA synthesis. The ethynyl group on EdU can form a stable triazole ring with fluorescent labeled small molecule Azide probes (such as FITC Azide, AF series dye and the like) under the catalysis of monovalent copper ions. This reaction is very rapid and is therefore known as Click reaction (Click reaction). By click reaction, the newly synthesized DNA is labeled with a corresponding fluorescent probe, and at this time, a fluorescent detection device can be used to detect the cell proliferation status.
3. The cultured SSC cells were labeled with EU (5-methyl-uretine) to give a fluorescent microscope, and the cells were found to have vigorous synthesis of nascent RNA, indicating that the cells had active proliferation and transcription (FIG. 6).
4. Immunofluorescent staining of the cultured SSC cells, labeled Ki67, showed that 50% or more of the cells expressed Ki67, indicating that the cells had active proliferation and division (fig. 7).
5. SSC cell growth rate and status remained stable with increasing passage times and prolonged culture time (FIG. 8), and cell number doubling time was still 36-48h (FIG. 9).
6. With increasing passage times and prolonged culture time, SSC cells did not show significant cycle arrest (fig. 10) and the number of apoptosis increased (fig. 11).
< morphological identification of human spermatogonial Stem cells >
Cultured SSC cells were collected by centrifugation and then pelleted, stained with toluidine blue (fig. 12), observed by scanning electron microscopy (fig. 13), and cell identity was determined based on the cell nucleus morphology, which showed that 90% or more of the cell morphology was consistent with aple having self-renewal capacity in human primary Spermatogenic Stem Cells (SSC), and this was designated aple like cells (ApLCs).
5 types of male germ cells (including two types of undifferentiated spermatogenic cells (aple and Adark), primary spermatocyte, secondary spermatocyte and sperm cell) can be obtained from testis tissue of a lower animal (e.g., a mouse or sheep), however, aple spermatogenic cells are relatively difficult to obtain from testis tissue of a human body, and SSC cells obtained by the human spermatogenic stem cell isolation culture method of the present invention show that more than 90% of cell morphology is consistent with aple spermatogenic cells.
< identification of expression Profile of human spermatogonial Stem cells >
1. Cell lines ApLCs of SSCs were isolated and cultured using testis tissue from different patients according to the method of the present invention, cells were collected by centrifugation and subjected to transcriptome sequencing, and main source analysis was performed together with transcriptome sequencing results of Adark cells, apale cells, spermatocytes, round sperm cells, and seminoma (seminoma) cell lines obtained by microdissection using human testis tissue, and it was revealed from the main source analysis diagram of the ApLCs and other human primary spermatogenic stem cell components of FIG. 14 that on a plane composed of coordinates PC1 and PC2 in three-dimensional coordinates, the transcriptome characteristics of ApLCs and Apale cells in human primary testis tissue were more similar to each other on coordinate PC1 (FIG. 14), indicating that ApLCs could realize a simulated human primary Apale cells on the expression profile.
2. Single cell sequencing of ApLCs showed that cells were divided into 5 sub-populations C1, C2, C3, C4, C5, with molecular characteristics closest to SPG4 reported in the literature, and dryness scores showed that more than 95% of cells had high dryness (fig. 15).
Single cell sequencing techniques (sham AN, zheng X, munyoki SK, ma Q, manske GL, green CD, sukhwani M, orwig KE, li JZ, hammoud ss.single-Cell RNA Sequencing of Human, macaque, and Mouse Testes Uncovers Conserved and Divergent Features of Mammalian spectugenin.dev cell.2020aug 24;54 (4): 529-547.e12.Doi:10.1016/j.devcel.2020.05.010.epub 2020Jun 5.PMID:32504559;PMCID:PMC7879256.) were applied in the literature, their pseudosequences were annotated as 6 groups, SPG1-6, respectively, in theory sequentially resting SSC, SSC in self-renewal, SSC in differentiation (reflecting the time-sequence change of SSC status during spermatogenesis). However, no document has been reported so far that it is possible to correlate conventional morphological Adark cells (resting SSCs) and Apale cells (SSCs in self-renewal) with the above transcriptional profile.
The inventors of the present invention successfully achieved that Adark cells and Apale cells were excised from human testis tissue and transcriptome sequenced, while the annotation method in the references (Aran D, looney AP, liu L, wu E, fong V, hsu A, chak S, naikawadi RP, wolters PJ, abate AR, butte AJ, bhattacharya M. Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macro-image. Nat immunol.2019Feb;20 (2): 163-172.Doi:10.1038/S41590-018-0276-y. Epub 2019Jan 14.PMID:30643263;PMCID:PMC6340744.) was compared to SPG1-6, and found that Adark cells corresponded predominantly to SPG2-3, apale cells corresponded to SPG 3-5.
The cell line ApLCs isolated and cultured in SSC according to the method of the present invention was subjected to BULK-RNA sequencing, and the transcription profile of the cell line Apale was found to be more similar to that of the Adark cells and Apale cells obtained as described above by combined analysis (PCA analysis) of the BULK-RNA data.
The single cell transcriptome sequencing of the cell line ApLCs from which SSCs were isolated and cultured according to the method of the present invention, was found to divide the cells into 5 sub-populations C1, C2, C3, C4, C5, each sub-population being in a different state of the cell cycle. Comparing the 5 subpopulations of cells with the data in the literature, the combination analysis using the method in the literature found that all 5 subpopulations of cells were annotated as SPG4.
Stem cells are not fully differentiated but still immature cells, but have a strong proliferative capacity, a highly differentiated capacity, and can be regenerated and proliferated into cells of various organs and tissues. Stem cells are generally considered to have two capacities: dryness (dryness) and functionality, which are mutually restricted, cancel each other out. "Stem" refers to the ability of stem cells to differentiate into a potential, while "functional" refers to the ability of stem cells to differentiate into mature cells toward a particular method. "Stem" of a stem cell refers to the property of a stem cell to remain undifferentiated, i.e., the potential of a stem cell to maintain self-renewal and differentiation. Stem cells undergo self-renewal to form cells identical to those of parent cells, while they have the ability to differentiate into a variety of terminal cells. As can be seen from the dryness fraction chart of FIG. 15, the isolated SSC of the method of the present invention has high dryness in 95% or more of the cells.
3. The single cell sequencing gene annotation analysis of the ApLCs showed that the ApLCs expressed SSC classical molecular markers, as well as other X-chromosome linked SSC specific genes, indicating that the ApLCs have the molecular characteristics of human primary Apale cells (FIG. 16).
4. ApLCs were isolated and cultured as spermatogonial stem cells by the method of the present invention using cell-preparation of ApLCs, immunofluorescent staining, confirming on protein level that ApLCs express the SSC classical molecular marker, and the cell proliferation molecular marker Ki67 (FIG. 17).
< functional identification of human spermatogonial Stem cells >
The seminiferous tubule xenograft experiments are gold standards to identify seminiferous cell stem properties. Will be 5X 10 5 ApLCs and control cells (primary mixed testis cells) were microinjected into seminiferous tubules of nude mice (22 cells each) of 1 month after busulfan treatment, DDX4 staining was performed by using a whole moving technique after 3 months, and the number of clones was calculated, showing that ApLCs isolated and cultured by the method of the present invention could be colonized in seminiferous tubules of nude mice and expanded to form clones, and the number of clones formed by the ApLCs group was 100 times or more than that of the control group, demonstrating that ApLCs isolated and cultured by the method of the present invention were seminiferous cells with high dryness (FIG. 18).
The invention comprehensively identifies the obtained human spermatogonial stem cell culture at the cell morphology, phenotype, molecular level and functional level, and proves that the human spermatogonial stem cell culture is the human spermatogonial stem cell with active cell cycle.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (14)
1. A method for obtaining human spermatogonial stem cells in vitro, comprising the steps of:
isolating human Spermatogonial Stem Cells (SSC) from human testis tissue;
the isolated human spermatogonial stem cells were placed in SSC medium for cell subculture.
2. The method of claim 1, wherein the step of isolating human Spermatogonial Stem Cells (SSC) from human testis tissue comprises: cutting testis under aseptic environment, removing white film in Hank's liquid, mechanically cutting testis tissue, treating with two-step enzyme digestion method, counting dead cells under 5% by trypan blue staining, filtering with 100 μm and 40 μm cell filter screen sequentially to remove impurities, centrifuging at 4deg.C for 5min, and sorting with ITGA6 magnetic beads.
3. The method according to claim 2, characterized in that the two-step enzymatic digestion process comprises the steps of: digestion was performed with 1mg/mL collagenase IV at 37℃for 15min, 0.25% trypsin/1. Mu.M EDTA, and after 15min, digestion was stopped with DMEM containing 10% fetal bovine serum.
4. The method of claim 1, wherein the step of subjecting the isolated human spermatogonial stem cells to cell subculture in SSC medium comprises: placing 5% suspension-grown single cells of human spermatogonial stem cells isolated from human testis tissue in SSC culture medium to obtain cell solution containing SSC, placing the cell solution in orifice plate, placing at 37deg.C and 5% O 2 7% CO 2 2D cell subculturing was performed in an incubator.
5. The method of claim 4, wherein the components of the SSC medium and the working concentrations of each component are as follows:
6. the method of claim 4, wherein the cellular concentration of the SSC-containing cellular fluid is (0.1-10) x 10 6 SSC cells/2 ml SSC medium, preferably 1X 10 6 SSC cells/2 ml SSC medium.
7. The method of claim 4, wherein the well plate is previously coated with laminin and the SSC medium is previously preheated at room temperature; after 2D cell subculture, the method further comprises the steps of: 1ml of SSC medium preheated at room temperature was supplemented on day 3 after passage and at 37℃with 5% O 2 7% CO 2 Continuously culturing in an incubator; on day 5 after passaging, cell passaging was performed again.
8. The method of any one of claims 1-7, further comprising the step of 3D cell clone formation, comprising: thawing corning basement membrane matrix, digesting with 0.25% trypsin/1 μm EDTA, centrifuging at 1000rpm, discarding supernatant, re-suspending cells with 1mL SSC medium, counting with a cell counter, and adjusting the concentration of cell suspension to (1-10) x 10 4 cells/mL; taking 10 mu L of cell suspension in an EP tube, centrifuging at 1000rpm, discarding supernatant, re-suspending 500 SSC cells with pre-cooled substrate membrane matrix of re-suspended cell Corning, dripping the obtained mixed solution of SSC cells and matrigel into an orifice plate, adding the SSC culture medium into the orifice plate, and then adding the orifice plate at 37deg.C and 5% O 2 7% CO 2 Incubator relayCulture was continued, and on days D0, D4, D7, D10 and D14, the clone formation state was observed by photographing with an OlympusIX83 microscope.
9. A cell population of isolated human spermatogonial stem cells obtained by the method of obtaining human spermatogonial stem cells in vitro according to any one of claims 1-8, said cell population of isolated human spermatogonial stem cells expressing at least one spermatogonial stem cell marker ApLCs (human spermatogonial stem cell line aple-like cells); the spermatogonial stem cell markers ApLCs are defined as follows: more than 90% of the isolated human spermatogonial stem cells are in cell morphology consistent with undifferentiated spermatogonial cells Apale with self-renewal capacity in human primary spermatogonial stem cells.
10. The isolated population of human spermatogonial stem cells according to claim 9, wherein the spermatogonial stem cell markers ApLCs (human spermatogonial stem cell line aple-like cells) are capable of long-term survival in vitro and are capable of differentiating into spermatogenic cells without forming tumor cells.
11. The isolated population of human spermatogonial stem cells according to claim 9, wherein single cell sequencing of the spermatogonial stem cell markers ApLCs (human spermatogonial stem cell line aple-like cells) reveals that the cells are divided into 5 sub-populations C1, C2, C3, C4, C5 in different states of the cell cycle.
12. The isolated population of human primary spermatogonial stem cells of claim 11, wherein the human primary spermatogonial stem cells are separated into 6 sub-populations of SPG1, SPG2, SPG3, SPG4, SPG5, SPG6 after single cell sequencing; wherein, the molecular characteristics of resting spermatogenic cells Adark in the human primary spermatogenic stem cells correspond to SPG2 and SPG3, and the molecular characteristics of spermatogenic cells Apale with self-renewal capacity in the human primary spermatogenic stem cells correspond to SPG3, SPG4 and SPG 5; the molecular characteristics of 5 subpopulations C1, C2, C3, C4, C5 obtained after single cell sequencing of the spermatogonial stem cell markers ApLCs (human spermatogonial stem cell line aple-like cells) were all annotated as SPG4.
13. A human spermatogonial stem cell culture obtainable by the method of in vitro obtaining human spermatogonial stem cells according to any one of claims 1-8, which is stably cultured in vitro for a long period of time.
14. The human spermatogonial stem cell culture capable of long term stable culture in vitro according to claim 13, wherein the human spermatogonial stem cell culture capable of long term stable culture in vitro comprises human spermatogonial stem cell line aple-like cells (ApLCs), more than 90% of the human spermatogonial stem cells obtained by the method for obtaining human spermatogonial stem cells in vitro according to any of claims 1 to 8 have a cell morphology consistent with the undifferentiated spermatogonial cells aple with self-renewal capacity in human primary spermatogonial stem cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311191974.8A CN117721072A (en) | 2023-09-15 | 2023-09-15 | Method for obtaining human spermatogonial stem cells in vitro and human spermatogonial stem cell culture capable of being stably cultured in vitro for long term |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311191974.8A CN117721072A (en) | 2023-09-15 | 2023-09-15 | Method for obtaining human spermatogonial stem cells in vitro and human spermatogonial stem cell culture capable of being stably cultured in vitro for long term |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117721072A true CN117721072A (en) | 2024-03-19 |
Family
ID=90209492
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311191974.8A Pending CN117721072A (en) | 2023-09-15 | 2023-09-15 | Method for obtaining human spermatogonial stem cells in vitro and human spermatogonial stem cell culture capable of being stably cultured in vitro for long term |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117721072A (en) |
-
2023
- 2023-09-15 CN CN202311191974.8A patent/CN117721072A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US12065671B2 (en) | Method for reproducible differentiation of clinical-grade retinal pigment epithelium cells | |
| CN108291206B (en) | MACS-based purification of stem cell-derived retinal pigment epithelium | |
| CN110396499B (en) | Method for inducing neural stem cells and application thereof | |
| CN109082411B (en) | Method for obtaining phagocytic macrophage by differentiation of pluripotent stem cells | |
| Karlmark et al. | Activation of ectopic Oct-4 and Rex-1 promoters in human amniotic fluid cells | |
| US20050239201A1 (en) | Methods of inducing differentiation of stem cells into a specific cell lineage | |
| CA2434362A1 (en) | Pluripotent adult stem cells derived from regenerative tissue | |
| US20130157365A1 (en) | Induced pluripotent stem cells from human umbilical cord tissue-derived cells | |
| US8673637B2 (en) | Human multipotent germ line stem cells expressing a germ line marker and a pluripotency marker produced by co-culture of testicular tissue | |
| CN115011553A (en) | Preparation method and application of stem neural crest-derived bone marrow mesenchymal stem cells | |
| US9163214B2 (en) | Method for culturing stem cells | |
| CN106754657B (en) | Serum-free medium for monkey embryonic stem cells | |
| US20050095708A1 (en) | Characterization and isolation of subsets of human embryonic stem cells (HES) and cells associated or derived therefrom | |
| CN117721072A (en) | Method for obtaining human spermatogonial stem cells in vitro and human spermatogonial stem cell culture capable of being stably cultured in vitro for long term | |
| EP1448763A1 (en) | Characterization and isolation of subsets of human embryonic stem cells (hes) and cells associated or derived therefrom | |
| KR102038503B1 (en) | Method for differentiation of hepatocyte | |
| US20190264174A1 (en) | Method of cultivation of human salivary gland cells | |
| Okubo et al. | Fabrication of three-dimensional lacrimal gland-like tissue organoids from human pluripotent stem cells | |
| WO2023027148A1 (en) | Method for producing sperm stem cell-like cells and sperm stem cell-like cell line | |
| US8445269B2 (en) | Method for generating pluripotent stem cells | |
| Li et al. | Rapid single-step separation of pluripotent mouse embryonic stem cells from mouse feeder fibroblasts | |
| CN118685360A (en) | Human dedifferentiated liposarcoma cell line and application | |
| WO2024168161A1 (en) | Potency assay and manufacturing method | |
| CN113667635A (en) | Xeno-free medium and method for amplifying mesenchymal stem cells using the same | |
| AU2003209830A1 (en) | Methods of inducing differentiation of stem cells into a specific cell lineage |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |