Extracellular vesicles (EVs) mediate many therapeutic effects of stem cells during cellular therapies. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) were manufactured to overexpress the human antiprotease alpha-1 antitrypsin (hAAT) and studied to compare the EV production compared to lentivirus treated control MSCs. The goal of this study was to compare protein profiles in the EVs/exosomes of control and hAAT-MSCs using unbiased, high resolution liquid chromatography and mass spectrometry to explore differences. Nanoparticle tracking analysis (NTA) showed that the particle size of the EVs from control MSCs or hAAT-MSCs ranged from 30 to 200 nm. Both MSCs and hAAT-MSCs expressed exosome-associated proteins, including CD63, CD81, and CD9. hAAT-MSCs also expressed high levels of hAAT. We next performed proteomic analysis of EVs from three healthy donor cell lines. Exosomes collected from cell supernatant were classified by GO analysis which showed proteins important to cell adhesion and extracellular matrix organization. However, there were differences between exosomes from control MSCs and hAAT-MSCs in cytokine signaling of the immune system, stem cell differentiation, and carbohydrate metabolism (p < 0.05). These results show that hAAT-MSC exosomes contain a different profile of paracrine effectors with altered immune function, impacts on MSC stemness, differentiation, and prevention of cell apoptosis and survival that could contribute to improved therapeutic functions.

Chronic pancreatitis (CP) is characterized by pancreatic inflammation, fibrosis, and abdominal pain that is challenging to treat. Mesenchymal stromal cells (MSCs) overexpressing human alpha-1 antitrypsin (hAAT-MSCs) showed improved mobility and protective functions over native MSCs in nonobese diabetic mice. We investigated whether hAAT-MSCs could mitigate CP and its associated pain using trinitrobenzene sulfonic acid (TNBS)-induced CP mouse models. CP mice were given native human MSCs or hAAT-MSCs (0.5 × 10⁶ cells/mouse, i.v., n = 6–8/group). The index of visceral pain was measured by graduated von Frey filaments. Pancreatic morphology and pancreatic mast cell count were analyzed by morphological stains. Nociceptor transient receptor potential vanilloid 1 (TRPV1) expression in dorsal root ganglia (DRG) was determined by immunohistochemistry. hAAT-MSC-treated CP mice best preserved pancreatic morphology and histology. MSC or hAAT-MSC infusion reduced abdominal pain sensitivities. hAAT-MSC therapy also suppressed TRPV1 expression in DRG and reduced pancreatic mast cell density induced by TNBS. Overall, hAAT-MSCs reduced pain and mitigated pancreatic inflammation in CP equal to MSCs with a trend toward a higher pancreatic weight and better pain relief in the hAAT-MSC group compared to the MSC group. Both MSCs and hAAT-MSCs might be used as a novel therapeutic tool for CP-related pain.

Browning of white adipose tissue (WAT) is suggested as a promising therapeutic approach to induce energy expenditure and counteract obesity and its associated complications. Systemic depletion of spinophilin (SPL) increases metabolism and improves energy balance in mice. In this study, we explored the mechanistic insight of SPL action in WAT browning. Gene expression and mitochondria tracker staining showed that visceral white adipose tissue (vWAT) harvested from SPL KO mice had a higher expression of classic browning-related genes, including uncoupling protein 1 (UCP1), Cell death inducing DFFA like effector A (CIDEA) and PR domain containing 16 (PRDM16), as well as a higher mtDNA level compared to vWAT from wild type (WT) control mice. When adipogenesis was induced in pre-adipocytes harvested from KO and WT mice ex vivo using the PPAR-γ agonist rosiglitazone (Rosi), SPL KO cells showed increased browning marker gene expression and mitochondria function compared to cells from WT mice. Increased PPAR-γ protein expression and nucleus retention in vWAT from SPL KO mice after Rosi treatment were also observed. The effect of SPL on vWAT browning was further confirmed in vivo when WT and KO mice were treated with Rosi. As a result, SPL KO mice lost body weight, which was associated with increased expression of browning maker genes in vWAT. In summary, our data demonstrate the critical role of SPL in the regulation of WAT browning.

Alpha-1 antitrypsin-overexpressing mesenchymal stromal/stem cells (AAT-MSCs) showed improved innate properties with a faster proliferation rate when studied for their protective effects in mouse models of diseases. Here, we investigated the potential mechanism(s) by which AAT gene insertion increases MSC proliferation. Human bone marrow-derived primary or immortalized MSCs (iMSCs) or AAT-MSCs (iAAT-MSCs) were used in the study. Cell proliferation was measured by cell counting and cell cycle analysis. Possible pathways involved in the pro-proliferation effect of AAT were investigated by measuring mRNA and protein expression of key cell cycle genes. Interval cell counting showed increased proliferation in AAT-MSCs or iAAT-MSCs compared to their corresponding MSC controls. Cell cycle analysis revealed more cells progressing into the S and G2/M phases in iAAT-MSCs, with a notable increase in the cell cycle protein, Cyclin D1. Moreover, treatment with Cyclin D1 inhibitors showed that the increase in proliferation is due to Cyclin D1 and that the AAT protein is upstream and a positive regulator of Cyclin D1. Furthermore, AAT’s effect on Cyclin D1 is independent of the Wnt signaling pathway as there were no differences in the expression of regulatory proteins, including GSK3β and β-Catenin in iMSC and iAAT-MSCs. In summary, our results indicate that AAT gene insertion in an immortalized MSC cell line increases cell proliferation and growth by increasing Cyclin D1 expression and consequently causing cells to progress through the cell cycle at a significantly faster rate.