Construction and Evaluation of Recombinant Chimeric Fibrillin and Elastin Fragment in Human Mesenchymal Stem Cells

2021 ◽  
Vol 28 ◽  
Author(s):  
Eui-Seung Jeong ◽  
Bo-Hyun Park ◽  
Sujin Lee ◽  
Jun-Hyeog Jang
Keyword(s):  
Stem Cells ◽  
Molecular Weight ◽  
Mesenchymal Stem Cells ◽  
Smooth Muscle ◽  
Stem Cell ◽  
Cell Adhesion ◽  
Cell Differentiation ◽  
Real Time ◽  
Real Time Pcr ◽  
Control Group

Background: Diverse extracellular matrix (ECM) proteins physically interact with stem cells and regulate stem cell function. However, the large molecular weight of the natural ECM renders large-scale fabrication of a similar functional structure challenging. Objective: The objective of this study was to construct a low molecular weight and multifunctional chimeric form of recombinant ECM to stimulate mesenchymal stem cell (MSC) for tissue repair. We engineered Fibrillin-1PF14 fused to an elastin-like polypeptide to develop a new biomimetic ECM for stem cell differentiation and investigated whether this recombinant chimeric Fibrillin-Elastin fragment (rcFE) was effective on human nasal inferior turbinate-derived mesenchymal stem cells (hTMSCs). Methods: hTMSCs were grown in the medium supplemented with rcFE, then the effect of the protein was confirmed through cell adhesion assay, proliferation assay, and real-time PCR. Results: rcFE enhanced the adhesion activity of hTMSCs by 2.7-fold at the optimal concentration, and the proliferation activity was 2.6-fold higher than that of the control group (non-treatment rcFE). In addition, when smooth muscle cell differentiation markers were identified by real-time PCR, Calponin increased about 6-fold, α-actin about 9-fold, and MYH11 about 10-fold compared to the control group. Conclusion: Chimeric rcFE enhanced cellular functions such as cell adhesion, proliferation, and smooth muscle differentiation of hTMSCs, suggesting that the rcFE can facilitate the induction of tissue regeneration.

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.

scholarly journals Tendon-Derived Stem Cell Differentiation in the Degenerative Tendon Microenvironment

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Chang Liu ◽  
Jing-wan Luo ◽  
Ke-ke Zhang ◽  
Long-xiang Lin ◽  
Ting Liang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Western Blot ◽  
Real Time ◽  
Cell Morphology ◽  
Real Time Pcr ◽  
Stem Cell Differentiation ◽  
Proliferation And Differentiation ◽  
Morphology Changes

Tendinopathy is prevalent in athletic and many occupational populations; nevertheless, the pathogenesis of tendinopathy remains unclear. Tendon-derived stem cells (TDSCs) were regarded as the key culprit for the development of tendinopathy. However, it is uncertain how TDSCs differentiate into adipocytes, chondrocytes, or osteocytes in the degenerative microenvironment of tendinopathy. So in this study, the regulating effects of the degenerative tendon microenvironment on differentiation of TDSCs were investigated. TDSCs were isolated from rat Achilles tendons and were grown on normal and degenerative (prepared by stress-deprived culture) decellularized tendon slices (DTSs). Immunofluorescence staining, H&E staining, real-time PCR, and Western blot were used to delineate the morphology, proliferation, and differentiation of TDSCs in the degenerative microenvironment. It was found that TDSCs were much more spread on the degenerative DTSs than those on normal DTSs. The tenocyte-related markers, COL1 and TNMD, were highly expressed on normal DTSs than the degenerative DTSs. The expression of chondrogenic and osteogenic markers, COL2, SOX9, Runx2, and ALP, was higher on the degenerative DTSs compared with TDSCs on normal DTSs. Furthermore, phosphorylated FAK and ERK1/2 were reduced on degenerative DTSs. In conclusion, this study found that the degenerative tendon microenvironment induced TDSCs to differentiate into chondrogenic and osteogenic lineages. It could be attributed to the cell morphology changes and reduced FAK and ERK1/2 activation in the degenerative microenvironment of tendinopathy.

Evaluation of Stemness Maintenance Properties of the Recombinant Human Laminin α2 LG1-3 Domains in Human Mesenchymal Stem Cells

2019 ◽  
Vol 26 (10) ◽  
pp. 785-791
Author(s):  
Ji-Eun Kim ◽  
Hye-Jin Seo ◽  
SuJin Lee ◽  
Jun-Hyeog Jang
Keyword(s):  
Escherichia Coli ◽  
Stem Cells ◽  
Molecular Weight ◽  
Mesenchymal Stem Cells ◽  
Cell Adhesion ◽  
Human Mesenchymal Stem Cells ◽  
Adhesion Assay ◽  
Proliferation Assay ◽  
Undifferentiated State ◽  
Laminin Α2

Background: Laminin, a member of the Extracellular Matrix (ECM), is a glycoprotein that is used as a factor that affects cell adhesion, proliferation, survival, and differentiation. Of these, five globular domains (LG domains) of the alpha chain play an important role in influencing the cell by binding to the integrin. Objective: This study aimed to evaluate the ability of globular domains 1-3 of laminin alpha2 (rhLAMA2LG1-3) in maintaining the pluripotency of human Mesenchymal Stem Cells (hMSCs), which are widely used in regenerative medicine. Methods: hMSCs were grown in the medium supplemented with rhLAMA2LG1-3, then the effect of the protein on hMSCs were confirmed through cell adhesion assay, proliferation assay and RTPCR. Results: rhLAMA2LG1-3 expressed in Escherichia coli has a molecular weight of 70 kDa, at 1 µg/ml concentration of rhLAMA2LG1-3, the attachment and proliferation of hMSCs were approximately 3.18-fold and 1.67-fold, respectively, more efficient than those of untreated controls. In addition, the undifferentiated state and degree of stemness of hMSCs were measured, on the basis of CD90 and CD105 levels. In the rhLAMA2LG1-3-treated hMSCs, the expression levels of CD90 and CD105 increased by 2.83-fold and 1.62-fold, respectively, compared to those in untreated controls. Conclusion: rhLAMA2LG1-3 can be potentially used in stem cell therapy to improve the viability and maintain the undifferentiated state of hMSCs.

scholarly journals Nuclear Export of Smads by RanBP3L Regulates Bone Morphogenetic Protein Signaling and Mesenchymal Stem Cell Differentiation

2015 ◽  
Vol 35 (10) ◽  
pp. 1700-1711 ◽  
Author(s):  
Fenfang Chen ◽  
Xia Lin ◽  
Pinglong Xu ◽  
Zhengmao Zhang ◽  
Yanzhen Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Mesenchymal Stem Cell ◽  
Nuclear Export ◽  
Stem Cell Differentiation ◽  
Bmp Signaling ◽  
Dependent Manner ◽  
Mesenchymal Stem Cell Differentiation

Bone morphogenetic proteins (BMPs) play vital roles in regulating stem cell maintenance and differentiation. BMPs can induce osteogenesis and inhibit myogenesis of mesenchymal stem cells. Canonical BMP signaling is stringently controlled through reversible phosphorylation and nucleocytoplasmic shuttling of Smad1, Smad5, and Smad8 (Smad1/5/8). However, how the nuclear export of Smad1/5/8 is regulated remains unclear. Here we report that the Ran-binding protein RanBP3L acts as a nuclear export factor for Smad1/5/8. RanBP3L directly recognizes dephosphorylated Smad1/5/8 and mediates their nuclear export in a Ran-dependent manner. Increased expression of RanBP3L blocks BMP-induced osteogenesis of mouse bone marrow-derived mesenchymal stem cells and promotes myogenic induction of C2C12 mouse myoblasts, whereas depletion of RanBP3L expression enhances BMP-dependent stem cell differentiation activity and transcriptional responses. In conclusion, our results demonstrate that RanBP3L, as a nuclear exporter for BMP-specific Smads, plays a critical role in terminating BMP signaling and regulating mesenchymal stem cell differentiation.

Magnetic field assisted stem cell differentiation – role of substrate magnetization in osteogenesis

2015 ◽  
Vol 3 (16) ◽  
pp. 3150-3168 ◽  
Author(s):  
Sunil Kumar Boda ◽  
Greeshma Thrivikraman ◽  
Bikramjit Basu
Keyword(s):  
Magnetic Field ◽  
Tissue Engineering ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Bone Tissue Engineering ◽  
Human Mesenchymal Stem Cells ◽  
Stem Cell Differentiation

Substrate magnetization as a tool for modulating the osteogenesis of human mesenchymal stem cells for bone tissue engineering applications.

scholarly journals The The Influence of Mesenchymal Stem Cell Wharton Jelly toward Prostaglandin E2 Gene Expression on Synoviocyte Cell Osteoarthritis

2019 ◽  
Vol 7 (8) ◽  
pp. 1252-1258 ◽  
Author(s):  
Vivi Sofia ◽  
Moch Saiful Bachri ◽  
Rizki Rahmadian
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Prostaglandin E2 ◽  
Expression Analysis ◽  
Control Group ◽  
E2 Gene

BACKGROUND: Pharmacological therapy in the management of OA causes many new health problems due to side effects caused by long-term use of drugs, such as long-term use of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) will cause gastric ulcers and impaired kidney function. In OA pathogenesis, PGE2 gene is involved in the inflammation process. AIM: This study aims to identify the influence of Wharton Jelly Mesenchymal Stem Cell (MSC-WJ) on PGE2 expression gene in synoviocyte by in vitro. MATERIAL AND METHODS: The method used in this study is the co-culture method of primary cells and stem cells in the appropriate media. This research is pure experimental research. The sample used came from synovial tissue of osteoarthritis patients who underwent Total Knee Replacement (TKR) surgery. This study was divided into 6 groups treated with 4 replications. The expression analysis of the Prostaglandin E2 gene was done using qPCR (Real-Time Polymerase Chain Reaction). The expression analysis of the Prostaglandin E2 gene was carried out before and after the co-culture with Wharton's Jelly and continued with the analysis of statistical data processing using the SPSS.15 program. PGE2 gene expression data were processed using the Kruskal-Wallis test and continued with the Mann-Whitney test with a 95% confidence level. RESULTS: The results showed that Mesenchymal Stem Cells Wharton Jelly could reduce the expression of Prostaglandin E2 gene after co-culture for 24 hours and 48 hours in synoviocyte cells osteoarthritis significantly compared with the control group. The administration of Mesenchymal Stem Cells for 24 hours reduced the expression level of PGE2 gene by 0.61 times compared to the control group (p < 0.05) and the administration of Mesenchymal Stem Cells for 48 hours decreased the expression level of PGE2 gene by 0, 47 times compared to the control group (p < 0.05). CONCLUSION: This study concluded that MSC-WJ in OA synoviocyte significantly reduced the expression of the PGE2 gene (p < 0.05).

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.

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