Human induced pluripotent stem (iPS) cells are generated by reprogramming somatic cells via the temporary overexpression of reprogramming factors, such as the Yamanaka factors. Traditionally, reprogramming conditions involve using mouse feeder layers and undefined media containing animal derived components. Recent improvements to make this process more defined include using
, a reprogramming media which is serum- and xeno-free, and is based on the published formulation
from Dr. James Thomson’s lab at the University of Wiscosin-Madison. Below you will find tips to help resolve two common issues encountered when reprogramming fibroblasts using TeSR™-E7™.
Problem 1: Fibroblast-like cell overgrowth.
Sometimes, when small to medium size iPS cell colonies arise during the induction phase, there may be a lot of densely packed fibroblast-like cells that appear to be growing around the colony (Figure 1A). As the latter could impact the expansion of the iPS cell colony prior to selection, it is advisable to clear away some of the densely packed fibroblast-like cells using a 22 gauge needle or a pulled-glass pipette, allowing the iPS cell colony more room to expand (Figure 1B).
Problem 2: When to pick colonies?
As iPS cell colonies arise at different rates during the induction phase, it is advisable that colonies be picked as they arise and transferred to
for further expansion and analysis.
The best way to determine whether a colony is ready to be picked is to identify colonies with ES-like morphology:
- well-defined colony borders
- tightly packed cells
- high nuclear-to-cytoplasmic ratio
- prominent nucleoli
Prior to picking an iPS cell colony, free the colony from the surrounding fibroblast-like cells by pulling the surrounding cells away, using a 22 gauge needle or a pulled-glass pipette.
If the iPS cell colony begins to shift or detach entirely when picking, transfer the entire colony to a vial and triturate before then transferring to a new well. Customers should consider leaving remaining colonies to grow more before selection.
For a detailed protocol on how to use TeSR™-E7™ with an episomal vector system for reprogramming fibroblasts, please refer to our
We have demonstrated successful reprogramming of fibroblasts, culture expanded hematopoietic cells, as well as mesenchymal stem cells in TeSR™-E7™ using an episomal vector system as shown in this
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