Batch Effects during Human Bone Marrow Stromal Cell Propagation Prevail Donor Variation and Culture Duration: Impact on Genotype, Phenotype and Function.

Gabriele Brachtl,Anna Raninger,Rodolphe Poupardin,Stephan Schlickeiser,Sven Geissler,Hans-Dieter Volk,Michaela Oeller,Dirk Strunk,Sarah Hochmann,Martin Wolf,Katharina Schallmoser,Mathias Streitz,Karsten Jürchott

doi:10.3390/cells11060946

Abstract

Donor variation is a prominent critical issue limiting the applicability of cell-based therapies. We hypothesized that batch effects during propagation of bone marrow stromal cells (BMSCs) in human platelet lysate (hPL), replacing fetal bovine serum (FBS), can affect phenotypic and functional variability. We therefore investigated the impact of donor variation, hPL- vs. FBS-driven propagation and exhaustive proliferation, on BMSC epigenome, transcriptome, phenotype, coagulation risk and osteochondral regenerative function. Notably, propagation in hPL significantly increased BMSC proliferation, created significantly different gene expression trajectories and distinct surface marker signatures, already after just one passage. We confirmed significantly declining proliferative potential in FBS-expanded BMSC after proliferative challenge. Flow cytometry verified the canonical fibroblastic phenotype in culture-expanded BMSCs. We observed limited effects on DNA methylation, preferentially in FBS-driven cultures, irrespective of culture duration. The clotting risk increased over culture time. Moreover, expansion in xenogenic serum resulted in significant loss of function during 3D cartilage disk formation and significantly increased clotting risk. Superior chondrogenic function under hPL-conditions was maintained over culture. The platelet blood group and isoagglutinins had minor impact on BMSC function. These data demonstrate pronounced batch effects on BMSC transcriptome, phenotype and function due to serum factors, partly outcompeting donor variation after just one culture passage.

Highlights

Important successes in skeletal regenerative medicine must not obscure the fact that a panacea for nonunion fractures has not yet been found
By rotational thromboelastometry (ROTEM), we found that bone marrow stromal cells (BMSCs) were less procoagulant in human plasma than stromal cells derived from white adipose tissue or umbilical cord [22]
Heparinized BM aspiration aliquots were divided in two equal parts and equilibrated directly in 500 mL BMSC expansion medium supplemented with either fetal bovine serum (FBS) or human platelet lysate (hPL) O/AB, to initiate P◦, in four-layered cell factories (CF-4) on 2528 cm2 growth area as described in [24,28]

Summary

Introduction

Important successes in skeletal regenerative medicine must not obscure the fact that a panacea for nonunion fractures has not yet been found. BMSCs, representing the prototype mesenchymal stromal cells, are a versatile source of cells for skeletal regeneration [3]. Culture conditions to direct reproducible bone and cartilage differentiation of BMSC are still not fully defined [4]. Organoid-like 3D BMSC differentiation models may provide more realistic readouts for studying osteochondral differentiation [5,6]. We have previously shown that cell culture-expanded BMSCs, but not stromal cells from white adipose tissue or umbilical cord, preserve their potential to create 3D organoid-like cartilage templates in vitro and fully re-establish bone and the hematopoietic marrow niche in vivo, in the presence of human platelet lysate (hPL) [7,8]. BMSC-derived scaffold-free cartilage organoid-like disk transplants could completely regenerate critically sized femur defects in a humanized mouse model. The skeletal regeneration competence was found to be predetermined by a discerning epigenetic landscape enabling common transcription factors to act on genes involved in ossification [9]

Objectives

Methods

Results

Conclusion