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reprogramming efficiency following somatic cell nuclear transfer is influenced by the differentiation and methylation state of the donor nucleus

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STEM CELLS 2006年第9期24卷 2007-2013页

作者： Blelloch, Robert Wang, Zhongde Meissner, Alex Pollard, Steven Smith, Austin Jaenisch, Rudolf MIT Whitehead Inst Biomed Res Cambridge MA USA MIT Dept Biol Cambridge MA USA Brigham & Womens Hosp Dept Pathol Boston MA 02115 USA Univ Edinburgh Ctr Dev Stem Cell Biol Inst Stem Cell Res Sch Biol Sci Edinburgh EH8 9YL Midlothian Scotland

reprogramming of a differentiated cell nucleus by somatic cell nuclear transplantation is an inefficient process. Following nuclear transfer, the donor nucleus often fails to express early embryonic genes and establish a normal embryonic pattern of chromatin modifications. These defects correlate with the low number of cloned embryos able to produce embryonic stem cells or develop into adult animals. Here, we show that the differentiation and methylation state of the donor cell influence the efficiency of genomic reprogramming. First, neural stem cells, when used as donors for nuclear transplantation, produce embryonic stem cells at a higher efficiency than blastocysts derived from terminally differentiated neuronal donor cells, demonstrating a correlation between the state of differentiation and cloning efficiency. Second, using a hypomorphic allele of DNA methyltransferase-1, we found that global hypomethylation of a differentiated cell genome improved cloning efficiency. Our results provide functional evidence that the differentiation and epigenetic state of the donor nucleus influences reprogramming efficiency.

关键词： embryonic stem cells DNA methylation nuclear transfer reprogramming

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reprogramming events of mammalian somatic cells induced by Xenopus laevis egg extracts

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MOLECULAR REPRODUCTION AND DEVELOPMENT 2007年第10期74卷 1268-1277页

作者： Miyamoto, Kei Furusawa, Tadashi Ohnuki, Mari Goel, Sandeep Tokunaga, Tomoyuki Minami, Naojiro Yamada, Masayasu Ohsumi, Keita Imai, Hiroshi Kyoto Univ Grad Sch Agr Reprod Biol Lab Kyoto 6068502 Japan Natl Inst Agrobiol Sci Dev & Differntiat Lab Tsukuba Ibaraki Japan Tokyo Inst Technol Lab Cell & Dev Biol Yokohama Kanagawa 227 Japan

It is known that differentiated cells can be reprogrammed to an undifferentiated state in oocyte cytoplasm after nuclear transfer. Recently, some reports suggested that Xenopus egg extracts have the ability to reprogram mammalian somatic cells. reprogramming events of mammalian cells after Xenopus egg extract treatment and after cell culture of extract-treated cells have not been elucidated. In this experiment, we examined reprogramming events in reversibly permeabilized or nonpermeabilized porcine fibroblast cells after Xenopus egg extract treatment. The Xenopus egg-specific histone 134 was assembled on porcine chromatin and nuclear lamin LIII was incorporated into nuclei. Deacetylation of histone H3 at lysine 9 in extract-treated cells was detected in nonpermeabilized cells, suggesting that a part of reprogramming may be induced even in nonpermeabilized cells. Following culture of extract-treated cells, the cells began to express the pluripotent marker genes such as POU5F1 (OCT4) and SOX2 and to form colonies. Reactivation of the OCT4 gene in extract-treated cells was also confirmed in bovine fibroblasts transformed with an OCT4-EGFP construct. These results suggest that nuclei of mammalian cells can be partially reprogrammed to an embryonic state by Xenopus egg extracts and the remodeled cells partly dedifferentiate after cell culture. A system using egg extracts may be useful for understanding the mechanisms and processes of dedifferentiation and reprogramming of mammalian somatic cells after nuclear transfer.

关键词： Xenopus egg extract histone B4 deacetylation pluripotent gene reprogramming

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reprogramming of Fibroblasts From Older Women With Pelvic Floor Disorders Alters Cellular Behavior Associated With Donor Age

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STEM CELLS TRANSLATIONAL MEDICINE 2013年第2期2卷 118-128页

作者： Wen, Yan Wani, Prachi Zhou, Lu Baer, Tom Phadnis, Smruti Madan Pera, Renee A. Reijo Chen, Bertha Stanford Univ Dept Obstet Gynecol Sch Med Stanford CA 94305 USA Stanford Univ Inst Stem Cell Biol & Regenerat Med Sch Med Stanford CA 94305 USA Stanford Univ Stanford Photon Res Ctr Stanford CA 94305 USA Maternal & Childrens Hosp Shenzhen Shenzhen Peoples R China

We aimed to derive induced pluripotent stem cell (iPSC) lines from vaginal fibroblasts from older women with pelvic organ prolapse. We examined the effect of donor age on iPSCs and on the cells redifferentiated from these iPSCs. Vaginal fibroblasts were isolated from younger and older subjects for reprogramming. iPSCs were generated simultaneously using an excisable polycistronic lentiviral vector expressing Oct4, Klf4, Sox2, and cMyc. The pluripotent markers of iPSCs were confirmed by immunocytochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Spectral karyotyping was performed. The ability of the iPSCs to differentiate into three germ layers was confirmed by embryoid body and teratoma formation. Senescence marker (p21, p53, and Bax) expressions were determined by qRT-PCR and Western blot. The iPSCs were redifferentiated to fibroblasts and were evaluated with senescence-associated beta-galactosidase (SA) activity and mitotic index using time-lapse dark-field microscopy. iPSCs derived from both the younger and older subjects expressed pluripotency markers and showed normal karyotype and positive teratoma assays. There was no significant difference in expression of senescence and apoptosis markers (p21, p53, and Bax) in iPSCs derived from the younger subject compared with the older subject. Furthermore, fibroblasts redifferentiated from these iPSCs did not differ in SA activity or mitotic index. We report successful derivation of iPSCs from women with pelvic organ prolapse. Older age did not interfere with successful reprogramming. Donor age differences were not observed in these iPSCs using standard senescence markers, and donor age did not appear to affect cell mitotic activity in fibroblasts redifferentiated from iPSCs. STEM CELLS TRANSLATIONAL MEDICINE 2013;2:118-128

关键词： p53 Induced pluripotent stem cells reprogramming

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reprogramming bladder cancer cells for studying cancer initiation and progression

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TUMOR BIOLOGY 2016年第10期37卷 13237-13245页

作者： Iskender, Banu Izgi, Kenan Canatan, Halit Erciyes Univ Dept Med Biol Fac Med TR-38039 Kayseri Turkey Erciyes Univ Betul Ziya Eren Genome & Stem Cell Ctr TR-38039 Kayseri Turkey Erciyes Univ Dept Med Biochem Fac Med TR-38039 Kayseri Turkey

The induced pluripotent stem cell (iPSC) technology is the forced expression of specific transcription factors in somatic cells resulting in transformation into self-renewing, pluripotent cells which possess the ability to differentiate into any type of cells in the human body. While malignant cells could also be reprogrammed into iPSC-like cells with lower efficiency due to the genetic and epigenetic barriers in cancer cells, only a limited number of cancer cell types could be successfully reprogrammed until today. In the present study, we aimed at reprogramming two bladder cancer cell lines HTB-9 and T24 using a non-integrating Sendai virus (SeV) system. We have generated six sub-clones using distinct combinations of four factors-OCT4, SOX2, KLF4 and c-MYC-in two bladder cancer cell lines. Only a single sub-clone, T24 transduced with 4Fs, gave rise to iPSC-like cells. Bladder cancer cell-derived T24 4F cells represent unique features of pluripotent cells such as epithelial-like morphology, colony-forming ability, expression of pluripotency-associated markers and bearing the ability to differentiate in vitro. This is the first study focusing on the reprogramming susceptibility of two different bladder cancer cell lines to nuclear reprogramming. Further molecular characterisation of T24 4F cells could provide a better insight for biomarker research in bladder carcinogenesis and could offer a valuable tool for the development of novel therapeutic approaches in bladder carcinoma.

关键词： reprogramming Pluripotency Induced pluripotent stem cells Bladder cancer T24 HTB-9

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reprogramming activity of NANOGP8, a NANOG family member widely expressed in cancer

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ONCOGENE 2014年第19期33卷 2513-2519页

作者： Palla, A. R. Piazzolla, D. Abad, M. Li, H. Dominguez, O. Schonthaler, H. B. Wagner, E. F. Serrano, M. Spanish Natl Canc Res Ctr CNIO Tumour Suppress Grp Mol Oncol Programme E-28029 Madrid Spain Spanish Natl Canc Res Ctr CNIO Genom Core Unit Biotechnol Programme E-28029 Madrid Spain Spanish Natl Canc Res Ctr CNIO F BBVA CNIO Canc Cell Biol Programme Genes Dev & Dis Grp Madrid Spain

NANOG is a key transcription factor for pluripotency in embryonic stem cells. The analysis of NANOG in human cells is confounded by the presence of multiple and highly similar paralogs. In particular, there are three paralogs encoding full-length proteins, namely, NANOG1, NANOG2 and NANOGP8, and at least eight additional paralogs that do not encode full-length NANOG proteins. Here, we have examined NANOG family expression in human embryonic stem cells (hESCs) and in human cancer cell lines using a multi-NANOG PCR that amplifies the three functional paralogs and most of the non-functional ones. As anticipated, we found that hESCs express large amounts of NANOG1 and, interestingly, they also express NANOG2. In contrast, most human cancer cells tested express NANOGP8 and the non-coding paralogs NANOGP4 and NANOGP5. Notably, in some cancer cell lines, the NANOG protein levels produced by NANOGP8 are comparable to those produced by NANOG1 in pluripotent cells. Finally, we show that NANOGP8 is as active as NANOG1 in the reprogramming of human and murine fibroblasts into induced pluripotent stem cells. These results show that cancer-associated NANOGP8 can contribute to promote de-differentiation and/or cellular plasticity.

关键词： NANOG NANOGP8 cancer reprogramming pluripotency

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reprogramming non-mammary and cancer cells in the developing mouse mammary gland

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SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY 2012年第5期23卷 591-598页

作者： Bruno, Robert D. Smith, Gilbert H. NCI Cell & Canc Biol Branch Ctr Canc Res Bethesda MD 20892 USA

The capacity of any portion of the murine mammary gland to produce a complete functional mammary outgrowth upon transplantation to an epithelium-divested fat pad is unaffected by the age or reproductive history of the donor. Likewise, through serial transplantations, no loss of potency is detected when compared to similar transplantations of the youngest mammary tissue tested. This demonstrates that stem cell activity is maintained intact throughout the lifetime of the animal despite aging and the repeated expansion and depletion of the mammary epithelium through multiple rounds of pregnancy, lactation and involution. These facts support the contention that mammary stem cells reside in protected tissue locales (niches), where their reproductive potency remains essentially unchanged through life. Disruption of the tissue, to produce dispersed cells results in the desecration of the protection afforded by the "niche" and leads to a reduced capacity of dispersed epithelial cells (in terms of the number transplanted) to recapitulate complete functional mammary structures. Our studies demonstrate that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary cells, including mouse and human cancer cells, may be sequestered and reprogrammed to perform mammary epithelial cell functions including those ascribed to mammary stem/progenitor cells. Published by Elsevier Ltd.

关键词： Transplantation Microenvironment Mammary Development reprogramming

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reprogramming of Urine-Derived Renal Epithelial Cells into iPSCs Using srRNA and Consecutive Differentiation into Beating Cardiomyocytes

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MOLECULAR THERAPY-NUCLEIC ACIDS 2019年 17卷 907-921页

作者： Steinle, Heidrun Weber, Marbod Behring, Andreas Mau-Holzmann, Ulrike von Ohle, Christiane Popov, Aron-Frederik Schlensak, Christian Wendel, Hans Peter Avci-Adali, Meltem Univ Hosp Tubingen Dept Thorac & Cardiovasc Surg Calwerstr 7-1 D-72076 Tubingen Germany Univ Hosp Tubingen Inst Med Genet & Appl Genom Calwerstr 7-1 D-72076 Tubingen Germany Univ Hosp Tubingen Ctr Dent Oral Med & Maxillofacial Surg Dept Conservat Dent & Periodontol Osianderstr 2-8 D-72076 Tubingen Germany

The generation of induced pluripotent stem cells (iPSCs) from patient's somatic cells and the subsequent differentiation into desired cell types opens up numerous possibilities in regenerative medicine and tissue engineering. Adult cardiomyocytes have limited self-renewal capacity;thus, the efficient, safe, and clinically applicable generation of autologous cardiomyocytes is of great interest for the treatment of damaged myocardium. In this study, footprint-free iPSCs were successfully generated from urine-derived renal epithelial cells through a single application of self-replicating RNA (srRNA). The expression of pluripotency markers and the in vitro as well as in vivo trilineage differentiation were demonstrated. Furthermore, the resulting iPSCs contained no residual srRNA, and the karyotyping analysis demonstrated no detectable anomalies. The cardiac differentiation of these iPSCs resulted in autologous contracting cardiomyocytes after 10 days. We anticipate that the use of urine as a non-invasive cell source to obtain patient cells and the use of srRNA for reprogramming into iPSCs will greatly improve the future production of clinically applicable cardiomyocytes and other cell types. This could allow the regeneration of tissues by generating sufficient quantities of autologous cells without the risk of immune rejection.

关键词： cardiomyocytes iPSCs regenerative medicine renal epithelial cells reprogramming self-replicating RNA urine

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reprogramming the tachycardia parameters with long-detection strategy in patients with pre-existing implantable cardioverter-defibrillator

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ACTA CARDIOLOGICA 2019年第3期74卷 246-251页

作者： Bozyel, Serdar Aktas, Mujdat Mutluer, Ferit Onur Guler, Turner Erdem Dervis, Emir Argan, Onur Celikyurt, Umut Agir, Aysen Vural, Ahmet Hlth Sci Univ Derince Training & Res Hosp Dept Cardiol TR-41900 Kocaeli Turkey Eregli State Hosp Dept Cardiol Zonguldak Turkey KocUniv Sch Med Dept Cardiol Istanbul Turkey Kocaeli Univ Sch Med Dept Cardiol Kocaeli Turkey Kocaeli State Hosp Dept Cardiol Kocaeli Turkey

Background: A long-detection interval (LDI) programming has been proved to reduce shock therapy in patients who underwent de novo implantable cardioverter defibrillator (ICD) implantation. We aimed to evaluate effectiveness and safety of this new strategy in old ICD recipients. Methods: We included 147 primary prevention patients with ischaemic and non-ischaemic aetiology. Conventional setting parameters (18 of 24 intervals to detect ventricular arrhythmias (VA's)) were reprogrammed with LDI strategy (30 of 40 intervals to detect VA's). One monitoring zone (between 360 and 330 ms) and two therapy zones were programmed, treating all rhythms of cycle length <330ms that met the duration criterion of 30/40 intervals and were discriminated as ventricular tachycardia/ventricular fibrillation (VT/VF). The supraventricular tachycardia (SVT) discriminators were used in all patients. Results: At a median follow-up of 24 months, 12.9% (n = 19) of patients received shock therapies (+/- antitachycardia pacing (ATP)). Appropriate and inappropriate shocks occurred in 7.5 and 5.4% of patients during follow-up, respectively. Only one patient experienced an arrhythmic syncope during the follow-up period. There was no death related to LDI programming. The LDI programming helped to stop unnecessary in 10 patients (6.8%), who otherwise would have been treated in the conventional programming. Conclusions: LDI programming was found safe and effective. Hence, old ICD recipients will benefit from this strategy.

关键词： Implantable cardioverter-defibrillator long detection strategy reprogramming shock reduction

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reprogramming Sacral Neuromodulation for Sub-Optimal Outcomes: Evidence and Recommendations for Clinical Practice

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NEUROMODULATION 2021年第7期24卷 1247-1257页

作者： Dudding, Thomas C. Lehur, Paul A. Sorensen, Michael Engelberg, Stefan Bertapelle, Maria Paola Chartier-Kastler, Emmanuel Everaert, Karel Van Kerrebroeck, Philip Knowles, Charles H. Lundby, Lilli Matzel, Klaus E. Munoz-Duyos, Arantxa Rydningen, Mona B. de Wachter, Stefan Univ Hosp Southampton NHS Fdn Trust Pelv Floor Unit Southampton SO16 6YD Hants England Osped Reg Lugano Coloproctol Unit Lugano Switzerland Hvidovre Univ Hosp Dept Surg & Med Gastroenterol Hvidovre Denmark Medtronic Intl Sarl Tolochenaz Switzerland Azienda Osped Citta Salute & Sci Neurourol Maria Adelaide Hosp Turin Italy Sorbonne Univ Dept Urol Acad Hosp Pitieal Salpetriere Paris France Univ Ghent Ghent Univ Hosp Dept Urol Ghent Belgium Maastricht Univ Med Ctr Pelv Care Ctr Maastricht Maastricht Netherlands Queen Mary Univ London & Barts Hlth NHS Trust Ctr Neurosci Surg & Trauma Blizard Inst London England Aarhus Univ Hosp Dept Surg Pelv Floor Unit Aarhus Denmark Univ Erlangen Nurnberg Sekt Koloproktol Chirurg Klin Erlangen Germany Univ Barcelona Hosp Univ Mutua Terrassa Coloproctol Unit Terrassa Spain Univ Hosp North Norway Norwegian Natl Advisory Unit Incontinence & Pelv Tromso Norway Univ Antwerp Univ Hosp Antwerpen Fac Hlth Sci Dept Urol Edegem Belgium

Objectives In some patients treated for urinary or fecal incontinence with sacral neuromodulation (SNM) persistence of symptoms, a reduction in efficacy or adverse effects of stimulation can occur. In such situations, further programming of the SNM device can help resolve problems. Infrequently hardware failure is detected. This article aims to provide practical guidance to solve sub-optimal outcomes (troubleshooting) occurring in the course of SNM therapy. Materials and Methods A systematic literature review was performed. Collective clinical experience from an expert multidisciplinary group was used to form opinion where evidence was lacking. Results Circumstances in which reprogramming is required are described. Actions to undertake include changes of electrode configuration, stimulation amplitude, pulse frequency, and pulse width. Guidance in case of loss of efficacy and adverse effects of stimulation, developed by a group of European experts, is presented. In addition, various hardware failure scenarios and their management are described. Conclusions reprogramming aims to further improve patient symptoms or ensure a comfortable delivery of the therapy. Initial changes of electrode configuration and adjustment of stimulation parameters can be performed at home to avoid unnecessary hospital visits. A logical and stepwise approach to reprogramming can improve the outcome of therapy and restore patient satisfaction.

关键词： Fecal incontinence reprogramming sacral neuromodulation troubleshooting urinary incontinence

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reprogramming of mouse retinal neurons and standardized quantification of their differentiation in 3D retinal cultures

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NATURE PROTOCOLS 2016年第10期11卷 1955-1976页

作者： Hiler, Daniel J. Barabas, Marie E. Griffiths, Lyra M. Dyer, Michael A. St Jude Childrens Res Hosp Dept Dev Neurobiol 332 N Lauderdale St Memphis TN 38105 USA Univ Tennessee Ctr Hlth Sci Dept Ophthalmol Memphis TN 38163 USA Howard Hughes Med Inst Chevy Chase MD 20815 USA

Postmitotic differentiated neurons are among the most difficult cells to reprogram into induced pluripotent stem cells (iPSCs)) because they have poor viability when cultured as dissociated cells. To overcome this, other protocols have required the inactivation of the p53 tumor suppressor to reprogram postmitotic neurons, which can result in tumorigenesis of the cells. We describe a method that does not require p53 inactivation but induces reprogramming in retinal cells from reprogrammable mice grown in aggregates with wild-type mouse retinal cells. After the first 10 d of reprogramming, the aggregates are then dispersed and plated on irradiated feeder cells to propagate and isolate individual iPSCs clones. The reprogramming efficiency of different neuronal populations at any stage of development can be quantified using this protocol. reprogramming retinal neurons using this protocol will take 56 d, and these retina-derived iPSCs can undergo retinal differentiation to produce retinae in 34 d. In addition, we describe a quantitative assessment of retinal differentiation from these neuron-derived iPSCs called STEM-RET. The procedure quantifies eye field specification, optic cup formation and retinal differentiation in 3D cultures using molecular, cellular and morphological criteria. An advanced level of cell culture experience is required to carry out this protocol.

关键词： Cell culture Differentiation reprogramming Retina

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