Tatiana Pinho Resende

The assessment of the regenerative capacity of the heart has been compromised by the lack of surface signatures to characterize cardiomyocytes. Here, combined multiparametric surface marker analysis with single cell transcriptional profiling and in vivo transplantation, identify the main fetal cardiac populations and their progenitors. We found that cardiomyocytes at different stages of differentiation co-exist during development. We identified a population of immature HSA/CD24 ⁺ cardiomyocytes that persists throughout life and that, unlike other cardiomyocyte subsets, actively proliferates up to one week of age and engraft cardiac tissue upon transplantation. In adult heart HSA/CD24 ⁺ cardiomyocytes appear as mononucleated cells that cycle and increase in frequency after infarction. Our work identified cell surface signatures that allow the prospective isolation of cardiomyocytes at any developmental stage and the detection of adult cardiomyocytes poised for activation in response to ischemic stimuli. This work opens new perspectives in the understanding and treatment of heart pathologies.

Although in flies the atypical cadherin Fat is an upstream regulator of Hippo signalling, the closest mammalian homologue, Fat4, has been shown to regulate tissue polarity rather than growth. Here we show in the mouse heart that Fat4 modulates Hippo signalling to restrict growth. Fat4 mutant myocardium is thicker, with increased cardiomyocyte size and proliferation, and this is mediated by an upregulation of the transcriptional activity of Yap1, an effector of the Hippo pathway. Fat4 is not required for the canonical activation of Hippo kinases but it sequesters a partner of Yap1, Amotl1, out of the nucleus. The nuclear translocation of Amotl1 is accompanied by Yap1 to promote cardiomyocyte proliferation. We, therefore, identify Amotl1, which is not present in flies, as a mammalian intermediate for non-canonical Hippo signalling, downstream of Fat4. This work uncovers a mechanism for the restriction of heart growth at birth, a process which impedes the regenerative potential of the mammalian heart.

All vertebrate species present a segmented body, easily observed in the vertebrate column and its associated components, which provides a high degree of motility to the adult body and efficient protection of the internal organs. The sequential formation of the segmented precursors of the vertebral column during embryonic development, the somites, is governed by an oscillating genetic network, the somitogenesis molecular clock. Herein, we provide an overview of the molecular clock operating during somite formation and its underlying molecular regulatory mechanisms. Human congenital vertebral malformations have been associated with perturbations in these oscillatory mechanisms. Thus, a better comprehension of the molecular mechanisms regulating somite formation is required in order to fully understand the origin of human skeletal malformations.

Introduction: Among the plethora of cells under investigation to restore a functional myocardium, mesenchymal stromal cells (MSCs) have been granted considerable interest. However, whereas the beneficial effects of bone marrow MSCs (BM-MSCs) in the context of the diseased heart are widely reported, data are still scarce on MSCs from the umbilical cord matrix (UCM-MSCs). Herein we report on the effect of UCM-MSC transplantation to the infarcted murine heart, seconded by the dissection of the molecular mechanisms at play. Methods: Human umbilical cord tissue-derived MSCs (UCX®), obtained by using a proprietary technology developed by ECBio, were delivered via intramyocardial injection to C57BL/6 females subjected to permanent ligation of the left descending coronary artery. Moreover, medium produced by cultured UCX® preconditioned under normoxia (CM) or hypoxia (CMH) was collected for subsequent in vitro assays. Results: Evaluation of the effects upon intramyocardial transplantation shows that UCX® preserved cardiac function and attenuated cardiac remodeling subsequent to myocardial infarction (MI). UCX® further led to increased capillary density and decreased apoptosis in the injured tissue. In vitro, UCX®-conditioned medium displayed (a) proangiogenic activity by promoting the formation of capillary-like structures by human umbilical vein endothelial cells (HUVECs), and (b) antiapoptotic activity in HL-1 cardiomyocytes subjected to hypoxia. Moreover, in adult murine cardiac Sca-1⁺ progenitor cells (CPCs), conditioned medium enhanced mitogenic activity while activating a gene program characteristic of cardiomyogenic differentiation. Conclusions: UCX® preserve cardiac function after intramyocardial transplantation in a MI murine model. The cardioprotective effects of UCX® were attributed to paracrine mechanisms that appear to enhance angiogenesis, limit the extent of the apoptosis, augment proliferation, and activate a pool of resident CPCs. Overall, these results suggest that UCX® should be considered an alternative cell source when designing new therapeutic approaches to treat MI.

Mammalian heart formation is a complex morphogenetic event that depends on the correct temporal and spatial contribution of distinct cell sources. During cardiac formation, cellular specification, differentiation, and rearrangement are tightly regulated by an intricate signaling network. Over the last years, many aspects of this network have been uncovered not only due to advances in cardiac development comprehension but also due to the use of embryonic stem cells (ESCs)in vitromodel system. Additionally, several of these pathways have been shown to be functional or reactivated in the setting of cardiac disease. Knowledge withdrawn from studying heart development, ESCs differentiation, and cardiac pathophysiology may be helpful to envisage new strategies for improved cardiac repair/regeneration. In this review, we provide a comparative synopsis of the major signaling pathways required for cardiac lineage commitment in the embryo and murine ESCs. The involvement and possible reactivation of these pathways following heart injury and their role in tissue recovery will also be discussed.

Putative cardiac progenitor cells (CPCs) have been identified in the myocardium and are regarded as promising candidates for cardiac cell-based therapies. Although two distinct populations of CPCs reached the clinical setting, more detailed studies are required to portray the optimal cell type and therapeutic setting to drive robust cell engraftment and cardiomyogenesis after injury. Owing to the scarcity of the CPCs and the need for reproducibility, the generation of faithful cellular models would facilitate this scrutiny. Here, we evaluate whether immortalized Lin ⁻ Sca-1 ⁺ CPCs (iCPC ^Sca-1 ) represent their native-cell counterpart, thereby constituting a robust in vitro model system for standardized investigation in the cardiac field. iCPC ^Sca-1 were established in vitro as plastic adherent cells endowed with robust self-renewal capacity while preserving a stable phenotype in long-term culture. iCPC ^Sca-1 differentiated into cardiomyocytic-, endothelial-, and smooth muscle-like cells when subjected to appropriate stimuli. The cell line consistently displayed features of Lin ⁻ Sca-1 ⁺ CPCs in vitro, as well as in vivo after intramyocardial delivery in the onset of myocardial infarction (MI). Transplanted iCPC ^Sca-1 significantly attenuated the functional and anatomical alterations caused by MI while promoting neovascularization. iCPC ^Sca-1 are further shown to engraft, establish functional connections, and differentiate in loco into cardiomyocyte- and vasculature-like cells. These data validate iCPC ^Sca-1 as an in vitro model system for Lin ⁻ Sca-1 ⁺ progenitors and for systematic dissection of mechanisms underlying CPC subsets engraftment/differentiation in vivo. Moreover, iCPC ^Sca-1 can be regarded as a ready-to-use CPCs source for pre-clinical bioengineering studies toward the development of novel strategies for restoration of the damaged myocardium.

Vertebrate embryo somite formation is temporally controlled by the cyclic expression of somitogenesis clock genes in the presomitic mesoderm (PSM). The somitogenesis clock is believed to be an intrinsic property of this tissue, operating independently of embryonic midline structures and the signaling molecules produced therein, namely Sonic hedgehog (Shh). This work revisits the notochord signaling contribution to temporal control of PSM segmentation by assessing the rate and number of somites formed and somitogenesis molecular clock gene expression oscillations upon notochord ablation. The absence of the notochord causes a delay in somite formation, accompanied by an increase in the period of molecular clock oscillations. Shh is the notochord-derived signal responsible for this effect, as these alterations are recapitulated by Shh signaling inhibitors and rescued by an external Shh supply. We have characterized chick smoothened expression pattern and have found that the PSM expresses both patched1 and smoothened Shh signal transducers. Upon notochord ablation, patched1 , gli1, and fgf8 are down-regulated, whereas gli2 and gli3 are overexpressed. Strikingly, notochord-deprived PSM segmentation rate recovers over time, concomitant with raldh2 overexpression. Accordingly, exogenous RA supplement rescues notochord ablation effects on somite formation. A model is presented in which Shh and RA pathways converge to inhibit PSM Gli activity, ensuring timely somite formation. Altogether, our data provide evidence that a balance between different pathways ensures the robustness of timely somite formation and that notochord-derived Shh is a component of the molecular network regulating the pace of the somitogenesis clock.

The spindle assembly checkpoint monitors the status of kinetochore–microtubule (K-MT) attachments and delays anaphase onset until full metaphase alignment is achieved. Recently, the role of spindle assembly checkpoint proteins was expanded with the discovery that BubR1 and Bub1 are implicated in the regulation of K-MT attachments. One unsolved question is whether Bub3, known to form cell cycle constitutive complexes with both BubR1 and Bub1, is also required for proper chromosome-to-spindle attachments. Using RNA interference and high-resolution microscopy, we analyzed K-MT interactions in Bub3-depleted cells and compared them to those in Bub1- or BubR1-depleted cells. We found that Bub3 is essential for the establishment of correct K-MT attachments. In contrast to BubR1 depletion, which severely compromises chromosome attachment and alignment, we found Bub3 and Bub1 depletions to produce defective K-MT attachments that, however, still account for significant chromosome congression. After Aurora B inhibition, alignment defects become severer in Bub3- and Bub1-depleted cells, while partially rescued in BubR1-depleted cells, suggesting that Bub3 and Bub1 depletions perturb K-MT attachments distinctly from BubR1. Interestingly, misaligned chromosomes in Bub3- and Bub1-depleted cells were found to be predominantly bound in a side-on configuration. We propose that Bub3 promotes the formation of stable end-on bipolar attachments.