To develop a clearer understanding of the pathophysiology of FH iPSC–derived hepatocytes, learn more we reprogrammed fibroblasts from JD, a 14-year-old boy with cutaneous

xanthomatosis and advanced cardiovascular disease.13 The choice to generate JD hiPSCs was considered historically relevant because Brown, Goldstein, and colleagues, in establishing the LDLR paradigm, studied JD fibroblasts extensively.10, 11 We produced several JD iPSC lines by transducing primary fibroblasts with lentiviral vectors encoding the transcription factors OCT4, SOX2, NANOG, and LIN2814 and demonstrated that they expressed characteristic markers of pluripotency (Fig. 1A). In each hiPSC line, we confirmed the retention of the JD LDLR mutations (Fig. 1B, Supporting Fig. 1), established that each had a normal karyotype (Fig. 1C), and determined that each JD hiPSC line could differentiate into derivatives of all three germ layers using teratoma assays AZD1208 (Fig. 1D). Using a previously described protocol (Fig. 2A), which we had shown could generate functional hepatocyte-like cells (referred to here as hepatocytes),4, 9 we demonstrated that each JD hiPSC clone was capable of directed differentiation toward a hepatic fate. On day 20 of differentiation, the morphology of both control hiPSC– and JD hiPSC–derived cells was indistinguishable

and closely resembled that of hepatocytes, including the presence of click here lipid vesicles, a high cytoplasmic to nuclear ratio, granular cytoplasm, and prominent nucleoli (Fig. 2B). In addition, the differentiated cells expressed hepatocyte markers, including hepatocyte nuclear factor 4a (HNF4a) and albumin (Fig. 2C). Flow cytometric analyses of hepatocytes from both control and JD hiPSCs confirmed that the cells differentiated into asialoglycoprotein receptor (ASGPR1)-positive

hepatocytes with comparable efficiency (Fig. 2D). Only cells expressing high levels of ASGPR1 were counted to avoid the possibility of counting false negatives. Finally, hepatocytes derived from control hESCs or hiPSCs as well as JD hiPSCs were found to express hepatic mRNAs at similar levels, whereas expression of each of these mRNAs was not detected in undifferentiated hESCs (Fig. 2E). Based on these data, we conclude that JD iPSCs could be directed to form cells with hepatocyte characteristics at efficiencies that were comparable to hESCs or control hiPSCs. The FH associated with JD is a consequence of compound heterozygosity at the LDLR locus. JD inherited a maternal allele containing a 5-kb deletion spanning part of exon 13 and all of exons 14 and 15 that results in the absence of functional protein.13 The inherited paternal allele contains an A>G transition within exon 17, which encodes a tyrosine>cysteine substitution at residue 807 in the LDLR cytoplasmic domain resulting in a mutant protein that can still bind LDL, but is inefficiently internalized.