scholarly journals Pluripotent Stem Cells for Transgenesis in the Rabbit: A Utopia?

2020 ◽  
Vol 10 (24) ◽  
pp. 8861
Author(s):  
Worawalan Samruan ◽  
Nathalie Beaujean ◽  
Marielle Afanassieff
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Self Renewal ◽  
Potential Barriers ◽  
Undifferentiated State ◽  
Early Embryos ◽  
Primed State ◽  
Induced Pluripotent ◽  
Intended Use

Pluripotent stem cells (PSCs) possess the following two main properties: self-renewal and pluripotency. Self-renewal is defined as the ability to proliferate in an undifferentiated state and pluripotency as the capacity to differentiate into cells of the three germ layers, i.e., ectoderm, mesoderm, and endoderm. PSCs are derived from early embryos as embryonic stem cells (ESCs) or are produced by reprogramming somatic cells into induced pluripotent stem cells (iPSCs). In mice, PSCs can be stabilized into two states of pluripotency, namely naive and primed. Naive and primed PSCs notably differ by their ability to colonize a host blastocyst to produce germline-competent chimeras; hence, naive PSCs are valuable for transgenesis, whereas primed PSCs are not. Thanks to its physiological and developmental peculiarities similar to those of primates, the rabbit is an interesting animal model for studying human diseases and early embryonic development. Both ESCs and iPSCs have been described in rabbits. They self-renew in the primed state of pluripotency and, therefore, cannot be used for transgenesis. This review presents the available data on the pluripotent state and the chimeric ability of these rabbit PSCs. It also examines the potential barriers that compromise their intended use as producers of germline-competent chimeras and proposes possible alternatives to exploit them for transgenesis.

308 Tet1 PLAYS IMPORTANT ROLES IN MAINTAINING CHARACTERIZATIONS OF PORCINE INDUCED PLURIPOTENT STEM CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 301
Author(s):  
A. R. Fan ◽  
K. Y. Ma ◽  
T. C. Zhao ◽  
P. P. An ◽  
B. Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Self Renewal ◽  
Qrt Pcr ◽  
Post Transfection ◽  
Fetal Fibroblasts ◽  
Induced Pluripotent

It was recently found that the ten-eleven-translocation (TET) family of Fe(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and thus promotes active demethylation of genomes. Tet1 is highly expressed in mouse embryonic stem cells (mESC) and has been demonstrated to involve in mESC maintenance. Here we used small interference RNA (siRNA) to transiently knockdown expression of Tet1 in porcine induced pluripotent stem cells (iPSC) in order to identify its functions. The fetal fibroblasts were isolated from a 30-day-old porcine fetus and induced into iPSC with defined transcription factors, namely Oct-4, Sox-2, Klf-4, and C-myc. The colonies appeared on Day 12 and were picked up on Day 14. These colonies had normal ES-like morphology and alkaline phosphatase activity. Specifically, they were positively stained for pluripotency-specific markers, including Oct4, Sox2, Nanog, Rex1, and SSEA1. When cultured in vitro, the cells formed embryoid bodies (EB), and all 3 germ layer markers (endoderm: AFP, alphaAT; mesoderm: BMP4, Enolase; ectoderm: GFAP, Neurod) were detected positively in EB. For siRNA transfections, iPSC from the colonies were transfected with 40 pmol of siRNA and 2 µL of Lipofectamine 2000 in 1 well of a 24-well plate. After transfection, iPSC were subjected to fluorescence-activated cell sorting to determine the fraction of FAM-positive cells in order to confirm transfection efficiency; the percentage of positive cells reached 48 ± 4.96. We observed obvious knockdown of Tet1 after short-term transfection of siRNA, and the knockdown efficiency was confirmed using qRT-PCR and immunofluorescence staining. Notably, knockdown of Tet1 resulted in morphological abnormality and loss of undifferentiated state of porcine iPSC. However, no obvious morphological changes were observed in the negative control (transfected with nonsense-siRNA), positive control (transfected with GAPDH-siRNA), or mock control (transfected with DEPC-treated water). To gain insight into the molecular mechanism underlying the self-renewal defect, we analysed the effects of Tet1 knockdown on the expression of key stem cell factors and differentiation markers of different embryonic layers using qRT-PCR. We found that knockdown of Tet1 resulted in downregulated expression of pluripotency-related genes, such as Lefty-2, Klf-2, and Sox-2 (the expression ratios of post-transfection to pre-transfection were 0.31 ± 0.21, 0.48 ± 0.072, and 0.65 ± 0.046, respectively), and upregulated expression of differentiation-related genes, including Pitx-2, Hand-1, Gata-6, and Lef-1 (the expression ratios of post-transfection to pre-transfection were 4.35 ± 1.36, 2.56 ± 0.68, 2.91 ± 1.47, and 2.33 ± 1.11, respectively). However, Oct-4, C-myc, Klf-4, and Nanog were not downregulated (the expression ratios of post-transfection to pre-transfection were 0.91 ± 0.15, 1.12 ± 0.26, 1.15 ± 0.21, and 1.08 ± 0.08, respectively). Taken together, Tet1 plays important roles in porcine iPSC self-renewal and characterization maintenance. This study was financed by National Basic Research Program of China (NO.2009CB941001).

scholarly journals Effects of TET1 knockdown on gene expression and DNA methylation in porcine induced pluripotent stem cells

Reproduction ◽  
2013 ◽  
Vol 146 (6) ◽  
pp. 569-579 ◽  
Author(s):  
Anran Fan ◽  
Kuiying Ma ◽  
Xinglan An ◽  
Yu Ding ◽  
Peipei An ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Differentiation Markers ◽  
Expression Of Genes ◽  
Undifferentiated State ◽  
Induced Pluripotent

TET1 is implicated in maintaining the pluripotency of embryonic stem cells. However, its precise effects on induced pluripotent stem cells (iPSCs), and particularly on porcine iPSCs (piPSCs), are not well defined. To investigate the role of TET1 in the pluripotency and differentiation of piPSCs, piPSCs were induced from porcine embryonic fibroblasts by overexpression ofPOU5F1(OCT4),SOX2,KLF4, andMYC(C-MYC). siRNAs targeting toTET1were used to transiently knockdown the expression ofTET1in piPSCs. Morphological abnormalities and loss of the undifferentiated state of piPSCs were observed in the piPSCs after the downregulation ofTET1. The effects ofTET1knockdown on the expression of key stem cell factors and differentiation markers were analyzed to gain insights into the molecular mechanisms underlying the phenomenon. The results revealed that knockdown ofTET1resulted in the downregulated expression of pluripotency-related genes, such asLEFTY2,KLF2, andSOX2, and the upregulated expression of differentiation-related genes includingPITX2,HAND1,GATA6, andLEF1. However,POU5F1,MYC,KLF4, andNANOGwere actually not downregulated. Further analysis showed that the methylation levels of the promoters forPOU5F1andMYCincreased significantly afterTET1downregulation, whereas there were no obvious changes in the promoters ofSOX2,KLF4, andNANOG. The methylation of the whole genome increased, while hydroxymethylation slightly declined. Taken together, these results suggest thatTET1may play important roles in the self-renewal of piPSCs and the maintenance of their characteristics by regulating the expression of genes and the DNA methylation.

scholarly journals Induced Pluripotent Stem Cells: Generation Strategy and Epigenetic Mystery behind Reprogramming

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Pengfei Ji ◽  
Sasicha Manupipatpong ◽  
Nina Xie ◽  
Yujing Li
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Clinical Medicine ◽  
Ethical Issues ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Self Renewal ◽  
Induced Pluripotent

Possessing the ability of self-renewal with immortalization and potential for differentiation into different cell types, stem cells, particularly embryonic stem cells (ESC), have attracted significant attention since their discovery. As ESC research has played an essential role in developing our understanding of the mechanisms underlying reproduction, development, and cell (de)differentiation, significant efforts have been made in the biomedical study of ESC in recent decades. However, such studies of ESC have been hampered by the ethical issues and technological challenges surrounding them, therefore dramatically inhibiting the potential applications of ESC in basic biomedical studies and clinical medicine. Induced pluripotent stem cells (iPSCs), generated from the reprogrammed somatic cells, share similar characteristics including but not limited to the morphology and growth of ESC, self-renewal, and potential differentiation into various cell types. The discovery of the iPSC, unhindered by the aforementioned limitations of ESC, introduces a viable alternative to ESC. More importantly, the applications of iPSC in the development of disease models such as neurodegenerative disorders greatly enhance our understanding of the pathogenesis of such diseases and also facilitate the development of clinical therapeutic strategies using iPSC generated from patient somatic cells to avoid an immune rejection. In this review, we highlight the advances in iPSCs generation methods as well as the mechanisms behind their reprogramming. We also discuss future perspectives for the development of iPSC generation methods with higher efficiency and safety.

scholarly journals Disruption of RING and PHD Domains of TRIM28 Evokes Differentiation in Human iPSCs

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1933
Author(s):  
Sylwia Mazurek ◽  
Urszula Oleksiewicz ◽  
Patrycja Czerwińska ◽  
Joanna Wróblewska ◽  
Marta Klimczak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Self Renewal ◽  
Human Ipscs ◽  
New Gene ◽  
Induced Pluripotent ◽  
The Impact ◽  
Phd Domain

TRIM28, a multi-domain protein, is crucial in the development of mouse embryos and the maintenance of embryonic stem cells’ (ESC) self-renewal potential. As the epigenetic factor modulating chromatin structure, TRIM28 regulates the expression of numerous genes and is associated with progression and poor prognosis in many types of cancer. Because of many similarities between highly dedifferentiated cancer cells and normal pluripotent stem cells, we applied human induced pluripotent stem cells (hiPSC) as a model for stemness studies. For the first time in hiPSC, we analyzed the function of individual TRIM28 domains. Here we demonstrate the essential role of a really interesting new gene (RING) domain and plant homeodomain (PHD) in regulating pluripotency maintenance and self-renewal capacity of hiPSC. Our data indicate that mutation within the RING or PHD domain leads to the loss of stem cell phenotypes and downregulation of the FGF signaling. Moreover, impairment of RING or PHD domain results in decreased proliferation and impedes embryoid body formation. In opposition to previous data indicating the impact of phosphorylation on TRIM28 function, our data suggest that TRIM28 phosphorylation does not significantly affect the pluripotency and self-renewal maintenance of hiPSC. Of note, iPSC with disrupted RING and PHD functions display downregulation of genes associated with tumor metastasis, which are considered important targets in cancer treatment. Our data suggest the potential use of RING and PHD domains of TRIM28 as targets in cancer therapy.

scholarly journals Efficient induction and sustenance of pluripotent stem cells from bovine somatic cells

Biology Open ◽  
2021 ◽  
Vol 10 (10) ◽  
Author(s):  
Viju Vijayan Pillai ◽  
Prasanthi P. Koganti ◽  
Tiffany G. Kei ◽  
Shailesh Gurung ◽  
W. Ronald Butler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
T Antigen ◽  
Bovine Embryo ◽  
Self Renewal ◽  
Induced Pluripotent

ABSTRACT Although derivation of naïve bovine embryonic stem cells is unachieved, the possibility for generation of bovine induced pluripotent stem cells (biPSCs) has been generally reported. However, attempts to sustain biPSCs by promoting self-renewal have not been successful. Methods established for maintaining murine and human induced pluripotent stem cells (iPSCs) do not support self-renewal of iPSCs for any bovid species. In this study, we examined methods to enhance complete reprogramming and concurrently investigated signaling relevant to pluripotency of the bovine blastocyst inner cell mass (ICM). First, we identified that forced expression of SV40 large T antigen together with the reprogramming genes (OCT4, SOX2, KLF4 and MYC) substantially enhanced the reprogramming efficacy of bovine fibroblasts to biPSCs. Second, we uncovered that TGFβ signaling is actively perturbed in the ICM. Inhibition of ALK4/5/7 to block TGFβ/activin/nodal signaling together with GSK3β and MEK1/2 supported robust in vitro self-renewal of naïve biPSCs with unvarying colony morphology, steady expansion, expected pluripotency gene expression and committed differentiation plasticity. Core similarities between biPSCs and stem cells of the 16-cell-stage bovine embryo indicated a stable ground state of pluripotency; this allowed us to reliably gain predictive understanding of signaling in bovine pluripotency using systems biology approaches. Beyond defining a high-fidelity platform for advancing biPSC-based biotechnologies that have not been previously practicable, these findings also represent a significant step towards understanding corollaries and divergent aspects of bovine pluripotency. This article has an associated First Person interview with the joint first authors of the paper.

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