Dataset Information

Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages.

ABSTRACT: The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson's correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson's correlation revealed high correlation (r > 0.9) between NF3 and NFopt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs.

SUBMITTER: Lee WJ

PROVIDER: S-EPMC4417979 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages.

Lee Won-Jae WJ Jeon Ryoung-Hoon RH Jang Si-Jung SJ Park Ji-Sung JS Lee Seung-Chan SC Baregundi Subbarao Raghavendra R Lee Sung-Lim SL Park Bong-Wook BW King William Allan WA Rho Gyu-Jin GJ

Stem cells international 20150420

The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used refer ...[more]

PMID: 25972899

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Project description:BackgroundTransdermal osseointegrated prosthesis have relatively high infection rates leading to implant revision or failure. A principle cause for this complication is the absence of a durable impervious biomechanical seal at the interface of the hard structure (implant) and adjacent soft tissues. This study explores the possibility of recapitulating an analogous cellular musculoskeletal-connective tissue interface, which is present at naturally occurring integumentary tissues where a hard structure exits the skin, such as the nail bed, hoof, and tooth.MethodsPorcine mesenchymal stromal cells (pMSCs) were derived from nine different porcine integumentary and connective tissues: hoof-associated superficial flexor tendon, molar-associated periodontal ligament, Achilles tendon, adipose tissue and skin dermis from the hind limb and abdominal regions, bone marrow and muscle. For all nine pMSCs, the phenotype, multi-lineage differentiation potential and their adhesiveness to clinical grade titanium was characterized. Transcriptomic analysis of 11 common genes encoding cytoskeletal proteins VIM (Vimentin), cell-cell and cell-matrix adhesion genes (Vinculin, Integrin β1, Integrin β2, CD9, CD151), and for ECM genes (Collagen-1a1, Collagen-4a1, Fibronectin, Laminin-α5, Contactin-3) in early passaged cells was performed using qRT-PCR.ResultsAll tissue-derived pMSCs were characterized as mesenchymal origin by adherence to plastic, expression of cell surface markers including CD29, CD44, CD90, and CD105, and lack of hematopoietic (CD11b) and endothelial (CD31) markers. All pMSCs differentiated into osteoblasts, adipocytes and chondrocytes, albeit at varying degrees, under specific culture conditions. Among the eleven adhesion genes evaluated, the cytoskeletal intermediate filament vimentin was found highly expressed in pMSC isolated from all tissues, followed by genes for the extracellular matrix proteins Fibronectin and Collagen-1a1. Expression of Vimentin was the highest in Achilles tendon, while Fibronectin and Col1agen-1a1 were highest in molar and hoof-associated superficial flexor tendon bone marrow, respectively. Achilles tendon ranked the highest in both multilineage differentiation and adhesion assessments to titanium metal.ConclusionsThese findings support further preclinical research of these tissue specific-derived MSCs in vivo in a transdermal osseointegration implant model.

Dataset Information

Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages.

Publications

Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages.

Similar Datasets

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