Cell cycle dependent telomere regulation by telomerase in human bone marrow mesenchymal stem cells

Human bone marrow mesenchymal stem cells (hMSCs) are a promising source for clinical stem cell transplantation. However, telomere regulation mechanisms, as one of the possible major mechanisms by which hMSCs sustain their stem cell characteristics, remain unknown. We isolated hMSCs by plastic adhesion and characterized these cells by morphology, immune phenotype and differentiation capacity. Telomerase was found negative in hMSCs, but slightly up-regulated in hMSC-derived adipocytes by the Telomeric Repeat Amplification Protocol (TRAP) assay. Moreover, hMSCs lack the alternative lengthening of telomeres (ALT) mechanism, because the hallmarks of ALT, such as very long and heterogeneous telomeres, extra-chromosome telomere repeat DNA (ECTR), and ALT-associated promyelocytic leukemia bodies (APBs), were not evident. However, when hMSCs were arrested in S phase with a combination of serum deprivation and aphidicolin, previously undetectable telomerase activity became predominantly positive. Meanwhile, the expression level of hTERT protein and mRNA increased, paralleled with the appearance of a large cohort of synchronized hMSCs at S phase. These findings provide a profile of telomere regulation by cell cycle dependent expression of telomerase in hMSCs and may lead to a better understanding of the stem cell nature of these cells.     

Telomere stability and telomerase in mesenchymal stem cells

Telomeres are repetitive genetic material that cap and thereby protect the ends of chromosomes. Each time a cell divides, telomeres get shorter. Telomere length is mainly maintained by telomerase. This enzyme is present in high concentrations in the embryonic stem cells and in fast growing embryonic cells, and declines with age. It is still unclear to what extent there is telomerase in adult stem cells, but since these are the founder cells of cells of all the tissues in the body, understanding the telomere dynamics and expression of telomerase in adult stem cells is very important. In the present communication we focus on telomere expression and telomere length in stem cells, with a special focus on mesenchymal stem cells. We consider different mechanisms by which stem cells can maintain telomeres and also focus on the dynamics of telomere length in mesenchymal stem cells, both the overall telomere length and the telomere length of individual chromosomes.     

Culture and neural differentiation of rat bone marrow mesenchymal stem cells in vitro

Adult bone marrow mesenchymal stem cells (MSCs) can differentiate into several types of mesenchymal cells, including osteocytes, chondrocytes, and adipocytes, but can also differentiate into non-mesenchymal cells, such as neural cells, under appropriate experimental conditions. Until now, many protocols for inducing neuro-differentiation in MSCs in vitro have been reported. But due to the differences in MSCs' isolation and culture conditions, the results of previous studies lacked consistency and comparability. In this study, we induced differentiation into neural phenotype in the same MSCs population by three different treatments: beta-mercaptoethanol, serum-free medium and co-cultivation with fetal mouse brain astrocytes. In all of the three treatments, MSCs could express neural markers such as NeuN or GFAP, associating with remarkable morphological modifications. But these treatments led to neural phenotype in a non-identical manner. In serum-free medium, MSCs mainly differentiated into neuron-like cells, expressing neuronal marker NeuN, and BME can promote this process. Differently, after co-culturing with astrocytes, MSCs leaned to differentiate into GFAP(+) cells. These data confirmed that MSCs can exhibit plastic neuro-differentiational potential in vitro, depending on the protocols of inducement.     

Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells

Several biological events are controlled by Hedgehog (Hh) signaling, including osteoblast phenotype development. This study aimed at evaluating the gene expression profile of human mesenchymal stem cells (hMSCs) treated with the Hh agonist, purmorphamine, focusing on Hh signaling and osteoblast differentiation. hMSCs from bone marrow were cultured in non‐osteogenic medium with or without purmorphamine (2 µM) for periods of up to 14 days. Purmorphamine up‐regulated gene expression of the mediators of Hh pathway, SMO, PTCH1, GLI1, and GLI2. The activation of Hh pathway by purmorphamine increased the expression of several genes (e.g., RUNX2 and BMPs) related to osteogenesis. Our results indicated that purmorphamine triggers Hh signaling pathway in hMSCs, inducing an increase in the expression of a set of genes involved in the osteoblast differentiation program. Thus, we conclude that Hh is a crucial pathway in the commitment of undifferentiated cells to the osteoblast lineage. J. Cell. Biochem. 113: 204–208, 2012. © 2011 Wiley Periodicals, Inc.     

Non-hematopoietic bone marrow stem cells: molecular control of expansion and differentiation

The first non-hematopoietic mesenchymal stem cells (MSCs) were discovered by Friedenstein in 1976, who described clonal, plastic adherent cells from bone marrow capable of differentiating into osteoblasts, adipocytes, and chondrocytes. More recently, investigators have now demonstrated that multi-potent MSCs can be recovered from a variety of other adult tissues and differentiate into numerous tissue lineages including myoblasts, hepatocytes and possibly even neural tissue. Because MSCs are multipotent and easily expanded in culture, there has been much interest in their clinical potential for tissue repair and gene therapy and as a result, numerous studies have been carried out demonstrating the migration and multi-organ engraftment potential of MSCs in animal models and in human clinical trials. This review describes the recent advances in the understanding of MSC biology.     

Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells

To clarify the mechanism of the stimulatory effect of statins on bone formation, we investigated the effect of simvastatin, a widely used statin, on osteoblastic and adipocytic differentiation in primary cultured mouse bone marrow stromal cells (BMSCs). Simvastatin treatment enhanced the expression level of mRNA for osteocalcin and protein for osteocalcin and osteopontin, and increased alkaline phosphatase activity significantly (p<0.05). After BMSCs were exposed to an adipocyte differentiation agonist, Oil Red O staining, fluorescence activated cell sorting, and decreased expression level of lipoprotein lipase mRNA showed that treatment with simvastatin significantly inhibits adipocytic differentiation compared to controls that did not receive simvastatin (p<0.05). Lastly, we found that simvastatin induces high expression of BMP(2) in BMSCs. These observations suggested that simvastatin acts on BMSCs to enhance osteoblastic differentiation and inhibits adipocytic differentiation; this effect is at least partially mediated by inducing BMP(2) expression in BMSCs.     

Conditioned medium from osteocytes stimulates the proliferation of bone marrow mesenchymal stem cells and their differentiation into osteoblasts

Osteocytes are the most abundant cells in bone and there is increasing evidence that they control bone remodeling via direct cell-to-cell contacts and by soluble factors. In the present study, we have used the MLO-Y4 cell line to study the effect of osteocytes on the proliferation, differentiation and bone-forming capacity of bone marrow mesenchymal stem cells (MSC). Conditioned media (CM) from osteocytic MLO-Y4 and osteoblastic MC3T3-E1 cell lines were collected and added on mouse bone marrow cultures, in which MSC were induced to osteoblasts. There was a significant increase in alkaline phosphatase activity and osteocalcin expression in the presence of MLO-Y4 CM. No such stimulus could be observed with MC3T3-E1 CM. There was almost 4-fold increase in bone formation and up to 2-fold increase in the proliferation of MSC with MLO-Y4 CM. The highly proliferating bone marrow cells were negative for ALP and OCN, suggesting that they could represent early osteoblast precursors. MLO-Y4 CM did not enhance the viability of mature osteoblasts nor protected them of apoptosis. This is the first study to describe soluble signals between osteocytes and osteoblasts and there most likely are several still unidentified or unknown factors in osteocyte CM. We conclude that osteocytes have an active stimulatory role in controlling bone formation.     

ESET histone methyltransferase regulates osteoblastic differentiation of mesenchymal stem cells during postnatal bone development

To investigate the effects of histone methyltransferase ESET (also known as SETDB1) on bone metabolism, we analyzed osteoblasts and osteoclasts in ESET knockout animals, and performed osteogenesis assays using ESET-null mesenchymal stem cells. We found that ESET deletion severely impairs osteoblast differentiation but has no effect on osteoclastogenesis, that co-transfection of ESET represses Runx2-mediated luciferase reporter while siRNA knockdown of ESET activates the luciferase reporter in mesenchymal cells, and that ESET is required for postnatal expression of Indian hedgehog protein in the growth plate. As the bone phenotype in ESET-null mice is 100% penetrant, these results support ESET as a critical regulator of osteoblast differentiation during bone development.     

Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

We developed and used real-time RT-PCR assays to investigate how the expression of typical osteoblast-related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor-2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, beta-glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein-2 (BMP-2), bone sialo protein-II (BSP), osteopontin (OP) and to a lower extent cbfa-1 increased with culture time in osteogenic medium (OM), both in CTR- and FGF/Dex-expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP-2, BSP, and OP were more expressed in FGF/Dex than in CTR-expanded BMSC (mRNA levels were, respectively, 9.5-, 14.9-, and 5.8-fold higher), unlike all the other investigated genes. Analysis of single-colony-derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex-expanded clones expressed higher mRNA levels of BMP-2, BSP, and OP than CTR-expanded clones. In conclusion, we provide evidence that mRNA levels of BMP-2, BSP, and OP, quantified using real-time RT-PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion.     

NT-3 promotes osteogenic differentiation of mouse bone marrow mesenchymal stem cells by regulating the Akt pathway

Objectives:To investigate the effect of neurotrophin-3 (NT-3) on osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).Methods:Osteogenic differentiation was detected by alkaline phosphatase (ALP) staining and alizarin red staining (ARS). Adipogenic differentiation was detected by oil red O (ORO) staining. The expression of bone-related genes (Runx2, Osterix, OCN, ALP) and lipogenic genes (FABP4, PPAR, CEBP, LPL) was detected by real-time quantitative polymerase chain reaction (real-time qPCR). The expression of p-Akt and Akt protein was detected by Western blot assay.Results:ALP staining and ARS staining showed that the overexpression of NT-3 could promote the differentiation into osteoblasts, while knockdown of NT-3 could inhibit that. Real-time qPCR showed that the overexpression of NT-3 could increase the expression of osteoblast genes, while knockdown of NT-3 could inhibit that. ORO staining showed that the overexpression of NT-3 could inhibit the differentiation into adipogenesis, while knockdown of NT-3 can promote that. Real-time qPCR showed that the overexpression of NT-3 could reduce the expression of lipogenic genes. while knockdown NT-3 could increase that. In addition, the overexpression of NT-3 increased p-Akt/Akt levels significantly, while knockdown NT-3 reduced that significantly.Conclusion:NT-3 could promote the differentiation of mouse BMSCs into osteoblasts and inhibit their differentiation into adipogenesis.     

Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation

Osteogenesis is a complex series of events involving the differentiation of mesenchymal stem cells to generate new bone. In this study, we examined the effect of pulsed electromagnetic fields (PEMFs) on cell proliferation, alkaline phosphatase (ALP) activity, mineralization of the extracellular matrix, and gene expression in bone marrow mesenchymal stem cells (BMMSCs) during osteogenic differentiation. Exposure of BMMSCs to PEMFs increased cell proliferation by 29.6% compared to untreated cells at day 1 of differentiation. Semi‐quantitative RT‐PCR indicated that PEMFs significantly altered temporal expression of osteogenesis‐related genes, including a 2.7‐fold increase in expression of the key osteogenesis regulatory gene cbfa1, compared to untreated controls. In addition, exposure to PEMFs significantly increased ALP expression during the early stages of osteogenesis and substantially enhanced mineralization near the midpoint of osteogenesis. These results suggest that PEMFs enhance early cell proliferation in BMMSC‐mediated osteogenesis, and accelerate the osteogenesis. Bioelectromagnetics 31:209–219, 2010. © 2009 Wiley‐Liss, Inc.     

A novel tripolymer coating demonstrating the synergistic effect of chitosan, collagen type 1 and hyaluronic acid on osteogenic differentiation of human bone marrow derived mesenchymal stem cells

The biomimetic approach mimicking in vivo micro environment is the key for developing functional tissue engineered constructs. In this study, we used a tripolymer combination consisting of a natural polymer, chitosan and two extracellular matrix components; collagen type 1 and hyaluronic acid to coat tissue culture plate to evaluate their effect on osteogenic differentiation of human bone marrow derived mesenchymal stem cells (hMSCs). The polymers were blended at different mixing ratios and the tissue culture plates were coated either by polyblend method or by surface modification method. hMSCs isolated from adult bone marrow were directed to osteoblast differentiation on the coated plates. Our results showed that the tripolymer coating of the tissue culture plate enhanced mineralization as evidenced by calcium quantification exhibiting significantly higher amount of calcium compared to the untreated or individual polymer coated plates. We found that the tripolymer coated plates having a 1:1 mixing ratio of chitosan and collagen type 1, surface modified with hyaluronic acid is an ideal combination to achieve the synergistic effect of these polymers on in vitro osteogenic differentiation of hMSCs. These results thus, establish a novel biomimetic approach of surface modification to enhance osteoblast differentiation and mineralization. Our findings hold great promise in implementing a biomimetic surface coating to improve osteoconductivity of implants and scaffolds for various orthopaedic and bone tissue engineering applications.     

Molecular pathways involved in neural in vitro differentiation of marrow stromal stem cells

In recent years several reports have claimed to demonstrate trans-differentiation, namely that stem cells have been derived from a given tissue and have differentiated into phenotypes characteristic of different tissues following transplantation or in vitro treatment. For example, the mesenchymal stem cells, also referred to as marrow stromal stem cells (MSCs), present in bone marrow, have been induced to differentiate into neurons. We decided to investigate this phenomenon more in depth by a molecular and morphological follow-up. We analyzed the biochemical pathways that are currently induced to trigger neuron-like commitment and maturation of MSCs. Our studies suggest that: (i) the increase in cAMP, induced to differentiate MSCs, activates the classical PKA pathway and not through the exchange protein directly activated by cAMP (EPAC), a guanine nucleotide exchange factor for the small GTPase Rap1 and Rap2; (ii) MEK-ERK signaling could contribute to neural commitment and differentiation; (iii) CaM KII activity seems dispensable for neuron differentiation. On the contrary, its inhibition could contribute to rescuing differentiating cells from death. Our research also indicates that the currently used in vitro differentiation protocols, while they allow the early steps of neural differentiation to take place, are not able to further sustain this process.     

Telomerase deficiency impairs differentiation of mesenchymal stem cells

Expression of telomerase activity presumably is involved in maintaining self-replication and the undifferentiated state of stem cells. Adult mouse bone marrow mesenchymal stem cells (mMSCs) are multipotential cells capable of differentiating into a variety of lineage cell types, including adipocytes and chondrocytes. Here we show that the lacking telomerase of mMSC lose multipotency and the capacity to differentiate. Primary cultures of mMSCs were obtained from both telomerase knockout (mTR(-/-)) and wild-type (WT) mice. The MSCs isolated from mTR(-/-) mice failed to differentiate into adipocytes and chondrocytes, even at early passages, whereas WT MSCs were capable of differentiation. Consistent with other cell types, late passages mTR(-/-)MSCs underwent senescence and were accompanied by telomere loss and chromosomal end-to-end fusions. These results suggest that in addition to its known role in cell replication, telomerase is required for differentiation of mMSCs in vitro. This work may be significant for further potentiating adult stem cells for use in tissue engineering and gene therapy and for understanding the significance of telomerase expression in the process of cell differentiation.