Localization Of Mesenchymal Stem Cells Research Articles

Transplantation of MSCs Overexpressing HGF into a Rat Model of Liver Fibrosis

The aim of this study is to evaluate the effect of overexpressing human hepatocyte growth factor (HGF) for mesenchymal stem cells (MSCs) in liver fibrosis regeneration and magnetic resonance (MR) tracking of MSCs in rat liver. MSCs were transfected with ad-HGF/ad-green fluorescent protein (GFP) and labeled with superparamagnetic iron oxide (SPIO). The characteristics of SPIO-HGF/MSCs were investigated. Prussian blue staining for iron assessment was conducted in vitro and in vivo. SPIO-HGF/MSCs (group A) or SPIO-GFP/MSCs (group B) were transplanted into a rat model of liver fibrosis, and MR imaging of the rat liver was performed. The signal to noise ratio (SNR) and R2* (1/T2*) value were measured. Prussian blue staining was performed to detect the in vivo distribution of MSCs, and liver Ki67 immunohistochemistry (IHC) staining was studied. The serum levels of HGF, alanine aminotransferase (ALT) and hyaluronic acid (HA) were determined. The positive rate of HGF transfection was 93.17 % and the HGF/MSCs were labeled with SPIO successfully (97.80 ± 1.06 %). Labeling of MSCs with SPIO did not alter cell proliferation in vitro. The signal intensity of liver T2* WI images decreased on day 1 after cell transplantation and recovered to pre-transplantation level on day 15 (group A) and day 13 (group B). The SNR of group A were significantly lower than that of group B (P = 0.006), and the R2* values of group A were significantly higher than those of group B (P < 0.001). The R2* value had a significantly negative correlation with SNR. There were more Prussian blue-positive cells in of group A were more than in group B in vivo. The positive rate of Ki67 was 16.11 ± 2.13 %, and the serum level of ALT/HA was decreased in group A. HGF transfection improved MSCs localization in the liver and aided liver repair. The R2* value might be a feasible index in addition to SNR to track the SPIO-MSC transplantation in the liver.

Mesenchymal Stem Cells Reduce Colitis in Mice via Release of TSG6, Independently of Their Localization to the Intestine

Mesenchymal stem cells (MSCs) are pluripotent cells that can promote expansion of immune regulatory cells and might be developed for the treatment of immune disorders, including inflammatory bowel diseases. MSCs were reported to reduce colitis in mice; we investigated whether MSC localization to the intestine and production of paracrine factors, including tumor necrosis factor-induced protein 6 (TSG6), were required for these effects. MSCs were isolated from bone marrow (BM-MSCs) of 4- to 6-week-old C57BL/6, C57BL/6-green fluorescent protein, or Balb/c Tsg6-/- male mice. Colitis was induced by ad libitum administration of dextran sulfate sodium for 10 days; after 5 days the mice were given intraperitoneal injections of BM-MSCs or saline (controls). Blood samples and intestinal tissues were collected 24, 48, 96, and 120 hours later; histologic and flow cytometry analyses were performed. Injection of BM-MSCs reduced colitis in mice, increasing body weight and reducing markers of intestinal inflammation, compared with control mice. However, fewer than 1% of MSCs reached the inflamed colon. Most of the BM-MSCs formed aggregates in the peritoneal cavity. The aggregates contained macrophages and B and T cells, and produced immune-regulatory molecules including FOXP3, interleukin (IL)10, transforming growth factor-β, arginase type II, chemokine (C-C motif) ligand 22 (CCL22), heme oxygenase-1, and TSG6. Serum from mice given BM-MSCs, compared with mice given saline, had increased levels of TSG6. Injection of TSG6 reduced the severity of colitis in mice, along with the numbers of CD45+ cells, neutrophils and metalloproteinase activity in the mucosa, while increasing the percentage of Foxp3CD45+ cells. TSG6 injection also promoted the expansion of regulatory macrophages that expressed IL10 and inducible nitric oxide synthase, and reduced serum levels of interferon-γ, IL6, and tumor necrosis factor. Tsg6-/- MSCs did not suppress the mucosal inflammatory response in mice with colitis. BM-MSCs injected into mice with colitis do not localize to the intestine but instead form aggregates in the peritoneum where they produce immunoregulatory molecules, including TSG6, that reduce intestinal inflammation. TSG6 is sufficient to reduce intestinal inflammation in mice with colitis.

Degradable hydrogels for spatiotemporal control of mesenchymal stem cells localized at decellularized bone allografts

The transplantation of cells, such as mesenchymal stem cells (MSCs), has numerous applications in the field of regenerative medicine. For cell transplantation strategies to be successful therapeutically, cellular localization and persistence must be controlled to maximize cell-mediated contributions to healing. Herein, we demonstrate that hydrolytic degradation of poly(ethylene glycol) (PEG) hydrogels can be used to spatiotemporally control encapsulated MSC localization to decellularized bone allografts, both in vitro and in vivo. By altering the number of hydrolytically degradable lactide repeat units within PEG-d,l-lactide-methacrylate macromers, a series of hydrogels was synthesized that degraded over ∼1, 2 and 3weeks. MSCs were encapsulated within these hydrogels formed around decellularized bone allografts, and non-invasive, longitudinal fluorescence imaging was used to track cell persistence both in vitro and in vivo. Spatiotemporal localization of MSCs to the exterior of bone allograft surfaces was similar to in vitro hydrogel degradation kinetics despite hydrogel mesh sizes being ∼2–3 orders of magnitude smaller than MSC size throughout the degradation process. Thus, localized, cell-mediated degradation and MSC migration from the hydrogels are suspected, particularly as ∼10% of the total transplanted MSC population was shown to persist in close proximity (within ∼650μm) to grafts 7weeks after complete hydrogel degradation. This work demonstrates the therapeutic utility of PEG-based hydrogels for controlling spatiotemporal cell transplantation for a myriad of regenerative medicine strategies.

Stromal Cell-Derived Factor 1α-Stimulated Mesenchymal Stem Cells Confer Enhanced Protection Against Light-Induced Retinal Degeneration in Rats

Purpose: Mesenchymal stem cells (MSCs) are currently considered to be modulators of repair in various tissues. After MSC transplant, photoreceptor rescue has been demonstrated in models of retinal degeneration. Herein, we evaluate the roles of MSCs in modulating the host reaction and photoreceptor preservation in rats suffering from light-induced retinal degeneration.Methods: Unstimulated and stromal cell-derived factor 1α (SDF-1α)-stimulated MSCs were intravenously transplanted into light-injured rats. Their photoreceptor rescue effect was compared with untreated light-injured rats and light-injured rats received only medium injection. Ciliary neurotrophic factor (CNTF) and glial fibrillary acidic protein (GFAP) expression was identified to assess host reaction post-transplantation. Retinal localization and integration of MSCs were determined by green fluorescence protein labeling.Results: MSCs were able to migrate and integrate into the host retina, and significantly inhibited retinal cell death. CNTF and GFAP were induced upregluation after MSC injection. SDF-1α stimulation elicited superior effects in both MSC migration and the inhibition of apoptosis. CNTF and GFAP expression in host retinas that received stimulated MSCs were stronger than in retinas that received unstimulated MSCs.Conclusions: Systemic administration of MSCs exerts a protective effect against light-induced retinal degeneration, and upregulates neurotrophin expression in the host retina. MSCs can be stimulated to enhance the therapeutic effect.

Immunohistochemical localization of mesenchymal stem cells in ossified human spinal ligaments

Mesenchymal stem cells (MSCs) have been isolated from various tissues and used for elucidating the pathogenesis of numerous diseases. In our previous in vitro study, we showed the existence of MSCs in human spinal ligaments and hypothesized that these MSCs contributed to the pathogenesis of ossification of spinal ligaments. The purpose of this study was to use immunohistochemical techniques to analyze the localization of MSCs in ossified human spinal ligaments in situ. Ossified (OLF) or non-ossified ligamentum flavum (non-OLF) samples from the thoracic vertebra were obtained from patients who had undergone posterior spinal surgery. Serial sections were prepared from paraffin-embedded samples, and double immunofluorescence staining was performed using antibodies against markers for MSCs (CD73, CD90 and CD105), endothelial cells (CD31), pericytes (α-smooth muscle actin), and chondrocytes (S100). Immunolocalization of MSCs was observed in the perivascular area and collagenous matrix in spinal ligaments. Markers for MSCs and pericytes were co-expressed in the perivascular area. Compared with non-OLF, OLF had a large amount of neovascularization in the fragmented ligament matrix, and a high accumulation of MSCs around blood vessels. The prevalence of MSCs in OLF within collagenous matrix was significantly higher than that in non-OLF. Chondrocytes near the ossification front in OLF also presented expression of MSC markers. MSCs may contribute to the ectopic ossification process of OLF through endochondral ossification.

Mesenchymal stem cells administered in the early phase of tumorigenesis inhibit colorectal tumor development in rats

To investigate the differences between the effects of mesenchymal stem cells (MSCs) administered in the early and late phases of tumorigenesis, MSCs were isolated from bone marrow and colorectal tumors were produced by exposing 7-week-old F344 rats to 1,2-dimethylhydrazine and dextran sulfate sodium. We evaluated tumor number and volume (week 25), MSC localization, number of aberrant crypt foci (ACF), transforming growth factor (TGF)-β1 protein levels in the rectum after administration of MSCs (week 5 or 15), and the effects of MSC-conditioned medium on ACL15 cell proliferation. Administered MSCs labeled with PKH26 were observed in the rectum. Administered MSCs in the early phase (week 5) before tumor occurrence (week 12) significantly decreased tumor number and volume (1.5 vs 4 and 21 mm3 vs 170 mm3; p<0.01), but not administered MSCs in the late phase (week 15). Administered MSCs in the early phase reduced ACF number on days 14 and 35 (1.9 vs 4.1 and 3.7 vs 7.3; p<0.01). Rectal TGF-β1 increased 1.3 fold on day 3, and MSC-conditioned medium containing TGF-β1 abundantly inhibited ACL15 cell proliferation. MSCs administered in the early phase but not late phase inhibited colorectal tumor development in a rat model.

Microfluidic artificial “vessels” for dynamic mechanical stimulation of mesenchymal stem cells

Cells in the cardiovascular system are constantly exposed to complex mechanical stimulation due to the pulsatile nature of blood flow and the haemodynamic forces that are key to the regulation of vascular development, remodeling and pathophysiology. Mechanical stretch can also modulate the differentiation of stem cells toward vascular cell lineages (i.e., vascular smooth muscle cells), and represent a critical factor in vascular tissue engineering. Here we report on the development of a microchip platform that can emulate several key aspects of the vascular mechanical environment, such as cyclic stimulation and circumferential strain. This chip consists of an array of microfluidic channels with widths ranging from 20 to 500 micrometers. These channels are covered by suspended deformable membranes, on which cells are cultured and stimulated by cyclic circumferential strain of up to 20% via hydrodynamic actuation of the fluid in the microfluidic channels, thereby mimicking the biomechanical conditions of small blood vessels. We show that human mesenchymal stem cells (MSCs) can be cultured and continuously stimulated by cyclic stretch over a period of 7 days with no evidence of device fatigue or performance degradation. We observed localization and alignment of MSCs when mechanical stretch is larger than 10%, indicating the importance of mechanical stimulation in modulating cellular behavior. We further demonstrated simultaneous detection of proteins in multiple signaling pathways, including SMAD1/SMAD2 and canonical Wnt/β-catenin. This microchip represents a generic and versatile platform for high-throughput and rapid screening of cellular responses, including signal transduction cascades, in response to mechanical cues. The system emulates the physiological conditions of blood vessels and other tissues that are subject to cyclic strain, and may have a wide range of applications in the fields of stem cell mechanobiology, vascular tissue engineering, and other areas of regenerative medicine.

Intravitreal delivery of mesenchymal stem cells loaded onto hydrogel affects the regulatory expression of endogenous NGF and BDNF in ischemic rat retina

The utility of a biodegradable hyaluronic acid (HyA)-based hydrogel as a tissue scaffold for intravitreal carriage of mesenchymal stem cells (MSCs) into the retina was tested. A rat model of retinal ischemia-reperfusion (IR) injury was used. DiI-labeled MSCs from three passages were loaded onto hydrogel and injected into the vitreous body of experimental and control eyeballs 1 week after IR. The neutral hydrogel was also modified to a basic pH. A control carrier, 0.01 M phosphate-buffered saline (pH 7.2; PBS) was compared. Retinal tissues were prepared 1 week and 2 weeks after MSCs application. MSCs localization and effects were evaluated on immunostained retinal preparations with confocal microscopy. MSCs localization was apparent in the retinas loaded onto hydrogels, adhering tightly to the inner limiting membrane, whereas those in PBS floated in the vitreous body. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were expressed in the end feet of Muller cells in the normal retina. NGF expression was slightly reduced 2 weeks IR, had expanded into the proximal processes 3 weeks IR, but it appeared reversely 1 week and 2 weeks after MSCs application, respectively. BDNF expression was higher in the ischemic and MSCs-treated ischemic retinas than in the normal and MSCs-treated control retinas, respectively. These findings demonstrate that HyA-based hydrogel is an efficient vehicle for intravitreal MSC transplantation into the retina, and that MSCs thus transplanted induce Muller glial cells to produce growth factors concerned with the survival of retinal ganglion cells.

Enhancing the Mesenchymal Stem Cell Therapeutic Response: Cell Localization and Support for Cartilage Repair

Articular cartilage is a complex, multilayered biological composite material, comprised of chondrocytes encapsulated in a water-based glycosaminoglycan matrix reinforced with collagen fibers. Once damaged by osteoarthritis or traumatic injury, this aneural, avascular tissue has little self-repair capacity. Over the last 20 years, cell therapies and tissue-engineering strategies have shown significant promise for the repair or regeneration of damaged cartilage. In particular, mesenchymal stem cells (MSCs) have great potential owing to their ability to create a reparative environment. Despite the fact that there have been great strides in the design and development of three-dimensional scaffolds, there is an upper limit to the number of viable cells that can be delivered using current approaches. To this end, this review examines current strategies for optimizing MSC localization, evaluates their limitations, and looks to other technologies to devise a combinatorial strategy for the creation of an MSC-seeded composite structure that addresses both the mechanical and biological property requirements for enhanced cartilage repair.

Abstract 403: Tracking the Migration of Porcine Mesenchymal Stem Cells with the MRI Contrast Agent Ferex in an AAA Model

Introduction: Mesenchymal stem cells (MSC) are being investigated in porcine abdominal aortic aneurysm (PAAA) models for their repair potential. This study uses MSCs labeled with the MRI contrast agent Ferex to non-invasively evaluate MSC migration in-vivo. Methods: MSCs from 6 pigs were isolated from bone marrow via Ficoll Paque separation and expanded in culture. Using a Lentiviral vector, MSC from all 6 pigs were transfected with green florescent protein (GFP). MSCs from 4 of these pigs were also labeled with 200μg/ml Ferex using Poly-L-Lysine and then analyzed for Ferex uptake and viability. Preservation of the MSC phenotype was confirmed using flow cytometry by detecting positive CD90 and negative CD45 and CD117. Transmission electron microscopy established that Ferex localized to lysosomes. MSCs were then injected into the adventitia of the PAAA. In-vivo MRI was performed using multiple echo gradient echo sequences. Effective transverse relaxation times (T2* values) were calculated on a pixel-by-pixel basis as a function of time post cell transplantation. Results: Ferex labeled MSCs were visible post transplantation at 4, 11, 15 and 21 days using MRI. The MRI signal void (decreased T2* values) correlated with the presence of Ferex within the PAAA. This signal loss progressively expanded circumferentially at each study interval representing cellular movement. MSC migration and localization were confirmed with GFP visualization on fluorescence microscopy and immunohistochemistry. In-vivo MRI signals also correlate with iron deposition on Perl’s stain. Conclusion: Ferex can be used as an in-vivo tracking agent of MSCs in PAAA models.

Recent progress toward understanding the physiological function of bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent cells that are being clinically explored as regenerative therapeutics. Cultured MSCs secrete various modulatory factors, which contribute to the immunosuppressive effects of transplanted MSCs as a therapy. Although the in vitro phenotype of MSCs has been well characterized, identification of MSCs in vivo is made difficult by the lack of specific markers. Current advances in murine MSC research provide valuable tools for studying the localization and function of MSCs in vivo. Recent findings suggest that MSCs exert diverse functions depending on tissue context and physiological conditions. This review focuses on bone marrow MSCs and their roles in haematopoiesis and immune responses.

Macrophage-Associated Mesenchymal Stem Cells Assume an Activated, Migratory, Pro-Inflammatory Phenotype with Increased IL-6 and CXCL10 Secretion

Mesenchymal stem cells (MSCs) exhibit tropism for sites of tissue injury and tumors. However, the influence of the microenvironment on MSC phenotype and localization remains incompletely characterized. In this study, we begin to define a macrophage-induced MSC phenotype. These MSCs secrete interleukin-6 (IL-6), CCL5, and interferon gamma-induced protein-10 (CXCL10) and exhibit increased mobility in response to multiple soluble factors produced by macrophages including IL-8, CCL2, and CCL5. The pro-migratory phenotype is dependent on activation of a c-Jun N-terminal kinase (JNK) pathway. This work begins to identify the influence of macrophages on MSC biology. These interactions are likely to play an important role in the tissue inflammatory response and may provide insight into the migratory potential of MSCs in inflammation and tissue injury.

Localization of mesenchymal stem cells grafted with a hyaluronan-based scaffold in the infarcted heart

BackgroundPermanence of grafted stem cells in the infarcted myocardial area has been suggested to be favored by tissue engineering strategies, including the application of a scaffold as a cell support. However, an estimation of how many cells remain localized in the site of transplantation has never been done. The aim of this work was to investigate the localization of mesenchymal stem cells (MSCs) grafted with a well cell-adhesive polymer in the scar region of the infarcted heart. Materials and methodsRat MSCs were engineered in a hyaluronan-based scaffold (HYAFF®11) for 3 wk. The hearts of donor rats were also explanted, subjected to coronary artery ligation, and grafted into the abdomen of syngeneic rats. Two wk after coronary ligation a small dish of the HYAFF®11/MSC construct was introduced into a pouch created in the ventricular wall of the infarct area and left for 2 wk. ResultsUnder ex vivo conditions, MSCs tightly adhered to the hyaluronan fibers and secreted abundant extracellular matrix. In contrast, HYAFF®11 was not more surrounded by the engrafted MSCs 2 wk after construct transplantation. Most MSCs migrated near the border zone of the infarcted area close to the coronary vessels. Moreover, the infarcted region of the heart was enriched in capillaries and the degree of fibrosis was attenuated. ConclusionsTwo wk after transplantation most MSCs grafted in the infarcted myocardium with HYAFF®11 had left the scaffold and moved to the border zone. Nevertheless, this treatment increased the myocardial vascularization and reduced the degree of fibrosis in the scar area.

Mesenchymal stem cells are short-lived and do not migrate beyond the lungs after intravenous infusion

Mesenchymal stem cells (MSC) are under investigation as a therapy for a variety of disorders. Although animal models show long term regenerative and immunomodulatory effects of MSC, the fate of MSC after infusion remains to be elucidated. In the present study the localization and viability of MSC was examined by isolation and re-culture of intravenously infused MSC. C57BL/6 MSC (500,000) constitutively expressing DsRed-fluorescent protein and radioactively labeled with Cr-51 were infused via the tail vein in wild-type C57BL/6 mice. After 5 min, 1, 24, or 72 h, mice were sacrificed and blood, lungs, liver, spleen, kidneys, and bone marrow removed. One hour after MSC infusion the majority of Cr-51 was found in the lungs, whereas after 24 h Cr-51 was mainly found in the liver. Tissue cultures demonstrated that viable donor MSC were present in the lungs up to 24 h after infusion, after which they disappeared. No viable MSC were found in the other organs examined at any time. The induction of ischemia-reperfusion injury in the liver did not trigger the migration of viable MSC to the liver. These results demonstrate that MSC are short-lived after i.v. infusion and that viable MSC do not pass the lungs. Cell debris may be transported to the liver. Long term immunomodulatory and regenerative effects of infused MSC must therefore be mediated via other cell types.

Mesenchymal stem cell isolation and characterization from human spinal ligaments

Mesenchymal stem cells (MSCs) have a fibroblast-like morphology, multilineage potential, long-term viability and capacity for self-renewal. While several articles describe isolating MSCs from various human tissues, there are no reports of isolating MSCs from human spinal ligaments, and their localization in situ. If MSCs are found in human spinal ligaments, they could be used to investigate hypertrophy or ossification of spinal ligaments. To isolate and characterize MSCs from human spinal ligaments, spinal ligaments were harvested aseptically from eight patients during surgery for lumbar spinal canal stenosis and ossification of the posterior longitudinal ligament. After collagenase digestion, nucleated cells were seeded at an appropriate density to avoid colony-to-colony contact. Cells were cultured in osteogenic, adipogenic or chondrogenic media to evaluate their multilineage differentiation potential. Immunophenotypic analysis of cell surface markers was performed by flow cytometry. Spinal ligaments were processed for immunostaining using MSC-related antibodies. Cells from human spinal ligaments could be extensively expanded with limited senescence. They were able to differentiate into osteogenic, adipogenic or chondrogenic cells. Flow cytometry revealed that their phenotypic characteristics met the minimum criteria of MSCs. Immunohistochemistry revealed the localization of CD90-positive cells in the collagenous matrix of the ligament, and in adjacent small blood vessels. We isolated and expanded MSCs from human spinal ligaments and demonstrated localization of MSCs in spinal ligaments. These cells may play an indispensable role in elucidating the pathogenesis of numerous spinal diseases.

Systemically Transplanted Human Bone Marrow Mesenchymal Stem Cells Primarily Traffic to Mesenteric Lymph Nodes

AbstractAbstract 2580Intravenously administered mesenchymal stem cells (MSCs) are trapped in pulmonary vascular bed and only few MSCs home to bone or other tissues in physiological or pathological conditions. Following intracardiac injection MSCs pass the lung barrier but their homing to bone and tissue localization is uncertain. The aim of the study was to investigate trafficking and exact localization of human MSCs following intracardiac injection into unchallenged mice and a xenograft bone tumor model. MSCs were isolated from human fetal bones (ABR Inc, Alameda CA) and expanded in DMEM-LG medium supplemented with 10% FBS. Global gene expression profiling revealed that the cultured MSCs were devoid of hematopoietic cells and expressed typical mesenchymal markers such as CD166, CD146 and CD90. We have previously shown that these MSCs are capable of differentiation into osteoblasts and adipocytes and retain their differentiation potential after multiple passages (Haematologica 2006). The MSCs were transduced with a luciferase/GFP reporter in a lentiviral vector and were maximally passaged 8 times before used in vivo. Detection of MSCs in mice was determined by live-animal imaging and ex vivo bioluminescence activity using the IVIS system, by microscopic examination of GFP-expressing cells and by immunohistochemistry for GFP. MSCs (1×106 cells/mouse) were intracardiacly injected into unconditioned SCID mice (n=8) using Dovetail Slide Micromanipulator that ensures accurate injection. Following 2 or 7 days after MSC injection to SCID mice, live-animal imaging revealed bioluminescence activity mainly in the mice abdomen but not bone, while ex vivo examination detected MSCs in various abdominal organs, primarily in reproductive organs, intestine and pancreas. Careful microscopic examination revealed localization of MSCs in draining lymph nodes attached to these organs by connective tissue. Immunohistochemistry showed GFP-expressing MSCs in the adjacent mesenteric lymph nodes but not within the organs. To confirm our findings, MSCs were intracardially injected into C57BL6 mice (n=6) that harbor functional lymph nodes. Evans blue dye which is known to accumulate in and identify lymph nodes, was injected into the rear footpad or lateral tail base of the mice, 3 hours after MSC injection and 30 minutes prior to bioluminescence and florescence analyses. The Evans blue dye and GFP positivity were co-localized, indicating specific trafficking of MSCs to lymph nodes. Culturing of the dissected lymph nodes resulted in release of GFP-expressing MSCs which regained their in vitro morphology. For testing MSCs trafficking in a xenograft model, we used our SCID-rab system constructed by implanting a 4-weeks old rabbit bone into which human myeloma cells were directly injected (Leukemia 2004; Blood 2007). In this model myeloma cells grow restrictively in the implanted bone. MSCs injected intracardiacly into SCID-rab mice were mostly found in mesenteric lymph nodes but were also detected in the myelomatous bone 72 hours after MSCs injection, validating the ability of tumor cells to attract MSCs and that these MSCs are capable of transmigration. We conclude that MSCs primarily traffic to draining lymph nodes, partially explaining their in vivo immunomodulatory activity, and that understanding the mechanism by which MSCs traffic to lymph nodes may help develop approaches to shift their homing to desired organs. Disclosures:No relevant conflicts of interest to declare.

Isolation and Perivascular Localization of Mesenchymal Stem Cells From Mouse Brain

Although originally isolated from the bone marrow, mesenchymal stem cells (MSCs) have recently been detected in other tissues. However, little is known about MSCs in the brain. To determine the extent to which cells with the features of MSCs exist in normal brain tissue and to determine the location of these cells in the brain. Single-cell suspensions from mouse brains were cultured according to the same methods used for culturing bone marrow-derived MSCs (BM-MSCs). These brain-derived cells were analyzed by fluorescence-activated cell sorting for surface markers associated with BM-MSCs (stem cell antigen 1 [Sca-1+], CD9+, CD45-, CD11b-, and CD31-). Brain-derived cells were exposed to mesenchymal differentiation conditions. To determine the locations of these cells within the brain, sections of normal brains were analyzed by immunostaining for Sca-1, CD31, and nerve/glial antigen 2. Cells morphologically similar to mouse BM-MSCs were identified and called brain-derived MSCs (Br-MSCs). Fluorescence-activated cell sorting indicated that the isolated cells had a surface marker profile similar to BM-MSCs, ie, Sca-1V+, CD9+, CD45-, and CD11b-. Like BM-MSCs, Br-MSCs were capable of differentiation into adipocytes, osteocytes, and chondrocytes. Immunostaining indicated that Sca-1+ Br-MSCs are located around blood vessels and may represent progenitor cells that serve as a source of mesenchymal elements (eg, pericytes) within the brain. Our results indicate that cells similar to BM-MSCs exist in the brain. These Br-MSCs appear to be located within the vascular niche and may provide the mesenchymal elements of this niche. Because MSCs may be part of the cellular response to tissue injury, Br-MSCs may represent targets in the therapy of pathological processes such as stroke, trauma, and tumorigenesis.

Enhancement of the survival of engrafted mesenchymal stem cells in the ischemic heart by TNFR gene transfectionThis paper is one of a selection of papers published in this special issue entitled “Second International Symposium on Recent Advances in Basic, Clinical, and Social Medicine” and has undergone the Journal's usual peer review process.

Autologous or allogeneic mesenchymal stem cells (MSCs) have been used as one of the potential cell sources for cellular cardiomyoplasty. The adverse microenvironment in acute myocardial infarction, however, is considered a deleterious factor for MSC transplantation and cell survival. Tumor necrosis factor (TNF)-alpha is an inflammatory mediator produced during ischemia that may affect the survival of MSCs. In this study, we investigated the enhancement of MSC survival by transfecting cells with the TNF receptor (TNFR) gene, leading to the overproduction of TNFR and the binding of TNF-alpha. Rats with acute myocardial infarction, induced by the occlusion of the left coronary artery, were transplanted with MSC or MSC-TNFR. After 2 weeks of acute myocardial infarction, cardiac function was assessed. Engrafted MSC survival and localization of TNF-alpha protein in infarction myocardium were evaluated. The levels of TNF-alpha and TNFR in the infarction zone were assessed. The results indicate that MSC-TNFR transplantation (1) improved left ventricular function; (2) enhanced engrafted MSC survival in the infarcted myocardium; (3) attenuated the level of TNF-alpha in serum and cardiac tissue; and (4) increased TNFR protein production in the infarcted myocardium. Our results showed that MSC modified by the TNFR gene improved cell viability and thereby has the potential to improve the efficiency of MSC transplantation therapy in the ischemic heart.

Isolation of Mesenchymal Stem Cells with Neurogenic Potential from the Mesoderm of the Amniotic Membrane

The amniotic membrane has been clinically applied as a therapeutic material in wound covering and corneal surface reconstruction. Recently, mesenchymal stem cells (MSCs) have been isolated from the placenta, specifically from the amniotic membrane. However, the localization of MSCs in the amniotic membrane has not been determined. In this study, term placenta was collected, and we performed immunohistochemical staining techniques to identify and localize MSCs in the mesoderm of the amniotic membrane in situ with MSC antibodies, including CD90 and CD105. We further directly cultured and characterized MSCs from the amniotic membrane mesoderm (AMSCs). The AMSCs were easily isolated and represented a homogenous fibroblastic morphology at early passages. In addition to MSC surface markers, AMSCs expressed Sox2, Oct-4 and Nanog. AMSCs could be induced into osteocytes, adipocytes and chondrocytes in vitro and show immunosuppressive effects on T-cell proliferation. Under appropriate conditions, AMSCs could differentiate into neuronal-like cells, which were identified by neuronal-specific markers and their ability to secrete dopamine. This study reveals that AMSCs provide a promising source for stem cell studies and also extend the clinical potential of the amniotic membrane in the field of regenerative medicine.

An optical imaging method to monitor stem cell migration in a model of immune-mediated arthritis

The objective of this work is to establish an optical imaging technique that would enable monitoring of the integration of mesenchymal stem cells (MSC) in arthritic joints. Our approach is based on first developing a labeling technique of MSC with the fluorescent dye DiD followed by tracking the cell migration kinetics from the spatial distribution of the DiD fluorescence in optical images (OI). The experimental approach involves first the in vitro OI of MSC labeled with DiD accompanied by fluorescence microscopy measurements to establish localization of the signal within the cells. Thereafter, DiD-labeled MSC were injected into polyarthritic, athymic rats and the signal localization within the experimental animals was monitored over several days. The experimental results indicate that DiD integrated into the cell membrane. DiD-labeled MSC localization in the arthritic ankle joints was observed with OI indicating that this method can be applied to monitor MSC in arthritic joints.