Impact of individual platelet lysates on isolation and growth of human mesenchymal stromal cells.

BACKGROUND AIMS: Culture medium for mesenchymal stromal cells (MSC) is frequently supplemented with fetal calf serum (FCS). FCS can induce xenogeneic immune reactions, transmit bovine pathogens and has a high lot-to-lot variability that hampers reproducibility of results. Several studies have demonstrated that pooled human platelet lysate (HPL) provides an attractive alternative for FCS. However, little is known about the variation between different platelet lysates. METHODS: We compared activities of individual HPL on initial fibroblastoid colony-forming units (CFU-F), proliferation, in vitro differentiation and long-term culture. These data were correlated with chemokine profiles of HPL. RESULTS: Isolation of MSC with either HPL or FCS resulted in similar CFU-F frequency, colony morphology, immunophenotype and adipogenic differentiation potential. Osteogenic differentiation was even more pronounced in HPL than FCS. There were significant differences in MSC proliferation with different HPL, but it was always higher in comparison with FCS. Cell growth correlated with the concentration of platelet-derived growth factor (PDGF) and there was a moderate association with platelet counts. All HPL facilitated expansion for more than 20 population doublings. CONCLUSIONS: Taken together, reliable long-term expansion was possible with all HPL, although there was some variation in platelet lysates of individual units. Therefore the use of donor recipient-matched or autologous HPL is feasible for therapeutic MSC products.

Influence of platelet-derived growth factor-AB on tissue development in autologous platelet-rich plasma gels.

Fibrin-based scaffolds are widely used in tissue engineering. We postulated that the use of platelet-rich plasma (PRP) in contrast to platelet-poor plasma and pure fibrinogen as the basic material leads to an increased release of autologous platelet-derived growth factor (PDGF)-AB, which may have a consequent positive effect on tissue development. Therefore, we evaluated the release of PDGF-AB during the production process and the course of PDGF release during cultivation of plasma gels with and w/o platelets. The influence of PDGF-AB on the proliferation rate of human umbilical cord artery smooth muscle cells (HUASMCs) was studied using XTT assay. The synthesis of extracellular matrix by HUASMCs in plasma- and fibrin gels was measured using hydroxyproline assay. The use of PRP led to an increase in autologous PDGF-AB release. Further, the platelet-containing plasma gels showed a prolonged release of growth factor during cultivation. Both PRP and platelet-poor plasma gels had a positive effect on the production of collagen. However, PDGF-AB as a supplement in medium and in pure fibrin gel had neither an effect on cell proliferation nor on the collagen synthesis rate. This observation may be due to an absence of PDGF receptors in HUASMCs as determined by flow cytometry. In conclusion, although the prolonged autologous production of PDGF-AB in PRP gels is possible, the enhanced tissue development by HUASMCs within such gels is not PDGF related.

Expansion of adipose mesenchymal stromal cells is affected by human platelet lysate and plating density.

The composition of mesenchymal stromal cells (MSCs) changes in the course of in vitro culture expansion. Little is known how these cell preparations are influenced by culture media, plating density, or passaging. In this study, we have isolated MSCs from human adipose tissue in culture medium supplemented with either fetal calf serum (FCS) or human platelet lysate (HPL). In addition, culture expansion was simultaneously performed at plating densities of 10 or 10,000 cells/cm(2). The use of FCS resulted in larger cells, whereas HPL significantly enhanced proliferation. Notably, HPL also facilitated expansion for more population doublings than FCS (43 ± 3 vs. 22 ± 4 population doubling; p < 0.001), while plating density did not have a significant effect on long-term growth curves. To gain further insight into population dynamics, we conceived a cellular automaton model to simulate expansion of MSCS. It is based on the assumptions that the number of cell divisions is limited and that due to contact inhibition proliferation occurs only at the rim of colonies. The model predicts that low plating densities result in more heterogeneity with regard to cell division history, and favor subpopulations of higher migratory activity. In summary, HPL is a suitable serum supplement for isolation of MSC from adipose tissue and facilitates more population doublings than FCS. Cellular automaton computer simulations provided additional insights into how complex population dynamics during long-term expansion are affected by plating density and migration.

Synergistic effects of growth factors and mesenchymal stromal cells for expansion of hematopoietic stem and progenitor cells.

OBJECTIVE: The number of hematopoietic stem and progenitor cells (HPCs) per cord blood unit is limited, and this can result in delayed engraftment or graft failure. In vitro expansion of HPCs provides a perspective to overcome these limitations. Cytokines as well as mesenchymal stromal cells (MSCs) have been shown to support HPCs ex vivo expansion, but a systematic analysis of their interplay remains elusive. MATERIALS AND METHODS: Twenty different combinations of growth factors (stem cell factor [SCF], thrombopoietin [TPO], fibroblast growth factor-1 [FGF-1], angiopoietin-like 5, and insulin-like growth factor-binding protein 2), either with or without MSC coculture were systematically compared for their ability to support HPC expansion. CD34(+) cells were stained with carboxyfluorescein diacetate N-succinimidyl ester to monitor cell division history in conjunction with immunophenotype. Colony-forming unit frequencies and hematopoietic reconstitution of nonobese diabetic severe combined immunodeficient mice were also assessed. RESULTS: Proliferation of HPCs was stimulated by coculture with MSCs. This was further enhanced in combination with SCF, TPO, and FGF-1. Moreover, these conditions maintained expression of primitive surface markers for more than four cell divisions. Colony-forming unit-initiating cells were not expanded without stromal support, whereas an eightfold increase was reached by simultaneous cytokine-treatment and MSC coculture. Importantly, in comparison to expansion without stromal support, coculture with MSCs significantly enhanced hematopoietic chimerism in a murine transplantation model. CONCLUSIONS: The supportive effect of MSCs on hematopoiesis can be significantly increased by addition of specific recombinant growth factors; especially in combination with SCF, TPO, and FGF-1.

Serum after autologous transplantation stimulates proliferation and expansion of human hematopoietic progenitor cells.

Regeneration after hematopoietic stem cell transplantation (HSCT) depends on enormous activation of the stem cell pool. So far, it is hardly understood how these cells are recruited into proliferation and self-renewal. In this study, we have addressed the question if systemically released factors are involved in activation of hematopoietic stem and progenitor cells (HPC) after autologous HSCT. Serum was taken from patients before chemotherapy, during neutropenia and after hematopoietic recovery. Subsequently, it was used as supplement for in vitro culture of CD34(+) cord blood HPC. Serum taken under hematopoietic stress (4 to 11 days after HSCT) significantly enhanced proliferation, maintained primitive immunophenotype (CD34(+), CD133(+), CD45(-)) for more cell divisions and increased colony forming units (CFU) as well as the number of cobblestone area-forming cells (CAFC). The stimulatory effect decays to normal levels after hematopoietic recovery (more than 2 weeks after HSCT). Chemokine profiling revealed a decline of several growth-factors during neutropenia, including platelet-derived growth factors PDGF-AA, PDGF-AB and PDGF-BB, whereas expression of monocyte chemotactic protein-1 (MCP-1) increased. These results demonstrate that systemically released factors play an important role for stimulation of hematopoietic regeneration after autologous HSCT. This feedback mechanism opens new perspectives for in vivo stimulation of the stem cell pool.