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Generation of iPS cells from four-factor induction of mEFs, NSCs, and ADCs. (a) Morphology of mEFs, NSCs, and ADCs in culture. P denotes passage number. (b) Flow cytometry cell cycle profiles. The x-axis denotes the intensity of propidium iodide (PI) fluorescence and the γ-axis denotes cell number. The different phases of the cell cycle are indicated by the different labeled peaks. ADCs show a higher proportion of cells at the G2/M phase compared with NSCs and mEFs. ES cells show a higher proportion of cells at both the S and G2/M phases. (c) Histogram showing the number of Oct4-GFP-positive colonies observed at day 12 postinduction with four factors. Columns indicate mean ± SD (n = 4). *Significantly different compared with mEF (p < 0.05, t-test). (d) Morphology of mEFs, NSCs, and ADCs at day 12 postinduction with Yamanaka's four factors. p0 indicates that Oct4-GFP-positive colonies have not been passaged since reactivation of GFP. (e) Phase and UV photographs of established iPS cell lines.
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Ectopic expression of key reprogramming transgenes in somatic cells enables them to adopt the characteristics of pluripotency. Such cells have been termed induced pluripotent stem (iPS) cells and have revolutionized the field of somatic cell reprogramming, as the need for embryonic material is obviated. One of the issues facing both the clinical tr...
Citations
... Overexpressing OCT4, KLF4, c-Myc and SOX2 transcription factor (TFs) on mouse fibroblast cells allowed Yamanaka and his group to successfully reprogram these cells into iPSCs in 2006 (Takahashi and Yamanaka, 2006). Since then, numerous research teams have analyzed the epigenetic and transcriptional modifications at various periods after inducing the factors in somatic cells to understand better the techniques and processes of somatic cell reprogramming better (Tat et al., 2010;Sun et al., 2009). Few analyses have been published so far on the topic of TF mediated transdifferentiation of ADSCs. ...
Mesenchymal stem cells are found to have the potential to differentiate into many lineages, thus regulating diverse signalling cascades. This unique property of stem cells, called trans differentiation/linear reprogramming, aided in regenerative medicine and tissue repair. The mechanism of such regeneration is still unclear and requires further analysis. Due to the use of external or oncogenic factors, one of the approaches for mending cardiac, renal, and neurological disorders after an injury by induced pluripotent stem cells in the form of reprogramming does not show much benefit in the clinical setting. Consequently, cellular reprogramming may enable the application of clinical research to cell therapy, disease modelling, drug screening, and the fabrication of artificial organs. Studies related to this distinctive phenomenon of stem cells, where the cells could reprogramme themselves into completely different cell lineages, showed a promising future in therapeutic applications. However, unrelenting development in cellular reprogramming has prepared the ways for novel strategies in which signalling pathway manipulation may decide cellular destiny. This cellular reprogramming has got bright prospects in the field of regenerative medicine. Therefore, understanding the relationship between stochasticity and defining cell fate can help decipher molecular regulatory mechanisms of cellular reprogramming.
... Conventionally, somatic cells can be reprogrammed to pluripotent stem cells by retrovirus-mediated ectopic overexpression of pluripotency factors Oct3/4, Sox2, Klf4, and c-Myc (OSKM) that in turn reactivate the endogenous pluripotency genes network, leading to the establishment of cells with ESC-like stemness, termed as induced pluripotent stem cells (iPSCs) . Since the first demonstration of iPSCs regeneration from mouse embryonic fibroblast and adult human dermal fibroblast cells (Takahashi et al. 2007) by ectopic overexpression of OSKM factors, various mouse and human cell types have been reprogrammed to bonafide iPSCs, including liver cells (Aoi et al. 2008), pancreatic beta cells , neural cells (Tat et al. 2010), blood cells (Wen et al. 2016), and mesenchymal stem cells (Oda et al. 2010). Subsequently, using a similar approach, iPSCs have also been derived from somatic cells of domesticated animal species, including canine (Koh and Piedrahita 2015), rabbit (Honda et al. 2013), bovine (Cao et al. 2012), goat (Sandmaier et al. 2015), porcine , and buffalo (Deng et al. 2012). ...
Stem cells provide novel approaches to improve animal health and productivity and thereby indirectly enrich human life. However, the use of stem cells, including the totipotent single–cell stage embryo, for the generation of genetically modified livestock and refinement of regenerative veterinary medicines has remained a less exploited domain until a few years ago, largely due to the nonavailability of efficient genetic manipulation tools. The inception of sequence-targeted genetic manipulation tools based on bacterial adaptive defense system, clustered regularly interspaced short palindromic repeat (CRISPR), and CRISPR-associated (Cas) protein (CRISPR/Cas) has enabled the bioengineers to harness the stem cell for the animal and human benefits in the way never done before. CRISPR/Cas-based genetic manipulation tools, due to its simplicity, high sequence specificity, and muliplexibility features, has dramatically broadened the dimension of stem cell applications in both the animal and human world, ranging from stem cell-based patient-specific therapeutics and anticancer vaccine development to the generation of genetically modified large animals with improved traits of agricultural and biomedical importance. This chapter provides an overview of various CRISPR/Cas-based gene editing and regulation tools that have been instrumented for genomic modulation of mammalian cells to date. It discusses the critical elements of a typical CRISPR/Cas-based genetic manipulation experiment for efficient modulation of mammalian cells. Based on the reported studies, this chapter sheds light on CRISPR/Cas tools’ potency to advance and accelerate the stem cell uses to benefit veterinary research.
... Conventionally, somatic cells can be reprogrammed to pluripotent stem cells by retrovirus-mediated ectopic overexpression of pluripotency factors Oct3/4, Sox2, Klf4, and c-Myc (OSKM) that in turn reactivate the endogenous pluripotency genes network, leading to the establishment of cells with ESC-like stemness, termed as induced pluripotent stem cells (iPSCs) . Since the first demonstration of iPSCs regeneration from mouse embryonic fibroblast and adult human dermal fibroblast cells (Takahashi et al. 2007) by ectopic overexpression of OSKM factors, various mouse and human cell types have been reprogrammed to bonafide iPSCs, including liver cells (Aoi et al. 2008), pancreatic beta cells , neural cells (Tat et al. 2010), blood cells (Wen et al. 2016), and mesenchymal stem cells (Oda et al. 2010). Subsequently, using a similar approach, iPSCs have also been derived from somatic cells of domesticated animal species, including canine (Koh and Piedrahita 2015), rabbit (Honda et al. 2013), bovine (Cao et al. 2012), goat (Sandmaier et al. 2015), porcine , and buffalo (Deng et al. 2012). ...
Mammary gland (MG) biology has attracted researchers’ attention in every mammalian species. Apart from the natural curiosity to understand the mammalian biology wherein newborns are nourished by milk, the incidence of benign and metastatic breast tumor further instigated the scientific community to understand the detailed cellular and molecular events so that the diagnosis, treatment, and prevention could be implemented. The mouse MG has served as the closest MG model to understand the anatomy and physiology despite several differences in the endocrine and reproductive systems of mice and humans. In bovine, the very immediate interest has been to improve milk production from dairy animals. Globally, the average milk yield from dairy animals has so far remained forwardly progressive. Nevertheless, disease like mastitis and the existence of low milk-yielding cows in population have necessitated accelerated research on understanding bovine mammary gland biology, host–pathogen relationship, cyclic changes in molecular and cellular physiology during puberty, pregnancy, lactation, and involution. Proteomics deals with studying many proteins together, thus deriving comprehensive information on cellular physiology. Along with antibodies, the application of mass spectrometry in the study of proteins has revolutionized the investigation method as never before. The complexity in tissue architecture of MG comprises many cell types, and their continuous turnover in the lifetime of an animal constantly challenged our mind and approach to discern cell identity correctly. It will not have been possible without the knowledge of cell type-specific marker proteins, which again owes a lot to proteomics. This chapter selectively discusses the application of mass spectrometry-based proteomics for mammary epithelial cells and mammary stem cells in MG in the context of cell-specific biomarkers, functional differentiation, and diseases.
... These methodologies offer the opportunity to produce individuals from the reprogramming of terminally differentiated cells in pluripotent lines or the trans-differentiation of adult stem cells into gametes [61]. To choose the cell lines to be used in reproductive biotech-niques, some important considerations are the ease of access to the tissue, ease of deriving and maintaining the lines in vitro, and the ability of these cells to reprogram/differentiate themselves [127]. Thus, it would be interesting for several types of cells to be stored and evaluate which ones are more prone to differentiation into germ cells, or even to reprogram into induced pluripotency stem cells (iPSC). ...
One of the most significant challenges in deer is the ability to maintain genetic diversity, avoiding inbreeding and sustaining population health and reproduction. Although our general knowledge of reproductive physiology is improving, it appears that the application of assisted reproductive technology (ART) will more efficiently advance wildlife conservation efforts and preserve genetic diversity. The purpose of this review is to present the most important results obtained with the use of ART in Neotropical deer. Thus, the state-of-the-art for estrus synchronization, semen technology, artificial insemination, and in vivo embryo production will be presented. In vitro embryo production (IVP) is also a biotechnology that is taking initial steps in deer. In this aspect, the approach with the proteomics of ovarian follicular fluid is being used as a tool for a better understanding of oocyte maturation. Finally, cell banks and the use of interspecific somatic cell nuclear transfer (iSCNT) as well as the use of stem cells for gametes differentiation are promising techniques.
... Different donor cells have different efficiency of reprogramming into iPSCs. It is generally believed that the efficiency of reprogramming adult stem cells into iPSCs is higher compared with fibroblasts (Tat et al., 2010). Adult stem cells are easy to reprogram mainly due to their specific features. ...
Past researches have shown that pluripotency maintenance of naive and primed-state pluripotent stem cells (PSCs) depends on different signaling pathways, and naive-state PSCs possess the ability to produce chimeras when they are introduced into a blastocyst. Considering porcine is an attractive model for preclinical studies, many researches about pig induced pluripotent stem cells (piPSCs) have been reported. Some cytokines and small molecule compounds could transform primed piPSCs into naive state. However, there are no suitable culture conditions for generation of naive-state piPSCs with high efficiency; other small molecule compounds need further exploration. In this study, we investigated whether p38 MAPK and JNK signal pathway inhibitor SB203580 and SP600125 could be of benefit for acquiring naive-state piPSCs. By comparing reprogramming efficiencies under conditions of different donor cells and culture environment, we found that porcine bone marrow mesenchymal stem cells (PBMSCs) have higher efficiency on piPSC induction, and the culture condition of CHIR99021+PD0325901(2i)+Lif+bFGF is more suitable for subculturing of piPSCs. Our results also indicate that SB203580 and SP600125 could promote reprogramming of PBMSCs into naive-like state piPSCs. These results provide guidance for choosing donor cells, culture conditions, and research of different state iPSCs during the process of reprogramming pig somatic cells.
... 173 This property has then been applied to umbilical cord blood cells, placental mesenchymal stromal cells, adipose-derived stem cells, and neural stem cells. 174,175 The improved proliferative function of these precursor cells over adult cells marks a key appealing feature of retrograde conversion. 173 Transplantation of iPSCs in stroke animals reduces infarction and enhances functional improvements, likely via observed downregulation of pro-inflammatory factors and upregulation of anti-inflammatory factors. ...
Hemorrhagic stroke is a global health crisis plagued by neuroinflammation in the acute and chronic phases. Neuroinflammation approximates secondary cell death, which in turn robustly contributes to stroke pathology. Both the physiological and behavioral symptoms of stroke correlate with various inflammatory responses in animal and human studies. That slowing the secondary cell death mediated by this inflammation may attenuate stroke pathology presents a novel treatment strategy. To this end, experimental therapies employing stem cell transplants support their potential for neuroprotection and neuroregeneration after hemorrhagic stroke. In this review, we evaluate experiments using different types of stem cell transplants as treatments for stroke-induced neuroinflammation. We also update this emerging area by examining recent preclinical and clinical trials that have deployed these therapies. While further investigations are warranted to solidify their therapeutic profile, the reviewed studies largely posit stem cells as safe and potent biologics for stroke, specifically owing to their mode of action for sequestering neuroinflammation and promoting neuroregenerative processes.
... iPSCs generated from mesenchymal stem cells or progenitor had shown better reprogramming efficiency and equivalent gene profiles to embryonic stem cells compared to fibroblasts [13,23]. ASCs demonstrated 8-and 38-fold superior reprogramming efficiency than neural stems cells and MEFs [24]. Sun et al. reported that the reprogramming of iPSCs by human ASCs is 20 times more efficient than IMR90 fibroblasts with reprogramming efficiencies of 0.01% on feeder-free Matrigel substrates to 0.2% on feeders with MEFs [10]. ...
Background:
Bone regeneration is a crucial and challenging issue in clinical practice. Bone tissue engineering (BTE) with an optimal cell source may provide an ideal strategy for the reconstruction of bone defects. This study examined whether induced pluripotent stem cells (iPSCs) derived from adipose-derived stem cells (ASCs) could act as an osteogenic substitute and whether these ASC-iPSCs yield more new bone formation than ASCs in hydrogel scaffolds.
Methods:
ASC-iPSCs were reprogrammed from ASCs through a retroviral system. ASCs were harvested and isolated from adipose tissue of humans. An aliquot of cell suspension (1 × 106 cells/mL) was seeded directly onto the nHAP-gelatin cryogel scaffolds. Nude mice back implantation of cell-seeded scaffolds was designed for in vivo comparison of osteogenic potentials between ASCs and ASC-iPSCs. Samples were harvested 4 and 8 weeks after implantation for further analysis based on histology and RT-PCR.
Results:
ASC-iPSCs were successfully obtained from human adipose-derived stem cells. PCR results also showed that specific genes of iPSCs with the ability to cause the differentiation of cells into the three germ layers were expressed. In our in vivo experiments, iPSCs were subcutaneously injected into nude mice to induce teratoma formation. The morphology of the three germ layers was confirmed by histological staining. ASC is an essential cell source for BTE with benefits of high volume and less-invasive acquisition. With additional transforming Yamanaka factors, ASC-iPSCs showed higher osteogenic differentiation and elevated expression of collagen type I (Col I), osteocalcin (OCN), alkaline phosphate (ALP), and runt-related transcription factor 2 (RunX-2).
Conclusions:
This report suggests that ASC-iPSCs could be a superior cell source in BTE with better osteogenic differentiation efficacy for future clinical applications.
... It is known that different starting cell types share distinct transcriptional features during iPS cell generation and, as a result, may exhibit different reprogramming dynamics and efficiency and yield iPS cells with different quality [17]. For example, reprogramming efficiencies of mouse adipose stem cell and neural stem cell were higher than that of mouse embryonic fibroblasts (MEFs) [18]. Pericytes, the microvascular mural cells, are abundant in the body and can differentiate into multiple cell types of the mesenchymal lineage, suggesting higher cellular plasticity than that of fibroblasts [19,20]. ...
Background:
Pigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown.
Methods:
iPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis.
Results:
In this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs. Both PC-iPS and PEF-iPS cells maintained the stable "dome-shaped" morphology and genome stability after long-term culture. The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint). PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro. The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively. Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance. And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-β, P53, and MAPK stem cell signaling pathways. Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses. Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%.
Conclusions:
Our findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo. However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low.
... Since then, many groups have studied the methods and mechanisms of the somatic cell reprogramming process by analyzing epigenetic and transcriptional changes at different time points after factor induction in different somatic cells. It has been reported that OSKM can reprogram ADSCs to iPSCs [104,105]. ...
Neurological diseases can severely compromise both physical and psychological health. Recently, adult mesenchymal stem cell- (MSC-) based cell transplantation has become a potential therapeutic strategy. However, most studies related to the transdifferentiation of MSCs into neural cells have had disappointing outcomes. Better understanding of the mechanisms underlying MSC transdifferentiation is necessary to make adult stem cells more applicable to treating neurological diseases. Several studies have focused on adipose-derived stromal/stem cell (ADSC) transdifferentiation. The purpose of this review is to outline the molecular characterization of ADSCs, to describe the methods for inducing ADSC transdifferentiation, and to examine factors influencing transdifferentiation, including transcription factors, epigenetics, and signaling pathways. Exploring and understanding the mechanisms are a precondition for developing and applying novel cell therapies.
... Differentiated somatic cells can be reprogrammed to pluripotency (iPSCs) by treatment with defined factors. Several groups have been able to induce pluripotency easily and non-invasively from different somatic cells including fibroblasts, bone marrow, adipose tissue, and peripheral leukocytes [87][88][89]. Chen et al. found in a rat focal ischemia model that after the combined subdural transplantation of iPSCs and fibrin glue, there was a significant reduction in the infarct volume and a greater functional recovery in the animals examined with the rotarod test [90]. It has also been reported in infarcted rats that the intracerebral transplantation of iPSCs in both the affected and non-affected hemispheres induced their migration to the damaged area and subsequent differentiation into neural cells that enhanced post-stroke sensorimotor function recovery [91,92]. ...
The use of advanced biomaterials as a structural and functional support for stem cells-based therapeutic implants has boosted the development of tissue engineering applications in multiple clinical fields. In relation to neurological disorders, we are still far from the clinical reality of restoring normal brain function in neurodegenerative diseases and cerebrovascular disorders. Hydrogel polymers show unique mechanical stiffness properties in the range of living soft tissues such as nervous tissue. Furthermore, the use of these polymers drastically enhances the engraftment of stem cells as well as their capacity to produce and deliver neuroprotective and neuroregenerative factors in the host tissue. Along this article, we review past and current trends in experimental and translational research to understand the opportunities, benefits, and types of tentative hydrogel-based applications for the treatment of cerebral disorders. Although the use of hydrogels for brain disorders has been restricted to the experimental area, the current level of knowledge anticipates an intense development of this field to reach clinics in forthcoming years.
