Generation of pig induced pluripotent stem cells using single factor, c-Myc

Dongchan Son,Kwang-Hwan Choi,Dong-Kyung Lee,Jong-Nam Oh,Seung-Hun Kim,Tae-Young Park,Chang-Kyu Lee 한국발생생물학회 2015 한국발생생물학회 학술발표대회 Vol.2015 No.9

The four transcription factors Oct4, Sox2, Klf4 and c-Myc have been used for making induced pluripotent stem cells. Many efforts have focused on reducing the number of transcription factors, especially c-Myc and Klf4 known as oncogene, for making induced pluripotent stem cells. Recently it have been demonstrated that Oct4 and Sox2 are able to reprogram human fibroblasts or cord blood cells to induced pluripotent stem cells and Oct4 has the ability to reprogram mouse and human neural stem cell to induced pluripotent stem cells. These researches imply cell types for reprogramming experiments have great influence on selection of reprogramming factors. Here we report that pig kidney cortex fibroblasts need only c-Myc factor when they are used for making induced pluripotent stem cells. We used two vector system including drug-inducible vector system and constitutive expression vector system. The two systems generate induced pluripotent stem cells from pig kidney fibroblasts successfully. These one-factor induced pluripotent stem cells are not only similar but also different to pig embryonic stem-like cells. These two one-factor induced pluripotent stem cell lines can express pluripotency related genes and be differentiated into all three germ layers in vitro. However, these two cell lines can be sub-cultured as a single cell by trypsin. Our results support that single factor, c-Myc, is sufficient to converting pig kidney cortex fibroblasts into induced pluripotent stem cells.

Dox-inducible induced pluripotent stem cell lines derived from porcine embryonic fibroblasts

Jin-Kyu Park,Dong-Chan Son,Kwang-Hwan Choi,Chang-Kyu Lee 한국발생생물학회 2013 한국발생생물학회 학술발표대회 Vol.2013 No.8

Pluripotent stem cells are cells that have a self-renewal capacity and the ability to differentiate into all lineages. These cells can be divided into naive- and primed-state pluripotent stem cells according to their pluripotent state. Only the naive state comprises a full pluripotency or ground state that contributes to germ-line transmission. Naive states are found in specific permissive strains or species, such as 129, C57BL/6 and BALB/C in mice. However, a number of attempts have been made to derive naive-state pluripotent stem cell lines from non-permissive species, including humans and pigs, using various exogenous factors including GSK3β and MEK inhibitors (2i), LIF, hypoxic conditions and up-regulation of Oct4 or Klf4. Therefore, in this study we investigated whether a naive pluripotent stem cell line could be derived from porcine embryonic fibroblasts (PEFs) via previously reported factors. Our mouse embryonic stem cell (mESC)-like cell lines expressed the pluripotency markers Oct4, Sox2 and Nanog and a stable mESC-like morphology for more than 50 passages. In addition, these cell lines could be sequentially reprogrammed into mESC-like induced pluripotent stem (iPS) cells from secondary or tertiary fibroblast-like cells differentiated from mESC-like iPS cells by addition of doxycycline (DOX), LIF and 2i. Our results suggest that, as a non-permissive species, porcine stem cells can be induced into mESC-like iPS cells from PEFs by various exogenous factors, including continuous transgene expression, 2i and LIF. However, further work that aims to effectively induce the activation of endogenous transcription factors is necessary to derive authentic naive-state pluripotent porcine stem cells.

Stem cell therapy in pain medicine

Han, Yong Hee,Kim, Kyung Hoon,Abdi, Salahadin,Kim, Tae Kyun The Korean Pain Society 2019 The Korean Journal of Pain Vol.32 No.4

Stem cells are attracting attention as a key element in future medicine, satisfying the desire to live a healthier life with the possibility that they can regenerate tissue damaged or degenerated by disease or aging. Stem cells are defined as undifferentiated cells that have the ability to replicate and differentiate themselves into various tissues cells. Stem cells, commonly encountered in clinical or preclinical stages, are largely classified into embryonic, adult, and induced pluripotent stem cells. Recently, stem cell transplantation has been frequently applied to the treatment of pain as an alternative or promising approach for the treatment of severe osteoarthritis, neuropathic pain, and intractable musculoskeletal pain which do not respond to conventional medicine. The main idea of applying stem cells to neuropathic pain is based on the ability of stem cells to release neurotrophic factors, along with providing a cellular source for replacing the injured neural cells, making them ideal candidates for modulating and possibly reversing intractable neuropathic pain. Even though various differentiation capacities of stem cells are reported, there is not enough knowledge and technique to control the differentiation into desired tissues in vivo. Even though the use of stem cells is still in the very early stages of clinical use and raises complicated ethical problems, the future of stem cells therapies is very bright with the help of accumulating evidence and technology.

줄기세포의 개요

허용준,김동욱 대한의사협회 2011 대한의사협회지 Vol.54 No.5

We are now in the middle of stem cell war. Each country is trying to invest a large amount of funds into stem cell research. This is due to a potentiality of stem cells. Stem cells are capable of proliferating in an undifferentiated manner and are able to differentiate into a desired cell lineage under certain conditions. These abilities make stem cells an appealing source for cell replacement therapies (regenerative medicine), the study of developmental biology and drug/toxin screening. In addition to embryonic and adult stem cells, induced pluripotent stem (iPS) cells has been recently generated through reprogramming from adult tissue cells such as fibroblasts. This technique has opened up new avenues to generate patient- and disease-specific pluripotent stem cells. Human iPS cells may be useful for gaining valuable insight into the pathophysiology of disease, as well as for discovering for new prognostic biomarkers and drug screening. Moreover,the iPS cell technology may play a major role in immune-matched clinical application in the future. In this chapter, we introduce general characteristics of various stem cells, clinical application of stem cells and future perspectives.

Stem cell therapy in pain medicine

Yong Hee Han,Kyung Hoon Kim,Salahadin Abdi,Tae Kyun Kim 대한통증학회 2019 The Korean Journal of Pain Vol.32 No.4

Stem cells are attracting attention as a key element in future medicine, satisfying the desire to live a healthier life with the possibility that they can regenerate tissue damaged or degenerated by disease or aging. Stem cells are defined as undifferentiated cells that have the ability to replicate and differentiate themselves into various tissues cells. Stem cells, commonly encountered in clinical or preclinical stages, are largely classified into embryonic, adult, and induced pluripotent stem cells. Recently, stem cell transplantation has been frequently applied to the treatment of pain as an alternative or promising approach for the treatment of severe osteoarthritis, neuropathic pain, and intractable musculoskeletal pain which do not respond to conventional medicine. The main idea of applying stem cells to neuropathic pain is based on the ability of stem cells to release neurotrophic factors, along with providing a cellular source for replacing the injured neural cells, making them ideal candidates for modulating and possibly reversing intractable neuropathic pain. Even though various differentiation capacities of stem cells are reported, there is not enough knowledge and technique to control the differentiation into desired tissues in vivo. Even though the use of stem cells is still in the very early stages of clinical use and raises complicated ethical problems, the future of stem cells therapies is very bright with the help of accumulating evidence and technology.

Comparative Analysis for In Vitro Differentiation Potential of Induced Pluripotent Stem Cells, Embryonic Stem Cells, and Multipotent Spermatogonial Stem Cells into Germ-lineage Cells

Go Young-Eun,Kim Hyung-Joon,Jo Jung-Hyun,Lee Hyun-Ju,Do Jeong-Tae,Ko Jung-Jae,Lee Dong-Ryul 한국발생생물학회 2011 발생과 생식 Vol.15 No.1

In the present study, embryoid bodies (EBs) obtained from induced pluripotent stem cells (iPSCs) were induced to differentiate into germ lineage cells by treatment with bone morphogenetic protein 4 (BMP4) and retinoic acid (RA). The results were compared to the results for embryonic stem cells (ESCs) and multipotent spermatogonial stem cells (mSSCs) and quantified using immunocytochemical analysis of germ cell-specific markers (integrin-, GFR-, CD90/Thy1), fluorescence activating cell sorting (FACS), and real time-RT-PCR. We show that the highest levels of germ cell marker-expressing cells were obtained from groups treated with 10 ng/ BMP4 or 0.01 RA. In the BMP4-treated group, GFR- and CD90/Thy-1 were highly expressed in the EBs of iPSCs and ESCs compared to EBs of mSSCs. The expression of Nanog was much lower in iPSCs compared to ESCs and mSSCs. In the RA treated group, the level of GFR- and CD90/Thy-1 expression in the EBs of mSSCs Induced pluripotent stem cells, Mouse embryonic stem cells, Multipotent spermatogonial stem cells, Germ cell lineage, Differentiation potential. was much higher than the levels found in the EBs of iPSCs and similar to the levels found in the EBs of ESCs. FACS analysis using integrin-, GFR-, CD90/Thy1 and immunocytochemistry using GFR- antibody showed similar gene expression results. Therefore our results show that iPSC has the potential to differentiate into germ cells and suggest that a protocol optimizing germ cell induction from iPSC should be developed because of their potential usefulness in clinical applications requiring patient-specific cells.

Comparative pluripotent characteristics of porcine induced pluripotent stem cells generated using different viral transduction systems

Sang-Ki Baek,In-Won Lee,Yeon-Ji Lee,Bo-Gyeong Seo,Jung-Woo Choi,Tae-Suk Kim,Cheol Hwangbo,Joon-Hee Lee 한국동물생명공학회(구 한국수정란이식학회) 2023 한국동물생명공학회지 Vol.38 No.4

Background: Porcine pluripotent stem cells (pPSCs) would provide enormous potential for agriculture and biomedicine. However, authentic pPSCs have not established yet because standards for pPSCs-specific markers and culture conditions are not clear. Therefore, the present study reports comparative pluripotency characteristics in porcine induced pluripotent stem cells (piPSCs) derived from different viral transduction and reprogramming factors [Lenti-iPSCs (OSKM), Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM)]. Methods: Porcine fibroblasts were induced into Lenti-iPSCs (OSKM) and Lenti-iPSCs (OSKMNL) by using Lentiviral vector and Sev-iPSCs (OSKM) by using Sendaiviral vector. Expressions of endogenous or exogenous pluripotency-associated genes, surface marker and in vitro differentiation in between Lenti-piPSCs (OSKM), Lenti-iPSCs (OSKMNL) and Sev-piPSCs (OSKM) were compared. Results: Colonial morphology of Lenti-iPSCs (OSKMNL) closely resembles the na?ve mouse embryonic stem cells colony for culture, whereas Sev-iPSCs (OSKM) colony is similar to the primed hESCs. Also, the activity of AP shows a distinct different in piPSCs (AP-positive (+) Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM), but AP-negative (-) LentiiPSCs (OSKM)). mRNAs expression of several marker genes (OCT-3/4, NANOG and SOX2) for pluripotency was increased in Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM), but Sev-iPSCs (OSKM). Interestingly, SSEA-1 of surface markers was expressed only in Sev-iPSCs (OSKM), whereas SSEA-4, Tra-1-60 and Tra-1-81 were positively expressed in Lenti-iPSCs (OSKMNL). Exogenous reprogramming factors continuously expressed in Lenti-iPSCs (OSKMNL) for passage 20, whereas Sev-iPSCs (OSKM) did not express any exogenous transcription factors. Finally, only Lenti-iPSCs (OSKMNL) express the three germ layers and primordial germ cells markers in aggregated EBs. Conclusions: These results indicate that the viral transduction system of reprograming factors into porcine differentiated cells display different pluripotency characteristics in piPSCs.

Pig Pluripotent Stem Cells as a Candidate for Biomedical Application

Kwang-Hwan Choi,Chang-Kyu Lee 한국동물생명공학회(구 한국동물번식학회) 2019 Journal of Animal Reproduction and Biotechnology Vol.34 No.3

Stem cells are progenitor cells that are capable of self-renewal and differentiation into various cells. Especially, pluripotent stem cells (PSCs) have in vivo and in vitro differentiation capacity into three germ layers and can proliferate infinitely. The differentiation ability of PSCs can be applied for regenerative medicine and tissue engineering. In domestic animals, their PSCs have a potential for preclinical therapy as well as the production of transgenic animals and agricultural usage such as cultured meat. Among several domestic animals, a pig is considered as an ideal model for biomedical and agricultural purposes mentioned above. In this reason, studies for pig PSCs including embryonic stem cells (ESCs), embryonic germ cells (EGCs) and induced pluripotent stem cells (iPSCs) have been conducted for decades. Therefore, this review will discuss the history of PSCs derived from various origins and recent progress in pig PSC research field.

Neural stem cells differentiated from iPS cells spontaneously regain pluripotency.

Choi, Hyun Woo,Kim, Jong Soo,Choi, Sol,Hong, Yean Ju,Kim, Min Jung,Seo, Han Geuk,Do, Jeong Tae AlphaMed Press 2014 Stem Cells Vol.32 No.10

<P>Differentiated somatic cells can be reprogrammed into pluripotent stem cells by transduction of exogenous reprogramming factors. After induced pluripotent stem (iPS) cells are established, exogenous genes are silenced. In the pluripotent state, retroviral genes integrated in the host genome are kept inactive through epigenetic transcriptional regulation. In this study, we tried to determine whether exogenous genes remain silenced or are reactivated upon loss of pluripotency or on differentiation using an in vitro system. We induced differentiation of iPS cells into neural stem cells (NSCs) in vitro; the NSCs appeared morphologically indistinguishable from brain-derived NSCs and stained positive for the NSC markers Nestin and Sox2. These iPS cell-derived NSCs (iPS-NSCs) were also capable of differentiating into all three neural subtypes. Interestingly, iPS-NSCs spontaneously formed aggregates on long-term culture and showed reactivation of the Oct4-GFP marker, which was followed by the formation of embryonic stem cell-like colonies. The spontaneously reverted green fluorescent protein (GFP)-positive (iPS-NSC-GFP(+) ) cells expressed high levels of pluripotency markers (Oct4 and Nanog) and formed germline chimeras, indicating that iPS-NSC-GFP(+) cells had the same pluripotency as the original iPS cells. The reactivation of silenced exogenous genes was tightly correlated with the downregulation of DNA methyltransferases (Dnmts) during differentiation of iPS cells. This phenomenon was not observed in doxycycline-inducible iPS cells, where the reactivation of exogenous genes could be induced only by doxycycline treatment. These results indicate that pluripotency can be regained through reactivation of exogenous genes, which is associated with dynamic change of Dnmt levels during differentiation of iPS cells.</P>

Deubiquitylating enzymes as cancer stem cell therapeutics

Haq, Saba,Suresh, Bharathi,Ramakrishna, Suresh Elsevier 2018 Biochimica et biophysica acta, Reviews on cancer Vol.1869 No.1

<P><B>Abstract</B></P> <P>The focus of basic and applied research on core stem cell transcription factors has paved the way to initial delineation of their characteristics, their regulatory mechanisms, and the applicability of their regulatory proteins for protein-induced pluripotent stem cells (protein-IPSC) generation and in further clinical settings. Striking parallels have been observed between cancer stem cells (CSCs) and stem cells. For the maintenance of stem cells and CSC pluripotency and differentiation, post translational modifications (i.e., ubiquitylation and deubiquitylation) are tightly regulated, as these modifications result in a variety of stem cell fates. The identification of deubiquitylating enzymes (DUBs) involved in the regulation of core stem cell transcription factors and CSC-related proteins might contribute to providing novel insights into the implications of DUB regulatory mechanisms for governing cellular reprogramming and carcinogenesis. Moreover, we propose the novel possibility of applying DUBs coupled with core transcription factors to improve protein-iPSC generation efficiency. Additionally, this review article further illustrates the potential of applying DUB inhibitors as a novel therapeutic intervention for targeting CSCs. Thus, defining DUBs as core pharmacological targets implies that future endeavors to develop their inhibitors may revolutionize our ability to regulate stem cell maintenance and differentiation, somatic cell reprogramming, and cancer stem cells.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Addressing the applications of deubiquitylating enzymes (DUBs) in applied stem cell research and cancer stem cells therapy </LI> <LI> Efficient protein-induced pluripotent stem cells generation by DUBs-mediated extension of half-life of Yamanaka factors </LI> <LI> Discussing the importance of cancer stem cells associated DUBs and developing inhibitors for CSCs-based therapy </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>