Upregulation of mitotic bookmarking factors during enhanced proliferation of human stromal cells in human platelet lysate

Abstract

BackgroundInnovative human stromal cell therapeutics require xeno-free culture conditions. Various formulations of human platelet lysate (HPL) are efficient alternatives for fetal bovine serum (FBS). However, a consistent lack of standardized manufacturing protocols and quality criteria hampers comparability of HPL-products. Aim of this study was to compare the biochemical composition of three differential HPL-preparations with FBS and to investigate their impact on stromal cell biology.MethodsStromal cells were isolated from bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC) and cultured in medium supplemented with pooled HPL (pHPL), fibrinogen-depleted serum-converted pHPL (pHPLS), mechanically fibrinogen-depleted pHPL (mcpHPL) and FBS. Biochemical parameters were analyzed in comparison to standard values in whole blood. Distinct growth factors and cytokines were measured by bead-based multiplex technology. Flow cytometry of stromal cell immunophenotype, in vitro differentiation, and mRNA expression analysis of transcription factors SOX2, KLF4, cMYC, OCT4 and NANOG were performed.ResultsBiochemical parameters were comparable in all pHPL preparations, but to some extent different to FBS. Total protein, glucose, cholesterol and Na+ were elevated in pHPL preparations, K+ and Fe3+ levels were higher in FBS. Compared to FBS, pHPL-based media significantly enhanced stromal cell propagation. Characteristic immunophenotype and in vitro differentiation potential were maintained in all four culture conditions. The analysis of growth factors and cytokines revealed distinct levels depending on the pre-existence in pHPL, consumption or secretion by the stromal cells. Interestingly, mRNA expression of the transcription and mitotic bookmarking factors cMYC and KLF4 was significantly enhanced in a source dependent manner in stromal cells cultured in pHPL- compared to FBS-supplemented media. SOX2 mRNA expression of all stromal cell types was increased in all pHPL culture conditions.ConclusionAll pHPL-supplemented media equally supported proliferation of WAT- and UC-derived stromal cells significantly better than FBS. Mitotic bookmarking factors, known to enable a quick re-entry to the cell cycle, were significantly enhanced in pHPL-expanded cells. Our results support a better characterization and standardization of humanized culture media for stromal cell-based medicinal products.

Human Platelet Lysate Supports Efficient Expansion and Stability of Wharton’s Jelly Mesenchymal Stromal Cells via Active Uptake and Release of Soluble Regenerative Factors

“…Some of these factors have been previously correlated with the production of immunoregulatory signals that downsize the immune response [ 29 ], facilitate efficient cell engraftment and support wound healing [ 30 ]. Moreover, our report is consistent with previous observations pointing to VEGF-A, HGF, GRO family, osteoprotegerin and IL-6 secretion by hPL-expanded MSC as crucial signals in promoting angiogenesis and formation of reparative M2 macrophages [ 22 , 31 ]. Specifically, WJ-MSC were showed to be capable of producing secretory signals depending on the microenvironment in which they were grown and promoted anti-inflammatory responses by activating the induction of PGE2, IDO, TGF beta and HGF, thereby contributing to suppression of T cell proliferation and induction of Treg lymphocytes [ 32 ].…”

“…Identified factors here, belong to the group of immunomodulatory molecules (MIF, IL-10, IL-6, TGF alpha, GM-CSF, G-CSF), chemokines (RANTES, GRO alpha and Beta) and pro-angiogenic and pro-survival factors (IGFBP-1 and 2, VEGF-A, PDFG-AA, AB and BB, FGF basic, EGF, MSP/MST1, HGF and osteoprotegerin). Consistent with previous studies, VEGF-A, TGF, FGF, EGF, RANTES, IL-6 and PDGF were significantly higher in hPL than in FBS [ 16 , 21 , 22 , 23 ]. Moreover, the secretome profile here obtained have been also recognized to contain critical cytokines and growth factors that promote angiogenic processes, cell repair and macrophage activation leading to wound healing [ 9 , 10 ].…”

“…In our study, neither hS nor FBS did show striking profiles of growth factor consumption, pointing to hPL as suitable source of critical factors to support WJ-MSC in the context of cell expansion. Noteworthily, PDGF and EGF were reported to influence the expression of cell cycle-associated genes such as cMyc and KLF4 during MSC expansion [ 22 , 25 , 26 , 27 , 28 ]. Furthermore, TGF alpha, HGF and EGF augmented cell survival and PDGF-BB reduced cell loss induced by apoptosis [ 16 ].…”

“…Here we observed that WJ-MSC expanded in hPL enhanced their proliferative capacity, observing an increase of 33.3% in PD and consequent reduction of PDT (by 38.1%), as compared to WJ-MSC expanded in hS or FBS. Several studies described mixed proliferation rates of different MSC types following hPL supplementation [ 13 , 22 , 31 , 33 ]. In particular Kandoi et al evidenced significantly faster cell growth of umbilical cord tissue-derived MSC in hPL-enriched cultures, reaching PDT of 20.95 h vs. PDT in FBS [ 5 ].…”

Influence of platelet storage time on human platelet lysates and platelet lysate-expanded mesenchymal stromal cells for bone tissue engineering

“…In the present study, the cytokine content of HPL was analyzed via a quantitative multiplex immunoassay to identify potentially relevant cytokines contributing to MSC osteogenesis. Although previous studies have measured cytokines in HPL via semi-quantitative assays [ 22 , 36 – 40 ], to our knowledge, only five studies have reported quantitative multiplex-based assessments [ 21 , 39 , 41 – 44 ]. Considerable differences in cytokine concentrations are observed across the different studies (Table 1 ).…”