Induced pluripotent stem cells (iPSCs) have unlimited proliferative capacity and multi-lineage differentiation potential. Autologous iPSC-derived cells reduce the risk of immune rejection, making them promising resources for regenerative medicine. However, iPSCs derived using conventional methods often show heterogeneity and defects in differentiation potential in a subset of cells. To overcome these limitations, we developed a novel reprogramming strategy, termed the “4CL-primed” strategy, that incorporates a transient early-pluripotency (4CL) treatment during reprogramming. The 4CL-primed iPSCs retained the core characteristics of conventional iPSCs, including normal morphology, pluripotency marker expression, and karyotypic stability, while exhibiting enhanced mesodermal and endodermal differentiation capacity. Furthermore, the cells showed superior performance in directed pancreatic lineage differentiation, generating a higher proportion of pancreatic endocrine β-like cells. Collectively, the 4CL-primed strategy offers a more practical approach for mesodermal and endodermal differentiation research, particularly pancreatic lineage differentiation, thereby providing a more efficient source of pancreatic lineage cells for regenerative medicine and cell-based therapy.
Abstract The mammalian lung is organized into anatomically distinct lobes, yet whether regional heterogeneity arises among different lobes remains poorly understood. Here, we performed comprehensive transcriptomic profiling of individual lung lobes at the pseudoglandular stage. Comparative analyses revealed that consistent inter-lobar divergence occurs primarily at the level of protein-coding genes. Notably, the left lung lobe exhibits enrichment of immune-response-associated gene expression and increased abundance of early general capillary (gCap) endothelial cells, whereas the right cranial lobe preferentially expresses genes associated with ventricular cardiac muscle morphogenesis and shows reduced gCap enrichment. Integration with publicly available datasets and experimental validation further confirmed the heterogeneous distribution of gCap cells. Together, our findings indicate that molecular and cellular inter-lobar heterogeneity emerges during embryonic development and highlight the significance of considering lobe identity in understanding pulmonary development and related congenital diseases.
Abstract Mechanical forces are recognized as regulators of organ development, homeostasis, and regeneration, yet how physical cues are sensed in a region-specific manner within the liver remains unclear. In a Science article, Zhang and colleagues identify DPP4-positive zone 2 hepatocytes as a mechanosensory population, demonstrating that mechanical stress triggers the regenerative response through PIEZO1-mediated mechanotransduction. PIEZO1 activation in these cells induces NFATC3-dependent IGFBP2 expression, establishing a molecular link between tissue mechanics and spatially coordinated proliferation. This work provides a mechanistic explanation for why regeneration originates from the mid-lobule and introduces a druggable mechanotransduction axis that might be enhanced to accelerate liver repair.
Vascular endothelial cells (ECs) exhibit significant diversity in different tissues. To elucidate the distinct physiological roles of individual EC subtypes, genetic mouse lines utilizing the Cre-loxP recombination system are crucial. However, there is still a lack of Cre mouse lines that specifically target muscle ECs. Here, through analysis of publicly available single-cell RNA sequencing datasets, C1q/tumor necrosis factor-related protein-9 (CTRP9) was identified as a highly selective marker for microvascular ECs in muscle and adipose tissues. A CTRP9-CreERT2-tdTomato knock-in mouse line was then generated using CRISPR/Cas9, with tdTomato serving as a reporter for endogenous CTRP9 expression. By breeding these mice with Rosa26LSL−EYFP reporter mice and administering tamoxifen, specific EYFP and tdTomato expression was observed in CD31+ ECs in the heart, skeletal muscle, tongue, and adipose tissue from postnatal to adult stages. The EYFP+ cells were mainly identified as microvascular ECs, rather than arterial or venous ECs. The CTRP9-CreERT2 mouse model allows for precise and effective genetic manipulation of this distinct endothelial population, providing a valuable tool for investigating their roles in muscle metabolism, regeneration, and pathophysiology.
Abstract Adult mammalian cardiomyocytes exhibit limited regenerative capacity, which remains a major challenge for functional recovery after myocardial infarction (MI). Our previous study identified a cocktail of five small molecules (5SM) that promotes cardiomyocyte proliferation and heart regeneration; however, the complexity of this five-compound mixture poses significant barriers to clinical translation and mechanistic studies. Here, we identified a simplified dual-compound cocktail (2SM) composed of phenylephrine hydrochloride (PE) and harmine (HM), which also robustly enhanced cardiomyocyte proliferation in vitro and in vivo. Transcriptomic and metabolomic analyses indicated that 2SM treatment activated cardiomyocyte cell-cycle programs and metabolic reprogramming. PE activated a fetal program and contributed to the up-regulation of CCND1 and WEE1 by activating mTOR signaling pathway, thus enabling cardiomyocytes to enter the G1/S phase but not G2/M phase. HM inhibited DYRK1A and activated MMB and FOXM1-MuvB complex, resulting in the upregulation of G2/M phase gene expression, of which the upregulated CDC25 proteins dephosphorylate CDK1, thereby relieving the inhibitory effect of PE. Sequential treatment of PE followed by HM was essential for completion of cytokinesis. In summary, we identified that 2SM, two compounds of 5SM, reprogrammed cardiomyocytes into an embryonic-like state, and precisely coordinated cell cycle progression for promoting cardiomyocyte proliferation.
