Abstract
Pooled human platelet lysate (pHPL) is increasingly used as replacement of animal serum for manufacturing of stromal cell therapeutics. Porcine heparin is commonly applied to avoid clotting of pHPL-supplemented medium but the influence of heparin on cell behavior is still unclear. Aim of this study was to investigate cellular uptake of heparin by fluoresceinamine-labeling and its impact on expression of genes, proteins and function of human stromal cells derived from bone marrow (BM), umbilical cord (UC) and white adipose tissue (WAT). Cells were isolated and propagated using various pHPL-supplemented media with or without heparin. Flow cytometry and immunocytochemistry showed differential cellular internalization and lysosomal accumulation of heparin. Transcriptome profiling revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis, depending on stromal cell origin. The influence of heparin on the WNT, PDGF, NOTCH and TGFbeta signaling pathways was further analyzed by a bead-based western blot revealing most alterations in BM-derived stromal cells. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells, indicating compatibility for clinically applied cell products.
Highlights
For clinical application, a prior ex vivo expansion of stromal cells is usually necessary
As heparin uptake has been observed for other cell types such as endothelial cells[35], lymphocytes[36], monocytes[37] and different cancer cells[34], we asked whether heparin is differentially internalized by stromal cells from various tissues
As the abundant amount of growth factors in Pooled human platelet lysate (pHPL) may cover an effect of heparin on cell proliferation, we reduced the pHPL concentration by titration (10%, 3.3% and 1.1%) in the cell culture medium and tested the impact of 2 and 4 IU/mL heparin on cell proliferation of stromal cells from umbilical cord (UC), bone marrow (BM) and white adipose tissue (WAT)
Summary
A prior ex vivo expansion of stromal cells is usually necessary. To avoid clotting of pHPL-supplemented cell culture medium, induced by plasma-derived fibrinogen, prior addition of 0.6–2 IU of porcine heparin per mL medium is common practice[8]. Ling et al showed that heparin affected multiple cell signaling components of human BM-STCs in a donor-dependent manner[26]. In these studies, human cells were cultured under FBS-supplemented conditions. A benefit of heparin for stromal cell propagation and clinical application is still under debate
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