Tissue engineering of the esophagus has been limited by stent dependance and poor muscle regeneration. Here we report an integrated strategy to engineer a 2.5-cm esophageal segment by microinjecting autologous pericyte-like myogenic precursors and fibroblasts in a decellularized porcine scaffold to repair circumferential defects in 10-kg minipigs ( n = 8), modeling pediatric use. Bioreactor maturation induced a proangiogenic phenotype, with in vivo support from biodegradable intraluminal stents and a vascularizing pleural wrap. This coordinated approach yielded safe and effective esophageal conduits; oral feeding supported normal growth, morbidity resembled that of clinical esophageal replacement and was endoscopically manageable, and 63% (5/8) survived to the 6-month endpoint. Comprehensive multimodal analyses demonstrated progressive recapitulation of native architecture, with increasing neuromuscular regeneration and vascularization, correlating with functional recovery, absence of symptomatic stricture and the presence of secondary peristalsis by 6 months. These results demonstrate that the combination of complementary regenerative, conditioning and surgical strategies enables a functionally integrated, contractile esophageal graft with ongoing structural maturation without immunosuppression.

Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets in vitro and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treated cells maintained their differentiation ability as examined by 3D-organotypic culture. However, these changes could be reversed and became indistinguishable between treated and untreated cells once ROCKi treatment was withdrawn. Further, the short-term ROCKi treatment did not reduce the number of KSCs. In addition, AKT and ERK pathways were rapidly activated upon ROCKi treatment. In conclusion, short-term ROCKi treatment can transiently and reversibly accelerate initial primary keratinocyte expansion while preserving the holoclone-forming cell population (KSCs), providing a safe avenue for clinical applications.

The dopaminergic system has key roles in human physiology and is implicated in a broad range of neurological and neuropsychiatric conditions that are increasingly investigated using induced pluripotent stem cell-derived midbrain models. To determine similarities of such models to human systems, here we undertake single-cell and spatial profiling of first and second trimester fetal midbrain and compare it to in vitro midbrain models. Histological examination reveals that, by the second trimester, fetal midbrain tissue exhibits structural complexity comparable to that of adults. At the molecular level, single-cell profiling uncovers differences in cellular composition across models, with brain organoids most closely resembling late first trimester tissue — an observation supported by meta-integration of existing midbrain datasets. By reconstructing developmental trajectories of neuronal and astrocytic lineages, we map gene expression dynamics associated with maturation. Importantly, integration of spatial transcriptomics provides critical context for aligning organoid models, revealing that their spatial organization and intercellular signaling resemble the architecture and microenvironment of the second trimester midbrain. Ultimately, we leverage our findings to study Dopamine Transporter Deficiency Syndrome progression in patient-derived midbrain organoids, validating their relevance. Understanding the extent of human tissue recapitulation in midbrain laboratory models is essential to justify their use as biological proxies.

A common task in single-cell transcriptomics is the identification of cell populations, often defined by their cell state or type, that are more abundant in one sample or condition than another. Existing methods, however, suffer from either low discovery rates or high rates of false positives, particularly when differences in abundance are confined to some cell states or types or stemming from rare cell populations. We introduce Dawnn, a pre-trained neural network able to find differential abundance with higher accuracy than current tools, both on simulated and biological datasets. Further, Dawnn recovers and extends published findings and discovers more cells in regions with localised differential abundance than existing methods for both common and rare cell types, including extremely uncommon ones such as pulmonary ionocytes. We thus advance the identification of abundance changes in cell populations, regardless of how prevalent the differences and populations are, promising novel biological insights at single-cell resolution.

Detects regions of differential abundance in single-cell transcriptomic data by applying a pre-trained neural network model to the labels of each cell's nearest neighbours. Tests for both local and global differential abundance, controlling the false discovery rate with the Benjamini-Yekutieli procedure. The method is described in Hall and Castellano (2023) .

Somatic stem cells can be cultured in-vitro and are attractive for cell and gene therapies, but their slow growth in in-vitro culture affects survival and stemness and hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome these obstacles. However, it risks changing the characteristics of stem cells. We found that primary keratinocyte stem cells (KSCs) cultured with the ROCKi Y-27632 for six days exhibited rapid proliferation while maintaining the ability to differentiate. Importantly, after discontinuation of ROCKi treatment, KSC numbers and characteristics were indistinguishable from those in non-treated cultures. We further confirmed that ROCKi treatment resulted in the activation of AKT and ERK pathways, which could support cell survival and proliferation in keratinocytes. We thus concluded that accelerating keratinocyte expansion with short-term ROCKi treatment does not exhaust KSCs’ self-renewal and differentiation capacities, presenting a safe avenue for clinical applications.