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Serum-Free Medium Enhances the Therapeutic Effects of Umbilical Cord Mesenchymal Stromal Cells on a Murine Model for Acute Colitis.

ABSTRACT: The usage of animal serum may ultimately prevent the application of ex vivo cultured mesenchymal stromal cells (MSCs) in a clinical setting due to safety concerns and batch-to-batch variability. Increasing regulatory pressure to limit use of animal serum has been issued and serum-free, xeno-free, and chemically defined media (S&XFM-CD) is encouraged to replace serum-containing media (SCM) in the stem cell preparation process. We previously developed a S&XFM-CD for the expansion of umbilical cord-derived MSCs (UCMSCs). Different culture conditions affect the function of MSCs, which may further affect the therapeutic efficiency and mechanisms of action. In this study, we compared the therapeutic effect and mechanism of UCMSCs in S&XFM-CD (UCMSCS&XFM-CD) in experimental colitis with those in SCM (UCMSCSCM). UCMSCS&XFM-CD exhibited better therapeutic effects than UCMSCSCM by body weight, disease activity index, and histological colitis score. UCMSCS&XFM-CD or UCMSCSCM migrated to the inflammation site of injured colon, but exhibited low levels of recruitment and persistence. Systemic depletion of endogenous macrophages impaired the therapeutic effects of UCMSCSCM and UCMSCS&XFM-CD. Furthermore, UCMSCS&XFM-CD more markedly promoted intestinal macrophage polarisation from M1 to M2 phenotype to produce higher levels of IL-10 and lower levels of TNF-? in colon tissue than UCMSCSCM, while a higher level of IL-4 was produced in UCMSCSCM-treated group. UCMSCS&XFM-CD cocultured with RAW264.7 cells in a transwell system promoted the release of TSG-6 and IL-6, whereas UCMSCSCM increased PGE2 levels. Taken together, we demonstrated that UCMSCs in S&XFM-CD exhibited improved therapeutic effects with altered cytokine secretion in an experimental acute colitis model.

SUBMITTER: Wu X

PROVIDER: S-EPMC7332562 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Serum-Free Medium Enhances the Therapeutic Effects of Umbilical Cord Mesenchymal Stromal Cells on a Murine Model for Acute Colitis.

Wu Xiaoyun X Wu Daocheng D Mu Yongxu Y Zhao Yuxia Y Ma Zhijie Z

Frontiers in bioengineering and biotechnology 20200626

The usage of animal serum may ultimately prevent the application of <i>ex vivo</i> cultured mesenchymal stromal cells (MSCs) in a clinical setting due to safety concerns and batch-to-batch variability. Increasing regulatory pressure to limit use of animal serum has been issued and serum-free, xeno-free, and chemically defined media (S&XFM-CD) is encouraged to replace serum-containing media (SCM) in the stem cell preparation process. We previously developed a S&XFM-CD for the expansion of umbilic ...[more]

PMID: 32671030

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Project description:Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval) of 15% non-activated platelet-rich concentrate (PRC) in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM). Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin) were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of Alizarin red stain. Collectively, these results demonstrate a great potential of PRC alone in inducing proliferation of hMSCs without any influence from other lineage-specific growth media. PRC alone has similar capacity to enhance hMSC osteogenic differentiation as a standard OM, without changing the temporal profile of the differentiation process. Thus, PRC could be used as a substitute medium to provide sufficient pool of pre-differentiated hMSCs for potential clinical application in bone regeneration.

Project description:Enhancing the immunomodulatory effects of mesenchymal stromal cells (MSCs) may increase their effects in sepsis. We tested the potential for overexpression of Interleukin-10 (IL-10) in human umbilical cord (UC) MSCs to increase MSC efficacy in Escherichia coli(E. coli) pneumosepsis and to enhance human macrophage function. Pneumonia was induced in rats by intratracheal instillation of E. coli ((2.0-3.0) × 109 Colony forming units (CFU)/kg). One hour later, animals were randomized to receive (a) vehicle; (b) naïve UC-MSCs; or (c) IL-10 overexpressing UC-MSCs (1 × 107 cells/kg). Lung injury severity, cellular infiltration, and E. coli colony counts were assessed after 48 h. The effects and mechanisms of action of IL-10 UC-MSCs on macrophage function in septic rodents and in humans were subsequently assessed. Survival increased with IL-10 (9/11 (82%)) and naïve (11/12 (91%)) UC-MSCs compared to vehicle (9/15 (60%, p = 0.03). IL-10 UC-MSCs-but not naïve UC-MSCs-significantly decreased the alveolar arterial gradient (455 93 and 520 81, mmHg, respectively) compared to that of vehicle animals (544 52, p = 0.02). Lung tissue bacterial counts were significantly increased in vehicle- and naïve-UC-MSC-treated animals but were not different from sham animals in those treated with IL-10 overexpressing UC-MSCs. IL-10 (but not naïve) UC-MSCs decreased alveolar neutrophils and increased alveolar macrophage percentages compared to vehicle. IL-10 UC-MSCs decreased structural lung injury compared to naïve UC-MSC or vehicle therapy. Alveolar macrophages from IL-10-UC-MSC-treated rats and from human volunteers demonstrated enhanced phagocytic capacity. This was mediated via increased macrophage hemeoxygenase-1, an effect blocked by prostaglandin E2 and lipoxygenase A4 blockade. IL-10 overexpression in UC-MSCs enhanced their effects in E. coli pneumosepsis and increased macrophage function. IL-10 UC-MSCs similarly enhanced human macrophage function, illustrating their therapeutic potential for infection-induced acute respiratory distress syndrome (ARDS).

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Serum-Free Medium Enhances the Therapeutic Effects of Umbilical Cord Mesenchymal Stromal Cells on a Murine Model for Acute Colitis.

Publications

Serum-Free Medium Enhances the Therapeutic Effects of Umbilical Cord Mesenchymal Stromal Cells on a Murine Model for Acute Colitis.

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