原发性硬化性胆管炎疾病源性iPSCs细胞模型的建立
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摘要
目的探讨原发性硬化性胆管炎(primary sclerosing cholangitis,PSC)疾病源性诱导性多能干细胞(induced pluripotent stem cells,iPSCs)细胞模型的建立。方法采用Oct3/4、Sox2、c-Myc和Klf4 4种转录因子,以仙台病毒为载体重编程PSC患者皮肤成纤维细胞,获得疾病特异性的iPSCs细胞,并对其生物学特性进行鉴定。结果 PSC疾病源性的iPSCs细胞呈典型胚胎干细胞样克隆,流式细胞分析显示高表达Oct4、TRA-1-81、SSEA和Nanog等胚胎干细胞标志性抗原,体外向内、中、外三个胚层诱导分化后均检测到各胚层标志抗原的表达,传代过程中保持稳定正常的染色体核型。结论应用Yamanaka因子和仙台病毒载体成功建立PSC疾病源性iPSCs细胞模型,后续进一步向胆管细胞诱导分化可用于研究疾病的发病机制。
Objective To explore the establishment of iPSCs cell model of primary sclerosing cholangitis(PSC). Methods Four transcription factors, Oct3/4, Sox2, c-Myc and Klf4, were used to reprogram skin fibroblasts of PSC patients with sendai virus as the vector to obtain disease-specific iPSCs cells and identify their biological characteristics. Results PSC disease-derived iPSCs were typical embryonic stem cell-like clones, and flow cytometry showed high expressions of Oct4, TRA-1-81, SSEA, Nanog. The expression of each germ layer marker was detected after induced differentiation into three endoderms in vitro. The chromosome karyotype remained stable and normal during passage. Conclusion The PSC disease-derived iPSCs cell model was successfully established by Yamanaka factor and sendai virus vector, and further induced differentiation into bile duct cells could be used to study the pathogenesis of the disease.
Objective To explore the establishment of iPSCs cell model of primary sclerosing cholangitis(PSC). Methods Four transcription factors, Oct3/4, Sox2, c-Myc and Klf4, were used to reprogram skin fibroblasts of PSC patients with sendai virus as the vector to obtain disease-specific iPSCs cells and identify their biological characteristics. Results PSC disease-derived iPSCs were typical embryonic stem cell-like clones, and flow cytometry showed high expressions of Oct4, TRA-1-81, SSEA, Nanog. The expression of each germ layer marker was detected after induced differentiation into three endoderms in vitro. The chromosome karyotype remained stable and normal during passage. Conclusion The PSC disease-derived iPSCs cell model was successfully established by Yamanaka factor and sendai virus vector, and further induced differentiation into bile duct cells could be used to study the pathogenesis of the disease.
引文
[1] LINDOR K D,KOWDLEY K V,HARRISON M E.ACG clinical guideline:primary sclerosing cholangitis [J].Am J Gastroenterol,2015,110(5):646-659.DOI:10.1038/ajg.2015.112.
[2] TAKAHASHI K,YAMANAKA S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors [J].Cell,2006,126 (4):663-676.DOI:10.1016/j.cell.2006.07.024.
[3] ESPEJEL S,ROLL G R,MCLAUGHLIN K J,et al.Induced pluripotent stem cell derived hepatocytes have the functional and proliferative capabilities needed for liver regeneration in mice [J].J Clin Invest,2010,120(9):3120-3126.DOI:10.1172/JCI43267.
[4] HUANG P,HE Z,JI S,et al.Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors [J].Nature,2011,475(7356):386-389.DOI:10.1038/nature10116.
[5] HEMAN-ACKAH S M,MANZANO R,HOOZEMANS J J M,et al.Alpha-synuclein induces the unfolded protein response in Parkinson’s disease SNCA triplication iPSC-derived neurons [J].Hum Mol Genet,2017,26(22):4441-4450.DOI:10.1093/hmg/ddx331.
[6] ISRAEL M A,YUAN S H,BARDY C,et al.Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells [J].Nature,2012,482(7384):216-220.DOI:10.1038/nature10821.
[7] YU J,VODYANIK M A,SMUGA-OTTO K,et al.Induced pluripotent stem cell lines derived from human somatic cells [J].Science,2007,318(5858):1917-1920.DOI:10.1126/science.1151526.
[8] YAMANAKA S.Induced pluripotent stem cells:past,present,and future [J].Cell Stem Cell,2012,10(6):678-684.DOI:10.1016/j.stem.2012.05.005.
[9] MIERE C,DEVITO L,ILIC D.Sendai virus-based reprogramming of mesenchymal stromal/stem cells from umbilical cord wharton’s jelly into induced pluripotent stem cells [J].Methods Mol Biol,2016,1357:33-44.DOI:10.1007/7651_2014_163.
[10] YUSA K,RAD R,TAKEDA J,et al.Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon [J].Nat Methods,2009,6(5):363-369.DOI:10.1038/nmeth.1323.
[11] ZHANG H,MA Y,GU J,et al.Reprogramming of somatic cells via TAT-mediated protein transduction of recombinant factors [J].Biomaterials,2012,33(20):5047-5055.DOI:10.1016/j.biomaterials.2012.03.061.
[12] LI D,WANG L,HOU J,et al.Optimized approaches for generation of integration-free iPSCs from human urine-derived cells with small molecules and autologous feeder [J].Stem Cell Reports,2016,6(5):717-728.DOI:10.1016/j.stemcr.2016.04.001.
[13] THATAVA T,NELSON T J,EDUKULLA R,et al.Indolactam V/GLP-1-mediated differentiation of human iPS cells into glucose-responsive insulin-secreting progeny [J].Gene Ther,2011,18(3):283-293.DOI:10.1038/gt.2010.145.
[14] LAZARIDIS K N,LARUSSO N F.The Cholangiopathies [J].Mayo Clin Proc,2015,90(6):791-800.DOI:10.1016/j.mayocp.2015.03.017.
[15] JALAN-SAKRIKAR N,HUEBERT R C.Ducts in a dish:Bioengineered biliary scaffolds for regenerative medicine [J].Hepatology,2018,67(4):1622-1624.DOI:10.1002/hep.29614.
[2] TAKAHASHI K,YAMANAKA S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors [J].Cell,2006,126 (4):663-676.DOI:10.1016/j.cell.2006.07.024.
[3] ESPEJEL S,ROLL G R,MCLAUGHLIN K J,et al.Induced pluripotent stem cell derived hepatocytes have the functional and proliferative capabilities needed for liver regeneration in mice [J].J Clin Invest,2010,120(9):3120-3126.DOI:10.1172/JCI43267.
[4] HUANG P,HE Z,JI S,et al.Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors [J].Nature,2011,475(7356):386-389.DOI:10.1038/nature10116.
[5] HEMAN-ACKAH S M,MANZANO R,HOOZEMANS J J M,et al.Alpha-synuclein induces the unfolded protein response in Parkinson’s disease SNCA triplication iPSC-derived neurons [J].Hum Mol Genet,2017,26(22):4441-4450.DOI:10.1093/hmg/ddx331.
[6] ISRAEL M A,YUAN S H,BARDY C,et al.Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells [J].Nature,2012,482(7384):216-220.DOI:10.1038/nature10821.
[7] YU J,VODYANIK M A,SMUGA-OTTO K,et al.Induced pluripotent stem cell lines derived from human somatic cells [J].Science,2007,318(5858):1917-1920.DOI:10.1126/science.1151526.
[8] YAMANAKA S.Induced pluripotent stem cells:past,present,and future [J].Cell Stem Cell,2012,10(6):678-684.DOI:10.1016/j.stem.2012.05.005.
[9] MIERE C,DEVITO L,ILIC D.Sendai virus-based reprogramming of mesenchymal stromal/stem cells from umbilical cord wharton’s jelly into induced pluripotent stem cells [J].Methods Mol Biol,2016,1357:33-44.DOI:10.1007/7651_2014_163.
[10] YUSA K,RAD R,TAKEDA J,et al.Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon [J].Nat Methods,2009,6(5):363-369.DOI:10.1038/nmeth.1323.
[11] ZHANG H,MA Y,GU J,et al.Reprogramming of somatic cells via TAT-mediated protein transduction of recombinant factors [J].Biomaterials,2012,33(20):5047-5055.DOI:10.1016/j.biomaterials.2012.03.061.
[12] LI D,WANG L,HOU J,et al.Optimized approaches for generation of integration-free iPSCs from human urine-derived cells with small molecules and autologous feeder [J].Stem Cell Reports,2016,6(5):717-728.DOI:10.1016/j.stemcr.2016.04.001.
[13] THATAVA T,NELSON T J,EDUKULLA R,et al.Indolactam V/GLP-1-mediated differentiation of human iPS cells into glucose-responsive insulin-secreting progeny [J].Gene Ther,2011,18(3):283-293.DOI:10.1038/gt.2010.145.
[14] LAZARIDIS K N,LARUSSO N F.The Cholangiopathies [J].Mayo Clin Proc,2015,90(6):791-800.DOI:10.1016/j.mayocp.2015.03.017.
[15] JALAN-SAKRIKAR N,HUEBERT R C.Ducts in a dish:Bioengineered biliary scaffolds for regenerative medicine [J].Hepatology,2018,67(4):1622-1624.DOI:10.1002/hep.29614.