Efficient Derivation and Genetic Modifications of Human Pluripotent Stem Cells on Engineered Human Feeder Cell Lines

Abstract

Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs, allowing us to efficiently target a transgene into the AAVS1 safe harbor locus in recently derived integration-free iPSCs. Our results have great implications in disease modeling and translational applications of human iPSCs, as these engineered human cell lines provide a more efficient tool for genetic modifications and a safer alternative for supporting self-renewal of human iPSCs and ESCs.

Diabetes mellitus and cellular replacement therapy: Expected clinical potential and perspectives

“…The transcription factors mediate β-cell renewing with diverse culturing methods [63] . In this context novel cellular strategies toward reprogramming may have better clinical prospects [64,65] . In has been expected that small molecules might be successful to be inducing pancreatic β-cell modification.…”

Efficient and Scalable Expansion of Human Pluripotent Stem Cells Under Clinically Compliant Settings: A View in 2013

“…For example, the hESCs used in the current RPE clinical trials were cultured on MEF feeder cells. 20 To eliminate xenogeneic contaminations, various types of human feeder cells from allogeneic muscle, skin, marrow, and endometrial cells, etc., 18,40,88,133 as well as from autogenic hESC-derived fibroblasts, 102,118 were developed in the early 2000s. Some of these new feeder cells also proved capable of supporting the derivation of new hPSC lines.…”

“…Some of these new feeder cells also proved capable of supporting the derivation of new hPSC lines. 40,118,133 …”

Promise and challenges of human iPSC-based hematologic disease modeling and treatment

“…Besides the virus-free integration-free reprogramming methods, conditions for mouse feeder-free iPSC culture are being constantly improved to make iPSCs safer and more suitable for clinical applications [37][38][39][40][41]. However, it is still a challenge to establish systems that can sufficiently support human iPSC self-renewal, genetic modifications and differentiation while compatible with clinical production under current Good Manufacturing Practice (cGMP) standards.…”