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Engineered ECMs modulate the expression profile of bone marrow-derived human MSC: implications for HSC self-renewal

ABSTRACT: The bone marrow harbours multipotent mesenchymal stromal cells (MSCs) that nurture haematopoietic stem cells (HSCs). The extracellular matrix (ECM) is an integral part of the bone marrow, and the aim of this study was therefore to (a) examine the effect of engineered ECM substrates on MSC gene expression over time, and (b) to determine the functional ability of ECM-cultured MSCs to support HSCs. ECMs were surface immobilised using films of maleic anhydride to covalently immobilise tropocollagen or fibrillar collagen type I to the substrate. Where indicated, collagen type 1 fibrils were supplemented with heparin or hyaluronic acid. All surfaces maintained MSC viability, though cell expansion was ECM dependent. Microarray analysis indicated that both time in culture and culture substrate were important factors regulating gene expression. Surprisingly, a number of MSC genes critical for the support of HSC self-renewal were down regulated during the study period. The functional effects of the different substrates were quantitatively assessed by determining cobblestone area-forming cell frequencies and colony-forming units (CFUs). Progenitor frequencies were similar for all substrates during the first 3 weeks in culture, but thereafter the reference material, plasma-treated polystyrene (PTP), outperformed ECM-based culture substrates, as predicted from expression studies. Likewise, PTP was able to maintain CFUs for extended periods, whereas HSCs rapidly exhausted themselves when cultured on any of the ECM substrates tested. The ability to regulate the expression of stromal factors using reconstituted ECM is exciting and warrants further studies to identify the ECM components/combinations that maximise the expansion of clonogenic HSCs. Keywords: time course, human MSC, extracellular matrix

ORGANISM(S): Homo sapiens

PROVIDER: GSE12635 | GEO | 2009/08/01

SECONDARY ACCESSION(S): PRJNA112813

REPOSITORIES: GEO

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Project description:Ex-vivo expanded mesenchymal stromal cells (MSCs) are increasingly used for paracrine support of hematopoietic stem cell (HSC) regeneration, but inconsistent outcomes have been the huddle for on-going clinical trials. Here, we hypothesized that the heterogeneity in the niche activity of manufactured MSCs can be a parameter for variable outcomes in MSC-based cell therapy. We first screened MSC culture medium and found that serum batches caused larger variations in colony forming unit-fibroblast (CFU-F) content of MSCs than individual donor variations. The culture conditions supporting high (stimulatory) and low (non-stimulatory) CFU-F caused distinct niche activity of MSCs; MSCs under stimulatory condition exhibited higher level expression of cross-talk molecules (Jagged-1 and CXCL-12) and higher support for HSCS during long-term culture than MSCs under non-stimulatory culture. Moreover, the effects of MSCs enhancing hematopoietic engraftment were only visible when HSCs were co-transplanted with MSCs expanded under stimulatory, but not non-stimulatory conditions. However, these differences of MSCs were readily reversed by switching the culture mediums, indicating their distinct functional state, rather than clonal heterogeneity. Accordingly, transcriptomic analysis showed distinct gene set enrichment between the different MSCs and revealed distinct upstream signaling pathways such as inhibition of P53 and activation of ATF4 for MSCs under stimulatory conditions. Taken together, our study shows that the heterogeneity in the niche activity of MSCs can be created during ex-vivo expansion to cause a difference in the hematopoietic engraftment and raise the possibility that MSCs can be pre-screened for more predictable outcomes in clinical trials of MSCs. Total RNA obtained from isolated human mesenchymal stromal cells. To compare stimulatory (SS) serum and non-stimulatory (NSS) serums, MSCs had been maintained in each serum media were sub-cultured for at least two passages before analysis.

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Engineered ECMs modulate the expression profile of bone marrow-derived human MSC: implications for HSC self-renewal

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