Rat Bone Marrow Marrow Mesenchymal Stem Cells Research Articles

Construction of calcitonin gene-related peptide-modified mesenchymal stem cells and analysis of their effects on the migration and proliferation of vascular smooth muscle cells.

Lentiviral expression vectors for calcitonin gene-related peptide (CGRP) were used to transfect rat bone marrow mesenchymal stem cells (MSCs). After assessing the biological characteristics of proliferation and aging in MSCs transfected with CGRP, we observed the effects of the CGRP-modified rat MSCs on the migration and proliferation of rat vascular smooth muscle cells (VSMCs) in vitro. Rat MSCs were isolated, cultured in vitro, and identified by flow cytometry. A CGRP recombinant lentivirus was transfected into MSCs. The transfection efficiency was determined by fluorescence microscopy and flow cytometry, and CGRP in MSCs was detected by real-time quantitative PCR, ELISA, and immunofluorescence. The proliferation and senescence of CGRP-modified MSCs were evaluated by MTT assay and beta-galactosidase staining. VSMCs were isolated, cultured in vitro, and identified by immunofluorescence. CGRP-modified MSCs and VSMCs were cocultured in a Transwell system. The proliferation and migration of VSMCs were evaluated by scratch testing and the MTT method. Rat bone marrow MSCs showed a spindle-shaped morphology, adherent growth in vitro, positive CD29 and CD90 expression, and negative CD45 expression. CGRP was stably expressed in MSCs after 48h of recombinant lentivirus transfection. CGRP mRNA and protein secretion in CGRP recombinant lentivirus-transfected MSCs were higher than that in control MSCs. Immunofluorescence showed that CGRP protein could be expressed in CGRP-modified MSCs. The proliferation ability and senescence rates did not differ between lentivirus-transfected MSCs and untransfected MSCs. Rat VSMCs expressed α-SMA protein and exhibited a spindle-shaped morphology and adherent growth in vitro. In Transwell coculture experiments, scratch testing of VSMCs showed that CGRP-modified MSCs could reduce VSMC proliferation and migration. The CGRP gene can be stably expressed in MSCs after CGRP recombinant lentivirus transfection. CGRP recombinant lentivirus transfection has little effect on the proliferation or senescence of MSCs, and CGRP-modified MSCs can inhibit the proliferation and migration of VSMCs. These results lay a foundation for research on the use of CGRP gene-engineered MSCs in restenosis therapy.

TREM-2 negatively regulates LPS-mediated inflammatory response in rat bone marrow-derived MSCs.

To the best of our knowledge, our previous study demonstrated the expression of triggering receptor expressed on myeloid cells 2 (TREM-2) in human bone marrow mesenchymal stem cells (MSCs) for the first time. However, the inflammation regulatory role of TREM-2 in MSCs remain elusive. The aim of the present study was to investigate the immune regulation and the underlying mechanism of TREM-2 in rat bone marrow MSCs. MSCs were divided into three groups: NullMSCs, TREM-2MSCs, and NormMSCs. TREM-2 was expressed in MSCs at the mRNA and protein level. Following stimulation by lipopolysaccharide (LPS), the gene transcription levels of TREM-2 and inflammatory cytokines were increased. The expression levels of inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), in the TREM-2MSCs lentiviral vector group were significantly downregulated, and the expression of IL-10 was significantly upregulated compared with the controls. Western blot analysis revealed that TREM-2 downregulated the LPS-induced inflammatory response in MSCs, which was probably associated with regulating AKT serine/threonine kinase and p38 mitogen-activated protein kinase downstream signaling proteins. The results of the current study demonstrated that TREM-2 negatively regulates the LPS-mediated inflammatory response in MSCs suggesting that TREM-2 is a potential target of immune regulation in rat MSCs.

Therapeutic effects of intranigral transplantation of mesenchymal stem cells in rat models of Parkinson's disease.

Stem cell transplantation is a promising tool for the treatment of neurodegenerative disorders, including Parkinson's disease (PD); however, the therapeutic routes and mechanisms of mechanical approaches to stem cell transplantation must be explored. This study tests the therapeutic effect of transplantation of rat bone marrow mesenchymal stem cells (MSCs) into the substantia nigra (SN) of the PD rat. 5-Bromo-2-deoxyuridine-labeled rat MSCs were transplanted into the SN of the 6-hydroxydopamine-injected side of PD rat brains. The behavioral changes in PD rats were examined before and 4 and 8 weeks after MSC transplantation. The expression of tyrosine hydroxylase (TH) in the SN and the striatum and the survival and differentiation of MSCs were assessed by immunohistochemical and double immunofluorescence techniques. Abnormal behavior of PD rats was significantly improved by the administration of bone marrow MSCs, and the number of TH-positive cells in the SN and the optical density of TH-positive fibers in the striatum were markedly increased. Transplanted MSCs can survive and migrate in the brain and differentiate into nestin-, neuron-specific enolase-, and GFAP-positive cells. Our findings suggest that transplantation of rat bone marrow MSCs into the SN of PD rats may provide therapeutic effects. © 2016 Wiley Periodicals, Inc.

Growth and expression of rat bone marrow mesenchymal stem cells modified by nerve growth factor in diabetic rat bladders

The aim of the present study was to determine whether rat bone marrow mesenchymal stem cells (MSCs) transfected with the nerve growth factor (NGF) gene and then transplanted into diabetic rat bladder tissues survive and continue to express NGF. A recombinant lentiviral vector carrying the NGF gene was constructed and transfected into rat bone marrow MSCs. BrdU‑labeled immunohistochemistry was used to observe NGF expression in the transfected MSCs. BrdU‑labeled and NGF‑transfected MSCs were transplanted into diabetic rat bladder tissues. BrdU‑labeled immunohistochemistry was used to observe the growth of NGF‑transfected MSCs in the tissue samples. NGF mRNA and protein expression levels in MSCs were analyzed using reverse transcription polymerase chain reaction (RT-PCR) and ELISA, respectively. The recombinant NGF gene lentiviral vector and NGF gene-modified rat bone marrow MSCs were successfully constructed. NGF gene-modified rat MSCs survived in the diabetic rat bladders 4 weeks following injection and NGF gene expression was increased. In the present study, NGF gene-modified MSCs were shown to be capable of survival in diabetic rat bladder tissues and stably expressed NGF.

Curcumin increases rat mesenchymal stem cell osteoblast differentiation but inhibits adipocyte differentiation

Background:Curcumin is a phenolic natural product isolated from the rhizome of Curcuma longa (turmeric) and has effects on bone health and fat formation. The bone marrow mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into osteoblasts and adipocytes. Osteoblast differentiation of MSCs can be a result of upregulation of heme oxygenase (HO)-1 expression. Curcumin can potently induce HO-1 expression.Objective:The present study describes the effects of curcumin on rat MSC (rMSCs) differentiation into osteoblasts and adipocytes.Materials and Methods:Rat bone marrow MSCs were isolated and treated with or without curcumin. Osteoblast differentiation was confirmed and determined by alkaline phosphatase (ALP) activity, mineralized nodule formation, the expression of Runx2 (runt-related transcription factor 2) and osteocalcin. Adipocyte differentiation was determined by Oil red O staining and the expression of peroxisome proliferator-activated receptor-γ 2 (PPARγ2) and CCAAT/enhancer-binding protein (C/EBP) α.Results:Curcumin increased ALP activity and osteoblast-specific mRNA expression of Runx2 and osteocalcin when rMSCs were cultured in osteogenic medium. In contrast, curcumin decreased adipocyte differentiation and inhibited adipocyte-specific mRNA expression of PPARγ2 and C/EBPα when rMSCs were cultured in adipogenic medium. HO-1 expression was increased during osteogenic differentiation of rMSCs.Conclusions:These findings demonstrate that curcumin can promote osteogenic differentiation of rMSCs and inhibit adipocyte formation. The effect of curcumin on osteogenic differentiation of rMSCs is correlated with HO-1 expression.

VEGF expression in mesenchymal stem cells promotes bone formation of tissue-engineered bones

The purpose of this study was to investigate the invivo vascularization and bone formation activity of tissue-engineered bone constructed using bone marrow mesenchymal stem cells (MSCs) transfected with vascular endothelial growth factor (VEGF). The expression of VEGF165 in rat bone marrow MSCs was confirmed using RT-PCR and immunohistochemistry. The MSCs were cultured together with nano-hydroxyapatite/collagen (NHAC) to form tissue-engineered bone. Untransfected MSCs were used as controls. The mice were sacrificed, and the bone xenografts were analyzed using immunohistochemistry and quantified for the degree of vascularization and new bone formation. Based on our results, expression of the VEGF165 gene was detected using RT-PCR and immunohistochemistry following transfection and 4 weeks of selection. The co-cultured NHAC- and VEGF-transfected MSCs had significantly higher alkaline phosphatase (AP) activity compared to the controls (P<0.05). In the mice that received the tissue-engineered bone xenografts, clumps of cartilage cells, irregular bone-like tissue and microvessels were observed. The growth of these structures progressed with time. In the control mice, however, only small amounts of bone-like and fibrotic tissue were observed. The differences between the control and experimental groups were statistically significant (P<0.05). In conclusion, VEGF165‑transfected bone marrow MSCs promotes vascularization of tissue-engineered bone and ectopic osteogenesis.

The differentiation of mesenchymal stem cells into inner ear hair cell-like cells in vitro

Conclusion. Bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into hair cells, and this method of culturing MSCs provides a useful tool for studies on mammalian cochlear hair cell regeneration. Objective: To investigate a method to induce bone marrow MSCs to differentiate into inner ear hair cells. Methods: Rat bone marrow MSCs were isolated from healthy rats and cultured in vitro. To make sure that the cultured cells were bone marrow MSCs, the expression of MSC markers such as SH2, CD31, CD34, and CD44 genes on the cultured cells was assessed by RT-PCR. Adipogenic cells and osteogenic cells were induced by the differentiation of the cultured cells, respectively, suggesting that the cultured cells have the characteristic of pluripotent differentiation. Then they were induced to differentiate into neural stem cells and hair cell progenitor cells. Immunohistochemistry experiments were carried out to detect the expression of molecular markers. Scanning electron microscope samples were prepared for observation of the morphology of the cells. Results: Rat bone marrow MSCs were successfully isolated, purified, cultured, and identified in vitro. They were also successfully induced to differentiate into neural progenitor cells and then hair cell-like cells that expressed myosin VIIa.

EMF acts on rat bone marrow mesenchymal stem cells to promote differentiation to osteoblasts and to inhibit differentiation to adipocytes

The use of electromagnetic fields (EMFs) to treat nonunion fractures developed from observations in the mid-1900s. Whether EMF directly regulates the bone marrow mesenchymal stem cells (MSCs), differentiating into osteoblasts or adipocytes, remains unknown. In the present study, we investigated the roles of sinusoidal EMF of 15 Hz, 1 mT in differentiation along these separate lineages using rat bone marrow MSCs. Our results showed that EMF promoted osteogenic differentiation of the stem cells and concurrently inhibited adipocyte formation. EMF increased alkaline phosphatase (ALP) activity and mineralized nodule formation, and stimulated osteoblast-specific mRNA expression of RUNX2, ALP, BMP2, DLX5, and BSP. In contrast, EMF decreased adipogenesis and inhibited adipocyte-specific mRNA expression of adipsin, AP-2, and PPARgamma2, and also inhibited protein expression of PPARgamma2. These observations suggest that commitment of MSCs into osteogenic or adipogenic lineages is influenced by EMF.

Chemically-Induced RAT Mesenchymal Stem Cells Adopt Molecular Properties of Neuronal-Like Cells but Do Not Have Basic Neuronal Functional Properties

Induction of adult rat bone marrow mesenchymal stem cells (MSC) by means of chemical compounds (β-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanizole) has been proposed to lead to neuronal transdifferentiation, and this protocol has been broadly used by several laboratories worldwide. Only a few hours of MSC chemical induction using this protocol is sufficient for the acquisition of neuronal-like morphology and neuronal protein expression. However, given that cell death is abundant, we hypothesize that, rather than true neuronal differentiation, this particular protocol leads to cellular toxic effects. We confirm that the induced cells with neuronal-like morphology positively stained for NF-200, S100, β-tubulin III, NSE and MAP-2 proteins. However, the morphological and molecular changes after chemical induction are also associated with an increase in the apoptosis of over 50% of the plated cells after 24 h. Moreover, increased intracellular cysteine after treatment indicates an impairment of redox circuitry during chemical induction, and in vitro electrophysiological recordings (patch-clamp) of the chemically induced MSC did not indicate neuronal properties as these cells do not exhibit Na+ or K+ currents and do not fire action potentials. Our findings suggest that a disruption of redox circuitry plays an important role in this specific chemical induction protocol, which might result in cytoskeletal alterations and loss of functional ion-gated channels followed by cell death. Despite the neuronal-like morphology and neural protein expression, induced rat bone marrow MSC do not have basic functional neuronal properties, although it is still plausible that other methods of induction and/or sources of MSC can achieve a successful neuronal differentiation in vitro.

Osteogenic growth peptide C-terminal pentapeptide [OGP(10–14)] acts on rat bone marrow mesenchymal stem cells to promote differentiation to osteoblasts and to inhibit differentiation to adipocytes

Cumulative evidence indicates that bone marrow mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating to osteogenic and adipogenic lineages when stimulated under appropriate conditions. Whether OGP(10–14) directly regulates the progenitor cells differentiating into osteoblasts or adipocytes remains unknown. In the present study, we investigated the roles of OGP(10–14) in differentiation along these separate lineages using rat bone marrow MSCs. Our results showed that OGP(10–14) promoted osteogenic differentiation of the stem cells and concurrently inhibited adipocyte formation. OGP(10–14) increased alkaline phosphatase (ALP) activity and mineralized nodule formation, and stimulated osteoblast-specific mRNA expression of core-binding factor 1 (cbfa1). In contrast, OGP(10–14) decreased adipocyte numbers and inhibited adipocyte-specific mRNA expression of peroxisome proliferator-activated receptor-γ 2 (PPARγ2). These observations suggest that commitment of MSCs into osteogenic or adipogenic lineages is regulated by OGP(10–14).

Vascular Endothelial Growth Factor Is Involved in Protective Effects of Mesenchymal Stem Cells on Cultured Neurons in Hypoxic Condition.

INTRODUCTION: Transplantation of autologous bone marrow mesenchymal stem cells (MSCs) is increasingly being considered in cell-based therapeutic strategies for traumatic brains. Pluripotent MSCs can differentiate into neural cells, but few reports completely address the therapeutic effect of transplanting the cells into damaged brains in vivo. The early remarkable functional effect of MSC-transplantation cannot be readily attributed to tissue regeneration from MSCs. Hypoxia is the major characteristic of ischemic microenvironment. Low oxygen tension is thought to be an integral component of the MSC native bone marrow microenvironment. Thus, we hypothesized that paracrine actions exerted by the cells through the release of soluble factors might be partly important mechanisms of tissue repair and functional improvement after injection of MSCs. In the current study, we demonstrate that hypoxia induces expression of vascular endothelial growth factor (VEGF) in rat bone marrow MSCs, and VEGF markedly inhibits apoptosis of neonatal rat neurons induced by hypoxia in vitro.METHODS: Adult Sprague-Dawley rat bone marrow MSCs and neonatal rat neurons were purified and cultured. MSCs were characterized by high expression of CD44, the lack of CD45 and CD11b molecules, their typical spindle-shaped morphology, together with their ability to differentiate into osteogenic, chondrogenic, and adipogenic cells, and neurons by expression of neuron specific enolase and neuronal nuclei marker NeuN. In the experiments, MSCs were co-cultured with or without neurons at the density of 2×105 cells/ml (ratio of 1:1) in DMEM/F12 containing 15% FBS. After 24 hours, cells were cultured with hypoxic DMEM/F12 containing 5% FBS in hypoxia condition (93% N2 + 2% O2 +5% CO2), and were treated with or without 0.2 nM VEGF and/or 100 nM SiRNA specific for VEGF mRNA. 72 hours after hypoxic culture, cells were harvested for analysis of cell apoptosis by flow cytometry. Level of the VEGF protein in medium was detected by ELISA.RESULTS: The apoptotic ratio of normoxic cultured neurons was (11.46±2.68)% at 72 hours. The apoptotic ratio of hypoxic cultured neurons increased to (38.95±4.23)% (P<0.01). The apoptotic ratio of neurons co-cultured with MSCs in hypoxic condition decreased to (17.52±3.19)% (P<0.01). VEGF protein in the co-cultured media was (178.15±11.93) pg/ml, which were undetectable in media of normoxic or hypoxic cultured neurons. However, specific SiRNA enhanced the apoptotic ratio of hypoxic-induced neurons co-cultured with MSCs (30.81±4.09)%, and inhibited expression of VEGF protein in MSCs. Moreover, 0.2 nM VEGF reversed the role of specific SiRNA, of which the apoptotic ratio was (16.83±3.16)% (P<0.01).CONCLUSION: These results showed that hypoxia induced expression of VEGF in bone marrow MSCs, and co-culture with MSCs improved the viability of neurons in hypoxia condition in vitro. We conclude that VEGF is involved in protective effects of Mesenchymal stem cells on cultured neurons in hypoxic condition. These evidences support paracrine hypothesis for mesenchymal stem cell-mediated acute neuroprotective effect and functional improvement, and the paracrine action is one of the multiple mechanisms of MSCs-based therapy for traumatic brains.