Generation and characterization of transgenic mouse mesenchymal stem cell lines expressing hIGF-1 or hG-CSF

Gonçalves, Gabrielle V. M.; Silva, Daniela Nunes da; Carvalho, Renata; Souza, Bruno S. F.; Silva, Kátia Nunes da; Vasconcelos, Juliana Fraga; Paredes, Bruno Diaz; Nonaka, Carolina Kymie Vasques; Ribeiro-dos-Santos, Ricardo; Soares, Milena Botelho Pereira

doi:10.1007/s10616-017-0131-2

Cited by 6 publications

(12 citation statements)

References 40 publications

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“…Isolation and culture of primary cells MSCs were isolated from the bone marrow of male C57Bl/6 mice, as previously described [9]. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA), with medium changes every 3-4 days, and maintained in an incubator at 37°C and humidified atmosphere with 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

“…A 2nd-generation lentiviral system was used, and nonreplicative lentiviral particles were produced by transient transfection of HEK293FT cells with psPAX2 (Addgene #12260), pMD2.G (Addgene #12259), and expression vector, at a 2:1:3 ratio, as previously described [9]. Three expression vectors were constructed: pFOXA2IP, pFHIG, and pCWFOXA2.…”

Section: Vector Productionmentioning

confidence: 99%

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Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

et al. 2020

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Background: Hepatocyte-like cells (iHEPs) generated by transcription factor-mediated direct reprogramming of somatic cells have been studied as potential cell sources for the development of novel therapies targeting liver diseases. The mechanisms involved in direct reprogramming, stability after long-term in vitro expansion, and safety profile of reprogrammed cells in different experimental models, however, still require further investigation.Methods: iHEPs were generated by forced expression of Foxa2/Hnf4a in mouse mesenchymal stromal cells and characterized their phenotype stability by in vitro and in vivo analyses.Results: The iHEPs expressed mixed hepatocyte and liver progenitor cell markers, were highly proliferative, and presented metabolic activities in functional assays. A progressive loss of hepatic phenotype, however, was observed after several passages, leading to an increase in alpha-SMA + fibroblast-like cells, which could be distinguished and sorted from iHEPs by differential mitochondrial content. The resulting purified iHEPs proliferated, maintained liver progenitor cell markers, and, upon stimulation with lineage maturation media, increased expression of either biliary or hepatocyte markers. In vivo functionality was assessed in independent pre-clinical mouse models. Minimal engraftment was observed following transplantation in mice with acute acetaminophen-induced liver injury. In contrast, upon transplantation in a transgenic mouse model presenting host hepatocyte senescence, widespread engraftment and uncontrolled proliferation of iHEPs was observed, forming islands of epithelial-like cells, adipocytelike cells, or cells presenting both morphologies. Conclusion: The results have significant implications for cell reprogramming, suggesting that iHEPs generated by Foxa2/Hnf4a expression have an unstable phenotype and depend on transgene expression for maintenance of hepatocyte-like characteristics, showing a tendency to return to the mesenchymal phenotype of origin and a compromised safety profile.

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Section: Methodsmentioning

confidence: 99%

Section: Vector Productionmentioning

confidence: 99%

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

et al. 2020

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“…Wild-type bone marrow-derived MSCs were obtained from male GFP transgenic C57BL/6 mice. A genetically modified MSC line with stable overexpression of hG-CSF (MSC_G-CSF) was previously generated and characterized by our group ( 15 ). MSCs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (ThermoFisher Scientific, Waltham, MA, USA) in a humidified incubator at 37°C and 5% CO 2 , with complete medium replacement every 3 days.…”

Section: Methodsmentioning

confidence: 99%

“…A non-template control and non-reverse transcription controls were also included. Quantification of parasites by qPCR was performed as previously described ( 15 ).…”

Section: Methodsmentioning

confidence: 99%

Granulocyte-Colony Stimulating Factor-Overexpressing Mesenchymal Stem Cells Exhibit Enhanced Immunomodulatory Actions Through the Recruitment of Suppressor Cells in Experimental Chagas Disease Cardiomyopathy

et al. 2018

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Genetic modification of mesenchymal stem cells (MSCs) is a promising strategy to improve their therapeutic effects. Granulocyte-colony stimulating factor (G-CSF) is a growth factor widely used in the clinical practice with known regenerative and immunomodulatory actions, including the mobilization of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Here we evaluated the therapeutic potential of MSCs overexpressing G-CSF (MSC_G-CSF) in a model of inflammatory cardiomyopathy due to chronic Chagas disease. C57BL/6 mice were treated with wild-type MSCs, MSC_G-CSF, or vehicle (saline) 6 months after infection with Trypanosoma cruzi. Transplantation of MSC_G-CSF caused an increase in the number of circulating leukocytes compared to wild-type MSCs. Moreover, G-CSF overexpression caused an increase in migration capacity of MSCs to the hearts of infected mice. Transplantation of either MSCs or MSC_G-CSF improved exercise capacity, when compared to saline-treated chagasic mice. MSC_G-CSF mice, however, were more potent than MSCs in reducing the number of infiltrating leukocytes and fibrosis in the heart. Similarly, MSC_G-CSF-treated mice presented significantly lower levels of inflammatory mediators, such as IFNγ, TNFα, and Tbet, with increased IL-10 production. A marked increase in the percentage of Tregs and MDSCs in the hearts of infected mice was seen after administration of MSC_G-CSF, but not MSCs. Moreover, Tregs were positive for IL-10 in the hearts of T. cruzi-infected mice. In vitro analysis showed that recombinant hG-CSF and conditioned medium of MSC_G-CSF, but not wild-type MSCs, induce chemoattraction of MDSCs in a transwell assay. Finally, MDSCs purified from hearts of MSC_G-CSF transplanted mice inhibited the proliferation of activated splenocytes in a co-culture assay. Our results demonstrate that G-CSF overexpression by MSCs potentiates their immunomodulatory effects in our model of Chagas disease and suggest that mobilization of suppressor cell populations such as Tregs and MDSCs as a promising strategy for the treatment of chronic Chagas disease. Finally, our results reinforce the therapeutic potential of genetic modification of MSCs, aiming at increasing their paracrine actions.

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“…The ability of MSCs to modulate immune responses and fibrosis has been demonstrated in the context of T. cruzi infection in mice [ 15 – 18 ]. We have previously generated and characterized a bone marrow-derived MSC cell line overexpressing IGF-1 [ 19 ]. In the present study, we investigated the therapeutic potential of IGF-1-overexpressing MSCs in the experimental model of chronic Chagas disease, by evaluating their immunomodulatory and proregenerative effects in the heart and skeletal muscles.…”

Section: Introductionmentioning

confidence: 99%

IGF-1-Overexpressing Mesenchymal Stem/Stromal Cells Promote Immunomodulatory and Proregenerative Effects in Chronic Experimental Chagas Disease

Silva

Souza

Azevedo

et al. 2018

Stem Cells International

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Mesenchymal stem/stromal cells (MSCs) have been investigated for the treatment of diseases that affect the cardiovascular system, including Chagas disease. MSCs are able to promote their beneficial actions through the secretion of proregenerative and immunomodulatory factors, including insulin-like growth factor-1 (IGF-1), which has proregenerative actions in the heart and skeletal muscle. Here, we evaluated the therapeutic potential of IGF-1-overexpressing MSCs (MSC_IGF-1) in a mouse model of chronic Chagas disease. C57BL/6 mice were infected with Colombian strain Trypanosoma cruzi and treated with MSCs, MSC_IGF-1, or vehicle (saline) six months after infection. RT-qPCR analysis confirmed the presence of transplanted cells in both the heart and skeletal muscle tissues. Transplantation of either MSCs or MSC_IGF-1 reduced the number of inflammatory cells in the heart when compared to saline controls. Moreover, treatment with MSCs or MSC_IGF-1 significantly reduced TNF-α, but only MSC treatment reduced IFN-γ production compared to the saline group. Skeletal muscle sections of both MSC- and MSC_IGF-1-treated mice showed a reduction in fibrosis compared to saline controls. Importantly, the myofiber area was reduced in T. cruzi-infected mice, and this was recovered after treatment with MSC_IGF-1. Gene expression analysis in the skeletal muscle showed a higher expression of pro- and anti-inflammatory molecules in MSC_IGF-1-treated mice compared to MSCs alone, which significantly reduced the expression of TNF-α and IL-1β. In conclusion, our results indicate the therapeutic potential of MSC_IGF-1, with combined immunomodulatory and proregenerative actions to the cardiac and skeletal muscles.

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Generation and characterization of transgenic mouse mesenchymal stem cell lines expressing hIGF-1 or hG-CSF

Cited by 6 publications

References 40 publications

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

Granulocyte-Colony Stimulating Factor-Overexpressing Mesenchymal Stem Cells Exhibit Enhanced Immunomodulatory Actions Through the Recruitment of Suppressor Cells in Experimental Chagas Disease Cardiomyopathy

IGF-1-Overexpressing Mesenchymal Stem/Stromal Cells Promote Immunomodulatory and Proregenerative Effects in Chronic Experimental Chagas Disease

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