人卵泡壁层颗粒细胞源性非整合诱导多能干细胞的制备
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摘要
目的探索将卵泡液中壁层颗粒细胞诱导为非整合的诱导多能干细胞(iPS细胞),并检测其颗粒细胞方向的分化能力。方法在取卵的操作过程中,收集废弃的壁层颗粒细胞,在颗粒细胞培养至第4天时,加入iPS仙台病毒,经过20余天的维持,挑取iPS细胞克隆,并扩增培养。对其多能基因表达情况、外源基因整合情况、自然分化能力、颗粒细胞定向分化能力进行鉴定,并与皮肤细胞来源的iPSC系进行平行分化能力比较。结果成功建立了壁层颗粒细胞来源的iPS细胞系,其通过了类似胚胎干细胞的多能性检测及体外三胚层的分化能力检测,尤其在定向分化为颗粒细胞时,分化出了大量FOXL2、CYP19A1和FSHR阳性的细胞,经ELISA试剂盒检测,发现该分化细胞可以分泌AMH并且能够将雄激素转化成雌激素;且颗粒细胞源iPS系较皮肤细胞源的iPS系在颗粒细胞方向的分化效率更高。结论提供了一种从人颗粒细胞建立iPSC的方法,并验证了其颗粒细胞方向分化的优势。该系统不仅可以用于建立生殖不孕疾病的iPSC库,还为颗粒细胞功能障碍不孕的患者提供了一种细胞治疗的新思路。
Objective:To induce human mural granulose cells to integration-free induced pluripotent stem cells(iPSC),and test the differentiation potential of granulosa cell-like(GC-like)cells.Methods:Human mural granulosa cells were collected during oocyte retrieval process from women undergoing infertility treatment.iPS Sendai virus was added to the 4th day of granulosa cell cultured.After20 days of maintenance,iPS cell clones were picked and cultured.The ability of pluripotent gene expression,the integration of foreign genes,and the ability of differentiation of GC-like cells were identified and compared with the iPSC derived from skin cells.Results: mGC-iPSCs were successfully generated,and passed the tests of similar pluripotency ofembryonic stem cells and differentiation ability of three germ layers in vitro.The differentiated GC-like cells expressed the granulosa cell specific marker FOXL2,CYP19A1 and FSHR.These GC-like cells were also capable of producing AMH and aromatizing testosterone to estradiol,which suggested that they were biologically functional.mGC-iPSCs have higher differentiation`efficiency thanbroblast-iPSCs.Conclusions:The study provides a safety method for generating human iPSCs from human mural granulosa cells,and finds that mGC-derived iPSCs reveal higher differentiation efficiency thanbroblastderived iPSCs.The system can not only be used to establish the iPSC library for reproductive infertility,but also to provide a new idea for cell therapy for the infertile patients with granule cell dysfunction.
Objective:To induce human mural granulose cells to integration-free induced pluripotent stem cells(iPSC),and test the differentiation potential of granulosa cell-like(GC-like)cells.Methods:Human mural granulosa cells were collected during oocyte retrieval process from women undergoing infertility treatment.iPS Sendai virus was added to the 4th day of granulosa cell cultured.After20 days of maintenance,iPS cell clones were picked and cultured.The ability of pluripotent gene expression,the integration of foreign genes,and the ability of differentiation of GC-like cells were identified and compared with the iPSC derived from skin cells.Results: mGC-iPSCs were successfully generated,and passed the tests of similar pluripotency ofembryonic stem cells and differentiation ability of three germ layers in vitro.The differentiated GC-like cells expressed the granulosa cell specific marker FOXL2,CYP19A1 and FSHR.These GC-like cells were also capable of producing AMH and aromatizing testosterone to estradiol,which suggested that they were biologically functional.mGC-iPSCs have higher differentiation`efficiency thanbroblast-iPSCs.Conclusions:The study provides a safety method for generating human iPSCs from human mural granulosa cells,and finds that mGC-derived iPSCs reveal higher differentiation efficiency thanbroblastderived iPSCs.The system can not only be used to establish the iPSC library for reproductive infertility,but also to provide a new idea for cell therapy for the infertile patients with granule cell dysfunction.
引文
[1]Takahashi K,Tanabe K,Ohnuki M,et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors[J].Cell,2007,131:861-872.
[2]Eggenschwiler R,Cantz T.Induced pluripotent stem cells generated without viral integration[J].Hepatology,2009,49:1048-1049.
[3]Okita K,Matsumura Y,Sato Y,et al.A more efficient method to generate integration-free human iPS cells[J].Nat Methods,2011,8:409-412.
[4]Kim D,Kim CH,Moon JI,et al.Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins[J].Cell Stem Cell,2009,4:472-476.
[5]Zhou H,Wu S,Joo JY,et al.Generation of induced pluripotent stem cells using recombinant proteins[J].Cell Stem Cell,2009,4:381-384.
[6]Warren L,Manos PD,Ahfeldt T,et al.Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA[J].Cell Stem Cell,2010,7:618-630.
[7]Lee HK,Morin P,Xia W.Peripheral blood mononuclear cellconverted induced pluripotent stem cells(iPSCs)from an early onset Alzheimer’s patient[J].Stem Cell Res,2016,16:213-215.
[8]Nishizawa M,Chonabayashi K,Nomura M,et al.Epigenetic variation between human induced pluripotent stem cell lines is an indicator of differentiation capacity[J].Cell Stem Cell,2016,19:341-354.
[9]Su YQ,Sugiura K,Eppig JJ.Mouse oocyte control of granulosa cell development and function:paracrine regulation of cumulus cell metabolism[J].Semin Reprod Med,2009,27:32-42.
[10]Lee G,Papapetrou EP,Kim H,et al.Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs[J].Nature,2009,461:402-406.
[11]Kossowska-Tomaszczuk K,De Geyter C.Cells with stem cell characteristics in somatic compartments of the ovary[J].Biomed Res Int,2013,2013:310859.
[12]Kossowska-Tomaszczuk K,De Geyter C,De Geyter M,et al.The multipotency of luteinizing granulosa cells collected from mature ovarian follicles[J].Stem Cells,2009,27:210-219.
[13]Mao J,Zhang Q,Ye X,et al.Efficient induction of pluripotent stem cells from granulosa cells by Oct4and Sox2[J].Stem Cells Dev,2014,23:779-789.
[14]Zhang J,Li H,Wu Z,et al.Differentiation of rat iPS cells and ES cells into granulosa cell-like cells in vitro[J].Acta Biochim Biophys Sin(Shanghai),2013,45:289-295.
[15]Lan CW,Chen MJ,Jan PS,et al.Differentiation of human embryonic stem cells into functional ovarian granulosa-like cells[J].J Clin Endocrinol Metab,2013,98:3713-3723.
[16]Woods DC,White YA,Niikura Y,et al.Embryonic stem cellderived granulosa cells participate in ovarian follicle formation in vitro and in vivo[J].Reprod Sci,2013,20:524-535.
[17]Shelling AN.Premature ovarian failure[J].Reproduction,2010,140:633-641.
[2]Eggenschwiler R,Cantz T.Induced pluripotent stem cells generated without viral integration[J].Hepatology,2009,49:1048-1049.
[3]Okita K,Matsumura Y,Sato Y,et al.A more efficient method to generate integration-free human iPS cells[J].Nat Methods,2011,8:409-412.
[4]Kim D,Kim CH,Moon JI,et al.Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins[J].Cell Stem Cell,2009,4:472-476.
[5]Zhou H,Wu S,Joo JY,et al.Generation of induced pluripotent stem cells using recombinant proteins[J].Cell Stem Cell,2009,4:381-384.
[6]Warren L,Manos PD,Ahfeldt T,et al.Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA[J].Cell Stem Cell,2010,7:618-630.
[7]Lee HK,Morin P,Xia W.Peripheral blood mononuclear cellconverted induced pluripotent stem cells(iPSCs)from an early onset Alzheimer’s patient[J].Stem Cell Res,2016,16:213-215.
[8]Nishizawa M,Chonabayashi K,Nomura M,et al.Epigenetic variation between human induced pluripotent stem cell lines is an indicator of differentiation capacity[J].Cell Stem Cell,2016,19:341-354.
[9]Su YQ,Sugiura K,Eppig JJ.Mouse oocyte control of granulosa cell development and function:paracrine regulation of cumulus cell metabolism[J].Semin Reprod Med,2009,27:32-42.
[10]Lee G,Papapetrou EP,Kim H,et al.Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs[J].Nature,2009,461:402-406.
[11]Kossowska-Tomaszczuk K,De Geyter C.Cells with stem cell characteristics in somatic compartments of the ovary[J].Biomed Res Int,2013,2013:310859.
[12]Kossowska-Tomaszczuk K,De Geyter C,De Geyter M,et al.The multipotency of luteinizing granulosa cells collected from mature ovarian follicles[J].Stem Cells,2009,27:210-219.
[13]Mao J,Zhang Q,Ye X,et al.Efficient induction of pluripotent stem cells from granulosa cells by Oct4and Sox2[J].Stem Cells Dev,2014,23:779-789.
[14]Zhang J,Li H,Wu Z,et al.Differentiation of rat iPS cells and ES cells into granulosa cell-like cells in vitro[J].Acta Biochim Biophys Sin(Shanghai),2013,45:289-295.
[15]Lan CW,Chen MJ,Jan PS,et al.Differentiation of human embryonic stem cells into functional ovarian granulosa-like cells[J].J Clin Endocrinol Metab,2013,98:3713-3723.
[16]Woods DC,White YA,Niikura Y,et al.Embryonic stem cellderived granulosa cells participate in ovarian follicle formation in vitro and in vivo[J].Reprod Sci,2013,20:524-535.
[17]Shelling AN.Premature ovarian failure[J].Reproduction,2010,140:633-641.