Low Oxygen Modulates Multiple Signaling Pathways, Increasing Self-Renewal, While Decreasing Differentiation, Senescence, and Apoptosis in Stromal MIAMI Cells.

Human bone marrow multipotent mesenchymal stromal cell (hMSC) number decreases with aging. Subpopulations of hMSCs can differentiate into cells found in bone, vasculature, cartilage, gut, and other tissues and participate in their repair. Maintaining throughout adult life such cell subpopulations should help prevent or delay the onset of age-related degenerative conditions. Low oxygen tension, the physiological environment in progenitor cell-rich regions of the bone marrow microarchitecture, stimulates the self-renewal of marrow-isolated adult multilineage inducible (MIAMI) cells and expression of Sox2, Nanog, Oct4a nuclear accumulation, Notch intracellular domain, notch target genes, neuronal transcriptional repressor element 1 (RE1)-silencing transcription factor (REST), and hypoxia-inducible factor-1 alpha (HIF-1α), and additionally, by decreasing the expression of (i) the proapoptotic proteins, apoptosis-inducing factor (AIF) and Bak, and (ii) senescence-associated p53 expression and ß-galactosidase activity. Furthermore, low oxygen increases canonical Wnt pathway signaling coreceptor Lrp5 expression, and PI3K/Akt pathway activation. Lrp5 inhibition decreases self-renewal marker Sox2 mRNA, Oct4a nuclear accumulation, and cell numbers. Wortmannin-mediated PI3K/Akt pathway inhibition leads to increased osteoblastic differentiation at both low and high oxygen tension. We demonstrate that low oxygen stimulates a complex signaling network involving PI3K/Akt, Notch, and canonical Wnt pathways, which mediate the observed increase in nuclear Oct4a and REST, with simultaneous decrease in p53, AIF, and Bak. Collectively, these pathway activations contribute to increased self-renewal with concomitant decreased differentiation, cell cycle arrest, apoptosis, and/or senescence in MIAMI cells. Importantly, the PI3K/Akt pathway plays a central mechanistic role in the oxygen tension-regulated self-renewal versus osteoblastic differentiation of progenitor cells.

Rac1b regulates NT3-stimulated Mek-Erk signaling, directing marrow-isolated adult multilineage inducible (MIAMI) cells toward an early neuronal phenotype.

Due to the limitations of neural stem cells to repair neuronal damage in the human brain, alternative approaches of repair using autologous adult stem cells have been examined for direct cell-replacement, or paracrine mediated neuroprotective effects. Human bone marrow-derived stromal cells (hMSCs) are a heterogeneous adult stem cell population with diverse immunomodulatory properties and the potential to differentiate into cells characteristic of all three germ layers. hMSCs are a renewable source of progenitor cells suitable for cell-based tissue repair. The marrow isolated adult multilineage inducible (MIAMI) cells developed by our laboratory are a developmentally immature homogeneous subpopulation of hMSCs that maintain self-renewal potential during ex vivo expansion, efficient differentiation capacity into neuron-like cells in vitro, as well as direct in vivo neuroprotection and functional recovery in animal models of neurological diseases. We now address the early signaling mechanisms regulating the neuron-like differentiation of MIAMI cells in vitro, in response to activation of the neurotrophic tyrosine-kinase receptor, type 3 (NTRK3) via neurotrophin 3 (NT3). We molecularly characterize a novel role for Rac1b mediating the neurogenic potential of MIAMI cells. Rac1b had an overall negative modulatory effect on the NT3-stimulated Mek1/2-Erk1/2 signaling pathway, proneuronal gene expression and neurite-like extensions. Rac1b was required for NT3-stimulated cell proliferation of MIAMI cells, yet was found to repress CCND1 and CCNB1 mRNA expression independent of NT3 stimulation, suggesting a dual neurotrophin dependent/independent function. Differential levels of Rac1b activity in hMSCs may explain the apparent contradictory reports regarding their neurogenic potential. These findings demonstrate the in vitro neurogenic potential of hMSCs as governed by Rac1b during NT3 stimulation.

Neuroprotective properties of marrow-isolated adult multilineage-inducible cells in rat hippocampus following global cerebral ischemia are enhanced when complexed to biomimetic microcarriers.

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 and rats subjected to asphyxial cardiac arrest. We next examined the value of combining fibronectin-coated biomimetic microcarriers (FN-BMMs) with epidermal growth factor (EGF)/basic fibroblast growth factor (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 oxygen and glucose deprivation and asphyxial cardiac arrest, 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 ßIII-tubulin suggesting that partial neuronal transdifferentiation may be a contributing factor to the neuroprotective mechanism of MIAMI cells.

Human marrow-isolated adult multilineage-inducible (MIAMI) cells protect against peripheral vascular ischemia in a mouse model.

BACKGROUND AIMS: The treatment of peripheral vascular disease (PVD) with stem cells potentially offers a promising strategy. We tested marrow-isolated adult multilineage-inducible (MIAMI) cells to induce neovascularization in a mouse model of critical hindlimb ischemia (CLI). METHODS: CLI was induced in the right hindlimb of Balb/C mice. One million MIAMI cells, normally grown at 3% O2, were injected in the adductor muscle along the ischemic region. All animals (n = 11 per group) were immunosuppressed with cyclosporine daily for the entire period. Human foreskin fibroblast (HFF) cells and phosphate-buffered saline (PBS) were used as controls. Blood perfusion in the ischemic right and non-ischemic left hindlimbs was measured. RESULTS: Compared with animals receiving HFF cells or PBS, MIAMI cells significantly improved blood perfusion, necrosis and inflammation in the ischemic limb. A fraction of injected MIAMI cells expressed CD31 and von Willebrand factor (vWF). MIAMI cells in vitro, under pro-angiogenic growth conditions, differentiated into endothelial-like cells and expressed endothelial markers such as CD31 and vWF, determined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and CD31 and kinase insert domain receptor (KDR), determined by immunofluorescence. Moreover, MIAMI cells formed vascular endothelial-like tubules in the presence of matrigel. Bioplex immunoassay analysis showed increased secretion of angiogenic/anti-inflammatory factors by the MIAMI cells under 3% O2 compared with 21% O2, including monocyte chemoattractant protein-1 (MCP-1), fractalkine (Ftk), growth-related oncogene (GRO), vascular endothelial growth factor (VEGF), interleukin (IL)-6 and IL-8. Furthermore, transcripts for anti-inflammatory molecules stanniocalcin-1 (STC-1) and tumor necrosis factor-α-stimulated gene 6 (TSG-6) were up-regulated several fold. CONCLUSIONS: MIAMI cells can be very useful for patients affected by CLI. MIAMI cells promote blood vessel formation and reduce inflammation and necrosis in ischemic tissue.

EF1alpha and RPL13a represent normalization genes suitable for RT-qPCR analysis of bone marrow derived mesenchymal stem cells.

BACKGROUND: RT-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. RESULTS: Eight genes including; ACTB, B2M, EF1alpha, GAPDH, RPL13a, YWHAZ, UBC and HPRT1 were tested as possible housekeeping genes based on their expression level and variability. EF1alpha 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. CONCLUSIONS: In 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 EF1alpha, 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.

Low dose combinations of 2-methoxyestradiol and docetaxel block prostate cancer cells in mitosis and increase apoptosis.

Clinical trials have shown that chemotherapy with docetaxel (Doc) combined with prednisone can improve survival of patients with androgen-independent prostate cancer (AI-PC). It is likely that the combination of Doc with other novel agents would also improve the survival of AI-PC patients. We investigated whether the combination of Doc and 2-methoxyestradiol (2ME2), an endogenous metabolite of estradiol promising for cancer therapy, can increase apoptotic cell death in prostate cancer cells. Low concentration 2ME2 (0.5-1 microM)+Doc (0.05-0.1 nM) combinations inhibit cell growth, increase G2/M cell cycle arrest, and increase apoptosis more effectively than the single concentrations in a variety of human AI-PC cells. Effects on apoptosis were associated with an increase in p53 protein and a decrease in cyclin A-dependent kinase activity. We then investigated whether the combination of 2ME2+Doc can increase apoptotic cell death and inhibit the growth of prostate tumors in the FG/Tag transgenic mouse model of AI-PC. Doses of 2ME2 and Doc that increase mitotic cell cycle arrest result in an increase in apoptosis and lower primary prostate tumor weights in FG/Tag mice. High dose 2ME2+Doc combinations did not increase G2/M cell cycle arrest or apoptosis in AI-PC cell lines and in the FG/Tag mice more than the single drugs. Overall, our data indicate that low dose 2ME2+Doc combinations may provide a treatment strategy that can improve therapeutic efficacy against AI-PC while reducing toxicity often seen in patients treated with Doc.

Increased expression of cyclin B1 sensitizes prostate cancer cells to apoptosis induced by chemotherapy.

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.

Sequential combination of flavopiridol and docetaxel reduces the levels of X-linked inhibitor of apoptosis and AKT proteins and stimulates apoptosis in human LNCaP prostate cancer cells.

Clinical trials have shown that chemotherapy with docetaxel combined with prednisone can improve survival of patients with androgen-independent prostate cancer. It is likely that the combination of docetaxel with other novel chemotherapeutic agents would also improve the survival of androgen-independent prostate cancer patients. We investigated whether the combination of docetaxel and flavopiridol, a broad cyclin-dependent kinase inhibitor, can increase apoptotic cell death in prostate cancer cells. Treatment of DU 145 prostate cancer cells with 500 nmol/L flavopiridol and 10 nmol/L docetaxel inhibited apoptosis probably because of their opposing effects on cyclin B1-dependent kinase activity. In contrast, when LNCaP prostate cancer cells were treated with flavopiridol for 24 hours followed by docetaxel for another 24 hours (FD), there was a maximal induction of apoptosis. However, there was greater induction of apoptosis in DU 145 cells when docetaxel was followed by flavopiridol or docetaxel. These findings indicate a heterogeneous response depending on the type of prostate cancer cell. Substantial decreases in X-linked inhibitor of apoptosis (XIAP) protein but not survivin, both being members of the IAP family, were required for FD enhanced apoptosis in LNCaP cells. Androgen ablation in androgen-independent LNCaP cells increased activated AKT and chemoresistance to apoptosis after treatment with FD. The proteasome inhibitor MG-132 blocked FD-mediated reduction of XIAP and AKT and antagonized apoptosis, suggesting that the activation of the proteasome pathway is one of the mechanisms involved. Overall, our data suggest that the docetaxel and flavopiridol combination requires a maximal effect on cyclin B1-dependent kinase activity and a reduction of XIAP and AKT prosurvival proteins for augmentation of apoptosis in LNCaP cells.