Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.
Mesenchymal stem cells have been increasingly introduced to have great potential in regenerative medicine, immunotherapy, and gene therapy due to their unique properties of self-renewal and differentiation into multiple cell lineages. Studies have shown that these properties may be limited and changed by senescence-associated growth arrest under different culture conditions. This study aimed to present the ability of some growth factors on human umbilical cord mesenchymal (hUCM) cells expansion and telomerase activity. To optimize hUCM cell growth, epidermal growth factor (EGF) and fibroblast growth factor (FGF) were utilized in culture media, and the ability of these growth factors on the expression of the telomerase reverse transcriptase (TERT) gene and cell cycle phases was investigated. TERT mRNA expression increased in the hUCM cells treated by EGF and FGF. So, the untreated hUCM cells expressed 30.49 ± 7.15% of TERT, while EGF-treated cells expressed 51.82 ± 12.96% and FGF-treated cells expressed 33.77 ± 11.55% of TERT. Exposure of hUCM cells to EGF or FGF also promoted the progression of cells from G1 to S phase of the cell cycle and induced them to decrease the number of cells entering the G2/M phase. Our study showed that EGF and, to a lesser extent, FGF amplify the proliferation and expansion of hUCM cells.
Objectives: Human umbilical cord mesenchymal cells (hUCM) can be easily obtained and processed in a laboratory. These cells may be considered as a suitable source in the repair of heart failure diseases. We, therefore, examined whether these cells may contribute to heart regeneration following an acute experimental myocardial infarction (MI). Methods: MI-induced animals received 5 × 106 hUCM cells, 5 × 106 5-azacytidine-treated cells (dhUCM), or PBS alone, subepicardially. A group of animals with MI and no other former intervention served as controls. dhUCM cells were assessed for F-actin, myogenin and troponin-I expression. Results: dhUCM cells appeared as binucleated cells with extensive cytoplasmic processes. These differentiated cells were F-actin and myogenin positive. Thirty days after LAD ligation, left ventricular ejection fraction and the percentage of fractional shortening improved significantly in cell-receiving animals. In addition, the amount of scar tissue was significantly reduced in hUCM and dhUCM groups compared to MI group (p < 0.05). These parameters were comparable between hUCM and dhUCM groups. Histopathological evaluations revealed that some engrafted cells adjacent to and remote from the MI area expressed troponin-I, F-actin and connexin43. Conclusion: These findings demonstrated the potential therapeutic use of either differentiated or undifferentiated hUCM cells in treatment of heart failure conditions.
Transplantation of neural-like cells is considered as a promising therapeutic strategy developed for neurodegenerative disease in particular for ischemic stroke. Since cell survival is a major concern following cell implantation, a number of studies have underlined the protective effects of preconditioning with hypoxia or hypoxia mimetic pharmacological agents such as deferoxamine (DFO), induced by activation of hypoxia inducible factor-1 (HIF-1) and its target genes. The present study has investigated the effects of DFO preconditioning on some factors involved in cell survival, angiogenesis, and neurogenesis of neural-like cells derived from human Wharton's jelly mesenchymal stem cells (HWJ-MSCs) in presence of hydrogen peroxide (H2O2). HWJ-MSCs were differentiated toward neural-like cells for 14 days and neural cell markers were identified using immunocytochemistry. HWJ-MSC-derived neural-like cells were then treated with 100 µM DFO, as a known hypoxia mimetic agent for 48 h. mRNA and protein expression of HIF-1 target genes including brain-derived neurotrophic factors (BDNF) and vascular endothelial growth factor (VEGF) significantly increased using RT-PCR and Western blotting which were reversed by HIF-1α inhibitor, while, gene expression of Akt-1, Bcl-2, and Bax did not change significantly but pAkt-1 was up-regulated as compared to poor DFO group. However, addition of H2O2 to DFO-treated cells resulted in higher resistance to H2O2-induced cell death. Western blotting analysis also showed significant up-regulation of HIF-1α, BDNF, VEGF, and pAkt-1, and decrease of Bax/Bcl-2 ratio as compared to poor DFO. These results may suggest that DFO preconditioning of HWJ-MSC-derived neural-like cells improves their tolerance and therapeutic potential and might be considered as a valuable strategy to improve cell therapy.
Many neural disorders are characterized by the loss of one or several types of neural cells. Human umbilical cord-derived mesenchymal cells (hUCMs) are capable of differentiating into neuron, astroglia-like and oligodendrocyte cell types. However, a reliable means of inducing the selective differentiation of hUCMs into neural cells in vitro has not yet been established. For induction of neural differentiation, hUCMs were seeded onto sterile glass slides and six various cocktails using a base medium (DMEM/LG) supplemented with 10 % FBS, retinoic acid (RA), dimethyl sulfoxide (DMSO), epidermal growth factor (EGF) and fibroblast growth factor (FGF) were used to compare their effect on neuronal, astrocyte and oligodandrocyte differentiation. The hUCMs were positive for mesenchymal markers, while they were negative for hematopoietic markers. Differentiation to adipogenic and osteogenic lineage was detected in these cells. Our data revealed that the cocktail consisting of DMEM/LG, FBS, RA, FGF, and EGF (DF/R/Fg/E group) induced hUCM cells to express the highest percentage of nestin, ß-tubulin III, neurofilament, and CNPase. The DF/Ds/Fg/E group led to the highest percentage of GFAP expression. While the expression levels of NF, GFAP, and CNPase were the lowest in the DF group. The least percentage of nestin and ß-tubulin III expression was observed in the DF/Ds group. We may conclude that FGF and EGF are important inducers for differentiation of hUCMs into neuron, astrocyte and oligodendrocyte. RA can induce hUCMs to differentiate into neuron and oligodendrocyte while for astrocyte differentiation DMSO had a pivotal role.
Human adipose-derived stem cells (hADSCs) are capable of differentiation into many cells including cardiac cells. Different types of scaffolds are used for cell differentiation but the best is yet to be determined. In this study, fibrin scaffold (3D) was fabricated using human plasma fibrinogen and compared with culture plates (2D) for the growth and differentiation of hADSCs into cardiomyocyte-like cells. For this purpose, after obtaining the properties of the isolated hADSCs and fibrin scaffold, four biochemical tests were employed to determine the relative growth rate of hADSCs in 2D and 3D cultures. To examine the effects of two different culture systems on cardiomyogenic differentiation, hADSCs were treated with 10 or 50 μM 5-azacytidine (5-Aza) for 24 h and followed until 10 weeks. The results indicated that the growth of hADSCs in 3D significantly increased after the seventh day (P < 0.05). Western blot, qRT-PCR and immunochemistry assays were used to evaluate the rate of cardiac differentiation, which showed significantly higher expression of special cardiac genes such as NKX2.5, Cx43, MLC2v, βMHC, HAND1, HAND2 and cTnI (P < 0.05) in the treated hADSCs with 50 μM 5-Aza in the 3D group. However, the expression level of the specific cardiac proteins in 3D was not significant using western blot and immunofluorescence staining. In conclusion, this study suggests that the fibrin scaffold with a compressive stress of 107.74 kPa can keep the cells alive for 10 weeks and also allows a higher and sooner differentiation of hADSCs into cardiomyocyte-like cells treated with 50 μM 5-Aza.
Purpose The main aim of this review was to provide an updated comprehensive report regarding isolation methods of MSCs from human extra embryonic tissues, including cord blood, amniotic fluid, and different parts of the placenta and umbilical cord, with respect to the efficacy of these methods. Results Extra embryonic tissues are the most available source for harvesting of mesenchymal stem cells (MSCs). They make a large number of cells accessible using non-invasive methods of isolation and the least immune-rejection reactions. A successful culture of primary cells requires obtaining a maximum yield of functional and viable cells from the tissues. In addition, there are many reports associated with their differentiation into various kinds of cells, and there are some clinical trials regarding their utilization for patients. Conclusion Currently, cord blood-MSCs have been tested for cartilage and lung diseases. Umbilical cord-MSCs were tested for liver and neural disorders. However, these MSCs can be isolated, expanded, and cryopreserved in a cell bank for patients in need.
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