Generation of Urine Cell-Derived Non-integrative Human iPSCs and iNSCs: A Step-by-Step Optimized Protocol

Abstract

Objective: Establishing a practical procedure to generate induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) from human urine cells (UCs). In this report, we optimized a non-integrative protocol to generate patient-specific iPSC and iNSC lines with high reprogramming efficiency.Methods: UCs were electroporated with the pEP4-EO2S-ET2K and pEP4-M2L plasmids containing the OCT4, SOX2, KLF4, SV40LT, c-MYC, and LIN28 genes, and then cultured with N2B27 medium plus four small molecule compounds (A83-01, PD0325901, Thiazovivin, and CHIR99021). When iPSC or iNSC clones emerged, the medium was replaced with mTeSR1 or neural growth medium. Morphological changes were seen at day 4–7. After day 10, the clones were picked up when the clone diameter exceeded 1 mm.Results: iPSCs and iNSCs were successfully derived from UCs with up to 80 clones/well. These iPSCs and iNSCs showed typical hESC or NSC morphology and were self-renewable. The iPSCs had pluripotency to differentiate into the three germinal layers and displayed high levels of expression of pluripotency markers SOX2, NANOG, OCT4, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase (AP). They maintained normal karyotype and had no transgene expression or genomic integration. The iNSCs were positive for NSC markers NESTIN, PAX6, SOX2, and OLIG2.Conclusion: The optimized protocol is an easy and fast procedure to yield both iPSC and iNSC lines from a convenient source of human urine in a single experiment.

Advances in Pluripotent Stem Cells: History, Mechanisms, Technologies, and Applications

“…Stem cells can be reprogrammed with various cocktails of small molecules such as the histone deacetylase inhibitor valproic acid [134,135], vitamin C [136], sodium butyrate [135], and the GSK-3 inhibitor CHiR99021 [127] [137],, among others. Valproic acid has been shown to dedifferentiate neonatal foreskin fibroblasts when used in conjugation with only Oct4 and Sox2; interestingly, valproic acid can be substituted for the proto-oncogene c-Myc to prevent tumor formation [134].…”

Advances in Regeneration of Retinal Ganglion Cells and Optic Nerves

“…As an excellent cell resource, patient-derived iPSCs allow the regeneration of different and sufficient quantities of autologous cell types almost without the risk of immune rejection and iPSCs have already been generated from patients with multifarious diseases [ 98 ]. In addition, the procedure to generate urine-derived iPSCs with high reprogramming efficiency has been established [ 99 ], which provides a promising noninvasive source of stem cells and can subsequently differentiate into desired cell types.…”

Urine-derived induced pluripotent/neural stem cells for modeling neurological diseases

“…Another study used a cocktail of four small molecules (A83-01, PD0325901, Thiazovivin, and CHIR99021) together with pEP4-EO2S-ET2K and pEP4-M2L plasmids containing OCT4 , SOX2 , KLF4 , SV40LT , c-MYC , and LIN28 genes to enhance the reprogramming efficiency of USCs into iPSCs and iNSCs. When using a higher concentration cocktail in the early stage of reprogramming, iPSCs appeared earlier (10 days) than iNSCs (12–15 days) [ 90 ]. This non-integrative method could generate iPSCs and iNSCs from USCs at twice the speed compared to reprogramming blood or skin cells.…”