Survival and Neuronal Differentiation of Mesenchymal Stem Cells Transplanted into the Rodent Brain Are Dependent upon Microenvironment

Introduction: The successful integration of stem cells in adult brain has become a central issue in modern neuroscience. In this study we sought to test the hypothesis that survival and neurodifferentiation of mesenchymal stem cells (MSCs) may be dependent upon microenvironmental conditions according to the site of implant in the brain. Methods: MSCs were isolated from adult rats and labeled with enhanced-green fluorescent protein (eGFP) lentivirus. A cell suspension was implanted stereotactically into the brain of 50 young rats, into one neurogenic area (hippocampus), and into another nonneurogenic area (striatum). Animals were sacrificed 6 or 12 weeks after surgery, and brains were stained for mature neuronal markers. Cells coexpressing NeuN (neuronal specific nuclear protein) and GFP (green fluorescent protein) were counted stereologically at both targets. Results: The isolated cell population was able to generate neurons positive for microtubule-associated protein 2 (MAP2), neuronal-specific nuclear protein (NeuN), and neurofilament 200 (NF200) in vitro. Electrophysiology confirmed expression of voltage-gated ionic channels. Once implanted into the hippocampus, cells survived for up to 12 weeks, migrated away from the graft, and gave rise to mature neurons able to synthesize neurotransmitters. By contrast, massive cell degeneration was seen in the striatum, with no significant migration. Induction of neuronal differentiation with increased cyclic adenosine monophosphate in the culture medium before implantation favored differentiation in vivo. Conclusions: Our data demonstrated that survival and differentiation of MSCs is strongly dependent upon a permissive microenvironment. Identification of the pro-neurogenic factors present in the hippocampus could subsequently allow for the integration of stem cells into nonpermissive areas of the central nervous system.

Shear Stress Induces Nitric Oxide-Mediated Vascular Endothelial Growth Factor Production in Human Adipose Tissue Mesenchymal Stem Cells

It has been demonstrated that human adipose tissue-derived mesenchymal stem cells (hASCs) enhance vascular density in ischemic tissues, suggesting that they can differentiate into vascular cells or release angiogenic factors that may stimulate neoangiogenesis. Moreover, there is evidence that shear stress (SS) may activate proliferation and differentiation of embryonic and endothelial precursor stem cells into endothelial cells (ECs). In this work, we investigated the effect of laminar SS in promoting differentiation of hASCs into ECs. SS (10 dyn/cm(2) up to 96 h), produced by a cone plate system, failed to induce EC markers (CD31, vWF, Flk-1) on hASC assayed by RT-PCR and flow cytometry. In contrast, there was a cumulative production of nitric oxide (determined by Griess Reaction) and vascular endothelial growth factor (VEGF; by ELISA) up to 96 h of SS stimulation ( NO(2)(-) in nmol/10(4) cells: static: 0.20 +/- 0.03; SS: 1.78 +/- 0.38, n = 6; VEGF in pg/10(4) cells: static: 191.31 +/- v35.29; SS: 372.80 +/- 46.74, n = 6, P < 0.05). Interestingly, the VEGF production was abrogated by 5 mM N(G)-L-nitro-arginine methyl ester (L-NAME) treatment (VEGF in pg/10(4) cells: SS: 378.80 +/- 46.74, n = 6; SS + L-NAME: 205.84 +/- 91.66, n = 4, P < 0.05). The results indicate that even though SS failed to induce EC surface markers in hASC under the tested conditions, it stimulated NO-dependent VEGF production.

Mesenchymal Stem Cells Derived From Canine Umbilical Cord Vein-A Novel Source for Cell Therapy Studies

The canine model provides a large animal system to evaluate many treatment modalities using stem cells (SCs). However, only bone marrow ( BM) protocols have been widely used in dogs for preclinical approaches. BM donation consists of an invasive procedure and the number and differentiation potential of its mesenchymal stem cells (MSCs) decline with age. More recently, umbilical cord was introduced as an alternative source to BM since it is obtained from a sample that is routinely discarded. Here, we describe the isolation of MSCs from canine umbilical cord vein (cUCV). These cells can be obtained from every cord received and grow successfully in culture. Their multipotent plasticity was demonstrated by their capacity to differentiate in adipocytic, chondrocytic, and osteocytic lineages. Furthermore, our results open possibilities to use cUCV cells in preclinical trials for many well-characterized canine model conditions homologs to human diseases.

Human fallopian tube: a new source of multipotent adult mesenchymal stem cells discarded in surgical procedures

Background: The possibility of using stem cells for regenerative medicine has opened a new field of investigation. The search for sources to obtain multipotent stem cells from discarded tissues or through non-invasive procedures is of great interest. It has been shown that mesenchymal stem cells (MSCs) obtained from umbilical cords, dental pulp and adipose tissue, which are all biological discards, are able to differentiate into muscle, fat, bone and cartilage cell lineages. The aim of this study was to isolate, expand, characterize and assess the differentiation potential of MSCs from human fallopian tubes (hFTs). Methods: Lineages of hFTs were expanded, had their karyotype analyzed, were characterized by flow cytometry and underwent in vitro adipogenic, chondrogenic, osteogenic, and myogenic differentiation. Results: Here we show for the first time that hFTs, which are discarded after some gynecological procedures, are a rich additional source of MSCs, which we designated as human tube MSCs (htMSCs). Conclusion: Human tube MSCs can be easily isolated, expanded in vitro, present a mesenchymal profile and are able to differentiate into muscle, fat, cartilage and bone in vitro.

Intracellular Ca(2+) Regulation During Neuronal Differentiation of Murine Embryonal Carcinoma and Mesenchymal Stem Cells

Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) play a central role in neuronal differentiation. However, Ca(2+) signaling in this process remains poorly understood and it is unknown whether embryonic and adult stem cells share the same signaling pathways. To clarify this issue, neuronal differentiation was analyzed in two cell lines: embryonic P19 carcinoma stem cells (CSCs) and adult murine bone-marrow mesenchymal stem cells (MSC). We studied Ca(2+) release from the endoplasmic reticulum via intracellular ryanodine-sensitive (RyR) and IP(3)-sensitive (IP(3)R) receptors. We observed that caffeine, a RyR agonist, induced a [Ca(2+)](i) response that increased throughout neuronal differentiation. We also demonstrated a functional coupling between RyRs and L-but not with N-, P-, or Q-type Ca(v)1 Ca(2+) channels, both in embryonal CSC and adult MSC. We also found that agonists of L-type channels and of RyRs increase neurogenesis and neuronal differentiation, while antagonists of these channels have the opposite effect. Thus, our data demonstrate that in both cell lines RyRs control internal Ca(2+) release following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels. This study shows that both in embryonal CSC and adult MSC [Ca(2+)](i) is controlled by a common pathway, indicating that coupling of L-type Ca(2+) channels and RyRs may be a conserved mechanism necessary for neuronal differentiation.

Limited Ca(2+) and PKA-pathway dependent neurogenic differentiation of human adult mesenchymal stem cells as compared to fetal neuronal stem cells

The ability of mesenchymal stem cells to generate functional neurons in culture is still a matter of controversy. In order to assess this issue, we performed a functional comparison between neuronal differentiation of human MSCs and fetal-derived neural stem cells (NSCs) based on morphological, immunocytochemical, and electrophysiological criteria. Furthermore, possible biochemical mechanisms involved in this process were presented. NF200 immunostaining was used to quantify the yield of differentiated cells after exposure to CAMP. The addition of a PKA inhibitor and Ca(2+) blockers to the differentiation medium significantly reduced the yield of differentiated cells. Activation of CREB was also observed on MSCs during maturation. Na(+)-, K(+)-, and Ca(2+)-voltage-dependent currents were recorded from MSCs-derived cells. In contrast, significantly larger Na(+) currents, firing activity, and spontaneous synaptic currents were recorded from NSCs. Our results indicate that the initial neuronal differentiation of MSCs is induced by CAMP and seems to be dependent upon Ca(2+) and the PKA pathway. However, compared to fetal neural stem cells, adult mesenchymal counterparts are limited in their neurogenic potential. Despite the similar yield of neuronal cells, NSCs achieved a more mature functional state. Description of the underlying mechanisms that govern MSCs` differentiation toward a stable neuronal phenotype and their limitations provides a unique opportunity to enhance our understanding of stem cell plasticity. (C) 2009 Elsevier Inc. All rights reserved.

Dual origin of mesenchymal stem cells contributing to organ growth and repair

In many adult tissues, mesenchymal stem cells (MSCs) are closely associated with perivascular niches and coexpress many markers in common with pericytes. The ability of pericytes to act as MSCs, however, remains controversial. By using genetic lineage tracing, we show that some pericytes differentiate into specialized tooth mesenchyme-derived cells-odontoblasts-during tooth growth and in response to damage in vivo. As the pericyte-derived mesenchymal cell contribution to odontoblast differentiation does not account for all cell differentiation, we identify an additional source of cells with MSC-like properties that are stimulated to migrate toward areas of tissue damage and differentiate into odontoblasts. Thus, although pericytes are capable of acting as a source of MSCs and differentiating into cells of mesenchymal origin, they do so alongside other MSCs of a nonpericyte origin. This study identifies a dual origin of MSCs in a single tissue and suggests that the pericyte contribution to MSC-derived mesenchymal cells in any given tissue is variable and possibly dependent on the extent of the vascularity.

Mesenchymal stem cells secretome as a modulator of the neurogenic niche: Basic insights and therapeutic opportunities

Neural stem cells (NSCs) and mesenchymal stem cells (MSCs) share few characteristics apart from self-renewal and multipotency. In fact, the neurogenic and osteogenic stem cell niches derive from two distinct embryonary structures; while the later originates from the mesoderm, as all the connective tissues do, the first derives from the ectoderm. Therefore, it is highly unlikely that stem cells isolated from one niche could form terminally differentiated cells from the other. Additionally, these two niches are associated to tissues/systems (e.g., bone and central nervous system) that have markedly different needs and display diverse functions within the human body. Nevertheless they do share common features. For instance, the differentiation of both NSCs and MSCs is intimately associated with the bone morphogenetic protein family. Moreover, both NSCs and MSCs secrete a panel of common growth factors, such as nerve growth factor (NGF), glial derived neurotrophic factor (GDNF), and brain derived neurotrophic factor (BDNF), among others. But it is not the features they share but the interaction between them that seem most important, and worth exploring; namely, it has already been shown that there are mutually beneficially effects when these cell types are co-cultured in vitro. In fact the use of MSCs, and their secretome, become a strong candidate to be used as a therapeutic tool for CNS applications, namely by triggering the endogenous proliferation and differentiation of neural progenitors, among other mechanisms. Quite interestingly it was recently revealed that MSCs could be found in the human brain, in the vicinity of capillaries. In the present review we highlight how MSCs and NSCs in the neurogenic niches interact. Furthermore, we propose directions on this field and explore the future therapeutic possibilities that may arise from the combination/interaction of MSCs and NSCs.

Epigenetic features of human mesenchymal stem cells determine their permissiveness for induction of relevant transcriptional changes by SYT-SSX1.

BACKGROUND: A characteristic SYT-SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas, a malignant soft tissue tumor widely believed to originate from as yet unidentified pluripotent stem cells. The resulting fusion protein has no DNA binding motifs but possesses protein-protein interaction domains that are believed to mediate association with chromatin remodeling complexes. Despite recent advances in the identification of molecules that interact with SYT-SSX and with the corresponding wild type SYT and SSX proteins, the mechanisms whereby the SYT-SSX might contribute to neoplastic transformation remain unclear. Epigenetic deregulation has been suggested to be one possible mechanism. METHODOLOGY/PRINCIPAL FINDINGS: We addressed the effect of SYT/SSX expression on the transcriptome of four independent isolates of primary human bone marrow mesenchymal stem cells (hMSC). We observed transcriptional changes similar to the gene expression signature of synovial sarcoma, principally involving genes whose regulation is linked to epigenetic factors, including imprinted genes, genes with transcription start sites within a CpG island and chromatin related genes. Single population analysis revealed hMSC isolate-specific transcriptional changes involving genes that are important for biological functions of stem cells as well as genes that are considered to be molecular markers of synovial sarcoma including IGF2, EPHRINS, and BCL2. Methylation status analysis of sequences at the H19/IGF2 imprinted locus indicated that distinct epigenetic features characterize hMSC populations and condition the transcriptional effects of SYT-SSX expression. CONCLUSIONS/SIGNIFICANCE: Our observations suggest that epigenetic features may define the cellular microenvironment in which SYT-SSX displays its functional effects.

Cardiac repair with allogeneic mesenchymal stem cells after myocardial infarction.

Over the past decade, use of autologous bone marrow-derived mononuclear cells (BMCs) has proven to be safe in phase-I/II studies in patients with myocardial infarction (MI). Taken as a whole, results support a modest yet significant improvement in cardiac function in cell-treated patients. Skeletal myoblasts, adipose-derived stem cells, and bone marrow-derived mesenchymal stem cells (MSCs) have also been tested in clinical studies. MSCs expand rapidly in vitro and have a potential for multilineage differentiation. However, their regenerative capacity decreases with aging, limiting efficacy in old patients. Allogeneic MSCs offer several advantages over autologous BMCs; however, immune rejection of allogeneic cells remains a key issue. As human MSCs do not express the human leukocyte antigen (HLA) class II under normal conditions, and because they modulate T-cell-mediated responses, it has been proposed that allogeneic MSCs may escape immunosurveillance. However, recent data suggest that allogeneic MSCs may switch immune states in vivo to express HLA class II, present alloantigen and induce immune rejection. Allogeneic MSCs, unlike syngeneic ones, were eliminated from rat hearts by 5 weeks, with a loss of functional benefit. Allogeneic MSCs have also been tested in initial clinical studies in cardiology patients. Intravenous allogeneic MSC infusion has proven to be safe in a phase-I trial in patients with acute MI. Endoventricular allogeneic MSC injection has been associated with reduced adverse cardiac events in a phase-II trial in patients with chronic heart failure. The long-term safety and efficacy of allogeneic MSCs for cardiac repair remain to be established. Ongoing phase-II trials are addressing these issues.

Obstructive apneas induce early activation of mesenchymal stem cells and enhancement of endothelial wound healing

Background: The aim was to test the hypothesis that the blood serum of rats subjected to recurrent airway obstructions mimicking obstructive sleep apnea (OSA) induces early activation of bone marrow-derived mesenchymal stem cells (MSC) and enhancement of endothelial wound healing. Methods: We studied 30 control rats and 30 rats subjected to recurrent obstructive apneas (60 per hour, lasting 15 s each, for 5 h). The migration induced in MSC by apneic serum was measured by transwell assays. MSC-endothelial adhesion induced by apneic serum was assessed by incubating fluorescent-labelled MSC on monolayers of cultured endothelial cells from rat aorta. A wound healing assay was used to investigate the effect of apneic serum on endothelial repair. Results: Apneic serum showed significant increase in chemotaxis in MSC when compared with control serum: the normalized chemotaxis indices were 2.20 +- 0.58 (m +- SE) and 1.00 +- 0.26, respectively (p < 0.05). MSC adhesion to endothelial cells was greater (1.75 +- 0.14 -fold; p < 0.01) in apneic serum than in control serum. When compared with control serum, apneic serum significantly increased endothelial wound healing (2.01 +- 0.24 -fold; p < 0.05). Conclusions: The early increases induced by recurrent obstructive apneas in MSC migration, adhesion and endothelial repair suggest that these mechanisms play a role in the physiological response to the challenges associated to OSA.

Isolation and culture of umbilical vein mesenchymal stem cells

Bone marrow contains a population of stem cells that can support hematopoiesis and can differentiate into different cell lines including adipocytes, osteocytes, chondrocytes, myocytes, astrocytes, and tenocytes. These cells have been denoted mesenchymal stem cells. In the present study we isolated a cell population derived from the endothelium and subendothelium of the umbilical cord vein which possesses morphological, immunophenotypical and cell differentiation characteristics similar to those of mesenchymal stem cells isolated from bone marrow. The cells were isolated from three umbilical cords after treatment of the umbilical vein lumen with collagenase. The cell population isolated consisted of adherent cells with fibroblastoid morphology which, when properly stimulated, gave origin to adipocytes and osteocytes in culture. Immunophenotypically, this cell population was found to be positive for the CD29, CD13, CD44, CD49e, CD54, CD90 and HLA-class 1 markers and negative for CD45, CD14, glycophorin A, HLA-DR, CD51/61, CD106, and CD49d. The characteristics described are the same as those presented by bone marrow mesenchymal stem cells. Taken together, these findings indicate that the umbilical cord obtained from term deliveries is an important source of mesenchymal stem cells that could be used in cell therapy protocols.

The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells

Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

CTLA4 enhances the osteogenic differentiation of allogeneic human mesenchymal stem cells in a model of immune activation

Allogeneic mesenchymal stem cells (allo-MSCs) have recently garnered increasing interest for their broad clinical therapy applications. Despite this, many studies have shown that allo-MSCs are associated with a high rate of graft rejection unless immunosuppressive therapy is administered to control allo-immune responses. Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is a co-inhibitory molecule expressed on T cells that mediates the inhibition of T-cell function. Here, we investigated the osteogenic differentiation potency of allo-MSCs in an activated immune system that mimics the in vivo allo-MSC grafting microenvironment and explored the immunomodulatory role of the helper T cell receptorCTLA4 in this process. We found that MSC osteogenic differentiation was inhibited in the presence of the activated immune response and that overexpression of CTLA4 in allo-MSCs suppressed the immune response and promoted osteogenic differentiation. Our results support the application of CTLA4-overexpressing allo-MSCs in bone tissue engineering.

Injection of adult mouse mesenchymal stem cells into the developing chick inner ear

This series of experiments attempted to characterize the abilities of stem cells derived from bone marrow and adipose tissue to integrate into the sensory epithelium of the inner ear and to differentiate into hair cells or neural cell types.