Cell Regeneration最新文献

Human umbilical cord mesenchymal stem cells (hUC-MSCs) have emerged as promising candidates for clinical applications in vascular disease therapy and in the in vitro modeling of vascular regeneration. However, the translational potential of hUC-MSCs requires direct differentiation into functional vascular lineage cells, particularly vascular endothelial cells (VECs) and endothelial progenitor cells (EPCs). A critical challenge is the lack of reliable sources that yield sufficient quantities of mature VECs/EPCs for therapeutic purposes. To address this limitation, we established an efficient protocol for generating VECs from hUC-MSCs. Preconditioning hUC-MSCs using small molecules with cytoprotective properties can enhance their potential for use in cell-based therapeutics. Through systematic screening, we identified CPP as a novel small chemical molecule that effectively induces the endothelial differentiation of hUC-MSCs. Remarkably, our CPP-based induction protocol achieved > 90% conversion to functionally competent VECs within 5 days, as evidenced by both in vitro assays and in vivo functional validation. Single-cell RNA sequencing (scRNA-seq) analysis further delineated the differentiation trajectory and confirmed the acquisition of endothelial-specific molecular signatures during lineage commitment. These findings establish CPP as a potent inducer of rapid endothelial differentiation, and provide mechanistic insights into stem cell fate determination.

Mesenchymal stromal cells (MSCs) possess well-described immunoregulatory properties, yet their capacity to drive regeneration in vertebrates is still debated and their mechanisms of action remain to be fully elucidated. In this study, we used zebrafish larvae, a highly regenerative vertebrate model to study the effects of MSC delivery on caudal fin fold regeneration and monitored macrophage dynamics through live imaging in fluorescent reporter lines. We found that MSCs enhanced fin regeneration by increasing the early recruitment of inflammatory (tnfa +) macrophages at 1-day-post-amputation (dpA), and accelerating resolution between 2 and 3 dpA. Given the established role of prostaglandin E2 (PGE2) in MSC-mediated immunoregulation, we examined its contribution using indomethacin, a cyclooxygenase inhibitor that suppresses PGE2 production in grafted MSCs. We observed that PGE2 inhibition abolished the pro-regenerative effect of MSCs and maintained elevated tnfa + macrophage levels. PGE2-inhibited MSCs were more susceptible to phagocytosis by both zebrafish and mammalian macrophages, while maintaining viability, indicating a loss of PGE2-mediated protection in treated cells. Together, these findings demonstrate that MSC-derived PGE2 is essential for MSC regenerative function by promoting MSC persistence and modulating macrophage behavior, highlight the zebrafish as a powerful in vivo platform to dissect stem cell-immune interactions and optimize MSC-based regenerative strategies.

Random-pattern skin flaps are widely employed in tissue reconstruction, however, their survival is frequently hindered by ischemia, leading to necrosis. Metabolic alterations have been implicated in playing critical roles in angiogenesis during tissue repair. Using RNA sequencing analysis in a mouse model, we identified significant disruptions in glutamine metabolism, which substantially impaired angiogenesis within random-pattern skin flaps. Although local glutamine repletion failed to alleviate ischemia, administering α-ketoglutarate (α-KG) markedly promoted angiogenesis, as evidenced at both gene and protein levels. In human umbilical vein endothelial cells,α-KG enhanced the stability of hypoxia-inducible factor (HIF-1) alpha through activation of the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway. Notably, α-KG treatment improved flap viability by augmenting blood perfusion, an effect correlated with upregulation of vascular endothelial growth factor expression. Together, these results reveal a novel mechanism by which α-KG enhances random-pattern skin flap viability via promoting angiogenesis through the PI3K/Akt/HIF-1α pathway, offering promising therapeutic insights for improving flap survival.

Advanced imaging and single-cell omics technologies are fundamentally transforming developmental biology research, shifting it from static observation to dynamic, spatially resolved systems biology. Super-resolution microscopy breaks the diffraction barrier to visualize nanoscale subcellular dynamics, while light-sheet fluorescence microscopy enables long-term, multi-scale volumetric imaging of living specimens. In parallel, single-cell omics (e.g., transcriptomics and proteomics) decipher molecular heterogeneity and lineage trajectories, and spatially resolved transcriptomics maps gene expression within native tissue contexts at subcellular resolution. However, each approach has inherent limitations: imaging lacks deep molecular profiling, while dissociation-based omics loses spatial context. This review highlights how the integration of these technologies bridges cellular behaviors with molecular mechanisms, providing unprecedented multi-scale perspectives on key developmental processes-including embryogenesis, organogenesis, neural patterning, and disease progression. By synergistically capturing the "when," "where," and "how" of developmental processes, this convergence resolves longstanding questions and establishes a new mechanistic and predictive paradigm in developmental biology.

Maintaining the odontogenic potential of dental mesenchymal cells (DMCs) in vitro remains a critical challenge in tooth regeneration research. Current culture systems fail to sustain DMC functionality beyond short-term periods, limiting their utility for tissue engineering applications. Here, we developed an optimized N2B27-based culture medium that preserves the odontogenic capacity of mouse DMCs (mDMCs) for up to 14 days with passaging-a significant improvement over conventional methods (≤ 24 h). Single-cell RNA sequencing (scRNA-seq) revealed distinct transcriptional profiles and cellular trajectories between traditionally cultured (FBS-based) and N2B27-cultured DMCs. Mechanistically, excessive BMP4 signaling in standard media suppressed odontogenesis, whereas elevated Spp1 (osteopontin, OPN) expression in the N2B27 system enhanced regenerative potential. We demonstrate that optimal maintenance of DMC functionality requires balanced BMP4 activity and is enhanced by high OPN levels. Notably, supplementation with recombinant OPN or all-trans retinoic acid (ATRA) partially restored tooth-forming capacity in suboptimal cultures. Our findings establish a robust in vitro platform for DMC expansion while preserving odontogenic competence, advancing both mechanistic studies of tooth development and the generation of clinically relevant cell sources for bioengineered dental tissues. This work provides key insights on the features of a regenerative tooth germ and its odontogenic microenvironment for future translational applications in tooth regeneration.

The interaction between hematopoietic stem and progenitor cell (HSPC) and its vascular niche is essential for supporting the homeostasis and reconstitution of hematopoietic system in adult bone marrow (BM), but a comprehensive atlas covering this HSPC-vascular niche crosstalk in multiple developmental stages and species is lacking. Here, we integrated single-cell transcriptomic data of HSPC and its vascular niches from fetal liver until aged BM, covering two species, two organs, and six developmental time points. Comparative analyses revealed dramatic differences in the gene expression, enriched pathway, and cell-cell communication between human fetal and adult BM. Notably, many of these differences were conserved between humans and mice. Multi-timepoint profiling of murine BM vascular niches revealed a stepwise maturation of gene expression, including critical niche factors such as SCF and CXCL12. Furthermore, analysis of this dynamic vascular niche atlas highlighted organ-specific features between fetal liver and BM niches, significant transcriptional changes in aged BM endothelial cells, and identified midkine as a previously unknown niche factor. Functional validation showed that transplanting HSPC into midkine knockout mice or treating with a midkine inhibitor (iMDK) enhanced hematopoietic reconstitution. In contrast, recombinant midkine suppressed HSPC differentiation. Together, our work presents a cross-species and multi-stage atlas of HSPC-vascular niche interactions, offering valuable insights into the dynamic changes of vascular niche through lifelong HSPC development and a platform to identify unknown niche factors.

Oct4 is a key transcription factor essential for maintaining pluripotency and self-renewal in embryonic stem cells (ESCs), where it activates pluripotency-related genes and represses differentiation-associated genes. While previous studies have identified OCT4 target genes using methods such as chromatin immunoprecipitation sequencing (ChIP-seq) and RNA interference (RNAi), these approaches may not fully capture direct transcriptional regulation. The auxin-inducible degron (AID) system, which enables rapid and reversible protein degradation, combined with nascent RNA sequencing, provides a refined method for identifying direct transcriptional targets by detecting immediate transcriptional changes in both protein-coding genes and non-coding RNAs. In this study, we utilized mouse Oct4-mAID ESCs and nascent RNA sequencing with 5-ethynyl uridine (5-EU) labeling to systematically identify direct OCT4 targets. Our results uncovered novel potential OCT4 targets, providing a dataset for further research into the functions and regulatory networks of Oct4 and related transcription factors.

Eukaryotic mRNAs are polyadenylated at their 3'-ends, and the poly(A) tails play critical roles in post-transcriptional gene regulation by influencing mRNA stability and translation. Here, we describe the biological processes and major protein factors that control poly(A) tail synthesis and shortening. We also discuss recent breakthroughs in poly(A) tail sequencing methods that enable high throughput and accurate measurement of poly(A) tail lengths. Finally, we review how poly(A)-tail regulators and poly(A)-tail-mediated post-transcriptional mechanisms affect stem cell fate and early embryonic development.

METTL3 is a crucial mRNA methyltransferase in mammals, essential for the regulation of gene expression and various biological processes. Here, we demonstrate that Mettl3 knockout (KO) in mouse embryonic stem cells (mESCs) leads to widespread upregulation of transposable elements (TEs) and 2-cell (2C)-like genes in a m⁶A enzyme activity-dependent manner, independent of culture conditions. Furthermore, embryo chimera experiments using a transient METTL3 degradation system (dTAG) revealed that METTL3-deficient mESCs can contribute to trophectoderm lineages at the blastocyst stage, indicative of expanded developmental potential. These findings highlight the role of METTL3-mediated m⁶A modifications in regulating the transcriptional and developmental plasticity of mESCs and suggest a link between m⁶A loss and the acquisition of a 2C-like state with features of extended potency.