A Comparative View on Human Somatic Cell Sources for iPSC Generation.
paper
Cited
Public
- Authors
- Raab, Stefanie; Klingenstein, Moritz; Liebau, Stefan; Linta, Leonhard
- Year
- 2014
- Journal
- Stem cells international
- PMID
- 25431601
- DOI
- 10.1155/2014/768391
- PMCID
- PMC4241335
The breakthrough of reprogramming human somatic cells was achieved in 2006 by the work of Yamanaka and Takahashi. From this point, fibroblasts are the most commonly used primary somatic cell type for the generation of induced pluripotent stem cells (iPSCs). Various characteristics of fibroblasts supported their utilization for the groundbreaking experiments of iPSC generation. One major advantage is the high availability of fibroblasts which can be easily isolated from skin biopsies. Furthermore, their cultivation, propagation, and cryoconservation properties are uncomplicated with respect to nutritional requirements and viability in culture. However, the required skin biopsy remains an invasive approach, representing a major drawback for using fibroblasts as the starting material. More and more studies appeared over the last years, describing the reprogramming of other human somatic cell types. Cells isolated from blood samples or urine, as well as more unexpected cell types, like pancreatic islet beta cells, synovial cells, or mesenchymal stromal cells from wisdom teeth, show promising characteristics for a reprogramming strategy. Here, we want to highlight the advantages of keratinocytes from human plucked hair as a widely usable, noninvasive harvesting method for primary material in comparison with other commonly used cell types.
Reprogramming of keratinocytes. (1) Plucked hair is cultured in flasks until outgrowth of keratinocytes. Those are transferred to a six-well plate. (2) Keratinocytes are infected with a lentivirus containing the four reprogramming factors OCT4, SOX2, KLF4, and C-MYC. (3) Infected keratinocytes are transferred to a plate with feeder cells (e.g., rat embryonic fibroblasts) in reprogramming medium. (4) After two to three weeks, stem cell colonies arise, and the uninfected keratinocytes do not proliferate in the reprogramming medium. (5) When the stem cell colonies reach a certain size, they are picked mechanically and a feeder-free system may be used for cultivating the human iPSCs.
Structure of a hair follicle embedded in skin. Human plucked scalp hair lies in the dermis. Under culture conditions, keratinocytes will grow out of the outer root sheath.
Comparison between plucked hair with good and bad roots. (a) Plucked hair with outer root sheath in the anagen growth phase transferred to a Matrigel coated culture flask. Keratinocytes grow out after 3β7 days. (b) Plucked hair without outer sheath root in katagen growth phase. No keratinocytes will grow out.
Culture conditions of keratinocytes in different media. (a) Keratinocytes proliferate over time in a low-calcium keratinocyte medium. (b) In reprogramming medium, the keratinocytes stop proliferating and get senescent while iPSCs form colonies and grow.
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