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.
While computational deconvolution is routinely used to estimate cell-type proportions from tissue mixtures, reconstructing cell-type-specific transcriptomes at single-sample resolution remains a fundamentally underdetermined algorithmic challenge. Consequently, accurate single-sample, gene-level inference is rarely achieved by existing tools. Here, we systematically benchmarked multiple deconvolution approaches across diverse biological contexts using both pseudo-bulk mixtures and real bulk RNA-seq datasets derived from multiple tissues. Evaluating the critical computational limitations in these models, we developed BayesPrism-DWLS, a framework that enables integrated inference of cell-type proportions and cell-type-specific expression at single-sample resolution. Applied to mouse colon bulk RNA-seq and spatial transcriptomics, BayesPrism-DWLS revealed cell-type-specific genes and pathways that were undetectable at the bulk or spot level. Therefore, this framework provides a robust, high-resolution tool for dissecting cell heterogeneity and supports mechanistic studies informed by cell-type-specific transcriptional programs using cost-effective sequencing data.
Skeletal muscle function deteriorates with injury, disease, or aging. While stem cell therapies offer therapeutic potential, concerns remain regarding the safety and efficacy of transplanted cells. Satellite cells (SCs), the primary endogenous stem cells responsible for muscle repair, demonstrate safe transplantation but limited clinical effectiveness. A deeper understanding of the mechanisms regulating SC activation and differentiation is critical for improving muscle regeneration strategies. We examined the role of CSRP3 in SC-mediated muscle repair using injury models and myoblast differentiation assays. We compared its function in pigs and mice, employing genetic approaches, CSRP3 knockdown (CSRP3KD), Csrp3-/- and overexpression (CSRP3OE), to assess its impact on muscle regeneration. Additionally, we investigated the AKT-SERCA2 signaling axis and its downstream effects on calcium homeostasis, mitochondrial function, and myogenic differentiation. CSRP3 was essential for muscle regeneration and SC differentiation in pigs but not in mice. Genetic ablation of CSRP3 (CSRP3KD) impaired porcine SC differentiation and reduced regenerative capacity, while CSRP3 overexpression (CSRP3OE) enhanced these processes. Mechanistically, CSRP3 deficiency disrupted AKT-SERCA2 signaling, leading to elevated intracellular Ca2⁺, mitochondrial dysfunction, and MYOG protein degradation. Pharmacological AKT activation rescued differentiation defects in CSRP3-deficient cells. Notably, Csrp3-/- mice showed normal SC differentiation and AKT-SERCA2 activity, highlighting a species-specific regulatory role for CSRP3. Our findings identify CSRP3 as a critical regulator of porcine muscle regeneration, with minimal impact in mice. These results suggest that strategically engineered porcine SCs, through CSRP3 modulation, could improve the effectiveness of xenotransplantation-based therapies for muscle repair.
The development of functional human vasculature is essential for tissue engineering, disease modeling, and regenerative medicine. Conventional differentiation protocols of vascular lineages often exhibit lineage heterogeneity and limited control over cellular ratios. Here, we describe a protocol for generating vascular organoids (VOs) via orthogonal forward programming of hPSCs. By utilizing doxycycline-inducible activation of the transcription factors ETV2 and NKX3.1, hPSCs are rapidly directed toward endothelial and mural cell lineages, respectively. This strategy enables the assembly of VOs with precisely tunable cellular compositions within six days. When combined with fluorescent reporter lines (PECAM1-mRuby3 and ACTA2-EGFP), vascular networks can be visualized in real time without the need for tissue clearing or immunostaining. We detail procedures for stable cell line engineering, 3D organoid assembly, in vitro angiogenesis assays for drug screening, and in vivo transplantation under the mouse kidney capsule to form perfusable human vasculature. This platform provides a flexible, standardized, and scalable tool for investigating vascular biology, modeling inherited vasculopathies, and enhancing the vascularization of co-transplant tissues.
