جستجوی مقالات مرتبط با کلیدواژه « wharton's jelly » در نشریات گروه « پزشکی »

To investigate the efficacy of Wharton's jelly mesenchymal stem cells (WJSCs) and their conditioned medium (CM) for corneal nerve regeneration in rats with diabetic keratopathy. Streptozotocin (STZ)-induced male diabetic (DM) rats (250–300 g) were divided to four groups (n=7/group): Control, DM, DM with WJSCs (DM+WJ), and DM with CM treatment (DM+CM). DM+WJ and DM+CM group received WJSCs or CM, respectively, topically by eye drops. Corneal sensibility, corneal epithelial layer integrity, histology, expression of GAP-43 and TUBB3 on mRNA level and their immunohistochemical expression were examined after two weeks of treatment. There were changes in corneal sensibility and corneal integrity between normal control and diabetic groups with/without WJSC or CM injection. Total central corneal thickness was significantly higher in DM+CM (249.81 ± 43.85 μm) than in control (174.72 ± 44.12 μm, p=0.004) and DM groups (190.15 ± 9.63 μm, p=0.03). GAP-43 mRNA expression levels of DM+WJ and DM+CM groups were higher compared with DM and control groups. TUBB3 mRNA level was increased after CM (p=0.047), but not after WJSCs treatment (p=1.00). GAP-43 and TUBB3 immunohistochemical expression of nerve fibers along the epithelial layer significantly increased in DM+WJ and DM+CM compared with DM group. Our findings showed that WJSCs and their CM improved corneal nerve regeneration in rats with diabetic keratopathy.

In utero xenotransplantation of stem cells in the abnormal fetuses is effectively used to treat several genetic illnesses. The current research was aimed to evaluate structural and morphological alterations in the liver of rabbit fetuses following xenotransplantation of human Wharton’s jelly-derived mesenchymal stromal/stem cells (hWJ-MSCs), using a stereological technique. hWJ-MSCs were isolated from human umbilical cord and their authenticity was established by flow-cytometry and differentiation. At gestational day 14, the rabbits were anesthetized and hWJ-MSCs were injected into uteri of 24 fetuses. 22 fetuses were born successfully. Ten rabbit liver specimens were prepared from injected fetuses including eight rabbits on day 3 following birth and two rabbits on the 21st post-natal day. The non-injected fetuses were considered as positive controls. The livers of the control and hWJ-MSCs-treated rabbits were fixed, processed, stained, and examined through stereological approaches. In the hWJ-MSCs-treated group, the mean of liver weight and volume enhanced ~42% and ~78% comparing with the control ones. The total volume of the hepatocytes increased ~63% and that of sinusoids almost triplicated in the treated rabbits. The total volume of the central veins increased ~70%. The total number corresponding to hepatocytes in the experimental group enhanced ~112% in comparing with control rabbits. The total volume of the hepatocyte nuclei in the experimental group enhanced ~117% in comparing with control rabbits. In conclusion, after xenotransplantation of human MSCs, host tissue microenvironments (here the rabbit liver) altered quantitative factors corresponding to the liver tissue and hepatocyte morphometric indices.

Mesenchymal stem cells (MSCs) enhance tissue repair through paracrine effects following transplantation. The versican protein is one of the important factors contributing to this repair mechanism. Using MSC conditioned medium for cultivating monocytes may increase versican protein production and could be a good alternative for transplantation of MSCs. This study investigates the effect of culture medium conditioned by human MSCs on the expression of the versican gene in peripheral blood mononuclear cells (PBMCs) under hypoxia-mimetic and normoxic conditions.

The conditioned media used were derived from either adipose tissue or from Wharton’s jelly (WJ). Flow cytometry for surface markers (CD105, CD73, and CD90) was used to confirm MSCs. The PBMCs were isolated and cultured with the culture media of the MSC derived from either the adipose tissue or WJ. Desferrioxamine and cobalt chloride (200 and 300 µM final concentrations, respectively) were added to monocytes media to induce hypoxia-mimetic conditions. Western blotting was applied to detect HIF-1α protein and confirm hypoxia-mimetic conditions in PBMC. Versican gene expression was assessed in PBMC using RT-PCR. Western blotting showed that the expression of HIF-1α in PBMC increased significantly (p < 0.01).

RT-PCR results demonstrated that the expression of the versican and VEGF genes in PBMC increased significantly (p < 0.01) in hypoxia-mimetic conditions as compared to normoxia.

Based on the findings in the present study, the secreted factors of MSCs can be replaced by direct use of MSCs to improve damaged tissues.

The β-catenin signaling pathway promises the potential for differentiation of stem cells into definitive endoderm (DE) cells as precursors of beta cells. Therefore, it can be considered as an inducer for cell replacement therapies in diabetes. The main goal of this research is to successfully culture and induce differentiation of human Wharton’s jelly mesenchymal stem cells (hWJMSCs) into Sox17-expressing cells using a Wnt/β-catenin pathway agonist (SKL2001) plus nanoparticles on a polylactic acid/chitosan (PLA/Cs) nanocomposite scaffold.

Materials And Methods:

In this experimental study, the nanocomposite was prepared through an electrospinning method and hWJMSCs were isolated through an explant technique. The morphology and the cell viability were evaluated by scanning electron microscopy (SEM) and 3-(4, 5- Dimethylthiazol-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Here, we present two differentiation protocols: the first one is induction with SKL2001; and the second one is with a combination of SKL2001 and zinc oxide nanoparticles (nZnO). Real-time quantitative reverse transcription (QRT-PCR) and immunocytochemistry analysis are carried out to examine the expression of specific markers in the differentiated cells.

The nanocomposite had appropriate biocompatibility for cell adhesion and growth. While the hWJMSCs cultured on the PLA/Cs scaffolds differentiated into DE cells in the presence of SKL2001, introducing nZnO to their environment increased the differentiation process. Analyses of DE-specific markers including SOX17, FOXA2, and gooscoid (GSC) genes in mRNA level, indicated significantly high levels of expression in the SKL2001/nZnO group, followed by SKL2001 group compared to the control.

Our results show the beneficial effects of the Wnt/β-catenin pathway agonist in three-dimensional (3D) cultures in cell replacement therapy for diabetes.

The past decade has witnessed a rapid growth in harnessing the potential of adult stem cells for regenerative medicine. An investigational new drug (IND) or a regenerative medicine advanced therapy (RMAT) product must fulfil many requirements, such as stability studies, after cryopreservation. Such studies are important to ascertain the utility of off-the-shelf allogeneic cells for clinical applications. The present work describes a complete characterisation of xenofree human Wharton’s Jelly mesenchymal stromal cells (hWJ-MSCs) before and up to 28 months post-cryopreservation.

In this experimental study, culture methods that involved plasma derived human serum and recombinant trypsin were used to develop clinical grade cells. Complete cell characterisation involved flow cytometry studies for viability, positive and negative markers, colony forming unit (CFU) potential, population doubling time (PDT), soft agar assay to evaluate in vitro tumourigenicity, karyotype analysis and differentiation studies which were performed before and at 6, 12, 18 and 28 months post-cryopreservation.

Our data showed consistency in the flow cytometry, CFU assay, PDT, soft agar assay, karyotyping and differentiation studies.

Using our protocols for extended xeno-free culture and cryopreservation of hWJ-MSCs, we could establish the shelf life of the cell-based product for up to 28 months.

Because of different potentials of T-cell subtypes in T-cell based cellular immunotherapy approaches such as CAR-T cell therapies; Regarding the high cost of the serum-free specific culture media, having distinct control on T-cell subset activation, expansion and differentiation seem crucial in T-cell expansion step of cell preparation methods. By the way, there was no clear data about the effect of acellular Wharton’s Jelly (AWJ) on T-cells expansion, activation or differentiation status. So, we have launched to study the effect of AWJ on T-cell’s immunobiological properties.

CD3+ T-cells were isolated from healthy bone marrow allogeneic donors, sorted by FACS method and cultured on either routine phyto-hemagglutinin complemented and different concentrations of AWJ, lag phase and doubling time of the cells calculated from cell growth curve. After 3, 7 and 14-days T-cell subtypes cell markers and cell activity related genes expression rate have been evaluated by flow cytometry and real-time polymerase chain reaction (PCR) methods respectively.

AWJ in a 1:1 ratio compared with contemporary lymphocyte culture media showed significant activating and proliferative capacities. The introduced condition has not affected the frequency of CD4+ subpopulation of T-cells, but significantly increased even CD8+ cells and immune-activator genes in T-cells. The regulatory and memory subsets of T-cells in this study have not affected significantly.

the study results revealed that AWJ can be utilized as a supportive substance to increase the memory properties of the T-cells, gives control to design a selective medium for expanding and differentiating memory T-cells, relatively.

Osteoarthritis (OA) is a chronic disease that attacks joints and bones
which can be caused by trauma or other joint diseases. Stem cell and Conditioned
Medium (CM) of stem cells are developed for OA therapy, which is minimally invasive. It can decrease inflammation and be a replacement for knee surgery. This study
aimed to utilize human Wharton’s Jelly-Mesenchymal Stem Cells (hWJMSCs) as an alternative OA therapy.

CM from hWJMSCs induced by IGF1 was collected. The OA cells model (IL1βCHON002) culture was treated as follows: 1) with hWJMSCs-CM 15% (v/v); 2) with
hWJMSCs-CM 30% (v/v); 3) with IGF1-hWJMSCs (IGF1-hWJMSCs-CM) 15% (v/v); 4)
with IGF1-hWJMSCs-CM 30% (v/v). Parameters including inflammatory cytokines
(IL10 and TNFα), extracellular matrix degradation (MMP3 expression), and chondrogenic marker (COL2 expression) were determined.

The most significant increase in COL2 chondrogenic markers was found in
IL1β-CHON002 treatment using 15% CM of hWJMSCs induced with IGF1. CM of hWJMSCs can reduce inflammatory cytokines (TNFα and IL10) and matrix degradation
mediator MMP3. Better result was gained from IGF1-induced hWJMSCs-CM.

CM of IGF1-hWJMSCs reduce inflammation while repairing injured joint in
the human chondrocyte OA model.

 Cervical cancer is one of the most common cancers of women in the world, which causes high mortality. The human umbilical cord Wharton's jelly stem cells (hWJSCs) can inhibit various cancer cells.

 This study aimed to investigate the effects of conditioned medium and cellular extract of human umbilical cord hWJSCs on cervical cancer cell line, Hela.

 After isolation and primary culture of hWJSCs, conditioned medium and cellular extracts of hWJSCs were prepared, and its anti-proliferative effects were evaluated on cervical cancer cells, Hela using micro-culture tetrazolium (MTT) assay. After total RNA extraction and cDNA synthesis, expression of apoptosis-related BCL-2 and BAX genes were evaluated using real-time PCR.

 The results showed that conditioned medium (55% concentration in 72 hours) and cellular extraction (10% concentration in 24 hours) caused death of 50% cancer cells (IC50). The anti-cancer effects of conditioned medium and cellular extraction were concentration- and time-dependent. The conditioned medium and cellular extract of hWJSCs significantly down-regulated and up-regulated mRNA expression of apoptosis-related BCL-2 and BAX genes, respectively.

 Our study showed that conditioned medium and cellular extract of human umbilical cord hWJSCs inhibit viability and proliferation of cervical cancer cells. However, further studies on animal models are necessary for more accurate results.
 

The ability of stem cells to differentiate into different cell types makes them a key component of healing damage in regenerative medicine. As human umbilical cord Wharton’s jelly (HUCWJ) is available non-invasively, HUCWJ does not raise any ethical issues with higher differentiation potential compared to adult stem cells. With the ability to express embryonic stem cell markers, HUCWJ can be considered as a good candidate in regenerative medicine applications. The objective of this study was to find if these cells form cell aggregates with the same features as that formed by embryonic stem cells (embryoid body) and could form three germ layers. Eighteen umbilical cords were of healthy infants with parent permission. The umbilical cords were cut into small pieces and the explants were cultured. At the third passage, 1000, 5000 and 10000 cells/ 20 µL were cultured in hanging drops for 3 days. Then, they were incubated for additional 3 days in non-adhesive dishes. As the center of cell aggregates formed from 5000 and 10000 cells were darker than those formed from 1000 cells, this study focused on the aggregates formed by 1000 cells for further assessments. The immunocytochemistry and flowcytometry were performed using 3 color antibodies to detect the markers for three germ cell lineages. The immunohistochemistry data showed that the embryoid-body-like aggregates expressed a low amount of ectodermal and endodermal markers and most of the cells expressed mesodermal markers. The flowcytometry percentage of the cells in each aggregate that expressed ectodermal marker Otx2 was17.1% and endodermal marker, Sox 17 was 5.49%. The frequency of cells expressing mesodermal marker Brachyury was high (75.0%). Flowcytometry also showed the percentages by mathematical evaluation and we did this three times for our result accuracy. These aggregates mainly kept their mesenchymal state and showed a poor differentiation potential toward ectoderm and endoderm identity.

Diabetes caused by insulin production disturbance is considered as the most common metabolic disorder all over the world. Diabetes may outbreak because of low insulin secretion by Islets of Langerhans β-cells, insulin resistance or both of them. In this way, using stem cells, which have the capability to differentiate into Pancreatic β-cells, is one of novel methods in this field. MSCs are the most important candidates for cellular therapy.
Insulin level was examined using ELIZA method. In order to examine the morphology of differentiated cells, they were stained by Dithizone. Insulin-producer cells are cells which turn into red as a result of staining. Specific gene involving insulin-producing cells was evaluated by Real Time-PCR method.
The ELISA results showed that the treated cells secreted more insulin than the control group. Moreover, we found differentiation of MSCs toward insulin-secreting cells. In order to evaluate insulin production in clusters on day 21 of differentiation, we used dithizone (DTZ) staining. PDX-1 gene was confirmed by RT- PCR analysis.
In this study, we differentiated MSCs into insulin-producing cells in vitro. It is concluded that MSCs may be considered as an excellent candidate in β-cell therapy in diabetes patients.

Wharton`s jelly-derived mesenchymal stem cells (WJ-MSCs), have a high proliferation valency and they do not produce teratogen or carcinogen after subsequent transplantation. They are known as regenerative medicine. Thus more research is needed on the isolation and characterization of mesenchymal stem cells. In this experimental study, we obtained Wharton's jelly tissues from mothers during normal vaginal delivery, after obtaining their informed consent. Mesenchymal stem cells were isolated from cultured Wharton`s jelly, cultured, and were then examined for their proliferation, immunophenotypes, and differentiation capacities. The immunophenotypes of WJ-MSCs were analyzed by flow cytometry. Differentiation was performed resulting in osteogenic, chondrogenic and adipogenic cells. WJ-MSCs formed a homogenous monolayer of adherent spindle-shaped cells. Our results showed the high capacity of the proliferation of WJ-MSCs. Immunophenotyping further confirmed the purity of the isolated cells; their surface antigen expression showed the phenotypical properties like those of WJ-MSCs. The expanded cells were positive for CD 90, CD105, and CD44; they were negative for CD34 and HLA-DR surface markers. The cells had the adipocytic, osteocytic and chondrogenic differentiation capacity. The isolation and characterization of WJ-MSCs with high purity had been conducted, and the results were obtained in a short span. The present study has revealed the feasibility of the culture medium with high glucose and 15% FBS in isolation and proliferation of WJ-MSCs. When Wharton`s jelly pieces were put in the dry bottom of the flask, very effective separation of the MSCs was achieved.

In this study, we describe an efficient approach for stable knockdown of adenosine kinase (ADK) using lentiviral system, in an astrocytoma cell line and in human Wharton’s jelly mesenchymal stem cells (hWJMSCs). These sources of stem cells besides having multilineage differentiation potential and immunomodulatory activities, are easily available in unlimited numbers, do not raise ethical concerns and are attractive for gene manipulation and cell-based gene therapy.
In this experimental study, we targeted adenosine kinase mRNA at 3' and performed coding sequences using eight miR-based expressing cassettes of anti-ADK short hairpin RNA (shRNAs). First, these cassettes with scrambled control sequences were cloned into expressing lentiviral pGIPZ vector. Quantitative real time-polymerase chain reaction (qRT-PCR) was used to screen multi-cassettes anti-ADK miR-shRNAs in stably transduced U-251 MG cell line and measuring ADK gene expression at mRNA level. Extracted WJMSCs were characterized using flow cytometry for expressing mesenchymal specific marker (CD44) and lack of expression of hematopoietic lineage marker (CD45-). Then, the lentiviral vector that expressed the most efficient anti-ADK miR-shRNA, was employed to stably transduce WJMSCs.
Transfection of anti-ADK miR-shRNAs in HEK293T cells using CaPO4 method showed high efficiency. We successfully transduced U-251 cell line by recombinant lentiviruses and screened eight cassettes of anti-ADK miR- shRNAs in stably transduced U-251 MG cell line by qRT-PCR. RNAi-mediated down-regulation of ADK by lentiviral system indicated up to 95% down-regulation of ADK. Following lentiviral transduction of WJMSCs with anti-ADK miR- shRNA expression cassette, we also implicated, down-regulation of ADK up to 95% by qRT-PCR and confirmed it by western blot analysis at the protein level.
Our findings indicate efficient usage of shRNA cassette for ADK knockdown. Engineered WJMSCs with genome editing methods like CRISPR/cas9 or more safe viral systems such as adeno-associated vectors (AAV) might be an attractive source in cell-based gene therapy and may have therapeutic potential for epilepsy.

Mesenchymal stem cells (MSCs) have been introduced for cell therapy strategies in osteoarthritis (OA). Despite of their capacity for differentiation into chondrocyte, there are some evidences about their life-threatening problem after transplantation. So, some researchers shifted on the application of stem cells conditioned medium. The goal of this study is to evaluate whether Wharton's jelly derived stem cell conditioned medium (WJSCs- CM) can enhance the gene expression profile by chondrocytes in monolayer and mass culture systems.
Conditioned medium was obtained from WJSCs at fourth passage. Isolated chondrocytes were plated at density of 1×106 for both monolayer and high density culture. Then cells in both groups were divided into control (received medium) and experiment group treated with WJ-CM for 3 and 6 days. Samples were prepared to evaluate gene expression profile of collagen II, aggrecan, cartilage oligomeric matrix protein (COMP) and sox-9 using real-time RT-PCR.
After 3 days, Chondrocytes treated with WJSCs-CM expressed significantly higher level of genes compared to the control group in both culture systems. After 6 days, the expression of genes in monolayer cultivated chondrocytes was decreased but that of the mass culture were up-regulated significantly.
WJ-SCs-CM can increase the expression of cartilage-specific genes and can be introduced as a promoting factor for cartilage regeneration.

Immunosuppressive property of mesenchymal stem cells (MSCs) has great attraction in regenerative medicine especially when dealing with tissue damage involving immune reactions. The most attractive tissue sources of human MSCs used in clinical applications are bone marrow (BM), adipose tissue (AT), and Wharton's jelly (WJ) of human umbilical cord. The current study has compared immunomodulatory properties of human BM, AT, and WJ-MSCs.
Three different types of human MSCs were isolated, cultured, and characterized by flow cytometry and differentiation potentials. The MSCs were co-cultured with allogeneic phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMCs). The proliferation of PBMCs was assessed by flow cytometry of carboxyfluorescein succinimidyl ester (CFSE) stained cells and compared to each other and to the growth of PBMCs in the absence of MSCs, 3 days post co-culture. Additionally, the growth suppression was indirectly assessed by using the transwell culture system.
the proliferation of PBMCs reduced to 6.2, 7 and 15.4- fold in cultures with AT-MSCs, WJ-MSCs, and BM-MSCs, respectively, compared to the PHA-activated cells. When the growth suppression was indirectly assessed by using the transwell culture system, it was revealed that AT-MSCs, WJ-MSCs, and BM-MSCs caused growth reduction in PBMCs to 3, 8, and 8 -fold, respectively, compared to the PHA-activated cells.
These data collectively conclude that the immunomodulatory effects of MSCs, which may mostly carry out through direct cell to cell contact, are different between various sources. Accordingly results of this study may contribute to the application of these cells in cell therapy and regenerative medicine.

Umbilical cord blood is a good source of the mesenchymal stem cells that can be banked, expanded and used in regenerative medicine. The objective of this study was to test whether amniotic membrane extract, as a rich source of growth factors such as basic-fibroblast growth factor, can promote the proliferation potential of the umbilical cord mesenchymal stem cells. The study design was interventional. Umbilical cord mesenchymal stem cells were isolated from voluntary healthy infants from hospitals in Shiraz, Iran, cultured in the presence of basic-fibroblast growth factor and amniotic membrane extracts (from pooled - samples), and compared with control cultures. Proliferation assay was performed and duplication number and time were calculated. The expression of stem cell’s specific markers and the differentiation capacity toward osteogenic and adipogenic lineages were evaluated. Amniotic membrane extract led to a significant increase in the proliferation rate and duplication number and a decrease in the duplication time without any change in the cell morphology. Both amniotic membrane extract and basic-fibroblast growth factor altered the expressing of CD44 and CD105 in cell population. Treating basic-fibroblast growth factor but not the amniotic membrane extract favored the differentiation potential of the stem cells toward osteogenic lineage. The amniotic membrane extract administration accelerated cell proliferation and modified the CD marker characteristics which may be due to the induction of differentiation toward a specific lineage. Amniotic membrane extract may enhance the proliferation rate and duplication number of the stem cell through changing the duplication time.

Human Wharton’s Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) are easily available cells without transplant rejection problems or ethical concerns compared to bone-marrow-derived MSCs for prospective clinical applications. These cells display immunosuppressive properties and may be able to play an important role in autoimmune disorders. Regulatory T-cells (Treg) are important to prevent autoimmune disease development. Interleukin 35 (IL-35) induces the proliferation of Treg cell populations and reduces the activity of T helper 17 (Th17) and T helper 1 (Th1) cells, which play a central role in initiation of inflammation and autoimmune disease.Recent studies identified IL-35 as a new inhibitory cytokine required for the suppressive function of Treg cells. We created IL-35-producing hWJ-MSCs as a good vehicle for reduction of inflammation and autoimmune diseases. We isolated hWJ-MSCs based on explant culture. HWJ-MSCs were transduced at MOI=50 (Multiplicity of Infection) with lentiviral particles harboring murine Interleukin 35 (mIL-35). Expression of IL-35 in hWJ-MSCs was quantified by an IL-35 ELISA kit.IL-35 bioactivity was analyzed by inhibiting the proliferation of mouse splenocytes using CFSE cell proliferation kit. Frequency of CD4+CD25+CD127low/neg Foxp3+ Treg cells was measured by flow cytometry. There was an up to 85% GFP positive transduction rate, and the cells successfully released a high level of mIL-35 protein (750 ng/ml). IL-35 managed to inhibit CD4+ T cell proliferation with PHA, and improved the frequency of Treg cells.Our data suggest that transduced hWJ-MSCs overexpressing IL-35 may provide a useful approach for basic research on gene therapy for autoimmune disorders.