Comparative Analysis of Targeted Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) and Human Embryonic Stem Cells Reveals Variability Associated With Incomplete Transgene Silencing in Retrovirally Derived hiPSC Lines

Abstract

Functional hepatocytes, cardiomyocytes, neurons, and retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) could provide a defined and renewable source of human cells relevant for cell replacement therapies, drug discovery, toxicology testing, and disease modeling. In this study, we investigated the differences between the differentiation potentials of three hESC lines, four retrovirally derived hiPSC lines, and one hiPSC line derived with the nonintegrating Sendai virus technology. Four independent protocols were used for hepatocyte, cardiomyocyte, neuronal, and RPE cell differentiation. Overall, cells differentiated from hESCs and hiPSCs showed functional similarities and similar expression of genes characteristic of specific cell types, and differences between individual cell lines were also detected. Reactivation of transgenic OCT4 was detected specifically during RPE differentiation in the retrovirally derived lines, which may have affected the outcome of differentiation with these hiPSCs. One of the hiPSC lines was inferior in all directions, and it failed to produce hepatocytes. Exogenous KLF4 was incompletely silenced in this cell line. No transgene expression was detected in the Sendai virus-derived hiPSC line. These findings highlight the problems related to transgene expression in retrovirally derived hiPSC lines.

Induced Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium: A Comparative Study Between Cell Lines and Differentiation Methods

“…Similar variation between lines has been previously reported for RPE 16,21,45 and other cell types derived from iPSC. [45][46][47][48][49] The SCAC method secreted higher levels of PEDF in iPSC-3-RPE compared to iPSC-1-RPE. The directed method showed superior mature RPE transcript expression and PEDF secretion in iPSC-3-RPE.…”

Biomimetic collagen I and IV double layer Langmuir–Schaefer films as microenvironment for human pluripotent stem cell derived retinal pigment epithelial cells

“…In this study, a difference in the functionality of hESC-RPE cells on double layer LS films was detected between the two investigated cell lines. Previously, it has been shown that individual hESC-lines behave slightly differently, which affects for instance to the appearance of pigmentation during RPE differentiation [56]. Despite the higher PEDF secretion in Regea 08/023 hESC-RPE, hESC-RPE cells derived from both cell lines were significantly more functional on the double layer LS films compared to the dip-coated controls.…”

New markers for tracking endoderm induction and hepatocyte differentiation from human pluripotent stem cells

“…This effort has led to advances in our basic understanding of human development and the generation of protocols for directed differentiation of hPSCs to pancreatic and hepatic fates that yield what seem to be highly enriched end-stage populations (Chen et al, 2012;Ogawa et al, 2013;Pagliuca et al, 2014;Rezania et al, 2014;Si-Tayeb et al, 2010). Despite this progress, efficiencies of differentiation vary considerably between different hPSC lines (Toivonen et al, 2013b;Vitale et al, 2012), even with the most advanced protocols, and end-stage populations are immature and, in some instances, contaminated with other cell types.…”