Derivation of naive-type induced pluripotent stem cells in cattle using piggyBac transposition of doxycycline-inducible transcription factors

2014 ◽  
Author(s):  
Takamasa Kawaguchi ◽  
Tomoyuki Tsukiyama ◽  
Naojiro Minami ◽  
Masayasu Yamada ◽  
Shuichi Matsuyama ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent

scholarly journals Biological importance of OCT transcription factors in reprogramming and development

2021 ◽  
Author(s):  
Kee-Pyo Kim ◽  
Dong Wook Han ◽  
Johnny Kim ◽  
Hans R. Schöler
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ectopic Expression ◽  
Donor Cell ◽  
Structural Components ◽  
Oct4 Expression ◽  
Reprogramming Factors ◽  
Induced Pluripotent

AbstractEctopic expression of Oct4, Sox2, Klf4 and c-Myc can reprogram somatic cells into induced pluripotent stem cells (iPSCs). Attempts to identify genes or chemicals that can functionally replace each of these four reprogramming factors have revealed that exogenous Oct4 is not necessary for reprogramming under certain conditions or in the presence of alternative factors that can regulate endogenous Oct4 expression. For example, polycistronic expression of Sox2, Klf4 and c-Myc can elicit reprogramming by activating endogenous Oct4 expression indirectly. Experiments in which the reprogramming competence of all other Oct family members tested and also in different species have led to the decisive conclusion that Oct proteins display different reprogramming competences and species-dependent reprogramming activity despite their profound sequence conservation. We discuss the roles of the structural components of Oct proteins in reprogramming and how donor cell epigenomes endow Oct proteins with different reprogramming competences.

Analysis of the transcription factors and their regulatory roles during a step-by-step differentiation of induced pluripotent stem cells into hepatocyte-like cells

2019 ◽  
Vol 15 (6) ◽  
pp. 383-398 ◽  
Author(s):  
Yannick Tauran ◽  
Stéphane Poulain ◽  
Myriam Lereau-Bernier ◽  
Mathieu Danoy ◽  
Marie Shinohara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Induced Pluripotent Stem Cells ◽  
Transcriptome Analysis ◽  
Pluripotent Stem Cells ◽  
Original Method ◽  
Induced Pluripotent

Human induced pluripotent stem cells have been investigated through a sequential in vitro step-by-step differentiation into hepatocyte-like cells using nanoCAGE, an original method for promoters, transcription factors, and transcriptome analysis.

Generation of Induced Pluripotent Stem Cells From Primary Chronic Myelogenous Leukemia Patient Sample.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1206-1206
Author(s):  
Keiki Kumano ◽  
Shunya Arai ◽  
Koki Ueda ◽  
Kumi Nakazaki ◽  
Yasuhiko Kamikubo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Induced Pluripotent Stem Cells ◽  
Chronic Myelogenous Leukemia ◽  
Pluripotent Stem Cells ◽  
Hematological Malignancies ◽  
Hematological Malignancy ◽  
Myelogenous Leukemia ◽  
Hematopoietic Progenitors ◽  
Induced Pluripotent

Abstract Abstract 1206 Introduction: Induced pluripotent stem cells (iPSCs) can be generated from various cell types by the expression of defined transcription factors. In addition to the regenerative medicine, iPSCs have been used for the study of the pathogenesis of inherited genetic disease. Recently, it was reported that iPSCs were generated not only from normal tissue, but also from malignant cells. In those cases, cancer cells themselves must be the starting material from which iPSCs are derived. However, in almost all the cases, they used the established cell lines (chronic myelogenous leukemia (CML), gastrointestinal cancers, and melanoma) except for the JAK2-V617F mutation (+) polycythemia vera (PV) patient. In this study, we established the iPSCs from primary CML patient sample. Results: After obtaining informed consent, bone marrow cells from CML patient were reprogrammed by introducing the transcription factors Oct3/4, Sox2, KLF4, and c-myc. To improve the efficiency of the development of iPSCs, we added valproic acid (VPA), a histone deacetylase inhibitor, to the culture. Two CML derived iPSCs (CML-iPSCs) were generated. CML-iPSCs expressed the pluripotency markers such as SSEA-4 and Tra-1-60, and the endogenous expression of embryonic stem cell (ESC) characteristic transcripts (Oct3/4, Sox2, KLF4, Nanog, LIN28, REX1) was confirmed by RT-PCR. Oct4 and Nanog promoter regions were demetylated in the CML-iPSCs. Although CML-iPSCs expressed bcr-abl, they were resistant to the imatinib. Then we differentiated them into hematopoietic progenitors within the ‘unique sac-like structures’ (iPS-sacs). This method was reported to be able to produce the hematopoietic progenitors with higher efficiency than the usual embryoid body formation method using human ESCs (Takayama et al., Blood, 111, 5298–306, 2008). The hematopoietic progenitors showed the hematopoietic marker CD45 and immature marker CD34, and recovered the sensitivity to the imatinib, which recapitulated the feature of initial CML disease. Then we investigated the mechanism of the resistance to the imatinib in CML-iPSCs. The phosphorylation state of ERK1/2, AKT, and STAT5, which are the essential for the survival of bcr-abl (+) hematopoietic progenitors, were evaluated after imatinib treatment in CML-iPSCs. The phosphorylation of ERK1/2 and AKT, which were also essential for the maintenance of iPSCs, were unchanged after treatment, although STAT5 was not activated both before and after treatment. These results showed that the signaling for iPSCs maintenance compensated for the inhibition of bcr-abl in CML-iPSCs and that the oncogene addiction was lost in CML-iPSCs. Conclusion: We generated the iPSCs from primary CML patient samples, re-differentiated them into hematopoietic lineage and showed the recapitulation of the features of initial disease. Primary samples of hematological malignancy are usually difficult to be expanded. However, if once they are reprogrammed to iPSCs, they can expand unlimitedly. As a result, we can obtain the genetically abnormal hematopoietic cells continuously by re-differentiating them into hematopoietic cells and use them for the studies which require the large number of living cells such as the analysis for leukemia stem cells or drug screening. Thus iPSCs technology would be useful for the study of hematological malignancy, especially for which animal model was not established such as myelodysplastic syndrome and be applicable for other cancers than hematological malignancies. We are now trying to establish the iPSCs derived from other hematological malignancies. Disclosures: No relevant conflicts of interest to declare.

337 PROMOTER-DEPENDENT SILENCING OF REPROGRAMMING TRANSCRIPTION FACTORS IN MOUSE INDUCED PLURIPOTENT STEM CELLS PRODUCED WITH SLEEPING BEAUTY TRANSPOSON VECTORS

2015 ◽  
Vol 27 (1) ◽  
pp. 257
Author(s):  
S. G. Petkov ◽  
W. A. Kues ◽  
H. Niemann
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Sleeping Beauty ◽  
Rt Pcr ◽  
Induced Pluripotent ◽  
Pluripotency Genes

Epigenetic silencing of the transgenes has been considered a prerequisite for complete reprogramming of mouse somatic cells to induced pluripotent stem cells (miPSC). Here, we examined the activity status of the reprogramming transcription factors in miPSC produced with Sleeping Beauty (SB) transposon vectors carrying expression cassettes with the porcine OCT4, SOX2, c-MYC, and KLF4 (pOSMK) under the control of doxycycline (DOX)-inducible (TetO) or constitutive (CAG) promoters. Mouse embryo fibroblasts (MEF) were electroporated with SB-TetO-rTA-SV40pA-TetO-pOSMK-IRES-tdTomato-bGHpA (TetO group) or with SB-loxP-CAG-pOSMK-IRES-tdTomato-SV40pA-loxP (CAG group) together with SB100x (SB transposase). The cells were cultured on mitotically inactivated MEF feeders with DMEM supplemented with 20% knockout serum replacement, 2 mM l-glutamine, penicillin-streptomycin, nonessential amino acids, 0.1 mM 2-mercaptoethanol, 1000 U mL–1 of ESGRO, and 5 µg mL–1 of DOX. The miPSC colonies were individually picked, disaggregated to single cells, and propagated further under the same culture conditions. Three cell lines from each experimental group were examined for pluripotency characteristics, and the activity of the transgenes was monitored by the presence of tdTomato fluorescence and by RT-PCR. The miPSC produced with TetO vector silenced the transgene expression within 11 days post-transfection (in the presence of DOX) and upregulated the endogenous pluripotency genes Oct4, Sox2, Nanog, Rex1, and Utf1. These cells showed typical miPSC morphology and ability to differentiate into cells from the 3 primary germ layers in vitro and in vivo (teratomas). At the same time, the miPSC from the CAG group did not silence the transgenes even after 20 passages of continuous propagation, although they upregulated the endogenous pluripotency genes similarly to the TetO group. Moreover, these cells also showed ability to differentiate in vitro into cells from the 3 germ layers (contracting cardiac myocytes, neurons, epithelia) expressing differentiation markers Afp, Sox17, Gata4, Gata6, cardiac troponin, nestin, and PGP 9.5. Following Cre-mediated excision of the reprogramming cassette, the miPSC from the CAG group continued to self-renew and the expression of pluripotency markers Oct4, Sox2, Nanog, and Rex1 did not change significantly, as evidenced by real-time RT PCR (all P > 0.1), showing that these cells were not dependent on the transgenes for maintaining their pluripotency characteristics. Currently, we are investigating the ability of the miPSC from the CAG group to differentiate in vivo by producing teratomas and chimeras. The results from our preliminary investigations suggest that porcine transcription factors can be used for production of miPSC and that the silencing of the reprogramming transcription factors in miPSC is promoter-dependent, but may not be absolutely necessary for complete reprogramming to pluripotency.

185 Directed Differentiation of Porcine Induced Pluripotent Stem Cells into all Three Germ Layers via Embryoid Body Formation

2018 ◽  
Vol 30 (1) ◽  
pp. 232
Author(s):  
W. Chakritbudsabong ◽  
S. Pamonsupornvichit ◽  
L. Sariya ◽  
R. Pronarkngver ◽  
S. Chaiwattanarungruengpaisan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Induced Pluripotent Stem Cells ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Caspase 3 ◽  
Embryoid Body ◽  
Germ Layers ◽  
Significant Difference ◽  
Induced Pluripotent

Human induced pluripotent stem cells (iPSC) have been generated by reprogramming somatic cells using a cocktail of stem cell transcription factors but the application has been limited in transplantation therapies. The pig represents an ideal model for human clinical research, in part because of its similarity to human physiology and immunology but also because of its use in assessing side effects in long-term preclinical studies. Porcine induced pluripotent stem cells (piPSC) have been established in many studies but their differentiation pattern has not been reported. The aim of this study was to estimate the efficiency and pattern of differentiated piPSC into all 3 germ layers using embryoid body (EB) formation. Two piPSC lines (VSMUi001-A and VSMUi001-D) were induced from porcine embryonic fibroblasts by retroviral overexpression of 5 human reprogramming transcription factors (OCT4, SOX2, KLF4, c-MYC, and LIN28). For EB formation, the piPSC were harvested by treating with TrypLE™ Select (Thermo Fisher Scientific, Waltham, MA, USA) and the cells were cultured in nonadherent 96-well plates in piPSC media without growth factors. Data are expressed as mean ± SEM of at least 3 independent experiments. Statistical analyses were evaluated with Student t-tests for comparison between the 2 cell lines. Statistical significance was set at a P-value of < 0.05. The percentages of EB formation, which were calculated as the number of wells containing EB on Day 3 of differentiation, were 95.3 ± 3.42 and 89.1 ± 5.34 (VSMUi001-A and VSMUi001-D, respectively). However, there was no significant difference between the percentages of EB formation derived from the 2 cell lines. For EB size measurement, 20 EB per experiment were taken after incubation for 3, 7, 14, and 21 days. Both EB sizes increased over time (average diameter of 238.1 ± 6.18, 297.9 ± 4.10, 438.6 ± 13.33, and 728.8 ± 24.92 mm from VSMUi001-A, and 255.8 ± 5.12, 357.9 ± 3.94, 459.6 ± 11.88, and 439.4 ± 20.31 mm from VSMUi001-D). Moreover, both EB displayed homogeneity in size and shape (Day 3, 7), exhibited a cystic structure (Day 14), and a vesicular cavity was present (Day 21). For immunohistochemical analysis, both EB had lower levels of cleaved caspase 3, a marker of apoptotic cells, on Day 3 but higher levels of cleaved caspase 3 from Day 7 through 21. On the contrary, EB showed higher levels of Ki67, a marker of proliferating cells, on Day 3 but lower levels of Ki67 on Days 7, 14, and 21, respectively. In gene expression assessment, EB exhibited ectoderm gene (NeuroD1), mesoderm genes (TNNT2 and TNNI1), and endoderm genes (SOX17 and Endolase) at Day 7 and 21 by using RT-PCR. In conclusion, we report the successful in vitro formation of cystic EB from 2 piPSC lines, indicating that the piPSC could differentiate into 3 germ layers. This will allow researchers to unveil the roadmap of molecular cues needed for piPSC differentiation. This research project is supported by grants from the Mahidol University, Thailand.

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