Repeated intravenous administration of hiPSC-MSCs enhance the efficacy of cell-based therapy in tissue regeneration.

We seek to demonstrate whether therapeutic efficacy can be improved by combination of repeated intravenous administration and local transplantation of human induced pluripotential stem cell derived MSCs (hiPSC-MSCs). In this study, mice model of hind-limb ischemia is established by ligation of left femoral artery. hiPSC-MSCs (5 × 105) is intravenously administrated immediately after induction of hind limb ischemia with or without following intravenous administration of hiPSC-MSCs every week or every 3 days. Intramuscular transplantation of hiPSC-MSCs (3 × 106) is performed one week after induction of hind-limb ischemia. We compare the therapeutic efficacy and cell survival of intramuscular transplantation of hiPSC-MSCs with or without a single or repeated intravenous administration of hiPSC-MSCs. Repeated intravenous administration of hiPSC-MSCs can increase splenic regulatory T cells (Tregs) activation, decrease splenic natural killer (NK) cells expression, promote the polarization of M2 macrophages in the ischemic area and improved blood perfusion in the ischemic limbs. The improved therapeutic efficacy of MSC-based therapy is due to both increased engraftment of intramuscular transplanted hiPSC-MSCs and intravenous infused hiPSC-MSCs. In conclusion, our study support a combination of repeated systemic infusion and local transplantation of hiPSC-MSCs for cardiovascular disease.

Liposome-encapsulated curcumin attenuates HMGB1-mediated hepatic inflammation and fibrosis in a murine model of Wilson's disease.

BACKGROUND AND AIMS: Wilson's disease (WD) is an inherited disorder of copper metabolism with predominant hepatic manifestations. Left untreated, it can be fatal. Current therapies focus on treating copper overload rather than targeting the pathophysiology of copper-induced liver injuries. We sought to investigate whether liposome-encapsulated curcumin (LEC) could attenuate the underlying pathophysiology of WD in a mouse model of WD. APPROACH AND RESULTS: Subcutaneous administration in a WD mouse model with ATP7B knockout (Atp7b-/-) resulted in robust delivery of LEC to the liver as determined by in-vitro and in-vivo imaging. Treatment with LEC attenuated hepatic injuries, restored lipid metabolism and decreased hepatic inflammation and fibrosis, and thus hepatosplenomegaly in Atp7b-/- mice. Mechanistically, LEC decreased hepatic immune cell and macrophage infiltration and attenuated the hepatic up-regulation of p65 by preventing cellular translocation of high-mobility group box-1 (HMGB-1). Moreover, decreased translocation of HMGB1 was associated with reduced splenic CD11b+/CD43+/Ly6CHi inflammatory monocyte expansion and circulating level of proinflammatory cytokines. Nevertheless there was no change in expression of oxidative stress-related genes or significant copper chelation effect of LEC in Atp7b-/- mice. CONCLUSION: Our results indicate that treatment with subcutaneous LEC can attenuate copper-induced liver injury in an animal model of WD via suppression of HMGB1-mediated hepatic and systemic inflammation. These findings provide important proof-of-principle data to develop LEC as a novel therapy for WD as well as other inflammatory liver diseases.

Generation of a human iPSC line CIBi010-A with a reporter for ASGR1 using CRISPR/Cas9.

ASGR1 is a liver-specific surface marker that has been used to purify human pluripotent stem cell (PSC)-derived hepatocytes (iHeps). Furthermore, ASGR1+ iHeps represents a more mature subpopulation of iHeps. To utilize this marker for optimizing iHep differentiation and purification, we substituted the stop coden of ASGR1 with a fluorescent reporter protein mCherry in a human iPSC line iPSN0052 via CRISPR/Cas9-mediated homologus recombination. The generated CIBi010-A enableds us to monitor ASGR1 expression during hepatic differentiation and thus can be used to optimize our hepatic differentiation procedures.

Generation of Human Liver Chimeric Mice and Harvesting of Human Hepatocytes from Mouse Livers.

Primary human hepatocytes (PHHs) are widely used as an in vitro model to evaluate various aspects of human hepatic physiology and pathology. However, PHHs isolated from the human liver have very limited ability for ex vivo expansion in culture. Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice are proven to be an ideal bioincubator for repopulation of PHHs. The human liver chimeric FRG mouse is not only a humanized animal model for disease study and drug screening in vivo, but also a potential source of PHHs for cellular therapy. This chapter describes experimental protocols to generate chimeric FRG mice with humanized liver and to isolate PHHs from human liver chimeric FRG mice. Using these methods, PHHs can be expanded to more than 100-fold for harvesting.

CRISPR-targeted genome editing of human induced pluripotent stem cell-derived hepatocytes for the treatment of Wilson's disease.

BACKGROUND & AIMS: Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism caused by loss-of-function mutations in ATP7B, which encodes a copper-transporting protein. It is characterized by excessive copper deposition in tissues, predominantly in the liver and brain. We sought to investigate whether gene-corrected patient-specific induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps) could serve as an autologous cell source for cellular transplantation therapy in WD. METHODS: We first compared the in vitro phenotype and cellular function of ATP7B before and after gene correction using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) in iHeps (derived from patients with WD) which were homozygous for the ATP7B R778L mutation (ATP7BR778L/R778L). Next, we evaluated the in vivo therapeutic potential of cellular transplantation of WD gene-corrected iHeps in an immunodeficient WD mouse model (Atp7b -/- / Rag2 -/- / Il2rg -/- ; ARG). RESULTS: We successfully created iPSCs with heterozygous gene correction carrying 1 allele of the wild-type ATP7B gene (ATP7BWT/-) using CRISPR/Cas9 and ssODNs. Compared with ATP7BR778L/R778L iHeps, gene-corrected ATP7BWT/- iHeps restored i n vitro ATP7B subcellular localization, its subcellular trafficking in response to copper overload and its copper exportation function. Moreover, in vivo cellular transplantation of ATP7BWT/- iHeps into ARG mice via intra-splenic injection significantly attenuated the hepatic manifestations of WD. Liver function improved and liver fibrosis decreased due to reductions in hepatic copper accumulation and consequently copper-induced hepatocyte toxicity. CONCLUSIONS: Our findings demonstrate that gene-corrected patient-specific iPSC-derived iHeps can rescue the in vitro and in vivo disease phenotypes of WD. These proof-of-principle data suggest that iHeps derived from gene-corrected WD iPSCs have potential use as an autologous ex vivo cell source for in vivo therapy of WD as well as other inherited liver disorders. LAY SUMMARY: Gene correction restored ATP7B function in hepatocytes derived from induced pluripotent stem cells that originated from a patient with Wilson's disease. These gene-corrected hepatocytes are potential cell sources for autologous cell therapy in patients with Wilson's disease.

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes.

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.