oa Hallmarks of regenerative medicine: Improved methods For reprogramming human fibroblasts using fluorescence activated cell sorting

Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on complex protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the combination of CD13NEGSSEA4POSTra-1-60POS cell surface markers between 7-10 days post infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and "contaminating" partially reprogrammed fibroblasts, substantially reducing the time and reagents required to generate iPSC lines. iPSC lines derived under this technology produced more unique and stable clones following retroviral infection than manual picking methods, expressed common markers of pluripotency at later passages, and possessed spontaneous differentiation potential in vitro and in vivo. FACS derivation produced iPSC lines had a normal karyotype and matched the parental DNA fingerprint. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines from a variety of 76 tissue sources that ranged in diseases from Cardiovascular disorders, neurological disorders and diabetes using either integrating (retroviral) or non-integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. This demonstrates feasibility of creating highly pure iPSCs for cellular therapeutics, drug screening, and understanding human disease phenotypes.