Patient-Induced Pluripotent Stem Cell-Derived Hepatostellate Organoids Establish a Basis for Liver Pathologies in Telomeropathies.

BACKGROUND & AIMS: Dyskeratosis congenita (DC) is a telomere biology disorder caused primarily by mutations in the DKC1 gene. Patients with DC and related telomeropathies resulting from premature telomere dysfunction experience multiorgan failure. In the liver, DC patients present with nodular hyperplasia, steatosis, inflammation, and cirrhosis. However, the mechanism responsible for telomere dysfunction-induced liver disease remains unclear. METHODS: We used isogenic human induced pluripotent stem cells (iPSCs) harboring a causal DC mutation in DKC1 or a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9)-corrected control allele to model DC liver pathologies. We differentiated these iPSCs into hepatocytes (HEPs) or hepatic stellate cells (HSCs) followed by generation of genotype-admixed hepatostellate organoids. Single-cell transcriptomics were applied to hepatostellate organoids to understand cell type-specific genotype-phenotype relationships. RESULTS: Directed differentiation of iPSCs into HEPs and stellate cells and subsequent hepatostellate organoid formation revealed a dominant phenotype in the parenchyma, with DC HEPs becoming hyperplastic and also eliciting a pathogenic hyperplastic, proinflammatory response in stellate cells independent of stellate cell genotype. Pathogenic phenotypes in DKC1-mutant HEPs and hepatostellate organoids could be rescued via suppression of serine/threonine kinase AKT (protein kinase B) activity, a central regulator of MYC-driven hyperplasia downstream of DKC1 mutation. CONCLUSIONS: Isogenic iPSC-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies.

High autophagic vesicle content marks facultative stem cells of the gut.

Understanding how macroautophagy/autophagy contributes to tissue homeostasis is essential for understanding organismal health. The intestinal epithelium is an ideal model to define mechanisms that regulate tissue homeostasis because it houses well-defined populations of intestinal stem cells. Active intestinal stem cells (a-ISCs) are defined by their active cycling and self-renewal during homeostasis, which supports continual tissue turnover in vivo. In vitro, this is observed as long-term organoid formation capacity. A second population of stem cells, called "facultative intestinal stem cells" (f-ISCs), are defined by their ability to 1) survive tissue damage that depletes the injury-sensitive a-ISCs and 2) reenter the cell cycle to repopulate the a-ISC compartment and regenerate the epithelium. The prospective identification of f-ISCs has been challenging, as cells expressing markers of multiple differentiated lineages, particularly secretory lineages, appear to function as f-ISCs in diverse injury contexts. We evaluated cell age (defined as time elapsed after cell cycle exit) and autophagic state (marked by autophagic vesicle content) as molecular features that may be related to f-ISC capacity. We found that autophagic state, but not cell age, prospectively identifies f-ISCs within multiple lineages. As such, we describe autophagy as a lineage-agnostic marker of f-ISC capacity in the mammalian intestine.

Autophagic state prospectively identifies facultative stem cells in the intestinal epithelium.

The intestinal epithelium exhibits a rapid and efficient regenerative response to injury. Emerging evidence supports a model where plasticity of differentiated cells, particularly those in the secretory lineages, contributes to epithelial regeneration upon ablation of injury-sensitive stem cells. However, such facultative stem cell activity is rare within secretory populations. Here, we ask whether specific functional properties predict facultative stem cell activity. We utilize in vivo labeling combined with ex vivo organoid formation assays to evaluate how cell age and autophagic state contribute to facultative stem cell activity within secretory lineages. Strikingly, we find that cell age (time elapsed since cell cycle exit) does not correlate with secretory cell plasticity. Instead, high autophagic vesicle content predicts plasticity and resistance to DNA damaging injury independently of cell lineage. Our findings indicate that autophagic status prior to injury serves as a lineage-agnostic marker for the prospective identification of facultative stem cells.

MTORC1 and the Rebirth of Stemness.

MTORC1 activity is critical for tissue regeneration in multiple organs and contexts. In this issue of Developmental Cell, Miao et al. describe upstream regulators of mTORC1 activity which promote paligenosis, a process where mature cells de-differentiate to acquire stem cell activity in the face of injury.

Activation of TRPA1 nociceptor promotes systemic adult mammalian skin regeneration.

Adult mammalian wounds, with rare exception, heal with fibrotic scars that severely disrupt tissue architecture and function. Regenerative medicine seeks methods to avoid scar formation and restore the original tissue structures. We show in three adult mouse models that pharmacologic activation of the nociceptor TRPA1 on cutaneous sensory neurons reduces scar formation and can also promote tissue regeneration. Local activation of TRPA1 induces tissue regeneration on distant untreated areas of injury, demonstrating a systemic effect. Activated TRPA1 stimulates local production of interleukin-23 (IL-23) by dermal dendritic cells, leading to activation of circulating dermal IL-17-producing γδ T cells. Genetic ablation of TRPA1, IL-23, dermal dendritic cells, or γδ T cells prevents TRPA1-mediated tissue regeneration. These results reveal a cutaneous neuroimmune-regeneration cascade triggered by topical TRPA1 activators that promotes adult mammalian tissue regeneration, presenting a new avenue for research and development of therapies for wounds and scars.