Abstract
Abstract 3628
Poster Board III-564
The bone marrow (BM) harbours haematopoietic stem/progenitor cells (HSCs) in anatomically distinct sites (niches) where HSCs are subjected to regulatory cues such as cytokines, cell-cell contacts and extra-cellular matrix (ECM) all of which control stem cell fate. In particular mesenchymal stromal cells (MSCs) are an integral part of the bone marrow and are known to be key regulators of the HSC niche. We have previously shown that bio-artificial scaffolds can have a significant impact on the in vitro behaviour of MSCs. Here, we are therefore focussing on the role of (native) ECM within the MSC-HSC microenvironment by building on our previous findings and published data (Seib et al.,Tissue Eng Part A., 2009 in press). Thus the aim of the current study is (a) to identify niche-specific ECM components and (b) the use of such ECMs for in vitro culture of BM-derived stem cells. To mimic the natural ECM composition of the BM, different ECM types were generated from BM-derived cells using (a) Dexter cultures, (b) standard MSC cultures, (c) MSCs subjected to osteogenic differentiation. After 10 days of culture those MSC-derived ECMs were decellularised using 0.5% Triton-X and 20mM NH4OH leaving only the ECM behind (verified by scanning electron microscopy). Those ECMs were used as a substrate for a second culture of MSCs, which were analysed for their proliferation and differentiation potential. Cell-free ECM from standard MSC cultures improved MSC proliferation compared to cells grown on regular tissue culture plastic (TCP) over the period of 8 days. Most notably, all cell-free ECM preparations lead to a significant difference in the cytoskeletal arrangement of MSCs during the first 2 days of culture compared to TCP controls. Cultivation of MSCs on native ECM provided a guiding structure for those cells to grow into, and helped to maintain an elongated cell shape compared to substantial cell spreading on TCP (roundness 0.2 versus 0.5 and cell area of 2.2 versus 8.2mm2, respectively, p<0.001, n=60. A factor of 1 was set to equate to a perfect circle). Next, we investigate if native ECM could either directly improve HSC cultures or maximise MSC feeder characteristics. For the latter set of studies MSCs were initially cultured for 7 days on cell-free ECM (from standard MSC cultures) and subsequently co-cultured with human peripheral blood CD34+ HSCs in serum free medium supplemented with cytokines (Tpo, Flt3, and SCF at 10ng/ml). Following a 14 day culture period up to 3.5-fold more CD34+ cells were present in ECM co-cultures compared to TCP co-cultures that was accompanied with an overall expansion of CD45+ cells of 109-fold versus 35-fold, respectively. Our data suggest that ECM preparations derived from MSCs might be useful to accomplish better expansion of HSCs under defined culture conditions. In addition, this system permits the identification of bimolecular key components that can be utilized in the future design of simple and robust carrier systems for improved HSC maintenance in vitro.
Figure HSC-MSC co-culture on preformed ECM substrates. (A) MSC-derived ECM (from standard MSC culture) following cell lysis (complete absence of cells). (B) Growth of a new set of MSCs on ECM substrates as shown in (A). (C) HSC-MSC co-culture on ECM substrates. Scale bars at 2μm. Arrow heads point out ECM structures.
Figure HSC-MSC co-culture on preformed ECM substrates. (A) MSC-derived ECM (from standard MSC culture) following cell lysis (complete absence of cells). (B) Growth of a new set of MSCs on ECM substrates as shown in (A). (C) HSC-MSC co-culture on ECM substrates. Scale bars at 2μm. Arrow heads point out ECM structures.
No relevant conflicts of interest to declare.
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