Human Induced Pluripotent Stem (hiPS) Cells from Urine Samples: A Non‐Integrative and Feeder‐Free Reprogramming Strategy

Abstract

Human induced pluripotent stem (hiPS) cell technology has already revolutionized some aspects of fundamental and applied research such as study of disease mechanisms and pharmacology screening. The first clinical trial using hiPS cell-derived cells began in Japan, only 10 years after the publication of the proof-of concept article. In this exciting context, strategies to generate hiPS cells have evolved quickly, tending towards non-invasive protocols to sample somatic cells combined with "safer" reprogramming strategies. In this unit, we describe a protocol combining both of these advantages to generate hiPS cells with episomal plasmid transfection from urine samples of individuals carrying the desired genotype. Based on previous published works, this simplified protocol requires minimal equipment and reagents, and is suitable both for scientists familiar with the hiPS cells technology and neophytes. HiPS cells displaying classical features of pluripotency and suitable for all desired downstream applications are generated rapidly (<10 weeks) and with high efficiency. © 2017 by John Wiley & Sons, Inc.

Urine-derived cells provide a readily accessible cell type for feeder-free mRNA reprogramming

“…Moreover, the u-d hiPSC did not correlate more with the parental urine-derived cells than hESCs or the f71 hiPSCs. Finally, urine-derived cells specific genes MCAM, PAX8 and NT5E 21 , 27 did not show persistence in u-d hiPSCs compared to hESCs or f71 hiPSCs (Fig. 2c ).…”

“…Finally, we repeated the reprogramming of patient 149 cells to establish hiPSC directly on Matrigel, a commonly used coating matrix. We were able to generate feeder-free hiPSC lines, as recently published for episomal reprogramming of urine-derived cells 21 (Fig. 1d ).…”

Urine-Derived Induced Pluripotent Stem Cells in Cardiovascular Disease

“…No free DNA was detected in iPSCs after 8 passages, which further indicated that iPSCs were not well integrated into the experiment [40]. Researchers have used free vectors to transfect transcription factors such as Oct4, Sox2, Lin28, l-myc, and Klf4 into urine cells, while added free vectors which mediated the EBNA1 gene expression, as well as a P53 knockout to improve reprogramming efficiency [24]. ese methods eventually generated successful iPSCs.…”

“…Furthermore, USCs do not express hematopoietic stem cell markers such as CD25, CD31, CD34, and CD45. Simultaneously, researchers have reported that USCs in the urine are the main source of the iPSCs [24].…”

“…Furthermore, it was reported that the type II clones grew faster (see Figure 2). Compared to dermal fibroblasts or adult adipose-derived mesenchymal stem cells, USCs demonstrate faster reprogramming kinetics, as well as higher efficiency [24,29], which may result from the high telomerase activity observed in the endogenous expression reprogramming factors c-myc, Klf4, and USCs [29].…”