No abstract

Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia1–6. Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD+ and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD+ without proton pumping7,8 was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor9,10 or NAD+ precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd, which maintains NAD+ regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD+ regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction.

One of the major drawbacks associated with autologous fat grafting is unpredictable graft retention. Various efforts to improve the survivability of these cells have been explored, but these methods are time-consuming, complex, and demand significant technical skill.In our study, we examine the use of cryopreserved amniotic membrane as a source of exogenous growth factors to improve adipocyte survivability under normal and hypoxic conditions. Human primary preadipocytes were cultured in a gelatin-ferulic acid (Gtn-FA) hydrogel with variable oxygen concentration and treated with amniotic membranederived condition medium (CM) for 7 days. This hydrogel provides a hypoxic environment and also creates a 3D cell culture to better mimic recipient site conditions. The O 2 concentration in the hydrogel was measured by electron paramagnetic resonance oxygen imaging (EPROI). The conjugation of FA was confirmed by FTIR and NMR spectroscopy.The cell viability and adipocyte differentiation were analyzed by alamarBlue™ assay, Oil Red O staining, and RT-qPCR. The expression of genes: Pref-1, C/EBP β, C/EBP α, PPAR-ƴ, SLC2A4, and VEGF-A were quantified. The cell viability results show that the 50% CM showed significantly higher cell pre-adipocyte cell viability. In addition, compared to normal conditions, hypoxia/CM provided higher PPAR-ƴ (p < .05), SLC2A4, and VEGF-A (p < .05) (early and terminal differentiating markers) mRNA expression. This finding demonstrates the efficacy of amniotic CM supplementation as a novel way to promote adipocyte survival and retention via the expression of key gene markers for differentiation and angiogenesis.

Skin tissue engineering is a developing technology to heal severe wounds. Combining polyvinyl alcohol (PVA) and silk fibroin (SF) nanofibers is a promising method of developing a skin scaffold because the resulting structure mimics collagen fibers. The aim of this research was to study the growth of human dermal fibroblasts (HDF) on a polyvinyl alcohol-silk fibroin (PVA-SF) nanofiber scaffold that was produced by electrospinning. Morphological characterization and chemical analysis of the scaffold were performed by scanning electron microscopy (SEM), Fourier transform infrared spectrophotometry (FTIR), and contact angle measurement. The biocompatibility of the scaffold was tested by MTT cytotoxicity assay, SEM analysis, adherence ratio calculation, and analysis of the HDF growth curve for 9 days. The FTIR results confirmed the presence of SF and PVA. The average fiber diameter and pore size of the PVA scaffold were greater than those of the PVA-SF scaffold. Both scaffolds had hydrophilic properties and were not cytotoxic. Thus, HDF can attach and grow on both types of scaffold better than HDF seeded on a polystyrene plate. In conclusion, the addition of SF to the PVA nanofibers caused bead formation, which affected the substrate topography, decreased hydrophilicity and also decreased the fiber diameter and pore size in the nanofiber scaffold compared to the PVA nanofiber scaffold without SF addition. SF addition increases cell attachment to the nanofiber scaffold and has potential to facilitate HDF cell growth.