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1
Abdal Dayem, Ahmed, Soo Bin Lee, Kyeongseok Kim, Kyung Min Lim, Tak-il Jeon, Jaekwon Seok, and Ssang-Goo Cho. "Production of Mesenchymal Stem Cells Through Stem Cell Reprogramming." International Journal of Molecular Sciences 20, no. 8 (April 18, 2019): 1922. http://dx.doi.org/10.3390/ijms20081922.
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Mesenchymal stem cells (MSCs) possess a broad spectrum of therapeutic applications and have been used in clinical trials. MSCs are mainly retrieved from adult or fetal tissues. However, there are many obstacles with the use of tissue-derived MSCs, such as shortages of tissue sources, difficult and invasive retrieval methods, cell population heterogeneity, low purity, cell senescence, and loss of pluripotency and proliferative capacities over continuous passages. Therefore, other methods to obtain high-quality MSCs need to be developed to overcome the limitations of tissue-derived MSCs. Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are considered potent sources for the derivation of MSCs. PSC-derived MSCs (PSC-MSCs) may surpass tissue-derived MSCs in proliferation capacity, immunomodulatory activity, and in vivo therapeutic applications. In this review, we will discuss basic as well as recent protocols for the production of PSC-MSCs and their in vitro and in vivo therapeutic efficacies. A better understanding of the current advances in the production of PSC-MSCs will inspire scientists to devise more efficient differentiation methods that will be a breakthrough in the clinical application of PSC-MSCs.
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Hsu, Yi-Chao, Yu-Ting Wu, Chia-Ling Tsai, and Yau-Huei Wei. "Current understanding and future perspectives of the roles of sirtuins in the reprogramming and differentiation of pluripotent stem cells." Experimental Biology and Medicine 243, no. 6 (March 2018): 563–75. http://dx.doi.org/10.1177/1535370218759636.
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In mammalian cells, there are seven members of the sirtuin protein family (SIRT1–7). SIRT1, SIRT6, and SIRT7 catalyze posttranslational modification of proteins in the nucleus, SIRT3, SIRT4, and SIRT5 are in the mitochondria and SIRT2 is in the cytosol. SIRT1 can deacetylate the transcription factor SOX2 and regulate induced pluripotent stem cells (iPSCs) reprogramming through the miR-34a–SIRT1–p53 axis. SIRT2 can regulate the function of pluripotent stem cells through GSK3β. SIRT3 can positively regulate PPAR gamma coactivator 1-alpha (PGC-1α) expression during the differentiation of stem cells. SIRT4 has no direct role in regulating reprogramming but may have the potential to prevent senescence of somatic cells and to facilitate the reprogramming of iPSCs. SIRT5 can deacetylate STAT3, which is an important transcription factor in regulating pluripotency and differentiation of stem cells. SIRT6 can enhance the reprogramming efficiency of iPSCs from aged skin fibroblasts through miR-766 and increase the expression levels of the reprogramming genes including Sox2, Oct4, and Nanog through acetylation of histone H3 lysine 56. SIRT7 plays a regulatory role in the process of mesenchymal-to-epithelial transition (MET), which has been suggested to be a crucial process in the generation of iPSCs from fibroblasts. In this review, we summarize recent findings of the roles of sirtuins in the metabolic reprogramming and differentiation of stem cells and discuss the bidirectional changes in the gene expression and activities of sirtuins in the commitment of differentiation of mesenchymal stem cells (MSCs) and reprogramming of somatic cells to iPSCs, respectively. Thus, understanding the molecular basis of the interplay between different sirtuins and mitochondrial function will provide new insights into the regulation of differentiation of stem cells and iPSCs formation, respectively, and may help design effective stem cell therapies for regenerative medicine. Impact statement This is an extensive review of the recent advances in our understanding of the roles of some members of the sirtuins family, such as SIRT1, SIRT2, SIRT3, and SIRT6, in the regulation of intermediary metabolism during stem cell differentiation and in the reprogramming of somatic cells to form induced pluripotent stem cells (iPSCs). This article provides an updated integrated view on the mechanisms by which sirtuins-mediated posttranslational protein modifications regulate mitochondrial biogenesis, bioenergetics, and antioxidant defense in the maintenance and differentiation of stem cells and in iPSCs formation, respectively.
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Umrath, Felix, Marbod Weber, Siegmar Reinert, Hans-Peter Wendel, Meltem Avci-Adali, and Dorothea Alexander. "iPSC-Derived MSCs Versus Originating Jaw Periosteal Cells: Comparison of Resulting Phenotype and Stem Cell Potential." International Journal of Molecular Sciences 21, no. 2 (January 16, 2020): 587. http://dx.doi.org/10.3390/ijms21020587.
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Induced pluripotent stem cell-derived mesenchymal stem cell-like cells (iMSCs) are considered to be a promising source of progenitor cells for approaches in the field of bone regeneration. In a previous study, we described the generation of footprint-free induced pluripotent stem cells (iPSCs) from human jaw periosteal cells (JPCs) by transfection of a self-replicating RNA (srRNA) and subsequent differentiation into functional osteogenic progenitor cells. In order to facilitate the prospective transfer into clinical practice, xeno-free reprogramming and differentiation methods were established. In this study, we compared the properties and stem cell potential of the iMSCs produced from JPC-derived iPSCs with the parental primary JPCs they were generated from. Our results demonstrated, on the one hand, a comparable differentiation potential of iMSCs and JPCs. Additionally, iMSCs showed significantly longer telomere lengths compared to JPCs indicating rejuvenation of the cells during reprogramming. On the other hand, proliferation, mitochondrial activity, and senescence-associated beta-galactosidase (SA-β-gal) activity indicated early senescence of iMSCs. These data demonstrate the requirement of further optimization strategies to improve mesenchymal development of JPC-derived iPSCs in order to take advantage of the best features of reprogrammed and rejuvenated cells.
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Wang, Aline Yen Ling. "Application of Modified mRNA in Somatic Reprogramming to Pluripotency and Directed Conversion of Cell Fate." International Journal of Molecular Sciences 22, no. 15 (July 29, 2021): 8148. http://dx.doi.org/10.3390/ijms22158148.
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Modified mRNA (modRNA)-based somatic reprogramming is an effective and safe approach that overcomes the genomic mutation risk caused by viral integrative methods. It has improved the disadvantages of conventional mRNA and has better stability and immunogenicity. The modRNA molecules encoding multiple pluripotent factors have been applied successfully in reprogramming somatic cells such as fibroblasts, mesenchymal stem cells, and amniotic fluid stem cells to generate pluripotent stem cells (iPSCs). Moreover, it also can be directly used in the terminal differentiation of stem cells and fibroblasts into functional therapeutic cells, which exhibit great promise in disease modeling, drug screening, cell transplantation therapy, and regenerative medicine. In this review, we summarized the reprogramming applications of modified mRNA in iPSC generation and therapeutic applications of functionally differentiated cells.
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Ramazzotti, Ratti, Fiume, Yung Follo, Billi, Rusciano, Owusu Obeng, Manzoli, Cocco, and Faenza. "Phosphoinositide 3 Kinase Signaling in Human Stem Cells from Reprogramming to Differentiation: A Tale in Cytoplasmic and Nuclear Compartments." International Journal of Molecular Sciences 20, no. 8 (April 24, 2019): 2026. http://dx.doi.org/10.3390/ijms20082026.
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Stem cells are undifferentiated cells that can give rise to several different cell types and can self-renew. Given their ability to differentiate into different lineages, stem cells retain huge therapeutic potential for regenerative medicine. Therefore, the understanding of the signaling pathways involved in stem cell pluripotency maintenance and differentiation has a paramount importance in order to understand these biological processes and to develop therapeutic strategies. In this review, we focus on phosphoinositide 3 kinase (PI3K) since its signaling pathway regulates many cellular processes, such as cell growth, proliferation, survival, and cellular transformation. Precisely, in human stem cells, the PI3K cascade is involved in different processes from pluripotency and induced pluripotent stem cell (iPSC) reprogramming to mesenchymal and oral mesenchymal differentiation, through different and interconnected mechanisms.
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Eguchi, Takanori, and Takuo Kuboki. "Cellular Reprogramming Using Defined Factors and MicroRNAs." Stem Cells International 2016 (2016): 1–12. http://dx.doi.org/10.1155/2016/7530942.
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Development of human bodies, organs, and tissues contains numerous steps of cellular differentiation including an initial zygote, embryonic stem (ES) cells, three germ layers, and multiple expertized lineages of cells. Induced pluripotent stem (iPS) cells have been recently developed using defined reprogramming factors such as Nanog, Klf5, Oct3/4 (Pou5f1), Sox2, and Myc. This outstanding innovation is largely changing life science and medicine. Methods of direct reprogramming of cells into myocytes, neurons, chondrocytes, and osteoblasts have been further developed using modified combination of factors such as N-myc, L-myc, Sox9, and microRNAs in defined cell/tissue culture conditions. Mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) are also emerging multipotent stem cells with particular microRNA expression signatures. It was shown that miRNA-720 had a role in cellular reprogramming through targeting the pluripotency factor Nanog and induction of DNA methyltransferases (DNMTs). This review reports histories, topics, and idea of cellular reprogramming.
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Moslem, Mohsen, Irina Eberle, Iuliia Weber, Reinhard Henschler, and Tobias Cantz. "Mesenchymal Stem/Stromal Cells Derived from Induced Pluripotent Stem Cells Support CD34posHematopoietic Stem Cell Propagation and Suppress Inflammatory Reaction." Stem Cells International 2015 (2015): 1–14. http://dx.doi.org/10.1155/2015/843058.
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Mesenchymal stem/stromal cells (MSCs) represent a promising cell source for research and therapeutic applications, but their restrictedex vivopropagation capabilities limit putative applications. Substantial self-renewing of stem cells can be achieved by reprogramming cells into induced pluripotent stem cells (iPSCs) that can be easily expanded as undifferentiated cells even in mass culture. Here, we investigated a differentiation protocol enabling the generation and selection of human iPSC-derived MSCs exhibiting relevant surface marker expression profiles (CD105 and CD73) and functional characteristics. We generated such iPSC-MSCs from fibroblasts and bone marrow MSCs utilizing two different reprogramming constructs. All such iPSC-MSCs exhibited the characteristics of normal bone marrow-derived (BM) MSCs. In direct comparison to BM-MSCs our iPSC-MSCs exhibited a similar surface marker expression profile but shorter doubling times without reaching senescence within 20 passages. Considering functional capabilities, iPSC-MSCs provided supportive feeder layer for CD34+hematopoietic stem cells’ self-renewal and colony forming capacities. Furthermore, iPSC-MSCs gained immunomodulatory function to suppress CD4+cell proliferation, reduce proinflammatory cytokines in mixed lymphocyte reaction, and increase regulatory CD4+/CD69+/CD25+T-lymphocyte population. In conclusion, we generated fully functional MSCs from various iPSC lines irrespective of their starting cell source or reprogramming factor composition and we suggest that such iPSC-MSCs allow repetitive cell applications for advanced therapeutic approaches.
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Mueller, Paula, Markus Wolfien, Katharina Ekat, Cajetan Immanuel Lang, Dirk Koczan, Olaf Wolkenhauer, Olga Hahn, et al. "RNA-Based Strategies for Cardiac Reprogramming of Human Mesenchymal Stromal Cells." Cells 9, no. 2 (February 22, 2020): 504. http://dx.doi.org/10.3390/cells9020504.
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Multipotent adult mesenchymal stromal cells (MSCs) could represent an elegant source for the generation of patient-specific cardiomyocytes needed for regenerative medicine, cardiovascular research, and pharmacological studies. However, the differentiation of adult MSC into a cardiac lineage is challenging compared to embryonic stem cells or induced pluripotent stem cells. Here we used non-integrative methods, including microRNA and mRNA, for cardiac reprogramming of adult MSC derived from bone marrow, dental follicle, and adipose tissue. We found that MSC derived from adipose tissue can partly be reprogrammed into the cardiac lineage by transient overexpression of GATA4, TBX5, MEF2C, and MESP1, while cells isolated from bone marrow, and dental follicle exhibit only weak reprogramming efficiency. qRT-PCR and transcriptomic analysis revealed activation of a cardiac-specific gene program and up-regulation of genes known to promote cardiac development. Although we did not observe the formation of fully mature cardiomyocytes, our data suggests that adult MSC have the capability to acquire a cardiac-like phenotype when treated with mRNA coding for transcription factors that regulate heart development. Yet, further optimization of the reprogramming process is mandatory to increase the reprogramming efficiency.
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Tran, Van Hong, Hoa Trong Nguyen, and Phuc Van Pham. "Conversion of human adipose derived stem cells into endothelial progenitor cells." Progress in Stem Cell 4, no. 3-4 (November 29, 2017): 217–27. http://dx.doi.org/10.15419/psc.v4i3.396.
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Introduction: Endothelial cells (ECs) or endothelial progenitor cells (EPCs) are essential cells for blood vascular regeneration and vascular tissue engineering. However, the source of EPCs are limited. Indeed, these cells only existence with low rate at some tissues such as bone marrow, umbilical cord blood and peripheral blood. This study aimed to produce EPCs from direct reprogramming of adipose tissue-derived mesenchymal stem cells (ADSCs) by ETV2 transfection in vitro.
Methods: ADSCs were isolated according to the published works. They were confirmed as mesenchymal stem cells (MSCs) with some characteristics included expression of CD44, CD73, CD90, negative of CD14, CD45, and HLA-DR; in vitro differentiation into adipocytes, and osteoblasts. ETV-2 mRNA was in vitro produced by commercial kit. ETV-2 mRNA molecules were transfected into ADSCs by Fugenes and Lipofectamine agents. These transfected cells were evaluated the expression of EPC properties included expression of CD31, VEGFR-2 in the cell surface by flow cytometry, immunocytochemistry, and in vitro vessel formation in the Matrigel.
Results: The results showed that ETV-2 could transform the ADSCs from mesenchymal cell phenotype into endothelial cell phenotype with 10% transfected ADSCs expressing the CD31 in their surface, they also could form the vessel structure in vitro.
Conclusion: Although the low efficacy of direct reprogramming, this study gave the new strategy to produce EPCs from the favorite cell sources as ADSCs.
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Teven, Chad M., Xing Liu, Ning Hu, Ni Tang, Stephanie H. Kim, Enyi Huang, Ke Yang, et al. "Epigenetic Regulation of Mesenchymal Stem Cells: A Focus on Osteogenic and Adipogenic Differentiation." Stem Cells International 2011 (2011): 1–18. http://dx.doi.org/10.4061/2011/201371.
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Stem cells are characterized by their capability to self-renew and terminally differentiate into multiple cell types. Somatic or adult stem cells have a finite self-renewal capacity and are lineage-restricted. The use of adult stem cells for therapeutic purposes has been a topic of recent interest given the ethical considerations associated with embryonic stem (ES) cells. Mesenchymal stem cells (MSCs) are adult stem cells that can differentiate into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Owing to their ease of isolation and unique characteristics, MSCs have been widely regarded as potential candidates for tissue engineering and repair. While various signaling molecules important to MSC differentiation have been identified, our complete understanding of this process is lacking. Recent investigations focused on the role of epigenetic regulation in lineage-specific differentiation of MSCs have shown that unique patterns of DNA methylation and histone modifications play an important role in the induction of MSC differentiation toward specific lineages. Nevertheless, MSC epigenetic profiles reflect a more restricted differentiation potential as compared to ES cells. Here we review the effect of epigenetic modifications on MSC multipotency and differentiation, with a focus on osteogenic and adipogenic differentiation. We also highlight clinical applications of MSC epigenetics and nuclear reprogramming.
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Umrath, Felix, Heidrun Steinle, Marbod Weber, Hans-Peter Wendel, Siegmar Reinert, Dorothea Alexander, and Meltem Avci-Adali. "Generation of iPSCs from Jaw Periosteal Cells Using Self-Replicating RNA." International Journal of Molecular Sciences 20, no. 7 (April 3, 2019): 1648. http://dx.doi.org/10.3390/ijms20071648.
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Jaw periosteal cells (JPCs) represent a suitable stem cell source for bone tissue engineering (BTE) applications. However, challenges associated with limited cell numbers, stressful cell sorting, or the occurrence of cell senescence during in vitro passaging and the associated insufficient osteogenic potential in vitro of JPCs and other mesenchymal stem/stromal cells (MSCs) are main hurdles and still need to be solved. In this study, for the first time, induced pluripotent stem cells (iPSCs) were generated from human JPCs to open up a new source of stem cells for BTE. For this purpose, a non-integrating self-replicating RNA (srRNA) encoding reprogramming factors and green fluorescent protein (GFP) as a reporter was used to obtain JPC-iPSCs with a feeder- and xeno-free reprogramming protocol to meet the highest safety standards for future clinical applications. Furthermore, to analyze the potential of these iPSCs as a source of osteogenic progenitor cells, JPC-iPSCs were differentiated into iPSC-derived mesenchymal stem/stromal like cells (iMSCs) and further differentiated to the osteogenic lineage under xeno-free conditions. The produced iMSCs displayed MSC marker expression and morphology as well as strong mineralization during osteogenic differentiation.
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Su, Xiaohu, Yu Ling, Chunxia Liu, Fanhua Meng, Junwei Cao, Li Zhang, Huanmin Zhou, Zongzheng Liu, and Yanru Zhang. "Isolation, Culture, Differentiation, and Nuclear Reprogramming of Mongolian Sheep Fetal Bone Marrow–Derived Mesenchymal Stem Cells." Cellular Reprogramming 17, no. 4 (August 2015): 288–96. http://dx.doi.org/10.1089/cell.2014.0109.
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He, Shuyang, Joseph Gleason, Ewa Fik-Rymarkiewicz, Andrea DiFiglia, Mini Bharathan, Andrew Morschauser, Ivana Djuretic, et al. "Human Placenta-Derived Mesenchymal Stromal-Like Cells Enhance Angiogenesis via T Cell-Dependent Reprogramming of Macrophage Differentiation." STEM CELLS 35, no. 6 (March 14, 2017): 1603–13. http://dx.doi.org/10.1002/stem.2598.
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Divvela, Satya Srirama Karthik, Patrick Nell, Markus Napirei, Holm Zaehres, Jiayu Chen, Wanda Maria Gerding, Huu Phuc Nguyen, Shaorong Gao, and Beate Brand-Saberi. "bHLH Transcription Factor Math6 Antagonizes TGF-β Signalling in Reprogramming, Pluripotency and Early Cell Fate Decisions." Cells 8, no. 6 (June 2, 2019): 529. http://dx.doi.org/10.3390/cells8060529.
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The basic helix-loop-helix (bHLH) transcription factor Math6 (Atonal homolog 8; Atoh8) plays a crucial role in a number of cellular processes during embryonic development, iron metabolism and tumorigenesis. We report here on its involvement in cellular reprogramming from fibroblasts to induced pluripotent stem cells, in the maintenance of pluripotency and in early fate decisions during murine development. Loss of Math6 disrupts mesenchymal-to-epithelial transition during reprogramming and primes pluripotent stem cells towards the mesendodermal fate. Math6 can thus be considered a regulator of reprogramming and pluripotent stem cell fate. Additionally, our results demonstrate the involvement of Math6 in SMAD-dependent TGF beta signalling. We furthermore monitor the presence of the Math6 protein during these developmental processes using a newly generated Math6Flag-tag mouse. Taken together, our results suggest that Math6 counteracts TGF beta signalling and, by this, affects the initiating step of cellular reprogramming, as well as the maintenance of pluripotency and early differentiation.
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Recchia, Kaiana, Laís Vicari de Figueiredo Pessôa, Naira Caroline Godoy Pieri, Pedro Ratto Lisboa Pires, and Fabiana Fernandes Bressan. "Influence of Cell Type in In Vitro Induced Reprogramming in Cattle." Life 12, no. 8 (July 28, 2022): 1139. http://dx.doi.org/10.3390/life12081139.
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Induced pluripotent stem cells (iPSCs) have been considered an essential tool in stem cell research due to their potential to develop new therapies and technologies and answer essential questions about mammalian early development. An important step in generating iPSCs is selecting their precursor cell type, influencing the reprogramming efficiency and maintenance in culture. In this study, we aim to characterize bovine mesenchymal cells from adipose tissue (bAdMSCs) and fetal fibroblasts (bFFs) and to compare the reprogramming efficiency of these cells when induced to pluripotency. The cells were characterized by immunostaining (CD90, SSEA1, SSEA3, and SSEA4), induced differentiation in vitro, proliferation rates, and were subjected to cell reprogramming using the murine OSKM transcription factors. The bFFs presented morphological changes resembling pluripotent cells after reprogramming and culture with different supplementation, and putative iPSCs were characterized by immunostaining (OCT4, SOX2, NANOG, and AP). In the present study, we demonstrated that cell line origin and cellular proliferation rate are determining factors for reprogramming cells into pluripotency. The generation of biPSCs is a valuable tool to improve both translational medicine and animal production and to study the different supplements required to maintain the pluripotency of bovine cells in vitro.
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Fink, Kyle D., Julien Rossignol, Ming Lu, Xavier Lévêque, Travis D. Hulse, Andrew T. Crane, Veronique Nerriere-Daguin, et al. "Survival and Differentiation of Adenovirus-Generated Induced Pluripotent Stem Cells Transplanted into the Rat Striatum." Cell Transplantation 23, no. 11 (November 2014): 1407–23. http://dx.doi.org/10.3727/096368913x670958.
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Induced pluripotent stem cells (iPSCs) offer certain advantages over embryonic stem cells in cell replacement therapy for a variety of neurological disorders. However, reliable procedures, whereby transplanted iPSCs can survive and differentiate into functional neurons, without forming tumors, have yet to be devised. Currently, retroviral or lentiviral reprogramming methods are often used to reprogram somatic cells. Although the use of these viruses has proven to be effective, formation of tumors often results following in vivo transplantation, possibly due to the integration of the reprogramming genes. The goal of the current study was to develop a new approach, using an adenovirus for reprogramming cells, characterize the iPSCs in vitro, and test their safety, survivability, and ability to differentiate into region-appropriate neurons following transplantation into the rat brain. To this end, iPSCs were derived from bone marrow-derived mesenchymal stem cells and tail-tip fibroblasts using a single cassette lentivirus or a combination of adenoviruses. The reprogramming efficiency and levels of pluripotency were compared using immunocytochemistry, flow cytometry, and real-time polymerase chain reaction. Our data indicate that adenovirus-generated iPSCs from tail-tip fibroblasts are as efficient as the method we used for lentiviral reprogramming. All generated iPSCs were also capable of differentiating into neuronal-like cells in vitro. To test the in vivo survivability and the ability to differentiate into region-specific neurons in the absence of tumor formation, 400,000 of the iPSCs derived from tail-tip fibroblasts that were transfected with the adenovirus pair were transplanted into the striatum of adult, immune-competent rats. We observed that these iPSCs produced region-specific neuronal phenotypes, in the absence of tumor formation, at 90 days posttransplantation. These results suggest that adenovirus-generated iPSCs may provide a safe and viable means for neuronal replacement therapies.
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Gu, Wenduo, Witold N. Nowak, Yao Xie, Alexandra Le Bras, Yanhua Hu, Jiacheng Deng, Shirin Issa Bhaloo, et al. "Single-Cell RNA-Sequencing and Metabolomics Analyses Reveal the Contribution of Perivascular Adipose Tissue Stem Cells to Vascular Remodeling." Arteriosclerosis, Thrombosis, and Vascular Biology 39, no. 10 (October 2019): 2049–66. http://dx.doi.org/10.1161/atvbaha.119.312732.
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Objective: Perivascular adipose tissue (PVAT) plays a vital role in maintaining vascular homeostasis. However, most studies ascribed the function of PVAT in vascular remodeling to adipokines secreted by the perivascular adipocytes. Whether mesenchymal stem cells exist in PVAT and play a role in vascular regeneration remain unknown. Approach and Results: Single-cell RNA-sequencing allowed direct visualization of the heterogeneous PVAT-derived mesenchymal stem cells (PV-ADSCs) at a high resolution and revealed 2 distinct subpopulations, among which one featured signaling pathways crucial for smooth muscle differentiation. Pseudotime analysis of cultured PV-ADSCs unraveled their smooth muscle differentiation trajectory. Transplantation of cultured PV-ADSCs in mouse vein graft model suggested the contribution of PV-ADSCs to vascular remodeling through smooth muscle differentiation. Mechanistically, treatment with TGF-β1 (transforming growth factor β1) and transfection of microRNA (miR)-378a-3p mimics induced a similar metabolic reprogramming of PV-ADSCs, including upregulated mitochondrial potential and altered lipid levels, such as increased cholesterol and promoted smooth muscle differentiation. Conclusions: Single-cell RNA-sequencing allows direct visualization of PV-ADSC heterogeneity at a single-cell level and uncovers 2 subpopulations with distinct signature genes and signaling pathways. The function of PVAT in vascular regeneration is partly attributed to PV-ADSCs and their differentiation towards smooth muscle lineage. Mechanistic study presents miR-378a-3p which is a potent regulator of metabolic reprogramming as a potential therapeutic target for vascular regeneration.
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Lee, Dong-Seol, Yeo Joon Song, Hye Ri Gug, Ji-Hyun Lee, Hyun Sook Bae, and Joo-Cheol Park. "Nuclear Factor I-C Regulates Stemness Genes and Proliferation of Stem Cells in Various Mineralized Tissue through Epithelial-Mesenchymal Interactions in Dental Epithelial Stem Cells." Stem Cells International 2022 (September 27, 2022): 1–19. http://dx.doi.org/10.1155/2022/1092184.
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Tooth development includes numerous cell divisions and cell-cell interactions generating the stem cell niche. After an indefinite number of divisions, pluripotent cells differentiate into various types of cells. Nuclear factor I (NFI) transcription factors are known as crucial regulators in various organ development and stem cell biology. Among its members, nuclear factor I-C (NFI-C) has been reported to play an essential role in odontogenesis. Nfic knockout mice show malformation in all mineralized tissues, but it remains unclear which stage of development Nfic is involved in. We previously reported that Nfic induces the differentiation of ameloblast, odontoblast, and osteoblast. However, the question remains whether Nfic participates in the late stage of development, perpetuating the proliferation of stem cells. This study aimed to elucidate the underlying mechanism of NFI-C function in stem cells capable of forming hard tissues. Here, we demonstrate that Nfic regulates Sox2 and cell proliferation in diverse mineralized tissue stem cells such as dental epithelial stem cells (DESCs), dental pulp stem cells, and bone marrow stem cells, but not in fibroblasts. It was also involved in the expression of pluripotency genes Lin28 and NANOG. Especially in DESCs, Nfic regulates the proliferation of epithelial cells via epithelial-mesenchymal interactions, which are the Fgf8-Nfic-Sox2 pathway in epithelium and Nfic-Fgf10 in the mesenchyme. Moreover, Nfic slightly increased reprogramming efficiency in induced pluripotent stem cells of mineralized tissues, but not in soft tissues. In conclusion, these results suggest that Nfic is a crucial factor for maintaining the stem cell niche of mineralized tissues and provides a possibility for Nfic as an additional factor in improving reprogramming efficiency.
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Choi, Da Hyeon, Kyeong Eun Lee, Jiwon Park, Yoon Jeong Park, Jue-Yeon Lee, and Yoon Shin Park. "Cell-Permeable Oct4 Gene Delivery Enhances Stem Cell-like Properties of Mouse Embryonic Fibroblasts." International Journal of Molecular Sciences 22, no. 17 (August 28, 2021): 9357. http://dx.doi.org/10.3390/ijms22179357.
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Direct conversion of one cell type into another is a trans-differentiation process. Recent advances in fibroblast research revealed that epithelial cells can give rise to fibroblasts by epithelial-mesenchymal transition. Conversely, fibroblasts can also give rise to epithelia by undergoing a mesenchymal to epithelial transition. To elicit stem cell-like properties in fibroblasts, the Oct4 transcription factor acts as a master transcriptional regulator for reprogramming somatic cells. Notably, the production of gene complexes with cell-permeable peptides, such as low-molecular-weight protamine (LMWP), was proposed to induce reprogramming without cytotoxicity and genomic mutation. We designed a complex with non-cytotoxic LMWP to prevent the degradation of Oct4 and revealed that the positively charged cell-permeable LMWP helped condense the size of the Oct4-LMWP complexes (1:5 N:P ratio). When the Oct4-LMWP complex was delivered into mouse embryonic fibroblasts (MEFs), stemness-related gene expression increased while fibroblast intrinsic properties decreased. We believe that the Oct4-LMWP complex developed in this study can be used to reprogram terminally differentiated somatic cells or convert them into stem cell-like cells without risk of cell death, improving the stemness level and stability of existing direct conversion techniques.
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Kocaefe, Çetin, Deniz Balcı, Burcu Balcı Hayta, and Alp Can. "Reprogramming of Human Umbilical Cord Stromal Mesenchymal Stem Cells for Myogenic Differentiation and Muscle Repair." Stem Cell Reviews and Reports 6, no. 4 (July 28, 2010): 512–22. http://dx.doi.org/10.1007/s12015-010-9177-7.
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Choi, K. H., D. Son, D. K. Lee, J. N. Oh, S. H. Kim, T. Y. Park, and C. K. Lee. "222 INCOMPLETE REPROGRAMMING OF INDUCED PLURIPOTENT STEM CELLS DERIVED FROM PORCINE FETAL FIBROBLASTS." Reproduction, Fertility and Development 28, no. 2 (2016): 242. http://dx.doi.org/10.1071/rdv28n2ab222.
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Cellular reprogramming of committed cells into a pluripotent state can be accomplished by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. However, during reprogramming, it has been verified that failures of reactivating endogenous genes and epigenetic remodelling lead to partially reprogrammed cells exhibiting features similar to those of fully reprogrammed cells. In this study, partially reprogrammed induced pluripotent stem cells (pre-iPSC) were derived from porcine fetal fibroblasts via drug-inducible vector carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study aimed to investigate characteristics of pre-iPSC and reprogramming mechanisms. The pre-iPSC were stably maintained over an extended period having in vitro differentiation ability into 3 germ layers. The pluripotent state of pre-iPSC was regulated by modulation of culture condition. They showed naive- or primed-like pluripotent state in leukemia inhibitory factor (LIF) or basic fibroblast growth factor (bFGF) supplemented culture conditions respectively. However, pre-iPSC could not be maintained without ectopic expression of transgenes. The cultured pre-iPSC expressed endogenous transcription factors (OCT4 and SOX2) except for NANOG known as gateway into complete reprogramming. In addition, endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occurred in female pre-iPSC. Given the results, we found that expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu for achieving faithful pluripotency in pig. Accordingly, investigating pre-iPSC could help us to improve and develop reprogramming methods via understanding reprogramming mechanisms in pig. This work was supported by the Next-generation BioGreen 21 Program (PJ0113002015), Rural Development Administration, Republic of Korea.
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Agrawal, Mona, Pratheepa Kumari Rasiah, Amandeep Bajwa, Johnson Rajasingh, and Rajashekhar Gangaraju. "Mesenchymal Stem Cell Induced Foxp3(+) Tregs Suppress Effector T Cells and Protect against Retinal Ischemic Injury." Cells 10, no. 11 (November 4, 2021): 3006. http://dx.doi.org/10.3390/cells10113006.
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Mesenchymal stem/stromal cells (MSC) are well known for immunomodulation; however, the mechanisms involved in their benefits in the ischemic retina are unknown. This study tested the hypothesis that MSC induces upregulation of transcription factor forkhead box protein P3 (Foxp3) in T cells to elicit immune modulation, and thus, protect against retinal damage. Induced MSCs (iMSCs) were generated by differentiating the induced pluripotent stem cells (iPSC) derived from urinary epithelial cells through a noninsertional reprogramming approach. In in-vitro cultures, iMSC transferred mitochondria to immune cells via F-actin nanotubes significantly increased oxygen consumption rate (OCR) for basal respiration and ATP production, suppressed effector T cells, and promoted differentiation of CD4+CD25+ T regulatory cells (Tregs) in coculture with mouse splenocytes. In in-vivo studies, iMSCs transplanted in ischemia-reperfusion (I/R) injured eye significantly increased Foxp3+ Tregs in the retina compared to that of saline-injected I/R eyes. Furthermore, iMSC injected I/R eyes significantly decreased retinal inflammation as evidenced by reduced gene expression of IL1β, VCAM1, LAMA5, and CCL2 and improved b-wave amplitudes compared to that of saline-injected I/R eyes. Our study demonstrates that iMSCs can transfer mitochondria to immune cells to suppress the effector T cell population. Additionally, our current data indicate that iMSC can enhance differentiation of T cells into Foxp3 Tregs in vitro and therapeutically improve the retina’s immune function by upregulation of Tregs to decrease inflammation and reduce I/R injury-induced retinal degeneration in vivo.
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Liu, Chang, Xu Hu, Yawen Li, Wenjie Lu, Wenlin Li, Nan Cao, Saiyong Zhu, Jinke Cheng, Sheng Ding, and Mingliang Zhang. "Conversion of mouse fibroblasts into oligodendrocyte progenitor-like cells through a chemical approach." Journal of Molecular Cell Biology 11, no. 6 (January 10, 2019): 489–95. http://dx.doi.org/10.1093/jmcb/mjy088.
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Abstract Transplantation of oligodendrocyte progenitor cells (OPCs) is a promising way for treating demyelinating diseases. However, generation of scalable and autologous sources of OPCs has proven difficult. We previously established a chemical condition M9 that could specifically initiate neural program in mouse embryonic fibroblasts. Here we found that M9 could induce the formation of colonies that undergo mesenchymal-to-epithelial transition at the early stage of reprogramming. These colonies may represent unstable and neural lineage-restricted intermediates that have not established a neural stem cell identity. By modulating the culture signaling recapitulating the principle of OPC development, these intermediate cells could be reprogrammed towards OPC fate. The chemical-induced OPC-like cells (ciOPLCs) resemble primary neural stem cell-derived OPCs in terms of their morphology, gene expression, and the ability of self-renewal. Upon differentiation, ciOPLCs could produce functional oligodendrocytes and myelinate the neuron axons in vitro, validating their OPC identity molecularly and functionally. Therefore, our study provides a non-integrating approach to OPC reprogramming that may ultimately provide an avenue to patient-specific cell-based or in situ regenerative therapy.
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Pessôa, Laís Vicari de Figueiredo, Pedro Ratto Lisboa Pires, Maite del Collado, Naira Caroline Godoy Pieri, Kaiana Recchia, Aline Fernanda Souza, Felipe Perecin, et al. "Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues." Stem Cells International 2019 (May 2, 2019): 1–15. http://dx.doi.org/10.1155/2019/1393791.
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Introduction. Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species. Objectives. We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes. Methods. Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed. Results. Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells. Conclusions. We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.
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Cherubini, Alessandro, Mario Barilani, Riccardo L. Rossi, Murtadhah M. K. Jalal, Francesco Rusconi, Giuseppe Buono, Enrico Ragni, et al. "FOXP1 circular RNA sustains mesenchymal stem cell identity via microRNA inhibition." Nucleic Acids Research 47, no. 10 (April 2, 2019): 5325–40. http://dx.doi.org/10.1093/nar/gkz199.
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Abstract Stem cell identity and plasticity are controlled by master regulatory genes and complex circuits also involving non-coding RNAs. Circular RNAs (circRNAs) are a class of RNAs generated from protein-coding genes by backsplicing, resulting in stable RNA structures devoid of free 5’ and 3’ ends. Little is known of the mechanisms of action of circRNAs, let alone in stem cell biology. In this study, for the first time, we determined that a circRNA controls mesenchymal stem cell (MSC) identity and differentiation. High-throughput MSC expression profiling from different tissues revealed a large number of expressed circRNAs. Among those, circFOXP1 was enriched in MSCs compared to differentiated mesodermal derivatives. Silencing of circFOXP1 dramatically impaired MSC differentiation in culture and in vivo. Furthermore, we demonstrated a direct interaction between circFOXP1 and miR-17–3p/miR-127–5p, which results in the modulation of non-canonical Wnt and EGFR pathways. Finally, we addressed the interplay between canonical and non-canonical Wnt pathways. Reprogramming to pluripotency of MSCs reduced circFOXP1 and non-canonical Wnt, whereas canonical Wnt was boosted. The opposing effect was observed during generation of MSCs from human pluripotent stem cells. Our results provide unprecedented evidence for a regulatory role for circFOXP1 as a gatekeeper of pivotal stem cell molecular networks.
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Kang, E. J., B. U. Park, H. J. Song, Y. I. Yang, M. J. Kim, K. H. Maeng, B. G. Jeon, S. L. Lee, and G. J. Rho. "279 COMPARISONS OF BONE MARROW AND PUTATIVE SKIN-DERIVED MESENCHYMAL STEM CELLS AS DONOR FOR NUCLEAR TRANSFER IN MINIATURE PIG." Reproduction, Fertility and Development 21, no. 1 (2009): 236. http://dx.doi.org/10.1071/rdv21n1ab279.
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Since the birth of the first cloned sheep was reported, fibroblasts are commonly used as donor cells for nuclear transfer. However, in some species there is a higher incidence of abnormal fetuses, still births, and neonatal deaths as a consequence of reprogramming disorders including DNA methylation and histone acetylation. Cloned embryos with mesenchymal stem cells (MSC) have shown higher developmental ability compared to fibroblast, suggesting that undifferentiated genome can required ease reprogramming. Because MSC are relatively difficult to collect from bone marrow, skin is an alternative source of the donor cells. However, molecular and functional analyses remain uncertain between MSC derived from bone marrow and skin stem cells isolated from ear. The present study compared the expression of early transcription factors (Oct-4, Nanog and Sox-2), and differentiation capability to osteocytes, adipocytes and chondrocytes of MSC isolated from bone marrow and skin-derived putative stem cells from miniature pig. Bone marrow was isolated by Ficoll density gradient method, and skin separated from epidermis and dermis was diced into 2-mm diameter explants, and attached to tissue culture dishes. Cells were then cultured in DMEM/F12 supplemented with 10% FBS, 10 ng mL–1 bFGF, 10 ng mL–1 EGF at 38.5°C, in a humidified atmosphere of 5% CO2 in air. Expression of Oct-4, Nanog, Sox2 was analysed by RT-PCR. Osteogenic and adipogenic differentiation were induced following the protocols described previously (Jin et al. 2007 Int. J. Dev. Biol. 51, 85–90; Mohana Kumar et al. 2007 Mol. Cells 24, 343–350) and compared by histological staining and RT-PCR. Osteocytes were defined by the formation of the mineral nodules of deposition of calcium by Von Kossa staining and differentiations into adipocytes and chondrocytes were identified by oil red O staining of lipid vacuoles and alcian blue of proteoglycan, respectively. Skin-derived MSC were revealed to similar mRNA expression of Oct-4, Nanog, Sox2 compared to bone marrow derived MSC. However, bone marrow derived MSC were higher mRNA expression about of osteocytic genes (osteoclacin and osteonectin), chondrocytic gene (collagen type) and adipocytic genes (aP2) than those of skin-derived MSC. In addition, bone marrow derived MSC were revealed greater deposition of calcium, proteoglycan, and lipid vacuoles than those of skin derived MSC by histological staining. The results of present study suggest that cells isolated from skin have lower potential than MSC isolated from bone marrow. However, skin-derived stem cells have properties of multi-lineage differentiation and can be obtained easily. These stem cells, therefore, can serve as easily accessible and expandable source possessing donor cells for cloning, potential animal model, and clinical applications. This work was supported by Grant No. 20070301034040 from Bio-organ, Republic of Korea.
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Sato, Yasushi, Hironobu Araki, Junji Kato, Kiminori Nakamura, Yutaka Kawano, Masayoshi Kobune, Tsutomu Sato, et al. "Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion." Blood 106, no. 2 (July 15, 2005): 756–63. http://dx.doi.org/10.1182/blood-2005-02-0572.
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Abstract Hepatic transdifferentiation of bone marrow cells has been previously demonstrated by intravenous administration of donor cells, which may recirculate to the liver after undergoing proliferation and differentiation in the recipient's bone marrow. In the present study, to elucidate which cellular components of human bone marrow more potently differentiate into hepatocytes, we fractionated human bone marrow cells into mesenchymal stem cells (MSCs), CD34+ cells, and non-MSCs/CD34- cells and examined them by directly xenografting to allylalcohol (AA)-treated rat liver. Hepatocyte-like cells, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), albumin (Alb), cytokeratin 19 (CK19), cytokeratin 18 (CK18), and asialoglycoprotein receptor (AGPR), and by reverse transcription-polymerase chain reaction (RT-PCR) for expression of AFP and Alb mRNA, were observed only in recipient livers with MSC fractions. Cell fusion was not likely involved since both human and rat chromosomes were independently identified by fluorescence in situ hybridization (FISH). The differentiation appeared to follow the process of hepatic ontogeny, reprogramming of gene expression in the genome of MSCs, as evidenced by expression of the AFP gene at an early stage and the albumin gene at a later stage. In conclusion, we have demonstrated that MSCs are the most potent component in hepatic differentiation, as revealed by directly xenografting into rat livers. (Blood. 2005;106:756-763)
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Janowicz, Krzysztof, Paul Mozdziak, Artur Bryja, Bartosz Kempisty, and Marta Dyszkiewicz-Konwińska. "Human Dental Pulp Stem Cells: recent findings and current research." Medical Journal of Cell Biology 7, no. 3 (November 8, 2019): 119–24. http://dx.doi.org/10.2478/acb-2019-0016.
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AbstractPrevalence of neurodegenerative diseases, most of which are life threatening and incurable, is an increasing clinical problem. To date, studies have demonstrated a superior proliferation rate of dental pulp stem cells (DPSCs) compared to other mesenchymal stem cells in vitro. DPSCs has recently been recognized as a novel treatment strategy for neurodegenerative disease, due to their advanced potential for neurogenic differentiation. The oral cavity has been described as a promising source of dental pulp stem cells. DPSCs are widely used in regenerative dentistry holding alternative capacity for osteogenic differentiation and therefore new promises for tissue and whole tooth regeneration. Dental stem cell banking offers a plentiful source of stem cells representing great potential for cell reprogramming and thus cell therapy. Recently, the association of pulp stem cells with three – dimensional scaffold templates allows for building up naturally derived implants. This review introduces to unique properties of DPSCs and biological factors influencing mineralization, proliferation and differentiation of pulp stem cells. Latest research studies are compared in terms of effectiveness and limitations of techniques for the isolation of pulp stem cells, including the enzymatic digestion and the explant culture methods. Moreover, a short overview of most recent findings and clinical application of DPSCs is proffered including progress of current research and limitations still to be addressed in the nearest future. Finally, the article presents new advances in the area of regenerative dentistry and regenerative medicine, including three dimensional printing and three dimensional analysis, emerged to deepen studies under procedures to replace the non patient specific artificial implants.Running title: DPSCs - review
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Su, Yue, Ling Wang, Zhiqiang Fan, Ying Liu, Jiaqi Zhu, Deborah Kaback, Julia Oudiz, et al. "Establishment of Bovine-Induced Pluripotent Stem Cells." International Journal of Molecular Sciences 22, no. 19 (September 28, 2021): 10489. http://dx.doi.org/10.3390/ijms221910489.
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Pluripotent stem cells (PSCs) have been successfully developed in many species. However, the establishment of bovine-induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs from bovine mesenchymal stem cells (bMSCs) by overexpression of lysine-specific demethylase 4A (KDM4A) and the other reprogramming factors OCT4, SOX2, KLF4, cMYC, LIN28, and NANOG (KdOSKMLN). These biPSCs exhibited silenced transgene expression at passage 10, and had prolonged self-renewal capacity for over 70 passages. The biPSCs have flat, primed-like PSC colony morphology in combined media of knockout serum replacement (KSR) and mTeSR, but switched to dome-shaped, naïve-like PSC colony morphology in mTeSR medium and 2i/LIF with single cell colonization capacity. These cells have comparable proliferation rate to the reported primed- or naïve-state human PSCs, with three-germ layer differentiation capacity and normal karyotype. Transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of integration into extra-embryonic tissues. Finally, at least 24.5% cloning efficiency could be obtained in nuclear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs.
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Ohneda, Osamu. "Isolation and characterization of mesenchymal stem cells derived from type 2 diabetes – research conducted in the Regenerative Medicine and Stem Cell Biology Lab (Ohneda Lab)." Impact 2019, no. 8 (November 26, 2019): 51–53. http://dx.doi.org/10.21820/23987073.2019.8.51.
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Stem cells are one of the leading methods through which we will gain the next generation of medical treatments. Given their ability to develop into almost any cell type, the medical applications are endless. Since the methods through which to reprogramme these master progenitors were established, many tests and clinical trials have taken place. However, as is often the case in biology, the picture is more complicated than simply reprogramming these cells with a cocktail of proteins. Stem cell expert Professor Osamu Ohneda is a Principal Investigator at the University of Tsukuba, Japan. 'Our focus is to achieve autologous transplantation whereby, mesenchymal stem cells are taken from the patient, encouraged to grow and reprogrammed before transplantation into the patient,' he observes. This process would be a crucial step in the personalisation of medicine, however, the more this process has been tested, the clearer it has become that additional variables are in play. 'There have been issues with nearly every step of the process after the acquirement of the raw mesenchymal stem cells (MSCs) necessary,' Ohneda highlights. 'The MSCs don't always respond as expected to growth stimulants and differentiation cues and transplants don't always take.' More in depth research is needed to overcome these issues and turn stem cell's potential into a reality. Investigating and solving these issues is the work of Ohneda and his research team, including Assistant Professor Vuong Cat Khanh and Assistant Professor Toshiharu Yamashita.
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Ospina-Muñoz, Natalia, and Jean-Paul Vernot. "Partial acquisition of stemness properties in tumorspheres obtained from interleukin-8-treated MCF-7 cells." Tumor Biology 42, no. 12 (December 2020): 101042832097943. http://dx.doi.org/10.1177/1010428320979438.
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The interleukin-8 is an important regulator of the tumor microenvironment, promoting the epithelial–mesenchymal transition and the acquisition of stem-like cell properties in cancer cells. The tumorsphere-formation assay has been used for the identification of cancer stem cell. Interleukin-8 induces the formation of larger tumorspheres in Michigan Cancer Foundation-7 (MCF-7) cells, suggesting cancer stem cell enrichment. In this work, we aimed to study the phenotypic and functional characteristics of the cells present within the tumorspheres of MCF-7 cells previously treated with interleukin-8. MCF-7 cells treated for 5 days or not with this cytokine were further cultivated in ultralow attachment plates for another 5 days to allow tumorspheres formation. We showed that the enhanced sphere formation by MCF-7 cells was not a consequence of higher cell proliferation by interleukin-8 stimulation. Despite maintaining an epithelial–mesenchymal transition phenotype with the presence of epithelial and mesenchymal markers, basic stemness properties were impaired in tumorspheres and in those treated with interleukin-8, while others were increased. Self-renewal capacity was increased in interleukin-8-treated cells only in the first generation of tumorspheres but was not sustained in consecutive assays. Accordingly, self-renewal and reprogramming gene expression, differentiation capacity to adipocytes, and clonogenicity were also impaired. We showed also that tumorspheres were enriched in differentiated luminal cells (EpCAM+/CD49f−). Nevertheless, cells were more quiescent and maintain a partial epithelial–mesenchymal transition, consistent with their increased resistance to Paclitaxel and Doxorubicin. They also presented higher migration and interleukin-8-directed invasion. Therefore, the breast cancer cell line MCF-7, having a low stemness index, might partially acquire some stem-like cell attributes after interleukin-8 stimulation, increasing its aggressiveness.
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Li, Wan-Ju. "Potential of Integration-free Induced Pluripotent Stem Cells in Musculoskeletal Regeneration." Ciencias Veterinarias 37, no. 3 (December 27, 2019): 27. http://dx.doi.org/10.15359/rcv.37-3.9.
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Stem cell therapies hold promise for the treatment of musculoskeletal disorders. Mesenchymal stem cells (MSCs) derived from adult tissues are the most common type of stem cells being investigated for biomedical applications among all stem cell types. However, studies have shown that MSC properties and functions are largely affected by age and health condition of the donor, which often causes inconsistency in therapeutic outcomes. This is a critical challenge that needs to be addressed before the promise of stem cells for therapies can be fulfilled. Our group has worked on tackling the challenge for more than a decade by developing strategies such as priming the cell with regulatory molecules or hypoxia culture. Recently, we successfully reprogramed human and pig somatic cells into induced pluripotent stem cells (iPSCs) using the integration-free episomal method and subsequently derived MSCs from iPSCs for evaluation of potential orthopedic applications. Our study results showed that through cellular reprogramming the capacity of cell propagation and multilineage differentiation of MSCs was greatly enhanced and the expression of aging-associated markers in the cell was significantly downregulated, suggesting that cellular reprogramming can rejuvenate MSCs to increase the regenerative capability, and our approach converting MSCs into iPSCs is promising for addressing the challenge of reduced therapeutic potential associated with MSC aging. In addition, we found that during chondrogenic induction reprogramed MSCs increasingly differentiated into hyaline chondrocytes expressing cartilage-specific markers, compared to control parental cells, suggesting that iPSC-derived MSCs are promising therapeutic agents for articular cartilage regeneration. In general, our findings highlight the potential of iPSCs in better understanding aging-associated musculoskeletal disorders and providing biological options for the treatment.
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Lim, S. M. L., I. Aksoy, K. G. C. Lim, J. Karuppasamy, U. Divakar, F. J. Ma, F. L. Lam, S. J. N. Remulla, and L. W. Stanton. "Re-establishing pluripotency in adult cells derived from breast stromal tissue." Journal of Clinical Oncology 29, no. 27_suppl (September 20, 2011): 227. http://dx.doi.org/10.1200/jco.2011.29.27_suppl.227.
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227 Background: Recent advances in pluripotent stem cell biology offer patient-specific disease models to investigate in vitro mechanisms of tumorigenesis. Induced pluripotent stem (iPS) cells were originally derived by reprogramming of human dermal fibroblasts through ectopic expression of pluripotency–associated transcription factors. A limitation to the use of dermal fibroblasts as the starting cell type for reprogramming is that it usually takes weeks to expand cells from a single biopsy, and the efficiency of the process is very low. In contrast, a large number of adipose-derived mesenchymal stromal cells (Ad-MSCs) can be easily obtained from the stroma of human breast tissue, without the time-consuming steps of cell expansion. Here we investigated the ability to induce pluripotency in committed, Ad-MSCs derived from the stroma of breast tissue. Methods: The aim of this study is to investigate the potential of using Ad-MSCs derived from surgically discarded breast stromal tissue to generate human iPS. Discarded tissue during surgical procedures was processed in vitro and Ad-MSCs were derived. These Ad-MSCs were then used to generate iPS cells by ectopic expression of “Yamanaka’s cocktail” containing OCT4, SOX2, KLF4 and c-MYC. Results: The success rate in generating iPS cells from human Ad-MSCs derived from breast stromal tissue is very high compared to the use of dermal fibroblasts. In our study, almost all human Ad-MSC cell lines can be reprogrammed into iPS cells, which share the same characteristics as skin fibroblast-derived iPS cells and human embryonic stem cells in their morphology, gene expression profile and differentiation capacities. Conclusions: We are now optimizing this approach and making it more clinically relevant by adopting an integration-free method to deliver the reprogramming factors. The successful reprogramming of breast stromal-derived Ad-MSCs into iPS cells may provide a valuable source of patient-specific iPS cells to study the mechanism of tumorigenesis in patients with breast cancer.
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Moro, L. N., G. Amin, V. Furmento, A. Waisman, G. Neiman, A. La Greca, N. L. Santin, et al. "188 MicroRNA characterization in equine induced pluripotent stem cells." Reproduction, Fertility and Development 31, no. 1 (2019): 218. http://dx.doi.org/10.1071/rdv31n1ab188.
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Cell reprogramming has been well described in mouse and human cells. The expression of specific microRNAs has demonstrated to be essential for pluripotent maintenance and cell differentiation, but not much information is available in domestic species. A single microRNA can regulate the expression of hundreds of mRNA targets, a property given by a short sequence (called “seed”) in positions 2 to 8 from the 5′ end that is complementary to the 3′ untranslated region (UTR) tail of specific mRNAs. We aimed to generate horse induced pluripotent stem cells (iPSC), characterise them, and evaluate the expression of different microRNAs (miR-302a, b, c, d, miR-205, miR-145, miR-9, miR-96, miR-125b, and miR-296) in pluripotency and differentiation. Both cell states were evaluated (pluripotency and differentiation) in order to understand more deeply the complex network of transcriptional regulation in different contexts but with the same genomic background. Two equine iPSC lines (named L2 and L3) were characterised after the reprogramming of equine fibroblasts with the 4 human Yamanaka factors (OCT-4, SOX-2, c-MYC, KLF4). The pluripotency of both lines was assessed by phosphatase alkaline activity, expression of OCT-4, NANOG, and REX1 by RT-PCR, and by immunofluorescence of OCT-4, SOX-2, and c-MYC. In vitro differentiation to embryo bodies (EB) showed the capacity of the iPSC to differentiate into ectodermal, endodermal, and mesodermal phenotypes. MicroRNA expression was analysed by quantitative RT-PCR and resulted in higher expression of the miR-302 family, miR-9, and miR-96 in L2 and L3v. fibroblasts (P ≤ 0.05), as previously shown in human pluripotent cells. Moreover, down-regulation of miR-145 and miR-205 was observed. After differentiation to EB, greater expression of miR-96 was observed in the EB compared with iPSC, and the expression of miR-205 was induced but only in the EB-L2. In addition, we performed in silico analysis of horse and human microRNAs. First, we compared the horse-miR-302/367 cluster with the human-miR-302/367 cluster and determined a 75% homology between them. Moreover, the seed region of the horse-miR-302 family resulted complementary to the 3′ UTR of horse cell cycle regulator genes CDK2, CYCLIN D1, and E2F1, and to the 3′ UTR of the RHOC gene, which is involved in the epithelial-mesenchymal transition. The miR-145 seed sequence was complementary to the 3′ UTR region of the OCT-4 and KLF-4 horse genes. With respect to miR-9 and miR-96, the seed sequence of these genes were complementary to the HES1 and PAX-6 genes. In all cases, the same gene targets were previously demonstrated in humans. In conclusion, we report the generation and characterization of equine iPSC and determined for the first time the expression of microRNAs in equine pluripotent cells. Moreover, several results led us to think that the horse microRNAs evaluated herein are highly conserved in sequence and function with respect to the human species. It will now be necessary to generate directed differentiations to derivatives of the 3 germ layers in order to strengthen our results. This is the first report to evaluate the expression and possible targets of microRNAs in pluripotent cells from domestic animals.
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Miyazaki, Yoshihiro, Nobuhito Mori, Yuka Akagi, Tatsuya Oda, and Yasuyuki S. Kida. "Potential Metabolite Markers for Pancreatic Cancer Identified by Metabolomic Analysis of Induced Cancer-Associated Fibroblasts." Cancers 14, no. 6 (March 8, 2022): 1375. http://dx.doi.org/10.3390/cancers14061375.
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Cancer-associated fibroblasts (CAFs) in the tumor microenvironment perform glycolysis to produce energy, i.e., ATP. Since the origin of CAFs is unidentified, it is not determined whether the intracellular metabolism transitions from oxidative phosphorylation (OXPHOS) to glycolysis when normal tissue fibroblasts differentiate into CAFs. In this study, we established an experimental system and induced the in vitro differentiation of mesenchymal stem cells (MSCs) to CAFs. Additionally, we performed metabolomic and RNA-sequencing analyses before and after differentiation to investigate changes in the intracellular metabolism. Consequently, we discovered that OXPHOS, which was the primary intracellular metabolism in MSCs, was reprogrammed to glycolysis. Furthermore, we analyzed the metabolites in pancreatic tumor tissues in a mice model. The metabolites extracted as candidates in the in vitro experiments were also detected in the in vivo experiments. Thus, we conclude that normal tissue fibroblasts that differentiate into CAFs undergo a metabolic reprogramming from OXPHOS to glycolysis. Moreover, we identified the CAF-specific metabolites expressed during metabolic reprogramming as potential future biomarkers for pancreatic cancer.
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Arora, Payal, Wen Li, Xiaobin Huang, Wenjing Yu, Ranran Huang, Qian Jiang, and Chider Chen. "Metabolic Reconfiguration Activates Stemness and Immunomodulation of PDLSCs." International Journal of Molecular Sciences 23, no. 7 (April 6, 2022): 4038. http://dx.doi.org/10.3390/ijms23074038.
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Periodontal ligament derived stem cells (PDLSC) are adult multipotent mesenchymal-like stem cells (MSCs) that can induce a promising immunomodulation to interact with immune cells for disease treatment. Metabolic reconfiguration has been shown to be involved in the immunomodulatory activity of MSCs. However, the underlying mechanisms are largely unknown, and it remains a challenging to establish a therapeutic avenue to enhance immunomodulation of endogenous stem cells for disease management. In the present study, RNA-sequencing (RNA-seq) analysis explores that curcumin significantly promotes PDLSC function through activation of MSC-related markers and metabolic pathways. In vitro stem cell characterization further confirms that self-renewal and multipotent differentiation capabilities are largely elevated in curcumin treated PDLSCs. Mechanistically, RNA-seq reveals that curcumin activates ERK and mTOR cascades through upregulating growth factor pathways for metabolic reconfiguration toward glycolysis. Interestingly, PDLSCs immunomodulation is significantly increased after curcumin treatment through activation of prostaglandin E2-Indoleamine 2,3 dioxygenase (PGE2-IDO) signaling, whereas inhibition of glycolysis activity by 2-deoxyglucose (2-DG) largely blocked immunomodulatory capacity of PDLSCs. Taken together, this study provides a novel pharmacological approach to activate endogenous stem cells through metabolic reprogramming for immunomodulation and tissue regeneration.
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Revilla, Giovanna, Rosa Corcoy, Antonio Moral, Joan Carles Escolà-Gil, and Eugenia Mato. "Cross-Talk between Inflammatory Mediators and the Epithelial Mesenchymal Transition Process in the Development of Thyroid Carcinoma." International Journal of Molecular Sciences 20, no. 10 (May 18, 2019): 2466. http://dx.doi.org/10.3390/ijms20102466.
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There is strong association between inflammatory processes and their main metabolic mediators, such as leptin, adiponectin secretion, and low/high-density lipoproteins, with the cancer risk and aggressive behavior of solid tumors. In this scenario, cancer cells (CCs) and cancer stem cells (CSCs) have important roles. These cellular populations, which come from differentiated cells and progenitor stem cells, have increased metabolic requirements when it comes to maintaining or expanding the tumors, and they serve as links to some inflammatory mediators. Although the molecular mechanisms that are involved in these associations remain unclear, the two following cellular pathways have been suggested: 1) the mesenchymal-epithelial transition (MET) process, which permits the differentiation of adult stem cells throughout the acquisition of cell polarity and the adhesion to epithelia, as well to new cellular lineages (CSCs); and, 2) a reverse process, termed the epithelial-mesenchymal transition (EMT), where, in pathophysiological conditions (tissue injury, inflammatory process, and oxidative stress), the differentiated cells can acquire a multipotent stem cell-like phenotype. The molecular mechanisms that regulate both EMT and MET are complex and poorly understood. Especially, in the thyroid gland, little is known regarding MET/EMT and the role of CCs or CSCs, providing an exciting, new area of knowledge to be investigated. This article reviews the progress to date in research on the role of inflammatory mediators and metabolic reprogramming during the carcinogenesis process of the thyroid gland and the EMT pathways.
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Palamaris, Kostas, Evangelos Felekouras, and Stratigoula Sakellariou. "Epithelial to Mesenchymal Transition: Key Regulator of Pancreatic Ductal Adenocarcinoma Progression and Chemoresistance." Cancers 13, no. 21 (November 4, 2021): 5532. http://dx.doi.org/10.3390/cancers13215532.
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Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies, characterized by aggressive biological behavior and a lack of response to currently available chemotherapy. Emerging evidence has identified epithelial to mesenchymal transition (EMT) as a key driver of PDAC progression and a central regulator in the development of drug resistance. EMT is a reversible transdifferentiation process controlled by complex interactions between multiple signaling pathways such as TGFb, Wnt, and Notch, which converge to a network of specific transcription factors. Activation of EMT transcriptional reprogramming converts cancer cells of epithelial differentiation into a more mesenchymal phenotypic state. EMT occurrence in pre-invasive pancreatic lesions has been implicated in early PDAC dissemination. Moreover, cancer cell phenotypic plasticity driven by EMT contributes to intratumoral heterogeneity and drug tolerance and is mechanistically associated with the emergence of cells exhibiting cancer stem cells (CSCs) phenotype. In this review we summarize the available data on the signaling cascades regulating EMT and the molecular isnteractions between pancreatic cancer and stromal cells that activate them. In addition, we provide a link between EMT, tumor progression, and chemoresistance in PDAC.
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Resar, Linda, Sandeep N. Shah, Candace Kerr, Leslie Cope, Elias Zambidis, Amy Belton, and David L. Huso. "HMGA1, a Factor Enriched in Hematopoietic Stem Cells, Embryonic Stem Cells, and Hematologic Malignancy, Enhances Cellular Reprogramming to a Pluripotent Stem-Like Cell." Blood 120, no. 21 (November 16, 2012): 2323. http://dx.doi.org/10.1182/blood.v120.21.2323.2323.
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Abstract Abstract 2323 Although recent studies have identified genes important in hematopoietic stem cells (HSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs), the molecular underpinnings of normal stem cell function are unclear. A better understanding of key stem cell pathways will be essential for the safe use of stem cells in regenerative medicine and should also uncover novel therapeutic targets in aggressive hematologic malignancies and other stem-like cancer cells. To elucidate the molecular underpinnings of “stemness”, we investigated transcriptional networks in pluripotent stem cells. Our focus is the high mobility group A1 (HMGA1) gene, which encodes the HMGA1a and HMGA1b chromatin remodeling proteins. These proteins bind to AT-rich regions of DNA and orchestrate the assembly of transcription factor complexes to alter chromatin structure and modulate gene expression. HMGA1 is highly expressed during embryogenesis with low or undetectable levels in adult, differentiated tissues. HMGA1 is also enriched in HSCs, hESCs, iPSCs, refractory leukemia, and poorly differentiated solid tumors. Our group discovered that HMGA1 functions as a potent oncogene in cultured cells and causes aggressive leukemia in transgenic mice. We also found that high levels of HMGA1 expression correlate with relapse in acute lymphoblastic leukemia. Together, these findings suggest that HMGA1 drives a stem cell phenotype during normal development, hematopoiesis, and malignant transformation. To further investigate the role of HMGA1 in a stem cell state, we compared its expression in iPSCs, hESCs, HSCs, and cancer cells. HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in HSCs and cancer cells, and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation in hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells (mesenchymal stem cells, HSCs, and fetal lung fibroblasts) to an iPSC together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC or OSKM). HMGA1 results in an increase in the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign, teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In summary, our findings uncover a key role for HMGA1 as a regulator of the stem cell state through transcriptional networks that induce pluripotency and an undifferentiated state. Further studies are needed to determine if HMGA1 pathways could be targeted in hematologic and other malignancies or exploited in regenerative medicine. Disclosures: No relevant conflicts of interest to declare.
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D'Aniello, Cristina, Federica Cermola, Eduardo Jorge Patriarca, and Gabriella Minchiotti. "Vitamin C in Stem Cell Biology: Impact on Extracellular Matrix Homeostasis and Epigenetics." Stem Cells International 2017 (2017): 1–16. http://dx.doi.org/10.1155/2017/8936156.
Full textAbstract:
Transcription factors and signaling molecules are well-known regulators of stem cell identity and behavior; however, increasing evidence indicates that environmental cues contribute to this complex network of stimuli, acting as crucial determinants of stem cell fate.L-Ascorbic acid (vitamin C (VitC)) has gained growing interest for its multiple functions and mechanisms of action, contributing to the homeostasis of normal tissues and organs as well as to tissue regeneration. Here, we review the main functions of VitC and its effects on stem cells, focusing on its activity as cofactor of Fe+2/αKG dioxygenases, which regulate the epigenetic signatures, the redox status, and the extracellular matrix (ECM) composition, depending on the enzymes’ subcellular localization. Acting as cofactor of collagen prolyl hydroxylases in the endoplasmic reticulum, VitC regulates ECM/collagen homeostasis and plays a key role in the differentiation of mesenchymal stem cells towards osteoblasts, chondrocytes, and tendons. In the nucleus, VitC enhances the activity of DNA and histone demethylases, improving somatic cell reprogramming and pushing embryonic stem cell towards the naive pluripotent state. The broad spectrum of actions of VitC highlights its relevance for stem cell biology in both physiology and disease.
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Gao, Dengke, Pengxiu Dai, Zhixin Fan, Jinglu Wang, and Yihua Zhang. "The Roles of Different Multigene Combinations of Pdx1, Ngn3, Sox9, Pax4, and Nkx2.2 in the Reprogramming of Canine ADSCs Into IPCs." Cell Transplantation 31 (January 2022): 096368972210814. http://dx.doi.org/10.1177/09636897221081483.
Full textAbstract:
Adipose-derived mesenchymal stem cells (ADSCs) are ideal sources for the treatment of diabetes, and the differentiation of ADSCs into insulin-producing cells (IPCs) through transfection of exogenous regulatory genes in vitro has been studied in depth. The differentiation of ADSCs is strictly regulated by a variety of transcription factors such as Pdx1, Ngn3, Pax4, Nkx2.2, and Sox9. However, whether these genes can coordinately regulate the differentiation of ADSCs into IPCs is still unknown. In this study, five multigene coexpressing adenovirus vectors ( pAdTrack-Pdx1-Ngn3-AdEasy, pAdTrack-Pdx1-Ngn3-Sox9-AdEasy, pAdTrack-Pdx1-Ngn3-Pax4-Sox9-AdEasy, pAdTrack-Pdx1-Ngn3-Nkx2.2-Sox9-AdEasy, and pAdTrack-Pdx1-Ngn3-Nkx2.2-Pax4-AdEasy) were constructed, and then the stocks of the packaged adenoviruses were used to infect the canine ADSCs (cADSCs). Based on results of morphological observation, dithizone staining, sugar-stimulated insulin secretion test, cellular insulin immunofluorescence assays, and the detection of pancreatic β-cell development–related genes in the induced cells, the best induction combination ( pAdTrack-Pdx1-Ngn3-Nkx2.2-Pax4-AdEasy) was identified after comparative screening. This study provides a theoretical reference and an experimental basis for further research on stem cell replacement therapy for diabetes.
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Li, S., T. Flisikowska, B. Kessler, T. Güngör, R. Kind, E. Wolf, A. Schnieke, and V. Zakhartchenko. "67 PRODUCTION OF CLONED TRANSGENIC RABBITS FROM MESENCHYMAL STEM CELLS." Reproduction, Fertility and Development 22, no. 1 (2010): 192. http://dx.doi.org/10.1071/rdv22n1ab67.
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Mesenchymal stem cells (MSC) are adult stem cells with fibroblast-like morphology, which can be easily isolated from bone marrow and expanded in culture. Mesenchymal stem cells are able to grow from a single cell into a cell clone, which makes them potentially useful for gene targeting. In our recent study we investigated the dynamics of epigenetic reprogramming following nuclear transfer (NT) with MSC and found that these cells can support development of cloned embryos as good as genetically identical fibroblasts (Brero et al. 2009 Cloning Stem Cells 11, 319-329). In the present study we tested whether live cloned rabbits can be produced from MSC. Nuclear donor cells were isolated from a 6-week-old transgenic Ali/Bas rabbit, expanded in culture, and assessed for their differentiation potential. Mesenchymal stem cells were transfected with a green fluorescent protein (GFP) reporter gene construct and stable cell clones were selected (GFP-MSC). The MSC and GFP-MSC were used for NT at passage 3 to 7 after serum starvation for 2 to 4 days. Nuclear transfer was performed essentially as described previously (Yang et al. 2007 Reproduction 133, 219-320). To assess the development to blastocyst, reconstructed embryos were cultured in B2 medium for 5 to 6 days, whereas for in vivo development embryos were cultured only overnight and then transferred into recipients at the 4- to 8-cell stage. In the MSC group, 844 oocytes were used, 793 (94%) of them fused, 698/786 (89%) cleaved, and 48/128 (38%) developed to blastocyst. After transfer of 483 cloned embryos into 13 recipients, 2 from 8 pregnant recipients gave birth to 10 (2.4%) rabbits, from which 2 and 1 survived for more than 7 days and 3 months, respectively. In the GFP-MSC group, 444 oocytes were used, 412 (93%) of them fused, 377/409 (92%) cleaved, and 97/178 (55%) developed to blastocyst. Transfer of 216 cloned embryos into 8 recipients resulted in 4 pregnancies. One recipient gave birth to 6 (3.7%) live and 2 stillborn rabbits, from which 2 and 1 rabbits survived for more than 3 days and 2 weeks, respectively. All cloned rabbits carried a GFP gene, and green fluorescence could be detected in the follicles of the skin under a fluorescence microscope (Zeiss Axiovert200, Carl Zeiss, Germany). Our study demonstrates that live cloned rabbits can be produced from genetically modified MSC, thus paving the way to generate gene targeted animals. This work is supported by Roche Diagnostic GmbH.
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Zhang, Y., C. Wei, P. F. Zhang, X. Li, Y. S. Li, Y. L. Zhang, and Y. H. Zhang. "190 EFFICIENT GENERATION OF INDUCED PLURIPOTENT STEM CELLS FROM PORCINE ADIPOSE-DERIVED STEM CELLS WITH A FEEDER-INDEPENDENT AND SERUM-FREE SYSTEM." Reproduction, Fertility and Development 26, no. 1 (2014): 209. http://dx.doi.org/10.1071/rdv26n1ab190.
Full textAbstract:
Somatic cells could be directly reprogrammed into stem state by ectopic expression of transcription factors, which share similar features of embryonic stem cells (ESC). Induced pluripotent stem cells (iPSC) possess promising application in producing genetically modified animals, whereas the generation of porcine offspring from iPSC is still difficult and controversial, and new materials are needed. In this study, we report the generation of iPSC from porcine adipose-derived stem cells (pADSC) using drug-inducible expression of defined human factors (Oct4, Sox2, Klf4, and c-Myc) and ‘2i’ plus leukemia inhibitory factor (LIF) culture system. pADSC were isolated from subcutaneous adipose tissue of a 28-day-old Danish Landrace, and subsequently characterised by high proliferation rate at low passages, long period passaging without significant replication senescence, mesenchymal stem cell-specific surface markers expression, including CD29 (0.995 ± 0.0577), CD44 (0.999 ± 0.0333), and CD90 (0.994 ± 0.0333), together with successful adipogenic and osteogenic differentiation ability in vitro. The reprogramming of iPSC from pADSC was evidently more efficient than the process from adult fibroblasts (P < 0.01), both of which were carried out under feeder-independent and serum-free conditions, and this may be due to the higher demethylation level of genomic DNA in pADSC. Two lines of porcine iPSC with naïve-like state were finally obtained through feeder-independent and serum-free conditions. The successful reprogramming of iPSC was demonstrated by short cell cycle interval, alkaline phosphatase (AP) staining positive, expression of stemness-related proteins including OCT-4, SOX2, NANOG, SSEA3, and SSEA4. Full reprogramming of iPSC was evaluated by the significant up-regulation of LIN28, ESRRB, UTF1, and DPPA5. Naïve-like state of porcine iPSC was further confirmed by the striking resemblance to naïve mESC, single-cell dissociation, LIF-dependency, up-regulation of STELLA and ERAS, and little translation of TRA-1-60 and TRA-1-81. In addition, porcine naïve-like iPSC possessed normal karyotypes, and could differentiate into cell types of all three germ layers in vitro and in vivo. Furthermore, in vivo studies to determine the capacity of these cells to integrate into the inner cell mass of blastocysts are still being undertaken for validation. Together, our study provided an efficient method to derive porcine naïve-like iPSC from pADSC, which may be useful for the production of living offspring. Y. Zhang and C. Wei contributed equally to this work. Y.-H. Zhang is the corresponding author. This work was supported by the National Natural Science Foundation Program 31272442.
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Echeverry, D., D. Rojas, C. Aguilera, L. Rodriguez-Alvarez, and F. Castro. "208 Effect of growth factors and reprogramming molecules on induction to multipotency of dermal fibroblasts from colocolo (Leopardus colocolo)." Reproduction, Fertility and Development 32, no. 2 (2020): 232. http://dx.doi.org/10.1071/rdv32n2ab208.
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Reprogramming of terminally differentiated cells to higher plasticity levels can be achieved with small molecules. This can be of value for somatic cell nucleus transfer, deriving multi and pluripotent cells and conservation purposes. Recently, induced mesenchymal stem cells were derived from differentiated human and mouse cells by using small molecules and growth factors. The pampas cat or colocolo (Leopardus colocolo) is a South American felid categorrized as near threatened by the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. Major historical threats to the pampas cat include illegal hunting, habitat loss or transformation, and conflict retaliation for poultry predation. Here, we tested 5-azacytidine (an epigenetic modifier) and A8301 (a potent inhibitor of transforming growth factor-β type I receptor superfamily), linked to platelet-rich plasma (PRP) and platelet-derived growth factor (PDGF-B) to induce changes in the expression of pluripotency genes and differentiation capacity of colocolo fibroblasts towards other mesodermal lineages. For this, dermal fibroblasts were treated with (I) 5-azacytidine + PRP + A8301 + VitC, or (II) 5- azacytidine + VitC + A8301 + PDFG for 12 days. On Days 0, 5, and 12 of reprogramming, expression of OCT4, NANOG, E-cadherin and SNAIL was evaluated by reverse transcription-PCR, and tri-lineage differentiation was induced. For treatment I, no statistical difference was found in the expression of OCT4 and NANOG. Chondrogenic and osteogenic differentiation was observed. In treatment II, significant expression of OCT4 and NANOG (P<0.05) was induced, and reprogrammed fibroblasts were differentiated into chondrogenic and osteogenic lineages. Immunohistochemistry positivity for OCT4 was detected in treatment II. In summary, we showed that dermal fibroblasts of pampas cat can be reprogrammed into cells with multipotent characteristics, particularly when a cocktail of 5-azacytidine + VitC + A8301 + PDFG was used. Treatment I probably failed because of other growth factors and proteins present in PRP, which might inhibit successful reprogramming or activate other pathways leading to a nonmultipotent phenotype. Further refinements of these protocols are required to improve the reprogramming protocol. This in turn should help us obtain cells that can be used in nucleus transfer or cellular therapies in endangered felid species.
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Mohamad-Fauzi, N., C. Feltrin, L. R. Bertolini, M. Bertolini, E. A. Maga, and J. D. Murray. "284 CHARACTERIZATION OF GOAT MESENCHYMAL STEM CELLS DERIVED FROM BONE MARROW AND ADIPOSE TISSUE." Reproduction, Fertility and Development 25, no. 1 (2013): 289. http://dx.doi.org/10.1071/rdv25n1ab284.
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Gene modification of cells in vitro followed by somatic cell nuclear transfer (SCNT) currently offers the best route for creating genetically modified livestock species. However, low cloning efficiencies in differentiated somatic cells have been attributed to the possibility of improper nuclear reprogramming. Adult stem cells may have greater developmental potential and better nuclear reprogramming potential following cloning. There is considerable interest in using goats as models for genetically engineering dairy animals and for using stem cells as therapeutics for bone and cartilage repair. Mesenchymal stem cells (MSC) are adult stem cells that that have been isolated and characterised from various species, but are poorly characterised in goats. Three MSC lines were isolated from bone marrow (9004 BM-MSC, 9003 BM-MSC) and adipose tissue (9003 A-MSC) of neonatal goats. In this study, these MSC lines were characterised to verify MSC-specific characteristics and assess their amenability to genetic modification in vitro. Passage 5 cells were evaluated for capacity to differentiate into osteogenic, adipogenic, and chondrogenic lineages, as well as for colony-forming efficiency after 10 days of culture from low-density plating. Expression of MSC-specific positive cell surface markers CD90, CD73, and CD105, as well as pluripotency markers Nanog, Oct-4, and Sox-2, was examined by RT-PCR. Oct-4 protein localization was examined by immunofluorescence. The MSC were also assessed for their potential for gene modification by nucleofection with circular and linearized plasmids expressing green fluorescent protein (GFP) and neomycin resistance. Differences between cell lines were statistically analysed using ANOVA. The 9003 BM-MSC cells were also utilised for SCNT. All 3 MSC lines showed a normal karyotype. The MSC lines were capable of undergoing osteogenic, adipogenic, and chondrogenic differentiation, with observed differences in capacities between the BM-MSC and A-MSC lines, as shown by staining with Alizarin Red S, Oil Red O, and Alcian Blue. Expression of CD90, CD73, CD105, Nanog, Oct-4, and Sox-2 was detected, and Oct-4 was localised in the cytoplasm. There were significant differences in clonability between the cell lines, with 9004 BM-MSC showing the highest colony-forming efficiency (61% ± 5.4; P < 0.05). There were no significant differences in the percentage of GFP-positive cells from transfections done with the circular plasmid, but 9003 BM-MSC yielded a significantly lower number of integrant colonies per 500 000 cells transfected with the linear plasmid and G418 selection (12.75 ± 3.24; P < 0.05). Somatic cell nuclear transfer was able to reprogram 9003 BM-MSC and produce pregnancies. One hundred forty-four embryos were reconstructed, 101 embryos were transferred into 8 recipients, and the resulting pregnancy rate was 73%. Our findings provide characterisation information on goat MSC, and show that significant differences can exist between MSC isolated from different tissues and from within the same tissue.
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Mao, Huimin, Andi Yang, Yunhe Zhao, Lang Lei, and Houxuan Li. "Succinate Supplement Elicited “Pseudohypoxia” Condition to Promote Proliferation, Migration, and Osteogenesis of Periodontal Ligament Cells." Stem Cells International 2020 (March 10, 2020): 1–14. http://dx.doi.org/10.1155/2020/2016809.
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Most mesenchymal stem cells reside in a niche of low oxygen tension. Iron-chelating agents such as CoCl2 and deferoxamine have been utilized to mimic hypoxia and promote cell growth. The purpose of the present study was to explore whether a supplement of succinate, a natural metabolite of the tricarboxylic acid (TCA) cycle, can mimic hypoxia condition to promote human periodontal ligament cells (hPDLCs). Culturing hPDLCs in hypoxia condition promoted cell proliferation, migration, and osteogenic differentiation; moreover, hypoxia shifted cell metabolism from oxidative phosphorylation to glycolysis with accumulation of succinate in the cytosol and its release into culture supernatants. The succinate supplement enhanced hPDLC proliferation, migration, and osteogenesis with decreased succinate dehydrogenase (SDH) expression and activity, as well as increased hexokinase 2 (HK2) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), suggesting metabolic reprogramming from oxidative phosphorylation to glycolysis in a normal oxygen condition. The succinate supplement in cell cultures promoted intracellular succinate accumulation while stabilizing hypoxia inducible factor-1α (HIF-1α), leading to a state of pseudohypoxia. Moreover, we demonstrate that hypoxia-induced proliferation was G-protein-coupled receptor 91- (GPR91-) dependent, while exogenous succinate-elicited proliferation involved the GPR91-dependent and GPR91-independent pathway. In conclusion, the succinate supplement altered cell metabolism in hPDLCs, induced a pseudohypoxia condition, and enhanced proliferation, migration, and osteogenesis of mesenchymal stem cells in vitro.
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Maioli, M., S. Rinaldi, S. Cruciani, A. Necas, V. Fontani, G. Corda, S. Santaniello, et al. "Antisenescence Effect of REAC Biomodulation to Counteract the Evolution of Myelodysplastic Syndrome." Physiological Research 71, no. 4 (August 31, 2022): 539–49. http://dx.doi.org/10.33549/physiolres.934903.
Full textAbstract:
About 30 percent of patients diagnosed with myelodysplastic syndromes (MDS) progress to acute myeloid leukemia (AML). The senescence of bone marrow‐derived mesenchymal stem cells (BMSCs) seems to be one of the determining factors in inducing this drift. Research is continuously looking for new methodologies and technologies that can use bioelectric signals to act on senescence and cell differentiation towards the phenotype of interest. The Radio Electric Asymmetric Conveyer (REAC) technology, aimed at reorganizing the endogenous bioelectric activity, has already shown to be able to determine direct cell reprogramming effects and counteract the senescence mechanisms in stem cells. Aim of the present study was to prove if the anti-senescence results previously obtained in different kind of stem cells with the REAC Tissue optimization – regenerative (TO-RGN) treatment, could also be observed in BMSCs, evaluating cell viability, telomerase activity, p19ARF, P21, P53, and hTERT gene expression. The results show that the REAC TO-RGN treatment may be a useful tool to counteract the BMSCs senescence which can be the basis of AML drift. Nevertheless, further clinical studies on humans are needed to confirm this hypothesis.
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Laplagne, Chloé, Marcin Domagala, Augustin Le Naour, Christophe Quemerais, Dimitri Hamel, Jean-Jacques Fournié, Bettina Couderc, Corinne Bousquet, Audrey Ferrand, and Mary Poupot. "Latest Advances in Targeting the Tumor Microenvironment for Tumor Suppression." International Journal of Molecular Sciences 20, no. 19 (September 23, 2019): 4719. http://dx.doi.org/10.3390/ijms20194719.
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The tumor bulk is composed of a highly heterogeneous population of cancer cells, as well as a large variety of resident and infiltrating host cells, extracellular matrix proteins, and secreted proteins, collectively known as the tumor microenvironment (TME). The TME is essential for driving tumor development by promoting cancer cell survival, migration, metastasis, chemoresistance, and the ability to evade the immune system responses. Therapeutically targeting tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), regulatory T-cells (T-regs), and mesenchymal stromal/stem cells (MSCs) is likely to have an impact in cancer treatment. In this review, we focus on describing the normal physiological functions of each of these cell types and their behavior in the cancer setting. Relying on the specific surface markers and secreted molecules in this context, we review the potential targeting of these cells inducing their depletion, reprogramming, or differentiation, or inhibiting their pro-tumor functions or recruitment. Different approaches were developed for this targeting, namely, immunotherapies, vaccines, small interfering RNA, or small molecules.
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Bosch, P., S. L. Pratt, E. Sherrer, C. A. Hodges, E. Ivy Hill, E. Kachline, and S. L. Stice. "30USE OF ADULT STEM/PROGENITOR CELLS AS NUCLEAR DONORS TO PRODUCE CLONED PORCINE EMBRYOS." Reproduction, Fertility and Development 16, no. 2 (2004): 137. http://dx.doi.org/10.1071/rdv16n1ab30.
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Incomplete or defective nuclear reprogramming may be responsible for low cloning efficiencies. Less differentiated stem cells are thought to be more easily reprogrammed, resulting in improved survival of cloned mice (Rideout WM III et al., 2000 Nat. Genet. 24, 109–110). Our objective was to establish porcine mesenchymal stem cell (MSC) cultures and use these as donor cells in nuclear transfer (NT). A bone marrow (BM) aspirate was collected from an anesthetized gilt. BM mononuclear cells were isolated by centrifugation over a density gradient (Histopaque-1077; Sigma, St. Louis, MO, USA), resuspended in low glucose DMEM (Gibco) plus 10% FBS and plated on flasks; fibroblast-like MSCs were later passaged. Ear skin fibroblast (SF) cultures from the same BM donor gilt were established. Cultures of MSC and SF were exposed to lipogenic, osteogenic or chondrogenic differentiation media (Pittenger MF et al., 1999 Science 284, 143–147) for 14 days. Cells cultured in DMEM with 10% FBS served as controls. Differentiation was assessed by histochemical methods. Calcium deposits and alkaline phosphatase (AP) activity (Vector Red AP Substrate Kit, Vector Labs) were indicative of osteogenic differentiation. MSCs cultured under osteogenic conditions were positive for AP activity and developed a black color after von Kossa staining, indicative of calcium deposition. Oil red O stain identified cellular lipid accumulation. When exposed to adipogenic differentiation media, 10–15% of MSCs developed an adipocyte phenotype with lipid droplet accumulation and oil red O staining. Lipogenic differentiation was not observed in SF and control cultures. Presence of acidic mucopolysaccharides associated with cartilage formation was determined by alcian blue stain. MSCs exposed to chondrogenic conditions were alcian blue-positive, and SF and control cultures were alcian blue negative. For NT, confluent (passage 2) MSC and SF cultures were exposed to roscovitine (15μM; Sigma) for 24h. In vitro-matured oocytes were enucleated and a single cell (MSC or SF) was transferred into the periviteline space. Cell-oocyte couplets were fused in Zimmerman’s medium with a single electric pulse (250V/mm for 20μs) delivered through a needle-type electrode. NT units were electrically activated (2 pulses of 100V/mm for 60μs separated by 5s) in a chamber 1h after fusion and transferred to NCSU-23 medium. Embryos were examined for cleavage and blastocyst formation at 2 and 7 days after NT, respectively. Cleavage rates were 53.3% (40/75) for MSC and 59.7% (46/77) for SF NT embryos. Development to blastocyst stage was 6.6% (5/75) in the MSC group and 1.2% (1/77) in SF group. In conclusion, we established an adult MSC line from a live animal using a minimally invasive BM aspiration technique. Additionally, MSC donor-derived NTs developed to the blastocyst stage. Further experiments will determine nuclear reprogramming in MSC-derived NT embryos.
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Liu, Li-Ping, Dong-Xu Zheng, Zheng-Fang Xu, Hu-Cheng Zhou, Yun-Cong Wang, Hang Zhou, Jian-Yun Ge, et al. "Transcriptomic and Functional Evidence Show Similarities between Human Amniotic Epithelial Stem Cells and Keratinocytes." Cells 11, no. 1 (December 27, 2021): 70. http://dx.doi.org/10.3390/cells11010070.
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Amniotic epithelial stem cells (AESCs) are considered as potential alternatives to keratinocytes (KCs) in tissue-engineered skin substitutes used for treating skin damage. However, their clinical application is limited since similarities and distinctions between AESCs and KCs remain unclear. Herein, a transcriptomics analysis and functional evaluation were used to understand the commonalities and differences between AESCs and KCs. RNA-sequencing revealed that AESCs are involved in multiple epidermis-associated biological processes shared by KCs and show more similarity to early stage immature KCs than to adult KCs. However, AESCs were observed to be heterogeneous, and some possessed hybrid mesenchymal and epithelial features distinct from KCs. A functional evaluation revealed that AESCs can phagocytose melanosomes transported by melanocytes in both 2D and 3D co-culture systems similar to KCs, which may help reconstitute pigmented skin. The overexpression of TP63 and activation of NOTCH signaling could promote AESC stemness and improve their differentiation features, respectively, bridging the gap between AESCs and KCs. These changes induced the convergence of AESC cell fate with KCs. In future, modified reprogramming strategies, such as the use of small molecules, may facilitate the further modulation human AESCs for use in skin regeneration.