Familial hypercholesterolemia (FH) is a hereditary disease primarily due to mutations in the low‐density lipoprotein receptor (LDLR) that lead to elevated cholesterol and premature development of cardiovascular disease. Homozygous FH patients (HoFH) with two dysfunctional LDLR alleles are not as successfully treated with standard hypercholesterol therapies, and more aggressive therapeutic approaches to control cholesterol levels must be considered. Liver transplant can resolve HoFH, and hepatocyte transplantation has shown promising results in animals and humans. However, demand for donated livers and high‐quality hepatocytes overwhelm the supply. Human pluripotent stem cells can differentiate to hepatocyte‐like cells (HLCs) with the potential for experimental and clinical use. To be of future clinical use as autologous cells, LDLR genetic mutations in derived FH‐HLCs need to be corrected. Genome editing technology clustered‐regularly‐interspaced‐short‐palindromic‐repeats/CRISPR‐associated 9 (CRISPR/Cas9) can repair pathologic genetic mutations in human induced pluripotent stem cells. Conclusion: We used CRISPR/Cas9 genome editing to permanently correct a 3‐base pair homozygous deletion in LDLR exon 4 of patient‐derived HoFH induced pluripotent stem cells. The genetic correction restored LDLR‐mediated endocytosis in FH‐HLCs and demonstrates the proof‐of‐principle that CRISPR‐mediated genetic modification can be successfully used to normalize HoFH cholesterol metabolism deficiency at the cellular level. (Hepatology Communications 2017;1:886–898)

the tumor microenvironment (tMe) is composed of a heterogeneous biological ecosystem of cellular and non-cellular elements including transformed tumor cells, endothelial cells, immune cells, activated fibroblasts or myofibroblasts, stem and progenitor cells, as well as the cytokines and matrix that they produce. The constituents of the TME stroma are multiple and varied, however cancer associated fibroblasts (CAF) and their contribution to the TME are important in tumor progression. CAF are hypothesized to arise from multiple progenitor cell types, including mesenchymal stem cells. Currently, isolation of tMe stroma from patients is complicated by issues such as limited availability of biopsy material and cell stress incurred during lengthy adaptation to atmospheric oxygen (20% O2) in cell culture, limiting pre-clinical studies of patient tumor stromal interactions. Here we describe a microenvironment mimetic in vitro cell culturing system that incorporates elements of the in vivo lung environment, including lung fibroblast derived extracellular matrix and physiological hypoxia (5% O2). Using this system, we easily isolated and rapidly expanded stromal progenitors from patient lung tumor resections without complex sorting methods or growth supplements. These progenitor populations retained expression of pluripotency markers, secreted factors associated with cancer progression, and enhanced tumor cell growth and metastasis. An understanding of the biology of these progenitor cell populations in a tMe-like environment may advance our ability to target these cells and limit their effects on promoting cancer metastasis.

Low-density lipoprotein (LDL) receptor (LDLR) mutations are the primary cause of familial hypercholesterolemia (FH). Class II LDLR mutations result in a misfolded LDLR retained in the endoplasmic reticulum (ER). We have developed a model of FH class II and CRISPR-corrected induced pluripotent stem cells (iPSC) capable of replicating mutant and repaired LDLR functions. We show here that iPSC and derived hepatocyte-like cells (HLC) replicate misfolded LDLR accumulation and restoration of LDLR function in CRISPRcorrected cells. It was reported that model cells overexpressing class II LDLR mutants result in endoplasmic reticulum (ER) accumulation of immature LDLR and activation of the unfolded protein response (UPR). We show here that statins induce a similar accumulation of immature LDLR that is resolved with class II correction. We also demonstrate that, although capable of UPR induction with tunicamycin treatment, unlike overexpression models, statin-treated class II iPSC and derived HLC do not induce the common UPR markers Grp78 (also known as HSPA5) or spliced XBP1 [XBP1 (S)]. Because statins are reported to inhibit UPR, we utilized lipoproteindeficient serum (LPDS) medium, but still did not detect UPR induction at the Grp78 and XBP1 (S) levels. Our study demonstrates the recapitulation of mutant and corrected class II LDLR function and suggests that overexpression models may not accurately predict statin-mediated class II protein biology.