Li, R. A. | Search bioRxiv

bioRxiv2025

p16.1 and p16.2, new HSPC markers, play redundant roles in zebrafish T-cell lymphopoiesis

In the recent years, the zebrafish model has become a first-choice animal model in the field of hematopoiesis, due to extra-uterine development and optical transparency that allow an easy observation of early stages of hematopoietic stem and progenitor cells (HSPCs) development. Here, we characterized the function of two undescribed genes, si:ch211-214p16.1 and si:ch211-214p16.2 (shortened p16.1 and p16.2) that are expressed by HSPCs, as early as their emergence. By combining different strategies, we have demonstrated that p16.1 and p16.2 play redundant roles in embryonic thymopoiesis. Knocking-down both genes at the same time - or one gene in the mutant background for the other gene - impaired HSPCs commitment towards lymphoid fate and homing to the thymus. However double mutant embryos did not show any phenotype. As these double-mutant animals were viable, their thymocytes were compared to their wild-type counterparts by RNA sequencing, revealing that double mutant thymocytes compensated the lack of p16.1 and p16.2 by upregulating genes involved in chemotaxis and cell migration. One of these transcripts, ccr9b, was indeed validated as an important regulator of thymic homing in double mutant embryos, only. In summary, we found two new markers of embryonic HSPCs, functionally linked to lymphoid fate. The description of such markers will be important for our understanding of heterogeneity among emerging HSPCs in the embryo.

developmental biology↗

bioRxiv2024

Reversal of contractile defects by mediating calcium homeostasis in human mini-heart models of heart failure with preserved ejection fraction (HFpEF) leads to first-in-human gene therapy clinical trial

AimsHeart failure with preserved ejection fraction (HFpEF), is a global health problem lacking disease-modifying therapeutic options, reflecting a lack of predictive models for preclinical drug testing. Aligned with FDA Modernization Act 2.0, we aimed to create the first in vitro human-specific mini-heart models of HFpEF, and to test the efficacy of a candidate gene therapy to improve cardiac kinetics and correct the disease phenotype. Methods and ResultsHealthy human pluripotent stem cell-derived ventricular cardiomyocytes were used to bioengineer beating cardiac tissue strips and pumping cardiac chambers. When conditioned with transforming growth factor-{beta}1 and endothelin-1, these mini-heart models exhibited signature disease phenotypes of significantly elevated diastolic force and tissue stiffness, and slowed contraction and relaxation kinetics, with no significant deficit in systolic force or ejection fraction versus unconditioned controls. Bioinformatic analysis of bulk RNA sequencing data from HFpEF mini-heart models and patient ventricular samples identified downregulation of SERCA2a of the calcium signalling pathway as a key differentially expressed gene. After dosage optimization, AAV-mediated expression of SERCA2a abrogated the disease phenotype and improved the cardiac kinetics in HFpEF mini-Hearts. ConclusionsThese findings contributed to FDA approval of an ongoing first-in-human gene therapy clinical trial for HFpEF, with Fast Track designation. We conclude that such human-based disease-specific mini-heart platforms are relevant for target discovery and validation that can facilitate clinical translation of novel cardiac therapies. Translational PerspectiveHeart failure with preserved ejection fraction (HFpEF) is a significant and growing global health concern lacking disease-modifying therapeutic options, reflecting inadequate preclinical models of the disease. Aligned with FDA Modernization Act 2.0, we created the first in vitro human-specific mini-heart models of HFpEF, demonstrated phenotypic disease characteristics of elevated stiffness and slowed kinetics, showed transcriptomic consistency with HFpEF patient data, identified SERCA2a as a key downregulated gene, performed dosing titration of SERCA2a gene therapy, and showed improvement of cardiac kinetics post-treatment. The findings contributed to FDA approval of an ongoing first-in-human gene therapy clinical trial for HFpEF.

bioengineering↗