Cargando…

Differentiation Potential of Early- and Late-Passage Adipose-Derived Mesenchymal Stem Cells Cultured under Hypoxia and Normoxia

With an increasing focus on the large-scale expansion of mesenchymal stem cells (MSCs) required for clinical applications for the treatment of joint and bone diseases such as osteoarthritis, the optimisation of conditions for in vitro MSC expansion requires careful consideration to maintain native M...

Descripción completa

Detalles Bibliográficos
Autores principales: Zhao, Ashley G., Shah, Kiran, Freitag, Julien, Cromer, Brett, Sumer, Huseyin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519987/
https://www.ncbi.nlm.nih.gov/pubmed/33014073
http://dx.doi.org/10.1155/2020/8898221
_version_ 1783587686648381440
author Zhao, Ashley G.
Shah, Kiran
Freitag, Julien
Cromer, Brett
Sumer, Huseyin
author_facet Zhao, Ashley G.
Shah, Kiran
Freitag, Julien
Cromer, Brett
Sumer, Huseyin
author_sort Zhao, Ashley G.
collection PubMed
description With an increasing focus on the large-scale expansion of mesenchymal stem cells (MSCs) required for clinical applications for the treatment of joint and bone diseases such as osteoarthritis, the optimisation of conditions for in vitro MSC expansion requires careful consideration to maintain native MSC characteristics. Physiological parameters such as oxygen concentration, media constituents, and passage numbers influence the properties of MSCs and may have major impact on their therapeutic potential. Cells grown under hypoxic conditions have been widely documented in clinical use. Culturing MSCs on large scale requires bioreactor culture; however, it is challenging to maintain low oxygen and other physiological parameters over several passages in large bioreactor vessels. The necessity to scale up the production of cells in vitro under normoxia may affect important attributes of MSCs. For these reasons, our study investigated the effects of normoxic and hypoxic culture condition on early- and late-passage adipose-derived MSCs. We examined effect of each condition on the expression of key stem cell marker genes POU5F1, NANOG, and KLF4, as well as differentiation genes RUNX2, COL1A1, SOX9, COL2A1, and PPARG. We found that expression levels of stem cell marker genes and osteogenic and chondrogenic genes were higher in normoxia compared to hypoxia. Furthermore, expression of these genes reduced with passage number, with the exception of PPARG, an adipose differentiation marker, possibly due to the adipose origin of the MSCs. We confirmed by flow cytometry the presence of cell surface markers CD105, CD73, and CD90 and lack of expression of CD45, CD34, CD14, and CD19 across all conditions. Furthermore, in vitro differentiation confirmed that both early- and late-passage adipose-derived MSCs grown in hypoxia or normoxia could differentiate into chondrogenic and osteogenic cell types. Our results demonstrate that the minimal standard criteria to define MSCs as suitable for laboratory-based and preclinical studies can be maintained in early- or late-passage MSCs cultured in hypoxia or normoxia. Therefore, any of these culture conditions could be used when scaling up MSCs in bioreactors for allogeneic clinical applications or tissue engineering for the treatment of joint and bone diseases such as osteoarthritis.
format Online
Article
Text
id pubmed-7519987
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher Hindawi
record_format MEDLINE/PubMed
spelling pubmed-75199872020-10-02 Differentiation Potential of Early- and Late-Passage Adipose-Derived Mesenchymal Stem Cells Cultured under Hypoxia and Normoxia Zhao, Ashley G. Shah, Kiran Freitag, Julien Cromer, Brett Sumer, Huseyin Stem Cells Int Research Article With an increasing focus on the large-scale expansion of mesenchymal stem cells (MSCs) required for clinical applications for the treatment of joint and bone diseases such as osteoarthritis, the optimisation of conditions for in vitro MSC expansion requires careful consideration to maintain native MSC characteristics. Physiological parameters such as oxygen concentration, media constituents, and passage numbers influence the properties of MSCs and may have major impact on their therapeutic potential. Cells grown under hypoxic conditions have been widely documented in clinical use. Culturing MSCs on large scale requires bioreactor culture; however, it is challenging to maintain low oxygen and other physiological parameters over several passages in large bioreactor vessels. The necessity to scale up the production of cells in vitro under normoxia may affect important attributes of MSCs. For these reasons, our study investigated the effects of normoxic and hypoxic culture condition on early- and late-passage adipose-derived MSCs. We examined effect of each condition on the expression of key stem cell marker genes POU5F1, NANOG, and KLF4, as well as differentiation genes RUNX2, COL1A1, SOX9, COL2A1, and PPARG. We found that expression levels of stem cell marker genes and osteogenic and chondrogenic genes were higher in normoxia compared to hypoxia. Furthermore, expression of these genes reduced with passage number, with the exception of PPARG, an adipose differentiation marker, possibly due to the adipose origin of the MSCs. We confirmed by flow cytometry the presence of cell surface markers CD105, CD73, and CD90 and lack of expression of CD45, CD34, CD14, and CD19 across all conditions. Furthermore, in vitro differentiation confirmed that both early- and late-passage adipose-derived MSCs grown in hypoxia or normoxia could differentiate into chondrogenic and osteogenic cell types. Our results demonstrate that the minimal standard criteria to define MSCs as suitable for laboratory-based and preclinical studies can be maintained in early- or late-passage MSCs cultured in hypoxia or normoxia. Therefore, any of these culture conditions could be used when scaling up MSCs in bioreactors for allogeneic clinical applications or tissue engineering for the treatment of joint and bone diseases such as osteoarthritis. Hindawi 2020-09-18 /pmc/articles/PMC7519987/ /pubmed/33014073 http://dx.doi.org/10.1155/2020/8898221 Text en Copyright © 2020 Ashley G. Zhao et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Zhao, Ashley G.
Shah, Kiran
Freitag, Julien
Cromer, Brett
Sumer, Huseyin
Differentiation Potential of Early- and Late-Passage Adipose-Derived Mesenchymal Stem Cells Cultured under Hypoxia and Normoxia
title Differentiation Potential of Early- and Late-Passage Adipose-Derived Mesenchymal Stem Cells Cultured under Hypoxia and Normoxia
title_full Differentiation Potential of Early- and Late-Passage Adipose-Derived Mesenchymal Stem Cells Cultured under Hypoxia and Normoxia
title_fullStr Differentiation Potential of Early- and Late-Passage Adipose-Derived Mesenchymal Stem Cells Cultured under Hypoxia and Normoxia
title_full_unstemmed Differentiation Potential of Early- and Late-Passage Adipose-Derived Mesenchymal Stem Cells Cultured under Hypoxia and Normoxia
title_short Differentiation Potential of Early- and Late-Passage Adipose-Derived Mesenchymal Stem Cells Cultured under Hypoxia and Normoxia
title_sort differentiation potential of early- and late-passage adipose-derived mesenchymal stem cells cultured under hypoxia and normoxia
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519987/
https://www.ncbi.nlm.nih.gov/pubmed/33014073
http://dx.doi.org/10.1155/2020/8898221
work_keys_str_mv AT zhaoashleyg differentiationpotentialofearlyandlatepassageadiposederivedmesenchymalstemcellsculturedunderhypoxiaandnormoxia
AT shahkiran differentiationpotentialofearlyandlatepassageadiposederivedmesenchymalstemcellsculturedunderhypoxiaandnormoxia
AT freitagjulien differentiationpotentialofearlyandlatepassageadiposederivedmesenchymalstemcellsculturedunderhypoxiaandnormoxia
AT cromerbrett differentiationpotentialofearlyandlatepassageadiposederivedmesenchymalstemcellsculturedunderhypoxiaandnormoxia
AT sumerhuseyin differentiationpotentialofearlyandlatepassageadiposederivedmesenchymalstemcellsculturedunderhypoxiaandnormoxia