RARα and RARγ reciprocally control K5+ progenitor cell expansion in developing salivary glands

Understanding the mechanisms of controlled expansion and differentiation of basal progenitor cell populations during organogenesis is essential for developing targeted regenerative therapies. Since the cytokeratin 5-positive (K5+) basal epithelial cell population in the salivary gland is regulated by retinoic acid signaling, we interrogated how isoform-specific retinoic acid receptor (RAR) signaling impacts the K5+ cell population during salivary gland organogenesis to identify RAR isoform-specific mechanisms that could be exploited in future regenerative therapies. In this study, we utilized RAR isoform-specific inhibitors and agonists with murine submandibular salivary gland organ explants. We determined that RARα and RARγ have opposing effects on K5+ cell cycle progression and cell distribution. RARα negatively regulates K5+ cells in both whole organ explants and in isolated epithelial rudiments. In contrast, RARγ is necessary but not sufficient to positively maintain K5+ cells, as agonism of RARγ alone failed to significantly expand the population. Although retinoids are known to stimulate differentiation, K5 levels were not inversely correlated with differentiated ductal cytokeratins. Instead, RARα agonism and RARγ inhibition, corresponding with reduced K5, resulted in premature lumenization, as marked by prominin-1. With lineage tracing, we demonstrated that K5+ cells have the capacity to become prominin-1+ cells. We conclude that RARα and RARγ reciprocally control K5+ progenitor cells endogenously in the developing submandibular salivary epithelium, in a cell cycle-dependent manner, controlling lumenization independently of keratinizing differentiation. Based on these data, isoform-specific targeting RARα may be more effective than pan-RAR inhibitors for regenerative therapies that seek to expand the K5+ progenitor cell pool.Summary statementRARα and RARγ reciprocally control K5+ progenitor cell proliferation and distribution in the developing submandibular salivary epithelium in a cell cycle-dependent manner while regulating lumenization independently of keratinizing differentiation

Single cell transcriptomics of human epidermis identifies basal stem cell transition states.

How stem cells give rise to epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find four spatially distinct stem cell populations at the top and bottom of rete ridges and transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling suggests that basal cell populations serve as crucial signaling hubs to maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest that transitional basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed transitional basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity

Single cell transcriptomics of human epidermis identifies basal stem cell transition states.

How stem cells give rise to epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find four spatially distinct stem cell populations at the top and bottom of rete ridges and transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling suggests that basal cell populations serve as crucial signaling hubs to maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest that transitional basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed transitional basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity

Single-cell transcriptomics of human-skin-equivalent organoids

Summary: Several methods for generating human-skin-equivalent (HSE) organoid cultures are in use to study skin biology; however, few studies thoroughly characterize these systems. To fill this gap, we use single-cell transcriptomics to compare in vitro HSEs, xenograft HSEs, and in vivo epidermis. By combining differential gene expression, pseudotime analyses, and spatial localization, we reconstruct HSE keratinocyte differentiation trajectories that recapitulate known in vivo epidermal differentiation pathways and show that HSEs contain major in vivo cellular states. However, HSEs also develop unique keratinocyte states, an expanded basal stem cell program, and disrupted terminal differentiation. Cell-cell communication modeling shows aberrant epithelial-to-mesenchymal transition (EMT)-associated signaling pathways that alter upon epidermal growth factor (EGF) supplementation. Last, xenograft HSEs at early time points post transplantation significantly rescue many in vitro deficits while undergoing a hypoxic response that drives an alternative differentiation lineage. This study highlights the strengths and limitations of organoid cultures and identifies areas for potential innovation

Single cell transcriptomics of human epidermis identifies basal stem cell transition states

The mechanisms regulating stem cells to give rise to human interfollicular epidermis are unclear. Here, the authors use single cell RNA sequencing to identify heterogeneity within the human neonatal interfollicular epidermis and distinct spatial positioning of at least four basal stem cell populations