Heparin Differentially Impacts Gene Expression of Stromal Cells from Various Tissues

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.

Heparin Anticoagulant for Human Bone Marrow Does Not Influence In Vitro Performance of Human Mesenchymal Stromal Cells

“…In the previous studies, isolated cells were expanded and cultured in the presence of heparin, which is a different approach. The results of the previous studies were briefly summarized in the introduction [11,12,14].…”

“…Here, transcriptome analyses revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation, and angiogenesis, depending on the stromal cell origin [14]. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells [14].…”

“…However, culture-expanded MSCs derived from these mononuclear cells were not investigated in the study [13]. In another analysis, human BM-MSCs as well as stromal cells from umbilical cord and white adipose tissue were isolated and propagated in human platelet lysate-supplemented media with or without heparin [14]. Here, transcriptome analyses revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation, and angiogenesis, depending on the stromal cell origin [14].…”

“…In another analysis, human BM-MSCs as well as stromal cells from umbilical cord and white adipose tissue were isolated and propagated in human platelet lysate-supplemented media with or without heparin [14]. Here, transcriptome analyses revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation, and angiogenesis, depending on the stromal cell origin [14]. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells [14].…”

Multiclass cancer classification in fresh frozen and formalin-fixed paraffin-embedded tissue by DigiWest multiplex protein analysis

“…Furthermore, DigiWest shows a similar sensitivity, reproducibility, and signal linearity as a high-end western blot system [26]. It has been effectively used in the analysis of signaling pathways and for the verification of biomarker candidates [27][28][29][30][31].…”