Constitutive expression of pluripotency-associated genes in mesodermal progenitor cells (MPCs)

Abstract

Background: We recently characterized a progenitor of mesodermal lineage (MPCs) from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation.

Methodology/principal findings: MPCs are cytofluorimetrically characterized and quantitative RT-PCR was performed to evaluate the gene expression profile, comparing it with MSCs and hESCs lines. Immunofluorescence and dot-blot analysis confirm qRT-PCR data. MPCs exhibit an increased expression of OCT4, NANOG, SALL4, FBX15, SPP1 and to a lesser extent c-MYC and KLF4, but lack LIN28 and SOX2. MPCs highly express SOX15.

Conclusions/significance: MPCs express many pluripotency-associated genes and show a peculiar Oct-4 molecular circuit. Understanding this unique molecular mechanism could lead to identifying MPCs as feasible, long telomeres, target cells for reprogramming with no up-regulation of the p53 pathway. Furthermore MPCs are easily and inexpensively harvested from human bone marrow.

Figure 1. Mesodermal Progenitor Cells (MPCs) differ from MSCs and show unusual immature gene expression pattern.

A) Mesodermal progenitor cells (MPCs) are easily distinguishable in culture from typical spindle-shaped MSCs by their characteristic fried egg-like shape (bars = 50 µm): rounded cells with a thick core region that is highly rifrangent. B) Cytofluorimetric analysis allows evaluation of the purity of selective cultures. MPCs show a high SSC signal, stain brightly with anti-SSEA-4 antibody and lack the mesenchymal markers CD90 and MSCA-1. MPCs share expression of CD105 with MSCs, but at a lower signal. C) Telomere length assay, values of medians TRF are reported, at the end of the lanes, for two paired samples (#1, #2, MW: molecular weight).

Figure 2. Quantitative RT-PCR analysis of 10 samples from MPCs (red squares) or MSCs (black dots).

A) Pluripotency-associated genes, B) Commitment-associated genes, C) Sox family genes and D) other genes of interest. Data are normalized and expressed as media +/− SEM (* p<0.05; ** p<0.01, *** p<0.001).

Figure 3. MPCs express Nanog, Oct-4 and Sox15 but no Sox2 in the nucleus.

Indirect immunofluorescent stain confirms the expression of Nanog, Oct-4 and Sox15 (green) in MPCs' nuclei (blue) but not in MSCs easily distinguishable from mesodermal progenitors by different spatial organization of F-actin (red). MPCs also differ from MSCs due to their unexpected high expression of well-organized Nestin filaments (green) as shown by the lower panels. Green fluorescent isotype controls reveal no specific signal (data not shown).

Figure 4. Comparison of median relative normalized expression of OCT4, NANOG, SOX2 and SOX15 among MPCs, MSCs and hESC lines.

qRT-PCR data from MPC (red dots) and MSC (black dots) populations were calibrated to be compared to hESC signals (blue dots).