Clonal Composition of Human Multipotent Mesenchymal Stromal Cells: Application of Genetic Barcodes in Research.

Clonal composition of human multipotent mesenchymal stromal cells (MMSCs) labeled with lentiviral vectors carrying genetic barcodes was studied. MMSCs were transduced with a cloned library of self-inactivating lentiviral vectors carrying 667 unique barcodes. At each cell culture passage, 120 cells were plated one cell per well in 96-well plates. The efficiency of cloning and labeling of the clonogenic cells was determined. DNA was extracted from the cell-derived colonies, and the barcodes were identified by Sanger sequencing. Also, DNA was extracted from the total MMSC population at each passage to analyze the diversity and representation of barcodes by deep sequencing using the Illumina platform. It was shown that the portion of MMSCs labeled with the lentiviral vectors remained stable in the passaged cells. Because of the high multiplicity of infection, the labeling procedure could decrease the proliferative potential of MMSCs. Identification of barcodes in individual cell clones confirmed the polyclonal character of the MMSC population. Clonal composition of MMSCs changed significantly with the passages due to the depletion of proliferative potential of most cells. Large clones were found at the first passage; at later passages, many small clones with a limited proliferative potential were detected in the population. The results of deep sequencing confirmed changes in the clonal composition of MMSCs. The polyclonal MMSC population contained only a small number of cells with a high proliferative potential, some of which could be stem cells. MMSCs with a high proliferative potential were detected more often in the earliest passages. In this regard, we would recommend to use MMSCs of early passages for regenerative medicine applications based on cell proliferation.

New genes for accurate normalization of qRT-PCR results in study of iPS and iPS-derived cells.

iPSC-derived cells (from induced pluripotent stem cells) are a useful source that provide a powerful and widely accepted tool for the study of various types of human cells in vitro. Indeed, iPSC-derived cells from patients with hereditary diseases have been shown to reproduce the hallmarks of these diseases in vitro, phenotypes that can then also be manipulated in vitro. Quantitative reverse transcription PCR (qRT-PCR) is often used to characterize the progress of iPSC differentiation, validate mature cell types and to determine levels of pathological markers. Quantitative reverse transcription PCR (qRT-PCR) is used to quantify mRNA levels. This method requires some way of normalizing the data, typically by relating the obtained levels of gene expression to the levels of expression of a "house keeping gene", a gene whose expression is presumed not to change during manipulation of the cells. In the literature, typically only one such reference gene is used and its stability of expression during cell manipulation is not demonstrated. We are not aware of any study systematically looking at the expression of such genes in human iPSC or during their differentiation into neurons. Here we compare the expression of 16 reference genes in iPSC, neural stem cells (NSC) and neurons derived from iPSC. The applications GeNorm and NormFinder were used to identify the most suitable reference genes. We showed that ACTb, C1orf43, PSMB4, GAPDH and HMBS have the most stable expression. The use of these reference genes allows an accurate normalization of qRT-PCR results in all the cell types discussed above. We hope that this report will help to enable the performance of proper qRT-PCR results normalization in studies with iPSC-derived cells and in disease-modeling reports.