Chin Cheong, J. | Search bioRxiv

bioRxiv2026

4D multimodal wound healing atlas reveals organ-level controls of repair phase transitions

Deep skin wounds demand tightly coordinated communication across diverse tissue systems, yet knowledge of the molecular logic governing organ-scale injury response remains incomplete. Existing wound atlases profile fragments of this process, capturing limited tissue groups and healing phases, obscuring how whole organs synchronize repair. Here, we present the Organ-Scale Wound Healing Atlas (OWHA), a 4D multimodal omnibus that integrates snRNA-seq, scRNA-seq, CITE-seq and high-definition spatial transcriptomics to reconstruct the complete spatial and temporal choreography of mammalian wound healing at single cell resolution. OWHA profiles over 725,000 murine single-cell and spatial transcriptomes encompassing the entire wound healing process from early to late healing phases across the vast skin microanatomical tissue niches. This omnibus overcomes long-standing technical limitations, enabling robust resolution of adipocytes, Schwann cells, fragile epithelial intermediates, and over 100 precisely annotated cell states, including populations missed in prior wound databases. This revealed that wound repair proceeds through sharp transcriptional and cellular inflection points driven by Central Orchestrator populations that coordinate healing via synchronized transcriptional activation and direct cross-tissue signaling. Key among these is a Sox6+ Tspear+ Il20ra+ keratinocyte subpopulation (Basal IV), detectable only through snRNA-seq but entirely missed by conventional wound atlasing. After injury, Basal IV cells deviate from canonical differentiation programs and adopt a neurovasculogenic signaling state during the proliferation phase, forming a transient spatially privileged regulatory hub at the wound edge. This epithelial-anchored niche spatially aligns Basal IV keratinocytes with proliferative endothelial cells, Pericytes, and Repair Schwann Cells, synchronizing re-epithelialization, angiogenesis, and neurite guidance. Mechanistically, this is orchestrated by a conserved Sema3C-Nrp1/Nrp2 axis that coordinates epithelial-vascular-neuronal crosstalk at the wound site. Cross-species integration confirms that the Basal IV/SEMA3C axis is conserved in human skin, yet undetected by conventional scRNA-seq human atlases due to dissociation-induced artifacts - underscoring the critical need for multimodal atlasing to accurately capture organ-scale physiology. Notably, the Basal IV/SEMA3C circuitry is selectively disrupted in human diabetic wounds, but topical Sema3C treatments restores peri-wound angiogenic sprouting and accelerates re-epithelialization of diabetic ulcers in vivo. OWHA establishes the first 4D, organ-scale molecular blueprint of mammalian wound healing, creating a foundational platform for decoding systems-level principles of repair and regeneration for tissue wounds.

cell biology↗

bioRxiv2020

Long-term high-yield skeletal muscle stem cell expansion through staged perturbation of cytokine signaling in a soft hydrogel culture platform

Muscle stem cells (MuSCs) are an essential stem cell population for skeletal muscle homeostasis and regeneration throughout adulthood. MuSCs are an ideal candidate for cell therapies for chronic and acute muscle injuries and diseases given their inherent ability to self-renew and generate progenitor cells capable of myogenic commitment and fusion. Given their rarity and propensity to lose stem-cell potential in prolonged culture, methods for ex vivo MuSC expansion that achieve clinical-scale stem cell yields represent a critical unmet need in muscle cell-therapeutic development. Here, we tested a microenvironment engineering approach to achieve long-term adult mouse MuSC expansion suitable for clinical demands through the combined optimization of techniques previously reported to achieve small-yield MuSC expansion in short-term cultures. We developed an optimized protocol for high-yield MuSC expansion through the combination of inflammatory cytokine and growth factor co-stimulation, temporally-staged inhibition of the p38/{beta} mitogen activated protein kinase signaling pathway, and modulation of substrate rigidity in long-term hydrogel cultures. We found that, on soft, muscle-mimicking (12 kPa) hydrogel substrates, a mixture of the cytokines TNF-, IL-1, IL-13, and IFN-{gamma} and the growth factor FGF2 stimulated robust exponential proliferation of adult MuSCs from both wildtype and mdx dystrophic mice for up to five weeks of culture that was accompanied by a phenotype shift towards committed myocytes. After observing that the temporal variation in myogenic commitment coincided with an oscillatory activation of p38/{beta} signaling, we tested a late-stage p38/{beta} inhibition strategy and found that blocking p38/{beta} signaling after three weeks, but not earlier, substantially enhanced cell yield, stem-cell phenotypes, and, critically, preserved transplantation potential for up to five weeks of FGF2/cytokine mix culture on soft hydrogels. Notably, this retention of transplant engraftment potency was not observed on traditional plastic substrates. We estimate that this protocol achieves >108-fold yield in Pax7+ stem cells from each starting MuSC, which represents a substantial improvement in stem-cell yield from long-term cultures compared to established methods. HighlightsO_LITNF-/IL-1/IL-13/IFN-{gamma} cytokine cocktail supports prolonged MuSC proliferation ex vivo but induces differentiation. C_LIO_LICytokine cocktail regulates cell signaling with varied prolonged activation signatures. C_LIO_LIEffects of p38/{beta} inhibition on cytokine-induced MuSC expansion are stage-dependent. C_LIO_LISoft hydrogels with late-stage p38/{beta} inhibition expand functional Pax7+ MuSCs long-term. C_LI Short summaryCosgrove and colleagues develop a long-term muscle stem cell expansion protocol by combining a tunable stiffness hydrogel substrate, an inflammatory cytokine cocktail, and targeted inhibition of p38 MAPK signaling. They show that soft, muscle-mimicking hydrogels with delayed p38 inhibition yield robust quantities of Pax7+ functional muscle stem cells.

bioengineering↗