Comparison of human bone marrow stromal cells cultured in human platelet growth factors and fetal bovine serum

Jiaqiang Ren,Dawn Ward,Katherine Tran,Ping Jin,Marianna Sabatino,Pamela G Robey,Steven Chen,David F Stroncek

doi:10.1186/s12967-018-1400-3

Abstract

BackgroundBone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with fetal bovine serum (FBS). As an alternative to FBS, pooled solvent detergent apheresis platelets, HPGF-C18, was evaluated for BMSC culture.MethodsA comparison of passage 2 BMSC growth revealed that 10% HPGF-C18 produced similar cell numbers as 20% FBS. Marrow aspirates from 5 healthy subjects were cultured for 4 passages in 10% HPGF-C18 or 20% FBS and were analyzed for proliferation, colony formation efficiency (CFE), surface marker expression, suppression of mixed lymphocyte reactions (MLRs), global gene and microRNA expression analysis. BMSC supernatant cytokine and growth factor concentrations were also compared.ResultsPrimary cultures of marrow aspirates in 10% HPGF-C18 and 20% FBS yielded similar numbers and CFE. After 4 passages, 10% HPGF-C18 and 20% FBS yielded similar numbers of BMSCs, surface marker expression patterns and immunosuppression effects. Gene and microRNA expression analysis revealed that BMSCs cultured under the two conditions had distinct expression profiles. Gene Set Enrichment Analysis (GSEA) revealed HPGF-C18-cultured BMSCs were enriched in metabolic processing and biosynthetic pathways, cell proliferation and cell cycle pathways, and immune response pathways. FBS-cultured BMSCs were enriched in MAPK signaling, TGF-beta signaling, cell adhesion and extracellular matrix pathways. Differently expressed microRNAs were related to the osteogenesis of BMSCs. The supernatant of HPGF-C18 BMSCs had higher levels of PEDF and TGFB1 and lower levels of IL6, VEGF, SDF1 and PLGF.ConclusionsTraditional measures, expansion, surface marker expression and inhibition of MLRs suggest that BMSC cultured in HPGF-C18 and FBS were similar, but analysis at the molecular level revealed many differences. BMSCs cultured in HPGF-C18 should be assessed in specific functional assays that reflect application-specific potency before substituting FBS with HPGF-C18.

Highlights

Bone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with fetal bovine serum (FBS)
Optimization of HPGF‐C18 concentration In order to determine the optimum concentration of HPGF-C18 to support the growth of BMSCs, cryopreserved passage 2 BMSCs prepared from 3 healthy subjects were thawed and cultured for 2 additional passages in αMEM supplemented with 5, 10, 15 and 20% HPGFC18 and 20% FBS
For all 3 donors, BMSCs cultured in 10% HPGF-C18 exhibited similar growth rate to those cultured in 20% FBS, and faster than those cultured in 5, 15 and 20% HPGF, except that 15% HPGF-C18 was more supportive for BMSC growth from donor 09FC49 (Fig. 1a)

Summary

Introduction

Bone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with fetal bovine serum (FBS). BMSCs have immune modulatory properties and can induce angiogenesis and tissue repair. They are being used in early phase clinical trials to treat steroid resistant acute graftversus-host-disease (GVHD) [4], inflammatory bowel disease [5, 6], ischemic vascular disease [7], acute lung injury [8] and traumatic brain injury [9]. Preliminary studies have shown that when BMSCs are cultured in media supplemented with the contents of lysed platelets, they express mesenchymal stromal cells (MSC) surface markers CD73, CD90 and CD105; they differentiate in vitro into adipose tissue, chondrocytes, and osteocytes; and, in general, they proliferate faster than cells grown in FBS [14,15,16,17,18,19,20]

Methods

Results

Discussion

Conclusion