Abstract
Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions, and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs.
Highlights
Since the derivation of human embryonic stem cells [1], their growth and maintenance in culture have remained challenging
The enhanced culture platform (ECP) was the combination of a feeder free culture system utilizing Geltrex [5], TeSR defined media [7], Accutase [8] to dissociate and detach cells and Rock-Inhibitor (Y-27632) [6] to stabilize the subsequent intermediate single cell state
In order to establish that the ECP was capable of maintaining the pluripotency and genetic integrity of hPSCs over extended culturing, we passaged HUES9 [9] and BJ-RiPSC [10] cells over 15 times using the ECP
Summary
Since the derivation of human embryonic stem cells [1], their growth and maintenance in culture have remained challenging. The ECP improved replating efficiencies and viability of single-cells when passaging hPSCs. This culture platform increased the viability of hPSCS after freezing and thawing.
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