Human fibroblasts were used for the reprogramming with Nucleofector 2b and Human dermal fibroblast Nucleofector kit. To Nucleofect human fibroblasts with Yamanaka episomal reprogramming plasmids, prepare Nucleofector solution plus DNA plasmids. Combine a total of 8 µg of the episomal vectors (pCXLE-hOCT3/4-shp53 (Addgene, cat. no. 27077), pCXLE-hSK (Addgene, cat. no. 27078) and pCXLE-EGFP (Addgene, cat. no. 27082)).
Induction of tissue-specific cell types via a conventional transdifferentiation strategy typically uses overexpression of the corresponding lineage-specific transcription factors. Alternatively, somatic cells can be temporarily activated via a common set of reprogramming factors into a transition state, which can then be directed into various cell types via soluble lineage-specific signals, without establishing a pluripotent state. Here, we provide protocols for the generation of cardiomyocytes, neural stem cells and hepatocytes from fibroblasts with such a cell activation (CACA) and signaling-directed (SD; CASCASCASD) strategy. In these protocols, beating cardiomyocytes can be induced from mouse fibroblasts in 2–5 weeks; expandable neural stem cells and definitive endoderm progenitors can be obtained from human fibroblasts as early as 2.5 weeks; and human definitive endoderm progenitors can be differentiated into functional hepatocytes in 2 weeks. Through further developments, the CASCASCASD strategy can serve as a unique avenue for generating diverse functional cell types for biomedical research and therapeutic applications.