Abstract
Background & Aim Background & Aim: The plasticity of mesenchymal stromal cells (MSCs) in responding to damage by promoting immunosuppression and tissue regeneration has been leveraged to treat conditions such as spinal cord injury. But can MSC plasticity be impacted by the culture conditions in which MSCs are manufactured? For instance, researchers speculate that fibrinogen depletion is necessary for MSCs to have an immunosuppressive effect. However, many supplements claim to promote growth of “bona fide” MSCs, capable of differentiation and immunosuppression. This begs the question, is one MSC phenotypically similar to another MSC grown in differing conditions? Our group examined the effect of media formulations on the proliferation, immunosuppressive ability, and transcriptomes of MSCs. Our aim was to evaluate the resulting phenotypic variation of MSCs and explore how this variety may impact therapeutic applications. Methods, Results & Conclusion Methods Adipose-derived MSCs were collected from normal healthy donors and grown in A-MEM media supplemented with either: fetal bovine serum (FBS), fibrinogen-depleted (FD) human platelet lysate (PL), PLTMax, PLTGold, Stemulate, UltraGRO-Pure, or StemMACS Xeno-Free (XF) Expansion Media. Proliferation was assessed using a NucLight Rapid Red assay detected with an IncuCyte Live-Cell Analysis Platform. Immunosuppressive ability was assessed by assaying indoleamine 2,3-deoxygenase (IDO) secreted by MSCs exposed to interferon gamma. RNA sequencing was performed by the Mayo Clinic Genome Analysis Core. Results The log phase doubling time of individual MSC lines remains relatively consistent across media supplements (Fig. A). However, the length of time spent in the log phase of growth was supplement-dependent. Of note, IDO secretion by donors varied within and across specific media formulations. IDO secretion was lower in FD-PL media than in other media supplements (Fig. B). Transcriptomic analyses revealed growth culture-dependent gene expression patterns of MSCs exposed to FBS, PL, or XF media (Fig. C), particularly among genes encoding cytokines, signal transduction molecules, and cell proliferation markers. Conclusion Manufacturing conditions can drive variances in gene expression, IDO secretion, and proliferation by MSCs. These results underscore the importance of media choice for MSC expansion and motivate the investigation of inherent diversity within MSC products manufactured in individual labs and how this diversity may impact therapeutic outcomes.
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