Isolation of progenitor cells from cord blood using adhesion matrices

Abstract

The aim of this study was to develop optimal conditions for selective adhesion and isolation of mesenchymal progenitor cells (MPCs) from cord blood and to determine their potential for osteogenic differentiation. Mononuclear cells (MNCs) were isolated by Ficoll-Paque gradient and plated onto 48-well culture plates precoated with: human or bovine collagen type I, human collagen type IV, fibronectin or matrigel. Cultures were incubated in aMEM containing fetal calf serum. Viability of the adherent cells was determined by alamarBlue1 assay after 2, 3, and 4 weeks. After 4 weeks in culture, cells were typsinized and replated. Primary cultures were analyzed by histochemistry and third passage cells by FACS. Isolated fibroblast-like cells were cultured in the presence of osteogenic factors and differentiation determined by Alizarin Red S staining, RT-PCR and electron dispersive spectroscopy (EDS). MNCs adhered to all types of matrices with the greatest adhesion rates on fibronectin. . Primary cultures expressed endothelial/macrophage RNA markers whether cultured on fibronectin or polystyrene and these markers decreased upon passage. The best osteogenic differentiation was observed in MPCs cultured in osteogenic medium containing vitamin D 3 and FGF9. These cells expressed the bonerelated mRNA, collagen type I, core binding factor I (Cbfa I), osteocalcin and osteopontin. EDS of deposits produced by these cells demonstrated a calcium/phosphate ratio parallel to hydroxyapatite. It was concluded that fibronectin increased adhesion rates and isolation potential of cord blood mesenchymal progenitor cells.

In vitro culture and differentiation of osteoblasts from human umbilical cord blood

“…This result may be explained by the fact that expression of osteocalcin is not correlated with the degree of cytochemical stains like AR, AP and VK (Sudo et al 2007). Therefore, an alternative solution was adopted suggested by Maurice et al (2007) who asserted that the enrichment of vitamin D 3 and FGF9 in their osteogenic medium was essential for the positive expression of osteocalcin. This finding was corroborated by other studies which stated that in vitro differentiation of embryonic stem cells (ES) and MSCs need a few important factors, among them are ascorbic acid, dexamethasone and 1,25 dihydroxyvitamin D 3 , essential elements for matrix deposition and mineralization (Purpura et al 2004;Duplomb et al 2007;Jono et al 1998;Long et al 1995).…”

“…Human MSCs have been isolated from various sources, such as adipose tissues, bone marrow (BM), umbilical cord blood (UCB), amniotic fluid, amniotic placenta, scalp tissue, amniotic membrane, and synovial tissue (Chao et al 2004;Koc and Lazarus 2001;Xu et al 2004;Minguell et al 2001;Song and Tuan 2004;Goodwin et al 2001;Rosada et al 2003;Kogler and Wernet 2006;Riordan et al 2007;Koblas et al 2005). Functional analysis has confirmed that they share a core of gene expression profiles (Musina et al 2007;Tsai et al 2007;Reddi 2006;Tuan and Chen 2006;Chang et al 2006;Maurice et al 2007;de Bari and Dell'Accio 2007;Liu et al 2007). MSC cultures in vitro are characterized by homogeneous colonies with a typical fibroblast spindle like shape Bieback et al 2004;Minguell et al 2001).…”

“…It is widely recognized that human UCB is a good source of hematopoietic stem cells and has been used as a valid alternative source for hematopoietic stem cell transplantation Park et al 2006;Bieback et al 2004;Chao et al 2004;Goodwin et al 2001;Rosada et al 2003;Jang et al 2006;Kang et al 2006;Musina et al 2007;Van de Ven et al 2007;Maurice et al 2007;Rebelatto et al 2008). However, the UCB MSCs are able to switch into different cell phenotypes including osteoblasts-osteoclasts, chondrocytes, adipocytes, keratinocytes and neurons Park et al 2006;Bieback et al 2004;Goodwin et al 2001;Rosada et al 2003;Kim et al 2004;Jang et al 2006;Kang et al 2006;Musina et al 2007; Van de Ven et al 2007;Maurice et al 2007).…”

“…However, the UCB MSCs are able to switch into different cell phenotypes including osteoblasts-osteoclasts, chondrocytes, adipocytes, keratinocytes and neurons Park et al 2006;Bieback et al 2004;Goodwin et al 2001;Rosada et al 2003;Kim et al 2004;Jang et al 2006;Kang et al 2006;Musina et al 2007; Van de Ven et al 2007;Maurice et al 2007). …”

Culture and differentiation of osteoblasts on coral scaffold from human bone marrow mesenchymal stem cells

“…However, the mechanism linked to transcriptor activators activity is mediated by histone acetyltransferases (HATs) which increase the target gene expression, conversely the transcriptor reversors are linked to histone deacetylases which downregulate target gene expression (HDACs) (Maurice et al 2007). There are a few members of HDAC family, HDAC3-4-5, that have been shown to be able to interact with Runx2, inhibiting its transcriptional activity in osteoblast differentiation process, particularly, HDAC 4 or 5 which are involved in TGF-b/ Sma and mad-related protein (Smad) and mediating the inhibition of Runx in osteogenesis and mineralization .…”