جستجوی مقالات مرتبط با کلیدواژه « cell differentiation » در نشریات گروه « پزشکی »

An optimal culture medium that can rapidly and efficiently proliferate cells ex vivo is very crucial for developing mesenchymal stem cells (MSCs)-based tissue engineering and regenerative medicine. We developed a set of MSCs ex vivo proliferation medium, mscGOTM XF, which consists of a basal medium and a screened human platelet lysate. In this study, the developed mscGOTM XF medium was prepared, and then testified by human MSCs isolated from bone marrow, umbilical cord, and fat tissue. The proliferation, surface markers, differentiation, and chromosomal stability of MSCs cultured in mscGOTM XF medium were investigated. The mscGOTM XF medium could sustain MSCs at a high proliferation rate, with the population doubling time of 16 to 39 hours (depending on the type and passage number of MSCs). The proliferated MSCs could express CD105, CD90, and CD73, lack expression of CD34 and CD45; and maintain the capacity to differentiate into adipocytes, osteoblasts, and chondrocytes. Additionally, G-Band karyotyping data confirmed chromosome stability in the duration of cell culture at passage 5 and passage 7. The mscGOTM XF medium could sustain MSCs proliferation ex vivo and exhibit the potential to be developed into a clinical-grade cell culture medium kit.

As the repair capacity of the nervous system is low, stem cell therapy is a trend for replacement therapy. Dental pulp stem cells (DPSCs) have the potential to differentiate into many tissues, such as neurons. Harmine (7-methoxy-1methyl-9H-pyrido[3,4-b] indole) is an alkaloidal component of medicinal plants with a long history in traditional medicine. Alginate is a biocompatible hydrogel widely used as a biomaterial base in various scaffolds.

This study investigated whether harmine and encapsulation of cells in alginate hydrogel could improve DPSCs differentiation into neural cells.

DPSCs were cultured under standard stem cell culture conditions, then encapsulated in alginate hydrogel, and treated with differentiation medium with and without harmine. After 14 days, cell proliferation and differentiation were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, real-time polymerase chain reaction (RT-PCR), and flow cytometry.

Harmine (5 and 10 M) significantly increased the proliferation and viability of DPSCs compared to the control group in both two-dimensional and three-dimensional culture systems (P < 0.05). The expression levels of three neural cell markers (nestin, microtubule-associated protein [MAP-2], and -tubulin III) in DPSCs-derived neural cells cultured in two-dimensional and three-dimensional culture systems were significantly increased in harmine-treated two-dimensionalandthree-dimensional culture systems compared to the control group (P < 0.05).

Either harmine or alginate hydrogel had an accelerating effect on DPSCs differentiation into neural cells. Harmine also increased the proliferation of the cells.

Sox2 (SRY box2) is an essential transcription factor that plays a vital role in spermatogenesis and regulates the genes in this process. Sox2 is important for pluripotency, self-renewal, and even spermatogonial stem cell differentiation. This gene is found in pluripotent and specialized cells, and it is involved in their biological activities.

Protein-protein interaction (PPI) network analysis was performed during spermatogenesis using NCBI, STRING, and Cytoscape databases. Then, after isolating spermatogonial stem cells from 6 C57BL/6 mice, mouse embryonic stem cells and ES-like cells were prepared. In the following, Sox2 expression was examined in differentiated and undifferentiated spermatogonia by immunohistochemistry (IMH), immunocytochemistry (ICC), and Fluidigm PCR (polymerase chain reaction). Finally, the results were compared using the Kruskal-Wallis and Dunn tests at the significance level of p<0.05.

The results of this experiment showed that contrary to expectations, Sox2 has cytoplasmic expression in undifferentiated cells and nuclear expression in differentiated cells in in vitro conditions. In addition, the expression of Sox2 increased during differentiation. Fluidigm PCR showed a significantly higher expression of Sox2 (p<0.05) in differentiated compared to undifferentiated spermatogonia. Sox2 has an interaction with other genes during spermatogenesis such as Oct4, Nanog, Klf4, Stra8, Smad1, Tcf3, and Osm.

Sox2, which is known as a pluripotency marker, has a vital role in spermatogenesis and could be a differential marker. Sox2 has strong connections with other genes such as Oct4, Nanog, Klf4, Tcf3, Osm, Stra8, Lim2, Smad1, Gdnf, and Kit.

Statement of the Problem:

 Currently, the reconstruction of bone defects with new platelet concentrates is considered a significant challenge in periodontics.

This study aimed to evaluate advanced- platelet rich fibrin (A-PRF) and leukocyte- and platelet rich fibrin’s (L-PRF) effects on the proliferation and differentiation of MG-63 cells.

In this in vitro study, blood samples of five healthy non-smoking volunteers were collected and immediately centrifuged according to the two protocols of Choukroun and Ghanaati, without adding any anticoagulants, to prepare L-PRF and A-PRF. After freezing the clots for one hour, they were crushed and centrifuged once more. After culturing MG-63 cells, the effects of 20%, 10%, 1%, and 0.5% concentrations of A-PRF and L-PRF extracts on cell proliferation and mineralization were evaluated by methyl thiazolyl tetrazolium (MTT) assay and Alizarin Red staining, respectively.

Generally, survival and proliferation in the L-PRF group at both time intervals were higher than the A-PRF group and increased with increasing the extract concentration. However, in the A-PRF group, there were no significant differences between the different concentrations, and only the number of cells increased over time. After three days, in the study on mineralization, nodule formation was observed only in the positive control group (osteogenic). In seven days, mineralized nodules were formed in all groups with different concentrations of A-PRF, but not in any of the L-PRF groups.

According to the results, L-PRF increased proliferation, and A-PRF exerted a positive effect on the differentiation of MG-63 cells.

For tissue engineering and clinical translation strategies, it is essential to have a reliable and safe lineage-specific differentiation of stem cells. To deal with several problems caused by growth factor delivery systems and growth factors, exosomes have been used as biomimetic tools to trigger the differentiation of stem cells. It is believed that cell type-specific exosomes can induce lineage-specific differentiation of stem cells. Exosomes trigger cell viability, cell proliferation and differentiation, embryonic implantation, and migration. They have been used successfully in regenerative medicine, such as liver fibrosis, renal diseases, cardiac ischemia, stroke, and skin injuries. The findings highlighted the necessity to take into account the condition and source of exosome donor cells before selecting them for therapeutic use.

Primordial germ cell (PGCs) lines are a source of a highly specialized type of cells, characteristically oocytes,during female germline development in vivo. The oocyte growth begins in the transition from the primary follicle. It isassociated with dynamic changes in gene expression, but the gene-regulating signals and transcription factors that control oocyte growth remain unknown. We aim to investigate the differentiation potential of mouse bone marrow mesenchymal stem cells (mMSCs) into female germ-like cells by testing several signals and transcription factors. In this experimental study, mMSCs were extracted from mice femur bone using the flushingtechnique. The cluster-differentiation (CD) of superficial mesenchymal markers was determined with flow cytometric analysis. We applied a set of transcription factors including retinoic acid (RA), titanium nanotubes (TNTs), and fibrin such as TNT-coated fibrin (F+TNT) formation and (RA+F+TNT) induction, and investigated the changes in gene, MVH/ DDX4, expression and functional screening using an in vitro mouse oocyte development condition. Germ cell markers expression, (MVH / DDX4), was analyzed with Immunocytochemistry staining, quantitative transcription-polymerase chain reaction (RT-qPCR) analysis, and Western blots. The expression of CD was confirmed by flow cytometry. The phase determination of the TNTs and F+TNT were confirmed using x-ray diffraction (XRD) and scanning electron microscope (SEM), respectively. Remarkably, applying these transcription factors quickly induced pluripotent stem cells into oocyte-like cells that were sufficient to generate female germlike cells, growth, and maturation from mMSCs differentiation. These transcription factors formed oocyte-like cells specification of stem cells, epigenetic reprogramming, or meiosis and indicate that oocyte meiosis initiation and oocyte growth are not separable from the previous epigenetic reprogramming in stem cells in vitro. Results suggested several transcription factors may apply for arranging oocyte-like cell growth and supplies an alternative source of in vitro maturation (IVM).

Nitric oxide (NO) is a signaling molecule that is required for the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). According to previous reports, high concentrations of sodium nitroprusside (SNP) inhibit the osteogenic differentiation of BMSCs, while its low concentration promotes this process.

The present investigation focused on evaluating the underlying mechanism of the osteogenic differentiation of BMSCs treated with low concentrations of SNP as an NO generating agent.

The BMSCs after the 3rd passage was differentiated to osteoblasts when treated with 100 µM for 1 hour every 48 hours until 5, 10, 15, and 20 days of incubation. Then, the matrix production was estimated by quantitative alizarin red assay and calcium determination. The expression of different genes involved in osteogenic differentiation was statistically determined using the reverse transcriptase polymerase chain reaction. Finally, alkaline phosphatase activity was measured by a commercial kit.

The exogenous NO caused a significant (P<0.05) increase in the matrix production of differentiated BMSCs from day 5 to 20. The results showed the elevation of alkaline phosphatase activity and the up-regulation of its gene. Eventually, an increase was observed in the expression of a cascade of other genes such as osteonectin, Bmp7, Smad1, Runx2, and Raf1 in treated BMSCs.

Overall, short-time treatment with a low concentration of exogenous NO increases the matrix production via gene up-regulation and protein production, which might open a new window in treating the low-density bone complication.

Dendritic cells (DCs) are a group of bone marrow-derived cells that play a crucial role in innate and acquired immune responses. Bone marrow-derived dendritic cells (BMDC) are used in many studies, so the efficiency and purity of the differentiated cells are essential. This study aimed to investigate the effect of several parameters, including the age of mice, cell culture medium, and swirling of the culture plate, to increase the efficiency of the induced cells, considering the standard protocols. Bone marrow-derived dendritic cells were induced from both juvenile and adult mice bone marrow cells. Then, the purity of CD11c+ cells was compared between juvenile mice BMDCs and adult mice BMDCs. Cells were cultured in an enriched and non-enriched medium, and some wells were swirled when changing the medium on the 3rd day. Then the effect of enriched medium and swirling before medium replacement were evaluated based on the expression of the CD11c marker. The efficiency of DCs differentiation (CD11c+ cells) was higher when juvenile mouse bone marrow precursors were used compared to adult mice; using the enriched media with supplements and swirling the well before media replacement significantly affected the purity of immature CD11c+ cells. Due to our results, using juvenile mice, an enriched culture medium, and physical removal of granulocyte cells could significantly improve the purity and efficiency of CD11c+ cells. Therefore, considering these three items in the production protocol of these cells can probably reduce the use of lymphocyte-removing antibodies and purification methods.

In vitro obtaining oocytes can be an appropriate alternative for patients with gonadal insufficiency or cancer survivors. The purpose of the current research was isolating stem cells from ovarian cortical tissue as well as evaluating the effectiveness of follicle stimulating hormone (FSH), basic fibroblast growth factor (bFGF), and neurotrophin 3 (NT3) in differentiating to oocyte-like cells.

A human ovary was dissected and cortical tissue pieces were cultured for cell isolation. Isolated cells were divided into 8 groups (3 cases in each group) of control, FSH, NT3, bFGF, FSH+NT3, FSH+bFGF, NT3+bFGF, and FSH+NT3+ bFGF. Pluripotency specific gene (OCT4-A and Nanog), initial germ cells (c-KIT and VASA) and PF growth initiators (GDF-9 and Lhx-8) were evaluated by qRT-PCR. Experiments were performed in triplicate and there were 3 samples in each group. The results were analyzed using one-way ANOVA and p-value less than 0.05 was considered statistically significant.

Flow cytometry results showed that cells isolated from the ovarian cortex expressed markers of pluripotency. The results showed that the expression of Nanog, OCT4, GDF-9 and VASA was significantly increased in FSH+NT3 group, while treatment with bFGF caused significant expression of c-KIT and Lhx-8 (p<0.05). Also, according to the results, isolated cells treated with NT3 significantly increased c-KIT expression.

According to our results, the ovarian cortex cells could be differentiated into primordial follicles if treated with the proper combination of FSH, bFGF, and NT3. These findings provided a new perspective for the future of in vitro gamete proudest.

The rates of invariant natural killer T (iNKT) cells in vivo are very low, and the amounts of cells obtained directly from the body are hard enough to fulfill their potential in clinical application. To overcome this problem, we subcutaneously injected alpha-galactosylceramide (α-GalCer) into DBA/1 mice and thymic single cells were isolated and cultured in vitro. Fluorescence-activated cell sorting was used to detect the iNKT cells and their subsets in the thymus after the injection of α-GalCer by different methods. In addition, in vitro changes of single-cell suspensions and their cytokines in culture supernatants were assessed. Compared with the α-GalCer multiple subcutaneous injection group, the rates of iNKT cells in the α-GalCer single subcutaneous injection group were markedly higher at each time point, while the highest levels of iNKT1 and iNKT2 cells were observed on day 4 and 8, respectively. In α-GalCer single subcutaneous injection for 8 days and thymic mononuclear cell cultured for 14 days group, the expansion rate of iNKT cells was significantly faster than the other groups, while it reached a peak for iNKT1 cells. Interferon-gamma was consistent with the development of iNKT1 cells, however no difference was found between the cultured iNKT cells in vitro and the natural iNKT cells in vivo in terms of cytokine production. Herein, we introduced a method in which antigenic stimulation in vivo and directed induction in vitro yielded high levels of iNKT cells with specific functions.

A wide variety of cytokines are released from human amniotic membrane cells (hAMCs), which can increase the rate of differentiation of mesenchymal stem cells into the neurons. We studied the effect of Retinoic Acid (RA) on the differentiation rate of human Umbilical Cord Mesenchymal Stem Cells (hUMSCs) which were co-cultured with hAMCs. 

In this experimental study, both hUMSCs and hAMCs were isolated from postpartum human umbilical cords and placenta respectively. The expression of mesenchymal (CD73, CD90 and CD105), hematopoietic and endothelial (CD34 and CD45) markers in hUMSCs were confirmed by flow cytometry. The hUMSCs were cultured in four distinct groups: group 1) Control, group 2) Co-culture with hAMCs, group 3) RA treatment and group 4) Co-culture with hAMCs treated by RA. Twelve days after culturing, the expression of NSE, MAP2 and ChAT differentiation genes and their related proteins were examined by real-time PCR and immunocytochemistry respectively. 

The flow-cytometry analysis indicated increased expression of mesenchymal markers and a low expression of both hematopoietic and endothelial markers (CD73:98.24%, CD90: 97.32%, CD105: 90.75%, CD34: 2.96%, and CD45:1.74%). Moreover, the expression of both NSE and MAP2 markers was increased significantly in all studied groups in comparison to the control group  On the other hand, the expression of ChAT had a significant increase in the group 2 and 4 (RA and RA+ co-culture).

RA can be used as an effective inducer to differentiate hUMSCs into cholinergic-like cells, and hAMCs could increase the number of differentiated cells as an effective factor.

Stem cell differentiation therapy is a promising strategy in cancer treatment. we show that protein cocktail prepared from serum starved fibroblasts has therapeutic potential based on this strategy.

The condition medium was prepared from foreskin isolated fibroblasts and analyzed by Liquid chromatography electrospray ionization mass spectrometry-mass spectrometry (LC-ESI-MS/MS). LA7 mammary gland cancer stem cells originated tumors were induced in Sprague Dawley rats. The rats treated subcutaneously with DMEM (group A), condition medium (group B), or normal saline (group C) once daily for 7 days. Then the tumors were removed and divided into the two parts, one part was used to quantify gene expression by stem-loop RT-qPCR assay and the other part was used for Hematoxylin & Eosin (H & E), Giemsa, and immunohistochemistry (IHC) staining.

All induced tumors appeared as sarcomatoid carcinoma (SC). Immunohistochemistry staining confirmed this conclusion by recognizing the tumor as Ki67+, cytokeratin+, vimentine+, and estrogen receptor negative SC. RT-qPCR analysis revealed that Oct4-, Sox-2, Nanog- gene expression was much reduced in the condition medium treated tumors versus proper controls (p< 0.05). Tissue necrosis was more prevalent in this group while tumors volume was diminished almost by 40%. The LC-ESI-MS/MS analysis unrevealed the stemness reducing and the cell death inducing proteins such as, pigment epithelium-derived factor (PEDF), insulin like growth factor binding protein-5 (IGFBP-5) and -7 (IGFBP-7) in the condition medium.

This study showed that the substances released from starved human fibroblasts were able to down-regulate the stemness-related genes and induce necrosis in LA7 derived tumors.

Since bone defects can result in different disabilities, many efforts have been made to bone tissue engineering. In this case, scaffolds play an important role as a key element of tissue engineering in providing three-dimensional structure for cell growth in vitro The aim of the present study was to provide the three-dimensional biological bioscaffold from the bovine femur dense bone and investigate the possibility of its potential for application in tissue engineering as biological 3D ECM bioscaffold via mesenchymal stem cells seeding and differentiation toward bone tissue. For the preparation of bioscaffolds, after cutting bovine femur bone into small pieces, demineralization and decellularization were done. Bioscaffolds biocompatibility was evaluated using an MTT assay. The morphological and cell adhesion characteristics of Bone marrow mesenchymal stem cells (BMSCs) on the bioscaffolds were evaluated using Scanning Electron Microscopy (SEM) technique. Finally, the cells were treated with an osteogenic differentiation medium and then evaluated for differentiation. Histological studies showed that the use of sodium dodecyl sulfate (2.5%) for 8 h eliminated the cells. Radiography and calcium oxalate test confirmed demineralization. MTT assay and SEM studies showed that the obtained bioscaffolds are biocompatible and could provide an optimum three-dimensional environment for cell adhesion and movement. Moreover, the Alizarin red staining showed a higher differentiation rate for BMSCs. In the present study, bone-derived 3D bioscaffold showed an important role in the growth and differentiation of BMSCs, due to the natural characteristics, cell adhesion properties, and potential to enhance differentiation toward bone tissue. It may have the potential for use as bioscaffold as supporting metrics for maintenance, growth in bone tissue engineering.

Low-level laser therapy (LLLT) has been reported to improve cell proliferation and differentiation. The stem cells derived from dental apical papilla (SCAPs) are a promising therapy because they are easily obtained from immature human teeth. The effect of LLLT over SCAPs is still unknown. This study aimed to evaluate the proliferation and osteogenic potential of the SCAPs stimulated with LLLT.

SCAPs were isolated from the third molars of a healthy donor and characterized according to the minimum established criteria. SCAPs were cultured for 24 hours before being exposed to LLLT. Cells were exposed to different doses, energy, and wavelengths for selecting the irradiation parameters. SCAPs proliferation was evaluated with the MTT assay at 24 hours and 7-day post-laser exposure. VEGF and TGF β 2 expression were assessed with a specific enzyme-linked immunosorbent assay (ELISA). The osteogenic differentiation potential was analyzed with alizarin red staining, and the nodule quantification was performed by the relative optical density (ROD) analysis using ImageJ software.

The cells isolated from the apical papilla showed phenotype and stem cell properties. SCAPs irradiated with one dose at 6 J/m 2 and 650 nm exhibited significantly higher proliferation ( P > 0.05) than the controls nonirradiated. LLLT stimulated SCAPs’ expression of factors VEGF and TGF β 2. Also, SCAPs irradiated showed higher osteogenic activity ( P < 0.05).

LLLT promotes proliferation, osteogenic differentiation, and VEGF and TGF β 2 expression on SCAPs. LLLT is a practical approach for the preconditioning of SCAPs in vitro for future regenerative therapies. More studies are needed to determine the underlying molecular processes that determine the mechanism of the LLLT.

Previously we reported functional leukocyte immunoglobulin-like receptor A3 (LILRA3) leads to susceptibility and sub-phenotypes of several autoimmune diseases. LILRA3 levels in blood serum and CD14+ monocytes enhanced in systemic lupus erythematosus and resulted in disease severity. However, the mechanism of LILRA3 in the pathogenesis of autoimmunity remains elusive. This study aims to explore the potential impact of LILRA3 on the differentiation, maturation, and function of monocyte-derived DCs (MoDCs). The human monocytic cell line (THP-1) was cultured to derive MoDCs in vitro. We performed plasmid transfection to examine the impact of LILRA3 on monocyte differentiation. Surface markers on MoDCs were measured using FACS. To assess the function of mature MoDCs, IL-12p70, IFN-γ and IL-4 levels were detected after the mixed leucocyte response by enzyme-linked immunosorbent assay. Western blot assay was employed in this study to determine the signaling pathways in MoDCs activation. LILRA3 promotes MoDCs maturation, our results showed significant up-regulation of CD40, CD80, CD86, CD209, and HLA-DR and increased production of pro-inflammatory cytokine IL-12. LILRA3-treated MoDCs exhibited a robust proliferation of allogeneic CD4+ T cells and induced naïve CD4+ T cell polarization into the Th1 phenotype. Furthermore, the preceding activation of MoDCs maturation and LILRA3 function might be attributed to p38 MAPK and STAT1 signaling pathway’s aberrant activation. This is the first study to report that LILRA3 played a critical role in promoting MoDCs maturation and directing MoDCs to modulate Th1 cell differentiation, which may have a role in the pathogenesis of autoimmune diseases.

Since photobiomodulation therapy (PBMT) favors in vitro mesenchymal stem cell (MSC) preconditioning before MSC transplantation, increasing the proliferation of these cells without molecular injuries by conserving their characteristics, in the present in vitro study we analyzed the effect of PBMT on the proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs).

Irradiation with an InGaAIP Laser (660 nm, 10 mW, 2.5 J/cm2, 0.08 cm2 spot size, and 10 s) was carried out. The cells were divided into four groups: CONTROL [cells grown in Dulbecco’s Modified Eagle Medium (DMEM)], OSTEO (cells grown in an osteogenic medium); PBMT (cells grown in DMEM+PBMT), and OSTEO+PBMT (cells grown in an osteogenic medium plus PBMT). The cell proliferation curve was obtained over periods of 24, 48 and 72 hours using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT) assay. Osteogenic differentiation was analyzed by the formation of calcium nodules over periods of 7, 14 and 21 days. Morphometric analysis was performed to quantify the total area of nodular calcification.

The highest cell proliferation and cell differentiation occurred in the OSTEO+PBMT group, followed by the PBMT, OSTEO and CONTROL groups respectively, at the observed times (P < 0.05).

PBMT enhanced the osteogenic proliferation and the differentiation of hUCMSCs during the periods tested, without causing damage to the cells and preserving their specific characteristics, a fact that may represent an innovative pretreatment in the application of stem cells.