Murine Bone Marrow-derived Mesenchymal Stem Research Articles

Substance P enhances the proliferation and migration potential of murine bone marrow-derived mesenchymal stem cell-like cell lines.

Due to the therapeutic characteristics of bone marrow (BM)-derived mesenchymal stem cells (MSCs), clinical trials are testing the use of autologous or allogeneic MSCs for the treatment of several conditions. These therapies require large numbers of MSCs and numerous studies are attempting to find substances that could enhance the egression of endogenous MSCs from the BM into the periphery and increase their proliferation in vivo and in vitro. It has been reported that substance P (SP) has the potential to increase the expansion of MSCs in vivo and to induce their mobilization from the BM into the periphery. The aim of the present study was to investigate the effects of SP on the migration and proliferation potential of two BM-derived MSC-like cell lines, ST2 and OP9. SP was found to induce the migration potential of ST2 cells in vitro. Furthermore, SP increased the proliferation of the MSCs cell line, OP9 cell line. Cyclin D1 expression was observed to increase in the OP9 cells, indicating the activation of the cell cycle in response to SP. The upstream signals involved in these phenomena have yet to be elucidated, although previous studies have proposed the activation of the extracellular signal-regulated kinase-1/2 and Wingless/β-catenin pathways as possible mediators of the cellular proliferation of human MSCs in response to SP. The present results therefore suggest that SP would facilitate the obtainment of higher numbers of endogenous MSCs from patients or donors and/or shorten the process of in vitro expansion that could cause the MSCs to undergo changes in their innate therapeutic characteristics prior to their use in therapy.

Dose-dependent effect of estrogen suppresses the osteo-adipogenic transdifferentiation of osteoblasts via canonical Wnt signaling pathway.

Fat infiltration within marrow cavity is one of multitudinous features of estrogen deficiency, which leads to a decline in bone formation functionality. The origin of this fat is unclear, but one possibility is that it is derived from osteoblasts, which transdifferentiate into adipocytes that produce bone marrow fat. We examined the dose-dependent effect of 17β-estradiol on the ability of MC3T3-E1 cells and murine bone marrow-derived mesenchymal stem cell (BMMSC)-derived osteoblasts to undergo osteo-adipogenic transdifferentiation. We found that 17β-estradiol significantly increased alkaline phosphatase activity (P<0.05); calcium deposition; and Alp, Col1a1, Runx2, and Ocn expression levels dose-dependently. By contrast, 17β-estradiol significantly decreased the number and size of lipid droplets, and Fabp4 and PPARγ expression levels during osteo-adipogenic transdifferentiation (P<0.05). Moreover, the expression levels of brown adipocyte markers (Myf5, Elovl3, and Cidea) and undifferentiated adipocyte markers (Dlk1, Gata2, and Wnt10b) were also affected by 17β-estradiol during osteo-adipogenic transdifferentiation. Western blotting and immunostaining further showed that canonical Wnt signaling can be activated by estrogen to exert its inhibitory effect of osteo-adipogenesis. This is the first study to demonstrate the dose-dependent effect of 17β-estradiol on the osteo-adipogenic transdifferentiation of MC3T3-E1 cells and BMMSCs likely via canonical Wnt signaling. In summary, our results indicate that osteo-adipogenic transdifferentiation modulated by canonical Wnt signaling pathway in bone metabolism may be a new explanation for the gradually increased bone marrow fat in estrogen-inefficient condition.

Effects of murine and human bone marrow-derived mesenchymal stem cells on cuprizone induced demyelination.

For the treatment of patients with multiple sclerosis there are no regenerative approaches to enhance remyelination. Mesenchymal stem cells (MSC) have been proposed to exert such regenerative functions. Intravenous administration of human MSC reduced the clinical severity of experimental autoimmune encephalomyelitis (EAE), an animal model mimicking some aspects of multiple sclerosis. However, it is not clear if this effect was achieved by systemic immunomodulation or if there is an active neuroregeneration in the central nervous system (CNS). In order to investigate remyelination and regeneration in the CNS we analysed the effects of intravenously and intranasally applied murine and human bone marrow-derived MSC on cuprizone induced demyelination, a toxic animal model which allows analysis of remyelination without the influence of the peripheral immune system. In contrast to EAE no effects of MSC on de- and remyelination and glial cell reactions were found. In addition, neither murine nor human MSC entered the lesions in the CNS in this toxic model. In conclusion, MSC are not directed into CNS lesions in the cuprizone model where the blood-brain-barrier is intact and thus cannot provide support for regenerative processes.

MicroRNA-322 (miR-322) and Its Target Protein Tob2 Modulate Osterix (Osx) mRNA Stability

Osteogenesis depends on a coordinated network of signals and transcription factors such as Runx2 and Osterix. Recent evidence indicates that microRNAs (miRNAs) act as important post-transcriptional regulators in a large number of processes, including osteoblast differentiation. In this study, we performed miRNA expression profiling and identified miR-322, a BMP-2-down-regulated miRNA, as a regulator of osteoblast differentiation. We report miR-322 gain- and loss-of-function experiments in C2C12 and MC3T3-E1 cells and primary cultures of murine bone marrow-derived mesenchymal stem cells. We demonstrate that overexpression of miR-322 enhances BMP-2 response, increasing the expression of Osx and other osteogenic genes. Furthermore, we identify Tob2 as a target of miR-322, and we characterize the specific Tob2 3'-UTR sequence bound by miR-322 by reporter assays. We demonstrate that Tob2 is a negative regulator of osteogenesis that binds and mediates degradation of Osx mRNA. Our results demonstrate a new molecular mechanism controlling osteogenesis through the specific miR-322/Tob2 regulation of specific target mRNAs. This regulatory circuit provides a clear example of a complex miRNA-transcription factor network for fine-tuning the osteoblast differentiation program.

Ligation of TLR2 and TLR4 on murine bone marrow-derived mesenchymal stem cells triggers differential effects on their immunosuppressive activity

Mesenchymal stem cells (MSCs) have potent regulatory effects on immune and inflammatory responses. Recently the findings of functional TLR expression on MSC implicates these receptors in the function established for MSCs. Here we specially investigated the effects of TLR2, 4 ligation in mice MSC on migration, modulation of allogeneic mixed lymphocytes reaction (allo-MLR) and inducing Treg cells. We demonstrated that ligation of TLR2, but not TLR4, could significantly inhibit migration of MSC, impair MSC-mediated immunosuppression on allo-MLR, and reduce MSC-mediated expansion of CD4+CD25+Foxp3+ regulatory T cells. Compared with TLR4 activated MSCs and non-TLR activated MSC, TLR2 activation induced a relatively lower level of CXCL-10 mRNA and protein expressions which has been elucidated to act in concert with other soluble factor in MSC-mediated immunomodulation. These data indicate that TLR2 and TLR4 ligation had different effects on immunomodulatory capability of murine BMSCs, which should be considered in their use for treating inflammatory diseases.

Systematic Chromosomal Aberrations Found in Murine Bone Marrow-Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are studied as a cellular source for the treatment of various diseases. In this work, we isolated and cultivated murine bone marrow-derived MSCs. After a first observation of a solid tumor in a mouse injected with these cells, we systematically explored their chromosomal stability. We observed in all the cytogenetically analyzed cases gross chromosomal alterations every time the MSCs went through the senescence crisis while the lymphocytes from the same animals showed a normal chromosome count. This observation was confirmed in different mouse strains, with different culture protocols, and even in short-term cultures after a hematopoietic cell negative immunodepletion performed in order to accelerate the isolation procedure. Therefore, we conclude that murine MSCs display high chromosomal instability and can generate tumors, and that care must be taken before using them for the evaluation of MSC therapeutic potential.

Distinct Roles of Glycogen Synthase Kinase (GSK)-3α and GSK-3β in Mediating Cardiomyocyte Differentiation in Murine Bone Marrow-derived Mesenchymal Stem Cells

The signaling mechanisms facilitating cardiomyocyte (CM) differentiation from bone marrow (BM)-derived mesenchymal stem cells (MSCs) are not well understood. 5-Azacytidine (5-Aza), a DNA demethylating agent, induces expression of cardiac-specific genes, such as Nkx2.5 and alpha-MHC, in mouse BM-derived MSCs. 5-Aza treatment caused significant up-regulation of glycogen synthase kinase (GSK)-3beta and down-regulation of beta-catenin, whereas it stimulated GSK-3alpha expression only modestly. The promoter region of GSK-3beta was heavily methylated in control MSCs, but was demethylated by 5-Aza. Although overexpression of GSK-3beta potently induced CM differentiation, that of GSK-3alpha induced markers of neuronal and chondrocyte differentiation. GSK-3 inhibitors, including LiCl, SB 216743, and BIO, abolished 5-Aza-induced up-regulation of CM-specific genes, suggesting that GSK-3 is necessary and sufficient for CM differentiation in MSCs. Although specific knockdown of endogenous GSK-3beta abolished 5-Aza-induced expression of cardiac specific genes, surprisingly, that of GSK-3alpha facilitated CM differentiation in MSCs. Although GSK-3beta is found in both the cytosol and nucleus in MSCs, GSK-3alpha is localized primarily in the nucleus. Nuclear-specific overexpression of GSK-3beta failed to stimulate CM differentiation. Down-regulation of beta-catenin mediates GSK-3beta-induced CM differentiation in MSCs, whereas up-regulation of c-Jun plays an important role in mediating CM differentiation induced by GSK-3alpha knockdown. These results suggest that GSK-3alpha and GSK-3beta have distinct roles in regulating CM differentiation in BM-derived MSCs. GSK-3beta in the cytosol induces CM differentiation of MSCs through down-regulation of beta-catenin. In contrast, GSK-3alpha in the nucleus inhibits CM differentiation through down-regulation of c-Jun.

Spontaneous Differentiation of Murine Bone Marrow-Derived Mesenchymal Stem Cells into Adipocytes without Malignant Transformation after Long-Term Culture

Some observations have suggested that extensive culture of adult stem cells can lead to malignant transformation. Therefore, it has become commonplace to use stem cells undergoing little or no in vitro culture to circumvent this presumptive limitation. Recently, a detailed study documented that malignant transformation of adult neural stem cells can be avoided under suitable culture conditions. Here, we report the first demonstration that murine bone marrow-derived mesenchymal stem cells (bMSCs) were propagated in vitro for up to 50 passages without any transformation sign under suitable conditions. However, it must be noted that although the long-term cultured bMSCs were comparable with short-term cultured bMSCs in proliferation, migration and invasion, they lost their pluripotent potential. The long-term cultured bMSCs could only differentiate into adipocytes but not into osteocytes or chondrocytes. In conclusion, murine bMSCs can be propagated in vitro for up to 50 passages with no malignant transformation sign under suitable conditions, but how to sustain their pluripotent potential requires further investigation.

Chromosomal Instability of Murine Adipose Tissue-Derived Mesenchymal Stem Cells in Long-Term Culture and Development of Cloned Embryos

Mice are the most commonly used laboratory animals for research, and some mouse stem cells, such as induced pluripotent stem cells, embryonic stem cells, and mesenchymal stem cells (MSCs), are also widely used in basic research. It is thus important to know if these stem cells maintain their genomic stability when cultured. Murine bone marrow-derived mesenchymal stem cells (BMSCs) appear to undergo spontaneous transformation in vitro. Murine adipose tissue-derived mesenchymal stem cells (ADSCs), like BMSCs, have the potential to differentiate into multiple lineages. In this study, we used G-banding, induction of multiple-lineage differentiation, flow cytometry, and nuclear transfer (NT), and found that murine ADSCs also displayed chromosomal instability in long-term culture. Furthermore, we performed NT using murine ADSCs to study the nuclear reprogramming ability of undifferentiated adult stem cells and to find a new efficient donor for NT. Using the stem cells did not increase the percentage of NT embryos that developed to the morula/blastocyst stage, compared with cloned embryos from cumulus cells. This may be because the stem cells displayed chromosomal instability. This is the first reported study of the use of ADSCs for NT in mice. ADSCs could provide an alternative donor cell type for NT in other species, with the advantages of easy harvesting involving little or no pain or trauma.

460. High-Efficiency Transduction of Primary Murine Bone Marrow-Derived Mesenchymal Stem Cells by Capsid Tyrosine-Mutant AAV2 Vectors

460. High-Efficiency Transduction of Primary Murine Bone Marrow-Derived Mesenchymal Stem Cells by Capsid Tyrosine-Mutant AAV2 Vectors

Murine bone marrow–derived mesenchymal stem cells as vehicles for interleukin‐12 gene delivery into Ewing sarcoma tumors

This study evaluated the therapeutic efficacy of interleukin 12 (IL-12) gene therapy in Ewing sarcoma and whether murine mesenchymal stem cells (MSCs) could serve as vehicles for IL-12 gene delivery. MSCs were isolated from murine bone marrow cells. Cells were phenotyped using flow cytometry. Cultured MSCs differentiated into osteocytes and adipocytes using the appropriate media. Freshly isolated MSCs were transfected with adenoviral vectors containing either the beta-galactosidase (Ad:beta-gal) or the IL-12 (Ad:IL-12) gene. Expression of IL-12 was confirmed using reverse transcription polymerase chain reaction. Mice with TC71 Ewing sarcoma tumors were then treated intravenously with MSCs transfected with Ad:beta-gal or Ad:IL-12. Tumors were measured and analyzed by immunohistochemical analysis for expression of IL-12 protein. Expression of both p35 and p40 IL-12 subunits was demonstrated in MSCs transfected in vitro with Ad:IL-12. IL-12 expression was seen in tumors from mice treated with MSCs transfected with Ad:IL-12. Tumor growth was also significantly inhibited compared with that in mice treated with MSCs transfected with Ad:beta-gal. MSCs can be transfected with the IL-12 gene. These transfected cells localize to tumors after intravenous injection and induce local IL-12 protein production and the regression of established tumors.

Circadian mechanisms in murine and human bone marrow mesenchymal stem cells following dexamethasone exposure

A core group of regulatory factors control circadian rhythms in mammalian cells. While the suprachiasmatic nucleus in the brain serves as the central core circadian oscillator, circadian clocks also exist within peripheral tissues and cells. A growing body of evidence has demonstrated that > 20% of expressed mRNAs in bone and adipose tissues oscillate in a circadian manner. The current manuscript reports evidence of the core circadian transcriptional apparatus within primary cultures of murine and human bone marrow-derived mesenchymal stem cells (BMSCs). Exposure of confluent, quiescent BMSCs to dexamethasone synchronized the oscillating expression of the mRNAs encoding the albumin D binding protein (dbp), brain–muscle arnt-like 1 (bmal1), period 3 (per3), rev-erb α (Rev A), and rev-erb β (Rev B). The genes displayed a mean oscillatory period of 22.2 to 24.3 h. The acrophase or peak expression of mRNAs encoding “positive” (bmal1) and “negative” (per3) components of the circadian regulatory apparatus were out of phase with each other by ~ 8–12 h, consistent with in vivo observations. In vivo, phosphyrylation by glycogen synthase kinase 3β (GSK3β) is known to regulate the turnover of per3 and components of the core circadian regulatory apparatus. In vitro addition of lithium chloride, a GSK3β inhibitor, significantly shifted the acrophase of all genes by 4.2–4.7 h oscillation in BMSCs; however, only the male murine BMSCs displayed a significant increase in the length of the period of oscillation. We conclude that human and murine BMSCs represent a valid in vitro model for the analysis of circadian mechanisms in bone metabolism and stem cell biology.

High-Yield Isolation, Expansion, and Differentiation of Murine Bone Marrow-Derived Mesenchymal Stem Cells Using Fibrin Microbeads (FMB)

Transplantation of adult mesenchymal stem cells (MSCs) could provide a basis for tissue regeneration. MSCs are typically isolated from bone marrow (BM) based on their preferential adherence to plastic, although with low efficiency in terms of yield and purity. Extensive expansion is needed to reach a significant number of MSCs for any application. Fibrin microbeads (FMB) were designed to attach mesenchymal cells and to provide a matrix for their expansion. The current study was aimed at isolating a high yield of purified BM-derived mouse MSCs based on their preferential adherence and proliferation on FMB in suspension cultures. MSCs could be downloaded to plastics or further expanded on FMB. The yield of MSCs obtained by the FMB isolation technique was about one order of magnitude higher than that achieved by plastic adherence, suggesting that these cells are more abundant than previously reported. FMB-isolated cells were classified as MSCs by their fibroblastic morphology, self-renewal ability, and expression profile of their surface antigens, as examined by flow cytometry and immunostaining. In cell culture, the isolated MSCs could be induced to differentiate into three different mesodermal lineages, as demonstrated by histochemical stains and by RT-PCR analyses of tissue-specific genes. MSCs were also able to differentiate into osteocytes while still cultured on FMB. Our results suggest that FMB might serve as an efficient platform for the isolation, expansion, and differentiation of mouse BM-derived MSCs to be subsequently implanted for tissue regeneration.

The fate of mesenchymal stem cells transplanted into immunocompetent neonatal mice: implications for skeletal gene therapy via stem cells

To explore the feasibility of skeletal gene and cell therapies, we transduced murine bone marrow-derived mesenchymal stem cells (MSCs) with a retrovirus carrying the enhanced green fluorescent protein and zeocin-resistance genes prior to transplantation into 2-day-old immunocompetent neonatal mice. Whole-body imaging of the recipient mice at 7 days post-systemic cell injection demonstrated a wide distribution of the cells in vivo. Twenty-five days posttransplantation, most of the infused cells were present in the lung as assessed by examination of the cells cultured from the lungs of the recipient mice. The cells persisted in lung and maintained a high level of gene expression and could be recovered from the recipient mice at 150 days after cell transplantation. A significant number of GFP-positive cells were also present in the bones of the recipient mice at 35 days post-cell transplantation. Recycling of the cells recovered from femurs of the recipient mice at 25 days posttransplantation by repeated injections into different neonatal mice resulted in the isolation of a clone of cells that was detected in bone and cartilage, but not in lung and liver after systemic injection. These data demonstrate that MSCs persist in immunocompetent neonatal mice, maintain a high level of gene expression, and may participate in skeletal growth and development of the recipient animals.