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

Sandra Laner-Plamberger,Michaela Oeller,Alina Sonderegger,Eva Rohde,Arnulf Hartl,Katharina Schallmoser,Cornelia Mrazek,Dirk Strunk

doi:10.1186/s12967-019-02183-0

Sandra Laner-Plamberger, Michaela Oeller + Show 6 more

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https://doi.org/10.1186/s12967-019-02183-0

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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.

Highlights

Innovative human stromal cell therapeutics require xeno-free culture conditions
Biochemical parameters tested for basal αMEM and αMEM supplemented with Pooled human platelet lysate (pHPL), pHPL ‘serum’ (pHPLS), mechanically fibrinogen-depleted pHPL (mcpHPL) and fetal bovine serum (FBS) are shown
In this study we investigated the impact of culture conditions on the expression of mitotic bookmarking factors SRY-box 2 (SOX2), Kruppel like factor 4 (KLF4), MYC proto-oncogene (cMYC), octamerbinding protein 4 (OCT4) and NANOG in stromal cells from different sources, as several studies have shown that PDGF-BB, which is a central component of human platelet lysate (HPL) [6], directly influences the expression of KLF4: The KLF4 promoter has three Sp1 binding sites, which are required for both baseline and PDGF-BB-induced KLF4 promoter activity in murine smooth muscle cells [50]

Summary

Introduction

Innovative human stromal cell therapeutics require xeno-free culture conditions. Various formulations of human platelet lysate (HPL) are efficient alternatives for fetal bovine serum (FBS). For manufacturing cell-based medicinal products fetal bovine serum (FBS) is still frequently used as medium supplement, bearing the risk of transmission of bovine pathogens and xeno-immunization [4, 5]. Ethical issues concerning the manufacturing process of FBS have to be considered [5,6,7] and in 2007 the European Medicine Agency (EMA) has discouraged the use of animal-derived raw materials for manufacturing of cell therapeutics [8]. The efficient use of human platelet lysate (HPL) for the expansion of stromal cells was introduced in 2005 [9] and confirmed by us and others [6, 10,11,12]. Containing abundant growth factors, cytokines and plasma proteins such as thrombin and fibrinogen [13,14,15], HPL is used for isolation and large scale expansion of stromal cells from different tissues [10, 11, 16,17,18]

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