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Derivation of a novel undifferentiated human fetal phenotype in serum-free cultures with BMP-2

Derivation of a novel undifferentiated human fetal phenotype in serum-free cultures with BMP-2
Derivation of a novel undifferentiated human fetal phenotype in serum-free cultures with BMP-2
Skeletal stem and progenitor populations provide a platform for cell-based tissue regeneration strategies. Optimized conditions for ex vivo expansion will be critical and use of serum-free culture may allow enhanced modelling of differentiation potential. Maintenance of human fetal femur-derived cells in a chemically defined medium (CDM) with Activin A and fibroblast growth factor 2 (FGF2) generated a unique undifferentiated cell population in comparison to basal cultures, with significantly reduced amino acid depletion, appearance and turnover, reduced alkaline phosphatase (ALP) activity and loss of type I and II collagen expression evidenced by fluorescence immunocytochemistry. Microarray analysis demonstrated up-regulation of CLU, OSR2, POSTN and RABGAP1 and down-regulation of differentiation-associated genes CRYAB, CSRP1, EPAS1, GREM1, MT1X and SRGN as validated by quantitative real-time polymerase chain reaction. Application of osteogenic conditions to CDM cultures demonstrated partial rescue of ALP activity. In contrast, the addition of bone morphogenetic protein-2 (BMP-2) resulted in reduced ALP levels, increased amino acid metabolism and, strikingly, a marked shift to a cobblestone-like cellular morphology, with expression of SOX2 and SOX9 but not STRO-1 as shown by immunocytochemistry, and significantly altered expression of metabolic genes (GFPT2, SC4MOL and SQLE), genes involved in morphogenesis (SOX15 and WIF1) and differentiation potential (C1orf19, CHSY-2, DUSP6, HMGCS1 and PPL). These studies demonstrate the use of an intermediary fetal cellular model for differentiation studies in chemically defined conditions and indicate the in vitro reconstruction of the mesenchymal condensation phenotype in the presence of BMP-2, with implications therein for rescue studies, screening assays and skeletal regeneration research
1582-1838
3541-3555
Mirmalek-Sani, S.H.
7eaa1681-73ff-4dbf-8f69-eec805d12a9c
Stokes, P.J.
225afb0e-ac6e-4c2d-828c-1020f6a78550
Tare, R.S.
587c9db4-e409-4e7c-a02a-677547ab724a
Ralph, E.J.
d1915f6b-8cd4-4948-bab3-898976ca04f9
Inglis, S.
cb950bb9-8b89-41c0-b547-3206580d16b4
Hanley, N.A.
ac5bcdcc-51dd-4f2c-ad06-fef6c1d50ebd
Houghton, F.D.
53946041-127e-45a8-9edb-bf4b3c23005f
Oreffo, R.O.
ff9fff72-6855-4d0f-bfb2-311d0e8f3778
Mirmalek-Sani, S.H.
7eaa1681-73ff-4dbf-8f69-eec805d12a9c
Stokes, P.J.
225afb0e-ac6e-4c2d-828c-1020f6a78550
Tare, R.S.
587c9db4-e409-4e7c-a02a-677547ab724a
Ralph, E.J.
d1915f6b-8cd4-4948-bab3-898976ca04f9
Inglis, S.
cb950bb9-8b89-41c0-b547-3206580d16b4
Hanley, N.A.
ac5bcdcc-51dd-4f2c-ad06-fef6c1d50ebd
Houghton, F.D.
53946041-127e-45a8-9edb-bf4b3c23005f
Oreffo, R.O.
ff9fff72-6855-4d0f-bfb2-311d0e8f3778

Mirmalek-Sani, S.H., Stokes, P.J., Tare, R.S., Ralph, E.J., Inglis, S., Hanley, N.A., Houghton, F.D. and Oreffo, R.O. (2009) Derivation of a novel undifferentiated human fetal phenotype in serum-free cultures with BMP-2. Journal of Cellular and Molecular Medicine, 13 (9B), 3541-3555. (doi:10.1111/j.1582-4934.2009.00742.x). (PMID:19438813)

Record type: Article

Abstract

Skeletal stem and progenitor populations provide a platform for cell-based tissue regeneration strategies. Optimized conditions for ex vivo expansion will be critical and use of serum-free culture may allow enhanced modelling of differentiation potential. Maintenance of human fetal femur-derived cells in a chemically defined medium (CDM) with Activin A and fibroblast growth factor 2 (FGF2) generated a unique undifferentiated cell population in comparison to basal cultures, with significantly reduced amino acid depletion, appearance and turnover, reduced alkaline phosphatase (ALP) activity and loss of type I and II collagen expression evidenced by fluorescence immunocytochemistry. Microarray analysis demonstrated up-regulation of CLU, OSR2, POSTN and RABGAP1 and down-regulation of differentiation-associated genes CRYAB, CSRP1, EPAS1, GREM1, MT1X and SRGN as validated by quantitative real-time polymerase chain reaction. Application of osteogenic conditions to CDM cultures demonstrated partial rescue of ALP activity. In contrast, the addition of bone morphogenetic protein-2 (BMP-2) resulted in reduced ALP levels, increased amino acid metabolism and, strikingly, a marked shift to a cobblestone-like cellular morphology, with expression of SOX2 and SOX9 but not STRO-1 as shown by immunocytochemistry, and significantly altered expression of metabolic genes (GFPT2, SC4MOL and SQLE), genes involved in morphogenesis (SOX15 and WIF1) and differentiation potential (C1orf19, CHSY-2, DUSP6, HMGCS1 and PPL). These studies demonstrate the use of an intermediary fetal cellular model for differentiation studies in chemically defined conditions and indicate the in vitro reconstruction of the mesenchymal condensation phenotype in the presence of BMP-2, with implications therein for rescue studies, screening assays and skeletal regeneration research

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Published date: September 2009
Organisations: Faculty of Medicine

Identifiers

Local EPrints ID: 69821
URI: https://eprints.soton.ac.uk/id/eprint/69821
ISSN: 1582-1838
PURE UUID: 75effb10-ffa2-4fcb-bc44-9b44efb688e0
ORCID for R.S. Tare: ORCID iD orcid.org/0000-0001-8274-8837
ORCID for R.O. Oreffo: ORCID iD orcid.org/0000-0001-5995-6726

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Date deposited: 08 Dec 2009
Last modified: 20 Jul 2019 01:08

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Contributors

Author: S.H. Mirmalek-Sani
Author: P.J. Stokes
Author: R.S. Tare ORCID iD
Author: E.J. Ralph
Author: S. Inglis
Author: N.A. Hanley
Author: F.D. Houghton
Author: R.O. Oreffo ORCID iD

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