Rat mesenchymal stem cells increase tyrosine hydroxylase expression and dopamine content in ventral mesencephalic cells in vitro

Mesenchymal stem cells (MSCs) are pluripotent adult stem cells. It has been shown that MSCs secrete neurotrophic factors involving nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Also, these neurotrophic factors can upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells and neural stem cells. Here, we investigated the effect of co-culturing rat E13.5 ventral mesencephalic cells (VMCs) with MSCs from rat bone marrow on TH expression and dopamine (DA) content. The study consisted of 3 groups: MSC, VMC and a combined MSC+VMC group. All groups were cultured in serum-free neuro-basal medium for 3 days. Thereafter, each group was analyzed by RT-PCR, western blotting, and HPLC. The co-culture group showed a higher expression at TH and DA than the VMC group. However, TH and DA were not present in the MSC group. These observations suggest that MSCs could be an alternative source for treating neurodegenerative diseases such as Parkinson's disease (PD).     

Dopaminergic mediation of Y-aminobutyric acid in the control of prolactin release: Plasma prolactin and brain tyrosine hydroxylase levels in ovariectomized conscious rats

The present experiments were carried out to further elucidate the mechanism by which dopamine mediates the actions of Y-aminobutyric acid on prolactin release from anterior pituitary following its intraventricular injection in overiectomized conscious rats, Y-Aminobutyric acid significantly suppressed the prolactin levels at 0.1 Μmol concentration while at 4 Μmol dose, the level was elevated. The activity of tyrosine hydroxylase was increased significantly in the anterior pituitary at the lower dose while the higher concentration of Y-aminobutyric acid did not bring about any change in the activity both in the hypothalamus and the anterior pituitary. The results presented suggest that intracellular dopamine in the anterior pituitary may directly inhibit prolactin release; the plasma prolactin level is elevated by Y-aminobutyric acid, by way of either inhibiting dopaminergic tone or possible stimulation of a physiological prolactin releasin g hormone.     

Galanin inhibits tyrosine hydroxylase expression in midbrain dopaminergic neurons

Galanin (GAL) inhibits midbrain dopamine (DA) activity in several experimental paradigms, yet the mechanism underlying this inhibition is unclear. We examined the effects of GAL on the expression of tyrosine hydroxylase (TH) in primary cultures of rat embryonic (E14) ventral mesencephalon (VM). One micromolar GAL had no effect on the number of TH-immunoreactive (ir) neurons in VM cultures. However, 1 micro m GAL reduced an approximately 100% increase in TH-ir neurons in 1 mm dibutyryl cAMP (dbcAMP)-treated cultures by approximately 50%. TH-ir neuron number in dbcAMP-treated VM cultures was dose-responsive to GAL and the GAL receptor antagonist M40 blocked GAL effects. Semi-quantitative RT-PCR and quantitative immunoblotting experiments revealed that GAL had no effect on TH mRNA levels in VM cultures but reduced TH protein. VM cultures expressed GALR1, GALR2, and GALR3 receptor mRNA. However, dbcAMP treatment resulted in a specific approximately 200% increase in GALR1 mRNA. GALR1 activity is linked to a pertussis toxin (PTX)-sensitive opening of G protein-gated K+ channels (GIRKs). GAL reduction of TH-ir neuron number in dbcAMP + GAL-treated cultures was sensitive to both PTX and tertiapin, a GIRK inhibitor. GAL inhibition of midbrain DA activity may involve a GALR1- mediated reduction of TH in midbrain dopaminergic neurons.     

Intrastriatal transplantation of stem cells from human exfoliated deciduous teeth reduces motor defects in Parkinsonian rats

Background

This study explored the neural differentiation and therapeutic effects of stem cells from human exfoliated deciduous teeth (SHED) in a rat model of Parkinson's disease (PD).

Study of differentiated human umbilical cord–derived mesenchymal stem cells transplantation on rat model of advanced parkinsonism

The aim of this study was to explore the curative effect of differentiated human umbilical cord–derived mesenchymal stem cells (hUC‐MSCs) transplantation on rat of advanced Parkinson disease (PD) model. Human umbilical cord–derived mesenchymal stem cells were cultured and induced differentiation in vitro. The PD rats were established and allocated randomly into 2 groups: differentiated hUC‐MSCs groups and physiological saline groups (the control group). Rotation test and immunofluorescence double staining were done. The result showed that hUC‐MSCs could differentiate into mature dopamine neurons. Frequency of rotation was significantly less in differentiated hUC‐MSCs groups than in normal saline group. After we transplanted these cells into the unilateral lesioned substantia nigra induced by striatal injection of 6‐hydroxydopamine and performed in the medial forebrain bundle and ventral tegmental area, nigral tyrosine hydroxylase–positive cells were observed and survival of at least 2 months. In addition, transplantation of hUC‐MSCs could make an obviously therapeutic effect on PD rats.     

Differential expression of tyrosine hydroxylase mRNA in the developing rat mesencephalon

1. With respect to the mesostriatal projection, the mesencephalon is composed of two dopaminergic (DA) cell populations, called dorsal tier and ventral tier. Strong evidence suggests differences in both the spatial and the temporal sequence of the innervation of the striatum between the two groups, with the ventral tier neurons innervating striatal patches prenatally and dorsal tier cells innervating striatal matrix postnatally. 2. Using in situ hybridization, we have examined the expression of the gene coding for tyrosine hydroxylase (TH) in mesencephalic DA neurons with respect to their postnatal development. Two ontogenic patterns of expression were observed: (a) dorsal tier neurons of the medial mesencephalon exhibited a sharp increase in expression beginning after birth, peaking on day 14, then decreasing and, finally, stabilizing; and (b) ventral tier neurons and dorsal tier cells from the lateral and the medial-dorsal mesencephalon showed only a slight increase in TH mRNA, reaching a plateau at P10. 3. The time course of the observed increase in TH gene expression in the first group, generally parallels the innervation of their target cells in the striatal matrix, suggesting that TH gene expression in these cells may be influenced by their postsynaptic cells or by the innervation process.     

Therapeutic potential of the immunomodulatory activities of adult mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) include a select population of resident cells within adult tissues, which retain the ability to differentiate along several tissue‐specific lineages under defined media conditions and have finite expansion potential in vitro. These adult progenitor populations have been identified in various tissues, but it remains unclear exactly what role both transplanted and native MSCs play in processes of disease and regeneration. Interestingly, increasing evidence reveals a unique antiinflammatory immunomodulatory phenotype shared among this population, lending support to the idea that MSCs play a central role in early tissue remodeling responses where a controlled inflammatory response is required. However, additional evidence suggests that MSCs may not retain infinite immune privilege and that the context with which these cells are introduced in vivo may influence their immune phenotype. Therefore, understanding this dynamic microenvironment in which MSCs participate in complex feedback loops acting upon and being influenced by a plethora of secreted cytokines, extracellular matrix molecules, and fragments will be critical to elucidating the role of MSCs in the intertwined processes of immunomodulation and tissue repair. Birth Defects Research (Part C) 90:67–74, 2010. © 2010 Wiley‐Liss, Inc.     

Dopaminergic‐like cells from epigenetically reprogrammed mesenchymal stem cells

A number of recent studies have examined the ability of stem cells derived from different sources to differentiate into dopamine‐producing cells and ameliorate behavioural deficits in Parkinsonian models. Recently, using the approach of cell reprogramming by small cell‐permeable biological active compounds that involved in the regulation of chromatin structure and function, and interfere with specific cell signalling pathways that promote neural differentiation we have been able to generate neural‐like cells from human bone marrow (BM)‐derived MSCs (hMSCs). Neurally induced hMSCs (NI‐hMSCs) exhibited several neural properties and exerted beneficial therapeutic effect on tissue preservation and locomotor recovery in spinal cord injured rats. In this study, we aimed to determine whether hMSCs neuralized by this approach can generate dopaminergic (DA) neurons. Immunocytochemisty studies showed that approximately 50–60% of NI‐hMSCs expressed early and late dopaminergic marker such as Nurr‐1 and TH that was confirmed by Western blot. ELISA studies showed that NI‐hMSCs also secreted neurotrophins and dopamine. Hypoxia preconditioning prior to neural induction increased hMSCs proliferation, viability, expression TH and the secretion level of dopamine induced by ATP. Taken together, these studies demonstrated that hMSCs neurally modified by this original approach can be differentiated towards DA‐like neurons.     

Multipotent mesenchymal stem cells from adult human eye conjunctiva stromal cells

Abstract Identification of mesenchymal stem cells (MSCs) derived from alternative sources has provided an exciting prospect for intensive investigation. This work focused on characterizing a new source of MSCs from stromal cells from human eye conjunctiva. In this study, after conjunctiva biopsies and culture of stromal segment of this tissue, fibroblast-like (SH2⁺, SH3⁺, CD29⁺, CD44⁺, CD166⁺, CD13⁺) human stromal cells, which can be differentiated toward the osteogenic, adipogenic, chondrogenic, and neurogenic lineages, were obtained. These cells expressed Oct-4, Nanog, Rex-1 genes, and some lineage-specific markers like cardiac actin and Keratin. Taken together, the results indicate that conjunctiva stromal-derived cells are a new source of multipotent MSCs and despite originating from an adult source, they express undifferentiated stem cell markers.     

Expression of tyrosine hydroxylase is epigenetically regulated in neural stem cells

Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in the biosynthesis of catecholamines, and its expression is regulated in a developmental stage- and cell type-specific manner. Our previous work suggested that the genetic elements responsible for cell type-specific expression of TH were in the repressor region of the TH promoter between −2187 and −1232 bp. To investigate the molecular mechanisms underlying the specificity of TH expression, the DNA methylation patterns of the CpG islands in the repressor region of the TH promoter were examined in human neural stem cells (NSCs) and dopaminergic neuron-like cells. Using a bisulfite sequencing method, we found that the cytosine residues of CpG islands within the NRSE-R site were specifically methylated in NSCs, but not in SH-SY5Y neuroblastoma cells. In NSCs, CpG methylation correlated with reduced TH gene expression, and inhibition of DNA methylation with 5-azacytidine restored TH expression. Furthermore, methyl-CpG binding domain proteins (MBDs) bound to the highly methylated X-1 and X-2 regions of the TH gene in NSCs. Taken together, these results suggest that region-specific methylation and MBDs play important roles in TH gene regulation in NSCs.     

Clonal mesenchymal stem cells derived from human bone marrow can differentiate into hepatocyte‐like cells in injured livers of SCID mice

There is increasing evidence that human mesenchymal stem cells (hMSCs) can be a valuable, transplantable source of hepatocytes. Most of the hMSCs preparations used in these studies were likely heterogeneous cell populations, isolated by adherence to plastic surfaces or by density gradient centrifugation. Therefore, the participation of other unknown trace cell populations cannot be rigorously discounted. Here we report the isolation and establishment of a cloned human MSC line (chMSC) from human bone marrow primary culture, through which we confirmed the hepatic differentiation capability of authentic hMSCs. chMSCs expressed markers of mesenchymal cells, but not markers of hematopoietic stem cells. In vitro, chMSCs can differentiate into either mesenchymal cells or cells exhibiting hepatocyte‐like phenotypes. When transplanted intrasplentically into carbon tetrachloride‐injured livers of SCID mice, EGFP‐tagged chMSCs engrafted into the host liver parenchyma, exhibited typical hepatocyte morphology, form a three‐dimensional architecture, and differentiate into hepatocyte‐like cells expressing human albumin and α‐1‐anti‐trypsin. By confocal microscopy, ultrafine intercellular nanotubular structures were visible between adjacent transplanted and host hepatocytes. We postulate that these structures may assist in the phenotype conversion of chMSCs, possibly by exchange of cytoplasmic components between native hepatocytes and transplanted cells. Thus, a clonal pure population of hMSCs, which can be expanded in culture, may have potential as a cellular source for substitution damaged cells in hepatic injury. J. Cell. Biochem. 108: 693–704, 2009. © 2009 Wiley‐Liss, Inc.     

Diencephalo-telencephalic changes of tyrosine hydroxylase in rats and grass frogs after sleep deprivation

Based on sleep deprivation-produced changes of electrographic parameters of the wakefulness-sleep cycle (WSC) in rats and frogs (Rana temporaria), dynamics of activity of tyrosine hydroxylase, the key enzyme of dopamine synthesis, was studied immunohistochemically in substantia nigra and nigrostriatal pathway in rats and in striatum, paraventricular organ, and extrahypothalamic pathways in frogs. Changes in dynamics of tyrosine hydroxylase in rats and in frogs are revealed after the 6-h sleep deprivation and after 2 h of postdeprivation sleep. This allows determining the degree of participation of corticostriatal neuroregulatory and hypothalamo-pituitary neurosecretory systems and their role in regulation of WSC. Possible evolutionary peculiarities of morphofunctional differences in homoiothermal and poikilothermal animals are discussed.     

Encapsulation of adult human mesenchymal stem cells within collagen-agarose microenvironments

Reliable control over the process of cell differentiation is a major challenge in moving stem cell-based therapies forward. The composition of the extracellular matrix (ECM) is known to play an important role in modulating differentiation. We have developed a system to encapsulate adult human mesenchymal stem cells (hMSC) within spherical three-dimensional (3D) microenvironments consisting of a defined mixture of collagen Type I and agarose polymers. These protein-based beads were produced by emulsification of liquid hMSC-matrix suspensions in a silicone fluid phase and subsequent gelation to form hydrogel beads, which were collected by centrifugation and placed in culture. Bead size and size distribution could be varied by changing the encapsulation parameters (impeller speed and blade separation), and beads in the range of 30-150 microns in diameter were reliably produced. Collagen concentrations up to 40% (wt/wt) could be incorporated into the bead matrix. Visible light and fluorescence microscopy confirmed that the collagen matrix was uniformly distributed throughout the beads. Cell viability post-encapsulation was in the range of 75-90% for all bead formulations (similar to control slab gels) and remained at this level for 8 days in culture. Fluorescent staining of the actin cytoskeleton revealed that hMSC spreading increased with increasing collagen concentration. This system of producing 3D microenvironments of defined matrix composition therefore offers a way to control cell-matrix interactions and thereby guide hMSC differentiation. The bead format allows the use of small amounts of matrix proteins, and such beads can potentially be used as a cell delivery vehicle in tissue repair applications.     

Limited plasticity of mesenchymal stem cells cocultured with adult cardiomyocytes

In order to assess, in a controlled in vitro model, the differentiation potential of adult bone marrow derived stem cells we have developed a coculture procedure using adult rat cardiomyocytes and mesenchymal stem cells (MSCs) from transgenic GFP positive rats. We investigated in the cocultured MSCs the time course of cellular processes that are difficult to monitor in in vivo experiments. Adult rat cardiomyocytes and adult rat MSCs were cocultured for up to 7 days and analyzed by confocal microscopy. Several markers were studied by immunofluorescence technique. The fluorescent ST-BODIPY-Dihydropyridine was used to label calcium channels in living cells. Intracellular calcium was monitored with the fluorescent probe X-Rhod-1. Immunofluorescence experiments showed the presence of connexin-43 between cardiomyocytes and MSCs and between MSCs, while no sarcomeric structures were observed at any time of the coculture. We looked at the expression of calcium channels and development of voltage-dependent calcium signaling in cocultured MSCs. MSCs showed a time-dependent increase of labeling of ST-BODIPY-Dihydropyridine, reaching a relatively strong level after 72 h of coculture. The treatment with a non-fluorescent DHP, Nifedipine, completely abolished ST-BODIPY labeling. We investigated whether depolarization could modulate intracellular calcium. Depolarization-induced calcium transients increased in MSCs in relation to the coculture time. We conclude that MSCs cocultured with adult cardiomyocytes present preliminary evidence of voltage-dependent calcium modulation uncoupled with the development of nascent or adult myofibrils, thus showing a limited lineage specification and a low plasticity to differentiate in a full cardiomyocyte-like phenotype.     

Ear mesenchymal stem cells (EMSC) can differentiate into spontaneously contracting muscle cells

We have previously shown that cells isolated from the outer ears of adult mice are a source of mesenchymal stem cells that can be induced to differentiate into adipo-, osteo-, and chondrocytes. In this study, we demonstrate that ear mesenchymal stem cells (EMSC) express stromal cell-associated markers (CD44, CD73) and stem cell marker Sca-1 and can be differentiated into spontaneously contracting muscle cells. Treatment of cells with epidermal growth factor (EGF) change their morphology from fibroblast shapes into stick-like structures that show repeated spontaneous contractions. Under conditions that promote myogenic differentiation, EMSC expressed mRNA for myoD and ventricular specific myosin light chain (MLC-2v) and protein for connexin 43, sarcomeric alpha-actinin, myocyte enhancer factor 2c (MEF2c), myosin heavy chain (MyHC), myogenin, and sarco-endoplasmic reticulum Ca(2+)ATPase (SERCA) 1. However, the cells were negative for Nkx2.5, GATA4, and ANP. Intracellular Ca(2+) transients in spontaneously beating EMSC, visualized by Fluo-3AM, showed a frequency of Ca(2+) oscillations ranging over 28-59/min (mean 41.17 +/- SEM 1.54). We also demonstrated that small pieces of ear tissues (ear punches) collected from live mice provide sufficient numbers of EMSC to isolate, culture and differentiate them into myocytes. Due to the ease of acquiring an expanding repertoire of differentiated EMSC cell types by a noninvasive surgical procedure, we conclude that the ear may prove to be a potential source of autologous cells for regenerative medicine, as supported by the fact that ears are one of the best sources of cells for somatic cell nuclear transfer (SCNT).     

Comparison of alterations in tyrosine hydroxylase,dopamine levels,and dopamine uptake in the striatum of the weaver mutant mouse

Previous reports have shown that among the markers for the nigro-striatal dopamine (DA) system measured in the striatum, dopamine uptake seems to be more severely affected than the others in the weaver mutant mouse. In the present study we examined DA levels, tyrosine hydroxylase (TH) activity, and high-affinity DA uptake to determine if the DA uptake is most affected when all the measurements are made in the same striatal homogenate in the same laboratory. We found that the DA uptake activity was most altered (93% lower) compared to DA levels (68% lower) and TH activity (64% lower). The DA uptake was so low in the weaver that we could not obtain reliable kinetic parameters. For TH activity we found that the Vmax was 36% lower while the Km forl-tyrosine was 92% higher in the weaver striatum. This lower affinity for substrate suggests that the TH enzyme itself may be altered in the nigro-striatal system of the weaver mutant mouse.Special issue dedicated to Dr. Morris H. Aprison.     

Comparative osteogenic transcription profiling of various fetal and adult mesenchymal stem cell sources

Human mesenchymal stem cells (MSC) from adult and fetal tissues are promising candidates for cell therapy but there is a need to identify the optimal source for bone regeneration. We have previously characterized MSC populations in first trimester fetal blood, liver, and bone marrow and we now evaluate their osteogenic differentiation potential in comparison to adult bone marrow MSC. Using quantitative real-time RT-PCR, we demonstrated that 16 osteogenic-specific genes (OC, ON, BSP, OP, Col1, PCE, Met2A, OPG, PHOS1, SORT, ALP, BMP2, CBFA1, OSX, NOG, IGFII) were expressed in both fetal and adult MSC under basal conditions and were up-regulated under osteogenic conditions both in vivo and during an in vitro 21-day time-course. However, under basal conditions, fetal MSC had higher levels of osteogenic gene expression than adult MSC. Upon osteogenic differentiation, fetal MSC produced more calcium in vitro and reached higher levels of osteogenic gene up-regulation in vivo and in vitro. Second, we observed a hierarchy within fetal samples, with fetal bone marrow MSC having greater osteogenic potential than fetal blood MSC, which in turn had greater osteogenic potential than fetal liver MSC. Finally, we found that the level of gene expression under basal conditions was positively correlated with both calcium secretion and gene expression after 21 days in osteogenic conditions. Our findings suggest that stem cell therapy for bone dysplasias such as osteogenesis imperfecta may benefit from preferentially using first trimester fetal blood or bone marrow MSC over fetal liver or adult bone marrow MSC.     

Therapeutic potential of adult bone marrow‐derived mesenchymal stem cells in diseases of the skeleton

Mesenchymal stem cells (MSCs) are the most popular among the adult stem cells in tissue engineering and regenerative medicine. Since their discovery and functional characterization in the late 1960s and early 1970s, MSCs or MSC‐like cells have been obtained from various mesodermal and non‐mesodermal tissues, although majority of the therapeutic applications involved bone marrow‐derived MSCs. Based on its mesenchymal origin, it was predicted earlier that MSCs only can differentiate into mesengenic lineages like bone, cartilage, fat or muscle. However, varied isolation and cell culturing methods identified subsets of MSCs in the bone marrow which not only differentiated into mesenchymal lineages, but also into ectodermal and endodermal derivatives. Although, true pluripotent status is yet to be established, MSCs have been successfully used in bone and cartilage regeneration in osteoporotic fracture and arthritis, respectively, and in the repair of cardiac tissue following myocardial infarction. Immunosuppressive properties of MSCs extend utility of MSCs to reduce complications of graft versus host disease and rheumatoid arthritis. Homing of MSCs to sites of tissue injury, including tumor, is well established. In addition to their ability in tissue regeneration, MSCs can be genetically engineered ex vivo for delivery of therapeutic molecule(s) to the sites of injury or tumorigenesis as cell therapy vehicles. MSCs tend to lose surface receptors for trafficking and have been reported to develop sarcoma in long‐term culture. In this article, we reviewed the current status of MSCs with special emphasis to therapeutic application in bone‐related diseases. J. Cell. Biochem. 111: 249–257, 2010. © 2010 Wiley‐Liss, Inc.