Regulation of the terminal event in cellular differentiation: biological mechanisms of the loss of proliferative potential

Human plasma has been demonstrated to contain factors that induce the sequential expression of nonterminal and terminal adipocyte differentiation in 3T3 T mesenchymal stem cells. We now report the development of methods for the isolation of purified populations of nonterminally differentiated cells and terminally differentiated cells, and we show that it is possible to experimentally induce transition from the nonterminal to the terminal state of differentiation. With this model system it is therefore now possible to examine the biological and molecular processes associated with the terminal event in differentiation, i.e., the irreversible loss of proliferative potential. In this regard, we demonstrate that transition from the nonterminal to terminal state of differentiation is a complex metabolic process that consists of at least two steps and that this process can be triggered by pulse exposure to an inducer for approximately 12 h but that approximately 24-48 h is required for the process to be completed. The data also establish that induction of the terminal event in differentiation requires protein synthesis but not RNA and DNA synthesis. These and additional results suggest that loss of proliferative potential associated with the terminal event in cellular differentiation is a distinct regulatory process, and we suggest that defects in this regulatory process may be of etiological significance in the pathogenesis of specific human diseases, especially cancer.     

Regulation of B cell maturation and differentiation. I. Suppression of pokeweed mitogen-induced B cell differentiation by tumor necrosis factor (TNF)

Growth and differentiation of B cells into Ig-secreting plasma cells is regulated by both T cells and macrophages and/or their secreted factors. Although the regulatory role of various cell-derived factors has been examined, the involvement of the macrophage-derived factor, TNF, in human B cell growth and differentiation has not yet been investigated. In the present study we examine the role of rTNF in polyclonal B cell response of human PBL induced by PWM. The addition of rTNF at the initiation of the culture resulted in the dose-dependent inhibition of the generation of both IgG and IgM PFC. Inhibition of PFC development followed the same dose response as rTNF-mediated cytotoxicity against a TNF-sensitive tumor target. The mechanism of rTNF-mediated suppression was examined in different experimental systems. Recombinant TNF did not affect the viability or proliferation of either the T cell or B cell subpopulations, suggesting that TNF does not mediate its suppressive effect by cytotoxic mechanisms. Kinetic studies in which rTNF was added at different times after initiation of culture indicated that inhibition can be observed as late as 4 days of culture and suggested that TNF acts at a late phase of the growth and differentiation pathway of B cells. In further studies we examined the cellular level of TNF-mediated suppression. The addition of rTNF to supernatants containing helper factors and enriched B cells resulted in no inhibition, suggesting that TNF does not act at the B cell level. This was confirmed by demonstrating that rTNF does not inhibit spontaneous PFC development by the CESS B cell line. The effect of TNF on T cell subpopulations was examined by using normal or irradiated T cells, which inactivate suppressor cells. Addition of rTNF to B cells combined with either T cell population suppressed both IgG and IgM PFC development, indicating that the target cell for suppression is the T helper cell but not ruling out an effect on macrophages or the T suppressor cells. Combined, the observed results demonstrate that rTNF suppresses PWM-induced B cell differentiation without affecting B cell proliferation. TNF appears to mediate the suppression by acting directly on T helper cells or else by regulating the production of factors controlling T cell activation and lymphokine secretion.     

Dose-specific effects of tumor necrosis factor alpha on osteogenic differentiation of mesenchymal stem cells

Objectives: To investigate tumor necrosis factor alpha (TNF‐α)‐induced changes in osteogenic differentiation from mesenchymal stem cells (MSCs). Materials and methods: Blockade of nuclear factor‐κB (NF‐κB) was achieved in ST2 murine MSCs via overexpression of the NF‐κB inhibitor, IκBα. Osteogenic differentiation was induced in IκBα‐overexpressing ST2 cells and normal ST2 cells when these cells were treated with TNF‐α at various concentrations. Expression levels of bone marker genes were determined using real time RT‐PCR and ALP activity assay. In vitro mineralization was performed to determine long‐term exposure to TNF‐α on mineral nodule formation. MTT assay was used to determine the changes in cell proliferation/survival. Results: Levels of Runx2, Osx, OC and ALP were up‐regulated in cell cultures treated with TNF‐α at lower concentrations, while down‐regulated in cell cultures treated with TNF‐α at higher concentrations. Blockade of NF‐κB signaling reversed the inhibitory effect observed in cell cultures treated with TNF‐α at higher concentrations, but showed no effect on cell cultures treated with TNF‐α at lower concentrations. In contrast, long‐term treatment of TNF‐α at all concentrations induced inhibitory effects on in vitro mineral nodule formation. MTT assay showed that TNF‐α inhibits proliferation/survival of mesenchymal stem cells when the NF‐κB signaling pathway is blocked. Conclusions: The binding of TNF‐α to its receptors results in the activation of multiple signaling pathways, which actively interact with each other to regulate the differentiation, proliferation, survival and apoptosis of MSCs.     

Differences and similarities between mesenchymal stem cell and endothelial progenitor cell immunoregulatory properties against T cells

Bone-marrow-derived mesenchymal stem cells and endothelial progenitor cells have some interesting biological properties that make them unique for cell therapy of degenerative and cardiovascular disorders. Although both cell populations have been already studied and used for their regenerative potentials, recently their special immunoregulatory features have brought much more attention. Mesenchymal stem cells and endothelial progenitor cells have both proangiogenic functions and have been shown to suppress the immune response, particularly T cell proliferation, activation, and cytokine production. This makes them suitable choices for allogeneic stem cell transplantation. Nevertheless, these two cells do not have equal immunoregulatory activities. Many elements including their extraction sources, age/passage, expression of different markers, secretion of bioactive mediators, and some others could change the efficiency of their immunosuppressive function. However, to our knowledge, no publication has yet compared mesenchymal stem cells and endothelial progenitor cells for their immunological interaction with T cells. This review aims to specifically compare the immunoregulatory effect of these two populations including their Tcell suppression, deactivation, cytokine production, and regulatory T cells induction capacities. Moreover, it evaluates the implications of the tumor necrosis factor alpha-tumor necrosis factor receptor 2 axis as an emerging immune checkpoint signaling pathway controlling most of their immunological properties.     

Specific expression of proteins and phosphoproteins in 3T3 T mesenchymal stem cells at distinct growth arrest and differentiation states

Murine mesenchymal stem cells can be induced to arrest their growth at a series of growth and differentiation states in the G1 phase of the cell cycle. These include the predifferentiation arrest state (GD) at which the integrated control of proliferation and differentiation is mediated, the growth factor/serum deficiency arrest state (GS), and the nutrient deficiency arrest state (GN). Cells at states of reversible nonterminal differentiation (GD') and irreversible terminal differentiation (TD) can also be isolated. In this paper we have employed 1- and 2-dimensional (D) gel electrophoresis to evaluate changes in specific proteins that occur during the various growth and differentiation states of 3T3 T mesenchymal stem cells. The protein composition of membrane, microsome and cytosol preparations of cells arrested at GD, GS and GN states was determined by 2-D gel electrophoresis. More than 50 distinct polypeptides could be identified for each arrest state in gels analysed by a silver staining procedure or by autoradiography following [35S]-methionine labelling. A second series of studies established that a more limited number of differences could be identified if phosphoproteins were analysed by 1-D gel electrophoresis in cells at the GS, GD, GD' and TD states. These results established that one distinct 37 kD phosphoprotein is present in all growth arrested cells and that two distinct differentiation-associated phosphoproteins with molecular weights of 29 kD and 72 kD are present in cells at the GD' and TD states. Thus, the composition of proteins and phosphoproteins in mesenchymal stem cells serves to characterize different states of growth arrest and differentiation.2+he identification of differential     

The p55 TNF receptor mediates TNF inhibition of osteoblast differentiation independently of apoptosis

After menopause, increased tumor necrosis factor-alpha (TNF-alpha) stimulates bone resorption while inhibiting differentiation of new bone-forming osteoblasts (OB). TNF receptors, p55 and p75, signal similar intracellular pathways, but only p55 activates apoptosis. To evaluate the relationship between the TNF receptor mediating inhibition of OB differentiation and the role of apoptosis, marrow stromal cells (MSC) were cultured from mice deficient in either or both receptors. Cells grown in ascorbate and beta-glycerophosphate produce alkaline phosphatase and osteocalcin and mineralize matrix. Treatment of wild-type or p55(+/+)/p75(-/-) MSC with murine TNF (binds p55 and p75) or human TNF (binds only p55) inhibited OB differentiation. TNF did not inhibit OB differentiation in p55(-/-) MSC. Expression of p75 modestly attenuated sensitivity to TNF. To determine the role of apoptosis, changes in total DNA, cell viability, caspase 3, and percentage of annexin V-positive cells were measured in MSC and preosteoblastic MC3T3 cells. TNF treatment that reduced differentiation by 50% did not decrease cell viability or increase apoptosis, as determined by alamar blue reduction, trypan blue exclusion, and percentage of annexin V-positive cells. TNF increased caspase 3 activity 1.5-fold in MC3T3 and insignificantly in MSC cells compared with > 4-fold after 4 h actinomycin D. Treatment of MSC or MC3T3 cells with three caspase inhibitors failed to reverse the inhibitory effect of TNF on OB differentiation despite inhibition of caspase activity. These results suggest that the p55 receptor is essential, and p75 dispensable, for TNF inhibition of OB differentiation through a mechanism that does not require apoptosis.     

IFN-γ-primed human bone marrow mesenchymal stem cells induce tumor cell apoptosis in vitro via tumor necrosis factor-related apoptosis-inducing ligand

Human mesenchymal stem cells hold promise as gene therapy vectors for delivery of various genes to solid tumors for either therapeutic or tumor-tracing purposes. However, whether Mesenchymal stem cells support or inhibit tumor growth remains unknown. Herein, we first observed that mesenchymal stem cells primed with IFN-γ selectively induced the death of tumor cell lines, but not normal cells. We further identified that IFN-γ-primed mesenchymal stem cells expressed tumor necrosis factor-related apoptosis-inducing ligand. Tumor-suppressive effect of IFN-γ-primed mesenchymal stem cells could be blocked by activity neutralization or expression reduction of tumor necrosis factor-related apoptosis-inducing ligand. Moreover, mesenchymal stem cells mediated apoptosis of tumor cells by activating caspase-3 in such cells, via a mechanism involving tumor necrosis factor-related apoptosis-inducing ligand. However, when IFN-γ-primed or non-primed mesenchymal stem cells were co-injected into nude mice along with H460 cells, tumor growth was much faster than that of the group receiving only tumor cells (p<0.01) because of the promoting vascularization effect of mesenchymal stem cells, although IFN-γ-primed mesenchymal stem cells also exerted a certain degree of tumor-suppressive effect compared with non-primed cells (2.79±0.9 g versus 2.03±0.6 g). Collectively, our findings show that IFN-γ-primed human mesenchymal stem cells could induce cancer cell apoptosis via TRAIL-mediated pathway. In addition, our data afford a novel explanation of the opposing effects of hMSCs presence on tumor growth in vitro and in vivo. Thus, more attention needs to be paid when seeking to exploit mesenchymal stem cells as a therapeutic option under the condition of malignant tumor.     

Suppression of tumor necrosis factor-mediated apoptosis by nuclear factor kappaB-independent bone morphogenetic protein/Smad signaling

The activation of nuclear factor kappaB (NF-kappa B) plays a pivotal role in the regulation of tumor necrosis factor (TNF)-mediated apoptosis. However, little is known about the regulation of TNF-mediated apoptosis by other signaling pathways or growth factors. Here, unexpectedly, we found that bone morphogenetic protein (BMP)-2 and BMP-4 inhibited TNF-mediated apoptosis by inhibition of caspase-8 activation in C2C12 cells, a pluripotent mesenchymal cell line that has the potential to differentiate into osteoblasts depending on BMP stimulation. Utilizing both a trans-dominant IkappaBalpha inhibitor of NF-kappaB expressed in C2C12 cells and IkappaB kinase beta-deficient embryonic mouse fibroblast, we show that BMP-mediated survival was independent of NF-kappaB activation. Rather, the antiapoptotic activity of BMPs functioned through the Smad signaling pathway. Thus, these findings provide the first report of a BMP/Smad signaling pathway that can inhibit TNF-mediated apoptosis, independent of the prosurvival activity of NF-kappaB. Our results suggest that BMPs not only stimulate osteoblast differentiation but can also promote cell survival during the induction of bone formation, offering new insight into the biological functions of BMPs.     

Polypeptide changes associated with loss of proliferative potential during the terminal event in differentiation

The differentiation of murine mesenchymal stem cells occurs in nonterminal and terminal phases. In previous reports we established the characteristics of nonterminally differentiated cells and showed that transition from the nonterminal to the terminal state of differentiation can be induced by human plasma. We also showed that this transition is blocked by protein synthesis inhibitors and other pharmacological agents. In this paper, we have employed two-dimensional gel electrophoresis to evaluate changes in specific polypeptides that are induced when cells lose proliferative capacity associated with the terminal event in differentiation. Using silver staining procedures for analysis of electrophoretograms, we detected only seven major polypeptide differences between nonterminally differentiated and terminally differentiated cells. Six polypeptides were expressed only in preparations of terminally differentiated cells; these included two polypeptides identified in cytosolic fractions and four polypeptides identified in nuclear fractions. One polypeptide was also found to be selectively expressed only in nuclear fractions of nonterminally differentiated cells. Based on these observations we conclude that the loss of proliferative potential that occurs during the terminal event in mesenchymal stem cell differentiation is associated with changes in the composition of a limited number of specific polypeptides. We suggest that one or more of these polypeptides may be important in the regulation of cellular proliferation.     

Transforming growth factor type beta is a specific inhibitor of 3T3 T mesenchymal stem cell differentiation

In the current studies we examined the effects of transforming growth factor type beta (TGF-beta) on the control of differentiation of BALB/c 3T3 T stem cells. We report that TGF-beta is a potent, reversible inhibitor of adipocyte differentiation (50% inhibition at approximately 0.06-0.08 ng/ml), while other biologically active polypeptides, such as epidermal growth factor (EGF), human growth hormone (hGH), and somatomedin C, have no specific effect on differentiation at even higher concentrations (200 ng/ml). We also report that TGF-beta inhibits differentiation in a cell cycle-dependent manner by its effect on a specific phase in the differentiation process. We therefore suggest that if TGF-beta is an important regulatory factor, one of its critical mechanisms of action may be its ability to inhibit the process of cell differentiation.     

Low frequency mechanical stimulation inhibits adipogenic differentiation of C3H10T1/2 mesenchymal stem cells

Oscillatory mechanical stimulation at relatively high frequencies (0.1 Hz) has been shown to inhibit adipogenic and promote osteogenic differentiation of mesenchymal stem cells. However, for physiological interpretations and ease of implementation it is of interest to know whether different rates of mechanical stimulation can produce similar results. We hypothesized that relatively low frequency mechanical stimulation (0.01 Hz) can inhibit adipogenic differentiation of C3H10T1/2 mouse mesenchymal stem cells, even in a potent adipogenic differentiation medium. C3H10T1/2 cells were cultured in adipogenic medium under control (non-mechanically stimulated) conditions and under oscillatory surface stretch with 10% amplitude and 0.01 Hz frequency for 6h per day for up to 5 days. Cell population was assessed by counting and adipogenic differentiation was assessed by real-time quantitative PCR (qPCR) analysis of peroxisome proliferator-activated receptor gamma (PPARγ) and fatty acid binding protein 4 (FABP4) after 3 and 5 days. Involvement of the ERK signaling pathway was assessed by Western blot. Low frequency mechanical stimulation significantly decreased expression of PPARγ after 3 days and FABP4 after 3 and 5 days versus non-stimulated culture. ERK signaling was decreased in mechanically-stimulated culture, indicating a role in the inhibition of adipogenic differentiation. Application of this study: Low frequency mechanical stimulation may provide a technically simple means for control of mesenchymal stem cell differentiation in cell-based therapies, particularly for inhibition of differentiation toward undesired adipogenic lineages.     

Expression of p107 and p130 during human adipose-derived stem cell adipogenesis

Within the first 24 h of hormonally stimulated adipocyte differentiation, murine 3T3-L1 preadipocytes undergo a mitotic expansion phase prior to terminal differentiation. During this time, the cell cycle regulatory proteins, p130 and p107 undergo dramatic differential expression and the transient increase in expression of p107 appears to be required for terminal differentiation. Recently, human adipose-derived human stem cells (hASC) of mesenchymal origin have been used as a model of human adipocyte differentiation and we sought to determine if differentiating hASC undergo clonal expansion and if the regulated expression of p130/p107 was similar to that observed during 3T3-L1 adipogenesis. Results indicate that differentiating hASC, unlike 3T3-L1 cells do not undergo clonal expansion and p130 expression gradually diminishes across differentiation. However, p107 expression is transiently increased during hASC differentiation in a manner analogous to 3T3-L1 cells suggesting a similar role for p107 in terminal differentiation in human adipocytes.     

Activation and promotion of adipose stem cells by tumour necrosis factor‐alpha preconditioning for bone regeneration

There is a major medical need for developing novel and effective approaches for repairing non‐union and critical‐sized bone defects. Although the mechanisms remain to be determined, it is known that inflammation plays a crucial role in initiating bone repair and regeneration. This study investigated the effect of short‐term (3 days) preconditioning with tumor necrosis factor‐alpha (TNF‐α) on proliferation, mobilization, and differentiation of adipose tissue‐derived mesenchymal stem cells (ASCs). We demonstrated that TNF‐α pre‐conditioning increased proliferation, mobilization, and osteogenic differentiation of ASCs and up‐regulated bone morphogenetic protein‐2 (BMP‐2) protein level. BMP‐2 silencing by siRNA partially inhibited osteogenic differentiation of ASCs induced by TNF‐α; BMP‐2 pre‐conditioning also significantly increased osteogenic differentiation of ASCs but the effects were significantly smaller than those observed for TNF‐α preconditioning. Furthermore, TNF‐α treatment promoted extracellular‐signal‐regulated kinases(Erk)1/2 and p38 mitogen‐activated protein kinase (MAPK) signaling pathways, but only Erk1/2 inhibition reduced the BMP‐2 levels and osteogenic differentiation induced by TNF‐α preconditioning. Together, these results support the hypothesis that inflammation contributes to bone regeneration by promoting proliferation, mobilization, and osteogenic differentiation of ASCs; 3 days of TNF‐α preconditioning, mimicking the short boost of inflammation normally occurring after bone injury, might serve as a feasible approach for directing stem cells into osteogenic differentiation. J. Cell. Physiol. 9999: XX–XX, 2013. © 2013 Wiley Periodicals, Inc. J. Cell. Physiol. 228: 1737–1744, 2013. © 2013 Wiley Periodicals, Inc.