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Current cell transplantation techniques are hindered by small graft size, requiring high cell doses to achieve therapeutic cardiac remuscularization. Enhancing the proliferation of transplanted human embryonic stem cell-derived cardiomyocytes (hESC-CMs) could address this, allowing an otherwise subtherapeutic cell dose to prevent disease progression after myocardial infarction. In this study, we designed a hydrogel that activates Notch signaling through 3D presentation of the Notch ligand Delta-1 to use as an injectate for transplanting hESC-CMs into the infarcted rat myocardium. After 4 weeks, hESC-CM proliferation increased 2-fold and resulted in a 3-fold increase in graft size with the Delta-1 hydrogel compared to controls. To stringently test the effect of Notch-mediated graft expansion on long-term heart function, a normally subtherapeutic dose of hESC-CMs was implanted into the infarcted myocardium and cardiac function was evaluated by echocardiography. Transplantation of the Delta-1 hydrogel + hESC-CMs augmented heart function and was significantly higher at 3 months compared to controls. Graft size and hESC-CM proliferation were also increased at 3 months post-implantation. Collectively, these results demonstrate the therapeutic approach of a Delta-1 functionalized hydrogel to reduce the cell dose required to achieve functional benefit after myocardial infarction by enhancing hESC-CM graft size and proliferation., Graphical Abstract, Transplantation of hESC-derived cardiomyocytes can remuscularize injured myocardium, but it requires a high therapeutic cell dose. Murry and colleagues developed a Notch-activating hydrogel used as a vehicle for transplantation of a subtherapeutic cell dose. When injected into injured rat myocardium, this hydrogel promoted hESC-cardiomyocyte proliferation, increased graft size, and prevented functional decline.

BackgroundVascular endothelial cells are a mesoderm-derived lineage with many essential functions, including angiogenesis and coagulation. However, the gene regulatory mechanisms that underpin endothelial specialization are largely unknown, as are the roles of 3D chromatin organization in regulating endothelial cell transcription.MethodsTo investigate the relationships between 3D chromatin organization and gene expression in endothelial cell differentiation, we induced endothelial cell differentiation from human pluripotent stem cells and performed Hi-C and RNA-seq assays at specific timepoints in differentiation.ResultsOur analyses reveal that long-range intrachromosomal contacts increase over the course of endothelial cell differentiation, as do genomic compartment transitions between active and inactive states. These compartmental states are tightly associated with endothelial transcription. Dynamic topologically associating domain (TAD) boundaries strengthen and converge on an endothelial cell state, and nascent TAD boundaries are linked to the expression of genes that support endothelial cell specification. Relatedly, chromatin pairwise point interactions (DNA loops) increase in frequency during differentiation and are linked to the expression of genes with essential roles in vascular biology, including MECOM, TFPI, and KDR. To identify forms of regulation specific to endothelial cell differentiation, we compared the functional chromatin dynamics of endothelial cells with those of developing cardiomyocytes. Cardiomyocytes exhibit greater long-range cis interactions than endothelial cells, whereas endothelial cells have increased local intra-TAD interactions and much more abundant pairwise point interactions.ConclusionsGenome topology changes dynamically during endothelial differentiation, including acquisition of long-range cis interactions and new TAD boundaries, interconversion of hetero- and euchromatin, and formation of DNA loops. These chromatin dynamics guide transcription in the development of endothelial cells and promote the divergence of endothelial cells from related cell types such as cardiomyocytes.

Purpose Myocardial infarction (MI) results in permanent cardiomyocyte loss, frequently leading to heart failure with 50% 5-year mortality. At subacute time points following MI, animal studies have shown ‘remuscularization’ of the damaged heart with human embryonic stem cell (hESC)-derived cardiomyocytes. We recently improved outcomes by co-delivering hESC-derived epicardium. Clinically, the main challenge remains chronic heart failure. However, hESC-cardiomyocytes alone, in the chronically infarcted heart show no benefit. We hypothesized that co-delivering hESC-epicardium or a species-matched therapy could regenerate the chronically infarcted heart. Methods 3 separate pilot studies on chronically infarcted rodents were conducted: I) Vehicle-control ii) P1/P2 neonatal rat cardiomyocytes (NRVM) iii) hESC-epicardium & cardiomyocytes (Epi+CM). Rats underwent temporary left anterior descending artery ligation, resulting in MI. At 28days post-MI, animals with fractional shortening (FS%) Results Overall, Vehicle-control persistently decreased in cardiac function, whilst species-matched therapy (NRVM) had sustained functional recovery. Initially, combination therapy (EPI+CM) sharply declined, with a trend towards recovery by 3months. Infarct sizes between groups were similar. At 3months, Epi+CM's cardiac graft size=1.2±1.3% of LV whilst NRVM=2.7±1.9% of LV. Both NRVM and Epi+CM grafts displayed increased sarcomeric alignment, maturation, connexin-43 organization and neovascularization, compared to previous reports. Conclusion Our serial observations of within-group trends showed functional recovery for species-matched therapy (NRVM), whereas combination cellular therapy (Epi+CM) attenuated cardiac dysfunction at 3months. hESC-combination cell therapy holds clinical promise for ‘remuscularising’ chronically infarcted hearts and needs validation in a randomized preclinical study.

Summary Genome editing in induced pluripotent stem cells is currently hampered by the laborious and expensive nature of identifying homology-directed repair (HDR)-modified cells. We present an approach where isolation of cells bearing a selectable, HDR-mediated editing event at one locus enriches for HDR-mediated edits at additional loci. This strategy, called co-targeting with selection, improves the probability of isolating cells bearing HDR-mediated variants and accelerates the production of disease models., Graphical Abstract, Highlights • Increases the efficiency of genome editing in human iPSCs • Enhances detectability of variants of interest derived by homology-directed repair • Is a simple, scalable, and adaptable strategy for knocking in variants of interest, The potential of CRISPR/Cas9-based genome editing in iPSCs is currently hampered by the laborious nature of identifying cells modified by the lesser-used homology-directed repair (HDR) pathway. Geurts, McDermott-Roe, and colleagues present a straightforward approach, co-targeting with selection, based on co-modification that enriches for cells undergoing HDR thereby improving targeting efficiencies.

Mutations of HSPB5 (also known as CRYAB or αB-crystallin), a bona fide heat shock protein and molecular chaperone encoded by the HSPB5 (crystallin, alpha B) gene, are linked to various multisystem disorders featuring variable combinations of cataracts, cardiomyopathy, and skeletal myopathy. This study aims at investigating the pathological mechanisms involved in an early onset myofibrillar myopathy manifesting in a child harboring a homozygous recessive mutation in HSPB5, 343delT. To study HSPB5 343delT protein dynamics, we utilize model cell culture systems including induced pluripotent stem cells (iPSCs) derived from the 343delT patient (343delT/343delT) along with isogenic, heterozygous, gene-corrected control cells (WT KI/343delT), and BHK21 cells, a cell line lacking endogenous HSPB5 expression. 343delT/343delT and WT KI/343delT iPSC-derived skeletal myotubes (iSKMs) and cardiomyocytes (iCMs) did not express detectable levels of 343delT protein, contributable to extreme insolubility of the mutant protein. Overexpression of HSPB5 343delT resulted in insoluble mutant protein aggregates and induction of a cellular stress response. Co-expression of 343delT with WT prevented visible aggregation of 343delT and improved its solubility. Additionally, in vitro refolding of 343delT in the presence of WT rescued its solubility. We demonstrate an interaction between WT and 343delT both in vitro and within cells. These data support a loss of function model for the myopathy observed in the patient, as the insoluble mutant would be unavailable to perform normal functions of HSPB5, though additional gain-of-function effects of the mutant protein cannot be excluded. Additionally, our data highlights the solubilization of 343delT by WT, concordant with the recessive inheritance of the disease and absence of symptoms in carrier individuals.

αB-Crystallin, or HspB5, is a small molecular-weight heat shock protein expressed highly in cardiac and skeletal muscle with multifaceted cellular roles including, chaperone function towards essential myofibrillar components. Insights into protective roles played by αB-crystallin, as well as mutations in the gene encoding αB-crystallin, CRYAB, which resulted in human pathologies, have highlighted the critical functions of αB-crystallin in both skeletal and cardiac muscle, inter alia. Various human mutations in CRYAB appear to have tissue-specific effects, with loss of αB-crystallin only impacting skeletal muscle under basal conditions. This review aims to highlight the roles of αB-crystallin in skeletal and cardiac muscle homeostasis as well as under conditions of stress and disease, drawing insights from human pathologies resulting from CRYAB mutations, and to discuss the potential of using induced pluripotent stem cells to model αB-crystallin-opathies in vitro.

Several mutations in αB-crystallin (CryAB), a heat shock protein with chaperone-like activities, are causally linked to skeletal and cardiac myopathies in humans. To better understand the underlying pathogenic mechanisms, we had previously generated transgenic (TG) mice expressing R120GCryAB, which recapitulated distinguishing features of the myopathic disorder (e.g., protein aggregates, hypertrophic cardiomyopathy). To determine whether induced pluripotent stem cell (iPSC)-derived cardiomyocytes, a new experimental approach for human disease modeling, would be relevant to aggregation-prone disorders, we decided to exploit the existing transgenic mouse model to derive iPSCs from tail tip fibroblasts. Several iPSC lines were generated from TG and non-TG mice and validated for pluripotency. TG iPSC-derived cardiomyocytes contained perinuclear aggregates positive for CryAB staining, whereas CryAB protein accumulated in both detergent-soluble and insoluble fractions. iPSC-derived cardiomyocytes identified by cardiac troponin T staining were significantly larger when expressing R120GCryAB at a high level in comparison with TG low expressor or non-TG cells. Expression of fetal genes such as atrial natriuretic factor, B-type natriuretic peptide, and α-skeletal α-actin, assessed by quantitative reverse transcription-polymerase chain reaction, were increased in TG cardiomyocytes compared with non-TG, indicating the activation of the hypertrophic genetic program in vitro. Our study demonstrates for the first time that differentiation of R120G iPSCs into cardiomyocytes causes protein aggregation and cellular hypertrophy, recapitulating in vitro key pathognomonic hallmarks found in both animal models and patients. Our findings pave the way for further studies exploiting this cell model system for mechanistic and therapeutic investigations. © AlphaMed Press.