A simple and reliable protocol for long-term culture of murine bone marrow stromal (mesenchymal) stem cells that retained their in vitro and in vivo stemness in long-term culture

Basem M Abdallah,Abdullah M Alzahrani,Ashraf M Abdel-Moneim,Moustapha Kassem,Nicholas Ditzel

doi:10.1186/s12575-019-0091-3

Abstract

BackgroundBone marrow derived stromal stem cells (BMSCs) are a clonogenic cell population that is characterized by self-renewal capacity and differentiation potential into osteoblasts, and other mesenchymal cell types. Mouse BMSCs (mBMSCs) are difficult to be cultured and propagated in vitro due to their replicative senescent phenotype, heterogeneity and high contamination with plastic adherent hematopoietic progenitors (HPCs). In this study, we described long-term culture of homogenous population of mBMSCs using simple and highly reproducible approach based on frequent subculturing (FS) at fixed split ratio in the presence of basic fibroblast growth factor (bFGF).ResultsCultured mBMSCs using this protocol (mBMSCs-FS) showed long-term survival in culture > 70 population doubling (PD) and retained their characteristic surface markers and differentiation capacity into osteoblast and adipocyte lineages. When compared to the clonal bone marrow-derived cell line ST2, mBMSCs-FS displayed more enhanced osteoblast differentiation potential and responsiveness to osteogenic factors including BMPs, IGF-1, PDGF, TGFβ1,3, FGF, cAMP, Wnt3a and VEGF. In addition, unlike ST2 cells, mBMSCs-FS maintained capacity to form ectopic bone and bone marrow stroma upon in vivo transplantation in immune-compromising mice, even at high PD levels. Interestingly, by applying the same FS + bFGF protocol, we succeeded to obtain long-term cultures of primary neonatal calvarial osteoprogenitor cells (OBs) that were cultured for more than 70 PD and maintained in vitro and in vivo osteoblast differentiation capacities.ConclusionsOur data provide a simple and reliable protocol for generating long-term cultures of mBMSCs and OBs with retained high in vitro and in vivo osteoblast differentiation capacities for use in pre-clinical and molecular mechanism studies.

Highlights

Bone marrow derived stromal stem cells (BMSCs) are a clonogenic cell population that is characterized by self-renewal capacity and differentiation potential into osteoblasts, and other mesenchymal cell types
Factors including the low frequencies of BMSCs in bone marrow, contamination with plastic adherent hematopoietic progenitors (HPCs) and replicative senescence phenotype hampered the culturing of large amount of Mouse BMSCs (mBMSCs) in pure yield that can be used efficiently in different molecular and cellular studies [3, 4]
Simple protocol for culturing and expanding murine BMSCs in long term We aimed to develop a simple, fast and highly reproducible protocol for culturing and expanding large number of mBMSCs for mechanistic and preclinical translational studies

Summary

Introduction

Bone marrow derived stromal stem cells (BMSCs) are a clonogenic cell population that is characterized by self-renewal capacity and differentiation potential into osteoblasts, and other mesenchymal cell types. Several techniques have been used to isolate BMSCs from mouse bone marrow, including using different plating density [5], negative and positive selection based on specific surface marker expression [6,7,8], frequent medium change in primary culture [9], passage-dependent reseeding following trypsinization [10], centrifugation on a Percoll gradient [11] and cell sorting for CD29 (Itgb1) and CD54 (Icam1) [12]. These methods are not simple, need special requirements and do not always result in long-term cultures of BMSCs

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