Abstract
Reprogramming a patient’s somatic cells into induced pluripotent stem cells (iPSCs) holds great promise for disease modeling and the development of autologous cellular therapeutics. However, it remains challenging to consistently reprogram primary human cells, as they are frequently aged, diseased, or in low abundance. Here we present a modified highly efficient and clinically relevant RNA-based method for reprogramming disease-associated and other difficult-to-reprogram human primary fibroblast lines into iPSCs. We also describe optimizations that can be employed for consistent reprogramming of these difficult-to-reprogram cells. With the provided protocol, integration-free iPSC lines can be successfully generated from a small number of primary human fibroblasts in approximately 5–7 weeks.
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Abbreviations
- bFGF:
-
basic fibroblast growth factor
- DPBS:
-
Dulbecco’s phosphate buffered saline
- FBS:
-
fetal bovine serum
- FEM:
-
fibroblasts expansion media
- iPSC:
-
induced pluripotent stem cell
- MEM:
-
minimum essential media
- mod-mRNA:
-
modified mRNA
- ReproM:
-
reprogramming media
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Acknowledgments
We are grateful for funding support from the National Institutes of Health (P30 AR057212 and R21 AR074642). We also thank Epidermolysis Bullosa (EB) Research Partnership, the EB Medical Research Foundation, the Cure EB Charity, Dystrophic Epidermolysis Bullosa Research Association (DEBRA) International and the Gates Frontiers Fund.
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McGrath, P.S., McGarvey, S.S., Kogut, I., Bilousova, G. (2020). Efficient RNA-Based Reprogramming of Disease-Associated Primary Human Fibroblasts into Induced Pluripotent Stem Cells. In: Kidder, B. (eds) Stem Cell Transcriptional Networks. Methods in Molecular Biology, vol 2117. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0301-7_17
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DOI: https://doi.org/10.1007/978-1-0716-0301-7_17
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