Identification of Appropriate Reference Genes for Human Mesenchymal Cells during Expansion and Differentiation

<div><p>Background</p><p>Quantitative real time polymerase chain reaction (qPCR) is an extremely powerful technique for monitoring gene expression. The quantity of the messenger ribonucleic acids (mRNA) of interest should be normalized using a reference gene, in order to avoid unreliable results originated by the obtained RNA quality and quantity, manipulation errors and inhibitory contaminants. A reference gene is any gene that is stably and consistently expressed under the conditions being studied. Completely false data can be generated if a reference gene is not chosen adequately.</p><p>Results</p><p>In the present study, we compared expression levels of five putative reference genes (HPRT1, ACTB, GAPDH, RPL13A and B2M) in primary cultures of four different human cells: mesenchymal stromal cells obtained from bone marrow, adipose tissue or umbilical cord Whartońs Jelly, and dermal fibroblasts, under different expansion and differentiation conditions. We observed that reference genes are not the same for different cells under the same culture conditions.</p><p>Conclusion</p><p>Most stable reference genes under our experimental conditions were: RPL13A for adipose tissue- and Whartońs Jelly-derived mesenchymal stromal cells, and HPRT1 for bone marrow-derived mesenchymal stromal cells and dermal fibroblasts. ACTB was the most unstable gene when evaluating adipose tissue- and Whartońs Jelly-derived mesenchymal stromal cells, whilst GAPDH and B2M were the most unstable genes for bone marrow-derived mesenchymal stromal cells and dermal fibroblasts, respectively.</p></div

Quantification of cell surface markers by flow cytometry (expressed in mean percentage ± standard deviation) in human adipose tissue- (AT-), bone marrow- (BM-), and Wharton's Jelly-mesenchymal stromal cells (WJ-MSC) when grown in α-MEM supplemented with 10% human platelet-rich plasma (PRP).

<p>Quantification of cell surface markers by flow cytometry (expressed in mean percentage ± standard deviation) in human adipose tissue- (AT-), bone marrow- (BM-), and Wharton's Jelly-mesenchymal stromal cells (WJ-MSC) when grown in α-MEM supplemented with 10% human platelet-rich plasma (PRP).</p

Q-values (arithmetic mean and standard deviation) obtained after statistical pair wise comparisons.

<p>AM: arithmetic mean; SD: standard deviation.</p

BestKeeper results obtained for bone marrow-derived mesenchymal stromal cells.

<p>The last column (BK, n = 3) shows the parameters obtained after combining all stably expressed genes (B2M, RPL13A and HPRT1), so they can be use as a single normalization factor. n: number of samples; BK: BestKeeper; GM [Cq]: geometric mean of Cq; AM [Cq]: arithmetic mean of Cq; Min [Cq]: minimum value of Cq; Max [Cq]: maximum value of Cq; SD [± Cq]: standard deviation of the Cq; CV [% Cq]: coefficient of variation expressed as a percentage on the Cq level; Min [x-fold]: minimum value of expression levels expressed as an absolute x-fold; Max [x-fold]: maximum value of expression levels expressed as an absolute x-fold; SD [± x-fold]: standard deviation of the absolute regulation coefficients. SD values obtained for ACTB (1.42) and GAPDH (1.03) are higher than cutoff (1.0), so both genes were defined as unstably expressed genes and therefore were not considered for BK (n = 3) calculations.</p

Effect of PRP supplementation on gene expression (expressed in mean ± standard deviation as the ratio between expression in PRP cultures and FBS cultures) for human adipose tissue- (AT-), bone marrow- (BM-) and Wharton's Jelly-mesenchymal stromal cells (WJ-MSC) when grown in α-MEM supplemented with 10% human platelet-rich plasma (PRP).

<p>*not detected in PRP sample;</p><p>**not detected in FBS sample;</p><p>***not detected in any sample.</p><p>Negative values mean downregulation on mRNA expression by PRP, when compared to FBS-cultured cells, and positive values, upregulation.</p

BestKeeper results obtained for adipose tissue-derived mesenchymal stromal cells.

<p>The last column (BK, n = 4) shows the parameters obtained after combining all stably expressed genes (B2M, RPL13A, HPRT1 and GAPDH), so they can be use as a single normalization factor. n: number of samples; BK: BestKeeper; GM [Cq]: geometric mean of Cq; AM [Cq]: arithmetic mean of Cq; Min [Cq]: minimum value of Cq; Max [Cq]: maximum value of Cq; SD [± Cq]: standard deviation of the Cq; CV [% Cq]: coefficient of variation expressed as a percentage on the Cq level; Min [x-fold]: minimum value of expression levels expressed as an absolute x-fold; Max [x-fold]: maximum value of expression levels expressed as an absolute x-fold; SD [± x-fold]: standard deviation of the absolute regulation coefficients. SD value obtained for ACTB (2.33) is higher than cutoff (1.0), so ACTB was defined as an unstably expressed gene and therefore was not considered for BK (n = 4) calculations.</p

Cytokine, growth factor and extracellular matrix protein concentration (expressed in mean pg/10⁶ cells/day ± standard deviation) in human adipose tissue- (AT-), bone marrow- (BM-) and Wharton's Jelly-mesenchymal stromal cells (WJ-MSC) supernatants when grown in α-MEM supplemented with 10% human platelet-rich plasma (PRP). nd: not determined (concentration below the detection limit).

<p>Cytokine, growth factor and extracellular matrix protein concentration (expressed in mean pg/10⁶ cells/day ± standard deviation) in human adipose tissue- (AT-), bone marrow- (BM-) and Wharton's Jelly-mesenchymal stromal cells (WJ-MSC) supernatants when grown in α-MEM supplemented with 10% human platelet-rich plasma (PRP). nd: not determined (concentration below the detection limit).</p

Cell differentiation assays of human bone marrow- (BM-), adipose tissue- (AT-) and Wharton's Jelly-derived mesenchymal stromal (WJ-MSC) into adipogenic, osteogenic and chondrogenic phenotypes under 10% human platelet-rich plasma (PRP) supplemented medium.

<p>Cell differentiation assays of human bone marrow- (BM-), adipose tissue- (AT-) and Wharton's Jelly-derived mesenchymal stromal (WJ-MSC) into adipogenic, osteogenic and chondrogenic phenotypes under 10% human platelet-rich plasma (PRP) supplemented medium.</p

Putative reference genes chosen in this study.

<p>Putative reference genes chosen in this study.</p

BestKeeper results obtained for dermal fibroblasts.

<p>The last column (BK, n = 5) shows the parameters obtained after combining all stably expressed genes (B2M, RPL13A and HPRT1), so they can be use as a single normalization factor. n: number of samples; BK: BestKeeper; GM [Cq]: geometric mean of Cq; AM [Cq]: arithmetic mean of Cq; Min [Cq]: minimum value of Cq; Max [Cq]: maximum value of Cq; SD [± Cq]: standard deviation of the Cq; CV [% Cq]: coefficient of variation expressed as a percentage on the Cq level; Min [x-fold]: minimum value of expression levels expressed as an absolute x-fold; Max [x-fold]: maximum value of expression levels expressed as an absolute x-fold; SD [± x-fold]: standard deviation of the absolute regulation coefficients. No unstably expressed gene detected.</p