Properties Of Multipotent Stem Cells Research Articles

Cellular Technologies in Traumatology: from Cells to Tissue Engineering

Severe injuries of the musculoskeletal system with damage of bone and cartilaginous tissue are associated with the necessity to find ways for replacement of defects. Current methods of treatment including the usage of autografts and modern synthetic materials are often not effective enough. The article deals with the prospects for developing the new areas in the traumatological pathology treatment based on the cell technologies application. Both medical and ethical aspects of the cell technology usage and the utilizing various sources of cell material for transplantation are discussed in detail. Special attention is devoted to application of the mesenchymal stem cells as the most suitable material for bone and cartilage repair. These cells possess the properties of multipotent stem cells that can adhere to plastic surfaces, differentiate into various cells, including chondroblasts, osteoblasts, and migrate to the damaged area. It is shown that the number and biological characteristics of the isolated cells depend on the tissue, the cell isolation method, and the culture medium. During the cultivation of mesenchymal stem cells, the true marker of which is Stro-1, it is necessary to monitor the duration and number of passages, because these values are inversely correlated with the differentiating potential of cells, and spontaneous transformation according to oncogenic pathway may occur. Thus, mesenchymal stem cells represent a current material for the reconstruction of hard tissues. Despite ethical and technological problems, cell technologies are a promising approach in regenerative medicine.

Influence of electromagnetic waves, with maxima in the green or red range, on the morphofunctional properties of multipotent stem cells.

This paper examines the effect of electromagnetic waves, with maxima in the green or red regions of the spectrum, on the morphofunctional state of multipotent mesenchymal stromal cells. The illumination regimes used in our experiments did not lead to any substantial heating of the samples; the physical parameters of the lighting were carefully monitored. When the samples were illuminated with a green light, no significant photostimulatory effect was observed. Red light, on the other hand, had an evident photostimulatory effect. It is shown that photostimulation with a red light decreases the enzymatic activities of mitochondrial dehydrogenases and enhances the viability of cells, their proliferative activity, and their ability to form bone tissue. It is also established that red light stimulates cell proliferation, while not activating the genes that increase the risk of the subsequent malignant transformation of cells or their death. This paper discusses the possible role of hydrogen peroxide in the processes examined.

Stem cells: Immunology and immunomodulation.

Multipotent progenitor cells have the ability to differentiate in vitro into tissue-specific cells under the proper cellular signaling conditions. Because of this intrinsic property, pluripotent derived stem cells from embryonic tissues, induced from genetic reprogramming of somatic cells or from mesenchymal cells, have become the focus of many regenerative medicine studies directed toward application to clinical settings. Generation of retinal pigment epithelium derived from patient-specific, induced pluripotent stem cells may create the ability to recapitulate the disease state and screen new therapeutics, improving upon the limited treatment strategies currently available for afflicted patients. However, the method of delivery, cell culture differentiation, and immunological rejection are some of the limitations that continue to influence ongoing attempts for clinical applications. Understanding stem cell properties and the immune responses these cells elicit in vitro and in vivo will be of importance for designing successful protocols to overcome these immunological limitations. This chapter examines the immunological and immunomodulatory properties of multipotent stem cells in an attempt to provide insights for successful cellular transplantation.

Mouse 3T3 fibroblasts under the influence of fibroblasts isolated from stroma of human basal cell carcinoma acquire properties of multipotent stem cells

Multipotent mesenchymal stem cells can participate in the formation of a microenvironment stimulating the aggressive behaviour of cancer cells. Moreover, cells exhibiting pluripotent ESC (embryonic stem cell) markers (Nanog and Oct4) have been observed in many tumours. Here, we investigate the role of cancer-associated fibroblasts in the formation of stem cell supporting properties of tumour stroma. We test the influence of fibroblasts isolated from basal cell carcinoma on mouse 3T3 fibroblasts, focusing on the expression of stem cell markers and plasticity in vitro by means of microarrays, qRT-PCR (quantitative real-time PCR) and immunohistochemistry. We demonstrate the biological activity of the cancer stromal fibroblasts by influencing the 3T3 fibroblasts to express markers such as Oct4, Nanog and Sox2 and to show differentiation potential similar to mesenchymal stem cells. The role of growth factors such as IGF2 (insulin-like growth factor 2), FGF7 (fibroblast growth factor 7), LEP (leptin), NGF (nerve growth factor) and TGFβ (transforming growth factor β), produced by the stromal fibroblasts, is established to participate in their bioactivity. Uninduced 3T3 do not express the stem cell markers and show minimal differentiation potential. Our observations indicate the pro-stem cell activity of cancer-associated fibroblasts and underline the role of epithelial-mesenchymal interaction in tumour biology.

Expansion of umbilical cord blood mesenchymal stem cells

7103 Background: Umbilical cord blood (UCB) is known to harbor 2 major types of stem cells, the hematopoietic stem cells (HSC) & the non-hematopoietic or mesenchymal stem cells (MSC). Under appropriate conditions, MSCs can give rise to cells of bone, fat, hepatic lineages, etc. Based on this potential, MSC hold promise for clinical applications in regenerative medicine. Methods: Stroma-free liquid culture: UCB cryopreserved mononuclear cells (MNC) were cultured in the presence of early growth factors: Flt-3 & SCF (25ng/ml), MGDF (10ng/ml) & human serum (10%). MNC derived adherent MSC were passaged at day 14 during HSC expansion & after enriching in MesenCult medium. Results: We developed a technology to generate & expand HSC & stromal/ MSC from all UCB units (5/5) at the same time using one culture system (stroma-free liquid culture). Following repeated passages, MSC count increased 357- 600-folds & CFU-Fibroblasts colonies (CFU-F) increased too (61–513 & 648–697) after 10 and 20 passages respectively. We used the CFU-F assay to demonstrate MSC activity in stromal cell formation in vitro. Phenotypically, MSC were negative for hematopoietic antigens (CD45, CD34 & CD14) & MHC class-II but >95% + for CD73, CD105, CD29, CD44 & MHC class I. To demonstrate MSC differentiation capacity in vitro, cells were incubated in various induction media to differentiate into adipocytes (fat)), osteoblasts (bone) and hepatocytes (liver) at passage 5. Following induction, positive staining with oil red O for cells of adipocyte and with alkaline phosphatase for cells of osteoblsts lineages was observed. The identity of hepatocytes was verified by the characteristic hexagonal hepatocytic shape as well as albumin, cytokeratin (CK) 18 and CK14 expression, as assessed by flow cytometry. Our data were corroborated by RT-PCR analysis. Conclusions: MSC described herein exhibit in vitro properties of multipotent stem cells. The established, stroma-free culture system facilitates expansion of MSC from all tested UCB units. Our data underline that it will be possible in the future to substitutes properly differentiated hepatocytes which might lead to efficient applications in patients suffering from various end stage liver disease. No significant financial relationships to disclose.

Expansion of Umbilical Cord Blood Hematopoietic Stem Cells for Clinical Use.

Umbilical cord blood (UCB) is an alternative donor source for allogeneic hematopoietic stem cell transplantation. However, transplantation in adults is frequently limited by the small number of cells available in a unit. We have previously developed the technology to expand hematopoietic stem cells (HSC) and stromal/mesenchymal stem cells (SMSC) from all UCB frozen samples (13, 3). The incubation of thawed UCB mononuclear cells (MNC) in the presence of SCF (25ng/ml), FLt-3 (25ng/ml), MGDF (10ng/ml) & IL-6 (20ng/ml) and 10% human serum in stroma-free liquid culture not only generated long term expansion of transplantable UCB HSC (non-adherent). Also, long-term expansion of SMSC (adherent cells) was successful. In order to upscale the expansion to use for clinical applications, we analyzed 3 frozen UCB comparing the expansion from 24-well plates (previous) versus expansion in bags (VueLife™) after 10 and 14 days of culture. Results show substantial expansion in total cell count (TCC, 4.8, 10.9: 2.4, 3.8 fold) at d10 and d14 from wells & bags respectively. TCC increased further in the presence of SMSC (38% & 33% in CD34+ cell count cultured in wells). CD133+CD34+CD38- HSC multiplied (11, 25 fold, d10/d14 & colony forming cells (CFC) 19 fold at d14 bags respectively). Heterogeneous cell populations were detected after d 14 in bags: T and B -lymphoid (%CD3/%CD19; 65/4:50w/3), megakaryocytic (%CD61; 7:4) and myeloid (%CD33; 31:43) at d0/d14 respectively. Further, expanded cells (250,000) containing a small number of CD133+CD34+CD38- (15,000–30,000) were injected into the liver of sub-lethally irradiated newborn Balb/C Rag2-Cγ−/− mice. Our preliminary data show no engraftment from cells expanded in wells and bags after 6 weeks of transplantation from d10 cultures (human CD45 + <1%). However, positive engraftment in mouse PB was detected from cells expanded for 14 d (wells, 1.16–2.5% & bags, 1.21–3.9%) as compared to control mice (CD45; 30% PB & 70% BM) receiving selected CD34 + (300,000 CD34+ at d0). Primitive repopulating cells (PRC), and multilineage human CFC were detected after transplantation. On d14 of HSC expansion, UCB MNC derived adherent cells (SMSC) were enriched by trypsinization. SMSC were established in serum free and serum plus culture as well. The immunophenotype of harvested SMSC was CD29+, CD44+ and CD45−, CD34− and CD133− at percentages + >90%. Following repeated trypsinization, SMSC count increased 41–96 folds. CFU-Fibroblast colonies (92–173) were generated from 104 SMSC after 2 weeks in MesenCult medium. We have previously differentiated SMSC into hepatocytes. Now we also generated adipocytes in an induction medium containing, Insulin, dexamethasone & indomethacin. SMSC formed Oil-droplet vacuoles in the cytoplasm in 3 weeks. The culture conditions we defined to maintain UCB PRC should be developed clinically. SMSC described herein exhibit in vitro properties of multipotent stem cells.

Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15–20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin α2β1 hi and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 5 μg/ml insulin (DMEM + 10% FBS + Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.

Multilineage Differentiation of Ectomesenchymal Cells Isolated from the First Branchial Arch

Cranial neural crest-derived ectomesenchymal cells may be pluripotent stem cells that are capable of generating a range of phenotypes. The fate of these cells appears to be determined in part by intrinsic genetic programs and also by the influence of extracellular signals in the local environment. The extent of lineage determination once neural crest cells have migrated to the first branchial arch is not clear, although branchial arch pattern is not thought to be the result of crest predetermination. The aim of the present study was to test the hypothesis that ectomesenchymal cells of the first branchial arch show properties of pluripotent stem cells, the lineage of which may be directed by specific molecular signaling. Ectomesenchymal cells were enzymatically isolated from the mandibular processes of BALB/c mice and maintained in an undifferentiated state while cultured with leukemia inhibitory factor or induced to differentiate by lineage-specific induction factors or growth conditions, including transforming growth factor beta, forskolin, and a mineralization-promoting medium. Morphological observations and immunocytochemistry demonstrated that cells could be induced to differentiate into smooth muscle cells, glial cells, and osteoblasts, respectively. In the presence of the mineralization-promoting medium, alkaline phosphatase activity increased significantly and mineralization nodules formed. The data reported support the concept that many, although not all, first branchial arch-derived ectomesenchymal cells show properties of multipotent stem cells, the subsequent fate of which can be influenced by induction factors and growth conditions. Some cells, however, showed a degree of commitment with respect to their fate. The possible application of first branchial arch-derived stem cells to tissue engineering of the orofacial tissues should involve consideration of the developmental stage of cell harvesting and the desired cell fate.

Putative intermediates in the nerve cell differentiation pathway in hydra have properties of multipotent stem cells

We have investigated the properties of nerve cell precursors in hydra by analyzing the differentiation and proliferation capacity of interstitial cells in the peduncle of Hydra oligactis, which is a region of active nerve cell differentiation. Our results indicate that about 50% of the interstitial cells in the peduncle can grow rapidly and also give rise to nematocyte precursors when transplanted into a gastric environment. If these cells were committed nerve cell precursors, one would not expect them to differentiate into nematocytes nor to proliferate apparently without limit. Therefore we conclude that cycling interstitial cells in peduncles are not intermediates in the nerve cell differentiation pathway but are stem cells. The remaining interstitial cells in the peduncle are in G1 and have the properties of committed nerve cell precursors ( Holstein and David, 1986). Thus, the interstitial cell population in the peduncle contains both stem cells and noncycling nerve precursors. The presence of stem cells in this region makes it likely that these cells are the immediate targets of signals which give rise to nerve cells.