BackgroundRT-qPCR analysis is a widely used method for the analysis of mRNA expression throughout the field of mesenchymal stromal cell (MSC) research. Comparison between MSC studies, both in vitro and in vivo, are challenging due to the varied methods of RT-qPCR data normalization and analysis. Therefore, this study focuses on putative housekeeping genes for the normalization of RT-qPCR data between heterogeneous commercially available human MSC, compared with more homogeneous populations of MSC such as MIAMI and RS-1 cells.ResultsEight genes including; ACTB, B2M, EF1α, GAPDH, RPL13a, YWHAZ, UBC and HPRT1 were tested as possible housekeeping genes based on their expression level and variability. EF1α and RPL13a were validated for RT-qPCR analysis of MIAMI cells during expansion in varied oxygen tensions, endothelial differentiation, neural precursor enrichment, and during the comparison with RS-1 cells and commercially available MSC. RPL13a and YWHAZ were validated as normalization genes for the cross-species analysis of MIAMI cells in an animal model of focal ischemia. GAPDH, which is one of the most common housekeeping genes used for the normalization of RT-qPCR data in the field of MSC research, was found to have the highest variability and deemed not suitable for normalization of RT-qPCR data.ConclusionsIn order to make comparisons between heterogeneous MSC populations, as well as adult stem cell like MSC which are used in different laboratories throughout the world, it is important to have a standardized, reproducible set of housekeeping genes for RT-qPCR analysis. In this study we demonstrate that EF1α, RPL13a and YWHAZ are suitable genes for the RT-qPCR analysis and comparison of several sources of human MSC during in vitro characterization and differentiation as well as in an ex vivo animal model of global cerebral ischemia. This will allow for the comparative RT-qPCR analysis of multiple MSC populations with the goal of future use in animal models of disease as well as tissue repair.

Chemotherapeutic drugs ideally should take advantage of the differences between transformed and normal cells and induce apoptosis only in cancer cells. One such difference may be the overexpression of cyclin B1 protein in cancer cells, which is required for the proper progression through mitosis. Previously, we showed that treatment of human prostate cancer cells with 2-methoxyestradiol (2-ME) or docetaxel results in an accumulation of cyclin B1 protein and an increase in cyclin B1 kinase activity, followed by induction of apoptotic cell death. Inhibition of cyclin B1 kinase lowers apoptosis induced by 2-ME and docetaxel. In this study, we established a positive correlation between cyclin B1 protein and apoptosis induced by chemotherapy in prostate cancer cells. There is minimal cyclin B1 and induction of apoptosis by chemotherapy in nontransformed cells. LNCaP and PC-3 prostate cancer cells stably overexpressing cyclin B1 are more sensitive to apoptosis induced by chemotherapy. LNCaP cells expressing cyclin B1 small interfering RNA to lower cyclin B1 protein or dominant negative cyclin-dependent kinase 1 to inhibit cyclin B1 kinase show a decrease in apoptosis. Increased sensitivity to apoptosis by overexpression of cyclin B1 may be due to lower Bcl-2, higher p53, and decreased neuroendocrine differentiation. We suggest that a cancer-specific mechanism whereby 2-ME and docetaxel may exert anti -prostate cancer activity is the deregulated activation of cyclin B1 kinase, leading to the induction of apoptotic cell death. Our results also suggest that higher levels of cyclin B1 in prostate cancer cells may be a good prognostic marker for chemotherapy. [Mol Cancer Ther 2007;6(5):1534 -43]

Vascular disease is one of the leading causes of death worldwide. Vascular repair, essential for tissue maintenance, is critically reduced during vascular disease and aging. Efficient vascular repair requires functional adult stem cells unimpaired by aging or mutation.One protein candidate for reducing stem cell–mediated vascular repair is progerin, an alternative splice variant of lamin A. Progerin results from erroneous activation of cryptic splice sites within the LMNA gene, and significantly increases during aging. Mutations triggering progerin overexpression cause the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS), in which patients die at approximately 13-years of age due to atherosclerosis-induced disease. Progerin expression affects tissues rich in cells that can be derived from marrow stromal cells (MSCs). Studies using various MSC subpopulations and models have led to discrepant results.Using a well-defined, immature subpopulation of MSCs, Marrow Isolated Adult Multilineage Inducible (MIAMI) cells, we find progerin significantly disrupts expression and localization of self-renewal markers, proliferation, migration, and membrane elasticity. One potential treatment, farnesyltransferase inhibitor, ameliorates some of these effects. Our results confirm proposed progerin-induced mechanisms and suggest novel ways in which progerin disturbs critical stem cell functions collectively required for proper tissue repair, offering promising treatment targets for future therapies.

Cell-based therapies for global cerebral ischemia represent promising approaches for neuronal damage prevention and tissue repair promotion. We examined the potential of Marrow-Isolated Adult Multilineage Inducible (MIAMI) cells, a homogeneous subpopulation of immature human mesenchymal stromal cell, injected into the hippocampus to prevent neuronal damage induced by global ischemia using rat organotypic hippocampal slices exposed to oxygen-glucose deprivation (OGD) and rats subjected to asphyxial cardiac arrest (ACA). We next examined the value of combining fibronectin-coated biomimetic microcarriers (FN-BMMs) with EGF/bFGF pre-treated MIAMI compared to EGF/bFGF pre-treated MIAMI cells alone, for their in vitro and in vivo neuroprotective capacity. Naïve and EGF/bFGF pre-treated MIAMI cells significantly protected the Cornu Ammonis layer 1 (CA1) against ischemic death in hippocampal slices and increased CA1 survival in rats. MIAMI cells therapeutic value was significantly increased when delivering the cells complexed with FN-BMMs, probably by increasing stem cell survival and paracrine secretion of pro-survival and/or anti-inflammatory molecules as concluded from survival, differentiation and gene expression analysis. Four days after OGD and ACA, few transplanted cells administered alone survived in the brain whereas stem cell survival improved when injected complexed with FN-BMMs. Interestingly, a large fraction of the transplanted cells administered alone or in complexes expressed βIII-Tubulin suggesting that partial neuronal transdifferentiation may be a contributing factor to the neuroprotective mechanism of MIAMI cells.