Loss of Stag2 cooperates with EWS-FLI1 to transform murine Mesenchymal stem cells

Abstract Background Ewing sarcoma is a malignancy of primitive cells, possibly of mesenchymal origin. It is probable that genetic perturbations other than EWS-FLI1 cooperate with it to produce the tumor. Sequencing studies identified STAG2 mutations in approximately 15% of cases in humans. In the present study, we hypothesize that loss of Stag2 cooperates with EWS-FLI1 in generating sarcomas derived from murine mesenchymal stem cells (MSCs). Methods Mice bearing an inducible EWS-FLI1 transgene were crossed to p53−/− mice in pure C57/Bl6 background. MSCs were derived from the bone marrow of the mice. EWS-FLI1 induction and Stag2 knockdown were achieved in vitro by adenovirus-Cre and shRNA-bearing pGIPZ lentiviral infection, respectively. The cells were then treated with ionizing radiation to 10 Gy. Anchorage independent growth in vitro was assessed by soft agar assays. Cellular migration and invasion were evaluated by transwell assays. Cells were injected with Matrigel intramuscularly into C57/Bl6 mice to test for tumor formation. Results Primary murine MSCs with the genotype EWS-FLI1 p53−/− were resistant to transformation and did not form tumors in syngeneic mice without irradiation. Stag2 inhibition increased the efficiency and speed of sarcoma formation significantly in irradiated EWS-FLI1 p53−/− MSCs. The efficiency of tumor formation was 91% for cells in mice injected with Stag2-repressed cells and 22% for mice receiving cells without Stag2 inhibition (p < .001). Stag2 knockdown reduced survival of mice in Kaplan-Meier analysis (p < .001). It also increased MSC migration and invasion in vitro but did not affect proliferation rate or aneuploidy. Conclusion Loss of Stag2 has a synergistic effect with EWS-FLI1 in the production of sarcomas from murine MSCs, but the mechanism may not relate to increased proliferation or chromosomal instability. Primary murine MSCs are resistant to transformation, and the combination of p53 null mutation, EWS-FLI1, and Stag2 inhibition does not confer immediate conversion of MSCs to sarcomas. Irradiation is necessary in this model, suggesting that perturbations of other genes beside Stag2 and p53 are likely to be essential in the development of EWS-FLI1-driven sarcomas from MSCs.

Download Full-text

Exosomal transfer of miR-769-5p promotes osteosarcoma proliferation and metastasis by targeting DUSP16

Cancer Cell International ◽

10.1186/s12935-021-02257-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Wanshun Liu ◽

Binyu Wang ◽

Ao Duan ◽

Kai Shen ◽

Qi Zhang ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Lines ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Clinical Specimens ◽

Proliferation And Metastasis

Abstract Background Osteosarcoma (OS) is a malignant tumor originating from mesenchymal stem cells, and has an extremely high fatality rate and ability to metastasize. Although mounting evidence suggests that miR-769-5p is strongly associated with the malignant progression and poor prognosis of various tumors, the exact role of miR-769-5p in OS is still unclear. Therefore, this study aimed to explore the relationship between miR-769-5p and the malignant progression of OS, and its underlying mechanism of action. Methods miR-769-5p expression was analyzed in GSE28423 from the GEO database and measured in OS clinical specimens and cell lines. The effects of miR-769-5p on OS proliferation, migration and invasion were measured both in vivo and in vitro. In addition, bioinformatics analyses and luciferase reporter assays were used to explore the target genes of miR-769-5p. Rescue experiments were also conducted. Moreover, a co-culture model was used to test the cell interaction between bone mesenchymal stem cells (BMSC) and OS cells. Results We found that miR-769-5p is highly expressed in OS clinical specimens and cell lines. In vivo and in vitro experiments also showed that miR-769-5p significantly promoted the proliferation, migration and invasion of OS cells. Dual-specific phosphatase 16 (DUSP16) was negatively associated with miR-769-5p expression in OS cells and tissue samples and was validated as the downstream target by luciferase reporter assay and western blotting. Rescue experiments showed that DUSP16 reverses the effect of miR-769-5p on OS cells by negatively regulating the JNK/p38 MAPK signaling pathway. Additionally, the results of the co-culture of BMSCs and OS cells confirmed that miR-769-5p was transferred from BMSCs to OS cells through exosomes. Conclusions In summary, this study demonstrates for the first time that BMSC-derived exosomal miR-769-5p promotes OS proliferation and metastasis by targeting DUSP16 and activating the JNK/p38 MAPK signaling pathway, which could provide rationale for a new therapeutic strategy for OS.

Download Full-text

Immortalized mesenchymal stem cells: a safe cell source for cellular or cell membrane-based treatment of glioma

10.21203/rs.3.rs-382529/v1 ◽

2021 ◽

Author(s):

Yuxuan Zhang ◽

Jie Liu ◽

Yunzhao Mo ◽

Zetao Chen ◽

Taoliang Chen ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Membranes ◽

Glioma Cells ◽

Soft Agar ◽

Proliferative Capacity ◽

Soft Agar Cloning ◽

The Brain

Abstract BACKGROUND: Mesenchymal stem cells (MSCs) have emerged as putative therapeutic tools due to their intrinsic tumour tropism, anti-tumour, and immunoregulatory properties. The limited passage and self-differentiation abilities of MSCs in vitro hinder preclinical studies of mesenchymal stem cells, and makes the MSC-based treatment of tumours lack a stable, uniform, and homogeneous source of cells. In this study, we focused on the safety of immortalised mesenchymal stem cells (im-MSCs) and, for the first time, studied the feasibility of im-MSCs as candidates for the treatment of glioma.METHODS: The im-MSCs were constructed by the lentiviral transfection of genes, and the proliferative capacity of immortalised MSCs and the proliferative phenotype of MSCs and MSCs co-cultured with glioma cells (U87) were measured using CCK-8 and EdU assays. After long-term culture, karyotype analysis of im-MSCs was conducted. In addition, the tumourigenicity of engineered MSCs was evaluated using soft agar cloning assays. Moreover, the engineered cells were injected into the brain of female BALB/c nude mice. Finally, the cell membranes of im-MSCs were labelled with DIO or DIR to detect their ability to be taken by glioma cells and target in situ gliomas using an IVIS system.RESULTS: Im-MSCs expressed CD73, CD90, CD105, CD29, and CD44 but did not express CD45, CD34, CD14, CD11b, or CD31. Engineered MSCs maintained the ability to differentiate into mesenchymal lineages in vitro. Im-MSCs showed stronger proliferative capacity than unengineered MSCs without colony formation in soft agar, no tumourigenicity in the brain, and normal chromosomes. MSCs or im-MSCs co-cultured with U87 cells showed enhanced proliferation ability, but did not show malignant characteristics in soft agar cloning experiments. Immortalised cells continued to express homing molecules. The cell membranes of im-MSCs were taken up by glioma cells and targeted in situ gliomas in vivo.CONCLUSIONS: Im-MSCs are promising candidates for cellular anti-glioma therapy. Im-MSCs provide a safe, adequate, quality-controlled, and continuous source of cells or cell membranes for the treatment of glioma.

Download Full-text

Toxicological Evaluation of Human Adipose-derived Mesenchymal Stem Cells (hADSCs) and hADSCs-derived Exosomes

10.21203/rs.3.rs-626854/v1 ◽

2021 ◽

Author(s):

Yang Zhou ◽

Bo Zhao ◽

Xin-Liao Zhang ◽

Yi-jun Lu ◽

Jian Cheng ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Human Adipose Tissue ◽

Soft Agar ◽

Toxicological Evaluation ◽

Toxicological Assessment ◽

Toxicological Profile ◽

Treatment Agent

Abstract Background: Human adipose tissue-derived stem cells (hADSCs) are considered an ideal source of cells for regenerative medicine. Mesenchymal stem cells derived-exosomes (MSC-Exos) are being opined as new cell-free therapeutics for numerous human diseases. For future clinical applications, the safety of allogenic hADSCs and hADSCs-derived exosomes (hADSCs-Exos) needs to be addressed and verified in pre-clinical animal models. This study sought to evaluate the toxicity of hADSCs and hADSCs-Exos by performing in vivo and in vitro toxicological assessments.Methods: We used IVIS to track the biodistribution of GFP-labeled hADSCs and the PKH26-labeled in a mouse model. The tumorigenicity of hADSCs and hADSCs-Exos was analyzed by soft agar colony formation assay and nude mice tumorigenicity test in vitro and in vivo. The acute animal toxicity and allergenicity test were used to explore the toxicological proﬁle of hADSCs and hADSCs-Exos in mice.Results: We found that hADSCs-Exos accumulated faster in the tissues of mice and were also cleared more rapidly compared to hADSCs. Both hADSCs and hADSCs-Exos have little risk of tumorigenicity, and hADSCs-Exos had lower toxicity and lower immunogenicity than hADSCs.Conclusion: Our study is the first to compare the safety between hADSCs and hADSCs-Exos, and revealed that hADSCs-Exos are safer for application as systemic therapy, without complications in toxicological assessment, and have a better prospective utility as a treatment agent and for drug delivery.

Download Full-text

Long-Term Cultured Human Term Placenta-Derived Mesenchymal Stem Cells of Maternal Origin Displays Plasticity

Stem Cells International ◽

10.1155/2012/174328 ◽

2012 ◽

Vol 2012 ◽

pp. 1-11 ◽

Cited By ~ 34

Author(s):

Vikram Sabapathy ◽

Saranya Ravi ◽

Vivi Srivastava ◽

Alok Srivastava ◽

Sanjay Kumar

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Flow Analysis ◽

Normal Karyotype ◽

Soft Agar ◽

Neural Cells ◽

Maternal Origin ◽

Pancreatic Progenitor

Mesenchymal stem cells (MSCs) are an alluring therapeutic resource because of their plasticity, immunoregulatory capacity and ease of availability. Human BM-derived MSCs have limited proliferative capability, consequently, it is challenging to use in tissue engineering and regenerative medicine applications. Hence, placental MSCs of maternal origin, which is one of richest sources of MSCs were chosen to establish long-term culture from the cotyledons of full-term human placenta. Flow analysis established bonafied MSCs phenotypic characteristics, staining positively for CD29, CD73, CD90, CD105 and negatively for CD14, CD34, CD45 markers. Pluripotency of the cultured MSCs was assessed by in vitro differentiation towards not only intralineage cells like adipocytes, osteocytes, chondrocytes, and myotubules cells but also translineage differentiated towards pancreatic progenitor cells, neural cells, and retinal cells displaying plasticity. These cells did not significantly alter cell cycle or apoptosis pattern while maintaining the normal karyotype; they also have limited expression of MHC-II antigens and are Naive for stimulatory factors CD80 and CD 86. Further soft agar assays revealed that placental MSCs do not have the ability to form invasive colonies. Taking together all these characteristics into consideration, it indicates that placental MSCs could serve as good candidates for development and progress of stem-cell based therapeutics.

Download Full-text

Mesenchymal Stem Cells With Cancer-Associated Fibroblast-Like Phenotype Stimulate SDF-1/CXCR4 Axis to Enhance the Growth and Invasion of B-Cell Acute Lymphoblastic Leukemia Cells Through Cell-to-Cell Communication

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.708513 ◽

2021 ◽

Vol 9 ◽

Author(s):

Chengyun Pan ◽

Qin Fang ◽

Ping Liu ◽

Dan Ma ◽

Shuyun Cao ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Migration And Invasion ◽

Integrin Β1 ◽

Cancer Associated Fibroblast ◽

Cell Acute Lymphoblastic Leukemia ◽

Cxcr4 Axis

Background: Bone marrow mesenchymal stem cells (BM-MSCs) are the stromal cells in the leukemia microenvironment, and can obtain cancer-associated fibroblast (CAF)-like phenotype under certain conditions to further promote leukemia progression. However, the mechanism of MSCs with CAF-like phenotype interacting with leukemia cells in B-cell acute lymphoblastic leukemia (B-ALL) and promoting the progression of B-ALL remains unclear.Methods: Mesenchymal stem cells with CAF-like phenotype were obtained by treating MSCs with recombinant human transforming growth factor-β (rhTGF-β), hereafter referred to as TGF-β conditioned MSCs. In vivo mouse model experiments, in vitro transwell chamber experiments, three-dimensional (3D) cell culture models, lentiviral transfection and other experimental methods were used to investigate the possible mechanism of the interaction between TGF-β conditioned MSCs and leukemia cells in promoting the growth, migration and invasion of B-ALL cells.Results: Compared with untreated MSCs, TGF-β conditioned MSCs significantly promoted the growth and proliferation of leukemia cells in mice, and increased the expression of CXCR4 in tumor tissues. In vitro cell experiments, TGF-β conditioned MSCs obviously promoted the migration and invasion of Nalm-6/RS4;11 cells, which were effectively blocked by the CXCR4 inhibitor AMD3100, thereby inhibiting the secretion of MMP-9 in TGF-β conditioned MSCs and inhibiting the activation of the PI3K/AKT signaling pathway in leukemia cells. Further, findings were made that the interaction between TGF-β conditioned MSCs and leukemia cells were mediated by the interaction between the integrin receptor α5β1 on the surface of leukemia cells and the increased expression of fibronectin on TGF-β conditioned MSCs. AMD3100 could weaken such effect by reducing the expression of integrin α5β1 on leukemia cells. Further regulation of integrin β1 could effectively interfere with the interaction between TGF-β conditioned MSCs and leukemia cells.Conclusion: Mesenchymal stem cells with CAF-like phenotype could be a key factor in promoting the growth and invasion of B-ALL cells, and the SDF-1/CXCR4 axis might be a significant factor in mediating the communication of MSCs with CAF-like phenotype and leukemia cells. To prevent the progression of B-ALL cells, blocking the SDF-1/CXCR4 axis with AMD3100 or targeting integrin β1 might be a potential therapeutic strategy.

Download Full-text

Histone Demethylase KDM5B Catalyzed H3k4me3 Demethylation to Promote Differentiation of Bone Marrow Mesenchymal Stem Cells into Cardiomyocytes

10.21203/rs.3.rs-1038705/v1 ◽

2021 ◽

Author(s):

Hongxiao li ◽

Chenlu Zhong ◽

Zhen Wang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Myocardial Cell ◽

Tumor Formation ◽

Control Group ◽

Pluripotency Gene ◽

Marrow Mesenchymal Stem Cells ◽

Differentiation Efficiency

Abstract BACKGROUND: Studies have shown that histone H3 methylation is involved in regulating the differentiation of Bone Marrow Mesenchymal Stem Cells (BMSCs). KDM5B can specifically reduce the level of histone 3 lysine 4 trimethylation (H3K4me3), thereby activating the expression of related genes and participating in biological processes such as cell differentiation, embryonic development and tumor formation. Whether KDM5B is involved in the regulation of BMSCs differentiation into cardiomyocytes through the above manner has not been reported.OBJECTIVE: To investigate the effect of KDM5B on the induction and differentiation of swine BMSCs into myocardial cells in vitro.METHODS: Swine bone marrow BMSCs were isolated and cultured, and the overexpression, interference expression and blank vector of KMD5B were constructed and transfected by lentivirus. BMSCs was induced to differentiate into cardiomyocytes by 5-azacytidine (5-AZA) in vitro, and the differentiation efficiency was compared by immunofluorescence, RT-PCR, Western Blot and whole-cell patch clamp detection.RESULT: Compared with the control group, the expression levels of histone H3K4me3 and pluripotency gene Nanog in KDM5B overexpression group were significantly decreased, while the expression level of key myocardial gene HCN4 was significantly increased, and the Na+ current density on the surface of differentiated myocardial cell membrane was significantly increased. Meanwhile, the corresponding results of the KDM5B silent expression group were just opposite. CONCLUSIONS: It indicated that enhanced KDM5B expression could promote the differentiation of BMSCs into cardiomyocytes and improve the differentiation efficiency by controlling H3K4 methylation levels..

Download Full-text

Migration of human mesenchymal stem cells stimulated with pulsed electric field and the dynamics of the cell surface glycosylation

10.1101/122382 ◽

2017 ◽

Author(s):

Katarzyna Jezierska-Wozniak ◽

Seweryn Lipiński ◽

Łukasz Grabarczyk ◽

Monika Barczewska ◽

Aleksandra Habich ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Surface ◽

Electric Field ◽

Human Mesenchymal Stem Cells ◽

Pulsed Electric Field ◽

Cellular Systems ◽

Cellular Migration

AbstractThe objective of our study was to develop novel techniques for investigations of cell motility, and to assess whether the electric field of the therapeutic spinal cord stimulation system used in vivo contributes to the migration of human mesenchymal stem cells (hMSCs) in vitro.We have investigated electrotaxis of bone marrow-derived MSCs using pulsed electric field (PEF) in range 16-80 mV/mm and frequency 130 Hz and 240 Hz. The PEF-related dynamics of the cell surface glycosylation was evaluated using six plant lectins.PEF at physiological levels (10mV/mm; 130 Hz) did not influence cellular motility in vitro, what may correspond to the maintenance of the transplanted cells at the lesion site in vivo. Increase of the PEF intensity and frequency above physiological levels resulted in the increase in the cellular migration rate in vitro. PEF elevated above physiological intensity and frequency (40-80 mV/mm; 240 Hz), but not at physiological levels, resulted in changes of the cell surface glycosylation.We find the described approach as convenient for investigations and for the in vitro modeling of the cellular systems intended for the regenerative cell transplantations in vivo. Probing cell surface glycomes may provide valuable biomarkers to assess competence of transplanted cells.

Download Full-text

Immunophenotyping of bone marrow-derived mesenchymal stem cells after transplantation in a heterologous in-vitro model using Laser Scanning Cytometry

The Thoracic and Cardiovascular Surgeon ◽

10.1055/s-2004-816714 ◽

2004 ◽

Vol 52 (S 1) ◽

Author(s):

J Garbade ◽

D Lenz ◽

A Rastan ◽

MJ Barten ◽

A Tarnok ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Laser Scanning ◽

In Vitro Model ◽

Laser Scanning Cytometry ◽

Vitro Model

Download Full-text

Autologous transplantation of mesenchymal stem cells into the portal vein of the miniature pig; a preliminary experiment for NOTES approach

Perspectives in Surgery ◽

10.33699/pis.2019.98.9.350-355 ◽

2019 ◽

Vol 98 (9) ◽

pp. 350-355

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Therapy ◽

Portal Vein ◽

Liver Parenchyma ◽

Miniature Pig ◽

Hepatic Diseases ◽

Study Results

Introduction: There is evidence that mesenchymal stem cells (MSCs) could trans-differentiate into the liver cells in vitro and in vivo and thus may be used as an unfailing source for stem cell therapy of liver disease. Combination of MSCs (with or without their differentiation in vitro) and minimally invasive procedures as laparoscopy or Natural Orifice Transluminal Endoscopic Surgery (NOTES) represents a chance for many patients waiting for liver transplantation in vain. Methods: Over 30 millions of autologous MSCs at passage 3 were transplanted via the portal vein in an eight months old miniature pig. The deposition of transplanted cells in liver parenchyma was evaluated histologically and the trans-differential potential of CM-DiI labeled cells was assessed by expression of pig albumin using immunofluorescence. Results: Three weeks after transplantation we detected the labeled cells (solitary, small clusters) in all 10 samples (2 samples from each lobe) but no diffuse distribution in the samples. The localization of CM-DiI+ cells was predominantly observed around the portal triads. We also detected the localization of albumin signal in CM-DiI labeled cells. Conclusion: The study results showed that the autologous MSCs (without additional hepatic differentiation in vitro) transplantation through the portal vein led to successful infiltration of intact miniature pig liver parenchyma with detectable in vivo trans-differentiation. NOTES as well as other newly developed surgical approaches in combination with cell therapy seem to be very promising for the treatment of hepatic diseases in near future.

Download Full-text