Generation of induced pluripotent stem cells and neural cells from a patient with critical illness polyneuropathy / 中华急诊医学杂志

ABSTRACT

Objective To establish disease-associated or cell type relevant neuron model generated from patients with Critical Illness Polyneuropathy (CIP) by making CIP patient-derived induced pluripotent stem (iPS) cell lines and neurons to provide a cell-based disease model of CIP.Methods Skin tissue of CIP patient was obtained clinically,and specific skin fibroblasts were isolated and cultured.The iPS cells were derived from CIP patient by introducing 4 transcription factors,namely Oct4,Klf4,Sox2,c-Myc,into patient-specific fibroblast cells by Millipore's Human STEMCCA Constitutive Polycistronic (OKSM) Lentivirus Kit.Colony morphology,alkaline phosphatase (AP) activity,immunofluorescence staining,quantitative reverse transcription polymerase chain reaction (RT-PCR),and differentiation ability were used to identify the pluripotency of these iPS cell lines.In addition,neurons were derived from these iPS cells by inhibiting SMAD pathway.Results The CIP-iPS cells presenting morphological and growth characteristics of human embryonic stem cell (hES) showed the presence of alkaline phosphatase detected by histochemical staining,and the expression of ESC-marker genes.The relative expressions of endogenous pluripotency genes,namely Sox2,REX1,NANOG and OCT4,in iPS cell lines were significantly increased compared with their primary fibroblasts (t values were-9.020,-10.753,-13.295,-12.677,P＜0.01).Subcutaneous injection of iPS cells into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers.Furthermore,cholinergic neurons were successfully induced from CIP-iPS cells.Conclusion The CIP patient-specific iPS cell line and cholinergic neurons were successfully established.Furthermore,the CIP-iPS cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for Critical Illness Polyneuropathy.