a-familial-hypercholesterolemia-human-liver-chimeric-mouse-model

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 ...

University of Hong Kong

Patients with rheumatoid arthritis (RA) are prone to premature atherosclerosis. We hypothesize that depletion of circulating endothelial progenitor cells (EPC) related to RA can contribute to the development of atherosclerosis.

Role of circulating endothelial progenitor cells in patients with rheumatoid arthritis with coronary calcification.

Embryonic stem cells (ESCs) can differentiate into functional cardiomyocytes and thus represent a promising cell source for cardiac regenerative therapy. Nevertheless, the therapeutic application of ESC-derived cardiomyocytes is limited by the low efficacy of the current protocol for cardiac differentiation and their immature phenotypes. Although thyroid hormone is essential for normal cardiac development and function, its role in the cardiac differentiation of ESCs, as well as the maturation of ESC-derived cardiomyocytes, remains unclear. In this study, we examined the cardiac differentiation of murine ESCs in the presence of T(3) for 7 d using flow cytometry, RT-PCR, cellular electrophysiology study, and confocal calcium imaging. Compared with control conditions, T(3) supplementation increased the number of ESC-derived cardiomyocytes and was accompanied by up-regulation of a panel of cardiac markers, including Nkx2.5, myosin light chain-2V, as well as alpha- and beta-myosin heavy chain. More importantly, electrophysiological study revealed that ESC-derived cardiomyocytes exhibited more adult-like phenotypes after T(3) supplementation based on action potential characteristics. They also exhibited more adult-like calcium homeostasis properties. These phenotypic changes were associated with up-regulation of sarco(endo)plasmic reticulum calcium ATPase-2a and ryanodine receptor-2 expression. In addition, the classical (genomic) pathway was shown to be involved in T(3)-induced cardiac differentiation of ESCs. Our results show that T(3) supplementation promotes cardiac differentiation of ESCs and enhances maturation of electrophysiological, as well as calcium homeostasis, properties of ESC-derived cardiomyocytes.

Triiodothyronine promotes cardiac differentiation and maturation of embryonic stem cells via the classical genomic pathway.

Vitamin D (Vit-D) deficiency is associated with type 2 diabetes mellitus (DM) and endothelial dysfunction. The relationship of Vit-D deficiency with circulating endothelial progenitor cells and endothelial dysfunction in type 2 DM patients nonetheless remains unclear.

Vitamin D deficiency is associated with depletion of circulating endothelial progenitor cells and endothelial dysfunction in patients with type 2 diabetes.

Suboptimal vitamin D status is associated with endothelial dysfunction and an increased risk of cardiovascular diseases but it is unclear whether vitamin D supplementation is beneficial. The aim was to investigate the effect of vitamin D supplementation on endothelial function in patients with type 2 diabetes mellitus (DM).

Randomized controlled trial of vitamin D supplement on endothelial function in patients with type 2 diabetes.

We identified an autosomal dominant non‐sense mutation (R225X) in exon 4 of the lamin A/C (LMNA) gene in a Chinese family spanning 3 generations with familial dilated cardiomyopathy (DCM). In present study, we aim to generate induced pluripotent stem cells derived cardiomyocytes (iPSC‐CMs) from an affected patient with R225X and another patient bearing LMNA frame‐shift mutation for drug screening.

Modeling of lamin A/C mutation premature cardiac aging using patient‐specific induced pluripotent stem cells.

Functional endothelial-like cells (EC) have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however, their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC), human embryonic stem cells (hESC-EC) and human induced pluripotent stem cells (hiPSC-EC), and compared their in-vitro tube formation, migration and cytokine expression profiles, and in-vivo capacity to attenuate hind-limb ischemia in mice. Successful differentiation of BM-EC was only achieved in 1/6 patient with severe coronary artery disease. Nevertheless, BM-EC, hESC-EC and hiPSC-EC exhibited typical cobblestone morphology, had the ability of uptaking DiI-labeled acetylated low-density-lipoprotein, and binding of Ulex europaeus lectin. In-vitro functional assay demonstrated that hiPSC-EC and hESC-EC had similar capacity for tube formation and migration as human umbilical cord endothelial cells (HUVEC) and BM-EC (P>0.05). While increased expression of major angiogenic factors including epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor, placental growth factor and stromal derived factor-1 were observed in all EC cultures during hypoxia compared with normoxia (P<0.05), the magnitudes of cytokine up-regulation upon hypoxic were more dramatic in hiPSC-EC and hESC-EC (P<0.05). Compared with medium, transplanting BM-EC (n = 6), HUVEC (n = 6), hESC-EC (n = 8) or hiPSC-EC (n = 8) significantly attenuated severe hind-limb ischemia in mice via enhancement of neovascularization. In conclusion, functional EC can be generated from hECS and hiPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases, and hESC-EC or iPSC-EC are readily available as "off-the-shelf" format for the treatment of tissue ischemia.

Attenuation of hind-limb ischemia in mice with endothelial-like cells derived from different sources of human stem cells.

Blood lipids are important risk factors for coronary artery disease (CAD). Here we perform an exome-wide association study by genotyping 12,685 Chinese, using a custom Illumina HumanExome BeadChip, to identify additional loci influencing lipid levels. Single-variant association analysis on 65,671 single nucleotide polymorphisms reveals 19 loci associated with lipids at exome-wide significance (P<2.69 × 10(-7)), including three Asian-specific coding variants in known genes (CETP p.Asp459Gly, PCSK9 p.Arg93Cys and LDLR p.Arg257Trp). Furthermore, missense variants at two novel loci-PNPLA3 p.Ile148Met and PKD1L3 p.Thr429Ser-also influence levels of triglycerides and low-density lipoprotein cholesterol, respectively. Another novel gene, TEAD2, is found to be associated with high-density lipoprotein cholesterol through gene-based association analysis. Most of these newly identified coding variants show suggestive association (P<0.05) with CAD. These findings demonstrate that exome-wide genotyping on samples of non-European ancestry can identify additional population-specific possible causal variants, shedding light on novel lipid biology and CAD. 

Exome-wide association analysis reveals novel coding sequence variants associated with lipid traits in Chinese.

Danon disease is an X-linked disorder that leads to fatal cardiomyopathy caused by a deficiency in lysosome-associated membrane protein-2 (LAMP2). In female patients, a later onset and less severe clinical phenotype have been attributed to the random inactivation of the X chromosome carrying the mutant diseased allele. We generated a patient-specific induced pluripotent stem cell (iPSCs)-based model of Danon disease to evaluate the therapeutic potential of Xi-chromosome reactivation using a DNA methylation inhibitor.

Amelioration of X-Linked Related Autophagy Failure in Danon Disease With DNA Methylation Inhibitor.

Familial hypercholesterolemia (FH) causes elevation of low-density lipoprotein cholesterol (LDL-C) in blood and carries an increased risk of early-onset cardiovascular disease. A caveat for exploration of new therapies for FH is the lack of adequate experimental models. We have created a comprehensive FH stem cell model with differentiated hepatocytes (iHeps) from human induced pluripotent stem cells (iPSCs), including genetically engineered iPSCs, for testing therapies for FH. We used FH iHeps to assess the effect of simvastatin and proprotein convertase subtilisin/kexin type 9 (PCSK9) antibodies on LDL-C uptake and cholesterol lowering in vitro. In addition, we engrafted FH iHeps into the liver of Ldlr/Rag2/Il2rg mice, and assessed the effect of these same medications on LDL-C clearance and endothelium-dependent vasodilation in vivo. Our iHep models recapitulate clinical observations of higher potency of PCSK9 antibodies compared with statins for reversing the consequences of FH, demonstrating the utility for preclinical testing of new therapies for FH patients.

Generation of Human Liver Chimeric Mice with Hepatocytes from Familial Hypercholesterolemia Induced Pluripotent Stem Cells.

Precision medicine is an emerging approach to disease treatment and prevention that takes into account individual variability in the environment, lifestyle, and genetic makeup of patients. Patient-specific human induced pluripotent stem cells hold promise to transform precision medicine into real-life clinical practice. Lamin A/C (LMNA)-related cardiomyopathy is the most common inherited cardiomyopathy in which a substantial proportion of mutations in the  gene are of nonsense mutation. PTC124 induces translational read-through over the premature stop codon and restores production of the full-length proteins from the affected genes. In this study we generated human induced pluripotent stem cells-derived cardiomyocytes from patients who harbored different  mutations (nonsense and frameshift) to evaluate the potential therapeutic effects of PTC124 in -related cardiomyopathy.

Modeling Treatment Response for Lamin A/C Related Dilated Cardiomyopathy in Human Induced Pluripotent Stem Cells.

Patients with Danon disease may suffer from severe cardiomyopathy, skeletal muscle dysfunction as well as varying degrees of mental retardation, in which the primary deficiency of lysosomal membrane-associated protein-2 (LAMP2) is considerably associated. Owing to the scarcity of human neurons, the pathological role of LAMP2 deficiency in neural injury of humans remains largely elusive. However, the application of induced pluripotent stem cells (iPSCs) may shed light on overcoming such scarcity. In this study, we obtained iPSCs derived from a patient carrying a mutated  gene that is associated with Danon disease. By differentiating such LAMP2-deficient iPSCs into cerebral cortical neurons and with the aid of various biochemical assays, we demonstrated that the LAMP2-deficient neurons are more susceptible to mild oxidative stress-induced injury. The data from MTT assay and apoptotic analysis demonstrated that there was no notable difference in cellular viability between the normal and LAMP2-deficient neurons under non-stressed condition. When exposed to mild oxidative stress (10 μM HO), the LAMP2-deficient neurons exhibited a significant increase in apoptosis. Surprisingly, we did not observe any aberrant accumulation of autophagic materials in the LAMP2-deficient neurons under such stress condition. Our results from cellular fractionation and inhibitor blockade experiments further revealed that oxidative stress-induced apoptosis in the LAMP2-deficient cortical neurons was caused by increased abundance of cytosolic cathepsin L. These results suggest the involvement of lysosomal membrane permeabilization in the LAMP2 deficiency associated neural injury.

Lysosomal membrane permeabilization is involved in oxidative stress-induced apoptotic cell death in LAMP2-deficient iPSCs-derived cerebral cortical neurons.

Article	Year
Role of circulating endothelial progenitor cells in patients with rheumatoid arthritis with coronary calcification. The Journal of rheumatology\| PubMed ID: 20080904	2010
Triiodothyronine promotes cardiac differentiation and maturation of embryonic stem cells via the classical genomic pathway. Molecular endocrinology (Baltimore, Md.)\| PubMed ID: 20667986	2010
Vitamin D deficiency is associated with depletion of circulating endothelial progenitor cells and endothelial dysfunction in patients with type 2 diabetes. The Journal of clinical endocrinology and metabolism\| PubMed ID: 21325459	2011
Randomized controlled trial of vitamin D supplement on endothelial function in patients with type 2 diabetes. Atherosclerosis\| PubMed ID: 23298824	2013
Modeling of lamin A/C mutation premature cardiac aging using patient‐specific induced pluripotent stem cells. Aging\| PubMed ID: 23362510	2012
Attenuation of hind-limb ischemia in mice with endothelial-like cells derived from different sources of human stem cells. PloS one\| PubMed ID: 23472116	2013
Exome-wide association analysis reveals novel coding sequence variants associated with lipid traits in Chinese. Nature communications\| PubMed ID: 26690388	2015
Amelioration of X-Linked Related Autophagy Failure in Danon Disease With DNA Methylation Inhibitor. Circulation\| PubMed ID: 27678261	2016
Generation of Human Liver Chimeric Mice with Hepatocytes from Familial Hypercholesterolemia Induced Pluripotent Stem Cells. Stem cell reports\| PubMed ID: 28262545	2017
Modeling Treatment Response for Lamin A/C Related Dilated Cardiomyopathy in Human Induced Pluripotent Stem Cells. Journal of the American Heart Association\| PubMed ID: 28754655	2017
Lysosomal membrane permeabilization is involved in oxidative stress-induced apoptotic cell death in LAMP2-deficient iPSCs-derived cerebral cortical neurons. Biochemistry and biophysics reports\| PubMed ID: 28955840	2016

Lai-Yung Wong

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