Differentiation and Characterization of Human MSCs

Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, LA, USA.
Methods in Molecular Biology (Impact Factor: 1.29). 02/2008; 449:93-107. DOI: 10.1007/978-1-60327-169-1_7
Source: PubMed


One of the hallmark characteristics of human MSCs (hMSCs) is their ability to differentiate into adipocytes, chondrocytes and osteocytes in culture. The default fate for hMSCs appears to be bone: if late-passage cultures are left in basic culture medium, the hMSCs will become confluent and produce mineral, an indication of bone formation. However, when grown under certain culture conditions or in media containing specific components, the cells can be driven to become a number of other specific cell types including neural cells, myocytes, and cardiomyocytes. The protocols given here are the basic differentiation procedures for inducing osteogenesis, adipogenesis, and chondrogenesis in cultures of hMSCs. Although there is still no clear consensus on the antigen expression pattern that will define hMSCs, a protocol is also presented for the flow cytometric analysis using a series of antibody panels. The analysis of these surface epitope patterns can aide in the isolation and characterization of hMSCs.


Available from: Hugh Alan Tucker, Feb 17, 2014
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    • "This expression profile is typical for ASCs except for CD45 and CD14. Phenotypic characterizations are summarized in Table 1[22,23]. In general, these results demonstrated successful ASC isolation. "
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    ABSTRACT: Adipose derived mesenchymal stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding the factors Insulin-like growth factor-1 (IGF-1), Transforming growth factor-b beta1 (TGF-beta1), Fibroblast growth factor 2 (FGF-2), and Sex determining region Y-box 9 (SOX9) either alone or in combinations. Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections (MOIs) of Ad.IGF-1, Ad.TGF-beta1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real time PCR (qRT-PCR) or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycans (GAGs) and collagen content, respectively. qRT-PCR expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage-matrix, proteoglycan, and collagen II (All with P = <0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen type II, with limited expression of collagens I and X demonstrated by histological analyses, and had significantly greater GAGs and collagen production than the positive control (P= <0.001). Western blot analyses also demonstrated for this combination, increased expression of collagen II, while expression of collagens I and X were undetectable and limited, respectively.
    Arthritis research & therapy 07/2013; 15(4):R80. DOI:10.1186/ar4260 · 3.75 Impact Factor
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    • "(CCM) comprising a-Minimum Essential Medium (a-MEM) supplemented with 16.5% (v/v) fetal bovine serum (FBS, Atlanta Biologicals, Inc., Lawrenceville, GA, USA), 2 mM L-glutamine (Gibco/Invitrogen, Carlsbad, CA, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco/Invitrogen, Carlsbad, CA, USA). The cells were seeded at a density of 3000 cells/cm 2 and placed in an incubator under 5% CO 2 [29] [30] "
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    ABSTRACT: Nanotopography changes human mesenchymal stem cells (hMSC) from their shape to their differentiation potential; however little is known about the underlying molecular mechanisms. Here we study the culture of hMSC on polydimethylsiloxane substrates with 350 nm grating topography and investigate the focal adhesion composition and dynamics using biochemical and imaging techniques. Our results show that zyxin protein plays a key role in the hMSC response to nanotopography. Zyxin expression is downregulated on 350 nm gratings, leading to smaller and more dynamic focal adhesion. Since the association of zyxin with focal adhesions is force-dependent, smaller zyxin-positive adhesion as well as its higher turnover rate suggests that the traction force in focal adhesion on 350 nm topography is decreased. These changes lead to faster and more directional migration on 350 nm gratings. These findings demonstrate that nanotopography decreases the mechanical forces acting on focal adhesions in hMSC and suggest that force-dependent changes in zyxin protein expression and kinetics underlie the focal adhesion remodeling in response to 350 nm grating topography, resulting in modulation of hMSC function.
    Biomaterials 04/2012; 33(20):4998-5003. DOI:10.1016/j.biomaterials.2012.03.053 · 8.56 Impact Factor
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    • "Complete kits designed for adiopcyte, chondrocyte, and osteocyte differentiation from MSC are commercially available or medias can be made in-house. The protocols described below are adapted from the commercially available Invitrogen protocols and the paper by Reger et al.[48] Figure 4 demonstrates a basic characterization of bone-marrow-derived MSC by FACS and their differentiation to adipocytes and osteocytes. In addition, human MSC have recently been shown to be an excellent source of SMC for arterial engineering.[4950] "
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    03/2012; 2(1):84-100. DOI:10.4103/2045-8932.94841
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