Combinatorial Microgels for 3D ECM Screening and Heterogeneous Microenvironmental Culture of Primary Human Hepatic Stellate Cells.

Nonalcoholic fatty liver disease affects 30% of the United States population and its progression can lead to nonalcoholic steatohepatitis (NASH), and increased risks for cirrhosis and hepatocellular carcinoma. NASH is characterized by a highly heterogeneous liver microenvironment created by the fibrotic activity of hepatic stellate cells (HSCs). While HSCs have been widely studied in 2D, further advancements in physiologically relevant 3D culture platforms for the in vitro modeling of these heterogeneous environments are needed. In this study, the use of stiffness-variable, extracellular matrix (ECM) protein-conjugated polyethylene glycol microgels as 3D cell culture scaffolds to modulate HSC activation is demonstrated. These microgels as a high throughput ECM screening system to identify HSC matrix remodeling and metabolic activities in distinct heterogeneous microenvironmental conditions are further employed. The 6 kPa fibronectin microgels are shown to significantly increase HSC matrix remodeling and metabolic activities in single or multiple-component microenvironments. Overall, heterogeneous microenvironments consisting of multiple distinct ECM microgels promoted a decrease in HSC matrix remodeling and metabolic activities compared to homogeneous microenvironments. The study envisions this ECM screening platform being adapted to a broad number of cell types to aid the identification of ECM microenvironments that best recapitulate the desired phenotype, differentiation, or drug efficacy.

Mitochondrial integrated stress response controls lung epithelial cell fate.

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.

Amniotic growth factors enhanced human pre-adipocyte cell viability and differentiation under hypoxia.

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 membrane-derived 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 O2 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 ß, C/EBP α, PPAR-Æ´, 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-Æ´ (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.

Matrix biophysical cues direct mesenchymal stromal cell functions in immunity.

Hydrogels have been used to design synthetic matrices that capture salient features of matrix microenvironments to study and control cellular functions. Recent advances in understanding of both extracellular matrix biology and biomaterial design have shown that biophysical cues are powerful mediators of cell biology, especially that of mesenchymal stromal cells (MSCs). MSCs have been tested in many clinical trials because of their ability to modulate immune cells in different pathological conditions. While roles of biophysical cues in MSC biology have been studied in the context of multilineage differentiation, their significance in regulating immunomodulatory functions of MSCs is just beginning to be elucidated. This review first describes design principles behind how biophysical cues in native microenvironments influence the ability of MSCs to regulate immune cell production and functions. We will then discuss how biophysical cues can be leveraged to optimize cell isolation, priming, and delivery, which can help improve the success of MSC therapy for immunomodulation. Finally, a perspective is presented on how implementing biophysical cues in MSC potency assay can be important in predicting clinical outcomes. STATEMENT OF SIGNIFICANCE: Stromal cells of mesenchymal origin are known to direct immune cell functions in vivo by secreting paracrine mediators. This property has been leveraged in developing mesenchymal stromal cell (MSC)-based therapeutics by adoptive transfer to treat immunological rejection and tissue injuries, which have been tested in over one thousand clinical trials to date, but with mixed success. Advances in biomaterial design have enabled precise control of biophysical cues based on how stromal cells interact with the extracellular matrix in microenvironments in situ. Investigators have begun to use this approach to understand how different matrix biophysical parameters, such as fiber orientation, porosity, dimensionality, and viscoelasticity impact stromal cell-mediated immunomodulation. The insights gained from this effort can potentially be used to precisely define the microenvironmental cues for isolation, priming, and delivery of MSCs, which can be tailored based on different disease indications for optimal therapeutic outcomes.