scholarly journals The Human IL-23 Decoy Receptor Inhibits T-Cells Producing IL-17 by Genetically Engineered Mesenchymal Stem Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-14
Author(s):  
Masoumeh Rostami ◽  
Kamran Haidari ◽  
Majid Shahbazi
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Mesenchymal Stem Cells ◽  
Autoimmune Diseases ◽  
Real Time ◽  
Real Time Pcr ◽  
Lentiviral Vector ◽  
T Helper ◽  
Mouse Splenocytes ◽  
Decoy Receptor

The immunomodulatory and self-renewable features of human adipose mesenchymal stem cells (hAD-MSCs) mark their importance in regenerative medicine. Interleukin 23 (IL- 23) as a proinflammatory cytokine suppresses T regulatory cells (Treg) and promotes the response of T helper 17 (Th17) and T helper 1 (Th1) cells. This pathway starts inflammation and immunosuppression in several autoimmune diseases. The current study for producing recombinant IL- 23 decoy receptor (RIL- 23R) using hAD-MSCs as a good candidate for ex vivo cell-based gene therapy purposes reducing inflammation in autoimmune diseases. hAD-MSCs was isolated from lipoaspirate and then characterized by differentiation. RIL- 23R was designed and cloned into a pCDH-813A- 1 lentiviral vector. The transduction of hAD-MSCs was performed at MOI (multiplicity of infection) = 50 with pCDH- EFI α- RIL- 23R- PGK copGFP. Expressions of RIL- 23R and octamer-binding transcription factor 4 (OCT- 4) were determined by real-time polymerase chain reaction (real time-PCR). Self-renewing properties were assayed with OCT- 4. Bioactivity of the designed RIL- 23R was evaluated by IL- 17 and IL- 10 expression of mouse splenocytes. Cell differentiation confirmed the true isolation of hAD-MSCs from lipoaspirate. Restriction of the enzyme digestion and sequencing verified the successful cloning of RIL- 23R in the CD813A-1 lentiviral vector. The green fluorescent protein (GFP) positive transduction rate was up to 90%, and real-time PCR showed the expression level of RIL-23R. Oct-4 had a similar expression pattern with nontransduced hAD-MSCs and transduced hAD-MSCs/ RIL-23R indicating that lentiviral vector did not affect hAD-MSCs characteristics. Downregulation of IL-17 and upregulation of IL-10 showed the correct activity of the engineered hAD-MSCs. The results showed that the transduced hAD-MSCs/ RIL- 23R, expressing IL-23 decoy receptor, can give a useful approach for a basic research on cell-based gene therapy for autoimmune disorders.

Effect of αCGRP on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Through Smad/Runx2 Signaling Pathway

2020 ◽  
Vol 10 (6) ◽  
pp. 868-873
Author(s):  
Shengxiang Huang ◽  
Haibo Mei ◽  
Rongguo He ◽  
Kun Liu ◽  
Jin Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Real Time Pcr ◽  
Caspase 3 ◽  
Alp Activity ◽  
Marrow Mesenchymal Stem Cells

The α-calcitonin gene-related peptide (α-CGRP) regulates bone metabolism and has potential applications in enhancing bone remodeling in vivo. However, α-CGRP's role in bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation remain unclear. Rat BMSCs were separated into control group, α-CGRP group and α-CGRP siRNA group, in which BMSCs were transfected with α-CGRP plasmid and α-CGRP siRNA respectively followed by analysis of α-CGRP level by real time PCR and ELISA, cell proliferation by MTT assay, Caspase 3 activity, ALP activity, formation of calcified nodules by alizarin red staining, Smad1 and Smad7 level by Western blot and Runx2 by real time PCR. αCGRP transfection into BMSCs significantly up-regulated CGRP, which could promote cell proliferation, inhibit Caspase 3 activity, promote ALP activity, increase calcified nodules formation and upregulate Smad1, Smad7 and Runx2 compared to control (P < 0.05); transfection of αCGRP siRNA significantly down-regulated CGRP in BMSCs, inhibited cell proliferation, promoted Caspase 3 activity, inhibited ALP activity, inhibited calcified nodules formation and downregulate Smad1, Smad7 and Runx2 (P < 0.05). αCGRP overexpression promotes the Smad/Runx2 signaling, which in turn promotes BMSCs proliferation and osteogenesis. Decreased αCGRP level inhibits Smad/Runx2 signaling, promotes BMSCs apoptosis, inhibits proliferation and osteogenic differentiation.

Effect of SOX9 on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Through WNTβ/Catenin Pathway

2019 ◽  
Vol 9 (10) ◽  
pp. 1429-1434
Author(s):  
Qing Yang ◽  
Cheng Li ◽  
Manli Yan ◽  
Chunhua Fang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Protein Expression ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Real Time Pcr ◽  
Alp Activity ◽  
Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) can be differentiated into different types of cells. SOX9 involves in the development and progression of various diseases. Our study aims to assess SOX9's effect on osteogenic differentiation of BMSCs and its related regulatory mechanisms. Rat BMSCs were isolated and randomly divided into control group, SOX9 group and SOX9 siRNA group, which was transfected with pcDNA-SOX9 plasmid or SOX9 siRNA respectively followed by analysis of SOX9 expression by Real time PCR, cell proliferation by MTT assay, Caspase3 and ALP activity, GSK-3β expression and Wntβ/Catenin Signaling pathway protein expression by Western blot, and expression of osteogenic genes Runx2 and BMP-2 by Real time PCR. Transfection of pcDNA-SOX9 plasmid into BMSCs significantly inhibited cell proliferation, promoted Caspase3 activity, decreased ALP activity and downregulated Runx2 and BMP-2, increased GSK-3β expression and decreased Wntβ/Catenin expression protein expression (P< 0.05). SOX9 siRNA transfection significantly promoted cell proliferation, inhibited Caspase3 activity, increased ALP activity and upregulated Runx2 and BMP-2, downregulated GSK-3β and increased Wntβ/Catenin expression. SOX9 regulates BMSCs proliferation and osteogenic differentiation through Wntβ/Catenin signaling pathway.

scholarly journals A Comparative Study on the Effect of Exosomes Secreted by Mesenchymal Stem Cells Derived from Adipose and Bone Marrow Tissues in the Treatment of Osteoarthritis-Induced Mouse Model

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Hoda Fazaeli ◽  
Naser Kalhor ◽  
Leila Naserpour ◽  
Faezeh Davoodi ◽  
Mohsen Sheykhhasan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
Histological Analysis ◽  
Collagen Type ◽  
Therapeutic Option ◽  
Health Condition ◽  
Histological Staining

Background. Exosomes as extracellular vesicles (EVs) are nanoscale intercellular messengers secreted from cells to deliver biological signals. Today, exosomes have become a new field of research in regenerative medicine and are considered as potential therapies to control inflammation and wound healing and enhance and improve healing in many diseases. Given the global burden of osteoarthritis (OA) as the fastest-growing health condition and one of the major causes of physical disability in the aging population, research to establish EVs as therapeutic products can meet the basic clinical needs in the management of osteoarthritis and provide a therapeutic solution. Objectives. The present study is aimed at evaluating the regenerative potentials of the exosomes secreted from adipose and bone marrow tissue-derived mesenchymal stem cells (AD- and BM-MSCs) in ameliorating the symptoms of OA. Method. In this experimental study, AD- and BM-MSCs were isolated and cultured in the laboratory until passage 3. Finally, these cells’ secreted exosomes were isolated from their conditioned medium. Ciprofloxacin-induced OA mouse models underwent intra-articular injection of exosomes from AD-MSCs and BM-MSCs. Finally, the expression levels of collagen I and II, sox9, and aggrecan genes using real-time PCR, histological analysis, and immunohistochemical (IHC) studies were performed. Results. Real-time PCR data showed that although the expression level of collagen type II was lower in both exosome-treated groups than the normal, but it was significantly increased in comparison with the sham and OA, with higher expression in BM-Exo rather than AD-Exo group. Similarly, the histological staining and IHC results have provided almost identical data, emphasizing on better therapeutic effect of BM-MSCs-exosome than AD-MSCs-exosome. Conclusion. BM-MSCs secreted exosomes in comparison with AD-MSCs could be considered as a better therapeutic option to improve osteoarthritis and exhibit potential as a disease-modifying osteoarthritis cell-free product.

scholarly journals Effects of single transplantation and multiple transplantation of human umbilical cord mesenchymal stem cells on the recovery of ovarian function in the treatment of premature ovarian failure in mice

2021 ◽  
Author(s):  
Xiaodan Lv ◽  
Chunyi Guan ◽  
Ying Li ◽  
Xing Su ◽  
lu Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Real Time ◽  
Umbilical Cord ◽  
Premature Ovarian Failure ◽  
Real Time Pcr ◽  
Ovarian Function ◽  
Follicle Stimulating Hormone ◽  
Ovarian Failure ◽  
Human Umbilical Cord

Abstract BackgroundAt present, there is no effective treatment for premature ovarian failure (POF), and stem cell therapy is considered the most promising treatment. Human umbilical cord blood mesenchymal stem cells (hUC-MSCs) have shown good regenerative ability in a variety of diseases including POI, but the method and dosage of hUC-MSCs to treat POI are not clear. This study aims to explore the treatment options of hUC-MSCs for POF by comparing single injection and multiple injections of hUC-MSCs on the ovarian function repair of POF caused by chemotherapy drugs.MethodsFemale mice were injected intraperitoneally with 30 mg/kg of busulfan and 120 mg/kg of cyclophosphamide to induce POF. In the single hUC-MSCs injection group, 7 days after the mice were injected with cyclophosphamide and busulfan, hUC-MSCs were transplanted into these mice. In the multiple injection group, hUC-MSCs were transplanted 7 days, 14 days and 21 days after the mice were injected with cyclophosphamide and busulfan. We evaluated ovarian morphology, fertility, follicle stimulating hormone and estradiol concentration, and follicle count, evaluated POF model and cell transplantation. In addition, real-time PCR, immunohistochemistry, miRNA chip and mRNA chip are used to evaluate the effect of cell therapy.ResultsCompared with the POF group, the ovarian size and primordial follicle count in the hUC-MSC group were significantly improved, and the fertility was also significantly improved. Immunohistochemistry showed that compared with the POF group, the anti-Mullerian hormone and Ki-67 in the ovary of the hUC-MSC group increased significantly, and ovulation was significantly restored. Real-time PCR showed that the expression of follicle stimulating hormone receptor, inhibin and inhibin in the hUC-MSCs group was significantly restored compared with the POF group. The results of mRNA and miRNA chips also showed that hUC-MSC restored ovarian function at the gene level. long-term treatment effect shows that the multiple transplantation hUC-MSCs group is better than the single transplantation hUC-MSCs group. 60 days after the mice were injected with cyclophosphamide and busulfan, the organ coefficient of multiple transplantation of hUC-MSCs increased compared with the POF group, the number of primary follicles increased, and hormone secretion increased. ConclusionThe results show that multiple trasplantation of hUC-MSCs can promote the recovery of ovarian function in POF mice more than a single transplantation. This study provides a basis for the therapeutic dose and therapeutic effect of hUC-MSCs on POF.

scholarly journals Osteogenic Differentiation of Pre-Conditioned Bone Marrow Mesenchymal Stem Cells with Nisin on the Modified Poly-L-Lactic-Acid Nanofibers

2021 ◽  
Author(s):  
Fariba Sadraei ◽  
Marzieh Ghollasi ◽  
Fatemeh Khakpai ◽  
Raheleh Halabian
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Lactic Acid ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Real Time Pcr ◽  
Alizarin Red ◽  
Marrow Mesenchymal Stem Cells

Abstract Background: Human bone marrow-derived mesenchymal stem (MSCs) cells are undifferentiated cells with the self-renewing ability and multi-lineage differentiation beneficial for regenerative medicine. Nano scaffolds are novel materials employed in bone repair and regeneration. Nisin is a prebiotic that can increase stem cells’ life span and proliferation. This study attempted to provide a proper strategy for bone marrow mesenchymal stem cells differentiation into the Osteocytes on a Poly‐L‐lactic‐acid scaffold (PLLA) after pretreating with probiotic Nisin. Methods: MSC osteogenic differentiation was evaluated by measuring Calcium, Alkaline phosphatase, and quantitative tests such as Real-Time PCR, Acridine Orange, Alizarin Red, Von Kossa, and others. Results: The result of the MTT test showed that the optimal dose of Nisin probiotic for the MSCs’ preconditioning was 200 IU/mL on the 1st, 3rd, and 5th days of culture. Real-time PCR data indicated that the expression rate of ALP, Osteonectin, Osteocalcin, and Collagen I have increased in the presence of Nisin, while the RUNX-2 gene expression has decreased. Furthermore, the results of Alizarin Red and Von Kossa tests, as well as Scanning electron microscopy (SEM), revealed that the cell proliferation in the preconditioned samples with Nisin increased significantly. Conclusions: The study concluded that the cell proliferation and differentiation increased in samples pretreated with Nisin on the PLLA Nano scaffolds.

scholarly journals Platelet Microparticles Accelerate Proliferation and Growth of Mesenchymal Stem Cells through Longevity-Related Genes

2021 ◽  
Vol 24 (8) ◽  
pp. 607-614
Author(s):  
Maryam Samareh Salavati Pour ◽  
Fatemeh Hoseinpour Kasgari ◽  
Alireza Farsinejad ◽  
Ahmad Fatemi ◽  
Gholamhossein Hassanshahi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
Treated Group ◽  
Population Doubling ◽  
Control Group ◽  
Population Doubling Time ◽  
Longevity Genes

Background: Due to their self-renewal and differentiation ability, the mesenchymal stem cells (MSCs) have been studied extensively. However, the MSCs lifespan is restricted; they undergo several divisions in vitro that cause several alternations in cellular features and relatively lessens their application. Thus, this study was aimed to assess the effect of platelet-derived microparticles (PMPs), a valuable source of proteins, microRNAs (miRNAs), and growth factors, on the expression of hTERT, c-MYC, p16, p53, and p21 as the most important aging and cell longevity genes alongside with population doubling time (PDT) of PMP-treated cells in comparison to a control group. Methods: Umbilical cord MSCs (UC-MSCs) were used in this study, whereby they reached a confluency of 30%. MSCs were treated by PMPs (50 µg/mL), and then, PDT was determined for both groups. Quantitative expression of hTERT, c-MYC, p16, p53, and p21 was examined through quantitative real-time PCR at various intervals (i.e. after five and thirty days as well as freezing-thawing process). Results: Our results demonstrated that the treated group had a shorter PDT in comparison to the control group (P<0.050). The real-Time PCR data also indicated that PMPs were able to remarkably up-regulate hTERT and c-MYC genes expression while down-regulating the expression of p16, p21, and p53 genes (P<0.050), especially following five days of treatment. Conclusion: According to these data, it appears that PMPs are a safe and effective candidate for prolonging the lifespan of UC-MSCs; however, further investigations are needed to corroborate this finding.

220 Exploring the use of mesenchymal stem cells for treatment of mastitis and metritis in cattle

2020 ◽  
Vol 32 (2) ◽  
pp. 238
Author(s):  
R. Singh ◽  
S. Saini ◽  
S. Ansari ◽  
S. Jamwal ◽  
D. Malakar
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Real Time ◽  
Real Time Pcr ◽  
Local Group ◽  
Control Group ◽  
Time Intervals ◽  
And Control

The present study was carried out to isolate mesenchymal stem cells (MSCs) from adipose tissue of cattle (Bos indicus), characterise them, and apply them for the treatment of mastitis and metritis in the cow. Cattle MSCs were isolated from adipose tissue near the loin region of cow. Isolated adipose tissue was subjected to enzymatic digestion using 2% collagenase with agitation at regular intervals. The cells obtained after digestion were resuspended in cell culture flasks containing growth enriched medium and cultured under standard culture conditions. Alkaline phosphatase staining was used as one of the parameters to confirm cultured putative MSCs. Bovine Ad-MSCs were further characterised using real time-PCR by amplification of MSC-specific markers: CD73, CD90, and CD105 as positive markers and CD34, CD45, and CD79a as negative markers. Immunocytochemistry showed the presence of CD73, CD90, and CD105 on the cell surface. Three groups-control (C), local (L), and intravenous (IV)-with 6 cows suffering from mastitis were taken in each group and subjected to MSC transplantation through local and intravenous routes. Control group animals were subjected to antibiotic treatment only. Similarly, another three groups were taken with 6 cows in each group suffering from metritis. Post-transplantation wound healing, tissue repair, and reduction in inflammation were monitored for 26 days, at different time intervals; that is, after Days 1, 3, 7, and 15. Blood samples were also collected from animals at the same time intervals for real time-PCR. A similar examination was also done in metritis groups along with the analysis of the reduction in turbidity of cervical fluid at the abovementioned time intervals. Real time-PCR was performed to determine relative expression of genes for proliferative factors, anti-inflammatory cytokines, and antimicrobial peptides on cells isolated from blood collected at different time intervals. Gene expression in the local group of mastitis subjected to MSC injection was significantly higher than that of the IV and control group. The somatic cell count declined in both local and IV groups compared with the control group. Whereas the expression of the same genes in the IV group of metritis was significantly higher than that of the local and control groups of cows. The turbidity of cervical fluid and mucus was reduced in the IV group compared with the local group. In conclusion, we demonstrated the healing potential of MSCs in a cow model via MSC injection. Promising results were obtained in curing mastitis in both local and IV groups, whereas healing in the case of metritis was significantly higher in the IV group compared with both the control and local groups of cows. The study indicates the potential use of MSc for treatment of mastitis and metritis in cattle through wound healing and decreasing microbial infection.

Down Regulation of GATA-2 and the Inversely-Correlated Expression of Adipogenic Specific Gene-PPARγ in Mesenchymal Stem Cells from Patients with Aplastic Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1414-1414
Author(s):  
Yinyan Xu ◽  
Yoshiyuki Takahashi ◽  
Yue Wang ◽  
Hirokazu Hidaka ◽  
Hiroshi Yagasaki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Aplastic Anemia ◽  
Cell Line ◽  
Real Time ◽  
Real Time Pcr ◽  
Specific Gene ◽  
Quantitative Real Time Pcr ◽  
Normal Individuals

Abstract (Introduction) Aplastic anemia (AA) is characterized by a reduced number of hematopoietic stem cells resulting in peripheral pancytopenia and fatty marrow replacement. Although immune-mediated suppression of hematopoiesis is considered to play an important role in most patients with AA, the mechanism of fatty marrow replacement remains unclear. Transcription factor GATA-2 is known to play important roles in hematopoiesis. Gene Chip analysis has implicated the decreased expression of GATA-2 in AA CD34 cells, a finding confirmed by quantitative real-time PCR analysis. In a mouse stromal preadipocyte cell line, the suppression of GATA-2 expression accelerated the differentiation of stromal preadipocytes. In order to explore the impact of GATA-2 expression on adipogenesis in AA, the expression of GATA-2, the adipogenic specific gene adipsin, CCAAT/enhancer binding protein alpha (C/EBPα), and peroxisome proliferator-activated receptor γ (PPARγ) was examined in mesenchymal stem cells (MSCs) derived from bone marrow from AA patients and normal individuals. (Patients and methods) Thirty-four AA patients and 15 healthy individuals were included in this study. The median patient age was 10 years, ranging from 2 to 21 years. Twelve patients were studied before immunosuppressive therapy (IST) or bone marrow transplantation. Twenty-two patients were studied after treatment, of whom 11 responded to IST and 11 did not respond. Mononuclear cells were separated from bone marrow aspirates and 1x107 cells used for MSCs culture. MesenCult (StemCell Technologies, USA) was used to culture and expand the MSCs in vitro, after which the cells retain their ability to differentiate along various lineages upon the addition of the appropriate stimulatory supplements. Total RNA was extracted from the third passage of MSCs and synthesis of cDNA was performed using the Thermoscript RT-PCR system. Quantitative real-time PCR was conducted using Taqman primers and probes. The K562 cell line for GATA-2, the EOL1 cell line for adipsin and the MOLM-13 cell line for C/EBPα and PPARγ were used as controls. (Results) GATA-2 expression by MSCs from patients with AA at diagnosis was significantly decreased compared with normal individuals (p=0.031). However, one of the adipogenic specific genes, PPARγ, was significantly increased in MSCs in AA at diagnosis (p=0.017). The expression of adipsin and C/EBPα in MSCs did not differ significantly between AA patients and healthy volunteers. Notably, in the 22 AA patients who received IST, the expression of GATA-2 and PPARγ did not significantly decrease if the patient had responded and hematopoiesis had improved (n=11). However, after the IST the 11 non-responders still had significantly lower GATA-2 and higher PPARγ expression compared to normal individuals. (Conclusion) The mechanism of fatty replacement in the bone marrow in AA may be explained by the down regulation of GATA-2 and the inversely-correlated expression of PPARγ in MSCs. The decreased expression of GATA-2 may be responsible for the pathogenesis and development of the clinical features of this disease.

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