A major challenge in developmental biology is to decipher gene expression controlling stemness and differentiation networks. A more practical goal is to identify among these genes those encoding membrane antigens such as receptors, which could be used as markers to track or select stem cells from the earliest pluripotent cells to the latest adult progenitor cells. In this study, we analyzed gene expression in pluripotent human Embryonic Stem Cell lines (hESCs), in human multipotent bone marrow Mesenchymal Stem Cells (hMSCs), and in hESC-derived MSCs (hES-MSCs). It is well known that hMSCs constitute a heterogeneous population that may include a subset of more primitive cells [1
]. Although many antibodies directed at cell membrane antigens have been used to select specific hMSC populations [4
], there is no cogent evidence that some of these antibodies may select for a more primitive population with enlarged differentiation potential and robust self-renewal capacity. Even in the mouse, where such a primitive population has been recently characterized by the expression of the cytoskeletal protein nestin [7
], separation according to membrane phenotype is not yet feasible.
In a previous study [8
], we observed that a 24
h pretreatment of Stro1+/GlycoA− or CD45−/GlycoA− hMSCs with a monoclonal antibody blocking the human type I interferon-alpha (IFNα
) receptor, or with a polyclonal anti-IFNα
antibody, resulted in a marked increase in the number of very large colonies (Colony-forming Units-fibroblasts containing more than 3000 cells). Interestingly, these activated High Proliferative Potential-Quiescent hMSCs (HPP-Q hMSCs) expressed SSEA-3 and -4 at a higher level than non-pretreated cells, suggesting that HPP-Q hMSCs constituted a more primitive cell population than nontreated cells.
A more direct way to characterize the most primitive hMSCs would be to select them from hESCs induced to differentiate into hMSCs, using various procedures that we and others have implemented [9
]. In the present work, we have studied hESC-derived MSCs (hES-MSCs) assuming that these cells might represent a more primitive hMSC population. We compared the expression profiles of differentiation genes and genes encoding cytokine and adhesion molecules and their receptors in the three populations hESCs, hES-MSCs and hMSCs, focusing on the genes that are differentially detected in hES-MSCs, and hMSCs. Our data confirmed previous results on hMSC gene expression [14
]. Moreover, the comparison of hES-MSCs with hMSCs has revealed subtle and significant variations in the expression of genes that may help characterize the more primitive hMSCs.
Our team and other investigators have shown that primitive hematopoietic stem cells or early keratinocytes which self-renew without differentiation, express low levels of receptors and respond to low concentrations of cytokines [21
]. In contrast, the same cytokines at high concentrations frequently induce differentiation. We address in this study the question of whether some Receptorlow
cells might represent within hES-MSCs the most primitive hMSC population with a greater mesodermal potential.
hMSCs are not only the stem cells for skeletal connective-tissue (osteoblasts, chondrocytes, and adipocytes), but are also important components of stem cell niches [1
]. Here we have studied the expression by hMSCs of transcripts for cytokines and extracellular matrix (ECM) components in the absence of their receptors, in keeping with the paracrine regulation of hematopoietic stem cells. We have also studied the possibility that hMSCs themselves express receptors to respond to cytokines produced by other cells of the stem cell niche.
The identification of marker genes expressed at low levels in primitive stem cells requires the application of highly sensitive and reliable molecular techniques. We chose a TaqMan Low Density Array (TLDA) to identify the differentially expressed genes. TaqMan assays are based on PCR, resulting in an increase of detectable reporter fluorescence in each cycle. This reliable technique, associated with statistical analyses, allows the identification of biological markers, which could have been overlooked by less sensitive techniques such as microarrays.
Finally, we propose to share with other laboratories our database to promote better understanding of the considerable clinical potential of hESCs, hES-MSCs, and hMSCs (see list of genes available in supplementary material available on line at doi:10.4061/2011/368192).