Technology

Reprogramming a somatic cell to a pluripotent stem cell

Human pluripotent stem cells including embryonic stem cells (hESC) and induced stem cells (hiPSC)
have unlimited ability for self-renewal and can differentiate to all cell types of the three germ layers,
which make them well suited for generating autologous and custom-tailored cells for regenerative
medicine and to produce in vitro disease models to study pathogenesis, treatments, drug screening,
and toxicology. While hESCs are derived from inner-cell mass of early embryos, hiPSCs are generated
by artificially converting non-pluripotent somatic cells to pluripotency by forced expression of specific
key pluripotent stem cell transcription factor genes including Oct-4, Sox2, c-MYC, and KLF4. Here, we
show a step-by-step production of iPSC from an established human foreskin fibroblast (ATCC # CRL
2522) using non-integrating Sendai virus reprogramming Kit (LifeTechnology # A13780-01).

 

  1. Plate 150-200,000 fibroblasts in 2 wells of 6-well plate
    Transfect with Sendai virus carrying Oct4, Sox2, KLF4, c-MYC
  2. After 7 days, harvest transfected cells and plate 200000, 100000, 50000 cells in each of 6 10-cm dishes coated with mouse embryonic fibroblasts (MEF) cells
  3. After 3-4 weeks, pick “good” colonies and re-plate in MEF coated 12-well plates (one colony in each well)
  4. After few days when the colonies are large, pick “good” colonies from 12-well plates and re-plate into 6-wellplates coated with MEF cells
  5. Harvest “good colonies” from each of 6-well plates into 10- cm dishes using collagenase enzyme (25-35 minutes). Collagenase concentration and time of incubation need to be optimized to detach only colonies leaving behind MEF cells.
  6. Collect colonies and cryo-store.

 

IPS progression 1 IPS progression 2 IPS progression 3 IPS progression 4
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Oct-4 expression of fully reprogrammed iPSC

Graphic Description of Process

reprogramming somatic cells