The growth and differentiation of adipose tissue-derived stem cells (ASCs) is stimulated and regulated by the adipose tissue (AT) microenvironment. In lipedema, both inflammation and hypoxia influence the expansion and differentiation of ASCs, resulting in hypertrophic adipocytes and deposition of collagen, a primary component of the extracellular matrix (ECM). The goal of this study was to characterize the adipogenic differentiation potential and assess the levels of expression of ECM-remodeling markers in 3D spheroids derived from ASCs isolated from both lipedema and healthy individuals. The data showed an increase in the expression of the adipogenic genes (ADIPOQ, LPL, PPAR-γ and Glut4), a decrease in matrix metalloproteinases (MMP2, 9 and 11), with no significant changes in the expression of ECM markers (collagen and fibronectin), or integrin A5 in 3D differentiated lipedema spheroids as compared to healthy spheroids. In addition, no statistically significant changes in the levels of expression of inflammatory genes were detected in any of the samples. However, immunofluorescence staining showed a decrease in fibronectin and increase in laminin and Collagen VI expression in the 3D differentiated spheroids in both groups. The use of 3D ASC spheroids provide a functional model to study the cellular and molecular characteristics of lipedema AT.

Three-dimensional (3D) hydrogel scaffolds are an attractive option for tissue regeneration applications because they allow for cell migration, fluid exchange, and can be synthesized to closely mimic the physical properties of the extracellular matrix environment. The material properties of hydrogels play a vital role in cellular migration and differentiation. In light of this, in-depth understanding of material properties is required before such scaffolds can be used to study their influence on cells. Herein, various blends and thicknesses of poly (ethylene glycol) dimethacrylate (PEGDMA) hydrogels were synthesized, flash frozen, and dried by lyophilization to create scaffolds with multiscale porosity. Environmental scanning electron microscopy (ESEM) images demonstrated that lyophilization induced microporous voids in the PEGDMA hydrogels while swelling studies show the hydrogels retain their innate swelling properties. Change in pore size was observed between drying methods, polymer blend, and thickness when imaged in the hydrated state. Human adipose-derived stem cells (hASCs) were seeded on lyophilized and non-lyophilized hydrogels to determine if the scaffolds would support cell attachment and proliferation of a clinically relevant cell type. Cell attachment and morphology of the hASCs were evaluated using fluorescence imaging. Qualitative observations in cell attachment and morphology of hASCs on the surface of the different hydrogel spatial configurations indicate these multiscale porosity hydrogels create a suitable scaffold for hASC culture. These findings offer another factor of tunability in creating biomimetic hydrogels for various tissue engineering applications including tissue repair, regeneration, wound healing, and controlled release of growth factors.

Human adipose-derived stem cells (ASCs) show immense promise for treating inflammatory diseases, attributed primarily to their potent paracrine signaling. Previous investigations demonstrated that short-term Rapamycin preconditioning of bone marrow-derived stem cells (BMSCs) elevated secretion of prostaglandin E2, a pleiotropic molecule with therapeutic effects in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), and enhanced immunosuppressive capacity in vitro. However, this has yet to be examined in ASCs. The present study examined the therapeutic potential of short-term Rapamycin-preconditioned ASCs in the EAE model. Animals were treated at peak disease with control ASCs (EAE-ASCs), Rapa-preconditioned ASCs (EAE-Rapa-ASCs), or vehicle control (EAE). Results show that EAE-ASCs improved clinical disease scores and elevated intact myelin compared to both EAE and EAE-Rapa-ASC animals. These results correlated with augmented CD4⁺ T helper (T_h) and T regulatory (T_reg) cell populations in the spinal cord, and increased gene expression of interleukin-10 (IL-10), an anti-inflammatory cytokine. Conversely, EAE-Rapa-ASC mice showed no improvement in clinical disease scores, reduced myelin levels, and significantly less T_h and T_reg cells in the spinal cord. These findings suggest that short-term Rapamycin preconditioning reduces the therapeutic efficacy of ASCs when applied to late-stage EAE.

Background: Alzheimer’s Disease (AD) is a prevalent age‐related neurodegenerative condition leading to dementia, yet factors regulating its polygenomic etiology and progression remain elusive. MicroRNAs (miRNAs), small RNA molecules regulating protein expression, play a role in neurodegeneration. MicroRNA‐34a (miR‐34a) is a crucial regulator of numerous genes associated with neurodegenerative disorders, protein aggregation and synaptic transmission genes. However, the lack of effective tools for in vivo study impedes comprehensive research in this area. We developed a global miR‐34a overexpressing mouse line in which cognitive deficits, altered amyloid and tau protein processing, and synaptic reorganization were noted.Method: We generated a CaMKIIα driven, excitatory neuron‐specific Tet‐inducible miR‐34a overexpressing mouse model. Male and female miR‐34a± mice aged 28‐46 weeks were treated with water or Doxycycline to induce miR‐34a overexpression for up to 90 days cognitive and neurobiological consequences were assessed. In this pilot study, we assessed the expression of predicted miR‐34a target genes and associated proteins (SIRT1, NMDAR2B). A subset of mice underwent sequencing analysis in one hemisphere to identify novel miR‐34a targets.Result: At 30 days of miR‐34a overexpression, we observed trends towards reduced levels of miR‐34a target genes (NMDAR2B, SHANK3) in the hippocampus of mice. However, these findings were non‐significant, likely attributed to the limited power or insufficient exposure duration. With 90 days of exposure, mice exhibited subtle behavior changes, characterized by a decreased percent of spontaneous alternations; no differences observed at 30 days. Distinct nervous system cell populations were isolated for miRNA analysis, hinting at region‐specific changes. Whole brain hemisphere bulk RNA sequencing of Doxy‐treated animals revealed upregulation of the genes Gh and Prl and downregulation of Avp and Drd1 compared to control.Conclusion: The inducible global miR‐34a overexpression mouse model exhibited modest dementia‐like characteristics and warrants further characterization. Further interrogation of contributions from other neural or peripheral cell types in the AD phenotype induced by global miR‐34a overexpression. The cell‐specific nature of our model provides valuable insights into miR‐34a’s role in AD pathology. These findings contribute to understanding the complex, polygenomic mechanisms underlying neurodegeneration in AD and may reveal novel miR‐focused therapeutic targets.