Abstract
Genomic manipulation of human pluripotent stem cells (hPSCs) has become essential to introduce genetic modifications and transgenes, and develop reporter lines. One of the major bottlenecks in gene editing is at the stage of single-cell cloning, which is thought to be variable across hPSC lines and is substantially reduced following a transfection. Due to the difficulty of performing fluorescent-assisted cell sorting (FACS) for single-cell isolation of hPSCs, previous approaches rely on manual colony picking, which is both time-consuming and labor-intensive. In this protocol, I provide a method for utilizing FACS to generate single-cell clones of hPSCs with efficiencies approaching 40% within 7–10 days. This can be achieved by sorting cells onto a feeder layer of MEFs in a stem cell defined medium with KSR and a Rock inhibitor, as early as 1–2 days following a transfection, streamlining the gene editing process. The approach described here provides a fundamental method for all researchers utilizing hPSCs for scientific studies.
Highlights
Human pluripotent stem cells, including human embryonic stem cells, and induced pluripotent stem cells, have become an essential cellular model system for research that span the spectrum of studies from developmental biology up through translational and therapeutic investigations (Yamanaka, 2012; Wu and Izpisua Belmonte, 2015; Rossant and Tam, 2017)
Serum-free, defined media formulations were developed such as mTESR (Ludwig et al, 2006), or HAIF (Wang et al, 2007; Singh et al, 2012), which is marketed as StemPRO human embryonic stem cells (hESCs) SFM by Thermo Fisher Scientific
We have found that cell viability is consistently reduced following these transfection approaches compared to un-transfected cells, and always results in lower single-cell cloning efficiency on Geltrex or Matrigel than on mouse embryonic fibroblasts (MEFs)
Summary
Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs), have become an essential cellular model system for research that span the spectrum of studies from developmental biology up through translational and therapeutic investigations (Yamanaka, 2012; Wu and Izpisua Belmonte, 2015; Rossant and Tam, 2017) These studies have been enabled, in part, due to considerable improvements in the techniques to passage and grow the cells. It should be noted that non-enzymatic approaches typically are more akin to manual passaging, in that cells are passaged as “clumps,” while enzymatic approaches, such as Accutase or TrypLE Select, permit single-cell disassociation prior to seeding of the cells Overall, these improvements in growing and passaging hPSCs have facilitated the use of these cells for numerous research disciplines.
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