Feeder- and serum-free establishment and expansion of human induced pluripotent stem cells.
Although human induced pluripotent stem cells (hiPSCs) hold great promise as a source of differentiated cells for vast therapeutic implications, many obstacles still need to be surmounted before this can become a reality. One obstacle, a robust feeder- and serum-free system to generate and expand hiPSCs in culture is still unavailable. Here, for the first time, we describe a novel establishment and maintenance culture technique that uses human dermal fibroblasts to generate hiPSCs by introducing four factors, Klf4, Oct4, Sox2, and c-Myc under serum- and feeder-independent conditions. We have used a serum replacement product, conditioned medium (CM), or feeder-free medium (FFM) supplemented with high elevated basic-fibroblast growth factor in the absence or presence of Matrigel. Our FFM system in the presence of Matrigel enhanced the efficiency of alkaline phosphatase-positive colonies at a frequency at least 10-fold greater than the conventional method on feeder cells. The established hiPSCs are similar to human embryonic stem cells in many aspects including morphology, passaging, surface and pluripotency markers, normal karyotype, gene expression, ultrastructure, and in vitro differentiation. Such hiPSCs could be useful particularly in the context of in vitro disease modeling, pharmaceutical screening and in cellular replacement therapies once the safety issues have been overcome.
Totonchi
Mehdi
M
Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Taei
Adeleh
A
Seifinejad
Ali
A
Tabebordbar
Mohammadsharif
M
Rassouli
Hassan
H
Farrokhi
Ali
A
Gourabi
Hamid
H
Aghdami
Nasser
N
Hosseini-Salekdeh
Ghasem
G
Baharvand
Hossein
H
D000328:Adult; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D007621:Karyotyping; D000090062:Kruppel-Like Factor 4; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016895:Culture Media, Serum-Free; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4