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Abstract Over the past decades, mesenchymal stromal cells (MSCs) have been extensively investigated as a potential therapeutic cell source for the treatment of various disorders. Differentiation of MSCs from human induced pluripotent stem cells (iMSCs) has provided a scalable approach for the biomanufacturing of MSCs and related biological products. Although iMSCs shared typical MSC markers and functions as primary MSCs (pMSCs), there is a lack of lineage specificity in many iMSC differentiation protocols. Here, a stepwise hiPSC‐to‐iMSC differentiation method is employed via intermediate cell stages of neural crest and cytotrophoblast to generate lineage‐specific MSCs with varying differentiation efficiencies and gene expression. Through a comprehensive comparison between early developmental cell types (hiPSCs, neural crest, and cytotrophoblast), two lineage‐specific iMSCs, and six source‐specific pMSCs, are able to not only distinguish the transcriptomic differences between MSCs and early developmental cells, but also determine the transcriptomic similarities of iMSC subtypes to postnatal or perinatal pMSCs. Additionally, it is demonstrated that different iMSC subtypes and priming conditions affected EV production, exosomal protein expression, and cytokine cargo. 

ABSTRACT Introduction:We hypothesized extracellular vesicles (EVs) from preconditioned human-induced pluripotent stem cell–derived mesenchymal stem cells (iMSCs) attenuate LPS-induced acute lung injury (ALI) and endotoxemia.Methods:iMSCs were incubated with cell stimulation cocktail (CSC) and EVs were isolated. iMSC-EVs were characterized by size and EV markers. Biodistribution of intratracheal (IT), intravenous, and intraperitoneal injection of iMSC-EVs in mice was examined using IVIS. Uptake of iMSC-EVs in lung tissue, alveolar macrophages, and RAW264.7 cells was also assessed. C57BL/6 mice were treated with IT/IP iMSC-EVs or vehicle ± IT/IP LPS to induce ALI/acute respiratory distress syndrome and endotoxemia. Lung tissues, plasma, and bronchoalveolar lavage fluid (BALF) were harvested at 24 h. Lung histology, BALF neutrophil/macrophage, cytokine levels, and total protein concentration were measured to assess ALI and inflammation. Survival studies were performed using IP LPS in mice for 3 days.Results:iMSC-EV route of administration resulted in differential tissue distribution. iMSC-EVs were taken up by alveolar macrophages in mouse lung and cultured RAW264.7 cells. IT LPS-treated mice demonstrated marked histologic ALI, increased BALF neutrophils/macrophages and protein, and increased BALF and plasma TNF-α/IL-6 levels. These parameters were attenuated by 2 h before or 2 h after treatment with IT iMSC-EVs in ALI mice. Interestingly, the IT LPS-induced increase in IL-10 was augmented by iMSC-EVs. Mice treated with IP LPS showed increases in TNF-α and IL-6 that were downregulated by iMSC-EVs and LPS-induced mortality was ameliorated by iMSC-EVs. Administration of IT iMSC-EVs 2 h after LPS downregulated the increase in proinflammatory cytokines (TNF-α/IL-6) by LPS and further increased IL-10 levels.Conclusions:iMSC-EVs attenuate the inflammatory effects of LPS on cytokine levels in ALI and IP LPS in mice. LPS-induced mortality was improved with administration of iMSC-EVs.