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, Nicholas Ditzel, Moustapha Kassem

*Corresponding author for this work

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Abstract

Background: 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. 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). Results: Cultured 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. Conclusions: Our 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.

Original languageEnglish
Article number3
JournalBiological Procedures Online
Volume21
Number of pages11
DOIs
Publication statusPublished - Feb 2019

Fingerprint

Mesenchymal Stromal Cells
Osteoblasts
Population
Fibroblast Growth Factor 2
Stromal Cells
In Vitro Techniques
Insulin-Like Growth Factor I
Adipocytes
Vascular Endothelial Growth Factor A
Cell Line

Keywords

  • bFGF
  • BMSC
  • Ectopic bone
  • Osteoblast
  • Stem cells

Cite this

@article{1b3cb100046d4bc7a14268f11c737cc7,
title = "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",
abstract = "Background: 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. 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). Results: Cultured 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. Conclusions: Our 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.",
keywords = "bFGF, BMSC, Ectopic bone, Osteoblast, Stem cells",
author = "Abdallah, {Basem M.} and Alzahrani, {Abdullah M.} and Abdel-Moneim, {Ashraf M.} and Nicholas Ditzel and Moustapha Kassem",
year = "2019",
month = "2",
doi = "10.1186/s12575-019-0091-3",
language = "English",
volume = "21",
journal = "Biological Procedures Online",
issn = "1480-9222",
publisher = "BioMed Central",

}

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

In: Biological Procedures Online, Vol. 21, 3, 02.2019.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - 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

AU - Abdallah, Basem M.

AU - Alzahrani, Abdullah M.

AU - Abdel-Moneim, Ashraf M.

AU - Ditzel, Nicholas

AU - Kassem, Moustapha

PY - 2019/2

Y1 - 2019/2

N2 - Background: 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. 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). Results: Cultured 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. Conclusions: Our 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.

AB - Background: 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. 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). Results: Cultured 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. Conclusions: Our 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.

KW - bFGF

KW - BMSC

KW - Ectopic bone

KW - Osteoblast

KW - Stem cells

U2 - 10.1186/s12575-019-0091-3

DO - 10.1186/s12575-019-0091-3

M3 - Journal article

VL - 21

JO - Biological Procedures Online

JF - Biological Procedures Online

SN - 1480-9222

M1 - 3

ER -