Jeanette Beers

Cardiovascular Pathology in Hutchinson-Gilford Progeria: Correlation With the Vascular Pathology of Aging

Objective—Children with Hutchinson-Gilford progeria syndrome (HGPS) exhibit dramatically accelerated cardiovascular disease (CVD), causing death from myocardial infarction or stroke between the ages of 7 and 20 years. We undertook the first histological comparative evaluation between genetically confirmed HGPS and the CVD of aging. Methods and Results—We present structural and immunohistological analysis of cardiovascular tissues from 2 children with HGPS who died of myocardial infarction. Both had features classically associated with the atherosclerosis of aging, as well as arteriolosclerosis of small vessels. In addition, vessels exhibited prominent adventitial fibrosis, a previously undescribed feature of HGPS. Importantly, although progerin was detected at higher rates in the HGPS coronary arteries, it was also present in non-HGPS individuals. Between the ages of 1 month and 97 years, progerin staining increased an average of 3.34% per year (P<0.0001) in coronary arteries. Conclusion—We find concordance among many aspects of cardiovascular pathology in both HGPS and geriatric patients. HGPS generates a more prominent adventitial fibrosis than typical CVD. Vascular progerin generation in young non-HGPS individuals, which significantly increases throughout life, strongly suggests that progerin has a role in cardiovascular aging of the general population.

Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions

This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (PSCs). In this protocol, passaging one six-well or 10-cm plate of cells takes about 6–7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization, centrifugation or drug treatment. It also allows for higher throughput, requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation, colony expansion, cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion, and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages, this procedure provides a consistent and universal approach to passaging human PSCs in E8 medium.

Disruption of Protein Arginine N-Methyltransferase 2 Regulates Leptin Signaling and Produces Leanness In Vivo Through Loss of STAT3 Methylation

Rationale: Arginine methylation by protein N-arginine methyltransferases (PRMTs) is an important posttranslational modification in the regulation of protein signaling. PRMT2 contains a highly conserved catalytic Ado-Met binding domain, but the enzymatic function of PRMT2 with respect to methylation is unknown. The JAK-STAT pathway is proposed to be regulated through direct arginine methylation of STAT transcription factors, and STAT3 signaling is known to be required for leptin regulation of energy balance. Objective: To identify the potential role of STAT3 arginine methylation by PRMT2 in the regulation of leptin signaling and energy homeostasis. Methods and Results: We identified that PRMT2−/− mice are hypophagic, lean, and have significantly reduced serum leptin levels. This lean phenotype is accompanied by resistance to food-dependent obesity and an increased sensitivity to exogenous leptin administration. PRMT2 colocalizes with STAT3 in hypothalamic nuclei, where it binds and methylates STAT3 through its Ado-Met binding domain. In vitro studies further clarified that the Ado-Met binding domain of PRMT2 induces STAT3 methylation at the Arg31 residue. Absence of PRMT2 results in decreased methylation and prolonged tyrosine phosphorylation of hypothalamic STAT3, which was associated with increased expression of hypothalamic proopiomelanocortin following leptin stimulation. Conclusions: These data elucidate a molecular pathway that directly links arginine methylation of STAT3 by PRMT2 to the regulation of leptin signaling, suggesting a potential role for PRMT2 antagonism in the treatment of obesity and obesity-related syndromes.

Neuropilin-1 Identifies Endothelial Precursors in Human and Murine Embryonic Stem Cells Before CD34 Expression

Background— In murine embryonic stem cells, the onset of vascular endothelial growth factor receptor 2 (VEGFR-2) expression identifies endothelial precursors. Undifferentiated human embryonic stem cells express VEGFR-2, and VEGFR-2 expression persists on differentiation. The objective of our study was to identify a single population of endothelial precursors with common identifying features from both human and murine embryonic stem cells. Methods and Results— We report that expression of the VEGF coreceptor neuropilin-1 (NRP-1) coincides with expression of Brachyury and VEGFR-2 and identifies endothelial precursors in murine and human embryonic stem cells before CD31 or CD34 expression. When sorted and differentiated, VEGFR-2+NRP-1+ cells form endothelial-like colonies that express CD31 and CD34 7-fold more efficiently than NRP-1 cells. Finally, antagonism of both the VEGF and Semaphorin binding functions of NRP-1 impairs the differentiation of vascular precursors to endothelial cells. Conclusions— The onset of NRP-1 expression identifies endothelial precursors in murine and human stem cells. The findings define the origin of a single population of endothelial precursors from human and murine stem cells to endothelial cells. Additionally, the function of both the VEGF and Semaphorin binding activities of NRP-1 has important roles in the differentiation of stem cells to endothelial cells, providing novel insights into the role of NRP-1 in a model of vasculogenesis.

A cost-effective and efficient reprogramming platform for large-scale production of integration-free human induced pluripotent stem cells in chemically defined culture.

Factors limiting the adoption of iPSC technology include the cost of developing lines and the time period that it takes to characterize and bank them, particularly when integration free, feeder free, and Xeno-free components are used. In this manuscript we describe our optimization procedure that enables a single technician to make 20–40 lines at a time in a 24–96 well format in a reliable and reproducible fashion. Improvements spanned the entire workflow and included using RNA virus, reducing cytotoxicity of reagents, developing improved transfection and freezing efficiencies, modifying the manual colony picking steps, enhancing passaging efficiency and developing early criteria of success. These modifications allowed us to make more than two hundred well-characterized lines per year.

Heparin Promotes Cardiac Differentiation of Human Pluripotent Stem Cells in Chemically Defined Albumin-Free Medium, Enabling Consistent Manufacture of Cardiomyocytes

Cardiomyocytes can be differentiated from human pluripotent stem cells (hPSCs) in defined conditions, but efficient and consistent cardiomyocyte differentiation often requires expensive reagents such as B27 supplement or recombinant albumin. Using a chemically defined albumin‐free (E8 basal) medium, we identified heparin as a novel factor that significantly promotes cardiomyocyte differentiation efficiency, and developed an efficient method to differentiate hPSCs into cardiomyocytes. The treatment with heparin helped cardiomyocyte differentiation consistently reach at least 80% purity (up to 95%) from more than 10 different hPSC lines in chemically defined Dulbeccos modified Eagles medium/F‐12‐based medium on either Matrigel or defined matrices like vitronectin and Synthemax. One of heparins main functions was to act as a Wnt modulator that helped promote robust and consistent cardiomyocyte production. Our study provides an efficient, reliable, and cost‐effective method for cardiomyocyte derivation from hPSCs that can be used for potential large‐scale drug screening, disease modeling, and future cellular therapies. Stem Cells Translational Medicine 2017;6:527–538

Induced Pluripotent Stem Cells for Disease Modeling and Evaluation of Therapeutics for Niemann-Pick Disease Type A

Niemann‐Pick disease type A (NPA) is a lysosomal storage disease caused by mutations in the SMPD1 gene that encodes acid sphingomyelinase (ASM). Deficiency in ASM function results in lysosomal accumulation of sphingomyelin and neurodegeneration. Currently, there is no effective treatment for NPA. To accelerate drug discovery for treatment of NPA, we generated induced pluripotent stem cells from two patient dermal fibroblast lines and differentiated them into neural stem cells. The NPA neural stem cells exhibit a disease phenotype of lysosomal sphingomyelin accumulation and enlarged lysosomes. By using this disease model, we also evaluated three compounds that reportedly reduced lysosomal lipid accumulation in Niemann‐Pick disease type C as well as enzyme replacement therapy with ASM. We found that α‐tocopherol, δ‐tocopherol, hydroxypropyl‐β‐cyclodextrin, and ASM reduced sphingomyelin accumulation and enlarged lysosomes in NPA neural stem cells. Therefore, the NPA neural stem cells possess the characteristic NPA disease phenotype that can be ameliorated by tocopherols, cyclodextrin, and ASM. Our results demonstrate the efficacies of cyclodextrin and tocopherols in the NPA cell‐based model. Our data also indicate that the NPA neural stem cells can be used as a new cell‐based disease model for further study of disease pathophysiology and for high‐throughput screening to identify new lead compounds for drug development.

Neural stem cells for disease modeling of Wolman disease and evaluation of therapeutics

BackgroundWolman disease (WD) is a rare lysosomal storage disorder that is caused by mutations in the LIPA gene encoding lysosomal acid lipase (LAL). Deficiency in LAL function causes accumulation of cholesteryl esters and triglycerides in lysosomes. Fatality usually occurs within the first year of life. While an enzyme replacement therapy has recently become available, there is currently no small-molecule drug treatment for WD.ResultsWe have generated induced pluripotent stem cells (iPSCs) from two WD patient dermal fibroblast lines and subsequently differentiated them into neural stem cells (NSCs). The WD NSCs exhibited the hallmark disease phenotypes of neutral lipid accumulation, severely deficient LAL activity, and increased LysoTracker dye staining. Enzyme replacement treatment dramatically reduced the WD phenotype in these cells. In addition, δ-tocopherol (DT) and hydroxypropyl-beta-cyclodextrin (HPBCD) significantly reduced lysosomal size in WD NSCs, and an enhanced effect was observed in DT/HPBCD combination therapy.ConclusionThe results demonstrate that these WD NSCs are valid cell-based disease models with characteristic disease phenotypes that can be used to evaluate drug efficacy and screen compounds. DT and HPBCD both reduce LysoTracker dye staining in WD cells. The cells may be used to further dissect the pathology of WD, evaluate compound efficacy, and serve as a platform for high-throughput drug screening to identify new compounds for therapeutic development.

Neural stem cells for disease modeling and evaluation of therapeutics for infantile (CLN1/PPT1) and late infantile (CLN2/TPP1) neuronal ceroid lipofuscinoses

BackgroundInfantile and late infantile neuronal ceroid lipofuscinoses (NCLs) are lysosomal storage diseases affecting the central nervous system (CNS). The infantile NCL (INCL) is caused by mutations in the PPT1 gene and late-infantile NCL (LINCL) is due to mutations in the TPP1 gene. Deficiency in PPT1 or TPP1 enzyme function results in lysosomal accumulation of pathological lipofuscin-like material in the patient cells. There is currently no small-molecular drug treatment for NCLs.ResultsWe have generated induced pluripotent stem cells (iPSC) from three patient dermal fibroblast lines and further differentiated them into neural stem cells (NSCs). Using these new disease models, we evaluated the effect of δ-tocopherol (DT) and hydroxypropyl-β-cyclodextrin (HPBCD) with the enzyme replacement therapy as the control. Treatment with the relevant recombinant enzyme or DT significantly ameliorated the lipid accumulation and lysosomal enlargement in the disease cells. A combination therapy of δ-tocopherol and HPBCD further improved the effect compared to that of either drug used as a single therapy.ConclusionThe results demonstrate that these patient iPSC derived NCL NSCs are valid cell- based disease models with characteristic disease phenotypes that can be used for study of disease pathophysiology and drug development.

Efficient differentiation of cardiomyocytes and generation of calcium-sensor reporter lines from nonhuman primate iPSCs

Nonhuman primate (NHP) models are more predictive than rodent models for developing induced pluripotent stem cell (iPSC)-based cell therapy, but robust and reproducible NHP iPSC-cardiomyocyte differentiation protocols are lacking for cardiomyopathies research. We developed a method to differentiate integration-free rhesus macaque iPSCs (RhiPSCs) into cardiomyocytes with >85% purity in 10 days, using fully chemically defined conditions. To enable visualization of intracellular calcium flux in beating cardiomyocytes, we used CRISPR/Cas9 to stably knock-in genetically encoded calcium indicators at the rhesus AAVS1 safe harbor locus. Rhesus cardiomyocytes derived by our stepwise differentiation method express signature cardiac markers and show normal electrochemical coupling. They are responsive to cardiorelevant drugs and can be successfully engrafted in a mouse myocardial infarction model. Our approach provides a powerful tool for generation of NHP iPSC-derived cardiomyocytes amenable to utilization in basic research and preclinical studies, including in vivo tissue regeneration models and drug screening.