Derivation of a novel undifferentiated human foetal phenotype in serum‐free cultures with BMP‐2

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 foetal femur-derived cells in a chemically defined medium (CDM) with activin A and fibroblast growth factor-2 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 demonstrated 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 SOX-2 and SOX-9 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 foetal 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.

Regionally-derived cell populations and skeletal stem cells from human foetal femora exhibit specific osteochondral and multi-lineage differentiation capacity in vitro and ex vivo

“…Foetal age was determined according to foot length and expressed as days post-conception. Samples ranged from 53 to 69 days (4 mm to 8.5 mm) [ 5 , 61 ]. Femora were dissected and surrounding soft tissues were removed by gentle rolling back and forth across sterile filter paper.…”

Regulation of proliferation and differentiation of human fetal bone cells

“…This result could be explained by the different composition of nutrients, for which those contained in αMEM are more appropriate for Characterization and regulation of human fetal bone cells the development and the physiology of fibroblastic-cell types. To date, only one other study has reported on the effect of several growth media on proliferation of fetal femur-derived cells (Mirmalek-Sani et al, 2009). However, these cells were taken at 8 weeks of development (only one in fetal development stage), which thus represents stem cells and not fetal (> 9 weeks) by definition.…”

CSRP1 Promotes Colon Adenocarcinoma Growth and Serves as an Independent Risk Biomarker for Worse Prognosis

“…In addition, CSRP1 has been reported to participate in cell differentiation. For example, stimulation of human fetal femur-derived cells with fibroblast growth factor (FGF)-2 resulted into a significantly decreased CSRP1 level and reduced amino acid turnover, suggesting an undifferentiated cell status [ 7 ].…”