Autologous cell using for the restoration of functional defects in patients with ischemic cerebrovascular accident

In studying the differentiation of stem cells along smooth muscle lineage, smooth muscle cell (SMC) contractile proteins serve as markers for the relative state of maturation. Yet, recent evidence suggests that some SMC markers are probably expressed in multipotent mesenchymal stem cells (MSCs). Such a paradox necessitates investigations to re-examine their role as differentiated markers in MSCs. We tried to detect the expression of four widely used SMC markers including α-smooth muscle actin (α-SMA), h1-calponin, desmin and smooth muscle myosin heavy chain (SM-MHC), as well as the other isoforms of calponin family in resting MSCs. Then we used three different conditions to initiate MSCs differentiation along SMC lineage, and examined the alternation of SMC markers expression at both the transcript level and protein level. Desmin and h1-calponin are expressed in MSCs, in the presence or absence of SMC induction conditions. Moreover, MSCs are shown to express all known isoforms of calponin. Double-staining reveals that h1-calponin +/α-SMA - cells constitute the majority of resting MSCs. Under differentiated conditions, expression of SM-MHC was initiated and expression of α-SMA was promoted. The expression of SM-MHC and upregulation of α-SMA are relatively reliable indications of a mature smooth muscle phenotype in MSCs. Given that the cells are particularly rich in calponins expression, we postulate possible roles of these proteins in regulating cellular function by taking part in actin cytoskeleton and signaling. These findings imply that an extensive study of the cell physiology of MSCs should focus on the functional roles for these proteins, rather than simply regard them as differentiated markers.

The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.

ABSTRACTBackground: The bone marrow has been considered as a main source for isolation of multipotent mesenchymal stem cells (MSCs). They are still the most frequently investigated cell type and often identified as the gold standard. However, a similar cell population has been also isolated from other tissues such as adipose tissue. Unlike bone marrow, the adipose tissue is abundantly accessible source of stem cell that can give a good yield in culture.Aim of the work: Was isolation of the rat bone marrow MSCs (BM-MSCs) and adipose tissue MSCs (AD-MSCs) and assessing their growth kinetics.Material and Methods: The rat bone marrow and adipose tissue were isolated from 10 male adult albino rats and cultured and expanded through 6 passages. BM-MSCs and AD-MSCs biological characteristics evaluated for cell therapy (morphology, flow cytometric analysis, colony-forming unit-fibroblast assay, proliferation capacity at passages 2, 4 and 6, population doubling time (PDT) and cell growth curves).Results: BM-MSCs and AD-MSCs attached to the culture flask and displayed spindle-shaped morphology, more evident in AD-MSCs. Proliferation rate of AD-MSCs in the analyzed passages was more than BM-MSCs. The increase in the PDT of both types of MSCs occurred with the increase in the number of passages.Conclusion: The rat AD- MSCs have growth kinetic advantages in the proliferative capacity, colony-forming unite fibroblast and population doubling time more than that of BM-MSCs. these advantages should be considered when choosing a stem cell source for specific clinical application.

Mesenchymal stem cells (MSCs), with high self-renewal ability and multipotency, are commonly used as the seed cells for tissue engineering. However, the reduction and loss of multipotential ability after necessary expansion in vitro set up a heavy obstacle to the clinical application of MSCs. Here in this study, we exploit the autologous crystallization ability of biocompatible poly (ε-caprolactone) (PCL) to obtain uniformly distributed nanoneedle arrays. By controlling the molecular weight of PCL, nanoneedle with a width of 2μm and height of 50nm, 80nm, and 100nm can be successfully fabricated. After surface chemical modification with polydopamine (PDA), the water contact angle of the fabricated PCL nanoneedle arrays are reduced from 84° to almost 60° with no significant change of the nanostructure. All the fabricated substrates are cultured with bone marrow MSCs (BMMSCs), and the adhesion, spreading, proliferation ability and multipotency of cells on different substrates are investigated. Compared with the BMMSCs cultured on pure PCL nanoneedle arrays, the decoration of PDA can improve the adhesion and spreading of cells and further change them from aggregated distribution to laminar distribution. Nevertheless, the laminar distribution of cultured cells leads to a weak cell-cell interaction, and hence the multipotency of BMMSCs cultured on the PCL-PDA substrates is decimated. On the contrary, the pure PCL nanoneedle arrays can be used to maintain the multipotency of BMMSCs via clustered growth, and the PCL1 nanoneedle array with a height of 50nm is more promising than the other 2 with regard to the highest proliferation rate and best multipotential differentiation ability of cultured cells. Interestingly, there is a positive correlation between the strength of cell-cell interaction and the multipotency of stem cells in vitro. In conclusion, we have successfully maintained the multipotency of BMMSCs by using the PCL nanoneedle arrays, especially the PCL1 nanoneedle array with a height of 50nm, as the substrates for in vitro extension, and further revealed the importance of cell-cell interaction on the multipotency of MSCs. The study provides a theoretical basis for the behavioral regulation of MSCs, and is instructive to the design of tissue engineering scaffolds.

In recent years, a large number of studies have contributed to our understanding of the immunomodulatory mechanisms used by multipotent mesenchymal stem cells (MSCs). Initially isolated from the bone marrow (BM), MSCs have been found in many tissues but the strong immunomodulatory properties are best studied in BM MSCs. The immunomodulatory effects of BM MSCs are wide, extending to T lymphocytes and dendritic cells, and are therapeutically useful for treatment of immune-related diseases including graft-versus-host disease as well as possibly autoimmune diseases. However, BM MSCs are very rare cells and require an invasive procedure for procurement. Recently, MSCs have also been found in fetal-stage embryo-proper and extra-embryonic tissues, and these human fetal MSCs (F-MSCs) have a higher proliferative profile, and are capable of multilineage differentiation as well as exert strong immunomodulatory effects. As such, these F-MSCs can be viewed as alternative sources of MSCs. We review here the current understanding of the mechanisms behind the immunomodulatory properties of BM MSCs and F-MSCs. An increase in our understanding of MSC suppressor mechanisms will offer insights for prevalent clinical use of these versatile adult stem cells in the near future.

Among the adult stem cells, multipotent mesenchymal stem cells (MSCs) turned out to be a promising option for cell-based therapies for the treatment of various diseases including autoimmune and cardiovascular disorders. MSCs bear a high proliferation and differentiation capability and exert immunomodulatory functions while being still clinically safe. As tissue-resident stem cells, MSCs can be isolated from various tissue including peripheral or umbilical cord blood, placenta, blood, fetal liver, lung, adipose tissue, and blood vessels, although the most commonly used source for MSCs is the bone marrow. However, the proportion of MSCs in primary isolates from adult tissue biopsies is rather low, and therefore MSCs must be intensively expanded in vitro before the MSCs find particular use in therapies that may require extensive and repetitive cell replacement. Therefore, more easily accessible sources of MSCs are needed. Here, we present a detailed protocol to generate tissue-typical MSCs by direct linage conversion using transcription factors defining target MSC identity from murine induced pluripotent stem cells (iPSCs).

We examined the presence of circulating plastic adherent multipotent mesenchymal stem cells (MSCs) in fracture patients. Three patient groups (n = 10-18) were evaluated, including elderly females with a femoral neck fracture treated with cemented hemiarthroplasty, an age- and sex-matched group with hip osteoarthritis (OA) treated with cemented total hip arthroplasty (THA), and younger adults with surgically treated lower extremity fractures. The presence of circulating MSCs pre- and postoperatively was compared to bone marrow (BM) MSCs from the same subjects. Criteria for identifying MSCs included cell surface markers (CD105+, CD73+, CD90+, CD45-, CD14-), proliferation through several passages as well as osteogenic, chondrogenic, and adipogenic differentiation. Plastic adherent MSCs were found in peripheral blood (PB) from 22% of hip fracture patients, 46% of younger fracture patients, and in none of 63 pre- and postmenopausal women with hip OA. When detectable, circulating MSCs appeared between 39 and 101 h after fracture. PB derived MSCs did not differ from BM derived MSCs, except for a small population (<15%) of CD34+ cells among PB derived MSCs. This initial study indicates mobilization of MSCs into the circulation in response to fracture, even in very old patients, while circulating MSCs were not detectable before or after elective THA.

Highly effective osteogenic conversion of mesenchymal Stem Cells by Cell Rev™ Supreme Osteo

Multipotential mesenchymal stem cells (MSCs) are found in human bone marrow and are shown to secrete hematopoietic cytokines and support hematopoietic progenitors in vitro. We hypothesized that infusion of autologous MSCs after myeloablative therapy would facilitate engraftment by hematopoietic stem cells, and we investigated the feasibility, safety, and hematopoietic effects of culture-expanded MSCs in breast cancer patients receiving autologous peripheral-blood progenitor-cell (PBPC) infusion. We developed an efficient method of isolating and culture-expanding a homogenous population of MSCs from a small marrow-aspirate sample obtained from 32 breast cancer patients. Twenty-eight patients were given high-dose chemotherapy and autologous PBPCs plus culture-expanded MSC infusion and daily granulocyte colony-stimulating factor. Human MSCs were successfully isolated from a mean +/- SD of 23.4 +/- 5.9 mL of bone marrow aspirate from all patients. Expansion cultures generated greater than 1 x 10(6) MSCs/kg for all patients over 20 to 50 days with a mean potential of 5.6 to 36.3 x 10(6) MSCs/kg after two to six passages, respectively. Twenty-eight patients were infused with 1 to 2.2 x 10(6) expanded autologous MSCs/kg intravenously over 15 minutes. There were no toxicities related to the infusion of MSCs. Clonogenic MSCs were detected in venous blood up to 1 hour after infusion in 13 of 21 patients (62%). Median time to achieve a neutrophil count greater than 500/microL and platelet count >/= 20,000/microL untransfused was 8 days (range, 6 to 11 days) and 8.5 days (range, 4 to 19 days), respectively. This report is the first describing infusion of autologous MSCs with therapeutic intent. We found that autologous MSC infusion at the time of PBPC transplantation is feasible and safe. The observed rapid hematopoietic recovery suggests that MSC infusion after myeloablative therapy may have a positive impact on hematopoiesis and should be tested in randomized trials.

Aim. To study the changes of splenic morphometric parameters in aged laboratory animals exposed to ionizing radiation in the dose of 4Gr after multipotent mesenchymal and hematopoietic stem cells transplantation. Methods. The experiments were conducted on 72 white male laboratory mice at the third year of life with the body weight of 50 g. Multipotent mesenchymal stem cells and hematopoietic stem cells were obtained from 8 laboratory female mice with the body weight of 30 g aged 3-4 months, the gestation term was 14 days. The first group (36 animals exposed to radiation) was subdivided to 2 subgroups of 18 animals each. The suspension of multipotent mesenchymal stem cells (6 000 000 cells/kg) and hematopoietic stem cells (330 000 cells/kg) was introduced as single intravenous injection to the experimental subgroup (18 animals) 1 hour after the animals were exposed to radiation. The animals of control subgroup (18 animals) were injected 0.2 ml of normal saline. The second group included two subgroups 18 mice each that underwent the same procedure without being exposed to radiation. 9 animals from each group were withdrawn from the study at 1st and 7th day each. The lymphoid follicle gross area, area of the T-cell and B-cell zones, general numbers of cells in the red pulp of spleen, including erythrocyte and lymphocyte count, were measured in splenic histologic specimens using the morphometric «BioVision 2008» software. Results. It was shown that on the 7th day after exposure to ionizing radiation followed by stem cells transplantation, the area of thymus-independent zone of lymphoid follicle restored back to normal ranges. The effect of multipotent mesenchymal stem cells and hematopoietic stem cells transplantation also resulted in the increase of the number of cells in the red pulp of spleen. There were no significant changes observed in numbers of erythroid cells and white blood cells in the spleen red pulp compared to control subgroup. At the same time, the leukocyte number in the red pulp of spleen restored to normal values. Conclusion. The restoration of the basic morphometric parameters in spleen of aged laboratory animals exposed to ionizing radiation may be explained be increased homing of splenic colony-forming units with subsequent activation of extramedullary hematopoiesis in spleen, and apoptosis-reducing effect of multipotent mesenchymal stem cells.

A significant complication in allogeneic stem cell transplantation is graft versus host disease (GVHD). The use of multipotential mesenchymal stem cells (MMSC) for the amelioration of GVHD has shown promise as a therapeutic intervention. Given that MMSC can suppress allogeneic immune responses, there is a concern that using these cells may promote leukemic relapse. We describe a murine model of GVHD in the presence of leukemic cells (L1210). Acute GVHD was induced in DBA mice by transplanting bone marrow and spleen cells from C57Bl/6J mice with or without prior injection of L1210 cells. The recipient mice were monitored for signs of GVHD. The mice were then treated with primary MMSC or a C57Bl bone marrow derived cloned mesenchymal cell line (OMA-AD). The results without L1210 cells, demonstrated that mice treated with primary MMSC that had developed moderate GVHD had increased long-term survival when compared to controls. The group treated with OMA-AD cells showed minimal GVHD so cloned OMA-AD MMSC cells provided a significant protective effect against GVHD, and the survival rate was superior to that of animals treated with primary MMSC on the same day. In the presence of L1210, the control mice all died by day 11, and the mice receiving OMA-AD and L1210 cells died by day 9. Both had minimal GVHD. Only the mice receiving primary MMSC that developed moderate to severe GVHD survived long term. It appears that although MMSC and OMA-AD cells can ameliorate GVHD; the greater immunosuppressive effect of OMA-AD cells permitted the re-growth of the leukemic cells. In contrast, the moderate GVHD that remained after primary MMSC treatment eliminated the leukemia in the majority of mice. These studies demonstrated that in the mouse model, as in man, administration of primary or cloned MMSC ameliorated GVHD. However, complete suppression of GVHD permitted leukemic relapse.

Bovine bone marrow (BM) and adipose tissue (AT) multipotent mesenchymal stem cells (MMSC) are believed to be a perspective material for the creation of new cellular systems and products for food biotechnology. We demonstrated the possibility of directed differentiation of BM and AT MMSC in those of muscle tissue by culturing them in the medium, containing inductors of myogenesis (5-azacytidine, 5-aza-2¢-deoxycytidine and retinoic acid). The comparative analysis of differentiation efficiency of these cells in the direction of myogenesis revealed morphological changes in BM MMSC on the 15-21 st day, and in AT MMSC — on the 20-25 th day of culturing in the medium with different inductors. The analysis of MYOD1 and MYOG gene expression in RT-PCR detected essential distinctions in the potency of these cells to differentiate into muscle tissue cells in vitro, and also showed that MMSC, isolated from BM, are more perspective source for muscle tissue engineering in vitro.

Resident Tissue-Specific Mesenchymal Progenitor Cells Contribute to Fibrogenesis in Human Lung Allografts

577 Aged Cardiac Patients Show Impaired Mesenchymal Stem Cell Differentiation to a Myogenic Phenotype Due to Depressed WNT/β-Catenin Signaling

Recent advancements in stem cell technology open a new door for patients suffering from diseases and disorders that have yet to be treated. Stem cell-based therapy, including human pluripotent stem cells (hPSCs) and multipotent mesenchymal stem cells (MSCs), has recently emerged as a key player in regenerative medicine. hPSCs are defined as self-renewable cell types conferring the ability to differentiate into various cellular phenotypes of the human body, including three germ layers. MSCs are multipotent progenitor cells possessing self-renewal ability (limited in vitro) and differentiation potential into mesenchymal lineages, according to the International Society for Cell and Gene Therapy (ISCT). This review provides an update on recent clinical applications using either hPSCs or MSCs derived from bone marrow (BM), adipose tissue (AT), or the umbilical cord (UC) for the treatment of human diseases, including neurological disorders, pulmonary dysfunctions, metabolic/endocrine-related diseases, reproductive disorders, skin burns, and cardiovascular conditions. Moreover, we discuss our own clinical trial experiences on targeted therapies using MSCs in a clinical setting, and we propose and discuss the MSC tissue origin concept and how MSC origin may contribute to the role of MSCs in downstream applications, with the ultimate objective of facilitating translational research in regenerative medicine into clinical applications. The mechanisms discussed here support the proposed hypothesis that BM-MSCs are potentially good candidates for brain and spinal cord injury treatment, AT-MSCs are potentially good candidates for reproductive disorder treatment and skin regeneration, and UC-MSCs are potentially good candidates for pulmonary disease and acute respiratory distress syndrome treatment.