title: Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons.
abstract: It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study, we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors, by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner, which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors, in a 2-dimensional or 3-dimensional environment. However, Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices, we observed mature Purkinje-like cells with right morphology and marker expression patterns, which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases.
authors.affiliation: 1] Sanofi-Xuanwu Joint Lab for Regenerative Medicine, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China [2] Center of Neural Injury and Repair, Beijing Institute for Brain Disorders, Beijing, China [3] Center of Parkinson's Disease, Beijing Institute for Brain Disorders, Beijing, China.
authors.forename: Shuyan
authors.initials: S
authors.lastname: Wang
authors.affiliation: Sanofi-Xuanwu Joint Lab for Regenerative Medicine, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Neurobiology, Beijing Key Laboratory of Major Brain Disorders, Beijing Institute of Brain Disorders, Capital Medical University, Beijing, 100069, China.
authors.forename: Na
authors.initials: N
authors.lastname: Pan
authors.affiliation: Sanofi-Xuanwu Joint Lab for Regenerative Medicine, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China.
authors.forename: Linlin
authors.initials: L
authors.lastname: Fu
authors.affiliation: Department of Neurobiology, Xuanwu Hosptial, Capital Medical University, Beijing, 100053, China.
authors.forename: Chaodong
authors.initials: C
authors.lastname: Wang
authors.affiliation: Sanofi-Xuanwu Joint Lab for Regenerative Medicine, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China.
authors.forename: Gongru
authors.initials: G
authors.lastname: Song
authors.affiliation: Sanofi-Xuanwu Joint Lab for Regenerative Medicine, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China.
authors.forename: Jing
authors.initials: J
authors.lastname: An
authors.affiliation: Sanofi-Xuanwu Joint Lab for Regenerative Medicine, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China.
authors.forename: Zhongfeng
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Sanofi-Xuanwu Joint Lab for Regenerative Medicine, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China.
authors.forename: Wanwan
authors.initials: W
authors.lastname: Zhu
authors.affiliation: Sanofi-Xuanwu Joint Lab for Regenerative Medicine, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China.
authors.forename: Yunqian
authors.initials: Y
authors.lastname: Guan
authors.affiliation: Department of Neurobiology, Beijing Key Laboratory of Major Brain Disorders, Beijing Institute of Brain Disorders, Capital Medical University, Beijing, 100069, China.
authors.forename: Zhi-Qing David
authors.initials: ZQ
authors.lastname: Xu
authors.affiliation: 1] Center of Parkinson's Disease, Beijing Institute for Brain Disorders, Beijing, China [2] Department of Neurobiology, Xuanwu Hosptial, Capital Medical University, Beijing, 100053, China.
authors.forename: Piu
authors.initials: P
authors.lastname: Chan
authors.affiliation: 1] Sanofi-Xuanwu Joint Lab for Regenerative Medicine, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China [2] Center of Neural Injury and Repair, Beijing Institute for Brain Disorders, Beijing, China [3] Center of Parkinson's Disease, Beijing Institute for Brain Disorders, Beijing, China.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Chen
authors.affiliation: 1] Sanofi-Xuanwu Joint Lab for Regenerative Medicine, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China [2] China R&D Center, Sanofi, Beijing 100022, China.
authors.forename: Y Alex
authors.initials: YA
authors.lastname: Zhang
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000378:metabolism / Q000473:pathology; D018920:Coculture Techniques; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D007136:Immunoglobulins / Q000378:metabolism; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D007328:Insulin / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000378:metabolism; D011689:Purkinje Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D051381:Rats; D014183:Transplantation, Heterologous; D014704:Veratrum Alkaloids / Q000494:pharmacology
chemical_list: D007136:Immunoglobulins; D007328:Insulin; C482646:KIRREL2 protein, human; D008565:Membrane Proteins; D014704:Veratrum Alkaloids; D016222:Fibroblast Growth Factor 2; C000541:cyclopamine