Isolation Of Mesenchymal Stem Cells Research Articles

Isolation and Characterization of Mesenchymal Stem Cells from Chicken Liver

Hepatic mesenchymal stem cells (HMSCs) are multipotent stem cells that is a vital part of the regeneration of hepatocytes after injury. In this study, HMSCs were isolated in embryonic livers from of 12-day-old chick embryo using collagenase, and the primary HMSCs were sub-cultured to passage. The protein markers of HMSCs, namely CD71, CD29 and CD44, were tested with immunofluorescence and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The proliferation of HMSCs in different passages was detected using growth curve, which shown a typically sigmoidal. And then, the pluripotent of HMSCs was analyzed, the results showed that HMSCs could directly induce to differentiate into neural-like cells, adipocytes, and osteoblasts. Our data illustrated that the chick HMSCs have same characteristics to those obtained from other species. The capacity of these cells for multilineage differentiation shows promise for many potential applications.

Autologous Adipose Tissue-Derived Mesenchymal Stem Cells Introduced by Biliary Stents or Local Immersion in Porcine Bile Duct Anastomoses.

Biliary complications (strictures and leaks) represent major limitations in living donor liver transplantation. Mesenchymal stem cells (MSCs) are a promising modality to prevent biliary complications because of immunosuppressive and angiogenic properties. Our goal was to evaluate the safety of adipose-derived MSC delivery to biliary anastomoses in a porcine model. Secondary objectives were defining the optimal method of delivery (intraluminal versus extraluminal) and to investigate MSC engraftment, angiogenesis, and fibrosis. Pigs were divided into 3 groups. Animals underwent adipose collection, MSC isolation, and expansion. Two weeks later, animals underwent bile duct transection, reanastomosis, and stent insertion. Group 1 received plastic stents wrapped in unseeded Vicryl mesh. Group 2 received stents wrapped in MSC-seeded mesh. Group 3 received unwrapped stents with the anastomosis immersed in an MSC suspension. Animals were killed 1 month after stent insertion when cholangiograms and biliary tissue were obtained. Serum was collected for liver biochemistries. Tissue was used for hematoxylin-eosin and trichrome staining and immunohistochemistry for MSC markers (CD44 and CD34) and for a marker of neoangiogenesis (CD31). There were no intraoperative complications. One pig died on postoperative day 3 due to acute cholangitis. All others recovered without complications. Cholangiography demonstrated no biliary leaks and minimal luminal narrowing. Surviving animals exhibited no symptoms, abnormal liver biochemistries, or clinically significant biliary stricturing. Group 3 showed significantly greater CD44 and CD34 staining, indicating MSC engraftment. Fibrosis was reduced at the anastomotic site in group 3 based on trichrome stain. CD31 staining of group 3 was more pronounced, supporting enhanced neoangiogenesis. In conclusion, adipose-derived MSCs were safely applied to biliary anastomoses. MSCs were locally engrafted within the bile duct and may have beneficial effects in terms of fibrosis and angiogenesis.

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs - including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Effects of VEGF-Notch Signaling Pathway on Proliferation and Apoptosis of Bone Marrow MSC in Patients with Aplastic Anemia

To evaluate the regulation of VEGF-Notch signaling pathway on proliferation and apoptosis of mesenchymal stem cells (MSC) in the patients with aplastic anemia (AA). The bone marrow specimens of AA patients were collected for isolation and identification of BM MSC. Westen blot was used to detect the expression of VEGF-Notch signaling pathway-related proteins (VEGF, VEGFR, Notch 1, Jagged 1, Delta-like 1 and Hes1). The VEGF (100 ng/ml) and DAPT (γ-secretase inhibitor, 10 μmol/L) were respectively added into MSC culture system in oder to activate and inhibit the signaling transduction of VEGF-Notch in BM MSC. The proliferation, apoptosis and cell cycle of MSC in AA patients were detected by CCK8 assay and flow cyfometry. The adipogenic differentiation of BM MSC was detected by oil red O staining. The VEGF-Notch signaling pathway was significantly inhibited in AA BM tissues and AA MSC (P＜0.05) detected by Western blot. The intervention of VEGF and DAPT significantly activated and inhibited VEGF-Notch signaling in AA MSC, respectively. CCK8 assay showed that VEGF intervention significantly promoted the proliferation of MSC in AA patients (P＜0.05). Flow cytometry showed that VEGF significantly inhibited apoptosis of MSCs by blocking S phase cells (P＜0.05). The activation of VEGF-Notch can restore the proliferation function of MSC in AA patients.

Effects of VEGF-Notch Signaling Pathway on Proliferation and Apoptosis of Bone Marrow MSC in Patients with Aplastic Anemia

To evaluate the regulation of VEGF-Notch signaling pathway on proliferation and apoptosis of mesenchymal stem cells (MSC) in the patients with aplastic anemia (AA). The bone marrow specimens of AA patients were collected for isolation and identification of BM MSC. Westen blot was used to detect the expression of VEGF-Notch signaling pathway-related proteins (VEGF, VEGFR, Notch 1, Jagged 1, Delta-like 1 and Hes1). The VEGF (100 ng/ml) and DAPT (γ-secretase inhibitor, 10 μmol/L) were respectively added into MSC culture system in oder to activate and inhibit the signaling transduction of VEGF-Notch in BM MSC. The proliferation, apoptosis and cell cycle of MSC in AA patients were detected by CCK8 assay and flow cyfometry. The adipogenic differentiation of BM MSC was detected by oil red O staining. The VEGF-Notch signaling pathway was significantly inhibited in AA BM tissues and AA MSC (P＜0.05) detected by Western blot. The intervention of VEGF and DAPT significantly activated and inhibited VEGF-Notch signaling in AA MSC, respectively. CCK8 assay showed that VEGF intervention significantly promoted the proliferation of MSC in AA patients (P＜0.05). Flow cytometry showed that VEGF significantly inhibited apoptosis of MSCs by blocking S phase cells (P＜0.05). The activation of VEGF-Notch can restore the proliferation function of MSC in AA patients.

Identification of Functionally Distinct Mx1+αSMA+ Periosteal Skeletal Stem Cells.

The periosteum is critical for bone maintenance and healing. However, the invivo identity and specific regulatory mechanisms of adult periosteum-resident skeletal stem cells are unknown. Here, we report animal models that selectively and durably label postnatal Mx1+αSMA+ periosteal stem cells (P-SSCs) and establish that P-SSCs are a long-term repopulating, functionally distinct SSC subset responsible forlifelong generation of periosteal osteoblasts. P-SSCs rapidly migrate toward an injury site, supply osteoblasts and chondrocytes, and recover new periosteum. Notably, P-SSCs specifically express CCL5 receptors, CCR3 and CCR5. Real-time intravital imaging revealed that the treatment with CCL5 induces P-SSC migration invivo and bone healing, while CCL5/CCR5 deletion, CCR5 inhibition, or local P-SSC ablation reduces osteoblast number and delays bone healing. Human periosteal cells express CCR5 and undergo CCL5-mediated migration. Thus, the adult periosteum maintains genetically distinct SSC subsets with a CCL5-dependent migratory mechanism required for bone maintenance and injury repair.

Isolation and characterization of mesenchymal stem cells derived from type 2 diabetes – research conducted in the Regenerative Medicine and Stem Cell Biology Lab (Ohneda Lab)

Stem cells are one of the leading methods through which we will gain the next generation of medical treatments. Given their ability to develop into almost any cell type, the medical applications are endless. Since the methods through which to reprogramme these master progenitors were established, many tests and clinical trials have taken place. However, as is often the case in biology, the picture is more complicated than simply reprogramming these cells with a cocktail of proteins. Stem cell expert Professor Osamu Ohneda is a Principal Investigator at the University of Tsukuba, Japan. 'Our focus is to achieve autologous transplantation whereby, mesenchymal stem cells are taken from the patient, encouraged to grow and reprogrammed before transplantation into the patient,' he observes. This process would be a crucial step in the personalisation of medicine, however, the more this process has been tested, the clearer it has become that additional variables are in play. 'There have been issues with nearly every step of the process after the acquirement of the raw mesenchymal stem cells (MSCs) necessary,' Ohneda highlights. 'The MSCs don't always respond as expected to growth stimulants and differentiation cues and transplants don't always take.' More in depth research is needed to overcome these issues and turn stem cell's potential into a reality. Investigating and solving these issues is the work of Ohneda and his research team, including Assistant Professor Vuong Cat Khanh and Assistant Professor Toshiharu Yamashita.

Functional comparison of beating cardiomyocytes differentiated from umbilical cord-derived mesenchymal/stromal stem cells and human foreskin-derived induced pluripotent stem cells.

In recent years, stem cell-based therapies shown to have promising effects on the clinical management of ischemic heart disease. Moreover, stem cells differentiation into cardiomyocytes (CMs) can overcome the cell source limitations. The current research involves the isolation and expansion of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), their differentiation into CMs and subsequent construction of tissue-engineered myocardium supported by random and aligned polycaprolactone (PCL) nanofibrous matrices (av. dia: 350-850 nm). Umbilical cord matrix (UCM)-derived MSCs were isolated successfully by routine enzymatic digestion and a nonenzymatic explant culture method and characterized by their morphology, differentiation into different lineages, and surface marker expression. Treatment of UCM-derived MSCs with 5-azacytidine (5 μM) induced their differentiation into putative cardiac cells, as revealed by the expression of cardiac-specific troponin T (cTnT), smooth muscles actin, myogenin (MYOG), smoothelin, cardiac α-actin genes and cTnT, α-actinin proteins by RT-PCR and immunocytochemistry, respectively. However, no beating cells were observed in differentiated MSCs. On the other hand, adult human foreskin-derived iPSCs cultured on Matrigel™-coated aligned PCL nanofibrous matrices showed anisotropic behavior along the PCL nanofibers and, upon differentiation, expressed cardiac-specific cTnT (23.34 vs. 32.55%) proteins and showed more synchronized beating than those differentiated on Matrigel™-coated tissue culture coated polystyrene surfaces. Moreover, aligned PCL nanofibers are able to promote cells orientation parallel to the fibers, thus providing an effective way to control anisotropic nature under in vitro condition.

Blood type and breed-associated differences in cell marker expression on equine bone marrow-derived mesenchymal stem cells including major histocompatibility complex class II antigen expression

BackgroundAs the search for an immune privileged allogeneic donor mesenchymal stem cell (MSC) line continues in equine medicine, the characterization of the cells between different sources becomes important. Our research seeks to more clearly define the MSC marker expression of different equine MSC donors.MethodsThe bone marrow-derived MSCs from two equine breeds and different blood donor-types were compared over successive culture passages to determine the differential expression of important antigens. Eighteen Thoroughbreds and 18 Standardbreds, including 8 blood donor (erythrocyte Aa, Ca, and Qa antigen negative) horses, were evaluated. Bone marrow was taken from each horse for isolation and culture of MSCs. Samples from passages 2, 4, 6, and 8 were labelled and evaluated by flow cytometry. The cell surface expression of CD11a/18, CD44, CD90 and MHC class II antigens were assessed. Trilineage assays for differentiation into adipogenic, chondrogenic and osteogenic lines were performed to verify characterization of the cells as MSCs.FindingsThere were significant differences in mesenchymal stem cell marker expression between breeds and blood antigen-type groups over time. Standardbred horses showed a significantly lower expression of MHC class II than did Thoroughbred horses at passages 2, 4 and 6. CD90 was significantly higher in universal blood donor Standardbreds as compared to non-blood donor Standardbreds over all time points. All MSC samples showed high expression of CD44 and low expression of CD11a/18.ConclusionsUniversal blood donor- type Standardbred MSCs from passages 2–4 show the most ideal antigen expression pattern of the horses and passages that we characterized for use as a single treatment of donor bone marrow-derived MSCs. Further work is needed to determine the significance of this differential expression along with the effect of the expression of MHC I on equine bone marrow-derived MSCs.

Improved Isolation of Mesenchymal Stem Cells Based on Interactions between N-Acetylglucosamine-Bearing Polymers and Cell-Surface Vimentin.

Mesenchymal stem cells (MSCs) in bone marrow and adipose tissues are expected to be effective tools for regenerative medicine to treat various diseases. To obtain MSCs that possess both high differentiation and tissue regenerative potential, it is necessary to establish an isolation system that does not require long-term culture. It has previously been reported that the cytoskeletal protein vimentin, expressed on the surfaces of multiple cell types, possesses N-acetylglucosamine- (GlcNAc-) binding activity. Therefore, we tried to exploit this interaction to efficiently isolate MSCs from rat bone marrow cells using GlcNAc-bearing polymer-coated dishes. Cells isolated by this method were identified as MSCs because they were CD34-, CD45-, and CD11b/c-negative and CD90-, CD29-, CD44-, CD54-, CD73-, and CD105-positive. Osteoblast, adipocyte, and chondrocyte differentiation was observed in these cells. In total, yields of rat MSCs were threefold to fourfold higher using GlcNAc-bearing polymer-coated dishes than yields using conventional tissue-culture dishes. Interestingly, MSCs isolated with GlcNAc-bearing polymer-coated dishes strongly expressed CD106, whereas those isolated with conventional tissue-culture dishes had low CD106 expression. Moreover, senescence-associated β-galactosidase activity in MSCs from GlcNAc-bearing polymer-coated dishes was lower than that in MSCs from tissue-culture dishes. These results establish an improved isolation method for high-quality MSCs.

STEM-24. THERAPEUTIC APPROACH OF STEM CELL CARRYING RETROVIRAL REPLICATING VECTORS IN HUMAN GLIOMA MODEL

Abstract Toca 511, an improved retroviral replicating vectors (RRVs) expressing a codon-optimized cytosine deaminase, has shown highly promising evidence of therapeutic benefit in preclinical and clinical studies for gene therapy of glioma. In the present study, we engineered human mesenchymal stem cells (MSC) as tumor-homing cellular carriers that produce and release RRV, and evaluated the effect of this mode of virus delivery on the time course of intratumoral RRV dissemination. Human MSC isolates were engineered to produce RRV (MSC-RRV). Cytotoxicity assays confirmed efficient prodrug activator function in U-87 glioma cells transduced with vectors produced from MSC-RRV. To evaluate intratumoral migration activity and tumor-homing migration activity in vivo, individual MSC isolates were injected either directly into human glioma xenografts, or into the contralateral brain hemisphere. MSC-RRV isolates showing the highest levels of intratumoral migration activity were selected, and the efficiency of intratumoral dissemination and tumoricidal activity achieved by these isolates was compared against injection of RRV virus preparations in subcutaneous glioma models. Compared to virus injection, MSC-RRV achieved 1.6x-higher levels of tumor transduction (p< 0.05), and 2x-reduced tumor growth (p=0.018) at earlier time points. In short-term survival studies using lower doses of MSC-RRV cells vs. RRV virus injected 14 days post-establishment of intracranial U-87 gliomas, a small but statistically significant prolongation of median survival was seen with intracranial tumors treated with MSC-RRV as compared to RRV (26 vs. 20 days, p< 0.05) after only a single cycle of 5-FC prodrug. Thus, MSC can be employed as mobile tumor-homing RRV-producer cells, which release RRV as they migrate to tumor foci in vivo and actively penetrate into individual tumor masses, resulting in more rapid tumor transduction and earlier therapeutic efficacy, which may be advantageous particularly for multi-focal and metastatic disease. This study was funded by the California Institute for Regenerative Medicine (TR2-01791).

Wharton's Jelly Mesenchymal Stem Cell: Various Protocols for Isolation and Differentiation of Hepatocyte-Like Cells; Narrative Review.

There are several differentiation methods for mesenchymal stem cells (MSCs) into hepatocyte-like cell. Investigators reported various hepatic differentiation protocols such as modifying culturing conditions or using various growth factors/cytokines. In this literature review, we compared different MSCs extraction and isolation protocols from Wharton’s jelly (WJ) and explored various MSCs differentiation methods. Various protocols have been recommended for MSCs isolated from WJ, such as enzymatic, enzymatic-explant, and explant methods. In the explant method, valuable time is wasted, but the cost and biological contaminations are reduced and the number of isolated cells is high. However, other features, such as immune phenotype and multiline-age differentiation capacity, do not differ from other methods. There are also several differentiation methods for hepatocyte-like cell including the induction of MSC by cytokines and growth factors, and the differentiation of MSC in 2- and 3-dimensional matrix (2D and 3D). Among several cytokines, hepatocyte growth factor (HGF) and fibroblast growth factor (FGF) are essential. In the early stage of the differentiation, 2D culture is useful, and in the development stage, 3D culture system with HGF and FGF cytokines are more effective in the process of differentiation. Some studies have used 3D culture system in biocompatible scaffolds, such as alginate, collagen, gelatin, and peptide-Gly-Leu-amide (PGLA). In conclusion, Wharton’s jelly-Mesenchymal stem cells (WJ-MSCs) can be considered as an appropriate source for hepatocyte differentiation. Moreover, we introduced the explant method as the most effective protocol. This review attempted to highlight factors in hepatocyte differentiation, but the most effective protocol is not still unknown.

Isolation and Characterization of Mesenchymal Stem Cells from Human Liposuction Aspirate

Background: Adipose derived stem cells (ADSCs) are a new type of mesenchymal stem cells (MSC) resemble bone marrow stem cells in their biologic activities. DScs is multipotent stem cells can differentiate into multiple cell types including osteoblasts, chondrocytes, adipocytes, myocytes, vascular endothelial cells, and neurons. Stromal vascular fraction (SVF) corresponds to ADSC and describes the cells obtained immediately from adipose tissues after the digestion of collagenase. Adipose tissue is a promising source of adult mesenchymal stem cells for therapeutic applications because it is available in large amounts (100 ml up to 1 liter) through liposuction and with minimal morbidity. Aim: To isolate and characterize the adipose-derived stem cells from human liposuction aspirate. Material and Methods: liposuction aspirate was used for the extraction of the stromal vascular fragment (SVF). SVF was used for culturing and isolation of hADSCs.ADSCs were cultured in tissue-culture flasks using collagenase digestion method. Flow cytometric analysis was used to identify ADSCs markers using fluorescence-activated cell sorting (FACS)against CD44, CD90, and CD45. Results: Human ADSCs were isolated successfully using collagenase digestion method. After 4 days the cells appeared to be spindle in shape and formed symmetric colonies. After 7 days, the cell sheet formed 60% confluence and completed to 90% confluence after 10 days incubation. FACS analysis of ADSCs surface markers revealed expression of CD44, and CD90 markers in most of the adherent cells. In contrast, most adherent cells were negative for CD45. Conclusion: Human ADSCs were isolated successfully from human lipoaspirate. Keywords: ADSCs; MSC; SVF

Isolation and characterization of deer-derived mesenchymal stem cells

Isolation and characterization of deer-derived mesenchymal stem cells

The effects of transplanted mesenchymal stem cells treated with 17-b estradiol on experimental autoimmune encephalomyelitis.

The present study was conducted aimed at exploring the modulatory effects of 17-b estradiol (17-bED) on mesenchymal stem cells (MSCs) in the EAE (experimental autoimmune encephalomyelitis) animal model of multiple sclerosis (MS). Following the isolation of bone marrow-derived MSCs from the bilateral femurs and tibias of the male Wistar rats, the cells were harvested and cultured in the presence of 100nM 17-bED for 24h. EAE was induced in male Wistar rats (8-12weeks old) using guinea pig spinal cord homogenate, in combination with the complete Freund's adjuvant. The MSC therapy was triggered when all of the animals obtained a disability score. The symptoms were monitored on a daily basis throughout the study until the rats were euthanized. The mRNA expression of cytokines, including IL-17, IFN-γ, TNF-α, IL-10, IL-4, and TGF-β together with MMP8 and MMP9 as the family members of matrix metalloproteinases(MMPs) in the brain and spinal cord tissues were examined using real-time PCR. The levels of splenocytes-originated IL-10 and IFN-γ cytokines were also measured by ELISA. The MTT-based research findings showed that the infiltration of lymphocytes into the spleen decreased considerably. It was also observed that the mRNA expression of proinflammatory cytokines decreased significantly, while the mRNA levels of anti-inflammatory cytokines increased remarkably. It was also found that the mRNA levels of the examined matrix metalloproteinases(MMP8 and MMP9) were downregulated significantly. The findings of the present study indicated that the administration of 17-bED enhanced the efficacy of MSCs transplantation and modulated immune responses relatively in the EAE model, via the regulation of either pro- or anti-inflammatory cytokines and matrix metalloproteinases.

The Effect of Mesenchymal Stem Cells Pulsed with 17 Beta-Estradiol in an Ameliorating Rat Model of Ulcerative Colitis

Background: Among the available supplements, 17 β-estradiol (E2) has strong proliferative effects on mesenchymal stem cells (MSCs). Objectives: The current study aimed at evaluating the effects of E2-primed MSCs therapy in a rat model of ulcerative colitis. Methods: After the isolation of MSCs, cells were co-cultured with E2 for 24 hours. Colitis was induced by acetic acid in four groups; the control colitis, MSCs-treated, E2-primed MSCs-treated (E2-MSCs), and normal. MSCs and E2-MSCs were injected into the peritoneum in two distinct groups. After 10 days, the rats were evaluated for the level of nitric oxide, malondialdehyde, myeloperoxidase, total protein, and proinflammatory cytokines (IL-6, IL-1β, and TNF-α) in colonic homogenates. Results: The cell therapy with E2-MSCs in rats with colitis had a more desirable outcome; indeed, it resulted in a favorable regression in clinical score and inflammatory profile in rats with colitis than the MSCs-treated. The levels of myeloperoxidase, nitric oxide, and malondialdehyde were significantly diminished and, conversely, the total protein levels were significantly increased in rats with ulcerative colitis receiving E2-MSCs compared to rats with colitis receiving untreated MSCs. On the same side, the level of proinflammatory cytokines reduced significantly in E2-MSCs rats than the MSCs group. Conclusions: E2-MSCs decrease colonic damage significantly, which is probably associated with the prominent reduction of lipid peroxidation, neutrophil infiltration, and the levels of proinflammatory cytokines in the colonic tissue. Therefore, it seems that E2 effectively surmounts the limitations of MSC application and makes it an ideal choice for cell therapy in inflammatory diseases such as ulcerative colitis.

Isolation of umbilical cord mesenchymal stem cells using human blood derivatives accompanied with explant method.

Mesenchymal stem cells (MSCs) are multipotent cells which can be isolated from many sources including umbilical cord. Isolation protocols are depended on either explant or enzymatic methods. Although fetal bovine serum (FBS) is used as a supplement in isolation and expansion of MSCs, human blood derivatives such as cord blood serum (CBS) and platelet lysate (PL) are attractive substitutes of FBS which overcome impediments of using FBS in clinical setups. Here we compared the effect of using CBS, PL and FBS supplemented media in isolation of umbilical cord tissue derived MSCs by using explant method. To do that we cultured umbilical cord tissue explant in either CBS or PL or FBS supplemented media. Isolated cells were quantified, their morphology was assessed and Cells in passage 3 were characterized based on their immunophenotyping and their potential for differentiation into adipocytes and osteocytes. Moreover, proliferation of cells was assessed by crystal violet staining. All the three media succeeded to isolate MSCs and maintain their stemness characteristics. However, the highest number of isolated cells were obtained using CBS, ~10-fold more than FBS, while the number of isolated cells obtained using PL was ~2-fold more than FBS. Moreover, crystal violet showed that both PL and CBS promote proliferation of MSCs more than FBS. Our data suggest that, although all supplements maintain stemness characteristics of MSCs when used to isolate those cells by explant method, using human blood derived supplements is more effective than FBS. In the same context CBS is more effective than PL.

45: The Effect of Transplantation of Undifferentiated Mesenchymal Stem Cells in Experimental Model of Myocardial Infarction: Preliminary Results

Introduction: Cardiovascular diseases like ischemic myocardial infarction remain worldwide with high lethality and mortality beside the extended researches for pharmaceutical therapies. Isolation of mesenchymal stem cells (ADSCs) from adipose tissue created a new spectrum of research. ADSCs are now known to be able to differentiate to myocardial cells and create cardiac tissue. In our protocol we use the genetic factors GATA-4 and nkx2,5 which are associated with the regeneration and differentiation of the myocardium respectively. Methods: We used 17 adult Wistar rats of conventional microbiological status (15 females – receivers and 2 males - donors) weighting 200-250gr. The adipose tissue was collected from the male donors and was immediately transferred to the laboratory at 4°C. The female-receivers were randomly allocated in 5 groups (3 animals per group). On Day -7 (7 days pre-operatively) we performed a SPECT–CT with injection of 99mTc-sestamibi to all female animals. On Day 0 (operation day) all groups underwent left thoracotomy and - besides sham operated - ligation of LAD for 45min with ECG monitoring confirming ischemia. Control 1: injection of N/S intramyocardially and removal of ligation. Control 2: injection of N/S intramyocardially without removal of ligation. Sham: no ligation. Experimental 1: injection of ADSCs intramyocardially and removal of ligation. Experimental 2: injection of ADSCs intramyocardially without removal of ligation. On Day +7 and Day +14 (7 and 14 days post-operatively) the animals that survived underwent new SPECT-CT followed by euthanasia on Day +16 (16 days postoperatively) together with blood sampling and heart harvesting for histological and immunohistochemical evaluation. Results: ADSCs were successfully engrafted into the myocardium and had beneficial effect on the ischemic myocardial area of the experimental groups with regeneration and increase of contractility especially in the 14th postoperative day compared with the control groups. Specifically imaging with SPECT-CT revealed significant absorption of 99Tc and viability of the myocardium on postoperative Day 14 in the experimental groups. The expression of GATA-4, nkx2,5 and IL-6 was significantly upregulated compared with the control groups. Conclusion: ADSCs are definitely the therapeutic approach of the future and GATA-4 as nkx2,5 are the genetic factors that are leading to an extended regeneration of the ischemic myocardium. The promising results so far are leading us to increase the number of animals in our protocol to further evaluate the clinical significance of ADSCs transplantation in cases of acute myocardial infarction.

Xenogen-free isolation and culture of human adipose mesenchymal stem cells.

Adipose-derived Stem Cells (ASCs) present great potential for reconstructive procedures. Currently, isolation by enzyme digestion and culturing using xenogenic substances remain the gold standard, impairing clinical use. Abdominal lipo-aspirate and blood samples were obtained from healthy patients. A novel mechanical isolation method for ASCs was compared to (the standard) collagenase digestion. ASCs are examined by flowcytometry and multilineage differentiation assays. Cell cultures were performed without xenogenic or toxic substances, using autologous plasma extracted from peripheral blood. After eGFP-transfection, an in vivo differentiation assay was performed. Mechanical isolation is more successful in isolating CD34+/CD31-/CD45-/CD13+/CD73+/CD146- ASCs from lipo-aspirate than isolation via collagenase digestion (p <0 .05). ASCs display multilineage differentiation potential in vitro. Autologous plasma is a valid additive for ASCs culturing. eGFP-ASCs, retrieved after 3 months in vivo, differentiated in adipocytes and endothelial cells. A practical method for human ASC isolation and culturing from abdominal lipo-aspirate, without the addition of xenogenic substances, is described. The mechanical protocol is more successful than the current gold standard protocol of enzyme digestion. These results are important in the translation of laboratory-based cell cultures to clinical reconstructive and aesthetic applications.