Tracking single hiPSC-derived cardiomyocyte contractile function using CONTRAX an efficient pipeline for traction force measurement.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are powerful in vitro models to study the mechanisms underlying cardiomyopathies and cardiotoxicity. Quantification of the contractile function in single hiPSC-CMs at high-throughput and over time is essential to disentangle how cellular mechanisms affect heart function. Here, we present CONTRAX, an open-access, versatile, and streamlined pipeline for quantitative tracking of the contractile dynamics of single hiPSC-CMs over time. Three software modules enable: parameter-based identification of single hiPSC-CMs; automated video acquisition of >200 cells/hour; and contractility measurements via traction force microscopy. We analyze >4,500 hiPSC-CMs over time in the same cells under orthogonal conditions of culture media and substrate stiffnesses; +/- drug treatment; +/- cardiac mutations. Using undirected clustering, we reveal converging maturation patterns, quantifiable drug response to Mavacamten and significant deficiencies in hiPSC-CMs with disease mutations. CONTRAX empowers researchers with a potent quantitative approach to develop cardiac therapies.

TRF2 rescues telomere attrition and prolongs cell survival in Duchenne muscular dystrophy cardiomyocytes derived from human iPSCs.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Heart failure, driven by cardiomyocyte death, fibrosis, and the development of dilated cardiomyopathy, is the leading cause of death in DMD patients. Current treatments decrease the mechanical load on the heart but do not address the root cause of dilated cardiomyopathy: cardiomyocyte death. Previously, we showed that telomere shortening is a hallmark of DMD cardiomyocytes. Here, we test whether prevention of telomere attrition is possible in cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPSC-CMs) and if preventing telomere shortening impacts cardiomyocyte function. We observe reduced cell size, nuclear size, and sarcomere density in DMD iPSC-CMs compared with healthy isogenic controls. We find that expression of just one telomere-binding protein, telomeric repeat-binding factor 2 (TRF2), a core component of the shelterin complex, prevents telomere attrition and rescues deficiencies in cell size as well as sarcomere density. We employ a bioengineered platform to micropattern cardiomyocytes for calcium imaging and perform Southern blots of telomere restriction fragments, the gold standard for telomere length assessments. Importantly, preservation of telomere lengths in DMD cardiomyocytes improves their viability. These data provide evidence that preventing telomere attrition ameliorates deficits in cell morphology, activation of the DNA damage response, and premature cell death, suggesting that TRF2 is a key player in DMD-associated cardiac failure.

Increased tissue stiffness triggers contractile dysfunction and telomere shortening in dystrophic cardiomyocytes.

Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disease that is associated with severe progressive muscle degeneration culminating in death due to cardiorespiratory failure. We previously observed an unexpected proliferation-independent telomere shortening in cardiomyocytes of a DMD mouse model. Here, we provide mechanistic insights using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using traction force microscopy, we show that DMD hiPSC-CMs exhibit deficits in force generation on fibrotic-like bioengineered hydrogels, aberrant calcium handling, and increased reactive oxygen species levels. Furthermore, we observed a progressive post-mitotic telomere shortening in DMD hiPSC-CMs coincident with downregulation of shelterin complex, telomere capping proteins, and activation of the p53 DNA damage response. This telomere shortening is blocked by blebbistatin, which inhibits contraction in DMD cardiomyocytes. Our studies underscore the role of fibrotic stiffening in the etiology of DMD cardiomyopathy. In addition, our data indicate that telomere shortening is progressive, contraction dependent, and mechanosensitive, and suggest points of therapeutic intervention.

Perturbation of single hematopoietic stem cell fates in artificial niches.

Hematopoietic stem cells (HSCs) are capable of extensive self-renewal in vivo and are successfully employed clinically to treat hematopoietic malignancies, yet are in limited supply as in culture this self-renewal capacity is lost. Using an approach at the interface of stem cell biology and bioengineering, here we describe a novel platform of hydrogel microwell arrays for assessing the effects of either secreted or tethered proteins characteristic of the in vivo microenvironment, or niche, on HSC fate in vitro. Time-lapse microscopic analyses of single cells were crucial to overcoming inevitable heterogeneity of FACS-enriched HSCs. A reduction in proliferation kinetics or an increase in asynchronous division of single HSCs in microwells in response to specific proteins (Wnt3a and N-Cadherin) correlated well with subsequent serial long-term blood reconstitution in mice in vivo. Single cells that divided once in the presence of a given protein were capable of in vivo reconstitution, providing evidence of self-renewal divisions of HSCs in vitro. These results validate the hydrogel microwell platform as a broadly applicable paradigm for dissecting the regulatory role of specific signals within a complex stem cell niche.

Extensive fusion of haematopoietic cells with Purkinje neurons in response to chronic inflammation.

Transplanted bone marrow-derived cells (BMDCs) have been reported to fuse with cells of diverse tissues, but the extremely low frequency of fusion has led to the view that such events are biologically insignificant. Nonetheless, in mice with a lethal recessive liver disease (tyrosinaemia), transplantation of wild-type BMDCs restored liver function by cell fusion and prevented death, indicating that cell fusion can have beneficial effects. Here we report that chronic inflammation resulting from severe dermatitis or autoimmune encephalitis leads to robust fusion of BMDCs with Purkinje neurons and formation of hundreds of binucleate heterokaryons per cerebellum, a 10-100-fold higher frequency than previously reported. Single haematopoietic stem-cell transplants showed that the fusogenic cell is from the haematopoietic lineage and parabiosis experiments revealed that fusion can occur without irradiation. Transplantation of rat bone marrow into mice led to activation of dormant rat Purkinje neuron-specific genes in BMDC nuclei after fusion with mouse Purkinje neurons, consistent with nuclear reprogramming. The precise neurological role of these heterokaryons awaits elucidation, but their frequency in brain after inflammation is clearly much higher than previously appreciated.