Deterministic direct reprogramming of somatic cells to pluripotency.

PubMed

Rais, Yoach; Zviran, Asaf; Geula, Shay; Gafni, Ohad; Chomsky, Elad; Viukov, Sergey; Mansour, Abed AlFatah; Caspi, Inbal; Krupalnik, Vladislav; Zerbib, Mirie; Maza, Itay; Mor, Nofar; Baran, Dror; Weinberger, Leehee; Jaitin, Diego A; Lara-Astiaso, David; Blecher-Gonen, Ronnie; Shipony, Zohar; Mukamel, Zohar; Hagai, Tzachi; Gilad, Shlomit; Amann-Zalcenstein, Daniela; Tanay, Amos; Amit, Ido; Novershtern, Noa; Hanna, Jacob H

2013-10-03

Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

Defined three-dimensional microenvironments boost induction of pluripotency

NASA Astrophysics Data System (ADS)

Caiazzo, Massimiliano; Okawa, Yuya; Ranga, Adrian; Piersigilli, Alessandra; Tabata, Yoji; Lutolf, Matthias P.

2016-03-01

Since the discovery of induced pluripotent stem cells (iPSCs), numerous approaches have been explored to improve the original protocol, which is based on a two-dimensional (2D) cell-culture system. Surprisingly, nothing is known about the effect of a more biologically faithful 3D environment on somatic-cell reprogramming. Here, we report a systematic analysis of how reprogramming of somatic cells occurs within engineered 3D extracellular matrices. By modulating microenvironmental stiffness, degradability and biochemical composition, we have identified a previously unknown role for biophysical effectors in the promotion of iPSC generation. We find that the physical cell confinement imposed by the 3D microenvironment boosts reprogramming through an accelerated mesenchymal-to-epithelial transition and increased epigenetic remodelling. We conclude that 3D microenvironmental signals act synergistically with reprogramming transcription factors to increase somatic plasticity.

Chemically Induced Reprogramming of Somatic Cells to Pluripotent Stem Cells and Neural Cells.

PubMed

Biswas, Dhruba; Jiang, Peng

2016-02-06

The ability to generate transplantable neural cells in a large quantity in the laboratory is a critical step in the field of developing stem cell regenerative medicine for neural repair. During the last few years, groundbreaking studies have shown that cell fate of adult somatic cells can be reprogrammed through lineage specific expression of transcription factors (TFs)-and defined culture conditions. This key concept has been used to identify a number of potent small molecules that could enhance the efficiency of reprogramming with TFs. Recently, a growing number of studies have shown that small molecules targeting specific epigenetic and signaling pathways can replace all of the reprogramming TFs. Here, we provide a detailed review of the studies reporting the generation of chemically induced pluripotent stem cells (ciPSCs), neural stem cells (ciNSCs), and neurons (ciN). We also discuss the main mechanisms of actions and the pathways that the small molecules regulate during chemical reprogramming.

Transcription Factors MYOCD, SRF, Mesp1 and SMARCD3 Enhance the Cardio-Inducing Effect of GATA4, TBX5, and MEF2C during Direct Cellular Reprogramming

PubMed Central

Christoforou, Nicolas; Chellappan, Malathi; Adler, Andrew F.; Kirkton, Robert D.; Wu, Tianyi; Addis, Russell C.; Bursac, Nenad; Leong, Kam W.

2013-01-01

Transient overexpression of defined combinations of master regulator genes can effectively induce cellular reprogramming: the acquisition of an alternative predicted phenotype from a differentiated cell lineage. This can be of particular importance in cardiac regenerative medicine wherein the heart lacks the capacity to heal itself, but simultaneously contains a large pool of fibroblasts. In this study we determined the cardio-inducing capacity of ten transcription factors to actuate cellular reprogramming of mouse embryonic fibroblasts into cardiomyocyte-like cells. Overexpression of transcription factors MYOCD and SRF alone or in conjunction with Mesp1 and SMARCD3 enhanced the basal but necessary cardio-inducing effect of the previously reported GATA4, TBX5, and MEF2C. In particular, combinations of five or seven transcription factors enhanced the activation of cardiac reporter vectors, and induced an upregulation of cardiac-specific genes. Global gene expression analysis also demonstrated a significantly greater cardio-inducing effect when the transcription factors MYOCD and SRF were used. Detection of cross-striated cells was highly dependent on the cell culture conditions and was enhanced by the addition of valproic acid and JAK inhibitor. Although we detected Ca2+ transient oscillations in the reprogrammed cells, we did not detect significant changes in resting membrane potential or spontaneously contracting cells. This study further elucidates the cardio-inducing effect of the transcriptional networks involved in cardiac cellular reprogramming, contributing to the ongoing rational design of a robust protocol required for cardiac regenerative therapies. PMID:23704920

Reprogramming of Sheep Fibroblasts into Pluripotency under a Drug-Inducible Expression of Mouse-Derived Defined Factors

PubMed Central

Li, Yang; Cang, Ming; Lee, Andrew Stephen; Zhang, Kehua; Liu, Dongjun

2011-01-01

Animal embryonic stem cells (ESCs) provide powerful tool for studies of early embryonic development, gene targeting, cloning, and regenerative medicine. However, the majority of attempts to establish ESC lines from large animals, especially ungulate mammals have failed. Recently, another type of pluripotent stem cells, known as induced pluripotent stem cells (iPSCs), have been successfully generated from mouse, human, monkey, rat and pig. In this study we show sheep fibroblasts can be reprogrammed to pluripotency by defined factors using a drug-inducible system. Sheep iPSCs derived in this fashion have a normal karyotype, exhibit morphological features similar to those of human ESCs and express AP, Oct4, Sox2, Nanog and the cell surface marker SSEA-4. Pluripotency of these cells was further confirmed by embryoid body (EB) and teratoma formation assays which generated derivatives of all three germ layers. Our results also show that the substitution of knockout serum replacement (KSR) with fetal bovine serum in culture improves the reprogramming efficiency of sheep iPSCs. Generation of sheep iPSCs places sheep on the front lines of large animal preclinical trials and experiments involving modification of animal genomes. PMID:21253598

Single cell qPCR reveals that additional HAND2 and microRNA-1 facilitate the early reprogramming progress of seven-factor-induced human myocytes

PubMed Central

Bektik, Emre; Dennis, Adrienne; Prasanna, Prateek; Madabhushi, Anant

2017-01-01

The direct reprogramming of cardiac fibroblasts into induced cardiomyocyte (CM)-like cells (iCMs) holds great promise in restoring heart function. We previously found that human fibroblasts could be reprogrammed toward CM-like cells by 7 reprogramming factors; however, iCM reprogramming in human fibroblasts is both more difficult and more time-intensive than that in mouse cells. In this study, we investigated if additional reprogramming factors could quantitatively and/or qualitatively improve 7-factor-mediated human iCM reprogramming by single-cell quantitative PCR. We first validated 46 pairs of TaqMan® primers/probes that had sufficient efficiency and sensitivity to detect the significant difference of gene expression between individual H9 human embryonic stem cell (ESC)-differentiated CMs (H9CMs) and human fibroblasts. The expression profile of these 46 genes revealed an improved reprogramming in 12-week iCMs compared to 4-week iCMs reprogrammed by 7 factors, indicating a prolonged stochastic phase during human iCM reprogramming. Although none of additional one reprogramming factor yielded a greater number of iCMs, our single-cell qPCR revealed that additional HAND2 or microRNA-1 could facilitate the silencing of fibroblast genes and yield a better degree of reprogramming in more reprogrammed iCMs. Noticeably, the more HAND2 expressed, the higher-level were cardiac genes activated in 7Fs+HAND2-reprogrammed iCMs. In conclusion, HAND2 and microRNA-1 could help 7 factors to facilitate the early progress of iCM-reprogramming from human fibroblasts. Our study provides valuable information to further optimize a method of direct iCM-reprogramming in human cells. PMID:28796841

Single cell qPCR reveals that additional HAND2 and microRNA-1 facilitate the early reprogramming progress of seven-factor-induced human myocytes.

PubMed

Bektik, Emre; Dennis, Adrienne; Prasanna, Prateek; Madabhushi, Anant; Fu, Ji-Dong

2017-01-01

The direct reprogramming of cardiac fibroblasts into induced cardiomyocyte (CM)-like cells (iCMs) holds great promise in restoring heart function. We previously found that human fibroblasts could be reprogrammed toward CM-like cells by 7 reprogramming factors; however, iCM reprogramming in human fibroblasts is both more difficult and more time-intensive than that in mouse cells. In this study, we investigated if additional reprogramming factors could quantitatively and/or qualitatively improve 7-factor-mediated human iCM reprogramming by single-cell quantitative PCR. We first validated 46 pairs of TaqMan® primers/probes that had sufficient efficiency and sensitivity to detect the significant difference of gene expression between individual H9 human embryonic stem cell (ESC)-differentiated CMs (H9CMs) and human fibroblasts. The expression profile of these 46 genes revealed an improved reprogramming in 12-week iCMs compared to 4-week iCMs reprogrammed by 7 factors, indicating a prolonged stochastic phase during human iCM reprogramming. Although none of additional one reprogramming factor yielded a greater number of iCMs, our single-cell qPCR revealed that additional HAND2 or microRNA-1 could facilitate the silencing of fibroblast genes and yield a better degree of reprogramming in more reprogrammed iCMs. Noticeably, the more HAND2 expressed, the higher-level were cardiac genes activated in 7Fs+HAND2-reprogrammed iCMs. In conclusion, HAND2 and microRNA-1 could help 7 factors to facilitate the early progress of iCM-reprogramming from human fibroblasts. Our study provides valuable information to further optimize a method of direct iCM-reprogramming in human cells.

Role of human oocyte-enriched factors in somatic cell reprograming.

PubMed

El-Gammal, Zaynab; AlOkda, Abdelrahman; El-Badri, Nagwa

2018-06-08

Cellular reprograming paves the way for creating functional patient-specific tissues to eliminate immune rejection responses by applying the same genetic profile. However, the epigenetic memory of a cell remains a challenge facing the current reprograming methods and does not allow transcription factors to bind properly. Because somatic cells can be reprogramed by transferring their nuclear contents into oocytes, introducing specific oocyte factors into differentiated cells is considered a promising approach for mimicking the reprograming process that occurs during fertilization. Mammalian metaphase II oocyte possesses a superior capacity to epigenetically reprogram somatic cell nuclei towards an embryonic stem cell-like state than the current factor-based reprograming approaches. This may be due to the presence of specific factors that are lacking in the current factor-based reprograming approaches. In this review, we focus on studies identifying human oocyte-enriched factors aiming to understand the molecular mechanisms mediating cellular reprograming. We describe the role of oocyte-enriched factors in metabolic switch, chromatin remodelling, and global epigenetic transformation. This is critical for improving the quality of resulting reprogramed cells, which is crucial for therapeutic applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Induced Pluripotency and Epigenetic Reprogramming

PubMed Central

Hochedlinger, Konrad; Jaenisch, Rudolf

2015-01-01

SUMMARY Induced pluripotency defines the process by which somatic cells are converted into induced pluripotent stem cells (iPSCs) upon overexpression of a small set of transcription factors. In this article, we put transcription factor–induced pluripotency into a historical context, review current methods to generate iPSCs, and discuss mechanistic insights that have been gained into the process of reprogramming. In addition, we focus on potential therapeutic applications of induced pluripotency and emerging technologies to efficiently engineer the genomes of human pluripotent cells for scientific and therapeutic purposes. PMID:26626939

Aging, Rejuvenation, and Epigenetic Reprogramming: Resetting the Aging Clock

PubMed Central

Rando, Thomas A.; Chang, Howard Y.

2012-01-01

The underlying cause of aging remains one of the central mysteries of biology. Recent studies in several different systems suggest that not only may the rate of aging be modified by environmental and genetic factors, but also that the aging clock can be reversed, restoring characteristics of youthfulness to aged cells and tissues. This Review focuses on the emerging biology of rejuvenation through the lens of epigenetic reprogramming. By defining youthfulness and senescence as epigenetic states, a framework for asking new questions about the aging process emerges. PMID:22265401

Reprogramming cells with synthetic proteins

PubMed Central

Yang, Xiaoxiao; Malik, Vikas; Jauch, Ralf

2015-01-01

Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to “read” genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies. PMID:25652623

Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kinoshita, Taisuke; Nagamatsu, Go, E-mail: gonag@sc.itc.keio.ac.jp; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012

2011-04-08

Highlights: {yields} iPS cells were induced with a fluorescence monitoring system. {yields} ATM-deficient tail-tip fibroblasts exhibited quite a low reprogramming efficiency. {yields} iPS cells obtained from ATM-deficient cells had pluripotent cell characteristics. {yields} ATM-deficient iPS cells had abnormal chromosomes, which were accumulated in culture. -- Abstract: During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellularmore » response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.« less

Discovery and progress of direct cardiac reprogramming.

PubMed

Kojima, Hidenori; Ieda, Masaki

2017-06-01

Cardiac disease remains a major cause of death worldwide. Direct cardiac reprogramming has emerged as a promising approach for cardiac regenerative therapy. After the discovery of MyoD, a master regulator for skeletal muscle, other single cardiac reprogramming factors (master regulators) have been sought. Discovery of cardiac reprogramming factors was inspired by the finding that multiple, but not single, transcription factors were needed to generate induced pluripotent stem cells (iPSCs) from fibroblasts. We first reported a combination of cardiac-specific transcription factors, Gata4, Mef2c, and Tbx5 (GMT), that could convert mouse fibroblasts into cardiomyocyte-like cells, which were designated as induced cardiomyocyte-like cells (iCMs). Following our first report of cardiac reprogramming, many researchers, including ourselves, demonstrated an improvement in cardiac reprogramming efficiency, in vivo direct cardiac reprogramming for heart regeneration, and cardiac reprogramming in human cells. However, cardiac reprogramming in human cells and adult fibroblasts remains inefficient, and further efforts are needed. We believe that future research elucidating epigenetic barriers and molecular mechanisms of direct cardiac reprogramming will improve the reprogramming efficiency, and that this new technology has great potential for clinical applications.

Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.

PubMed

Saha, Krishanu; Mei, Ying; Reisterer, Colin M; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C; Alexander, Morgan R; Langer, Robert; Anderson, Daniel G; Jaenisch, Rudolf

2011-11-15

The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming.

PubMed

Schmitt, Christopher E; Morales, Blanca M; Schmitz, Ellen M H; Hawkins, John S; Lizama, Carlos O; Zape, Joan P; Hsiao, Edward C; Zovein, Ann C

2017-06-05

Non-integrating episomal vectors have become an important tool for induced pluripotent stem cell reprogramming. The episomal vectors carrying the "Yamanaka reprogramming factors" (Oct4, Klf, Sox2, and L-Myc + Lin28) are critical tools for non-integrating reprogramming of cells to a pluripotent state. However, the reprogramming process remains highly stochastic, and is hampered by an inability to easily identify clones that carry the episomal vectors. We modified the original set of vectors to express spectrally separable fluorescent proteins to allow for enrichment of transfected cells. The vectors were then tested against the standard original vectors for reprogramming efficiency and for the ability to enrich for stoichiometric ratios of factors. The reengineered vectors allow for cell sorting based on reprogramming factor expression. We show that these vectors can assist in tracking episomal expression in individual cells and can select the reprogramming factor dosage. Together, these modified vectors are a useful tool for understanding the reprogramming process and improving induced pluripotent stem cell isolation efficiency.

Second generation codon optimized minicircle (CoMiC) for nonviral reprogramming of human adult fibroblasts.

PubMed

Diecke, Sebastian; Lisowski, Leszek; Kooreman, Nigel G; Wu, Joseph C

2014-01-01

The ability to induce pluripotency in somatic cells is one of the most important scientific achievements in the fields of stem cell research and regenerative medicine. This technique allows researchers to obtain pluripotent stem cells without the controversial use of embryos, providing a novel and powerful tool for disease modeling and drug screening approaches. However, using viruses for the delivery of reprogramming genes and transcription factors may result in integration into the host genome and cause random mutations within the target cell, thus limiting the use of these cells for downstream applications. To overcome this limitation, various non-integrating techniques, including Sendai virus, mRNA, minicircle, and plasmid-based methods, have recently been developed. Utilizing a newly developed codon optimized 4-in-1 minicircle (CoMiC), we were able to reprogram human adult fibroblasts using chemically defined media and without the need for feeder cells.

Reprogramming cancer cells: a novel approach for cancer therapy or a tool for disease-modeling?

PubMed

Yilmazer, Açelya; de Lázaro, Irene; Taheri, Hadiseh

2015-12-01

Chromatin dynamics have been the major focus of many physiological and pathological processes over the past 20 years. Epigenetic mechanisms have been shown to be reshaped during both cellular reprogramming and tumorigenesis. For this reason, cancer cell reprogramming can provide a powerful tool to better understand both regenerative and cancer-fate processes, with a potential to develop novel therapeutic approaches. Recent studies showed that cancer cells can be reprogrammed to a pluripotent state by the overexpression of reprogramming transcription factors. Activation of transcription factors and modification of chromatin regulators may result in the remodeling of epigenetic status and refueling of tumorigenicity in these reprogrammed cancer cells. However, studies focusing on cancer cell reprogramming are contradictory; some studies reported increased tumor progression whereas others showed that cellular reprogramming has a treatment potential for cancer. In this review, first, the current knowledge on the epigenetic mechanisms involved during cancer development and cellular reprogramming will be presented. Later, different reports and key factors about pluripotency-based reprogramming of cancer cells will be reviewed in detail. New insights will be provided on cancer biology and therapy in the light of cellular reprogramming. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

PubMed Central

Hiramatsu, Kunihiko; Sasagawa, Satoru; Outani, Hidetatsu; Nakagawa, Kanako; Yoshikawa, Hideki; Tsumaki, Noriyuki

2011-01-01

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells. PMID:21293062

Elixir of Life: Thwarting Aging With Regenerative Reprogramming.

PubMed

Beyret, Ergin; Martinez Redondo, Paloma; Platero Luengo, Aida; Izpisua Belmonte, Juan Carlos

2018-01-05

All living beings undergo systemic physiological decline after ontogeny, characterized as aging. Modern medicine has increased the life expectancy, yet this has created an aged society that has more predisposition to degenerative disorders. Therefore, novel interventions that aim to extend the healthspan in parallel to the life span are needed. Regeneration ability of living beings maintains their biological integrity and thus is the major leverage against aging. However, mammalian regeneration capacity is low and further declines during aging. Therefore, modalities that reinforce regeneration can antagonize aging. Recent advances in the field of regenerative medicine have shown that aging is not an irreversible process. Conversion of somatic cells to embryonic-like pluripotent cells demonstrated that the differentiated state and age of a cell is not fixed. Identification of the pluripotency-inducing factors subsequently ignited the idea that cellular features can be reprogrammed by defined factors that specify the desired outcome. The last decade consequently has witnessed a plethora of studies that modify cellular features including the hallmarks of aging in addition to cellular function and identity in a variety of cell types in vitro. Recently, some of these reprogramming strategies have been directly used in animal models in pursuit of rejuvenation and cell replacement. Here, we review these in vivo reprogramming efforts and discuss their potential use to extend the longevity by complementing or augmenting the regenerative capacity. © 2017 American Heart Association, Inc.

Vectorology and Factor Delivery in Induced Pluripotent Stem Cell Reprogramming

PubMed Central

2014-01-01

Induced pluripotent stem cell (iPSC) reprogramming requires sustained expression of multiple reprogramming factors for a limited period of time (10–30 days). Conventional iPSC reprogramming was achieved using lentiviral or simple retroviral vectors. Retroviral reprogramming has flaws of insertional mutagenesis, uncontrolled silencing, residual expression and re-activation of transgenes, and immunogenicity. To overcome these issues, various technologies were explored, including adenoviral vectors, protein transduction, RNA transfection, minicircle DNA, excisable PiggyBac (PB) transposon, Cre-lox excision system, negative-sense RNA replicon, positive-sense RNA replicon, Epstein-Barr virus-based episomal plasmids, and repeated transfections of plasmids. This review provides summaries of the main vectorologies and factor delivery systems used in current reprogramming protocols. PMID:24625220

Retinoic Acid Inducible Gene 1 Protein (RIG1)-like Receptor Pathway is Required for Efficient Nuclear Reprogramming

PubMed Central

Sayed, Nazish; Ospino, Frank; Himmati, Farhan; Lee, Jieun; Chanda, Palas; Mocarski, Edward S.; Cooke, John P.

2017-01-01

We have revealed a critical role for innate immune signaling in nuclear reprogramming to pluripotency, and in the nuclear reprogramming required for somatic cell transdifferentiation. Activation of innate immune signaling causes global changes in the expression and activity of epigenetic modifiers to promote epigenetic plasticity. In our previous papers, we focused on the role of toll-like receptor 3 (TLR3) in this signaling pathway. Here we define the role of another innate immunity pathway known to participate in the response to viral RNA, the retinoic acid-inducible gene 1 receptor (RIG-1)-like receptor (RLR) pathway. This pathway is represented by the sensors of viral RNA, RIG-1, LGP2 and MDA5. We first found that TLR3 deficiency only causes a partial inhibition of nuclear reprogramming to pluripotency in mouse tail-tip fibroblasts, which motivated us to determine the contribution of RLR. We found that knockdown of iPS-1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming induced by retroviral or by mmRNA expression of Oct 4, Sox2, KLF4 and cMYC (OSKM). Importantly a double knockdown of both RLR and TLR3 pathway led to a further decrease in iPSC colonies suggesting an additive effect of both these pathways on nuclear reprogramming. Furthermore, in murine embryonic fibroblasts expressing a dox-inducible cassette of the genes encoding OSKM, an RLR agonist increased the yield of iPSCs. Similarly, the RLR agonist enhanced nuclear reprogramming by cell permeant peptides of the Yamanaka factors. Finally, in the dox-inducible system, RLR activation promotes activating histone marks in the promoter region of pluripotency genes. To conclude, innate immune signaling mediated by RLR plays a critical role in nuclear reprogramming. Manipulation of innate immune signaling may facilitate nuclear reprogramming to achieve pluripotency. PMID:28276156

Direct reprogramming and biomaterials for controlling cell fate.

PubMed

Kim, Eunsol; Tae, Giyoong

2016-01-01

Direct reprogramming which changes the fate of matured cell is a very useful technique with a great interest recently. This approach can eliminate the drawbacks of direct usage of stem cells and allow the patient specific treatment in regenerative medicine. Overexpression of diverse factors such as general reprogramming factors or lineage specific transcription factors can change the fate of already differentiated cells. On the other hand, biomaterials can provide physical and topographical cues or biochemical cues on cells, which can dictate or significantly affect the differentiation of stem cells. The role of biomaterials on direct reprogramming has not been elucidated much, but will be potentially significant to improve the efficiency or specificity of direct reprogramming. In this review, the strategies for general direct reprogramming and biomaterials-guided stem cell differentiation are summarized with the addition of the up-to-date progress on biomaterials for direct reprogramming.

Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.

PubMed

Slamecka, Jaroslav; Salimova, Lilia; McClellan, Steven; van Kelle, Mathieu; Kehl, Debora; Laurini, Javier; Cinelli, Paolo; Owen, Laurie; Hoerstrup, Simon P; Weber, Benedikt

2016-01-01

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

Epigenetic regulation leading to induced pluripotency drives cancer development in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohnishi, Kotaro; Department of Medicine, Gifu University Graduate School of Medicine, Gifu 501-1194; Semi, Katsunori

Highlights: • Epigenetic regulation of failed reprogramming-associated cancer cells is discussed. • Similarity between pediatric cancer and reprogramming-associated cancer is discussed. • Concept for epigenetic cancer is discussed. - Abstract: Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by the transient expression of reprogramming factors. During the reprogramming process, somatic cells acquire the ability to undergo unlimited proliferation, which is also an important characteristic of cancer cells, while their underlying DNA sequence remains unchanged. Based on the characteristics shared between pluripotent stem cells and cancer cells, the potential involvement of the factors leading to reprogramming toward pluripotencymore » in cancer development has been discussed. Recent in vivo reprogramming studies provided some clues to understanding the role of reprogramming-related epigenetic regulation in cancer development. It was shown that premature termination of the in vivo reprogramming result in the development of tumors that resemble pediatric cancers. Given that epigenetic modifications play a central role during reprogramming, failed reprogramming-associated cancer development may have provided a proof of concept for epigenetics-driven cancer development in vivo.« less

Induced Pluripotent Stem Cells: Generation, Characterization, and Differentiation--Methods and Protocols.

PubMed

Graversen, Veronica Kon; Chavala, Sai H

2016-01-01

Reprogramming fibroblasts into induced pluripotent stem cells (iPSC) remains a promising technique for cell replacement therapy. Diverse populations of somatic cells have been examined for their reprogramming potential. Recently, ocular ciliary body epithelial cells (CECs) have been reprogrammed with high reprogramming efficiency and single transcription factor reprogramming, making them an exciting candidate for cellular reprogramming strategies.

A deterministic method for estimating free energy genetic network landscapes with applications to cell commitment and reprogramming paths.

PubMed

Olariu, Victor; Manesso, Erica; Peterson, Carsten

2017-06-01

Depicting developmental processes as movements in free energy genetic landscapes is an illustrative tool. However, exploring such landscapes to obtain quantitative or even qualitative predictions is hampered by the lack of free energy functions corresponding to the biochemical Michaelis-Menten or Hill rate equations for the dynamics. Being armed with energy landscapes defined by a network and its interactions would open up the possibility of swiftly identifying cell states and computing optimal paths, including those of cell reprogramming, thereby avoiding exhaustive trial-and-error simulations with rate equations for different parameter sets. It turns out that sigmoidal rate equations do have approximate free energy associations. With this replacement of rate equations, we develop a deterministic method for estimating the free energy surfaces of systems of interacting genes at different noise levels or temperatures. Once such free energy landscape estimates have been established, we adapt a shortest path algorithm to determine optimal routes in the landscapes. We explore the method on three circuits for haematopoiesis and embryonic stem cell development for commitment and reprogramming scenarios and illustrate how the method can be used to determine sequential steps for onsets of external factors, essential for efficient reprogramming.

A deterministic method for estimating free energy genetic network landscapes with applications to cell commitment and reprogramming paths

PubMed Central

Olariu, Victor; Manesso, Erica

2017-01-01

Depicting developmental processes as movements in free energy genetic landscapes is an illustrative tool. However, exploring such landscapes to obtain quantitative or even qualitative predictions is hampered by the lack of free energy functions corresponding to the biochemical Michaelis–Menten or Hill rate equations for the dynamics. Being armed with energy landscapes defined by a network and its interactions would open up the possibility of swiftly identifying cell states and computing optimal paths, including those of cell reprogramming, thereby avoiding exhaustive trial-and-error simulations with rate equations for different parameter sets. It turns out that sigmoidal rate equations do have approximate free energy associations. With this replacement of rate equations, we develop a deterministic method for estimating the free energy surfaces of systems of interacting genes at different noise levels or temperatures. Once such free energy landscape estimates have been established, we adapt a shortest path algorithm to determine optimal routes in the landscapes. We explore the method on three circuits for haematopoiesis and embryonic stem cell development for commitment and reprogramming scenarios and illustrate how the method can be used to determine sequential steps for onsets of external factors, essential for efficient reprogramming. PMID:28680655

Boosters and barriers for direct cardiac reprogramming.

PubMed

Talkhabi, Mahmood; Zonooz, Elmira Rezaei; Baharvand, Hossein

2017-06-01

Heart disease is currently the most significant cause of morbidity and mortality worldwide, which accounts for approximately 33% of all deaths. Recently, a promising and alchemy-like strategy has been developed called direct cardiac reprogramming, which directly converts somatic cells such as fibroblasts to cardiac lineage cells such as cardiomyocytes (CMs), termed induced CMs or iCMs. The first in vitro cardiac reprogramming study, mediated by cardiac transcription factors (TFs)-Gata4, Tbx5 and Mef2C-, was not enough efficient to produce an adequate number of fully reprogrammed, functional iCMs. As a result, numerous combinations of cardiac TFs exist for direct cardiac reprogramming of mouse and human fibroblasts. However, the efficiency of direct cardiac reprogramming remains low. Recently, a number of cellular and molecular mechanisms have been identified to increase the efficiency of direct cardiac reprogramming and the quality of iCMs. For example, microgrooved substrate, cardiogenic growth factors [VEGF, FGF, BMP4 and Activin A], and an appropriate stoichiometry of TFs boost the direct cardiac reprogramming. On the other hand, serum, TGFβ signaling, activators of epithelial to mesenchymal transition, and some epigenetic factors (Bmi1 and Ezh2) are barriers for direct cardiac reprogramming. Manipulating these mechanisms by the application of boosters and removing barriers can increase the efficiency of direct cardiac reprogramming and possibly make iCMs reliable for cell-based therapy or other potential applications. In this review, we summarize the latest trends in cardiac TF- or miRNA-based direct cardiac reprogramming and comprehensively discuses all molecular and cellular boosters and barriers affecting direct cardiac reprogramming. Copyright © 2017 Elsevier Inc. All rights reserved.

Small molecule-induced cellular fate reprogramming: promising road leading to Rome.

PubMed

Li, Xiang; Xu, Jun; Deng, Hongkui

2018-05-29

Cellular fate reprogramming holds great promise to generate functional cell types for replenishing new cells and restoring functional loss. Inspired by transcription factor-induced reprogramming, the field of cellular reprogramming has greatly advanced and developed into divergent streams of reprogramming approaches. Remarkably, increasing studies have shown the power and advantages of small molecule-based approaches for cellular fate reprogramming, which could overcome the limitations of conventional transgenic-based reprogramming. In this concise review, we discuss these findings and highlight the future potentiality with particular focus on this new trend of chemical reprogramming. Copyright © 2018 Elsevier Ltd. All rights reserved.

Induced pluripotent stem (iPS) cells from human fetal stem cells.

PubMed

Guillot, Pascale V

2016-02-01

Pluripotency defines the ability of stem cells to differentiate into all the lineages of the three germ layers and self-renew indefinitely. Somatic cells can regain the developmental potential of embryonic stem cells following ectopic expression of a set of transcription factors or, in certain circumstances, via modulation of culture conditions and supplementation with small molecule, that is, induced pluripotent stem (iPS) cells. Here, we discuss the use of fetal tissues for reprogramming, focusing in particular on stem cells derived from human amniotic fluid, and the development of chemical reprogramming. We next address the advantages and disadvantages of deriving pluripotent cells from fetal tissues and the potential clinical applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chemical Enhancement of In Vitro and In Vivo Direct Cardiac Reprogramming.

PubMed

Mohamed, Tamer M A; Stone, Nicole R; Berry, Emily C; Radzinsky, Ethan; Huang, Yu; Pratt, Karishma; Ang, Yen-Sin; Yu, Pengzhi; Wang, Haixia; Tang, Shibing; Magnitsky, Sergey; Ding, Sheng; Ivey, Kathryn N; Srivastava, Deepak

2017-03-07

Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells in situ represents a promising strategy for cardiac regeneration. A combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), can convert fibroblasts into induced cardiomyocyte-like cells, albeit with low efficiency in vitro. We screened 5500 compounds in primary cardiac fibroblasts to identify the pathways that can be modulated to enhance cardiomyocyte reprogramming. We found that a combination of the transforming growth factor-β inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency 8-fold when added to GMT-overexpressing cardiac fibroblasts. The small molecules also enhanced the speed and quality of cell conversion; we observed beating cells as early as 1 week after reprogramming compared with 6 to 8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared with those exposed to only GMT. Human cardiac reprogramming was similarly enhanced on transforming growth factor-β and WNT inhibition and was achieved most efficiently with GMT plus myocardin. Transforming growth factor-β and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration. © 2016 American Heart Association, Inc.

Rational Development of A Polycistronic Plasmid with A CpG-Free Bacterial Backbone as A Potential Tool for Direct Reprogramming.

PubMed

Dormiani, Kianoush; Mir Mohammad Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Forouzanfar, Mahboobeh; Baharvand, Hossein; Ghaedi, Kamran; Nasr-Esfahani, Mohammad Hossein

2017-01-01

Induced pluripotent stem cells are generated from somatic cells by direct reprogramming. These reprogrammed pluripotent cells have different applications in biomedical fields such as regenerative medicine. Although viral vectors are widely used for efficient reprogramming, they have limited applications in the clinic due to the risk for immunogenicity and insertional mutagenesis. Accordingly, we designed and developed a small, non-integrating plasmid named pLENSO/Zeo as a 2A-mediated polycistronic expression vector. In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human LIN28, NANOG, SOX2 and OCT4 along with an EGFP reporter gene. Each reprogramming factor is separated by an intervening sequence that encodes a 2A self-processing peptide. The reprogramming cassette is located downstream of a CMV promoter. The vector is easily propagated in the E. coli GT115 strain through a CpG-depleted vector backbone. We evaluated the stability of the constructed vector bioinformatically, and its ability to stoichiometric expression of the reprogramming factors using quantitative molecular methods analysis after transient transfection into HEK293 cells. In the present study, we developed a nonviral episomal vector named pLENSO/ Zeo. Our results demonstrated the general structural stability of the plasmid DNA. This relatively small vector showed concomitant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical parameters for efficient and consistent reprogramming. According to our experimental results, this stable extrachromosomal plasmid expresses reliable amounts of four reprogramming factors simultaneously. Consequently, these promising results encouraged us to evaluate the capability of pLENSO/Zeo as a simple and feasible tool for generation of induced pluripotent stem cells from primary cells in the future.

Heart repair by reprogramming non-myocytes with cardiac transcription factors

PubMed Central

Song, Kunhua; Nam, Young-Jae; Luo, Xiang; Qi, Xiaoxia; Tan, Wei; Huang, Guo N.; Acharya, Asha; Smith, Christopher L.; Tallquist, Michelle D.; Neilson, Eric G.; Hill, Joseph A.; Bassel-Duby, Rhonda; Olson, Eric N.

2012-01-01

The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodeling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, Hand2, MEF2C and Tbx5 can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodeling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules. PMID:22660318

Biological computational approaches: new hopes to improve (re)programming robustness, regenerative medicine and cancer therapeutics.

PubMed

Ebrahimi, Behnam

2016-01-01

Hundreds of transcription factors (TFs) are expressed and work in each cell type, but the identity of the cells is defined and maintained through the activity of a small number of core TFs. Existing reprogramming strategies predominantly focus on the ectopic expression of core TFs of an intended fate in a given cell type regardless of the state of native/somatic gene regulatory networks (GRNs) of the starting cells. Interestingly, an important point is that how much products of the reprogramming, transdifferentiation and differentiation (programming) are identical to their in vivo counterparts. There is evidence that shows that direct fate conversions of somatic cells are not complete, with target cell identity not fully achieved. Manipulation of core TFs provides a powerful tool for engineering cell fate in terms of extinguishment of native GRNs, the establishment of a new GRN, and preventing installation of aberrant GRNs. Conventionally, core TFs are selected to convert one cell type into another mostly based on literature and the experimental identification of genes that are differentially expressed in one cell type compared to the specific cell types. Currently, there is not a universal standard strategy for identifying candidate core TFs. Remarkably, several biological computational platforms are developed, which are capable of evaluating the fidelity of reprogramming methods and refining existing protocols. The current review discusses some deficiencies of reprogramming technologies in the production of a pure population of authentic target cells. Furthermore, it reviews the role of computational approaches (e.g. CellNet, KeyGenes, Mogrify, etc.) in improving (re)programming methods and consequently in regenerative medicine and cancer therapeutics. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Induction of human cardiomyocyte-like cells from fibroblasts by defined factors.

PubMed

Wada, Rie; Muraoka, Naoto; Inagawa, Kohei; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Kaneda, Ruri; Suzuki, Tomoyuki; Kamiya, Kaichiro; Tohyama, Shugo; Yuasa, Shinsuke; Kokaji, Kiyokazu; Aeba, Ryo; Yozu, Ryohei; Yamagishi, Hiroyuki; Kitamura, Toshio; Fukuda, Keiichi; Ieda, Masaki

2013-07-30

Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca(2+) oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2'-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.

Laterally confined growth of cells induces nuclear reprogramming in the absence of exogenous biochemical factors.

PubMed

Roy, Bibhas; Venkatachalapathy, Saradha; Ratna, Prasuna; Wang, Yejun; Jokhun, Doorgesh Sharma; Nagarajan, Mallika; Shivashankar, G V

2018-05-22

Cells in tissues undergo transdifferentiation programs when stimulated by specific mechanical and biochemical signals. While seminal studies have demonstrated that exogenous biochemical factors can reprogram somatic cells into pluripotent stem cells, the critical roles played by mechanical signals in such reprogramming process have not been well documented. In this paper, we show that laterally confined growth of fibroblasts on micropatterned substrates induces nuclear reprogramming with high efficiency in the absence of any exogenous reprogramming factors. We provide compelling evidence on the induction of stem cell-like properties using alkaline phosphatase assays and expression of pluripotent markers. Early onset of reprogramming was accompanied with enhanced nuclear dynamics and changes in chromosome intermingling degrees, potentially facilitating rewiring of the genome. Time-lapse analysis of promoter occupancy by immunoprecipitation of H3K9Ac chromatin fragments revealed that epithelial, proliferative, and reprogramming gene promoters were progressively acetylated, while mesenchymal promoters were deacetylated by 10 days. Consistently, RNA sequencing analysis showed a systematic progression from mesenchymal to stem cell transcriptome, highlighting pathways involving mechanisms underlying nuclear reprogramming. We then demonstrated that these mechanically reprogrammed cells could be maintained as stem cells and can be redifferentiated into multiple lineages with high efficiency. Importantly, we also demonstrate the induction of cancer stemness properties in MCF7 cells grown in such laterally confined conditions. Collectively, our results highlight an important generic property of somatic cells that, when grown in laterally confined conditions, acquire stemness. Such mechanical reprogramming of somatic cells demonstrated here has important implications in tissue regeneration and disease models. Copyright © 2018 the Author(s). Published by PNAS.

Artificial acceleration of mammalian cell reprogramming by bacterial proteins.

PubMed

Ikeda, Takashi; Uchiyama, Ikuo; Iwasaki, Mio; Sasaki, Tetsuhiko; Nakagawa, Masato; Okita, Keisuke; Masui, Shinji

2017-10-01

The molecular mechanisms of cell reprogramming and differentiation involve various signaling factors. Small molecule compounds have been identified to artificially influence these factors through interacting cellular proteins. Although such small molecule compounds are useful to enhance reprogramming and differentiation and to show the mechanisms that underlie these events, the screening usually requires a large number of compounds to identify only a very small number of hits (e.g., one hit among several tens of thousands of compounds). Here, we show a proof of concept that xenospecific gene products can affect the efficiency of cell reprogramming to pluripotency. Thirty genes specific for the bacterium Wolbachia pipientis were forcibly expressed individually along with reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) that can generate induced pluripotent stem cells in mammalian cells, and eight were found to affect the reprogramming efficiency either positively or negatively (hit rate 26.7%). Mechanistic analysis suggested one of these proteins interacted with cytoskeleton to promote reprogramming. Our results raise the possibility that xenospecific gene products provide an alternative way to study the regulatory mechanism of cell identity. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors.

PubMed

Koblas, Tomas; Leontovyc, Ivan; Loukotova, Sarka; Kosinova, Lucie; Saudek, Frantisek

2016-05-17

Direct reprogramming of pancreatic nonendocrine cells into insulin-producing β-cells represents a promising approach for the treatment of insulin-dependent diabetes. However, its clinical application is limited by the potential for insertional mutagenesis associated with the viral vectors currently used for cell reprogramming. With the aim of developing a nonintegrative reprogramming strategy for derivation of insulin-producing cells, here, we evaluated a new approach utilizing synthetic messenger RNAs encoding reprogramming transcription factors. Administration of synthetic mRNAs encoding three key transcription regulators of β-cell differentiation-Pdx1, Neurogenin3, and MafA-efficiently reprogrammed the pancreatic exocrine cells into insulin-producing cells. In addition to the insulin genes expression, the synthetic mRNAs also induced the expressions of genes important for proper pancreatic β-cell function, including Sur1, Kir6.2, Pcsk1, and Pcsk2. Pretreating cells with the chromatin-modifying agent 5-Aza-2'-deoxycytidine further enhanced reprogramming efficiency, increasing the proportion of insulin-producing cells from 3.5 ± 0.9 to 14.3 ± 1.9% (n = 4). Moreover, 5-Aza-2'-deoxycytidine pretreatment enabled the reprogrammed cells to respond to glucose challenge with increased insulin secretion. In conclusion, our results support that the reprogramming of pancreatic exocrine cells into insulin-producing cells, induced by synthetic mRNAs encoding pancreatic transcription factors, represents a promising approach for cell-based diabetes therapy.

Growth Factor-Activated Stem Cell Circuits and Stromal Signals Cooperatively Accelerate Non-Integrated iPSC Reprogramming of Human Myeloid Progenitors

PubMed Central

Park, Tea Soon; Huo, Jeffrey S.; Peters, Ann; Talbot, C. Conover; Verma, Karan; Zimmerlin, Ludovic; Kaplan, Ian M.; Zambidis, Elias T.

2012-01-01

Nonviral conversion of skin or blood cells into clinically useful human induced pluripotent stem cells (hiPSC) occurs in only rare fractions (∼0.001%–0.5%) of donor cells transfected with non-integrating reprogramming factors. Pluripotency induction of developmentally immature stem-progenitors is generally more efficient than differentiated somatic cell targets. However, the nature of augmented progenitor reprogramming remains obscure, and its potential has not been fully explored for improving the extremely slow pace of non-integrated reprogramming. Here, we report highly optimized four-factor reprogramming of lineage-committed cord blood (CB) myeloid progenitors with bulk efficiencies of ∼50% in purified episome-expressing cells. Lineage-committed CD33+CD45+CD34− myeloid cells and not primitive hematopoietic stem-progenitors were the main targets of a rapid and nearly complete non-integrated reprogramming. The efficient conversion of mature myeloid populations into NANOG+TRA-1-81+ hiPSC was mediated by synergies between hematopoietic growth factor (GF), stromal activation signals, and episomal Yamanaka factor expression. Using a modular bioinformatics approach, we demonstrated that efficient myeloid reprogramming correlated not to increased proliferation or endogenous Core factor expressions, but to poised expression of GF-activated transcriptional circuits that commonly regulate plasticity in both hematopoietic progenitors and embryonic stem cells (ESC). Factor-driven conversion of myeloid progenitors to a high-fidelity pluripotent state was further accelerated by soluble and contact-dependent stromal signals that included an implied and unexpected role for Toll receptor-NFκB signaling. These data provide a paradigm for understanding the augmented reprogramming capacity of somatic progenitors, and reveal that efficient induced pluripotency in other cell types may also require extrinsic activation of a molecular framework that commonly regulates self-renewal and differentiation in both hematopoietic progenitors and ESC. PMID:22905176

Direct Reprogramming of Murine Fibroblasts to Hematopoietic Progenitor Cells

PubMed Central

Batta, Kiran; Florkowska, Magdalena; Kouskoff, Valerie; Lacaud, Georges

2014-01-01

Summary Recent reports have shown that somatic cells, under appropriate culture conditions, could be directly reprogrammed to cardiac, hepatic, or neuronal phenotype by lineage-specific transcription factors. In this study, we demonstrate that both embryonic and adult somatic fibroblasts can be efficiently reprogrammed to clonal multilineage hematopoietic progenitors by the ectopic expression of the transcription factors ERG, GATA2, LMO2, RUNX1c, and SCL. These reprogrammed cells were stably expanded on stromal cells and possessed short-term reconstitution ability in vivo. Loss of p53 function facilitated reprogramming to blood, and p53−/− reprogrammed cells efficiently generated erythroid, megakaryocytic, myeloid, and lymphoid lineages. Genome-wide analyses revealed that generation of hematopoietic progenitors was preceded by the appearance of hemogenic endothelial cells expressing endothelial and hematopoietic genes. Altogether, our findings suggest that direct reprogramming could represent a valid alternative approach to the differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) for disease modeling and autologous blood cell therapies. PMID:25466247

Advances in reprogramming somatic cells to induced pluripotent stem cells.

PubMed

Patel, Minal; Yang, Shuying

2010-09-01

Traditionally, nuclear reprogramming of cells has been performed by transferring somatic cell nuclei into oocytes, by combining somatic and pluripotent cells together through cell fusion and through genetic integration of factors through somatic cell chromatin. All of these techniques changes gene expression which further leads to a change in cell fate. Here we discuss recent advances in generating induced pluripotent stem cells, different reprogramming methods and clinical applications of iPS cells. Viral vectors have been used to transfer transcription factors (Oct4, Sox2, c-myc, Klf4, and nanog) to induce reprogramming of mouse fibroblasts, neural stem cells, neural progenitor cells, keratinocytes, B lymphocytes and meningeal membrane cells towards pluripotency. Human fibroblasts, neural cells, blood and keratinocytes have also been reprogrammed towards pluripotency. In this review we have discussed the use of viral vectors for reprogramming both animal and human stem cells. Currently, many studies are also involved in finding alternatives to using viral vectors carrying transcription factors for reprogramming cells. These include using plasmid transfection, piggyback transposon system and piggyback transposon system combined with a non viral vector system. Applications of these techniques have been discussed in detail including its advantages and disadvantages. Finally, current clinical applications of induced pluripotent stem cells and its limitations have also been reviewed. Thus, this review is a summary of current research advances in reprogramming cells into induced pluripotent stem cells.

Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta

2013-05-24

Highlights: •We investigate reprogramming of gene expression in multinucleate single cells. •Cells of two differentiation control mutants are fused. •Fused cells proceed to alternative gene expression patterns. •The population of nuclei damps stochastic fluctuations in gene expression. •Dynamic processes of cellular reprogramming can be observed by repeated sampling of a cell. -- Abstract: Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states.more » The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level.« less

Development of Defective and Persistent Sendai Virus Vector

PubMed Central

Nishimura, Ken; Sano, Masayuki; Ohtaka, Manami; Furuta, Birei; Umemura, Yoko; Nakajima, Yoshiro; Ikehara, Yuzuru; Kobayashi, Toshihiro; Segawa, Hiroaki; Takayasu, Satoko; Sato, Hideyuki; Motomura, Kaori; Uchida, Eriko; Kanayasu-Toyoda, Toshie; Asashima, Makoto; Nakauchi, Hiromitsu; Yamaguchi, Teruhide; Nakanishi, Mahito

2011-01-01

The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However, this is a slow and inefficient process, depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover, once cell reprogramming is accomplished, these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However, no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus, which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes, deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore, interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ∼1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus, this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research. PMID:21138846

Engineering kidney cells: reprogramming and directed differentiation to renal tissues.

PubMed

Kaminski, Michael M; Tosic, Jelena; Pichler, Roman; Arnold, Sebastian J; Lienkamp, Soeren S

2017-07-01

Growing knowledge of how cell identity is determined at the molecular level has enabled the generation of diverse tissue types, including renal cells from pluripotent or somatic cells. Recently, several in vitro protocols involving either directed differentiation or transcription-factor-based reprogramming to kidney cells have been established. Embryonic stem cells or induced pluripotent stem cells can be guided towards a kidney fate by exposing them to combinations of growth factors or small molecules. Here, renal development is recapitulated in vitro resulting in kidney cells or organoids that show striking similarities to mammalian embryonic nephrons. In addition, culture conditions are also defined that allow the expansion of renal progenitor cells in vitro. Another route towards the generation of kidney cells is direct reprogramming. Key transcription factors are used to directly impose renal cell identity on somatic cells, thus circumventing the pluripotent stage. This complementary approach to stem-cell-based differentiation has been demonstrated to generate renal tubule cells and nephron progenitors. In-vitro-generated renal cells offer new opportunities for modelling inherited and acquired renal diseases on a patient-specific genetic background. These cells represent a potential source for developing novel models for kidney diseases, drug screening and nephrotoxicity testing and might represent the first steps towards kidney cell replacement therapies. In this review, we summarize current approaches for the generation of renal cells in vitro and discuss the advantages of each approach and their potential applications.

Somatic cell reprogramming informed by the oocyte.

PubMed

Gonzalez-Munoz, Elena; Cibelli, Jose B

2018-05-08

The successful production of animals and embryonic stem cells (ESCs) using somatic cell nuclear transfer (SCNT) has demonstrated the unmatched nuclear reprogramming capacity of the oocyte and helped prove the degree of plasticity of differentiated cells. The introduction of transcription factors to generate induced pluripotent stem cells (iPSCs) displaced SCNT and, due to its ease of implementation, became the method of choice for cell reprogramming. Nonetheless, iPSC derivation remains inefficient and stochastic. This review article focuses on using the oocyte as a source of reprogramming factors, comparing the SCNT and iPSC mechanisms for remodeling chromatin and acquiring pluripotency.

c-MYC independent nuclear reprogramming favors cardiogenic potential of induced pluripotent stem cells

PubMed Central

Martinez-Fernandez, Almudena; Nelson, Timothy J.; Ikeda, Yasuhiro; Terzic, Andre

2010-01-01

Induced pluripotent stem cell (iPS) technology has launched a new platform in regenerative medicine aimed at deriving unlimited replacement tissue from autologous sources through somatic cell reprogramming using stemness factor sets. In this way, authentic cardiomyocytes have been obtained from iPS and recently demonstrated in proof-of-principle studies to repair infarcted heart. Optimizing the cardiogenic potential of iPS progeny would ensure a maximized yield of bioengineered cardiac tissue. Here, we reprogrammed fibroblasts in the presence or absence of c-MYC to determine if the acquired cardiogenicity is sensitive to the method of nuclear reprogramming. Using lentiviral constructs that expressed stemness factors SOX2, OCT4, and KLF4 with or without c-MYC, iPS clones generated through fibroblast reprogramming demonstrated indistinguishable characteristics for 5 days of differentiation with similar cell morphology, growth rates, and chimeric embryo integration. However, 4-factor c-MYC dependent nuclear reprogramming produced iPS progeny that consistently prolonged the expression of pluripotent Oct-4 and Fgf4 genes and repressed cardiac differentiation. In contrast, 3-factor c-MYC-less iPS clones efficiently up-regulated pre-cardiac (CXCR4, Flk-1, and Mesp1/2) and cardiac (Nkx2.5, Mef2c, and Myocardin) gene expression patterns. In fact, 3-factor iPS progeny demonstrated early and robust cardiogenesis during in vitro differentiation with consistent beating activity, sarcomere maturation, and rhythmical intracellular calcium dynamics. Thus, nuclear reprogramming independent of c-MYC enhances production of pluripotent stem cells with innate cardiogenic potential. PMID:20221419

Identifying Candidate Reprogramming Genes in Mouse Induced Pluripotent Stem Cells.

PubMed

Gao, Fang; Li, Jingyu; Zhang, Heng; Yang, Xu; An, Tiezhu

2017-08-01

Factor-based induced reprogramming approaches have tremendous potential for human regenerative medicine, but the efficiencies of these approaches are still low. In this study, we analyzed the global transcriptional profiles of mouse induced pluripotent stem cells (miPSCs) and mouse embryonic stem cells (mESCs) from seven different labs and present here the first successful clustering according to cell type, not by lab of origin. We identified 2131 different expression genes (DEs) as candidate pluripotency-associated genes by comparing mESCs/miPSCs with somatic cells and 720 DEs between miPSCs and mESCs. Interestingly, there was a significant overlap between the two DE sets. Therefore, we defined the overlap DEs as "consensus DEs" including 313 miPSC-specific genes expressed at a higher level in miPSCs versus mESCs and 184 mESC-specific genes in total and reasoned that these may contribute to the differences in pluripotency between mESCs and miPSCs. A classification of "consensus DEs" according to their different expression levels between somatic cells and mESCs/miPSCs shows that 86% of the miPSC-specific genes are more highly expressed in somatic cells, while 73% of mESC-specific genes are highly expressed in mESCs/miPSCs, indicating that the miPSCs have not efficiently silenced the expression pattern of the somatic cells from which they are derived and failed to completely induce the genes with high expression levels in mESCs. We further revealed a strong correlation between oocyte-enriched factors and insufficiently induced mESC-specific genes and identified 11 hub genes via network analysis. In light of these findings, we postulated that these key hub genes might not only drive somatic cell nuclear transfer (SCNT) reprogramming but also augment the efficiency and quality of miPSC reprogramming.

Reprogramming Human Retinal Pigmented Epithelial Cells to Neurons Using Recombinant Proteins

PubMed Central

Hu, Qirui; Chen, Renwei; Teesalu, Tambet; Ruoslahti, Erkki

2014-01-01

Somatic cells can be reprogrammed to an altered lineage by overexpressing specific transcription factors. To avoid introducing exogenous genetic material into the genome of host cells, cell-penetrating peptides can be used to deliver transcription factors into cells for reprogramming. Position-dependent C-end rule (CendR) cell- and tissue-penetrating peptides provide an alternative to the conventional cell-penetrating peptides, such as polyarginine. In this study, we used a prototypic, already active CendR peptide, RPARPAR, to deliver the transcription factor SOX2 to retinal pigmented epithelial (RPE) cells. We demonstrated that RPE cells can be directly reprogrammed to a neuronal fate by introduction of SOX2. Resulting neuronal cells expressed neuronal marker mRNAs and proteins and downregulated expression of RPE markers. Cells produced extensive neurites and developed synaptic machinery capable of dye uptake after depolarization with potassium. The RPARPAR-mediated delivery of SOX2 alone was sufficient to allow cell lineage reprogramming of both fetal and stem cell-derived RPE cells to become functional neurons. PMID:25298373

Non-stochastic reprogramming from a privileged somatic cell state

PubMed Central

Guo, Shangqin; Zi, Xiaoyuan; Schulz, Vincent P.; Cheng, Jijun; Zhong, Mei; Koochaki, Sebastian H.J.; Megyola, Cynthia M.; Pan, Xinghua; Heydari, Kartoosh; Weissman, Sherman M.; Gallagher, Patrick G.; Krause, Diane S.; Fan, Rong; Lu, Jun

2014-01-01

SUMMARY Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient, and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a non-stochastic manner. Subsets of murine hematopoietic progenitors are privileged, whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after 4–5 divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ~8 hours. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression, and is increased by p53-knockdown. This ultrafast-cycling population accounts for >99% of the bulk reprogramming activity in wildtype or p53-knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state, and suggest that cell cycle acceleration toward a critical threshold is an important bottleneck for reprogramming. PMID:24486105

Improvements in cloning efficiencies may be possible by increasing uniformity in recipient oocytes and donor cells.

PubMed

Miyoshi, Kazuchika; Rzucidlo, S Jacek; Pratt, Scott L; Stice, Steven L

2003-04-01

The low efficiency of somatic cell cloning is the major obstacle to widespread use of this technology. Incomplete nuclear reprogramming following the transfer of donor nuclei into recipient oocytes has been implicated as a primary reason for the low efficiency of the cloning procedure. The mechanisms and factors that affect the progression of the nuclear reprogramming process have not been completely elucidated, but the identification of these factors and their subsequent manipulation would increase cloning efficiency. At present, many groups are studying donor nucleus reprogramming. Here, we present an approach in which the efficiency of producing viable offspring is improved by selecting recipient oocytes and donor cells that will produce cloned embryos with functionally reprogrammed nuclei. This approach will produce information useful in future studies aimed at further deciphering the nuclear reprogramming process.

Transdifferentiation and reprogramming: Overview of the processes, their similarities and differences.

PubMed

Cieślar-Pobuda, Artur; Knoflach, Viktoria; Ringh, Mikael V; Stark, Joachim; Likus, Wirginia; Siemianowicz, Krzysztof; Ghavami, Saeid; Hudecki, Andrzej; Green, Jason L; Łos, Marek J

2017-07-01

Reprogramming, or generation of induced pluripotent stem (iPS) cells (functionally similar to embryonic stem cells or ES cells) by the use of transcription factors (typically: Oct3/4, Sox2, c-Myc, Klf4) called "Yamanaka factors" (OSKM), has revolutionized regenerative medicine. However, factors used to induce stemness are also overexpressed in cancer. Both, ES cells and iPS cells cause teratoma formation when injected to tissues. This raises a safety concern for therapies based on iPS derivates. Transdifferentiation (lineage reprogramming, or -conversion), is a process in which one mature, specialized cell type changes into another without entering a pluripotent state. This process involves an ectopic expression of transcription factors and/or other stimuli. Unlike in the case of reprogramming, tissues obtained by this method do not carry the risk of subsequent teratomagenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors

PubMed Central

Cheng, Hui; Ang, Heather Yin-Kuan; A. EL Farran, Chadi; Li, Pin; Fang, Hai Tong; Liu, Tong Ming; Kong, Say Li; Chin, Michael Lingzi; Ling, Wei Yin; Lim, Edwin Kok Hao; Li, Hu; Huber, Tara; Loh, Kyle M.; Loh, Yuin-Han; Lim, Bing

2016-01-01

Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into ‘induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase+ megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit+ hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors. PMID:27869129

Residual Expression of the Reprogramming Factors Prevents Differentiation of iPSC Generated from Human Fibroblasts and Cord Blood CD34+ Progenitors

PubMed Central

Ramos-Mejía, Verónica; Montes, Rosa; Bueno, Clara; Ayllón, Verónica; Real, Pedro J.; Rodríguez, René; Menendez, Pablo

2012-01-01

Human induced pluripotent stem cells (hiPSC) have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB)-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM). The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation. PMID:22545141

Genetic reprogramming of host cells by bacterial pathogens.

PubMed

Tran Van Nhieu, Guy; Arbibe, Laurence

2009-10-29

During the course of infection, pathogens often induce changes in gene expression in host cells and these changes can be long lasting and global or transient and of limited amplitude. Defining how, when, and why bacterial pathogens reprogram host cells represents an exciting challenge that opens up the opportunity to grasp the essence of pathogenesis and its molecular details.

SCL, LMO1 and Notch1 Reprogram Thymocytes into Self-Renewing Cells

PubMed Central

Rojas-Sutterlin, Shanti; Herblot, Sabine; Hébert, Josée; Sauvageau, Guy; Lemieux, Sébastien; Lécuyer, Eric; Veiga, Diogo F. T.; Hoang, Trang

2014-01-01

The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive NOTCH1 allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive NOTCH1 cannot reprogram thymocytes on its own, despite the fact that NOTCH1 is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating NOTCH1 triggers a parallel pathway involving Hes1 and Myc that dramatically enhances the activity of SCL-LMO1 We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, LYL1 and LMO1 or LMO2 are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network described here may be relevant to a majority of human T-ALL. PMID:25522233

Generation of Patient-Specific induced Pluripotent Stem Cell from Peripheral Blood Mononuclear Cells by Sendai Reprogramming Vectors.

PubMed

Quintana-Bustamante, Oscar; Segovia, Jose C

2016-01-01

Induced pluripotent stem cells (iPSC) technology has changed preclinical research since their generation was described by Shinya Yamanaka in 2006. iPSCs are derived from somatic cells after being reprogrammed back to an embryonic state by specific combination of reprogramming factors. These reprogrammed cells resemble all the characteristic of embryonic stem cells (ESC). The reprogramming technology is even more valuable to research diseases biology and treatment by opening gene and cell therapies in own patient's iPSC. Patient-specific iPSC can be generated from a large variety of patient cells by any of the myriad of reprogramming platforms described. Here, we describe the generation of patient-specific iPSC from patient peripheral blood mononuclear cells by Sendai Reprogramming vectors.

Deterministic versus stochastic model of reprogramming: new evidence from cellular barcoding technique

PubMed Central

Yunusova, Anastasia M.; Fishman, Veniamin S.; Vasiliev, Gennady V.

2017-01-01

Factor-mediated reprogramming of somatic cells towards pluripotency is a low-efficiency process during which only small subsets of cells are successfully reprogrammed. Previous analyses of the determinants of the reprogramming potential are based on average measurements across a large population of cells or on monitoring a relatively small number of single cells with live imaging. Here, we applied lentiviral genetic barcoding, a powerful tool enabling the identification of familiar relationships in thousands of cells. High-throughput sequencing of barcodes from successfully reprogrammed cells revealed a significant number of barcodes from related cells. We developed a computer model, according to which a probability of synchronous reprogramming of sister cells equals 10–30%. We conclude that the reprogramming success is pre-established in some particular cells and, being a heritable trait, can be maintained through cell division. Thus, reprogramming progresses in a deterministic manner, at least at the level of cell lineages. PMID:28446707

Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.

PubMed

Gallego-Perez, Daniel; Otero, Jose J; Czeisler, Catherine; Ma, Junyu; Ortiz, Cristina; Gygli, Patrick; Catacutan, Fay Patsy; Gokozan, Hamza Numan; Cowgill, Aaron; Sherwood, Thomas; Ghatak, Subhadip; Malkoc, Veysi; Zhao, Xi; Liao, Wei-Ching; Gnyawali, Surya; Wang, Xinmei; Adler, Andrew F; Leong, Kam; Wulff, Brian; Wilgus, Traci A; Askwith, Candice; Khanna, Savita; Rink, Cameron; Sen, Chandan K; Lee, L James

2016-02-01

Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage. In the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs. Copyright © 2016 Elsevier Inc. All rights reserved.

Efficient generation of induced pluripotent stem cells from human bone marrow mesenchymal stem cells.

PubMed

Yulin, X; Lizhen, L; Lifei, Z; Shan, F; Ru, L; Kaimin, H; Huang, H

2012-01-01

Ectopic expression of defined sets of genetic factors can reprogramme somatic cells to induced pluripotent stem cells (iPSCs) that closely resemble embryonic stem cells. However, the low reprogramming efficiency is a significant handicap for mechanistic studies and potential clinical application. In this study, we used human bone marrow-derived mesenchymal stem cells (hBMMSCs) as target cells for reprogramming and investigated efficient iPSC generation from hBMMSCs using the compounds of p53 siRNA, valproic acid (VPA) and vitamin C (Vc) with four transcription factors OCT4, SOX2, KLF4, and c-MYC (compound induction system). The synergetic mechanism of the compounds was studied. Our results showed that the compound induction system could efficiently reprogramme hBMMSCs to iPSCs. hBMMSC-derived iPSC populations expressed pluripotent markers and had multi-potential to differentiate into three germ layer-derived cells. p53 siRNA, VPA and Vc had a synergetic effect on cell reprogramming and the combinatorial use of these substances greatly improved the efficiency of iPSC generation by suppressing the expression of p53, decreasing cell apoptosis, up-regulating the expression of the pluripotent gene OCT4 and modifying the cell cycle. Therefore, our study highlights a straightforward method for improving the speed and efficiency of iPSC generation and provides versatile tools for investigating early developmental processes such as haemopoiesis and relevant diseases. In addition, this study provides a paradigm for the combinatorial use of genetic factors and molecules to improve the efficiency of iPSC generation.

Targeting Hypoxia-Inducible Factor-1α/Pyruvate Dehydrogenase Kinase 1 Axis by Dichloroacetate Suppresses Bleomycin-induced Pulmonary Fibrosis.

PubMed

Goodwin, Justin; Choi, Hyunsung; Hsieh, Meng-Hsiung; Neugent, Michael L; Ahn, Jung-Mo; Hayenga, Heather N; Singh, Pankaj K; Shackelford, David B; Lee, In-Kyu; Shulaev, Vladimir; Dhar, Shanta; Takeda, Norihiko; Kim, Jung-Whan

2018-02-01

Hypoxia has long been implicated in the pathogenesis of fibrotic diseases. Aberrantly activated myofibroblasts are the primary pathological driver of fibrotic progression, yet how various microenvironmental influences, such as hypoxia, contribute to their sustained activation and differentiation is poorly understood. As a defining feature of hypoxia is its impact on cellular metabolism, we sought to investigate how hypoxia-induced metabolic reprogramming affects myofibroblast differentiation and fibrotic progression, and to test the preclinical efficacy of targeting glycolytic metabolism for the treatment of pulmonary fibrosis. Bleomycin-induced pulmonary fibrotic progression was evaluated in two independent, fibroblast-specific, promoter-driven, hypoxia-inducible factor (Hif) 1A knockout mouse models and in glycolytic inhibitor, dichloroacetate-treated mice. Genetic and pharmacological approaches were used to explicate the role of metabolic reprogramming in myofibroblast differentiation. Hypoxia significantly enhanced transforming growth factor-β-induced myofibroblast differentiation through HIF-1α, whereas overexpression of the critical HIF-1α-mediated glycolytic switch, pyruvate dehydrogenase kinase 1 (PDK1) was sufficient to activate glycolysis and potentiate myofibroblast differentiation, even in the absence of HIF-1α. Inhibition of the HIF-1α/PDK1 axis by genomic deletion of Hif1A or pharmacological inhibition of PDK1 significantly attenuated bleomycin-induced pulmonary fibrosis. Our findings suggest that HIF-1α/PDK1-mediated glycolytic reprogramming is a critical metabolic alteration that acts to promote myofibroblast differentiation and fibrotic progression, and demonstrate that targeting glycolytic metabolism may prove to be a potential therapeutic strategy for the treatment of pulmonary fibrosis.

Chemical Enhancement of In Vitro and In Vivo Direct Cardiac Reprogramming

PubMed Central

Mohamed, Tamer M. A.; Stone, Nicole R.; Berry, Emily C.; Radzinsky, Ethan; Huang, Yu; Pratt, Karishma; Ang, Yen-Sin; Yu, Pengzhi; Wang, Haixia; Tang, Shibing; Magnitsky, Sergey; Ding, Sheng; Ivey, Kathryn N.; Srivastava, Deepak

2017-01-01

Background Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs) in situ represents a promising strategy for cardiac regeneration. A combination of three cardiac transcription factors, Gata4, Mef2c and Tbx5 (GMT), can convert fibroblasts into iCMs, albeit with low efficiency in vitro. Methods We screened 5,500 compounds in primary cardiac fibroblasts to identify the pathways that can be modulated to enhance cardiomyocyte reprogramming. Results We found that a combination of the transforming growth factor (TGF)-β inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency eight-fold when added to GMT-overexpressing cardiac fibroblasts. The small-molecules also enhanced the speed and the quality of cell conversion, as we observed beating cells as early as 1 week after reprogramming compared to 6–8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared to those exposed to only GMT. Human cardiac reprogramming was similarly enhanced upon TGF-β and WNT inhibition and was achieved most efficiently with GMT plus Myocardin. Conclusions Thus, TGF-β and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration. PMID:27834668

Cellular trajectories and molecular mechanisms of iPSC reprogramming.

PubMed

Apostolou, Effie; Stadtfeld, Matthias

2018-06-16

The discovery of induced pluripotent stem cells (iPSCs) has solidified the concept of transcription factors as major players in controlling cell identity and provided a tractable tool to study how somatic cell identity can be dismantled and pluripotency established. A number of landmark studies have established hallmarks and roadmaps of iPSC formation by describing relative kinetics of transcriptional, protein and epigenetic changes, including alterations in DNA methylation and histone modifications. Recently, technological advancements such as single-cell analyses, high-resolution genome-wide chromatin assays and more efficient reprogramming systems have been used to challenge and refine our understanding of the reprogramming process. Here, we will outline novel insights into the molecular mechanisms underlying iPSC formation, focusing on how the core reprogramming factors OCT4, KLF4, SOX2 and MYC (OKSM) drive changes in gene expression, chromatin state and 3D genome topology. In addition, we will discuss unexpected consequences of reprogramming factor expression in in vitro and in vivo systems that may point towards new applications of iPSC technology. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

LET-418/Mi2 and SPR-5/LSD1 cooperatively prevent somatic reprogramming of C. elegans germline stem cells.

PubMed

Käser-Pébernard, Stéphanie; Müller, Fritz; Wicky, Chantal

2014-04-08

Throughout their journey to forming new individuals, germline stem cells must remain totipotent, particularly by maintaining a specific chromatin structure. However, the place epigenetic factors occupy in this process remains elusive. So far, "sensitization" of chromatin by modulation of histone arrangement and/or content was believed to facilitate transcription-factor-induced germ cell reprogramming. Here, we demonstrate that the combined reduction of two epigenetic factors suffices to reprogram C. elegans germ cells. The histone H3K4 demethylase SPR-5/LSD1 and the chromatin remodeler LET-418/Mi2 function together in an early process to maintain germ cell status and act as a barrier to block precocious differentiation. This epigenetic barrier is capable of limiting COMPASS-mediated H3K4 methylation, because elevated H3K4me3 levels correlate with germ cell reprogramming in spr-5; let-418 mutants. Interestingly, germ cells deficient for spr-5 and let-418 mainly reprogram as neurons, suggesting that neuronal fate might be the first to be derepressed in early embryogenesis.

LET-418/Mi2 and SPR-5/LSD1 Cooperatively Prevent Somatic Reprogramming of C. elegans Germline Stem Cells

PubMed Central

Käser-Pébernard, Stéphanie; Müller, Fritz; Wicky, Chantal

2014-01-01

Summary Throughout their journey to forming new individuals, germline stem cells must remain totipotent, particularly by maintaining a specific chromatin structure. However, the place epigenetic factors occupy in this process remains elusive. So far, “sensitization” of chromatin by modulation of histone arrangement and/or content was believed to facilitate transcription-factor-induced germ cell reprogramming. Here, we demonstrate that the combined reduction of two epigenetic factors suffices to reprogram C. elegans germ cells. The histone H3K4 demethylase SPR-5/LSD1 and the chromatin remodeler LET-418/Mi2 function together in an early process to maintain germ cell status and act as a barrier to block precocious differentiation. This epigenetic barrier is capable of limiting COMPASS-mediated H3K4 methylation, because elevated H3K4me3 levels correlate with germ cell reprogramming in spr-5; let-418 mutants. Interestingly, germ cells deficient for spr-5 and let-418 mainly reprogram as neurons, suggesting that neuronal fate might be the first to be derepressed in early embryogenesis. PMID:24749077

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos

PubMed Central

Miyamoto, Kei; Nagai, Kouhei; Kitamura, Naoya; Nishikawa, Tomoaki; Ikegami, Haruka; Binh, Nguyen T.; Tsukamoto, Satoshi; Matsumoto, Mai; Tsukiyama, Tomoyuki; Minami, Naojiro; Yamada, Masayasu; Ariga, Hiroyoshi; Miyake, Masashi; Kawarasaki, Tatsuo; Matsumoto, Kazuya; Imai, Hiroshi

2011-01-01

Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti–DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer. PMID:21482765

Spin glass model for cell reprogramming

NASA Astrophysics Data System (ADS)

Pusuluri, Sai Teja; Castillo, Horacio E.

2014-03-01

Recent experiments show that differentiated cells can be reprogrammed to become pluripotent stem cells. The possible cell fates can be modeled as attractors in a dynamical system, the ``epigenetic landscape.'' Both cellular differentiation and reprogramming can be described in the landscape picture as motion from one attractor state to another attractor state. We use a simple model based on spin glass theory that can construct a simulated epigenetic landscape starting from the experimental genomic data. We modify the model to incorporate experimental reprogramming protocols. Our simulations successfully reproduce several reprogramming experiments. We probe the robustness of the results against random changes in the model, explore the importance of asymmetric interactions between transcription factors and study the importance of histone modification errors in reprogramming.

Fatty acid oxidation promotes reprogramming by enhancing oxidative phosphorylation and inhibiting protein kinase C.

PubMed

Lin, Zhaoyu; Liu, Fei; Shi, Peiliang; Song, Anying; Huang, Zan; Zou, Dayuan; Chen, Qin; Li, Jianxin; Gao, Xiang

2018-02-26

Changes in metabolic pathway preferences are key events in the reprogramming process of somatic cells to induced pluripotent stem cells (iPSCs). The optimization of metabolic conditions can enhance reprogramming; however, the detailed underlying mechanisms are largely unclear. By comparing the gene expression profiles of somatic cells, intermediate-phase cells, and iPSCs, we found that carnitine palmitoyltransferase (Cpt)1b, a rate-limiting enzyme in fatty acid oxidation, was significantly upregulated in the early stage of the reprogramming process. Mouse embryonic fibroblasts isolated from transgenic mice carrying doxycycline (Dox)-inducible Yamanaka factor constructs were used for reprogramming. Various fatty acid oxidation-related metabolites were added during the reprogramming process. Colony counting and fluorescence-activated cell sorting (FACS) were used to calculate reprogramming efficiency. Fatty acid oxidation-related metabolites were measured by liquid chromatography-mass spectrometry. Seahorse was used to measure the level of oxidative phosphorylation. We found that overexpression of cpt1b enhanced reprogramming efficiency. Furthermore, palmitoylcarnitine or acetyl-CoA, the primary and final products of Cpt1-mediated fatty acid oxidation, also promoted reprogramming. In the early reprogramming process, fatty acid oxidation upregulated oxidative phosphorylation and downregulated protein kinase C activity. Inhibition of protein kinase C also promoted reprogramming. We demonstrated that fatty acid oxidation promotes reprogramming by enhancing oxidative phosphorylation and inhibiting protein kinase C activity in the early stage of the reprogramming process. This study reveals that fatty acid oxidation is crucial for the reprogramming efficiency.

Esrrb Unlocks Silenced Enhancers for Reprogramming to Naive Pluripotency.

PubMed

Adachi, Kenjiro; Kopp, Wolfgang; Wu, Guangming; Heising, Sandra; Greber, Boris; Stehling, Martin; Araúzo-Bravo, Marcos J; Boerno, Stefan T; Timmermann, Bernd; Vingron, Martin; Schöler, Hans R

2018-06-11

Transcription factor (TF)-mediated reprogramming to pluripotency is a slow and inefficient process, because most pluripotency TFs fail to access relevant target sites in a refractory chromatin environment. It is still unclear how TFs actually orchestrate the opening of repressive chromatin during the long latency period of reprogramming. Here, we show that the orphan nuclear receptor Esrrb plays a pioneering role in recruiting the core pluripotency factors Oct4, Sox2, and Nanog to inactive enhancers in closed chromatin during the reprogramming of epiblast stem cells. Esrrb binds to silenced enhancers containing stable nucleosomes and hypermethylated DNA, which are inaccessible to the core factors. Esrrb binding is accompanied by local loss of DNA methylation, LIF-dependent engagement of p300, and nucleosome displacement, leading to the recruitment of core factors within approximately 2 days. These results suggest that TFs can drive rapid remodeling of the local chromatin structure, highlighting the remarkable plasticity of stable epigenetic information. Copyright © 2018 Elsevier Inc. All rights reserved.

Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA.

PubMed

Kim, Bo-Eun; Choi, Soon Won; Shin, Ji-Hee; Kim, Jae-Jun; Kang, Insung; Lee, Byung-Chul; Lee, Jin Young; Kook, Myoung Geun; Kang, Kyung-Sun

2018-01-01

Neural stem cells (NSCs) are a prominent cell source for understanding neural pathogenesis and for developing therapeutic applications to treat neurodegenerative disease because of their regenerative capacity and multipotency. Recently, a variety of cellular reprogramming technologies have been developed to facilitate in vitro generation of NSCs, called induced NSCs (iNSCs). However, the genetic safety aspects of established virus-based reprogramming methods have been considered, and non-integrating reprogramming methods have been developed. Reprogramming with in vitro transcribed (IVT) mRNA is one of the genetically safe reprogramming methods because exogenous mRNA temporally exists in the cell and is not integrated into the chromosome. Here, we successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via transfection with IVT mRNA encoding SOX2 (SOX2 mRNA) with properly optimized conditions. We confirmed that generated human UCB-MSC-derived iNSCs (UM-iNSCs) possess characteristics of NSCs, including multipotency and self-renewal capacity. Additionally, we transfected human dermal fibroblasts (HDFs) with SOX2 mRNA. Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited neural reprogramming with similar morphologies and NSC-enriched mRNA levels, but they showed limited proliferation ability. Our results demonstrated that human UCB-MSCs can be used for direct reprogramming into NSCs through transfection with IVT mRNA encoding a single factor, which provides an integration-free reprogramming tool for future therapeutic application.

LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming

PubMed Central

Huang, Delun; Wang, Ling; Duan, Jingyue; Huang, Chang; Tian, Xiuchun (Cindy); Zhang, Ming

2018-01-01

ABSTRACT The regulatory process of naïve-state induced pluripotent stem cell (iPSC) generation is not well understood. Leukemia inhibitory factor (LIF)-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb) serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs). However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs), and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation. PMID:29212799

Targeted release of transcription factors for cell reprogramming by a natural micro-syringe.

PubMed

Berthoin, Lionel; Toussaint, Bertrand; Garban, Frédéric; Le Gouellec, Audrey; Caulier, Benjamin; Polack, Benoît; Laurin, David

2016-11-20

Ectopic expression of defined transcription factors (TFs) for cell fate handling has proven high potential interest in reprogramming differentiated cells, in particular for regenerative medicine, ontogenesis study and cell based modelling. Pluripotency or transdifferentiation induction as TF mediated differentiation is commonly produced by transfer of genetic information with safety concerns. The direct delivery of proteins could represent a safer alternative but still needs significant advances to be efficient. We have successfully developed the direct delivery of proteins by an attenuated bacterium with a type 3 secretion system that does not require challenging and laborious steps for production and purification of recombinant molecules. Here we show that this natural micro-syringe is able to inject TFs to primary human fibroblasts and cord blood CD34 + hematopoietic stem cells. The signal sequence for vectorization of the TF Oct4 has no effect on DNA binding to its nucleic target. As soon as one hour after injection, vectorized TFs are detectable in the nucleus. The injection process is not associated with toxicity and the bacteria can be completely removed from cell cultures. A three days targeted release of Oct4 or Sox2 embryonic TFs results in the induction of the core pluripotency genes expression in fibroblasts and CD34 + hematopoietic stem cells. This micro-syringe vectorization represents a new strategy for TF delivery and has potential applications for cell fate reprogramming. Copyright © 2016 Elsevier B.V. All rights reserved.

Ten years of progress and promise of induced pluripotent stem cells: historical origins, characteristics, mechanisms, limitations, and potential applications.

PubMed

Omole, Adekunle Ebenezer; Fakoya, Adegbenro Omotuyi John

2018-01-01

The discovery of induced pluripotent stem cells (iPSCs) by Shinya Yamanaka in 2006 was heralded as a major breakthrough of the decade in stem cell research. The ability to reprogram human somatic cells to a pluripotent embryonic stem cell-like state through the ectopic expression of a combination of embryonic transcription factors was greeted with great excitement by scientists and bioethicists. The reprogramming technology offers the opportunity to generate patient-specific stem cells for modeling human diseases, drug development and screening, and individualized regenerative cell therapy. However, fundamental questions have been raised regarding the molecular mechanism of iPSCs generation, a process still poorly understood by scientists. The efficiency of reprogramming of iPSCs remains low due to the effect of various barriers to reprogramming. There is also the risk of chromosomal instability and oncogenic transformation associated with the use of viral vectors, such as retrovirus and lentivirus, which deliver the reprogramming transcription factors by integration in the host cell genome. These challenges can hinder the therapeutic prospects and promise of iPSCs and their clinical applications. Consequently, extensive studies have been done to elucidate the molecular mechanism of reprogramming and novel strategies have been identified which help to improve the efficiency of reprogramming methods and overcome the safety concerns linked with iPSC generation. Distinct barriers and enhancers of reprogramming have been elucidated, and non-integrating reprogramming methods have been reported. Here, we summarize the progress and the recent advances that have been made over the last 10 years in the iPSC field, with emphasis on the molecular mechanism of reprogramming, strategies to improve the efficiency of reprogramming, characteristics and limitations of iPSCs, and the progress made in the applications of iPSCs in the field of disease modelling, drug discovery and regenerative medicine. Additionally, this study appraises the role of genomic editing technology in the generation of healthy iPSCs.

Ten years of progress and promise of induced pluripotent stem cells: historical origins, characteristics, mechanisms, limitations, and potential applications

PubMed Central

2018-01-01

The discovery of induced pluripotent stem cells (iPSCs) by Shinya Yamanaka in 2006 was heralded as a major breakthrough of the decade in stem cell research. The ability to reprogram human somatic cells to a pluripotent embryonic stem cell-like state through the ectopic expression of a combination of embryonic transcription factors was greeted with great excitement by scientists and bioethicists. The reprogramming technology offers the opportunity to generate patient-specific stem cells for modeling human diseases, drug development and screening, and individualized regenerative cell therapy. However, fundamental questions have been raised regarding the molecular mechanism of iPSCs generation, a process still poorly understood by scientists. The efficiency of reprogramming of iPSCs remains low due to the effect of various barriers to reprogramming. There is also the risk of chromosomal instability and oncogenic transformation associated with the use of viral vectors, such as retrovirus and lentivirus, which deliver the reprogramming transcription factors by integration in the host cell genome. These challenges can hinder the therapeutic prospects and promise of iPSCs and their clinical applications. Consequently, extensive studies have been done to elucidate the molecular mechanism of reprogramming and novel strategies have been identified which help to improve the efficiency of reprogramming methods and overcome the safety concerns linked with iPSC generation. Distinct barriers and enhancers of reprogramming have been elucidated, and non-integrating reprogramming methods have been reported. Here, we summarize the progress and the recent advances that have been made over the last 10 years in the iPSC field, with emphasis on the molecular mechanism of reprogramming, strategies to improve the efficiency of reprogramming, characteristics and limitations of iPSCs, and the progress made in the applications of iPSCs in the field of disease modelling, drug discovery and regenerative medicine. Additionally, this study appraises the role of genomic editing technology in the generation of healthy iPSCs. PMID:29770269

Metabolic reprogramming in glioblastoma: the influence of cancer metabolism on epigenetics and unanswered questions

PubMed Central

Agnihotri, Sameer; Zadeh, Gelareh

2016-01-01

A defining hallmark of glioblastoma is altered tumor metabolism. The metabolic shift towards aerobic glycolysis with reprogramming of mitochondrial oxidative phosphorylation, regardless of oxygen availability, is a phenomenon known as the Warburg effect. In addition to the Warburg effect, glioblastoma tumor cells also utilize the tricarboxylic acid cycle/oxidative phosphorylation in a different capacity than normal tissue. Altered metabolic enzymes and their metabolites are oncogenic and not simply a product of tumor proliferation. Here we highlight the advantages of why tumor cells, including glioblastoma cells, require metabolic reprogramming and how tumor metabolism can converge on tumor epigenetics and unanswered questions in the field. PMID:26180081

Immortalized prairie vole-derived fibroblasts (VMF-K4DTs) can be transformed into pluripotent stem cells and provide a useful tool with which to determine optimal reprogramming conditions

PubMed Central

KATAYAMA, Masafumi; HIRAYAMA, Takashi; KIYONO, Tohru; ONUMA, Manabu; TANI, Tetsuya; TAKEDA, Satoru; NISHIMORI, Katsuhiko; FUKUDA, Tomokazu

2017-01-01

The cellular conditions required to establish induced pluripotent stem cells (iPSCs), such as the number of reprogramming factors and/or promoter selection, differ among species. The establishment of iPSCs derived from cells of previously unstudied species therefore requires the extensive optimization of programming conditions, including promoter selection and the optimal number of reprogramming factors, through a trial-and-error approach. While the four Yamanaka factors Oct3/4, Sox2, Klf4, and c-Myc are sufficient for iPSC establishment in mice, we reported previously that six reprogramming factors were necessary for the creation of iPSCs from primary prairie vole-derived cells. Further to this study, we now show detailed data describing the optimization protocol we developed in order to obtain iPSCs from immortalized prairie vole-derived fibroblasts. Immortalized cells can be very useful tools in the optimization of cellular reprogramming conditions, as cellular senescence is known to dramatically decrease the efficiency of iPSC establishment. The immortalized prairie vole cells used in this optimization were designated K4DT cells as they contained mutant forms of CDK4, cyclin D, and telomerase reverse transcriptase (TERT). We show that iPSCs derived from these immortalized cells exhibit the transcriptional silencing of exogenous reprogramming factors while maintaining pluripotent cell morphology. There were no observed differences between the iPSCs derived from primary and immortalized prairie vole fibroblasts. Our data suggest that cells that are immortalized with mutant CDK4, cyclin D, and TERT provide a useful tool for the determination of the optimal conditions for iPSC establishment. PMID:28331164

Immortalized prairie vole-derived fibroblasts (VMF-K4DTs) can be transformed into pluripotent stem cells and provide a useful tool with which to determine optimal reprogramming conditions.

PubMed

Katayama, Masafumi; Hirayama, Takashi; Kiyono, Tohru; Onuma, Manabu; Tani, Tetsuya; Takeda, Satoru; Nishimori, Katsuhiko; Fukuda, Tomokazu

2017-06-21

The cellular conditions required to establish induced pluripotent stem cells (iPSCs), such as the number of reprogramming factors and/or promoter selection, differ among species. The establishment of iPSCs derived from cells of previously unstudied species therefore requires the extensive optimization of programming conditions, including promoter selection and the optimal number of reprogramming factors, through a trial-and-error approach. While the four Yamanaka factors Oct3/4, Sox2, Klf4, and c-Myc are sufficient for iPSC establishment in mice, we reported previously that six reprogramming factors were necessary for the creation of iPSCs from primary prairie vole-derived cells. Further to this study, we now show detailed data describing the optimization protocol we developed in order to obtain iPSCs from immortalized prairie vole-derived fibroblasts. Immortalized cells can be very useful tools in the optimization of cellular reprogramming conditions, as cellular senescence is known to dramatically decrease the efficiency of iPSC establishment. The immortalized prairie vole cells used in this optimization were designated K4DT cells as they contained mutant forms of CDK4, cyclin D, and telomerase reverse transcriptase (TERT). We show that iPSCs derived from these immortalized cells exhibit the transcriptional silencing of exogenous reprogramming factors while maintaining pluripotent cell morphology. There were no observed differences between the iPSCs derived from primary and immortalized prairie vole fibroblasts. Our data suggest that cells that are immortalized with mutant CDK4, cyclin D, and TERT provide a useful tool for the determination of the optimal conditions for iPSC establishment.

Transcriptome analyses of rhesus monkey preimplantation embryos reveal a reduced capacity for DNA double-strand break repair in primate oocytes and early embryos

PubMed Central

Wang, Xinyi; Liu, Denghui; He, Dajian; Suo, Shengbao; Xia, Xian; He, Xiechao; Han, Jing-Dong J.; Zheng, Ping

2017-01-01

Preimplantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA), and cell-fate commitment. The molecular basis of these processes remains obscure in primates in which there is a high rate of embryo wastage. Thus, understanding the factors involved in genome reprogramming and ZGA might help reproductive success during this susceptible period of early development and generate induced pluripotent stem cells with greater efficiency. Moreover, explaining the molecular basis responsible for embryo wastage in primates will greatly expand our knowledge of species evolution. By using RNA-seq in single and pooled oocytes and embryos, we defined the transcriptome throughout preimplantation development in rhesus monkey. In comparison to archival human and mouse data, we found that the transcriptome dynamics of monkey oocytes and embryos were very similar to those of human but very different from those of mouse. We identified several classes of maternal and zygotic genes, whose expression peaks were highly correlated with the time frames of genome reprogramming, ZGA, and cell-fate commitment, respectively. Importantly, comparison of the ZGA-related network modules among the three species revealed less robust surveillance of genomic instability in primate oocytes and embryos than in rodents, particularly in the pathways of DNA damage signaling and homology-directed DNA double-strand break repair. This study highlights the utility of monkey models to better understand the molecular basis for genome reprogramming, ZGA, and genomic stability surveillance in human early embryogenesis and may provide insights for improved homologous recombination-mediated gene editing in monkey. PMID:28223401

Global transcriptomic analysis of induced cardiomyocytes predicts novel regulators for direct cardiac reprogramming.

PubMed

Talkhabi, Mahmood; Razavi, Seyed Morteza; Salari, Ali

2017-06-01

Heart diseases are the most significant cause of morbidity and mortality in the world. De novo generated cardiomyocytes (CMs) are a great cellular source for cell-based therapy and other potential applications. Direct cardiac reprogramming is the newest method to produce CMs, known as induced cardiomyocytes (iCMs). During a direct cardiac reprogramming, also known as transdifferentiation, non-cardiac differentiated adult cells are reprogrammed to cardiac identity by forced expression of cardiac-specific transcription factors (TFs) or microRNAs. To this end, many different combinations of TFs (±microRNAs) have been reported for direct reprogramming of mouse or human fibroblasts to iCMs, although their efficiencies remain very low. It seems that the investigated TFs and microRNAs are not sufficient for efficient direct cardiac reprogramming and other cardiac specific factors may be required for increasing iCM production efficiency, as well as the quality of iCMs. Here, we analyzed gene expression data of cardiac fibroblast (CFs), iCMs and adult cardiomyocytes (aCMs). The up-regulated and down-regulated genes in CMs (aCMs and iCMs) were determined as CM and CF specific genes, respectively. Among CM specific genes, we found 153 transcriptional activators including some cardiac and non-cardiac TFs that potentially activate the expression of CM specific genes. We also identified that 85 protein kinases such as protein kinase D1 (PKD1), protein kinase A (PRKA), calcium/calmodulin-dependent protein kinase (CAMK), protein kinase C (PRKC), and insulin like growth factor 1 receptor (IGF1R) that are strongly involved in establishing CM identity. CM gene regulatory network constructed using protein kinases, transcriptional activators and intermediate proteins predicted some new transcriptional activators such as myocyte enhancer factor 2A (MEF2A) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A), which may be required for qualitatively and quantitatively efficient direct cardiac reprogramming. Taken together, this study provides new insights into the complexity of cell fate conversion and better understanding of the roles of transcriptional activators, signaling pathways and protein kinases in increasing the efficiency of direct cardiac reprogramming and maturity of iCMs.

Bovine trophectoderm cell lines induced from bovine fibroblasts with reprogramming factors

USDA-ARS?s Scientific Manuscript database

Bovine trophectoderm (TE) cells were induced [induced bovine trophectoderm-like (iBT)] from bovine fetal liver-derived fibroblasts, and other bovine fetal fibroblasts, after viral-vector transduction with either four or six reprogramming factors (RF), including POU5F1, KLF4, SOX2, C-MYC, SV40 large ...

MiR-590 Promotes Transdifferentiation of Porcine and Human Fibroblasts Toward a Cardiomyocyte-Like Fate by Directly Repressing Specificity Protein 1.

PubMed

Singh, Vivek P; Mathison, Megumi; Patel, Vivekkumar; Sanagasetti, Deepthi; Gibson, Brian W; Yang, Jianchang; Rosengart, Todd K

2016-11-10

Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells represents a promising potential new therapy for treating heart disease, inducing significant improvements in postinfarct ventricular function in rodent models. Because reprogramming factors effective in transdifferentiating rodent cells are not sufficient to reprogram human cells, we sought to identify reprogramming factors potentially applicable to human studies. Lentivirus vectors expressing Gata4, Mef2c, and Tbx5 (GMT); Hand2 (H), Myocardin (My), or microRNA (miR)-590 were administered to rat, porcine, and human cardiac fibroblasts in vitro. induced cardiomyocyte-like cell production was then evaluated by assessing expression of the cardiomyocyte marker, cardiac troponin T (cTnT), whereas signaling pathway studies were performed to identify reprogramming factor targets. GMT administration induced cTnT expression in ≈6% of rat fibroblasts, but failed to induce cTnT expression in porcine or human cardiac fibroblasts. Addition of H/My and/or miR-590 to GMT administration resulted in cTNT expression in ≈5% of porcine and human fibroblasts and also upregulated the expression of the cardiac genes, MYH6 and TNNT2. When cocultured with murine cardiomyocytes, cTnT-expressing porcine cardiac fibroblasts exhibited spontaneous contractions. Administration of GMT plus either H/My or miR-590 alone also downregulated fibroblast genes COL1A1 and COL3A1. miR-590 was shown to directly suppress the zinc finger protein, specificity protein 1 (Sp1), which was able to substitute for miR-590 in inducing cellular reprogramming. These data support porcine studies as a surrogate for testing human cardiac reprogramming, and suggest that miR-590-mediated repression of Sp1 represents an alternative pathway for enhancing human cardiac cellular reprogramming. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Inducing pluripotency using in vivo gene therapy.

PubMed

Gardlik, Roman

2012-08-01

Since the original study of Takahashi and Yamanaka in 2006 [1], the field of induced pluripotent stem (iPS) cells has made a great progress. Since then, a number of different cell types have been successfully brought to a state of pluripotency and a different set of transcription factors have been reported to be sufficient to reprogram mouse and human somatic cells. Although still with low efficiency of reprogramming, the patient- and disease-specific therapy represents the most valuable outcome of the whole area of iPS cells. Herein we hypothesize that inducing pluripotency in vivo might be an interesting alternative to the standard ex vivo methods. In vivo reprogramming would benefit from the direct administration of the DNA encoding the reprogramming factors into the target tissue/organ of an individual. The target cells that are to be reprogrammed would be transduced in their natural environment that can provide all the necessary molecular and spatial factors that could be missing during ex vivo reprogramming. However, since no available data exist on in vivo induced pluripotency, it is difficult to predict if testing the hypothesis will provide any promising results. On the way to this point, a number of pilot experiments have to be performed to overcome many limitations and pitfalls that are arising from such a risky concept. Safety issues, such as the risk of somatic tumor formation, will likely be the crucial point to focus on during the process of proving the validity of the hypothesis. However, initial data from the study on inflammatory bowel disease suggest that there might be some beneficial effect of in vivo gene therapy based on reprogramming the target cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Direct Reprogramming of Human Fibroblasts toward a Cardiomyocyte-like State

PubMed Central

Fu, Ji-Dong; Stone, Nicole R.; Liu, Lei; Spencer, C. Ian; Qian, Li; Hayashi, Yohei; Delgado-Olguin, Paul; Ding, Sheng; Bruneau, Benoit G.; Srivastava, Deepak

2013-01-01

Summary Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4, MEF2C, and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However, GMT alone appears insufficient in human fibroblasts, at least in vitro. Here, we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells, fetal heart, and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming, including sarcomere formation, calcium transients, and action potentials, although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore, we found that transforming growth factor β signaling was important for, and improved the efficiency of, human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage, and lay the foundation for future refinements in vitro and in vivo. PMID:24319660

A Blueprint for a Synthetic Genetic Feedback Controller to Reprogram Cell Fate.

PubMed

Del Vecchio, Domitilla; Abdallah, Hussein; Qian, Yili; Collins, James J

2017-01-25

To artificially reprogram cell fate, experimentalists manipulate the gene regulatory networks (GRNs) that maintain a cell's phenotype. In practice, reprogramming is often performed by constant overexpression of specific transcription factors (TFs). This process can be unreliable and inefficient. Here, we address this problem by introducing a new approach to reprogramming based on mathematical analysis. We demonstrate that reprogramming GRNs using constant overexpression may not succeed in general. Instead, we propose an alternative reprogramming strategy: a synthetic genetic feedback controller that dynamically steers the concentration of a GRN's key TFs to any desired value. The controller works by adjusting TF expression based on the discrepancy between desired and actual TF concentrations. Theory predicts that this reprogramming strategy is guaranteed to succeed, and its performance is independent of the GRN's structure and parameters, provided that feedback gain is sufficiently high. As a case study, we apply the controller to a model of induced pluripotency in stem cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

NRF2 Orchestrates the Metabolic Shift during Induced Pluripotent Stem Cell Reprogramming

PubMed Central

Hawkins, Kate E.; Joy, Shona; Delhove, Juliette M.K.M.; Kotiadis, Vassilios N.; Fernandez, Emilio; Fitzpatrick, Lorna M.; Whiteford, James R.; King, Peter J.; Bolanos, Juan P.; Duchen, Michael R.; Waddington, Simon N.; McKay, Tristan R.

2016-01-01

Summary The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα) activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation. PMID:26904936

Alkaline phosphatase and OCT-3/4 as useful markers for predicting susceptibility of human deciduous teeth-derived dental pulp cells to reprogramming factor-induced iPS cells.

PubMed

Inada, Emi; Saitoh, Issei; Kubota, Naoko; Soda, Miki; Matsueda, Kazunari; Murakami, Tomoya; Sawami, Tadashi; Kagoshima, Akiko; Yamasaki, Youichi; Sato, Masahiro

2017-11-01

The aim of the present study was to prove that primary cells enriched with stem cells are more easily reprogrammed to generate induced pluripotent stem (iPS) cells than those with scarce numbers of stem cells. We surveyed the alkaline phosphatase (ALP) activity in five primarily-isolated human deciduous teeth-derived dental pulp cells (HDDPC) with cytochemical staining to examine the possible presence of stem cells. Next, the expression of stemness-specific factors, such as OCT(Octumer-binding transcription factor)3/4, NANOG, SOX2(SRY (sex determining region Y)-box 2), CD90, muscle segment homeodomain homeobox (MSX) 1, and MSX2, was assessed with a reverse transcription polymerase chain reaction method. Finally, these isolated HDDPC were transfected with plasmids carrying genes coding Yamanaka factors to determine whether these cells could be reprogrammed to generate iPS cells. Of the five primarily-isolated HDDPC, two (HDDPC-1 and -5) exhibited higher degrees of ALP activity. OCT-3/4 expression was also prominent in those two lines. Furthermore, these two lines proliferated faster than the other three lines. The transfection of HDDPC with Yamanaka factors resulted in the generation of iPS cells from HDDPC-1 and -5. The number of cells with the stemness property of HDDPC differs among individuals, which suggests that HDDPC showing an increased expression of both ALP and OCT-3/4 can be more easily reprogrammed to generate iPS cells after the forced expression of reprogramming factors. © 2016 John Wiley & Sons Australia, Ltd.

Deubiquitylating enzymes as cancer stem cell therapeutics.

PubMed

Haq, Saba; Suresh, Bharathi; Ramakrishna, Suresh

2018-01-01

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. Copyright © 2017 Elsevier B.V. All rights reserved.

Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes

PubMed Central

Zhou, Huanyu; Dickson, Matthew E.; Kim, Min Soo; Bassel-Duby, Rhonda; Olson, Eric N.

2015-01-01

Conversion of fibroblasts to functional cardiomyocytes represents a potential approach for restoring cardiac function after myocardial injury, but the technique thus far has been slow and inefficient. To improve the efficiency of reprogramming fibroblasts to cardiac-like myocytes (iCMs) by cardiac transcription factors [Gata4, Hand2, Mef2c, and Tbx5 (GHMT)], we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process in three different types of fibroblasts (mouse embryo, adult cardiac, and tail tip). Approximately 50% of reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 wk of induction by Akt plus GHMT. Furthermore, addition of Akt1 to GHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Insulin-like growth factor 1 (IGF1) and phosphoinositol 3-kinase (PI3K) acted upstream of Akt whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3 (Foxo3a) acted downstream of Akt to influence fibroblast-to-cardiomyocyte reprogramming. These findings provide insights into the molecular basis of cardiac reprogramming and represent an important step toward further application of this technique. PMID:26354121

Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1

PubMed Central

Moon, Jai-Hee; Heo, June Seok; Kim, Jun Sung; Jun, Eun Kyoung; Lee, Jung Han; Kim, Aeree; Kim, Jonggun; Whang, Kwang Youn; Kang, Yong-Kook; Yeo, Seungeun; Lim, Hee-Joung; Han, Dong Wook; Kim, Dong-Wook; Oh, Sejong; Yoon, Byung Sun; Schöler, Hans R; You, Seungkwon

2011-01-01

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4. PMID:21709693

A model for genetic and epigenetic regulatory networks identifies rare pathways for transcription factor induced pluripotency

NASA Astrophysics Data System (ADS)

Artyomov, Maxim; Meissner, Alex; Chakraborty, Arup

2010-03-01

Most cells in an organism have the same DNA. Yet, different cell types express different proteins and carry out different functions. This is because of epigenetic differences; i.e., DNA in different cell types is packaged distinctly, making it hard to express certain genes while facilitating the expression of others. During development, upon receipt of appropriate cues, pluripotent embryonic stem cells differentiate into diverse cell types that make up the organism (e.g., a human). There has long been an effort to make this process go backward -- i.e., reprogram a differentiated cell (e.g., a skin cell) to pluripotent status. Recently, this has been achieved by transfecting certain transcription factors into differentiated cells. This method does not use embryonic material and promises the development of patient-specific regenerative medicine, but it is inefficient. The mechanisms that make reprogramming rare, or even possible, are poorly understood. We have developed the first computational model of transcription factor-induced reprogramming. Results obtained from the model are consistent with diverse observations, and identify the rare pathways that allow reprogramming to occur. If validated, our model could be further developed to design optimal strategies for reprogramming and shed light on basic questions in biology.

Cell reprogramming: Therapeutic potential and the promise of rejuvenation for the aging brain.

PubMed

López-León, Micaela; Outeiro, Tiago F; Goya, Rodolfo G

2017-11-01

Aging is associated with a progressive increase in the incidence of neurodegenerative diseases, with Alzheimer's (AD) and Parkinson's (PD) disease being the most conspicuous examples. Within this context, the absence of efficacious therapies for most age-related brain pathologies has increased the interest in regenerative medicine. In particular, cell reprogramming technologies have ushered in the era of personalized therapies that not only show a significant potential for the treatment of neurodegenerative diseases but also promise to make biological rejuvenation feasible. We will first review recent evidence supporting the emerging view that aging is a reversible epigenetic phenomenon. Next, we will describe novel reprogramming approaches that overcome some of the intrinsic limitations of conventional induced-pluripotent-stem-cell technology. One of the alternative approaches, lineage reprogramming, consists of the direct conversion of one adult cell type into another by transgenic expression of multiple lineage-specific transcription factors (TF). Another strategy, termed pluripotency factor-mediated direct reprogramming, uses universal TF to generate epigenetically unstable intermediates able to differentiate into somatic cell types in response to specific differentiation factors. In the third part we will review studies showing the potential relevance of the above approaches for the treatment of AD and PD. Copyright © 2017 Elsevier B.V. All rights reserved.

p53 isoform Δ133p53 promotes efficiency of induced pluripotent stem cells and ensures genomic integrity during reprogramming.

PubMed

Gong, Lu; Pan, Xiao; Chen, Haide; Rao, Lingjun; Zeng, Yelin; Hang, Honghui; Peng, Jinrong; Xiao, Lei; Chen, Jun

2016-11-22

Human induced pluripotent stem (iPS) cells have great potential in regenerative medicine, but this depends on the integrity of their genomes. iPS cells have been found to contain a large number of de novo genetic alterations due to DNA damage response during reprogramming. Thus, to maintain the genetic stability of iPS cells is an important goal in iPS cell technology. DNA damage response can trigger tumor suppressor p53 activation, which ensures genome integrity of reprogramming cells by inducing apoptosis and senescence. p53 isoform Δ133p53 is a p53 target gene and functions to not only antagonize p53 mediated apoptosis, but also promote DNA double-strand break (DSB) repair. Here we report that Δ133p53 is induced in reprogramming. Knockdown of Δ133p53 results 2-fold decrease in reprogramming efficiency, 4-fold increase in chromosomal aberrations, whereas overexpression of Δ133p53 with 4 Yamanaka factors showes 4-fold increase in reprogamming efficiency and 2-fold decrease in chromosomal aberrations, compared to those in iPS cells induced only with 4 Yamanaka factors. Overexpression of Δ133p53 can inhibit cell apoptosis and promote DNA DSB repair foci formation during reprogramming. Our finding demonstrates that the overexpression of Δ133p53 not only enhances reprogramming efficiency, but also results better genetic quality in iPS cells.

Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhai, Yingying; Chen, Xi; Yu, Dehai

2015-09-10

Histone deacetylase inhibitor valproic acid (VPA) has been used to increase the reprogramming efficiency of induced pluripotent stem cell (iPSC) from somatic cells, yet the specific molecular mechanisms underlying this effect is unknown. Here, we demonstrate that reprogramming with lentiviruses carrying the iPSC-inducing factors (Oct4-Sox2-Klf4-cMyc, OSKM) caused senescence in mouse fibroblasts, establishing a stress barrier for cell reprogramming. Administration of VPA protected cells from reprogramming-induced senescent stress. Using an in vitro pre-mature senescence model, we found that VPA treatment increased cell proliferation and inhibited apoptosis through the suppression of the p16/p21 pathway. In addition, VPA also inhibited the G2/M phasemore » blockage derived from the senescence stress. These findings highlight the role of VPA in breaking the cell senescence barrier required for the induction of pluripotency. - Highlights: • Histone deacetylase inhibitor valproic acid enhances iPSC induction. • Valproic acid suppresses reprogramming-induced senescence stress. • Valproic acid downregulates the p16/p21 pathway in reprogramming. • This study demonstrates a new mechanistic role of valproic acid in enhancing reprogramming.« less

Systematic optimization of human pluripotent stem cells media using Design of Experiments

NASA Astrophysics Data System (ADS)

Marinho, Paulo A.; Chailangkarn, Thanathom; Muotri, Alysson R.

2015-05-01

Human pluripotent stem cells (hPSC) are used to study the early stages of human development in vitro and, increasingly due to somatic cell reprogramming, cellular and molecular mechanisms of disease. Cell culture medium is a critical factor for hPSC to maintain pluripotency and self-renewal. Numerous defined culture media have been empirically developed but never systematically optimized for culturing hPSC. We applied design of experiments (DOE), a powerful statistical tool, to improve the medium formulation for hPSC. Using pluripotency and cell growth as read-outs, we determined the optimal concentration of both basic fibroblast growth factor (bFGF) and neuregulin-1 beta 1 (NRG1β1). The resulting formulation, named iDEAL, improved the maintenance and passage of hPSC in both normal and stressful conditions, and affected trimethylated histone 3 lysine 27 (H3K27me3) epigenetic status after genetic reprogramming. It also enhances efficient hPSC plating as single cells. Altogether, iDEAL potentially allows scalable and controllable hPSC culture routine in translational research. Our DOE strategy could also be applied to hPSC differentiation protocols, which often require numerous and complex cell culture media.

Sequential EMT-MET induces neuronal conversion through Sox2

PubMed Central

He, Songwei; Chen, Jinlong; Zhang, Yixin; Zhang, Mengdan; Yang, Xiao; Li, Yuan; Sun, Hao; Lin, Lilong; Fan, Ke; Liang, Lining; Feng, Chengqian; Wang, Fuhui; Zhang, Xiao; Guo, Yiping; Pei, Duanqing; Zheng, Hui

2017-01-01

Direct neuronal conversion can be achieved with combinations of small-molecule compounds and growth factors. Here, by studying the first or induction phase of the neuronal conversion induced by defined 5C medium, we show that the Sox2-mediated switch from early epithelial–mesenchymal transition (EMT) to late mesenchymal–epithelial transition (MET) within a high proliferation context is essential and sufficient for the conversion from mouse embryonic fibroblasts (MEFs) to TuJ+ cells. At the early stage, insulin and basic fibroblast growth factor (bFGF)-induced cell proliferation, early EMT, the up-regulation of Stat3 and Sox2, and the subsequent activation of neuron projection. Up-regulated Sox2 then induced MET and directed cells towards a neuronal fate at the late stage. Inhibiting either stage of this sequential EMT-MET impaired the conversion. In addition, Sox2 could replace sequential EMT-MET to induce a similar conversion within a high proliferation context, and its functions were confirmed with other neuronal conversion protocols and MEFs reprogramming. Therefore, the critical roles of the sequential EMT-MET were implicated in direct cell fate conversion in addition to reprogramming, embryonic development and cancer progression. PMID:28580167

Increased reprogramming of human fetal hepatocytes compared with adult hepatocytes in feeder-free conditions.

PubMed

Hansel, Marc C; Gramignoli, Roberto; Blake, William; Davila, Julio; Skvorak, Kristen; Dorko, Kenneth; Tahan, Veysel; Lee, Brian R; Tafaleng, Edgar; Guzman-Lepe, Jorge; Soto-Gutierrez, Alejandro; Fox, Ira J; Strom, Stephen C

2014-01-01

Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus, hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these, 37 hiPSC lines were generated from fetal hepatocytes, 2 hiPSC lines from normal hepatocytes, and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar syndrome, type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression, flow cytometry, immunocytochemistry, and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors, while fetal hepatocytes could be reprogrammed with three (OCT4, SOX2, NANOG) or four factors (OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, KLF4, C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes, although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation.

Optical reprogramming of human somatic cells using ultrashort Bessel-shaped near-infrared femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada; Breunig, Hans Georg; Batista, Ana; König, Karsten

2015-11-01

We report a virus-free optical approach to human cell reprogramming into induced pluripotent stem cells with low-power nanoporation using ultrashort Bessel-shaped laser pulses. Picojoule near-infrared sub-20 fs laser pulses at a high 85 MHz repetition frequency are employed to generate transient nanopores in the membrane of dermal fibroblasts for the introduction of four transcription factors to induce the reprogramming process. In contrast to conventional approaches which utilize retro- or lentiviruses to deliver genes or transcription factors into the host genome, the laser method is virus-free; hence, the risk of virus-induced cancer generation limiting clinical application is avoided.

Perspective for special Gurdon issue for differentiation: can cell fusion inform nuclear reprogramming?

PubMed

Burns, David; Blau, Helen M

2014-07-01

Nuclear reprogramming was first shown to be possible by Sir John Gurdon over a half century ago. The process has been revolutionized by the production of induced pluripotent cells by overexpression of the four transcription factors discovered by Shinya Yamanaka, which now enables mammalian applications. Yet, reprogramming by a few transcription factors remains incomplete and inefficient, whether to pluripotent or differentiated cells. We propose that a better understanding of mechanistic insights based on developmental principles gained from heterokaryon studies may inform the process of directing cell fate, fundamentally and clinically. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells.

PubMed

Bhise, Nupura S; Wahlin, Karl J; Zack, Donald J; Green, Jordan J

2013-01-01

Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups. This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming.

Comprehensive Identification of Krüppel-Like Factor Family Members Contributing to the Self-Renewal of Mouse Embryonic Stem Cells and Cellular Reprogramming.

PubMed

Jeon, Hyojung; Waku, Tsuyoshi; Azami, Takuya; Khoa, Le Tran Phuc; Yanagisawa, Jun; Takahashi, Satoru; Ema, Masatsugu

2016-01-01

Pluripotency is maintained in mouse embryonic stem (ES) cells and is induced from somatic cells by the activation of appropriate transcriptional regulatory networks. Krüppel-like factor gene family members, such as Klf2, Klf4 and Klf5, have important roles in maintaining the undifferentiated state of mouse ES cells as well as in cellular reprogramming, yet it is not known whether other Klf family members exert self-renewal and reprogramming functions when overexpressed. In this study, we examined whether overexpression of any representative Klf family member, such as Klf1-Klf10, would be sufficient for the self-renewal of mouse ES cells. We found that only Klf2, Klf4, and Klf5 produced leukemia inhibitory factor (LIF)-independent self-renewal, although most KLF proteins, if not all, have the ability to occupy the regulatory regions of Nanog, a critical Klf target gene. We also examined whether overexpression of any of Klf1-Klf10 would be sufficient to convert epiblast stem cells into a naïve pluripotent state and found that Klf5 had such reprogramming ability, in addition to Klf2 and Klf4. We also delineated the functional domains of the Klf2 protein for LIF-independent self-renewal and reprogramming. Interestingly, we found that both the N-terminal transcriptional activation and C-terminal zinc finger domains were indispensable for this activity. Taken together, our comprehensive analysis provides new insight into the contribution of Klf family members to mouse ES self-renewal and cellular reprogramming.

An inherently bi-functional subset of Foxp3+ T helper cells is controlled by the transcription factor Eos

PubMed Central

Sharma, Madhav D.; Huang, Lei; Choi, Jeong-Hyeon; Lee, Eun-Joon; Wilson, James M.; Lemos, Henrique; Pan, Fan; Blazar, Bruce R.; Pardoll, Drew M.; Mellor, Andrew L; Shi, Huidong; Munn, David H.

2013-01-01

SUMMARY At sites of inflammation, certain regulatory T cells (Treg cells) can undergo rapid reprogramming into helper-like cells, without loss of the transcription factor Foxp3. We show that reprogramming is controlled by down-regulation of the transcription factor Eos (Ikzf4), an obligate co-repressor for Foxp3. Reprogramming was restricted to a specific subset of “Eoslabile” Treg cells which were present in the thymus and identifiable by characteristic surface markers and DNA methylation. Mice made deficient in this subset became impaired in their ability to provide help for presentation of new antigens to naive T cells. Down-regulation of Eos required the pro-inflammatory cytokine IL-6, and mice lacking IL-6 had impaired development and function of the Eos-labile subset. Conversely, the immunoregulatory enzyme IDO blocked loss of Eos, and prevented the Eos-labile Treg cells from reprogramming. Thus, the Foxp3+ lineage contains a committed subset of Treg cells capable of rapid conversion into biologically important helper cells. PMID:23684987

Overcoming reprogramming resistance of Fanconi anemia cells

PubMed Central

Müller, Lars U. W.; Milsom, Michael D.; Harris, Chad E.; Vyas, Rutesh; Brumme, Kristina M.; Parmar, Kalindi; Moreau, Lisa A.; Schambach, Axel; Park, In-Hyun; London, Wendy B.; Strait, Kelly; Schlaeger, Thorsten; DeVine, Alexander L.; Grassman, Elke; D'Andrea, Alan; Daley, George Q.

2012-01-01

Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs. PMID:22371882

Transcriptional Mechanisms of Proneural Factors and REST in Regulating Neuronal Reprogramming of Astrocytes

PubMed Central

Masserdotti, Giacomo; Gillotin, Sébastien; Sutor, Bernd; Drechsel, Daniela; Irmler, Martin; Jørgensen, Helle F.; Sass, Steffen; Theis, Fabian J.; Beckers, Johannes; Berninger, Benedikt; Guillemot, François; Götz, Magdalena

2015-01-01

Summary Direct lineage reprogramming induces dramatic shifts in cellular identity, employing poorly understood mechanisms. Recently, we demonstrated that expression of Neurog2 or Ascl1 in postnatal mouse astrocytes generates glutamatergic or GABAergic neurons. Here, we take advantage of this model to study dynamics of neuronal cell fate acquisition at the transcriptional level. We found that Neurog2 and Ascl1 rapidly elicited distinct neurogenic programs with only a small subset of shared target genes. Within this subset, only NeuroD4 could by itself induce neuronal reprogramming in both mouse and human astrocytes, while co-expression with Insm1 was required for glutamatergic maturation. Cultured astrocytes gradually became refractory to reprogramming, in part by the repressor REST preventing Neurog2 from binding to the NeuroD4 promoter. Notably, in astrocytes refractory to Neurog2 activation, the underlying neurogenic program remained amenable to reprogramming by exogenous NeuroD4. Our findings support a model of temporal hierarchy for cell fate change during neuronal reprogramming. PMID:26119235

The Effect of Substrate Topography on Direct Reprogramming of Fibroblasts to Induced Neurons

PubMed Central

Kulangara, Karina; Adler, Andrew F.; Wang, Hong; Chellappan, Malathi; Hammett, Ellen; Yasuda, Ryohei; Leong, Kam W.

2014-01-01

Cellular reprogramming holds tremendous potential for cell therapy and regenerative medicine. Recently, fibroblasts have been directly converted into induced neurons (iNs) by overexpression of the neuronal transcription factors Ascl1, Brn2 and Myt1L. Hypothesizing that cell-topography interactions could influence the fibroblast-to-neuron reprogramming process, we investigated the effects of various topographies on iNs produced by direct reprogramming. Final iN purity and conversion efficiency were increased on micrograting substrates. Neurite branching was increased on microposts and decreased on microgratings, with a simplified dendritic arbor characterized by the reduction of MAP2+ neurites. Neurite outgrowth increased significantly on various topographies. DNA microarray analysis detected 20 differentially expressed genes in iNs reprogrammed on smooth versus microgratings, and quantitative PCR (qPCR) confirmed the upregulation of Vip and downregulation of Thy1 and Bmp5 on microgratings. Electrophysiology and calcium imaging verified the functionality of these iNs. This study demonstrates the potential of applying topographical cues to optimize cellular reprogramming. PMID:24709523

RNA splicing and its connection with other regulatory layers in somatic cell reprogramming.

PubMed

Zavolan, Mihaela; Kanitz, Alexander

2018-06-01

Understanding how cell identity is established and maintained is one of the most exciting challenges of molecular biology today. Recent work has added a conserved layer of RNA splicing and other post-transcriptional regulatory processes to the transcriptional and epigenetic networks already known to cooperate in the establishment and maintenance of cell identity. Here we summarize these findings, highlighting specifically the multitude of splicing factors that can modulate the efficiency of somatic cell reprogramming. Distinct patterns of gene expression dynamics of these factors during reprogramming suggest that further improvements in efficiency could be obtained through optimal timing of overexpression or knockdown of individual regulators. Copyright © 2017 Elsevier Ltd. All rights reserved.

Epigenetics of cell fate reprogramming and its implications for neurological disorders modelling.

PubMed

Grzybek, Maciej; Golonko, Aleksandra; Walczak, Marta; Lisowski, Pawel

2017-03-01

The reprogramming of human induced pluripotent stem cells (hiPSCs) proceeds in a stepwise manner with reprogramming factors binding and epigenetic composition changes during transition to maintain the epigenetic landscape, important for pluripotency. There arises a question as to whether the aberrant epigenetic state after reprogramming leads to epigenetic defects in induced stem cells causing unpredictable long term effects in differentiated cells. In this review, we present a comprehensive view of epigenetic alterations accompanying reprogramming, cell maintenance and differentiation as factors that influence applications of hiPSCs in stem cell based technologies. We conclude that sample heterogeneity masks DNA methylation signatures in subpopulations of cells and thus believe that beside a genetic evaluation, extensive epigenomic screening should become a standard procedure to ensure hiPSCs state before they are used for genome editing and differentiation into neurons of interest. In particular, we suggest that exploitation of the single-cell composition of the epigenome will provide important insights into heterogeneity within hiPSCs subpopulations to fast forward development of reliable hiPSC-based analytical platforms in neurological disorders modelling and before completed hiPSC technology will be implemented in clinical approaches. Copyright © 2016 Elsevier Inc. All rights reserved.

In vivo reprogramming for heart regeneration: A glance at efficiency, environmental impacts, challenges and future directions.

PubMed

Ebrahimi, Behnam

2017-07-01

Replacing dying or diseased cells of a tissue with new ones that are converted from patient's own cells is an attractive strategy in regenerative medicine. In vivo reprogramming is a novel strategy that can circumvent the hurdles of autologous/allogeneic cell injection therapies. Interestingly, studies have demonstrated that direct injection of cardiac transcription factors or specific miRNAs into the infarct border zone of murine hearts following myocardial infarction converts resident cardiac fibroblasts into functional cardiomyocytes. Moreover, in vivo cardiac reprogramming not only drives cardiac tissue regeneration, but also improves cardiac function and survival rate after myocardial infarction. Thanks to the influence of cardiac microenvironment and the same developmental origin, cardiac fibroblasts seem to be more amenable to reprogramming toward cardiomyocyte fate than other cell sources (e.g. skin fibroblasts). Thus, reprogramming of cardiac fibroblasts to functional induced cardiomyocytes in the cardiac environment holds great promises for induced regeneration and potential clinical purposes. Application of small molecules in future studies may represent a major advancement in this arena and pharmacological reprogramming would convey reprogramming technology to the translational medicine paradigm. This study reviews accomplishments in the field of in vitro and in vivo mouse cardiac reprogramming and then deals with strategies for the enhancement of the efficiency and quality of the process. Furthermore, it discusses challenges ahead and provides suggestions for future research. Human cardiac reprogramming is also addressed as a foundation for possible application of in vivo cardiac reprogramming for human heart regeneration in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression.

PubMed

Umei, Tomohiko C; Yamakawa, Hiroyuki; Muraoka, Naoto; Sadahiro, Taketaro; Isomi, Mari; Haginiwa, Sho; Kojima, Hidenori; Kurotsu, Shota; Tamura, Fumiya; Osakabe, Rina; Tani, Hidenori; Nara, Kaori; Miyoshi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2017-08-19

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming.

Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression

PubMed Central

Umei, Tomohiko C.; Yamakawa, Hiroyuki; Muraoka, Naoto; Sadahiro, Taketaro; Isomi, Mari; Haginiwa, Sho; Kojima, Hidenori; Kurotsu, Shota; Tamura, Fumiya; Osakabe, Rina; Tani, Hidenori; Nara, Kaori; Miyoshi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2017-01-01

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming. PMID:28825623

Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro.

PubMed

Qian, Li; Berry, Emily C; Fu, Ji-dong; Ieda, Masaki; Srivastava, Deepak

2013-06-01

Cardiac fibroblasts can be reprogrammed to cardiomyocyte-like cells by the introduction of three transcription factors: Gata4, Mef2c and Tbx5 (collectively referred to here as GMT). Resident cardiac fibroblasts can be converted in vivo into induced cardiomyocyte-like cells (iCMs) that closely resemble endogenous cardiomyocytes and electrically integrate with the host myocardium. In contrast, in vitro reprogramming yields many partially reprogrammed iCMs, with a few that reprogram fully into contracting myocytes (~3 out of 10,000 GMT-transduced cells). iCMs can be observed as early as 3 d after viral infection, and they continue to mature over 2 months before beating is observed. Despite the success of multiple groups, the inefficiency of in vitro reprogramming has made it challenging for others. However, given the advantages of in vitro iCMs for performing mechanistic studies and, if refined, for testing drugs or small molecules for personalized medicine and modeling cardiac disease in a dish, it is important to standardize the protocol to improve reproducibility and enhance the technology further. Here we describe a detailed step-by-step protocol for in vitro cardiac reprogramming using retroviruses encoding GMT.

Pluripotent Conversion of Muscle Stem Cells Without Reprogramming Factors or Small Molecules.

PubMed

Bose, Bipasha; Shenoy P, Sudheer

2016-02-01

Muscle derived stem cells (MDSCs) are multipotent stem cells that can differentiate into several lineages including skeletal muscle precursor cells. Here, we show that MDSCs from myostatin null mice (Mstn (-/-) ) can be readily induced into pluripotent stem cells without using reprogramming factors. Microarray studies revealed a strong upregulation of markers like Leukemia Inhibitory factor (LIF) and Leukemia Inhibitory factor receptor (LIFR) in Mstn (-/-) MDSCs as compared to wild type MDSCs (WT-MDSCs). Furthermore when cultured in mouse embryonic stem cell media with LIF for 95 days, Mstn (-/-) MDSCs formed embryonic stem cell (ES) like colonies. We termed such ES like cells as the culture-induced pluripotent stem cells (CiPSC). CiPSCs from Mstn (-/-) MDSCs were phenotypically similar to ESCs, expressed high levels of Oct4, Nanog, Sox2 and SSEA-1, maintained a normal karyotype. Furthermore, CiPSCs formed embryoid bodies and teratomas when injected into immunocompromised mice. In addition, CiPSCs differentiated into somatic cells of all three lineages. We further show that culturing in ES cell media, resulted in hypermethylation and downregulation of BMP2 in Mstn(-/-) MDSCs. Western blot further confirmed a down regulation of BMP2 signaling in Mstn (-/-) MDSCs in supportive of pluripotent reprogramming. Given that down regulation of BMP2 has been shown to induce pluripotency in cells, we propose that lack of myostatin epigenetically reprograms the MDSCs to become pluripotent stem cells. Thus, here we report the successful establishment of ES-like cells from adult stem cells of the non-germline origin under culture-induced conditions without introducing reprogramming genes.

Robust Differentiation of mRNA-Reprogrammed Human Induced Pluripotent Stem Cells Toward a Retinal Lineage.

PubMed

Sridhar, Akshayalakshmi; Ohlemacher, Sarah K; Langer, Kirstin B; Meyer, Jason S

2016-04-01

The derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has allowed for the development of novel approaches to studies of human development and disease. However, traditional methods of generating hiPSCs involve the risks of genomic integration and potential constitutive expression of pluripotency factors and often exhibit low reprogramming efficiencies. The recent description of cellular reprogramming using synthetic mRNA molecules might eliminate these shortcomings; however, the ability of mRNA-reprogrammed hiPSCs to effectively give rise to retinal cell lineages has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNA-reprogrammed hiPSCs to yield retinal cell types in a directed, stepwise manner. hiPSCs were generated from human fibroblasts via mRNA reprogramming, with parallel cultures of isogenic human fibroblasts reprogrammed via retroviral delivery of reprogramming factors. New lines of mRNA-reprogrammed hiPSCs were established and were subsequently differentiated into a retinal fate using established protocols in a directed, stepwise fashion. The efficiency of retinal differentiation from these lines was compared with retroviral-derived cell lines at various stages of development. On differentiation, mRNA-reprogrammed hiPSCs were capable of robust differentiation to a retinal fate, including the derivation of photoreceptors and retinal ganglion cells, at efficiencies often equal to or greater than their retroviral-derived hiPSC counterparts. Thus, given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes, such methods likely represent a promising new approach for retinal stem cell research, in particular, those for translational applications. In the current report, the ability to derive mRNA-reprogrammed human induced pluripotent stem cells (hiPSCs), followed by the differentiation of these cells toward a retinal lineage, including photoreceptors, retinal ganglion cells, and retinal pigment epithelium, has been demonstrated. The use of mRNA reprogramming to yield pluripotency represents a unique ability to derive pluripotent stem cells without the use of DNA vectors, ensuring the lack of genomic integration and constitutive expression. The studies reported in the present article serve to establish a more reproducible system with which to derive retinal cell types from hiPSCs through the prevention of genomic integration of delivered genes and should also eliminate the risk of constitutive expression of these genes. Such ability has important implications for the study of, and development of potential treatments for, retinal degenerative disorders and the development of novel therapeutic approaches to the treatment of these diseases. ©AlphaMed Press.

Biophysical regulation of epigenetic state and cell reprogramming

NASA Astrophysics Data System (ADS)

Downing, Timothy L.; Soto, Jennifer; Morez, Constant; Houssin, Timothee; Fritz, Ashley; Yuan, Falei; Chu, Julia; Patel, Shyam; Schaffer, David V.; Li, Song

2013-12-01

Biochemical factors can help reprogram somatic cells into pluripotent stem cells, yet the role of biophysical factors during reprogramming is unknown. Here, we show that biophysical cues, in the form of parallel microgrooves on the surface of cell-adhesive substrates, can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells’ epigenetic state. Specifically, decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)—a subunit of H3 methyltranferase—by microgrooved surfaces lead to increased histone H3 acetylation and methylation. We also show that microtopography promotes a mesenchymal-to-epithelial transition in adult fibroblasts. Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves, suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state. These findings have important implications in cell biology and in the optimization of biomaterials for cell-engineering applications.

Direct Reprogramming of Human Amniotic Fluid Stem Cells by OCT4 and Application in Repairing of Cerebral Ischemia Damage

PubMed Central

Qin, Mingde; Chen, Ruihua; Li, Hong; Liang, Hansi; Xue, Qun; Li, Fang; Chen, Ying; Zhang, Xueguang

2016-01-01

Amniotic fluid stem cells (AFSCs) are a type of fetal stem cell whose stemness encompasses both embryonic and adult stem cells, suggesting that they may be easily and efficiently reprogrammed into induced pluripotent stem cells (iPSCs). To further simplify the reprogramming process, the creation of AFSC-derived iPSCs using a single factor is desirable. Here we report the generation of one-factor human AFSC-iPSCs (AiPSCs) from human AFSCs by ectopic expression of the transcription factor OCT4. Just like human embryonic stem cells, AiPSCs exhibited similar epigenetic status, global gene expression profiles, teratoma formation and in vitro & in vivo pluripotency. Our results indicate that the OCT4 is necessary and sufficient to directly reprogram human AFSCs into pluripotent AiPSCs. Moreover, reflecting the similar memory characteristics of AFSCs and neural stem cells, we show that AiPSC membrane-derived vesicles (MVs) repair cerebral ischemia damage. We anticipate that the successful generation of one-factor AiPSCs will facilitate the creation of patient-specific pluripotent stem cells without the need for transgenic expression of oncogenes. Moreover, MVs from tissue-specific AiPSCs have potential in tissue repair, representing a novel application of iPSCs. PMID:27019637

Generation and genetic engineering of human induced pluripotent stem cells using designed zinc finger nucleases.

PubMed

Ramalingam, Sivaprakash; London, Viktoriya; Kandavelou, Karthikeyan; Cebotaru, Liudmila; Guggino, William; Civin, Curt; Chandrasegaran, Srinivasan

2013-02-15

Zinc finger nucleases (ZFNs) have become powerful tools to deliver a targeted double-strand break at a pre-determined chromosomal locus in order to insert an exogenous transgene by homology-directed repair. ZFN-mediated gene targeting was used to generate both single-allele chemokine (C-C motif) receptor 5 (CCR5)-modified human induced pluripotent stem cells (hiPSCs) and biallele CCR5-modified hiPSCs from human lung fibroblasts (IMR90 cells) and human primary cord blood mononuclear cells (CBMNCs) by site-specific insertion of stem cell transcription factor genes flanked by LoxP sites into the endogenous CCR5 locus. The Oct4 and Sox2 reprogramming factors, in combination with valproic acid, induced reprogramming of human lung fibroblasts to form CCR5-modified hiPSCs, while 5 factors, Oct4/Sox2/Klf4/Lin28/Nanog, induced reprogramming of CBMNCs. Subsequent Cre recombinase treatment of the CCR5-modified IMR90 hiPSCs resulted in the removal of the Oct4 and Sox2 transgenes. Further genetic engineering of the single-allele CCR5-modified IMR90 hiPSCs was achieved by site-specific addition of the large CFTR transcription unit to the remaining CCR5 wild-type allele, using CCR5-specific ZFNs and a donor construct containing tdTomato and CFTR transgenes flanked by CCR5 homology arms. CFTR was expressed efficiently from the endogenous CCR5 locus of the CCR5-modified tdTomato/CFTR hiPSCs. These results suggest that it might be feasible to use ZFN-evoked strategies to (1) generate precisely targeted genetically well-defined patient-specific hiPSCs, and (2) then to reshape their function by targeted addition and expression of therapeutic genes from the CCR5 chromosomal locus for autologous cell-based transgene-correction therapy to treat various recessive monogenic human diseases in the future.

Direct Cardiac Reprogramming as a Novel Therapeutic Strategy for Treatment of Myocardial Infarction.

PubMed

Ma, Hong; Wang, Li; Liu, Jiandong; Qian, Li

2017-01-01

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds great promise as a novel therapy for the treatment of heart failure, a common and morbid disease that is usually caused by irreversible loss of functional cardiomyocytes (CMs). Recently, we and others showed that in a murine model of acute myocardial infarction, delivery of three transcription factors, Gata4, Mef2c, and Tbx5 converted endogenous cardiac fibroblasts into functional iCMs. These iCMs integrated electrically and mechanically with surrounding myocardium, resulting in a reduction in scar size and an improvement in heart function. Our findings suggest that iCM reprogramming may be a means of regenerating functional CMs in vivo for patients with heart disease. However, because relatively little is known about the factors that regulate iCM reprogramming, the applicability of iCM reprogramming is currently limited to the experimental settings in which it has been attempted. Specific hurdles include the relatively low conversion rate of iCMs and the need for reprogramming to occur in the context of acute injury. Therefore, before this treatment can become a viable therapy for human heart disease, the optimal condition for efficient iCM generation must be determined. Here, we provide a detailed protocol for both in vitro and in vivo iCM generation that has been optimized so far in our lab. We hope that this protocol will lay a foundation for future further improvement of iCM generation and provide a platform for mechanistic studies.

Simple and effective generation of transgene-free induced pluripotent stem cells using an auto-erasable Sendai virus vector responding to microRNA-302.

PubMed

Nishimura, Ken; Ohtaka, Manami; Takada, Hitomi; Kurisaki, Akira; Tran, Nhi Vo Kieu; Tran, Yen Thi Hai; Hisatake, Koji; Sano, Masayuki; Nakanishi, Mahito

2017-08-01

Transgene-free induced pluripotent stem cells (iPSCs) are valuable for both basic research and potential clinical applications. We previously reported that a replication-defective and persistent Sendai virus (SeVdp) vector harboring four reprogramming factors (SeVdp-iPS) can efficiently induce generation of transgene-free iPSCs. This vector can express all four factors stably and simultaneously without chromosomal integration and can be eliminated completely from reprogrammed cells by suppressing vector-derived RNA-dependent RNA polymerase. Here, we describe an improved SeVdp-iPS vector (SeVdp(KOSM)302L) that is automatically erased in response to microRNA-302 (miR-302), uniquely expressed in pluripotent stem cells (PSCs). Gene expression and genome replication of the SeVdp-302L vector, which contains miRNA-302a target sequences at the 3' untranslated region of L mRNA, are strongly suppressed in PSCs. Consequently, SeVdp(KOSM)302L induces expression of reprogramming factors in somatic cells, while it is automatically erased from cells successfully reprogrammed to express miR-302. As this vector can reprogram somatic cells into transgene-free iPSCs without the aid of exogenous short interfering RNA (siRNA), the results we present here demonstrate that this vector may become an invaluable tool for the generation of human iPSCs for future clinical applications. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Concise review: reprogramming strategies for cardiovascular regenerative medicine: from induced pluripotent stem cells to direct reprogramming.

PubMed

Budniatzky, Inbar; Gepstein, Lior

2014-04-01

Myocardial cell-replacement therapies are emerging as novel therapeutic paradigms for myocardial repair but are hampered by the lack of sources of autologous human cardiomyocytes. The recent advances in stem cell biology and in transcription factor-based reprogramming strategies may provide exciting solutions to this problem. In the current review, we describe the different reprogramming strategies that can give rise to cardiomyocytes for regenerative medicine purposes. Initially, we describe induced pluripotent stem cell technology, a method by which adult somatic cells can be reprogrammed to yield pluripotent stem cells that could later be coaxed ex vivo to differentiate into cardiomyocytes. The generated induced pluripotent stem cell-derived cardiomyocytes could then be used for myocardial cell transplantation and tissue engineering strategies. We also describe the more recent direct reprogramming approaches that aim to directly convert the phenotype of one mature cell type (fibroblast) to another (cardiomyocyte) without going through a pluripotent intermediate cell type. The advantages and shortcomings of each strategy for cardiac regeneration are discussed, along with the hurdles that need to be overcome on the road to clinical translation.

Direct In Vivo Reprogramming with Sendai Virus Vectors Improves Cardiac Function after Myocardial Infarction.

PubMed

Miyamoto, Kazutaka; Akiyama, Mizuha; Tamura, Fumiya; Isomi, Mari; Yamakawa, Hiroyuki; Sadahiro, Taketaro; Muraoka, Naoto; Kojima, Hidenori; Haginiwa, Sho; Kurotsu, Shota; Tani, Hidenori; Wang, Li; Qian, Li; Inoue, Makoto; Ide, Yoshinori; Kurokawa, Junko; Yamamoto, Tsunehisa; Seki, Tomohisa; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2018-01-04

Direct cardiac reprogramming holds great promise for regenerative medicine. We previously generated directly reprogrammed induced cardiomyocyte-like cells (iCMs) by overexpression of Gata4, Mef2c, and Tbx5 (GMT) using retrovirus vectors. However, integrating vectors pose risks associated with insertional mutagenesis and disruption of gene expression and are inefficient. Here, we show that Sendai virus (SeV) vectors expressing cardiac reprogramming factors efficiently and rapidly reprogram both mouse and human fibroblasts into integration-free iCMs via robust transgene expression. SeV-GMT generated 100-fold more beating iCMs than retroviral-GMT and shortened the duration to induce beating cells from 30 to 10 days in mouse fibroblasts. In vivo lineage tracing revealed that the gene transfer of SeV-GMT was more efficient than retroviral-GMT in reprogramming resident cardiac fibroblasts into iCMs in mouse infarct hearts. Moreover, SeV-GMT improved cardiac function and reduced fibrosis after myocardial infarction. Thus, efficient, non-integrating SeV vectors may serve as a powerful system for cardiac regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Induced Pluripotent Stem Cell Technology in Regenerative Medicine and Biology

NASA Astrophysics Data System (ADS)

Pei, Duanqing; Xu, Jianyong; Zhuang, Qiang; Tse, Hung-Fat; Esteban, Miguel A.

The potential of human embryonic stem cells (ESCs) for regenerative medicine is unquestionable, but practical and ethical considerations have hampered clinical application and research. In an attempt to overcome these issues, the conversion of somatic cells into pluripotent stem cells similar to ESCs, commonly termed nuclear reprogramming, has been a top objective of contemporary biology. More than 40 years ago, King, Briggs, and Gurdon pioneered somatic cell nuclear reprogramming in frogs, and in 1981 Evans successfully isolated mouse ESCs. In 1997 Wilmut and collaborators produced the first cloned mammal using nuclear transfer, and then Thomson obtained human ESCs from in vitro fertilized blastocysts in 1998. Over the last 2 decades we have also seen remarkable findings regarding how ESC behavior is controlled, the importance of which should not be underestimated. This knowledge allowed the laboratory of Shinya Yamanaka to overcome brilliantly conceptual and technical barriers in 2006 and generate induced pluripotent stem cells (iPSCs) from mouse fibroblasts by overexpressing defined combinations of ESC-enriched transcription factors. Here, we discuss some important implications of human iPSCs for biology and medicine and also point to possible future directions.

Identification of Ccr4-Not Complex Components as Regulators of Transition from Partial to Genuine Induced Pluripotent Stem Cells

PubMed Central

Kamon, Masayoshi; Katano, Miyuki; Hiraki-Kamon, Keiko; Hishida, Tomoaki; Nakachi, Yutaka; Mizuno, Yosuke; Okazaki, Yasushi; Suzuki, Ayumu; Hirasaki, Masataka; Ueda, Atsushi; Nishimoto, Masazumi; Kato, Hidemasa

2014-01-01

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by defined factors. However, substantial cell numbers subjected to iPSC induction stray from the main reprogramming route and are immortalized as partial iPSCs. These partial iPSCs can become genuine iPSCs by exposure to the ground state condition. However, such conversion is only possible for mouse partial iPSCs, and it is not applicable to human cells. Moreover, the molecular basis of this conversion is completely unknown. Therefore, we performed genome-wide screening with a piggyBac vector to identify genes involved in conversion from partial to genuine iPSCs. This screening led to identification of Cnot2, one of the core components of the Ccr4-Not complex. Subsequent analyses revealed that other core components, Cnot1 and Cnot3, also contributed to the conversion. Thus, our data have uncovered a novel role of core components of the Ccr4-Not complex as regulators of transition from partial to genuine iPSCs. PMID:24200330

Proteomic Analysis of Mouse Oocytes Identifies PRMT7 as a Reprogramming Factor that Replaces SOX2 in the Induction of Pluripotent Stem Cells.

PubMed

Wang, Bingyuan; Pfeiffer, Martin J; Drexler, Hannes C A; Fuellen, Georg; Boiani, Michele

2016-08-05

The reprogramming process that leads to induced pluripotent stem cells (iPSCs) may benefit from adding oocyte factors to Yamanaka's reprogramming cocktail (OCT4, SOX2, KLF4, with or without MYC; OSK(M)). We previously searched for such facilitators of reprogramming (the reprogrammome) by applying label-free LC-MS/MS analysis to mouse oocytes, producing a catalog of 28 candidates that are (i) able to robustly access the cell nucleus and (ii) shared between mature mouse oocytes and pluripotent embryonic stem cells. In the present study, we hypothesized that our 28 reprogrammome candidates would also be (iii) abundant in mature oocytes, (iv) depleted after the oocyte-to-embryo transition, and (v) able to potentiate or replace the OSKM factors. Using LC-MS/MS and isotopic labeling methods, we found that the abundance profiles of the 28 proteins were below those of known oocyte-specific and housekeeping proteins. Of the 28 proteins, only arginine methyltransferase 7 (PRMT7) changed substantially during mouse embryogenesis and promoted the conversion of mouse fibroblasts into iPSCs. Specifically, PRMT7 replaced SOX2 in a factor-substitution assay, yielding iPSCs. These findings exemplify how proteomics can be used to prioritize the functional analysis of reprogrammome candidates. The LC-MS/MS data are available via ProteomeXchange with identifier PXD003093.

Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells.

PubMed

Tammam, Salma; Malak, Peter; Correa, Daphne; Rothfuss, Oliver; Azzazy, Hassan M E; Lamprecht, Alf; Schulze-Osthoff, Klaus

2016-06-21

Protein-based reprogramming of somatic cells is a non-genetic approach for the generation of induced pluripotent stem cells (iPSCs), whereby reprogramming factors, such as OCT4, SOX2, KLF4 and c-MYC, are delivered as functional proteins. The technique is considered safer than transgenic methods, but, unfortunately, most protein-based protocols provide very low reprogramming efficiencies. In this study, we developed exemplarily a nanoparticle (NP)-based delivery system for the reprogramming factor OCT4. To this end, we expressed human OCT4 in Sf9 insect cells using a baculoviral expression system. Recombinant OCT4 showed nuclear localization in Sf9 cells indicating proper protein folding. In comparison to soluble OCT4 protein, encapsulation of OCT4 in nuclear-targeted chitosan NPs strongly stabilized its DNA-binding activity even under cell culture conditions. OCT4-loaded NPs enabled cell treatment with high micromolar concentrations of OCT4 and successfully delivered active OCT4 into human fibroblasts. Chitosan NPs therefore provide a promising tool for the generation of transgene-free iPSCs.

Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

PubMed Central

Rodriguez-Osorio, Nelida; Wang, Zhongde; Kasinathan, Poothappillai; Page, Grier P; Robl, James M; Memili, Erdogan

2009-01-01

Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT). Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively) have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF) than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively). However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research. PMID:19393066

Regenerating the human heart: direct reprogramming strategies and their current limitations.

PubMed

Ghiroldi, Andrea; Piccoli, Marco; Ciconte, Giuseppe; Pappone, Carlo; Anastasia, Luigi

2017-10-27

Cardiovascular diseases are the leading cause of death in the Western world. Unfortunately, current therapies are often only palliative, consequently essentially making heart transplantation necessary for many patients. However, several novel therapeutic approaches in the past two decades have yielded quite encouraging results. The generation of induced pluripotent stem cells, through the forced expression of stem cell-specific transcription factors, has inspired the most promising strategies for heart regeneration by direct reprogramming of cardiac fibroblasts into functional cardiomyocytes. Initial attempts at this reprogramming were conducted using a similar approach to the one used with transcription factors, but during years, novel strategies have been tested, e.g., miRNAs, recombinant proteins and chemical molecules. Although preliminary results on animal models are promising, the low reprogramming efficiency, as well as the incomplete maturation of the cardiomyocytes, still represents important obstacles. This review covers direct transdifferentiation strategies that have been proposed and developed and illustrates the pros and cons of each approach. Indeed, as described in the manuscript, there are still many unanswered questions and drawbacks that require a better understanding of the basic signaling pathways and transcription factor networks before functional cells, suitable for cardiac regeneration and safe for the patients, can be generated and used for human therapies.

Porcine induced pluripotent stem cells produce chimeric offspring.

PubMed

West, Franklin D; Terlouw, Steve L; Kwon, Dae Jin; Mumaw, Jennifer L; Dhara, Sujoy K; Hasneen, Kowser; Dobrinsky, John R; Stice, Steven L

2010-08-01

Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology.

Fractional Factorial Design to Investigate Stromal Cell Regulation of Macrophage Plasticity

PubMed Central

Barminko, Jeffrey A.; Nativ, Nir I.; Schloss, Rene; Yarmush, Martin L.

2018-01-01

Understanding the regulatory networks which control specific macrophage phenotypes is essential in identifying novel targets to correct macrophage mediated clinical disorders, often accompanied by inflammatory events. Since mesenchymal stromal cells (MSCs) have been shown to play key roles in regulating immune functions predominantly via a large number of secreted products, we used a fractional factorial approach to streamline experimental evaluation of MSC mediated inflammatory macrophage regulation. Our macrophage reprogramming metrics, human bone marrow MSC attenuation of macrophage pro-inflammatory M1 TNFα secretion and simultaneous enhanced expression of the M2 macrophage marker, CD206, were used as analysis endpoints. Objective evaluation of a panel of MSC secreted mediators indicated that PGE2 alone was sufficient in facilitating macrophage reprogramming, while IL4 only provided partial reprogramming. Inhibiting stromal cell PGE2 secretion with Indomethacin, reversed the macrophage reprogramming effect. PGE2 reprogramming was mediated through the EP4 receptor and indirectly through the CREB signaling pathway as GSK3 specific inhibitors induced M1 macrophages to express CD206. This reprogramming pathway functioned independently from the M1 suppression pathway, as neither CREB nor GSK3 inhibition reversed PGE2 TNF-α secretion attenuation. In conclusion, fractional factorial experimental design identified stromal derived PGE2 as the factor most important in facilitating macrophage reprogramming, albeit via two unique pathways. PMID:24891120

SIRT3 opposes reprogramming of cancer cell metabolism through HIF1α destabilization

PubMed Central

Finley, Lydia W.S.; Carracedo, Arkaitz; Lee, Jaewon; Souza, Amanda; Egia, Ainara; Zhang, Jiangwen; Teruya-Feldstein, Julie; Moreira, Paula I.; Cardoso, Sandra M.; Clish, Clary B.; Pandolfi, Pier Paolo; Haigis, Marcia C.

2011-01-01

Summary Tumor cells exhibit aberrant metabolism characterized by high glycolysis even in the presence of oxygen. This metabolic reprogramming, known as the Warburg effect, provides tumor cells with the substrates required for biomass generation. Here, we show that the mitochondrial NAD-dependent deacetylase SIRT3 is a crucial regulator of the Warburg effect. Mechanistically, SIRT3 mediates metabolic reprogramming by destabilizing hypoxia-inducible factor-1α (HIF1α), a transcription factor that controls glycolytic gene expression. SIRT3 loss increases reactive oxygen species production, leading to HIF1α stabilization. SIRT3 expression is reduced in human breast cancers, and its loss correlates with the upregulation of HIF1α target genes. Finally, we find that SIRT3 overexpression represses glycolysis and proliferation in breast cancer cells, providing a metabolic mechanism for tumor suppression. PMID:21397863

Alternative sources of pluripotency: science, ethics, and stem cells.

PubMed

Kastenberg, Zachary J; Odorico, Jon S

2008-07-01

Despite many advances in human embryonic stem cell (hESC) technology the ethical dilemma involving the destruction of a human embryo is one factor that has limited the development of hESC based clinical therapies. Two recent reports describing the production of pluripotent stem cells following the in vitro reprogramming of human somatic cells with certain defined factors illustrate one potential method of bypassing the ethical debate surrounding hESCs (Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007 Dec;318(5858):1917-1920; Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007 Nov;131(5): 861-872.). Other alternative methods include nuclear transfer, altered nuclear transfer, and parthenogenesis; each with its own set of advantages and disadvantages. This review discusses recent advances in these technologies with specific focus on the issues of embryo destruction, oocyte recovery, and the potential of each technology to produce large scale, patient specific cell transplantation therapies that would require little or no immunosuppression.

Reprogramming: A Preventive Strategy in Hypertension Focusing on the Kidney

PubMed Central

Tain, You-Lin; Joles, Jaap A.

2015-01-01

Adulthood hypertension can be programmed in response to a suboptimal environment in early life. However, developmental plasticity also implies that one can prevent hypertension in adult life by administrating appropriate compounds during early development. We have termed this reprogramming. While the risk of hypertension has been assessed in many mother-child cohorts of human developmental programming, interventions necessary to prove causation and provide a reprogramming strategy are lacking. Since the developing kidney is particularly vulnerable to environmental insults and blood pressure is determined by kidney function, renal programming is considered key in developmental programming of hypertension. Common pathways, whereby both genetic and acquired developmental programming converge into the same phenotype, have been recognized. For instance, the same reprogramming interventions aimed at shifting nitric oxide (NO)-reactive oxygen species (ROS) balance, such as perinatal citrulline or melatonin supplements, can be protective in both genetic and developmentally programmed hypertension. Furthermore, a significantly increased expression of gene Ephx2 (soluble epoxide hydrolase) was noted in both genetic and acquired animal models of hypertension. Since a suboptimal environment is often multifactorial, such common reprogramming pathways are a practical finding for translation to the clinic. This review provides an overview of potential clinical applications of reprogramming strategies to prevent programmed hypertension. We emphasize the kidney in the following areas: mechanistic insights from human studies and animal models to interpret programmed hypertension; identified risk factors of human programmed hypertension from mother-child cohorts; and the impact of reprogramming strategies on programmed hypertension from animal models. It is critical that the observed effects on developmental reprogramming in animal models are replicated in human studies. PMID:26712746

Optimization of Direct Fibroblast Reprogramming to Cardiomyocytes Using Calcium Activity as a Functional Measure of Success

PubMed Central

Addis, Russell C.; Ifkovits, Jamie L.; Pinto, Filipa; Kellam, Lori D.; Esteso, Paul; Rentschler, Stacey; Christoforou, Nicolas; Epstein, Jonathan A.; Gearhart, John D.

2013-01-01

Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persists for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods. PMID:23591016

A site-specific genetic modification for induction of pluripotency and subsequent isolation of derived lung alveolar epithelial type II cells.

PubMed

Yan, Qing; Quan, Yuan; Sun, Huanhuan; Peng, Xinmiao; Zou, Zhengyun; Alcorn, Joseph L; Wetsel, Rick A; Wang, Dachun

2014-02-01

Human induced pluripotent stem cells (hiPSCs) have great therapeutic potential in repairing defective lung alveoli. However, genetic abnormalities caused by vector integrations and low efficiency in generating hiPSCs, as well as difficulty in obtaining transplantable hiPSC-derived cell types are still major obstacles. Here we report a novel strategy using a single nonviral site-specific targeting vector with a combination of Tet-On inducible gene expression system, Cre/lox P switching gene expression system, and alveolar epithelial type II cell (ATIIC)-specific Neomycin(R) transgene expression system. With this strategy, a single copy of all of the required transgenes can be specifically knocked into a site immediately downstream of β-2-microglobulin (B2M) gene locus at a high frequency, without causing B2M dysfunction. Thus, the expression of reprogramming factors, Oct4, Sox2, cMyc, and Klf4, can be precisely regulated for efficient reprogramming of somatic cells into random integration-free or genetic mutation-free hiPSCs. The exogenous reprogramming factor transgenes can be subsequently removed after reprogramming by transient expression of Cre recombinase, and the resulting random integration-free and exogenous reprogramming factor-free hiPSCs can be selectively differentiated into a homogenous population of ATIICs. In addition, we show that these hiPSC-derived ATIICs exhibit ultrastructural characteristics and biological functions of normal ATIICs. When transplanted into bleomycin-challenged mice lungs, hiPSC-derived ATIICs efficiently remain and re-epithelialize injured alveoli to restore pulmonary function, preventing lung fibrosis and increasing survival without tumorigenic side effect. This strategy allows for the first time efficient generation of patient-specific ATIICs for possible future clinical applications. © 2013 AlphaMed Press.

A site-specific genetic modification for induction of pluripotency and subsequent isolation of derived lung alveolar epithelial type II cells

PubMed Central

Yan, Qing; Quan, Yuan; Sun, Huanhuan; Peng, Xinmiao; Zou, Zhengyun; Alcorn, Joseph L.; Wetsel, Rick A.; Wang, Dachun

2013-01-01

Human induced pluripotent stem cells (hiPSCs) have great therapeutic potential in repairing defective lung alveoli. However, genetic abnormalities caused by vector-integrations and low efficiency in generating hiPSCs, as well as difficulty in obtaining transplantable hiPSC-derived cell types, are still major obstacles. Here we report a novel strategy using a single non-viral site-specific-targeting vector with a combination of Tet-On inducible gene expression system, Cre/lox P switching gene expression system, and alveolar epithelial type II cell (ATIIC)-specific NeomycinR trangene expression system. With this strategy, a single copy of all of the required transgenes can be specifically knocked into a site immediately downstream of beta-2-microglobulin (B2M) gene locus at a high frequency, without causing B2M dysfunction. Thus, the expression of reprogramming factors, Oct4, Sox2, cMyc and Klf4, can be precisely regulated for efficient reprogramming of somatic cells into random-integration-free or genetic mutation-free hiPSCs. The exogenous reprogramming factor transgenes can be subsequently removed after reprogramming by transient expression of Cre recombinase, and the resulting random-integration-free and exogenous reprogramming-factor-free hiPSCs can be selectively differentiated into a homogenous population of ATIICs. In addition, we show that these hiPSC-derived ATIICs exhibit ultra-structural characteristics and biological functions of normal ATIICs. When transplanted into bleomycin-challenged mice lungs, hiPSC-derived ATIICs efficiently remain and re-epithelialize injured alveoli to restore pulmonary function, preventing lung fibrosis and increasing survival without tumorigenic side effect. This strategy allows for the first time efficient generation of patient-specific ATIICs for possible future clinical applications. PMID:24123810

Chick derived induced pluripotent stem cells by the poly-cistronic transposon with enhanced transcriptional activity.

PubMed

Katayama, Masafumi; Hirayama, Takashi; Tani, Tetsuya; Nishimori, Katsuhiko; Onuma, Manabu; Fukuda, Tomokazu

2018-02-01

Induced pluripotent stem (iPS) cell technology lead terminally differentiated cells into the pluripotent stem cells through the expression of defined reprogramming factors. Although, iPS cells have been established in a number of mammalian species, including mouse, human, and monkey, studies on iPS cells in avian species are still very limited. To establish chick iPS cells, six factors were used within the poly-cistronic reprogramming vector (PB-R6F), containing M3O (MyoD derived transactivation domain fused with Oct3/4), Sox2, Klf4, c-Myc, Lin28, and Nanog. The PB-R6F derived iPS cells were alkaline-phosphatase and SSEA-1 positive, which are markers of pluripotency. Elevated levels of endogenous Oct3/4 and Nanog genes were detected in the established iPS cells, suggesting the activation of the FGF signaling pathway is critical for the pluripotent status. Histological analysis of teratoma revealed that the established chick iPS cells have differentiation ability into three-germ-layer derived tissues. This is the first report of establishment of avian derived iPS cells with a single poly-cistronic transposon based expression system. The establishment of avian derived iPS cells could contribute to the genetic conservation and modification of avian species. © 2017 Wiley Periodicals, Inc.

Defining Translational Reprogramming in Tuberous Sclerosis Complex

DTIC Science & Technology

2015-07-01

Genomics & Development, Cornell University, Ithaca, New York, USA. 3Department of Chemistry, The Scripps Research Inst tute, Jupiter , Florida, USA...4Molecular Therapeutics and Natural Products Library Initiative, The Scripps Research Institute, Jupiter , Florida, USA. 5Present address

Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

PubMed Central

Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

2016-01-01

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells.

PubMed

Trevisan, Marta; Desole, Giovanna; Costanzi, Giulia; Lavezzo, Enrico; Palù, Giorgio; Barzon, Luisa

2017-01-20

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.

MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

PubMed

Muraoka, Naoto; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Isomi, Mari; Nakashima, Hanae; Akiyama, Mizuha; Wada, Rie; Inagawa, Kohei; Nishiyama, Takahiko; Kaneda, Ruri; Fukuda, Toru; Takeda, Shu; Tohyama, Shugo; Hashimoto, Hisayuki; Kawamura, Yoshifumi; Goshima, Naoki; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2014-07-17

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming. © 2014 The Authors.

MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures

PubMed Central

Muraoka, Naoto; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Isomi, Mari; Nakashima, Hanae; Akiyama, Mizuha; Wada, Rie; Inagawa, Kohei; Nishiyama, Takahiko; Kaneda, Ruri; Fukuda, Toru; Takeda, Shu; Tohyama, Shugo; Hashimoto, Hisayuki; Kawamura, Yoshifumi; Goshima, Naoki; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2014-01-01

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming. PMID:24920580

MFG-E8 Reprogramming of Macrophages Promotes Wound Healing by Increased bFGF Production and Fibroblast Functions.

PubMed

Laplante, Patrick; Brillant-Marquis, Frédéric; Brissette, Marie-Joëlle; Joannette-Pilon, Benjamin; Cayrol, Romain; Kokta, Victor; Cailhier, Jean-François

2017-09-01

Macrophages are essential for tissue repair. They have a crucial role in cutaneous wound healing, participating actively in the inflammation phase of the process. Unregulated macrophage activation may, however, represent a source of excessive inflammation, leading to abnormal wound healing and hypertrophic scars. Our research group has shown that apoptotic endothelial and epithelial cells secrete MFG-E8, which has the ability to reprogram macrophages from an M1 (proinflammatory) to an M2 (anti-inflammatory, pro-repair) phenotype. Hence, we tested whether modulation of macrophage reprogramming would promote tissue repair. Using a mouse model of wound healing, we showed that the presence and/or addition of MFG-E8 favors wound closure associated with an increase in CD206-positive cells and basic fibroblast growth factor production in healing tissues. More importantly, adoptive transfer of ex vivo MFG-E8-treated macrophages promoted wound closure. We also observed that MFG-E8-treated macrophages produced basic fibroblast growth factor that is responsible for fibroblast migration and proliferation. Taken together, our results strongly suggest that MFG-E8 plays a key role in macrophage reprogramming in tissue healing through induction of an anti-inflammatory M2 phenotype and basic fibroblast growth factor production, leading to fibroblast migration and wound closure. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Enhancer Analysis Unveils Genetic Interactions between TLX and SOX2 in Neural Stem Cells and In Vivo Reprogramming.

PubMed

Islam, Mohammed M; Smith, Derek K; Niu, Wenze; Fang, Sanhua; Iqbal, Nida; Sun, Guoqiang; Shi, Yanhong; Zhang, Chun-Li

2015-11-10

The orphan nuclear receptor TLX is a master regulator of postnatal neural stem cell (NSC) self-renewal and neurogenesis; however, it remains unclear how TLX expression is precisely regulated in these tissue-specific stem cells. Here, we show that a highly conserved cis-element within the Tlx locus functions to drive gene expression in NSCs. We demonstrate that the transcription factors SOX2 and MYT1 specifically interact with this genomic element to directly regulate Tlx enhancer activity in vivo. Knockdown experiments further reveal that SOX2 dominantly controls endogenous expression of TLX, whereas MYT1 only plays a modulatory role. Importantly, TLX is essential for SOX2-mediated in vivo reprogramming of astrocytes and itself is also sufficient to induce neurogenesis in the adult striatum. Together, these findings unveil functional genetic interactions among transcription factors that are critical to NSCs and in vivo cell reprogramming.

Practical Integration-Free Episomal Methods for Generating Human Induced Pluripotent Stem Cells.

PubMed

Kime, Cody; Rand, Tim A; Ivey, Kathryn N; Srivastava, Deepak; Yamanaka, Shinya; Tomoda, Kiichiro

2015-10-06

The advent of induced pluripotent stem (iPS) cell technology has revolutionized biomedicine and basic research by yielding cells with embryonic stem (ES) cell-like properties. The use of iPS-derived cells for cell-based therapies and modeling of human disease holds great potential. While the initial description of iPS cells involved overexpression of four transcription factors via viral vectors that integrated within genomic DNA, advances in recent years by our group and others have led to safer and higher quality iPS cells with greater efficiency. Here, we describe commonly practiced methods for non-integrating induced pluripotent stem cell generation using nucleofection of episomal reprogramming plasmids. These methods are adapted from recent studies that demonstrate increased hiPS cell reprogramming efficacy with the application of three powerful episomal hiPS cell reprogramming factor vectors and the inclusion of an accessory vector expressing EBNA1. Copyright © 2015 John Wiley & Sons, Inc.

Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells

PubMed Central

Lee, Kunwoo; Yu, Pengzhi; Lingampalli, Nithya; Kim, Hyun Jin; Tang, Richard; Murthy, Niren

2015-01-01

The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation. PMID:25834424

High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins.

PubMed

Chen, Fanfan; Zhang, Guoqiang; Yu, Ling; Feng, Yanye; Li, Xianghui; Zhang, Zhijun; Wang, Yongting; Sun, Dapeng; Pradhan, Sriharsa

2016-07-30

Induced pluripotent mesenchymal stem cells (iPMSCs) are novel candidates for drug screening, regenerative medicine, and cell therapy. However, introduction of transcription factor encoding genes for induced pluripotent stem cell (iPSC) generation which could be used to generate mesenchymal stem cells is accompanied by the risk of insertional mutations in the target cell genome. We demonstrate a novel method using an inactivated viral particle to package and deliver four purified recombinant Yamanaka transcription factors (Sox2, Oct4, Klf4, and c-Myc) resulting in reprogramming of human primary fibroblasts. Whole genome bisulfite sequencing was used to analyze genome-wide CpG methylation of human iPMSCs. Western blot, quantitative PCR, immunofluorescence, and in-vitro differentiation were used to assess the pluripotency of iPMSCs. The resulting reprogrammed fibroblasts show high-level expression of stem cell markers. The human fibroblast-derived iPMSC genome showed gains in DNA methylation in low to medium methylated regions and concurrent loss of methylation in previously hypermethylated regions. Most of the differentially methylated regions are close to transcription start sites and many of these genes are pluripotent pathway associated. We found that DNA methylation of these genes is regulated by the four iPSC transcription factors, which functions as an epigenetic switch during somatic reprogramming as reported previously. These iPMSCs successfully differentiate into three embryonic germ layer cells, both in vitro and in vivo. Following multipotency induction in our study, the delivered transcription factors were degraded, leading to an improved efficiency of subsequent programmed differentiation. Recombinant transcription factor based reprogramming and derivatization of iPMSC offers a novel high-efficiency approach for regenerative medicine from patient-derived cells.

Crosstalk between Two bZIP Signaling Pathways Orchestrates Salt-Induced Metabolic Reprogramming in Arabidopsis Roots

PubMed Central

Hartmann, Laura; Pedrotti, Lorenzo; Weiste, Christoph; Fekete, Agnes; Schierstaedt, Jasper; Göttler, Jasmin; Kempa, Stefan; Krischke, Markus; Dietrich, Katrin; Mueller, Martin J.; Vicente-Carbajosa, Jesus; Hanson, Johannes; Dröge-Laser, Wolfgang

2015-01-01

Soil salinity increasingly causes crop losses worldwide. Although roots are the primary targets of salt stress, the signaling networks that facilitate metabolic reprogramming to induce stress tolerance are less understood than those in leaves. Here, a combination of transcriptomic and metabolic approaches was performed in salt-treated Arabidopsis thaliana roots, which revealed that the group S1 basic leucine zipper transcription factors bZIP1 and bZIP53 reprogram primary C- and N-metabolism. In particular, gluconeogenesis and amino acid catabolism are affected by these transcription factors. Importantly, bZIP1 expression reflects cellular stress and energy status in roots. In addition to the well-described abiotic stress response pathway initiated by the hormone abscisic acid (ABA) and executed by SnRK2 (Snf1-RELATED-PROTEIN-KINASE2) and AREB-like bZIP factors, we identify a structurally related ABA-independent signaling module consisting of SnRK1s and S1 bZIPs. Crosstalk between these signaling pathways recruits particular bZIP factor combinations to establish at least four distinct gene expression patterns. Understanding this signaling network provides a framework for securing future crop productivity. PMID:26276836

Nanos promotes epigenetic reprograming of the germline by down-regulation of the THAP transcription factor LIN-15B

PubMed Central

Lee, Chih-Yung Sean; Lu, Tu

2017-01-01

Nanos RNA-binding proteins are required for germline development in metazoans, but the underlying mechanisms remain poorly understood. We have profiled the transcriptome of primordial germ cells (PGCs) lacking the nanos homologs nos-1 and nos-2 in C. elegans. nos-1nos-2 PGCs fail to silence hundreds of transcripts normally expressed in oocytes. We find that this misregulation is due to both delayed turnover of maternal transcripts and inappropriate transcriptional activation. The latter appears to be an indirect consequence of delayed turnover of the maternally-inherited transcription factor LIN-15B, a synMuvB class transcription factor known to antagonize PRC2 activity. PRC2 is required for chromatin reprogramming in the germline, and the transcriptome of PGCs lacking PRC2 resembles that of nos-1nos-2 PGCs. Loss of maternal LIN-15B restores fertility to nos-1nos-2 mutants. These findings suggest that Nanos promotes germ cell fate by downregulating maternal RNAs and proteins that would otherwise interfere with PRC2-dependent reprogramming of PGC chromatin. PMID:29111977

Nanos promotes epigenetic reprograming of the germline by down-regulation of the THAP transcription factor LIN-15B.

PubMed

Lee, Chih-Yung Sean; Lu, Tu; Seydoux, Geraldine

2017-11-07

Nanos RNA-binding proteins are required for germline development in metazoans, but the underlying mechanisms remain poorly understood. We have profiled the transcriptome of primordial germ cells (PGCs) lacking the nanos homologs nos-1 and nos-2 in C. elegans. nos-1nos-2 PGCs fail to silence hundreds of transcripts normally expressed in oocytes. We find that this misregulation is due to both delayed turnover of maternal transcripts and inappropriate transcriptional activation. The latter appears to be an indirect consequence of delayed turnover of the maternally-inherited transcription factor LIN-15B, a synMuvB class transcription factor known to antagonize PRC2 activity. PRC2 is required for chromatin reprogramming in the germline, and the transcriptome of PGCs lacking PRC2 resembles that of nos-1nos-2 PGCs. Loss of maternal LIN-15B restores fertility to nos-1nos-2 mutants. These findings suggest that Nanos promotes germ cell fate by downregulating maternal RNAs and proteins that would otherwise interfere with PRC2-dependent reprogramming of PGC chromatin.

DNA methylation at a bovine alpha satellite I repeat CpG site during development following fertilization and somatic cell nuclear transfer.

PubMed

Couldrey, Christine; Wells, David N

2013-01-01

Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a differentiated somatic cell is reset to a state resembling totipotency, the efficiency of SCNT is likely to remain low.

DNA Methylation at a Bovine Alpha Satellite I Repeat CpG Site during Development following Fertilization and Somatic Cell Nuclear Transfer

PubMed Central

Couldrey, Christine; Wells, David N.

2013-01-01

Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a differentiated somatic cell is reset to a state resembling totipotency, the efficiency of SCNT is likely to remain low. PMID:23383311

JMJD3 aids in reprogramming of bone marrow progenitor cells to hepatic phenotype through epigenetic activation of hepatic transcription factors

PubMed Central

Kochat, Veena; Equbal, Zaffar; Baligar, Prakash; Kumar, Vikash; Srivastava, Madhulika; Mukhopadhyay, Asok

2017-01-01

The strictly regulated unidirectional differentiation program in some somatic stem/progenitor cells has been found to be modified in the ectopic site (tissue) undergoing regeneration. In these cases, the lineage barrier is crossed by either heterotypic cell fusion or direct differentiation. Though studies have shown the role of coordinated genetic and epigenetic mechanisms in cellular development and differentiation, how the lineage fate of adult bone marrow progenitor cells (BMPCs) is reprogrammed during liver regeneration and whether this lineage switch is stably maintained are not clearly understood. In the present study, we wanted to decipher genetic and epigenetic mechanisms that involve in lineage reprogramming of BMPCs into hepatocyte-like cells. Here we report dynamic transcriptional change during cellular reprogramming of BMPCs to hepatocytes and dissect the epigenetic switch mechanism of BM cell-mediated liver regeneration after acute injury. Genome-wide gene expression analysis in BM-derived hepatocytes, isolated after 1 month and 5 months of transplantation, showed induction of hepatic transcriptional program and diminishing of donor signatures over the time. The transcriptional reprogramming of BM-derived cells was found to be the result of enrichment of activating marks (H3K4me3 and H3K9Ac) and loss of repressive marks (H3K27me3 and H3K9me3) at the promoters of hepatic transcription factors (HTFs). Further analyses showed that BMPCs possess bivalent histone marks (H3K4me3 and H3K27me3) at the promoters of crucial HTFs. H3K27 methylation dynamics at the HTFs was antagonistically regulated by EZH2 and JMJD3. Preliminary evidence suggests a role of JMJD3 in removal of H3K27me3 mark from promoters of HTFs, thus activating epigenetically poised hepatic genes in BMPCs prior to partial nuclear reprogramming. The importance of JMJD3 in reprogramming of BMPCs to hepatic phenotype was confirmed by inhibiting catalytic function of the enzyme using small molecule GSK-J4. Our results propose a potential role of JMJD3 in lineage conversion of BM cells into hepatic lineage. PMID:28328977

Receptor Signaling Directs Global Recruitment of Pre-existing Transcription Factors to Inducible Elements.

PubMed

Cockerill, Peter N

2016-12-01

Gene expression programs are largely regulated by the tissue-specific expression of lineage-defining transcription factors or by the inducible expression of transcription factors in response to specific stimuli. Here I will review our own work over the last 20 years to show how specific activation signals also lead to the wide-spread re-distribution of pre-existing constitutive transcription factors to sites undergoing chromatin reorganization. I will summarize studies showing that activation of kinase signaling pathways creates open chromatin regions that recruit pre-existing factors which were previously unable to bind to closed chromatin. As models I will draw upon genes activated or primed by receptor signaling in memory T cells, and genes activated by cytokine receptor mutations in acute myeloid leukemia. I also summarize a hit-and-run model of stable epigenetic reprograming in memory T cells, mediated by transient Activator Protein 1 (AP-1) binding, which enables the accelerated activation of inducible enhancers.

Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ostrup, Olga, E-mail: osvarcova@gmail.com; Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo; Norwegian Center for Stem Cell Research, Oslo

Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression.more » This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.« less

Efficient Production of Retroviruses Using PLGA/bPEI-DNA Nanoparticles and Application for Reprogramming Somatic Cells

PubMed Central

Do, Eun Kyoung; Cheon, Hyo Cheon; Heo, Soon Chul; Kwon, Yang Woo; Jeong, Geun Ok; Kim, Ba Reun; Kim, Jae Ho

2013-01-01

Reprogramming of somatic cells to pluripotent cells requires the introduction of factors driving fate switches. Viral delivery has been the most efficient method for generation of induced pluripotent stem cells. Transfection, which precedes virus production, is a commonly-used process for delivery of nucleic acids into cells. The aim of this study is to evaluate the efficiency of PLGA/ bPEI nanoparticles in transfection and virus production. Using a modified method of producing PLGA nanoparticles, PLGA/bPEI-DNA nanoparticles were examined for transfection efficiency and virus production yield in comparison with PLGA-DNA, bPEI-DNA nanoparticles or liposome-DNA complexes. After testing various ratios of PLGA, bPEI, and DNA, the ratio of 6:3:1 (PLGA:bPEI:DNA, w/w/w) was determined to be optimal, with acceptable cellular toxicity. PLGA/bPEI-DNA (6:3:1) nanoparticles showed superior transfection efficiency, especially in multiple gene transfection, and viral yield when compared with liposome-DNA complexes. The culture supernatants of HEK293FT cells transfected with PLGA/bPEI-DNA of viral constructs containing reprogramming factors (Oct4, Sox2, Klf4, or c-Myc) successfully and more efficiently generated induced pluripotent stem cell colonies from mouse embryonic fibroblasts. These results strongly suggest that PLGA/bPEI-DNA nanoparticles can provide significant advantages in studying the effect of multiple factor delivery such as in reprogramming or direct conversion of cell fate. PMID:24098810

Cloning from stem cells: different lineages, different species, same story.

PubMed

Oback, Björn

2009-01-01

Following nuclear transfer (NT), the most stringent measure of extensive donor cell reprogramming is development into viable offspring. This is referred to as cloning efficiency and quantified as the proportion of cloned embryos transferred into surrogate mothers that survive into adulthood. Cloning efficiency depends on the ability of the enucleated recipient cell to carry out the reprogramming reactions ('reprogramming ability') and the ability of the nuclear donor cell to be reprogrammed ('reprogrammability'). It has been postulated that reprogrammability of the somatic donor cell epigenome is inversely proportional to its differentiation status. In order to test this hypothesis, reprogrammability was compared between undifferentiated stem cells and their differentiated isogenic progeny. In the mouse, cells of divergent differentiation status from the neuronal, haematopoietic and skin epithelial lineage were tested. In cattle and deer, skeletal muscle and antler cells, respectively, were used as donors. No conclusive correlation between differentiation status and cloning efficiency was found, indicating that somatic donor cell type may not be the limiting factor for cloning success. This may reflect technical limitations of the NT-induced reprogramming assay. Alternatively, differentiation status and reprogrammability may be unrelated, making all cells equally difficult to reprogramme once they have left the ground state of pluripotency.

Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family.

PubMed

Hysolli, Eriona; Tanaka, Yoshiaki; Su, Juan; Kim, Kun-Yong; Zhong, Tianyu; Janknecht, Ralf; Zhou, Xiao-Ling; Geng, Lin; Qiu, Caihong; Pan, Xinghua; Jung, Yong-Wook; Cheng, Jijun; Lu, Jun; Zhong, Mei; Weissman, Sherman M; Park, In-Hyun

2016-07-12

Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Generation of human β-thalassemia induced pluripotent cell lines by reprogramming of bone marrow-derived mesenchymal stromal cells using modified mRNA.

PubMed

Varela, Ioanna; Karagiannidou, Angeliki; Oikonomakis, Vasilis; Tzetis, Maria; Tzanoudaki, Marianna; Siapati, Elena-Konstantina; Vassilopoulos, George; Graphakos, Stelios; Kanavakis, Emmanuel; Goussetis, Evgenios

2014-12-01

Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with β-thalassemia (β-thal) with the aim to generate trangene-free β-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of five iPSC colonies, from which three were picked up and expanded in β-thal-iPSC lines. After 10 serial passages in vitro, β-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%, but with a decreased hematopoietic colony-forming capability. In conclusion, we report herein the generation of transgene-free β-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-based reprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs.

Bovine oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP).

PubMed

Tani, Tetsuya; Shimada, Hiroaki; Kato, Yoko; Tsunoda, Yukio

2007-01-01

Despite the long-held assumption that reprogramming factors are present in mammalian oocytes at the second metaphase stage, the molecular nature of these factors is not known. Here, we demonstrated that oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP). Injection of TCTP double-stranded RNA into germinal vesicle oocytes decreased the potential of nuclear-transferred (NT) oocytes, but not in vitro fertilized oocytes, to develop into blastocysts. Phosphorylated TCTP is considered to facilitate the first step of somatic cell reprogramming. After transfer of blastocysts that developed from NT oocytes fused with cumulus cells in which phosphorylated TCTP peptide was previously incorporated, the recipient pregnancy rate (47%) increased and the abortion rate (13%) decreased. Moreover, all seven cloned calves survived for at least 1 month after parturition, and had no morphologic abnormalities. The present study demonstrated that pretreatment of donor cells with phosphorylated TCTP peptide has a beneficial effect on the potential of bovine somatic cell nuclei to develop into normal cloned calves. Before widespread application of TCTP for bovine cloning, however, a large-scale embryo transfer study using different donor cell lines of various origins is necessary.

Systemic evaluation of cellular reprogramming processes exploiting a novel R-tool: eegc.

PubMed

Zhou, Xiaoyuan; Meng, Guofeng; Nardini, Christine; Mei, Hongkang

2017-08-15

Cells derived by cellular engineering, i.e. differentiation of induced pluripotent stem cells and direct lineage reprogramming, carry a tremendous potential for medical applications and in particular for regenerative therapies. These approaches consist in the definition of lineage-specific experimental protocols that, by manipulation of a limited number of biological cues-niche mimicking factors, (in)activation of transcription factors, to name a few-enforce the final expression of cell-specific (marker) molecules. To date, given the intricate complexity of biological pathways, these approaches still present imperfect reprogramming fidelity, with uncertain consequences on the functional properties of the resulting cells. We propose a novel tool eegc to evaluate cellular engineering processes, in a systemic rather than marker-based fashion, by integrating transcriptome profiling and functional analysis. Our method clusters genes into categories representing different states of (trans)differentiation and further performs functional and gene regulatory network analyses for each of the categories of the engineered cells, thus offering practical indications on the potential lack of the reprogramming protocol. eegc R package is released under the GNU General Public License within the Bioconductor project, freely available at https://bioconductor.org/packages/eegc/. christine.nardini.rsrc@gmail.com or hongkang.k.mei@gsk.com. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Systemic evaluation of cellular reprogramming processes exploiting a novel R-tool: eegc

PubMed Central

Zhou, Xiaoyuan; Meng, Guofeng; Nardini, Christine; Mei, Hongkang

2017-01-01

Abstract Motivation Cells derived by cellular engineering, i.e. differentiation of induced pluripotent stem cells and direct lineage reprogramming, carry a tremendous potential for medical applications and in particular for regenerative therapies. These approaches consist in the definition of lineage-specific experimental protocols that, by manipulation of a limited number of biological cues—niche mimicking factors, (in)activation of transcription factors, to name a few—enforce the final expression of cell-specific (marker) molecules. To date, given the intricate complexity of biological pathways, these approaches still present imperfect reprogramming fidelity, with uncertain consequences on the functional properties of the resulting cells. Results We propose a novel tool eegc to evaluate cellular engineering processes, in a systemic rather than marker-based fashion, by integrating transcriptome profiling and functional analysis. Our method clusters genes into categories representing different states of (trans)differentiation and further performs functional and gene regulatory network analyses for each of the categories of the engineered cells, thus offering practical indications on the potential lack of the reprogramming protocol. Availability and Implementation eegc R package is released under the GNU General Public License within the Bioconductor project, freely available at https://bioconductor.org/packages/eegc/. Contact christine.nardini.rsrc@gmail.com or hongkang.k.mei@gsk.com Supplementary information Supplementary data are available at Bioinformatics online. PMID:28398503

Generation and characterization of human iPSC line generated from mesenchymal stem cells derived from adipose tissue.

PubMed

Zapata-Linares, Natalia; Rodriguez, Saray; Mazo, Manuel; Abizanda, Gloria; Andreu, Enrique J; Barajas, Miguel; Prosper, Felipe; Rodriguez-Madoz, Juan R

2016-01-01

In this work, mesenchymal stem cells derived from adipose tissue (ADSCs) were used for the generation of the human-induced pluripotent stem cell line G15.AO. Cell reprogramming was performed using retroviral vectors containing the Yamanaka factors, and the generated G15.AO hiPSC line showed normal karyotype, silencing of the exogenous reprogramming factors, induction of the typical pluripotency-associated markers, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation ability to the three germ layers. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

DNA methylation as a dynamic regulator of development and disease processes: spotlight on the prostate.

PubMed

Keil, Kimberly P; Vezina, Chad M

2015-01-01

Prostate development, benign hyperplasia and cancer involve androgen and growth factor signaling as well as stromal-epithelial interactions. We review how DNA methylation influences these and related processes in other organ systems such as how proliferation is restricted to specific cell populations during defined temporal windows, how androgens elicit their actions and how cells establish, maintain and remodel DNA methylation in a time and cell specific fashion. We also discuss mechanisms by which hormones and endocrine disrupting chemicals reprogram DNA methylation in the prostate and elsewhere and examine evidence for a reawakening of developmental epigenetic pathways as drivers of prostate cancer and benign prostate hyperplasia.

DNA methylation as a dynamic regulator of development and disease processes: spotlight on the prostate

PubMed Central

Keil, Kimberly P; Vezina, Chad M

2015-01-01

Prostate development, benign hyperplasia and cancer involve androgen and growth factor signaling as well as stromal–epithelial interactions. We review how DNA methylation influences these and related processes in other organ systems such as how proliferation is restricted to specific cell populations during defined temporal windows, how androgens elicit their actions and how cells establish, maintain and remodel DNA methylation in a time and cell specific fashion. We also discuss mechanisms by which hormones and endocrine disrupting chemicals reprogram DNA methylation in the prostate and elsewhere and examine evidence for a reawakening of developmental epigenetic pathways as drivers of prostate cancer and benign prostate hyperplasia. PMID:26077429

Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming.

PubMed

West, Jason A; Cook, April; Alver, Burak H; Stadtfeld, Matthias; Deaton, Aimee M; Hochedlinger, Konrad; Park, Peter J; Tolstorukov, Michael Y; Kingston, Robert E

2014-08-27

Chromatin structure determines DNA accessibility. We compare nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs) and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach to identify regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. Surprisingly, most chromatin remains unchanged; a majority of rearrangements appear to affect a single nucleosome. RoDs are enriched at genes and regulatory elements, including enhancers associated with pluripotency and differentiation. RoDs co-localize with binding sites of key developmental regulators, including the reprogramming factors Klf4, Oct4/Sox2 and c-Myc. Nucleosomal landscapes in ESC enhancers are extensively altered, exhibiting lower nucleosome occupancy in pluripotent cells than in somatic cells. Most changes are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes.

Enhancer Analysis Unveils Genetic Interactions between TLX and SOX2 in Neural Stem Cells and In Vivo Reprogramming

PubMed Central

Islam, Mohammed M.; Smith, Derek K.; Niu, Wenze; Fang, Sanhua; Iqbal, Nida; Sun, Guoqiang; Shi, Yanhong; Zhang, Chun-Li

2015-01-01

Summary The orphan nuclear receptor TLX is a master regulator of postnatal neural stem cell (NSC) self-renewal and neurogenesis; however, it remains unclear how TLX expression is precisely regulated in these tissue-specific stem cells. Here, we show that a highly conserved cis-element within the Tlx locus functions to drive gene expression in NSCs. We demonstrate that the transcription factors SOX2 and MYT1 specifically interact with this genomic element to directly regulate Tlx enhancer activity in vivo. Knockdown experiments further reveal that SOX2 dominantly controls endogenous expression of TLX, whereas MYT1 only plays a modulatory role. Importantly, TLX is essential for SOX2-mediated in vivo reprogramming of astrocytes and itself is also sufficient to induce neurogenesis in the adult striatum. Together, these findings unveil functional genetic interactions among transcription factors that are critical to NSCs and in vivo cell reprogramming. PMID:26607952

Feeder-free reprogramming of human fibroblasts with messenger RNA.

PubMed

Warren, Luigi; Wang, Jiwu

2013-11-13

This unit describes a feeder-free protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts by transfection of synthetic mRNA. The reprogramming of somatic cells requires transient expression of a set of transcription factors that collectively activate an endogenous gene regulatory network specifying the pluripotent phenotype. The necessary ectopic factor expression was first effected using retroviruses; however, as viral integration into the genome is problematic for cell therapy applications, the use of footprint-free vectors such as mRNA is increasingly preferred. Strong points of the mRNA approach include high efficiency, rapid kinetics, and obviation of a clean-up phase to purge the vector. Still, the method is relatively laborious and has, up to now, involved the use of feeder cells, which brings drawbacks including poor applicability to clinically oriented iPSC derivation. Using the methods described here, mRNA reprogramming can be performed without feeders at much-reduced labor and material costs relative to established protocols. Copyright © 2013 John Wiley & Sons, Inc.

Pluripotency, Differentiation, and Reprogramming: A Gene Expression Dynamics Model with Epigenetic Feedback Regulation

PubMed Central

Miyamoto, Tadashi; Furusawa, Chikara; Kaneko, Kunihiko

2015-01-01

Embryonic stem cells exhibit pluripotency: they can differentiate into all types of somatic cells. Pluripotent genes such as Oct4 and Nanog are activated in the pluripotent state, and their expression decreases during cell differentiation. Inversely, expression of differentiation genes such as Gata6 and Gata4 is promoted during differentiation. The gene regulatory network controlling the expression of these genes has been described, and slower-scale epigenetic modifications have been uncovered. Although the differentiation of pluripotent stem cells is normally irreversible, reprogramming of cells can be experimentally manipulated to regain pluripotency via overexpression of certain genes. Despite these experimental advances, the dynamics and mechanisms of differentiation and reprogramming are not yet fully understood. Based on recent experimental findings, we constructed a simple gene regulatory network including pluripotent and differentiation genes, and we demonstrated the existence of pluripotent and differentiated states from the resultant dynamical-systems model. Two differentiation mechanisms, interaction-induced switching from an expression oscillatory state and noise-assisted transition between bistable stationary states, were tested in the model. The former was found to be relevant to the differentiation process. We also introduced variables representing epigenetic modifications, which controlled the threshold for gene expression. By assuming positive feedback between expression levels and the epigenetic variables, we observed differentiation in expression dynamics. Additionally, with numerical reprogramming experiments for differentiated cells, we showed that pluripotency was recovered in cells by imposing overexpression of two pluripotent genes and external factors to control expression of differentiation genes. Interestingly, these factors were consistent with the four Yamanaka factors, Oct4, Sox2, Klf4, and Myc, which were necessary for the establishment of induced pluripotent stem cells. These results, based on a gene regulatory network and expression dynamics, contribute to our wider understanding of pluripotency, differentiation, and reprogramming of cells, and they provide a fresh viewpoint on robustness and control during development. PMID:26308610

Optimization of direct fibroblast reprogramming to cardiomyocytes using calcium activity as a functional measure of success.

PubMed

Addis, Russell C; Ifkovits, Jamie L; Pinto, Filipa; Kellam, Lori D; Esteso, Paul; Rentschler, Stacey; Christoforou, Nicolas; Epstein, Jonathan A; Gearhart, John D

2013-07-01

Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persist for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Reprogramming human gallbladder cells into insulin-producing β-like cells

PubMed Central

Benedetti, Eric; Wang, Yuhan; Pelz, Carl; Schug, Jonathan; Kaestner, Klaus H.; Grompe, Markus

2017-01-01

The gallbladder and cystic duct (GBCs) are parts of the extrahepatic biliary tree and share a common developmental origin with the ventral pancreas. Here, we report on the very first genetic reprogramming of patient-derived human GBCs to β-like cells for potential autologous cell replacement therapy for type 1 diabetes. We developed a robust method for large-scale expansion of human GBCs ex vivo. GBCs were reprogrammed into insulin-producing pancreatic β-like cells by a combined adenoviral-mediated expression of hallmark pancreatic endocrine transcription factors PDX1, MAFA, NEUROG3, and PAX6 and differentiation culture in vitro. The reprogrammed GBCs (rGBCs) strongly induced the production of insulin and pancreatic endocrine genes and these responded to glucose stimulation in vitro. rGBCs also expressed an islet-specific surface marker, which was used to enrich for the most highly reprogrammed cells. More importantly, global mRNA and microRNA expression profiles and protein immunostaining indicated that rGBCs adopted an overall β-like state and these rGBCs engrafted in immunodeficient mice. Furthermore, comparative global expression analyses identified putative regulators of human biliary to β cell fate conversion. In summary, we have developed, for the first time, a reliable and robust genetic reprogramming and culture expansion of primary human GBCs—derived from multiple unrelated donors—into pancreatic β-like cells ex vivo, thus showing that human gallbladder is a potentially rich source of reprogrammable cells for autologous cell therapy in diabetes. PMID:28813430

Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.

PubMed

Merkl, Claudia; Saalfrank, Anja; Riesen, Nathalie; Kühn, Ralf; Pertek, Anna; Eser, Stefan; Hardt, Markus Sebastian; Kind, Alexander; Saur, Dieter; Wurst, Wolfgang; Iglesias, Antonio; Schnieke, Angelika

2013-01-01

Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.

Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis.

PubMed

Feng, Dingqing; Yan, Keqin; Zhou, Ying; Liang, Haiyan; Liang, Jing; Zhao, Weidong; Dong, Zhongjun; Ling, Bin

2016-10-04

The human papillomavirus (HPV) oncoproteins E6 and E7 are risk factors that are primarily responsible for the initiation and progression of cervical cancer, and they play a key role in immortalization and transformation by reprogramming differentiating host epithelial cells. It is unclear how cervical epithelial cells transform into tumor-initiating cells (TICs). Here, we observed that the germ stem cell protein Piwil2 is expressed in pre-cancerous and malignant lesions of the cervix and cervical cancer cell lines with the exception of the non-HPV-infected C33a cell line. Knockdown of Piwil2 by shRNA led to a marked reduction in proliferation and colony formation, in vivo tumorigenicity, chemo-resistance, and the proportion of cancer stem-like cells. In contrast, Piwil2 overexpression induced malignant transformation of HaCaT cells and the acquisition of tumor-initiating capabilities. Gene-set enrichment analysis revealed embryonic stem cell (ESC) identity, malignant biological behavior, and specifically, activation targets of the cell reprogramming factors c-Myc, Klf4, Nanog, Oct4, and Sox2 in Piwil2-overexpressing HaCaT cells. We further confirmed that E6 and E7 reactivated Piwil2 and that E6 and E7 overexpression resulted in a similar gene-set enrichment pattern as Piwil2 overexpression in HaCaT cells. Moreover, Piwil2 overexpression or E6 and E7 activation induced H3K9 acetylation but reduced H3K9 trimethylation, which contributed to the epigenetic reprogramming and ESC signature maintenance, as predicted previously. Our study demonstrates that Piwil2, reactivated by the HPV oncoproteins E6 and E7, plays an essential role in the transformation of cervical epithelial cells to TICs via epigenetics-based cell reprogramming.

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation*

PubMed Central

Bernard, Karen; Logsdon, Naomi J.; Ravi, Saranya; Xie, Na; Persons, Benjamin P.; Rangarajan, Sunad; Zmijewski, Jaroslaw W.; Mitra, Kasturi; Liu, Gang; Darley-Usmar, Victor M.; Thannickal, Victor J.

2015-01-01

Contraction is crucial in maintaining the differentiated phenotype of myofibroblasts. Contraction is an energy-dependent mechanism that relies on the production of ATP by mitochondria and/or glycolysis. Although the role of mitochondrial biogenesis in the adaptive responses of skeletal muscle to exercise is well appreciated, mechanisms governing energetic adaptation of myofibroblasts are not well understood. Our study demonstrates induction of mitochondrial biogenesis and aerobic glycolysis in response to the differentiation-inducing factor transforming growth factor β1 (TGF-β1). This metabolic reprogramming is linked to the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Inhibition of p38 MAPK decreased accumulation of active peroxisome proliferator-activated receptor γ coactivator 1α in the nucleus and altered the translocation of mitochondrial transcription factor A to the mitochondria. Genetic or pharmacologic approaches that block mitochondrial biogenesis or glycolysis resulted in decreased contraction and reduced expression of TGF-β1-induced α-smooth muscle actin and collagen α-2(I) but not of fibronectin or collagen α-1(I). These data indicate a critical role for TGF-β1-induced metabolic reprogramming in regulating myofibroblast-specific contractile signaling and support the concept of integrating bioenergetics with cellular differentiation. PMID:26318453

Reprogrammed mouse astrocytes retain a "memory" of tissue origin and possess more tendencies for neuronal differentiation than reprogrammed mouse embryonic fibroblasts.

PubMed

Tian, Changhai; Wang, Yongxiang; Sun, Lijun; Ma, Kangmu; Zheng, Jialin C

2011-02-01

Direct reprogramming of a variety of somatic cells with the transcription factors Oct4 (also called Pou5f1), Sox2 with either Klf4 and Myc or Lin28 and Nanog generates the induced pluripotent stem cells (iPSCs) with marker similarity to embryonic stem cells. However, the difference between iPSCs derived from different origins is unclear. In this study, we hypothesized that reprogrammed cells retain a "memory" of their origins and possess additional potential of related tissue differentiation. We reprogrammed primary mouse astrocytes via ectopic retroviral expression of OCT3/4, Sox2, Klf4 and Myc and found the iPSCs from mouse astrocytes expressed stem cell markers and formed teratomas in SCID mice containing derivatives of all three germ layers similar to mouse embryonic stem cells besides semblable morphologies. To test our hypothesis, we compared embryonic bodies (EBs) formation and neuronal differentiation between iPSCs from mouse embryonic fibroblasts (MEFsiPSCs) and iPSCs from mouse astrocytes (mAsiPSCs). We found that mAsiPSCs grew slower and possessed more potential for neuronal differentiation compared to MEFsiPSCs. Our results suggest that mAsiPSCs retain a "memory" of the central nervous system, which confers additional potential upon neuronal differentiation.

Signals that regulate the oncogenic fate of neural stem cells and progenitors

PubMed Central

Swartling, Fredrik J.; Bolin, Sara; Phillips, Joanna J.; Persson, Anders I.

2013-01-01

Brain tumors have frequently been associated with a neural stem cell (NSC) origin and contain stem-like tumor cells, so-called brain tumor stem cells (BTSCs) that share many features with normal NSCs. A stem cell state of BTSCs confers resistance to radiotherapy and treatment with alkylating agents. It is also a hallmark of aggressive brain tumors and is maintained by transcriptional networks that are also active in embryonic stem cells. Advances in reprogramming of somatic cells into induced pluripotent stem (iPS) cells have further identified genes that drive stemness. In this review, we will highlight the possible drivers of stemness in medulloblastoma and glioma, the most frequent types of primary malignant brain cancer in children and adults, respectively. Signals that drive expansion of developmentally defined neural precursor cells are also active in corresponding brain tumors. Transcriptomal subgroups of human medulloblastoma and glioma match features of NSCs but also more restricted progenitors. Lessons from genetically-engineered mouse (GEM) models show that temporally and regionally defined NSCs can give rise to distinct subgroups of medulloblastoma and glioma. We will further discuss how acquisition of stem cell features may drive brain tumorigenesis from a non-NSC origin. Genetic alterations, signaling pathways, and therapy-induced changes in the tumor microenvironment can drive reprogramming networks and induce stemness in brain tumors. Finally, we propose a model where dysregulation of microRNAs (miRNAs) that normally provide barriers against reprogramming plays an integral role in promoting stemness in brain tumors. PMID:23376224

Chemical compound-based direct reprogramming for future clinical applications

PubMed Central

Takeda, Yukimasa; Harada, Yoshinori; Yoshikawa, Toshikazu; Dai, Ping

2018-01-01

Recent studies have revealed that a combination of chemical compounds enables direct reprogramming from one somatic cell type into another without the use of transgenes by regulating cellular signaling pathways and epigenetic modifications. The generation of induced pluripotent stem (iPS) cells generally requires virus vector-mediated expression of multiple transcription factors, which might disrupt genomic integrity and proper cell functions. The direct reprogramming is a promising alternative to rapidly prepare different cell types by bypassing the pluripotent state. Because the strategy also depends on forced expression of exogenous lineage-specific transcription factors, the direct reprogramming in a chemical compound-based manner is an ideal approach to further reduce the risk for tumorigenesis. So far, a number of reported research efforts have revealed that combinations of chemical compounds and cell-type specific medium transdifferentiate somatic cells into desired cell types including neuronal cells, glial cells, neural stem cells, brown adipocytes, cardiomyocytes, somatic progenitor cells, and pluripotent stem cells. These desired cells rapidly converted from patient-derived autologous fibroblasts can be applied for their own transplantation therapy to avoid immune rejection. However, complete chemical compound-induced conversions remain challenging particularly in adult human-derived fibroblasts compared with mouse embryonic fibroblasts (MEFs). This review summarizes up-to-date progress in each specific cell type and discusses prospects for future clinical application toward cell transplantation therapy. PMID:29739872

Histone chaperone APLF regulates induction of pluripotency in murine fibroblasts.

PubMed

Syed, Khaja Mohieddin; Joseph, Sunu; Mukherjee, Ananda; Majumder, Aditi; Teixeira, Jose M; Dutta, Debasree; Pillai, Madhavan Radhakrishna

2016-12-15

Induction of pluripotency in differentiated cells through the exogenous expression of the transcription factors Oct4, Sox2, Klf4 and cellular Myc involves reprogramming at the epigenetic level. Histones and their metabolism governed by histone chaperones constitute an important regulator of epigenetic control. We hypothesized that histone chaperones facilitate or inhibit the course of reprogramming. For the first time, we report here that the downregulation of histone chaperone Aprataxin PNK-like factor (APLF) promotes reprogramming by augmenting the expression of E-cadherin (Cdh1), which is implicated in the mesenchymal-to-epithelial transition (MET) involved in the generation of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Downregulation of APLF in MEFs expedites the loss of the repressive MacroH2A.1 (encoded by H2afy) histone variant from the Cdh1 promoter and enhances the incorporation of active histone H3me2K4 marks at the promoters of the pluripotency genes Nanog and Klf4, thereby accelerating the process of cellular reprogramming and increasing the efficiency of iPSC generation. We demonstrate a new histone chaperone (APLF)-MET-histone modification cohort that functions in the induction of pluripotency in fibroblasts. This regulatory axis might provide new mechanistic insights into perspectives of epigenetic regulation involved in cancer metastasis. © 2016. Published by The Company of Biologists Ltd.

Metabolome Profiling of Partial and Fully Reprogrammed Induced Pluripotent Stem Cells.

PubMed

Park, Soon-Jung; Lee, Sang A; Prasain, Nutan; Bae, Daekyeong; Kang, Hyunsu; Ha, Taewon; Kim, Jong Soo; Hong, Ki-Sung; Mantel, Charlie; Moon, Sung-Hwan; Broxmeyer, Hal E; Lee, Man Ryul

2017-05-15

Acquisition of proper metabolomic fate is required to convert somatic cells toward fully reprogrammed pluripotent stem cells. The majority of induced pluripotent stem cells (iPSCs) are partially reprogrammed and have a transcriptome different from that of the pluripotent stem cells. The metabolomic profile and mitochondrial metabolic functions required to achieve full reprogramming of somatic cells to iPSC status have not yet been elucidated. Clarification of the metabolites underlying reprogramming mechanisms should enable further optimization to enhance the efficiency of obtaining fully reprogrammed iPSCs. In this study, we characterized the metabolites of human fully reprogrammed iPSCs, partially reprogrammed iPSCs, and embryonic stem cells (ESCs). Using capillary electrophoresis time-of-flight mass spectrometry-based metabolomics, we found that 89% of analyzed metabolites were similarly expressed in fully reprogrammed iPSCs and human ESCs (hESCs), whereas partially reprogrammed iPSCs shared only 74% similarly expressed metabolites with hESCs. Metabolomic profiling analysis suggested that converting mitochondrial respiration to glycolytic flux is critical for reprogramming of somatic cells into fully reprogrammed iPSCs. This characterization of metabolic reprogramming in iPSCs may enable the development of new reprogramming parameters for enhancing the generation of fully reprogrammed human iPSCs.

Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences

PubMed Central

Yu, Junying; Hu, Kejin; Smuga-Otto, Kim; Tian, Shulan; Stewart, Ron; Slukvin, Igor I.; Thomson, James A.

2009-01-01

Reprogramming differentiated human cells to induced pluripotent stem (iPS) cells has applications in basic biology, drug development, and transplantation. Human iPS cell derivation previously required vectors that integrate into the genome, which can create mutations and limit the utility of the cells in both research and clinical applications. Here we describe the derivation of human iPS cells using non-integrating episomal vectors. After removal of the episome, iPS cells completely free of vector and transgene sequences are derived that are similar to human embryonic stem (ES) cells in proliferative and developmental potential. These results demonstrate that reprogramming human somatic cells does not require genomic integration or the continued presence of exogenous reprogramming factors, and removes one obstacle to the clinical application of human iPS cells. PMID:19325077

Defining Translational Reprogramming in Tuberous Sclerosis Complex

DTIC Science & Technology

Inactivating mutations in the TSC1 and TSC2 tumor suppressor genes lead to the disease tuberous sclerosis complex (TSC). The TSC1/TSC2complex...integrates multiple cues to regulate protein translation and cell growth via mammalian target of rapamycin complex 1 (mTORC1). Loss of TSC functions leads to

c-MYC inhibition impairs hypoxia response in glioblastoma multiforme

PubMed Central

Falchetti, Maria Laura; Illi, Barbara; Bozzo, Francesca; Valle, Cristiana; Helmer-Citterich, Manuela; Ferrè, Fabrizio; Nasi, Sergio; Levi, Andrea

2016-01-01

The c-MYC oncoprotein is a DNA binding transcription factor that enhances the expression of many active genes. c-MYC transcriptional signatures vary according to the transcriptional program defined in each cell type during differentiation. Little is known on the involvement of c-MYC in regulation of gene expression programs that are induced by extracellular cues such as a changing microenvironment. Here we demonstrate that inhibition of c-MYC in glioblastoma multiforme cells blunts hypoxia-dependent glycolytic reprogramming and mitochondria fragmentation in hypoxia. This happens because c-MYC inhibition alters the cell transcriptional response to hypoxia and finely tunes the expression of a subset of Hypoxia Inducible Factor 1-regulated genes. We also show that genes whose expression in hypoxia is affected by c-MYC inhibition are able to distinguish the Proneural subtype of glioblastoma multiforme, thus potentially providing a molecular signature for this class of tumors that are the least tractable among glioblastomas. PMID:27119353

Directed Differentiation of Embryonic Stem Cells Into Cardiomyocytes by Bacterial Injection of Defined Transcription Factors.

PubMed

Bai, Fang; Ho Lim, Chae; Jia, Jingyue; Santostefano, Katherine; Simmons, Chelsey; Kasahara, Hideko; Wu, Weihui; Terada, Naohiro; Jin, Shouguang

2015-10-09

Forced expression of defined transcriptional factors has been well documented as an effective method for cellular reprogramming or directed differentiation. However, transgene expression is not amenable for therapeutic application due to potential insertional mutagenesis. Here, we have developed a bacterial type III secretion system (T3SS)-based protein delivery tool and shown its application in directing pluripotent stem cell differentiation by a controlled delivery of transcription factors relevant to early heart development. By fusing to an N-terminal secretion sequence for T3SS-dependent injection, three transcriptional factors, namely Gata4, Mef2c, and Tbx5 (abbreviated as GMT), were translocated into murine embryonic stem cells (ESCs), where the proteins are effectively targeted to the nucleus with an average intracellular half-life of 5.5 hours. Exogenous GMT protein injection activated the cardiac program, and multiple rounds of GMT protein delivery significantly improved the efficiency of ESC differentiation into cardiomyocytes. Combination of T3SS-mediated GMT delivery and Activin A treatment showed an additive effect, resulting in on average 60% of the ESCs differentiated into cardiomyocytes. ESC derived cardiomyocytes displayed spontaneous rhythmic contractile movement as well as normal hormonal responses. This work serves as a foundation for the bacterial delivery of multiple transcription factors to direct cell fate without jeopardizing genomic integrity.

Directed Differentiation of Embryonic Stem Cells Into Cardiomyocytes by Bacterial Injection of Defined Transcription Factors

PubMed Central

Bai, Fang; Ho Lim, Chae; Jia, Jingyue; Santostefano, Katherine; Simmons, Chelsey; Kasahara, Hideko; Wu, Weihui; Terada, Naohiro; Jin, Shouguang

2015-01-01

Forced expression of defined transcriptional factors has been well documented as an effective method for cellular reprogramming or directed differentiation. However, transgene expression is not amenable for therapeutic application due to potential insertional mutagenesis. Here, we have developed a bacterial type III secretion system (T3SS)-based protein delivery tool and shown its application in directing pluripotent stem cell differentiation by a controlled delivery of transcription factors relevant to early heart development. By fusing to an N-terminal secretion sequence for T3SS-dependent injection, three transcriptional factors, namely Gata4, Mef2c, and Tbx5 (abbreviated as GMT), were translocated into murine embryonic stem cells (ESCs), where the proteins are effectively targeted to the nucleus with an average intracellular half-life of 5.5 hours. Exogenous GMT protein injection activated the cardiac program, and multiple rounds of GMT protein delivery significantly improved the efficiency of ESC differentiation into cardiomyocytes. Combination of T3SS-mediated GMT delivery and Activin A treatment showed an additive effect, resulting in on average 60% of the ESCs differentiated into cardiomyocytes. ESC derived cardiomyocytes displayed spontaneous rhythmic contractile movement as well as normal hormonal responses. This work serves as a foundation for the bacterial delivery of multiple transcription factors to direct cell fate without jeopardizing genomic integrity. PMID:26449528

Chromatin reprogramming in breast cancer.

PubMed

Swinstead, Erin E; Paakinaho, Ville; Hager, Gordon

2018-04-24

Reprogramming of the chromatin landscape is a critical component to the transcriptional response in breast cancer. Effects of sex hormones such as estrogens and progesterone have been well described to have a critical impact on breast cancer proliferation. However, the complex network of the chromatin landscape, enhancer regions, and mode of function of steroid receptors (SRs) and other transcription factors (TFs), is an intricate web of signaling and functional processes that is still largely misunderstood at the mechanistic level. In this review, we describe what is currently known about the dynamic interplay between TFs with chromatin and the reprogramming of enhancer elements. Emphasis has been placed on characterizing the different modes of action of TFs in regulating enhancer activity, specifically, how different SRs target enhancer regions and reprogram chromatin in breast cancer cells. In addition, we discuss current techniques employed to study enhancer function at a genome-wide level. Further, we have noted recent advances in live cell imaging technology. These single cell approaches enable the coupling of population based assays with real-time studies to address many unsolved questions about SRs and chromatin dynamics in breast cancer.

Environmental signals and transgenerational epigenetics

PubMed Central

Skinner, Michael K; Guerrero-Bosagna, Carlos

2010-01-01

The ability of an environmental factor or toxicant to promote a phenotype or disease state not only in the individual exposed, but also in subsequent progeny for multiple generations, is termed transgenerational inheritance. The majority of environmental agents do not promote genetic mutations or alterations in DNA sequence, but do have the capacity to alter the epigenome. Although most environmental exposures will influence somatic cells and not allow the transgenerational transmission of a phenotype, the ability of an environmental factor to reprogram the germline epigenome can promote a transgenerational inheritance of phenotypes and disease states. A limited number of critical developmental periods exist when environmental signals can produce a significant epigenetic reprogramming of the germline. In this review, the ability of environmental factors or toxicants to promote epigenetic transgenerational phenotypes is reviewed. PMID:20563319

Alternative Splicing of MBD2 Supports Self-Renewal in Human Pluripotent Stem Cells

PubMed Central

Lu, Yu; Loh, Yuin-Han; Li, Hu; Cesana, Marcella; Ficarro, Scott B.; Parikh, Jignesh R.; Salomonis, Nathan; Toh, Cheng-Xu Delon; Andreadis, Stelios T.; Luckey, C. John; Collins, James J.; Daley, George Q.; Marto, Jarrod A.

2014-01-01

Summary Alternative RNA splicing (AS) regulates proteome diversity, including isoform-specific expression of several pluripotency genes. Here, we integrated global gene expression and proteomic analyses and identified a molecular signature suggesting a central role for AS in maintaining human pluripotent stem cell (hPSC) self-renewal. We demonstrate the splicing factor SFRS2 is an OCT4 target gene required for pluripotency. SFRS2 regulates AS of the methyl-CpG-binding protein MBD2, whose isoforms play opposing roles in maintenance of, and reprogramming to, pluripotency. While both MDB2a and MBD2c are enriched at the OCT4 and NANOG promoters, MBD2a preferentially interacts with repressive NuRD chromatin remodeling factors and promotes hPSC differentiation, whereas overexpression of MBD2c enhances reprogramming of fibroblasts to pluripotency. The miR-301 and miR-302 families provide additional regulation by targeting SFRS2 and MDB2a. These data suggest that OCT4, SFRS2, and MBD2 participate in a positive feedback loop, regulating proteome diversity complexity in support of hPSC self-renewal and reprogramming. PMID:24813856

Distinct gene regulatory programs define the inhibitory effects of liver X receptors and PPARG on cancer cell proliferation.

PubMed

Savic, Daniel; Ramaker, Ryne C; Roberts, Brian S; Dean, Emma C; Burwell, Todd C; Meadows, Sarah K; Cooper, Sara J; Garabedian, Michael J; Gertz, Jason; Myers, Richard M

2016-07-11

The liver X receptors (LXRs, NR1H2 and NR1H3) and peroxisome proliferator-activated receptor gamma (PPARG, NR1C3) nuclear receptor transcription factors (TFs) are master regulators of energy homeostasis. Intriguingly, recent studies suggest that these metabolic regulators also impact tumor cell proliferation. However, a comprehensive temporal molecular characterization of the LXR and PPARG gene regulatory responses in tumor cells is still lacking. To better define the underlying molecular processes governing the genetic control of cellular growth in response to extracellular metabolic signals, we performed a comprehensive, genome-wide characterization of the temporal regulatory cascades mediated by LXR and PPARG signaling in HT29 colorectal cancer cells. For this analysis, we applied a multi-tiered approach that incorporated cellular phenotypic assays, gene expression profiles, chromatin state dynamics, and nuclear receptor binding patterns. Our results illustrate that the activation of both nuclear receptors inhibited cell proliferation and further decreased glutathione levels, consistent with increased cellular oxidative stress. Despite a common metabolic reprogramming, the gene regulatory network programs initiated by these nuclear receptors were widely distinct. PPARG generated a rapid and short-term response while maintaining a gene activator role. By contrast, LXR signaling was prolonged, with initial, predominantly activating functions that transitioned to repressive gene regulatory activities at late time points. Through the use of a multi-tiered strategy that integrated various genomic datasets, our data illustrate that distinct gene regulatory programs elicit common phenotypic effects, highlighting the complexity of the genome. These results further provide a detailed molecular map of metabolic reprogramming in cancer cells through LXR and PPARG activation. As ligand-inducible TFs, these nuclear receptors can potentially serve as attractive therapeutic targets for the treatment of various cancers.

Klf4 reverts developmentally programmed restriction of ground state pluripotency

PubMed Central

Guo, Ge; Yang, Jian; Nichols, Jennifer; Hall, John Simon; Eyres, Isobel; Mansfield, William; Smith, Austin

2009-01-01

Summary Mouse embryonic stem (ES) cells derived from pluripotent early epiblast contribute functionally differentiated progeny to all foetal lineages of chimaeras. By contrast, epistem cell (EpiSC) lines from post-implantation epithelialised epiblast are unable to colonise the embryo even though they express the core pluripotency genes Oct4, Sox2 and Nanog. We examined interconversion between these two cell types. ES cells can readily become EpiSCs in response to growth factor cues. By contrast, EpiSCs do not change into ES cells. We exploited PiggyBac transposition to introduce a single reprogramming factor, Klf4, into EpiSCs. No effect was apparent in EpiSC culture conditions, but in ground state ES cell conditions a fraction of cells formed undifferentiated colonies. These EpiSC-derived induced pluripotent stem (Epi-iPS) cells activated expression of ES cell-specific transcripts including endogenous Klf4, and downregulated markers of lineage specification. X chromosome silencing in female cells, a feature of the EpiSC state, was erased in Epi-iPS cells. They produced high-contribution chimaeras that yielded germline transmission. These properties were maintained after Cre-mediated deletion of the Klf4 transgene, formally demonstrating complete and stable reprogramming of developmental phenotype. Thus, re-expression of Klf4 in an appropriate environment can regenerate the naïve ground state from EpiSCs. Reprogramming is dependent on suppression of extrinsic growth factor stimuli and proceeds to completion in less than 1% of cells. This substantiates the argument that EpiSCs are developmentally, epigenetically and functionally differentiated from ES cells. However, because a single transgene is the minimum requirement to attain the ground state, EpiSCs offer an attractive opportunity for screening for unknown components of the reprogramming process. PMID:19224983

Developmental Programming of Renal Function and Re-Programming Approaches.

PubMed

Nüsken, Eva; Dötsch, Jörg; Weber, Lutz T; Nüsken, Kai-Dietrich

2018-01-01

Chronic kidney disease affects more than 10% of the population. Programming studies have examined the interrelationship between environmental factors in early life and differences in morbidity and mortality between individuals. A number of important principles has been identified, namely permanent structural modifications of organs and cells, long-lasting adjustments of endocrine regulatory circuits, as well as altered gene transcription. Risk factors include intrauterine deficiencies by disturbed placental function or maternal malnutrition, prematurity, intrauterine and postnatal stress, intrauterine and postnatal overnutrition, as well as dietary dysbalances in postnatal life. This mini-review discusses critical developmental periods and long-term sequelae of renal programming in humans and presents studies examining the underlying mechanisms as well as interventional approaches to "re-program" renal susceptibility toward disease. Clinical manifestations of programmed kidney disease include arterial hypertension, proteinuria, aggravation of inflammatory glomerular disease, and loss of kidney function. Nephron number, regulation of the renin-angiotensin-aldosterone system, renal sodium transport, vasomotor and endothelial function, myogenic response, and tubuloglomerular feedback have been identified as being vulnerable to environmental factors. Oxidative stress levels, metabolic pathways, including insulin, leptin, steroids, and arachidonic acid, DNA methylation, and histone configuration may be significantly altered by adverse environmental conditions. Studies on re-programming interventions focused on dietary or anti-oxidative approaches so far. Further studies that broaden our understanding of renal programming mechanisms are needed to ultimately develop preventive strategies. Targeted re-programming interventions in animal models focusing on known mechanisms will contribute to new concepts which finally will have to be translated to human application. Early nutritional concepts with specific modifications in macro- or micronutrients are among the most promising approaches to improve future renal health.

Induced pluripotent stem cells: Mechanisms, achievements and perspectives in farm animals

PubMed Central

Kumar, Dharmendra; Talluri, Thirumala R; Anand, Taruna; Kues, Wilfried A

2015-01-01

Pluripotent stem cells are unspecialized cells with unlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem (iPS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived iPS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming mRNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of iPS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of iPS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals. PMID:25815117

Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1.

PubMed

Arczewska, Katarzyna D; Tomazella, Gisele G; Lindvall, Jessica M; Kassahun, Henok; Maglioni, Silvia; Torgovnick, Alessandro; Henriksson, Johan; Matilainen, Olli; Marquis, Bryce J; Nelson, Bryant C; Jaruga, Pawel; Babaie, Eshrat; Holmberg, Carina I; Bürglin, Thomas R; Ventura, Natascia; Thiede, Bernd; Nilsen, Hilde

2013-05-01

Transcription-blocking oxidative DNA damage is believed to contribute to aging and to underlie activation of oxidative stress responses and down-regulation of insulin-like signaling (ILS) in Nucleotide Excision Repair (NER) deficient mice. Here, we present the first quantitative proteomic description of the Caenorhabditis elegans NER-defective xpa-1 mutant and compare the proteome and transcriptome signatures. Both methods indicated activation of oxidative stress responses, which was substantiated biochemically by a bioenergetic shift involving increased steady-state reactive oxygen species (ROS) and Adenosine triphosphate (ATP) levels. We identify the lesion-detection enzymes of Base Excision Repair (NTH-1) and global genome NER (XPC-1 and DDB-1) as upstream requirements for transcriptomic reprogramming as RNA-interference mediated depletion of these enzymes prevented up-regulation of genes over-expressed in the xpa-1 mutant. The transcription factors SKN-1 and SLR-2, but not DAF-16, were identified as effectors of reprogramming. As shown in human XPA cells, the levels of transcription-blocking 8,5'-cyclo-2'-deoxyadenosine lesions were reduced in the xpa-1 mutant compared to the wild type. Hence, accumulation of cyclopurines is unlikely to be sufficient for reprogramming. Instead, our data support a model where the lesion-detection enzymes NTH-1, XPC-1 and DDB-1 play active roles to generate a genomic stress signal sufficiently strong to result in transcriptomic reprogramming in the xpa-1 mutant.

Restoration of Mitochondrial NAD+ Levels Delays Stem Cell Senescence and Facilitates Reprogramming of Aged Somatic Cells.

PubMed

Son, Myung Jin; Kwon, Youjeong; Son, Taekwon; Cho, Yee Sook

2016-12-01

The fundamental tenet that aging is irreversible has been challenged by the development of reprogramming technology that can restore molecular and cellular age by reversing the progression of aging. The use of cells from aged individuals as sources for reprogramming or transplantation creates a major barrier in stem cell therapy with respect to cell quality and quantity. Here, we investigated the molecular features underlying senescence and rejuvenation during aged cell reprogramming and identified novel factors that can overcome age-associated barriers. Enzymes, such as nicotinamide nucleotide transhydrogenase (NNT) and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3), that control mitochondrial NAD + levels appear to be susceptible to aging. In aged cells, mitochondrial NAD + levels decrease, accompanied by reduced SIRT3 activity; these changes severely impede cell fate transition. However, in cells collected from aged p16 knockout mice, which exhibit delayed cellular senescence, no changes in NNT or NMNAT3 expression were found. Importantly, restoring mitochondrial NAD + levels by overexpressing NNT and NMNAT3 enhanced reprogramming efficiency of aged somatic cells and extended the lifespan of human mesenchymal stem cells by delaying replicative senescence. These results demonstrate that maintenance of mitochondrial NAD + levels is critical for reversing the mechanisms of aging and ensuring that cells collected from aged individuals are of high quality. Stem Cells 2016;34:2840-2851. © 2016 AlphaMed Press.

Programmable genetic switches to control transcriptional machinery of pluripotency.

PubMed

Pandian, Ganesh N; Sugiyama, Hiroshi

2012-06-01

Transcriptional activators play a central role in the regulation of gene expression and have the ability to manipulate the specification of cell fate. Pluripotency is a transient state where a cell has the potential to develop into more than one type of mature cell. The induction of pluripotency in differentiated cells requires extensive chromatin reorganization regulated by core transcriptional machinery. Several small molecules have been shown to enhance the efficiency of somatic cell reprogramming into pluripotent stem cells. However, entirely chemical-based reprogramming remains elusive. Recently, we reported that selective DNA-binding hairpin pyrrole-imidazole polyamides conjugated with histone deacetylase inhibitor could mimic natural transcription factors and epigenetically activate certain pluripotency-associated genes. Here, we review the need to develop selective chromatin-modifying transcriptional activators for somatic genome reprogramming. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reprogramming mediated radio-resistance of 3D-grown cancer cells.

PubMed

Xue, Gang; Ren, Zhenxin; Grabham, Peter W; Chen, Yaxiong; Zhu, Jiayun; Du, Yarong; Pan, Dong; Li, Xiaoman; Hu, Burong

2015-07-01

In vitro 3D growth of tumors is a new cell culture model that more closely mimics the features of the in vivo environment and is being used increasingly in the field of biological and medical research. It has been demonstrated that cancer cells cultured in 3D matrices are more radio-resistant compared with cells in monolayers. However, the mechanisms causing this difference remain unclear. Here we show that cancer cells cultured in a 3D microenvironment demonstrated an increase in cells with stem cell properties. This was confirmed by the finding that cells in 3D cultures upregulated the gene and protein expression of the stem cell reprogramming factors such as OCT4, SOX2, NANOG, LIN28 and miR-302a, compared with cells in monolayers. Moreover, the expression of β-catenin, a regulating molecule of reprogramming factors, also increased in 3D-grown cancer cells. These findings suggest that cancer cells were reprogrammed to become stem cell-like cancer cells in a 3D growth culture microenvironment. Since cancer stem cell-like cells demonstrate an increased radio-resistance and chemo-resistance, our results offer a new perspective as to why. Our findings shed new light on understanding the features of the 3D growth cell model and its application in basic research into clinical radiotherapy and medicine. © The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Nucleosome organizations in induced pluripotent stem cells reprogrammed from somatic cells belonging to three different germ layers.

PubMed

Tao, Yu; Zheng, Weisheng; Jiang, Yonghua; Ding, Guitao; Hou, Xinfeng; Tang, Yitao; Li, Yueying; Gao, Shuai; Chang, Gang; Zhang, Xiaobai; Liu, Wenqiang; Kou, Xiaochen; Wang, Hong; Jiang, Cizhong; Gao, Shaorong

2014-12-21

Nucleosome organization determines the chromatin state, which in turn controls gene expression or silencing. Nucleosome remodeling occurs during somatic cell reprogramming, but it is still unclear to what degree the re-established nucleosome organization of induced pluripotent stem cells (iPSCs) resembles embryonic stem cells (ESCs), and whether the iPSCs inherit some residual gene expression from the parental fibroblast cells. We generated genome-wide nucleosome maps in mouse ESCs and in iPSCs reprogrammed from somatic cells belonging to three different germ layers using a secondary reprogramming system. Pairwise comparisons showed that the nucleosome organizations in the iPSCs, regardless of the iPSCs' tissue of origin, were nearly identical to the ESCs, but distinct from mouse embryonic fibroblasts (MEF). There is a canonical nucleosome arrangement of -1, nucleosome depletion region, +1, +2, +3, and so on nucleosomes around the transcription start sites of active genes whereas only a nucleosome occupies silent transcriptional units. Transcription factor binding sites possessed characteristic nucleosomal architecture, such that their access was governed by the rotational and translational settings of the nucleosome. Interestingly, the tissue-specific genes were highly expressed only in the parental somatic cells of the corresponding iPS cell line before reprogramming, but had a similar expression level in all the resultant iPSCs and ESCs. The re-established nucleosome landscape during nuclear reprogramming provides a conserved setting for accessibility of DNA sequences in mouse pluripotent stem cells. No persistent residual expression program or nucleosome positioning of the parental somatic cells that reflected their tissue of origin was passed on to the resulting mouse iPSCs.

Reprogramming of a defense signaling pathway in rough lemon and sweet orange is a critical element of the early response to ‘Candidatus Liberibacter asiaticus’

PubMed Central

Yu, Qibin; Chen, Chunxian; Du, Dongliang; Huang, Ming; Yao, Jiqiang; Yu, Fahong; Brlansky, Ronald H; Gmitter, Frederick G.

2017-01-01

Huanglongbing (HLB) in citrus infected by Candidatus Liberibacter asiaticus (CLas) has caused tremendous losses to the citrus industry. No resistant genotypes have been identified in citrus species or close relatives. Among citrus varieties, rough lemon (Citrus jambhiri) has been considered tolerant due to its ability to produce a healthy flush of new growth after infection. The difference between tolerance and susceptibility is often defined by the speed and intensity of a plant’s response to a pathogen, especially early defense responses. RNA-seq data were collected from three biological replicates of CLas- and mock-inoculated rough lemon and sweet orange at week 0 and 7 following infection. Functional analysis of the differentially expressed genes (DEGs) indicated that genes involved in the mitogen activated protein kinase (MAPK) signaling pathway were highly upregulated in rough lemon. MAPK induces the transcription of WRKY and other transcription factors which potentially turn on multiple defense-related genes. A Subnetwork Enrichment Analysis further revealed different patterns of regulation of several functional categories, suggesting DEGs with different functions were subjected to reprogramming. In general, the amplitude of the expression of defense-related genes is much greater in rough lemon than in sweet orange. A quantitative disease resistance response may contribute to the durable tolerance level to HLB observed in rough lemon. PMID:29214028

Protein phosphatase AP2C1 negatively regulates basal resistance and defense responses to Pseudomonas syringae.

PubMed

Shubchynskyy, Volodymyr; Boniecka, Justyna; Schweighofer, Alois; Simulis, Justinas; Kvederaviciute, Kotryna; Stumpe, Michael; Mauch, Felix; Balazadeh, Salma; Mueller-Roeber, Bernd; Boutrot, Freddy; Zipfel, Cyril; Meskiene, Irute

2017-02-01

Mitogen-activated protein kinases (MAPKs) mediate plant immune responses to pathogenic bacteria. However, less is known about the cell autonomous negative regulatory mechanism controlling basal plant immunity. We report the biological role of Arabidopsis thaliana MAPK phosphatase AP2C1 as a negative regulator of plant basal resistance and defense responses to Pseudomonas syringae. AP2C2, a closely related MAPK phosphatase, also negatively controls plant resistance. Loss of AP2C1 leads to enhanced pathogen-induced MAPK activities, increased callose deposition in response to pathogen-associated molecular patterns or to P. syringae pv. tomato (Pto) DC3000, and enhanced resistance to bacterial infection with Pto. We also reveal the impact of AP2C1 on the global transcriptional reprogramming of transcription factors during Pto infection. Importantly, ap2c1 plants show salicylic acid-independent transcriptional reprogramming of several defense genes and enhanced ethylene production in response to Pto. This study pinpoints the specificity of MAPK regulation by the different MAPK phosphatases AP2C1 and MKP1, which control the same MAPK substrates, nevertheless leading to different downstream events. We suggest that precise and specific control of defined MAPKs by MAPK phosphatases during plant challenge with pathogenic bacteria can strongly influence plant resistance. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Molecular Mechanisms of Induced Pluripotency

PubMed Central

Muchkaeva, I.A.; Dashinimaev, E.B.; Terskikh, V.V.; Sukhanov, Y.V.; Vasiliev, A.V.

2012-01-01

In this review the distinct aspects of somatic cell reprogramming are discussed. The molecular mechanisms of generation of induced pluripotent stem (iPS) cells from somatic cells via the introduction of transcription factors into adult somatic cells are considered. Particular attention is focused on the generation of iPS cells without genome modifications via the introduction of the mRNA of transcription factors or the use of small molecules. Furthermore, the strategy of direct reprogramming of somatic cells omitting the generation of iPS cells is considered. The data concerning the differences between ES and iPS cells and the problem of epigenetic memory are also discussed. In conclusion, the possibility of using iPS cells in regenerative medicine is considered. PMID:22708059

NGF reprograms metastatic melanoma to a bipotent glial-melanocyte neural crest-like precursor

PubMed Central

Kasemeier-Kulesa, Jennifer C.; Romine, Morgan H.; Morrison, Jason A.; Bailey, Caleb M.; Welch, Danny R.

2018-01-01

ABSTRACT Melanoma pathogenesis from normal neural crest-derived melanocytes is often fatal due to aggressive cell invasion throughout the body. The identification of signals that reprogram de-differentiated, metastatic melanoma cells to a less aggressive and stable phenotype would provide a novel strategy to limit disease progression. In this study, we identify and test the function of developmental signals within the chick embryonic neural crest microenvironment to reprogram and sustain the transition of human metastatic melanoma to a neural crest cell-like phenotype. Results reveal that co-culture of the highly aggressive and metastatic human melanoma cell line C8161 upregulate a marker of melanosome formation (Mart-1) in the presence of embryonic day 3.5 chick trunk dorsal root ganglia. We identify nerve growth factor (NGF) as the signal within this tissue driving Mart-1 re-expression and show that NGF receptors trkA and p75 cooperate to induce Mart-1 re-expression. Furthermore, Mart-1 expressing C8161 cells acquire a gene signature of poorly aggressive C81-61 cells. These data suggest that targeting NGF signaling may yield a novel strategy to reprogram metastatic melanoma toward a benign cell type. PMID:29175861

Interferons direct Th2 cell reprogramming to generate a stable GATA-3(+)T-bet(+) cell subset with combined Th2 and Th1 cell functions.

PubMed

Hegazy, Ahmed N; Peine, Michael; Helmstetter, Caroline; Panse, Isabel; Fröhlich, Anja; Bergthaler, Andreas; Flatz, Lukas; Pinschewer, Daniel D; Radbruch, Andreas; Löhning, Max

2010-01-29

Current T cell differentiation models invoke separate T helper 2 (Th2) and Th1 cell lineages governed by the lineage-specifying transcription factors GATA-3 and T-bet. However, knowledge on the plasticity of Th2 cell lineage commitment is limited. Here we show that infection with Th1 cell-promoting lymphocytic choriomeningitis virus (LCMV) reprogrammed otherwise stably committed GATA-3(+) Th2 cells to adopt a GATA-3(+)T-bet(+) and interleukin-4(+)interferon-gamma(+) "Th2+1" phenotype that was maintained in vivo for months. Th2 cell reprogramming required T cell receptor stimulation, concerted type I and type II interferon and interleukin-12 signals, and T-bet. LCMV-triggered T-bet induction in adoptively transferred virus-specific Th2 cells was crucial to prevent viral persistence and fatal immunopathology. Thus, functional reprogramming of unfavorably differentiated Th2 cells may facilitate the establishment of protective immune responses. Stable coexpression of GATA-3 and T-bet provides a molecular concept for the long-term coexistence of Th2 and Th1 cell lineage characteristics in single memory T cells. Copyright 2010 Elsevier Inc. All rights reserved.

S-phase Synchronization Facilitates the Early Progression of Induced-Cardiomyocyte Reprogramming through Enhanced Cell-Cycle Exit.

PubMed

Bektik, Emre; Dennis, Adrienne; Pawlowski, Gary; Zhou, Chen; Maleski, Danielle; Takahashi, Satoru; Laurita, Kenneth R; Deschênes, Isabelle; Fu, Ji-Dong

2018-05-04

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds a great promise for regenerative medicine and has been studied in several major directions. However, cell-cycle regulation, a fundamental biological process, has not been investigated during iCM-reprogramming. Here, our time-lapse imaging on iCMs, reprogrammed by Gata4, Mef2c, and Tbx5 (GMT) monocistronic retroviruses, revealed that iCM-reprogramming was majorly initiated at late-G1- or S-phase and nearly half of GMT-reprogrammed iCMs divided soon after reprogramming. iCMs exited cell cycle along the process of reprogramming with decreased percentage of 5-ethynyl-20-deoxyuridine (EdU)⁺/α-myosin heavy chain (αMHC)-GFP⁺ cells. S-phase synchronization post-GMT-infection could enhance cell-cycle exit of reprogrammed iCMs and yield more GFP high iCMs, which achieved an advanced reprogramming with more expression of cardiac genes than GFP low cells. However, S-phase synchronization did not enhance the reprogramming with a polycistronic-viral vector, in which cell-cycle exit had been accelerated. In conclusion, post-infection synchronization of S-phase facilitated the early progression of GMT-reprogramming through a mechanism of enhanced cell-cycle exit.

Assessing the risks of genotoxicity in the therapeutic development of induced pluripotent stem cells.

PubMed

Hong, So Gun; Dunbar, Cynthia E; Winkler, Thomas

2013-02-01

Induced pluripotent stem cells (iPSCs) have great potential for regenerative medicine as well as for basic and translational research. However, following the initial excitement over the enormous prospects of this technology, several reports uncovered serious concerns regarding its safety for clinical applications and reproducibility for laboratory applications such as disease modeling or drug screening. In particular, the genomic integrity of iPSCs is the focus of extensive research. Epigenetic remodeling, aberrant expression of reprogramming factors, clonal selection, and prolonged in vitro culture are potential pathways for acquiring genomic alterations. In this review, we will critically discuss current reprogramming technologies particularly in the context of genotoxicity, and the consequences of these alternations for the potential applications of reprogrammed cells. In addition, current strategies of genetic modification of iPSCs, as well as applicable suicide strategies to control the risk of iPSC-based therapies will be introduced.

Somatic cell cloning: the ultimate form of nuclear reprogramming?

PubMed

Piedrahita, Jorge A; Mir, Bashir; Dindot, Scott; Walker, Shawn

2004-05-01

With the increasing difficulties associated with meeting the required needs for organs used in transplantation, alternative approaches need to be considered. These include the use of stem cells as potential sources of specialized cells, the ability to transdifferentiate cell types in culture, and the development of complete organs that can be used in humans. All of the above goals will require a complete understanding of the factors affecting cell differentiation and nuclear reprogramming. To make this a reality, however, techniques associated with cloning and genetic modifications in somatic cells need to be continued to be developed and optimized. This includes not only an enhancement of the rate of homologous recombination in somatic cells, but also a thorough understanding of the nuclear reprogramming process taking place during nuclear transfer. The understanding of this process is likely to have an effect beyond the area of nuclear transfer and assist with better methods for transdifferentiation of mammalian cells.

Spermatogenesis disruption by dioxins: Epigenetic reprograming and windows of susceptibility.

PubMed

Pilsner, J Richard; Parker, Mikhail; Sergeyev, Oleg; Suvorov, Alexander

2017-04-01

Dioxins are a group of highly persistent chemicals that are generated as by-products of industrial and natural processes. Reduction in sperm counts is among the most sensitive endpoints of dioxin toxicity. The exact mechanism by which dioxins reduce sperm counts is not known. Recent data implicate the role of epididymal factors rather than disruption of spermatogenesis. Studies reviewed here demonstrate that dioxins induce the transfer of environmental conditions to the next generation via male germline following exposures during the window of epigenetic reprogramming of primordial germ cells. Increased incidence of birth defects in offspring of male veterans exposed to dioxin containing, Agent Orange, suggest that dioxins may induce epigenomic changes in male germ cells of adults during spermatogenesis. This is supported by recent animal data that show that environmental conditions can cause epigenetic dysregulation in sperm in the context of specific windows of epigenetic reprogramming during spermatogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimada, Hidenori; Hashimoto, Yoshiya; Nakada, Akira

2012-01-13

Highlights: Black-Right-Pointing-Pointer Very rapid generation of human iPS cells under optimized conditions. Black-Right-Pointing-Pointer Five chemical inhibitors under hypoxia boosted reprogramming. Black-Right-Pointing-Pointer We performed genome-wide DNA methylation analysis. -- Abstract: Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain largemore » amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1 month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of iPS cell technology to biomedical research.« less

Cell reprogramming by 3D bioprinting of human fibroblasts in polyurethane hydrogel for fabrication of neural-like constructs.

PubMed

Ho, Lin; Hsu, Shan-Hui

2018-04-01

3D bioprinting is a technique which enables the direct printing of biodegradable materials with cells into 3D tissue. So far there is no cell reprogramming in situ performed with the 3D bioprinting process. Forkhead box D3 (FoxD3) is a transcription factor and neural crest marker, which was reported to reprogram human fibroblasts into neural crest stem-like cells. In this study, we synthesized a new biodegradable thermo-responsive waterborne polyurethane (PU) gel as a bioink. FoxD3 plasmids and human fibroblasts were co-extruded with the PU hydrogel through the syringe needle tip for cell reprogramming. The rheological properties of the PU hydrogel including the modulus, gelation time, and shear thinning were optimized for the transfection effect of FoxD3 in situ. The corresponding shear rate and shear stress were examined. Results showed that human fibroblasts could be reprogrammed into neural crest stem-like cells with high cell viability during the extrusion process under an average shear stress ∼190 Pa. We further translated the method to the extrusion-based 3D bioprinting, and demonstrated that human fibroblasts co-printed with FoxD3 in the thermo-responsive PU hydrogel could be reprogrammed and differentiated into a neural-tissue like construct at 14 days after induction. The neural-like tissue construct produced by 3D bioprinting from human fibroblasts may be applied to personalized drug screening or neuroregeneration. There is no study so far on cell reprogramming in situ with 3D bioprinting. In this manuscript, a new thermoresponsive polyurethane bioink was developed and employed to deliver FoxD3 plasmid into human fibroblasts by the extrusion-based bioprinting. When the polyurethane gel was extruded through the syringe tip, the shear stress generated may have caused the transient membrane permeability for transfection. The shear stress was optimized for transfection in situ by 3D bioprinting. We demonstrated that human fibroblasts could be reprogrammed into neural crest-like stem cells by 3D bioprinting with the gel, and the reprogrammed cells underwent neural differentiation in the printed structure after induction. The neural-like tissue engineering constructs fabricated by 3D bioprinting from human fibroblasts may be applied for neuroregeneration or further developed as mini-brain for basic research and drug screening. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Efficient Direct Reprogramming of Mature Amniotic Cells into Endothelial Cells by ETS Factors and TGFβ Suppression

PubMed Central

Ginsberg, Michael; James, Daylon; Ding, Bi-Sen; Nolan, Daniel; Geng, Fuqiang; Butler, Jason M; Schachterle, William; Pulijaal, Venkat R; Mathew, Susan; Chasen, Stephen T; Xiang, Jenny; Rosenwaks, Zev; Shido, Koji; Elemento, Olivier; Rabbany, Sina Y; Rafii, Shahin

2012-01-01

ETS transcription factors ETV2, FLI1 and ERG1 specify pluripotent stem cells into endothelial cells (ECs). However, these ECs are unstable and drift towards non-vascular cell fates. We show that human mid-gestation c-Kit− lineage-committed amniotic cells (ACs) can be readily reprogrammed into induced vascular endothelial cells (iVECs). Transient ETV2 expression in ACs generated proliferative but immature iVECs, while co-expression with FLI1/ERG1 endowed iVECs with a vascular repertoire and morphology matching mature stable ECs. Brief TGFβ-inhibition functionalized VEGFR2 signaling, augmenting specification of ACs to iVECs. Genome-wide transcriptional analyses showed that iVECs are similar to adult ECs in which vascular-specific genes are turned on and non-vascular genes are silenced. Functionally, iVECs form long-lasting patent vasculature in Matrigel plugs and regenerating livers. Thus, short-term ETV2 expression and TGFβ-inhibition along with constitutive ERG1/FLI1 co-expression reprogram mature ACs into durable and functional iVECs with clinical-scale expansion potential. Public banking of HLA-typed iVECs would establish a vascular inventory for treatment of genetically diverse disorders. PMID:23084400

Paternal stress exposure alters sperm microRNA content and reprograms offspring HPA stress axis regulation

PubMed Central

Rodgers, Ali B.; Morgan, Christopher P.; Bronson, Stefanie L.; Revello, Sonia; Bale, Tracy L.

2013-01-01

Neuropsychiatric disease frequently presents with an underlying hypo- or hyper- reactivity of the HPA stress axis, suggesting an exceptional vulnerability of this circuitry to external perturbations. Parental lifetime exposures to environmental challenges are associated with increased offspring neuropsychiatric disease risk, and likely contribute to stress dysregulation. While maternal influences have been extensively examined, much less is known regarding the specific role of paternal factors. To investigate the potential mechanisms by which paternal stress may contribute to offspring hypothalamic-pituitary-adrenal (HPA) axis dysregulation, we exposed mice to six weeks of chronic stress prior to breeding. As epidemiological studies support variation in paternal germ cell susceptibility to reprogramming across the lifespan, male stress exposure occurred either throughout puberty or in adulthood. Remarkably, offspring of sires from both paternal stress groups displayed significantly reduced HPA axis stress responsivity. Gene set enrichment analyses in offspring stress regulating brain regions, the paraventricular nucleus (PVN) and the bed nucleus of stria terminalis (BNST), revealed global pattern changes in transcription suggestive of epigenetic reprogramming and consistent with altered offspring stress responsivity, including increased expression of glucocorticoid-responsive genes in the PVN. In examining potential epigenetic mechanisms of germ cell transmission, we found robust changes in sperm miRNA (miR) content, where nine specific miRs were significantly increased in both paternal stress groups. Overall, these results demonstrate that paternal experience across the lifespan can induce germ cell epigenetic reprogramming and impact offspring HPA stress axis regulation, and may therefore offer novel insight into factors influencing neuropsychiatric disease risk. PMID:23699511

Delayed transition to new cell fates during cellular reprogramming

PubMed Central

Cheng, Xianrui; Lyons, Deirdre C.; Socolar, Joshua E. S.; McClay, David R.

2014-01-01

In many embryos specification toward one cell fate can be diverted to a different cell fate through a reprogramming process. Understanding how that process works will reveal insights into the developmental regulatory logic that emerged from evolution. In the sea urchin embryo, cells at gastrulation were found to reprogram and replace missing cell types after surgical dissections of the embryo. Non-skeletogenic mesoderm (NSM) cells reprogrammed to replace missing skeletogenic mesoderm cells and animal caps reprogrammed to replace all endomesoderm. In both cases evidence of reprogramming onset was first observed at the early gastrula stage, even if the cells to be replaced were removed earlier in development. Once started however, the reprogramming occurred with compressed gene expression dynamics. The NSM did not require early contact with the skeletogenic cells to reprogram, but the animal cap cells gained the ability to reprogram early in gastrulation only after extended contact with the vegetal halves prior to that time. If the entire vegetal half was removed at early gastrula, the animal caps reprogrammed and replaced the vegetal half endomesoderm. If the animal caps carried morpholinos to either hox11/13b or foxA (endomesoderm specification genes), the isolated animal caps failed to reprogram. Together these data reveal that the emergence of a reprogramming capability occurs at early gastrulation in the sea urchin embryo and requires activation of early specification components of the target tissues. PMID:24780626

Discovery of survival factor for primitive chronic myeloid leukemia cells using induced pluripotent stem cells

PubMed Central

Suknuntha, Kran; Ishii, Yuki; Tao, Lihong; Hu, Kejin; McIntosh, Brian E.; Yang, David; Swanson, Scott; Stewart, Ron; Wang, Jean Y.J.; Thomson, James; Slukvin, Igor

2016-01-01

A definitive cure for chronic myeloid leukemia (CML) requires identifying novel therapeutic targets to eradicate leukemia stem cells (LSCs). However, the rarity of LSCs within the primitive hematopoietic cell compartment remains a major limiting factor for their study in humans. Here we show that primitive hematopoietic cells with typical LSC features, including adhesion defect, increased long-term survival and proliferation, and innate resistance to tyrosine kinase inhibitor (TKI) imatinib, can be generated de novo from reprogrammed primary CML cells. Using CML iPSC-derived primitive leukemia cells, we discovered olfactomedin 4 (OLFM4) as a novel factor that contributes to survival and growth of somatic lin−CD34+ cells from bone marrow of patients with CML in chronic phase, but not primitive hematopoietic cells from normal bone marrow. Overall, this study shows the feasibility and advantages of using reprogramming technology to develop strategies for targeting primitive leukemia cells. PMID:26561938

Transcript levels of several epigenome regulatory genes in bovine somatic donor cells are not correlated with their cloning efficiency.

PubMed

Zhou, Wenli; Sadeghieh, Sanaz; Abruzzese, Ronald; Uppada, Subhadra; Meredith, Justin; Ohlrichs, Charletta; Broek, Diane; Polejaeva, Irina

2009-09-01

Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

Cooperative Regulatory Functions of miR858 and MYB83 during Cyst Nematode Parasitism1[OPEN

PubMed Central

Piya, Sarbottam; Kihm, Christina; Baum, Thomas J.

2017-01-01

MicroRNAs (miRNAs) recently have been established as key regulators of transcriptome reprogramming that define cell function and identity. Nevertheless, the molecular functions of the greatest number of miRNA genes remain to be determined. Here, we report cooperative regulatory functions of miR858 and its MYB83 transcription factor target gene in transcriptome reprogramming during Heterodera cyst nematode parasitism of Arabidopsis (Arabidopsis thaliana). Gene expression analyses and promoter-GUS fusion assays documented a role of miR858 in posttranscriptional regulation of MYB83 in the Heterodera schachtii-induced feeding sites, the syncytia. Constitutive overexpression of miR858 interfered with H. schachtii parasitism of Arabidopsis, leading to reduced susceptibility, while reduced miR858 abundance enhanced plant susceptibility. Similarly, MYB83 expression increases were conducive to nematode infection because overexpression of a noncleavable coding sequence of MYB83 significantly increased plant susceptibility, whereas a myb83 mutation rendered the plants less susceptible. In addition, RNA-seq analysis revealed that genes involved in hormone signaling pathways, defense response, glucosinolate biosynthesis, cell wall modification, sugar transport, and transcriptional control are the key etiological factors by which MYB83 facilitates nematode parasitism of Arabidopsis. Furthermore, we discovered that miR858-mediated silencing of MYB83 is tightly regulated through a feedback loop that might contribute to fine-tuning the expression of more than a thousand of MYB83-regulated genes in the H. schachtii-induced syncytium. Together, our results suggest a role of the miR858-MYB83 regulatory system in finely balancing gene expression patterns during H. schachtii parasitism of Arabidopsis to ensure optimal cellular function. PMID:28512179

Cancer drug addiction is relayed by an ERK2-dependent phenotype switch.

PubMed

Kong, Xiangjun; Kuilman, Thomas; Shahrabi, Aida; Boshuizen, Julia; Kemper, Kristel; Song, Ji-Ying; Niessen, Hans W M; Rozeman, Elisa A; Geukes Foppen, Marnix H; Blank, Christian U; Peeper, Daniel S

2017-10-12

Observations from cultured cells, animal models and patients raise the possibility that the dependency of tumours on the therapeutic drugs to which they have acquired resistance represents a vulnerability with potential applications in cancer treatment. However, for this drug addiction trait to become of clinical interest, we must first define the mechanism that underlies it. We performed an unbiased CRISPR-Cas9 knockout screen on melanoma cells that were both resistant and addicted to inhibition of the serine/threonine-protein kinase BRAF, in order to functionally mine their genome for 'addiction genes'. Here we describe a signalling pathway comprising ERK2 kinase and JUNB and FRA1 transcription factors, disruption of which allowed addicted tumour cells to survive on treatment discontinuation. This occurred in both cultured cells and mice and was irrespective of the acquired drug resistance mechanism. In melanoma and lung cancer cells, death induced by drug withdrawal was preceded by a specific ERK2-dependent phenotype switch, alongside transcriptional reprogramming reminiscent of the epithelial-mesenchymal transition. In melanoma cells, this reprogramming caused the shutdown of microphthalmia-associated transcription factor (MITF), a lineage survival oncoprotein; restoring this protein reversed phenotype switching and prevented the lethality associated with drug addiction. In patients with melanoma that had progressed during treatment with a BRAF inhibitor, treatment cessation was followed by increased expression of the receptor tyrosine kinase AXL, which is associated with the phenotype switch. Drug discontinuation synergized with the melanoma chemotherapeutic agent dacarbazine by further suppressing MITF and its prosurvival target, B-cell lymphoma 2 (BCL-2), and by inducing DNA damage in cancer cells. Our results uncover a pathway that underpins drug addiction in cancer cells, which may help to guide the use of alternating therapeutic strategies for enhanced clinical responses in drug-resistant cancers.

Resetting the transcription factor network reverses terminal chronic hepatic failure

PubMed Central

Nishikawa, Taichiro; Bell, Aaron; Brooks, Jenna M.; Setoyama, Kentaro; Melis, Marta; Han, Bing; Fukumitsu, Ken; Handa, Kan; Tian, Jianmin; Kaestner, Klaus H.; Vodovotz, Yoram; Locker, Joseph; Soto-Gutierrez, Alejandro; Fox, Ira J.

2015-01-01

The cause of organ failure is enigmatic for many degenerative diseases, including end-stage liver disease. Here, using a CCl4-induced rat model of irreversible and fatal hepatic failure, which also exhibits terminal changes in the extracellular matrix, we demonstrated that chronic injury stably reprograms the critical balance of transcription factors and that diseased and dedifferentiated cells can be returned to normal function by re-expression of critical transcription factors, a process similar to the type of reprogramming that induces somatic cells to become pluripotent or to change their cell lineage. Forced re-expression of the transcription factor HNF4α induced expression of the other hepatocyte-expressed transcription factors; restored functionality in terminally diseased hepatocytes isolated from CCl4-treated rats; and rapidly reversed fatal liver failure in CCl4-treated animals by restoring diseased hepatocytes rather than replacing them with new hepatocytes or stem cells. Together, the results of our study indicate that disruption of the transcription factor network and cellular dedifferentiation likely mediate terminal liver failure and suggest reinstatement of this network has therapeutic potential for correcting organ failure without cell replacement. PMID:25774505

The WRKY transcription factor family and senescence in switchgrass

USDA-ARS?s Scientific Manuscript database

Background: Early aerial senescence in switchgrass (Panicum virgatum) can significantly limit biomass yields. WRKY transcription factors that can regulate senescence could be used to reprogram senescence and enhance biomass yields. Methods: All potential WRKY genes present in the version 1.0 of the...

SoxC Transcription Factors Are Required for Neuronal Differentiation in Adult Hippocampal Neurogenesis

PubMed Central

Mu, Lifang; Berti, Lucia; Masserdotti, Giacomo; Covic, Marcela; Michaelidis, Theologos M.; Doberauer, Kathrin; Merz, Katharina; Rehfeld, Frederick; Haslinger, Anja; Wegner, Michael; Sock, Elisabeth; Lefebvre, Veronique; Couillard-Despres, Sebastien; Aigner, Ludwig; Berninger, Benedikt; Lie, D. Chichung

2012-01-01

Neural stem cells (NSCs) generate new hippocampal dentate granule neurons throughout adulthood. The genetic programs controlling neuronal differentiation of adult NSCs are only poorly understood. Here we show that, in the adult mouse hippocampus, expression of the SoxC transcription factors Sox4 and Sox11 is initiated around the time of neuronal commitment of adult NSCs and is maintained in immature neurons. Overexpression of Sox4 and Sox11 strongly promotes in vitro neurogenesis from adult NSCs, whereas ablation of Sox4/Sox11 prevents in vitro and in vivo neurogenesis from adult NSCs. Moreover, we demonstrate that SoxC transcription factors target the promoters of genes that are induced on neuronal differentiation of adult NSCs. Finally, we show that reprogramming of astroglia into neurons is dependent on the presence of SoxC factors. These data identify SoxC proteins as essential contributors to the genetic network controlling neuronal differentiation in adult neurogenesis and neuronal reprogramming of somatic cells. PMID:22378879

Non-Viral Generation of Marmoset Monkey iPS Cells by a Six-Factor-in-One-Vector Approach

PubMed Central

Debowski, Katharina; Warthemann, Rita; Lentes, Jana; Salinas-Riester, Gabriela; Dressel, Ralf; Langenstroth, Daniel; Gromoll, Jörg; Sasaki, Erika; Behr, Rüdiger

2015-01-01

Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS) cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus) as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80) and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement therapies in preclinical settings. PMID:25785453

Non-viral generation of marmoset monkey iPS cells by a six-factor-in-one-vector approach.

PubMed

Debowski, Katharina; Warthemann, Rita; Lentes, Jana; Salinas-Riester, Gabriela; Dressel, Ralf; Langenstroth, Daniel; Gromoll, Jörg; Sasaki, Erika; Behr, Rüdiger

2015-01-01

Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS) cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus) as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80) and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement therapies in preclinical settings.

Induced pluripotent stem (iPS) cells: a new source for cell-based therapeutics?

PubMed

de Lázaro, Irene; Yilmazer, Açelya; Kostarelos, Kostas

2014-07-10

The generation of induced pluripotent stem (iPS) cells from somatic cells by the ectopic expression of defined transcription factors has provided the regenerative medicine field with a new tool for cell replacement strategies. The advantages that these pluripotent cells can offer in comparison to other sources of stem cells include the generation of patient-derived cells and the lack of embryonic tissue while maintaining a versatile differentiation potential. The promise of iPS cell derivatives for therapeutic applications is encouraging albeit very early in development, with the first clinical study currently ongoing in Japan. Many challenges are yet to be circumvented before this technology can be clinically translated widely though. The delivery and expression of the reprogramming factors, the genomic instability, epigenetic memory and impact of cell propagation in culture are only some of the concerns. This article aims to critically discuss the potential of iPS cells as a new source of cell therapeutics. Copyright © 2014 Elsevier B.V. All rights reserved.

Transposable elements at the center of the crossroads between embryogenesis, embryonic stem cells, reprogramming, and long non-coding RNAs.

PubMed

Hutchins, Andrew Paul; Pei, Duanqing

Transposable elements (TEs) are mobile genomic sequences of DNA capable of autonomous and non-autonomous duplication. TEs have been highly successful, and nearly half of the human genome now consists of various families of TEs. Originally thought to be non-functional, these elements have been co-opted by animal genomes to perform a variety of physiological functions ranging from TE-derived proteins acting directly in normal biological functions, to innovations in transcription factor logic and influence on epigenetic control of gene expression. During embryonic development, when the genome is epigenetically reprogrammed and DNA-demethylated, TEs are released from repression and show embryonic stage-specific expression, and in human and mouse embryos, intact TE-derived endogenous viral particles can even be detected. A similar process occurs during the reprogramming of somatic cells to pluripotent cells: When the somatic DNA is demethylated, TEs are released from repression. In embryonic stem cells (ESCs), where DNA is hypomethylated, an elaborate system of epigenetic control is employed to suppress TEs, a system that often overlaps with normal epigenetic control of ESC gene expression. Finally, many long non-coding RNAs (lncRNAs) involved in normal ESC function and those assisting or impairing reprogramming contain multiple TEs in their RNA. These TEs may act as regulatory units to recruit RNA-binding proteins and epigenetic modifiers. This review covers how TEs are interlinked with the epigenetic machinery and lncRNAs, and how these links influence each other to modulate aspects of ESCs, embryogenesis, and somatic cell reprogramming.

Generation and genetic modification of induced pluripotent stem cells.

PubMed

Schambach, Axel; Cantz, Tobias; Baum, Christopher; Cathomen, Toni

2010-07-01

The generation of induced pluripotent stem cells (iPSCs) enabled by exogenous expression of the canonical Oct4, Sox2, Klf4 and c-Myc reprogramming factors has opened new ways to create patient- or disease-specific pluripotent cells. iPSCs represent an almost inexhaustible source of cells for targeted differentiation into somatic effector cells and hence are likely to be invaluable for therapeutic applications and disease-related research. After an introduction on the biology of reprogramming we cover emerging technological advances, including new reprogramming approaches, small-molecule compounds and tailored genetic modification, and give an outlook towards potential clinical applications of iPSCs. Although this field is progressing rapidly, reprogramming is still an inefficient process. The reader will learn about innovative tools to generate patient-specific iPSCs and how to modify these established lines in a safe way. Ideally, the disease-causing mutation is edited directly in the genome using novel technologies based on artificial nucleases, such as zinc-finger nucleases. Human iPSCs create fascinating options with regard to disease modeling, drug testing, developmental studies and therapeutic applications. However, important hurdles have to be taken and more efficient protocols to be established to achieve the ambitious goal of bringing iPSCs into clinical use.

Chromosome microduplication in somatic cells decreases the genetic stability of human reprogrammed somatic cells and results in pluripotent stem cells.

PubMed

Yu, Yang; Chang, Liang; Zhao, Hongcui; Li, Rong; Fan, Yong; Qiao, Jie

2015-05-12

Human pluripotent stem cells, including cloned embryonic and induced pluripotent stem cells, offer a limitless cellular source for regenerative medicine. However, their derivation efficiency is limited, and a large proportion of cells are arrested during reprogramming. In the current study, we explored chromosome microdeletion/duplication in arrested and established reprogrammed cells. Our results show that aneuploidy induced by somatic cell nuclear transfer technology is a key factor in the developmental failure of cloned human embryos and primary colonies from implanted cloned blastocysts and that expression patterns of apoptosis-related genes are dynamically altered. Overall, ~20%-53% of arrested primary colonies in induced plurpotent stem cells displayed aneuploidy, and upregulation of P53 and Bax occurred in all arrested primary colonies. Interestingly, when somatic cells with pre-existing chromosomal mutations were used as donor cells, no cloned blastocysts were obtained, and additional chromosomal mutations were detected in the resulting iPS cells following long-term culture, which was not observed in the two iPS cell lines with normal karyotypes. In conclusion, aneuploidy induced by the reprogramming process restricts the derivation of pluripotent stem cells, and, more importantly, pre-existing chromosomal mutations enhance the risk of genome instability, which limits the clinical utility of these cells.

Epigenetic reprogramming in plant sexual reproduction.

PubMed

Kawashima, Tomokazu; Berger, Frédéric

2014-09-01

Epigenetic reprogramming consists of global changes in DNA methylation and histone modifications. In mammals, epigenetic reprogramming is primarily associated with sexual reproduction and occurs during both gametogenesis and early embryonic development. Such reprogramming is crucial not only to maintain genomic integrity through silencing transposable elements but also to reset the silenced status of imprinted genes. In plants, observations of stable transgenerational inheritance of epialleles have argued against reprogramming. However, emerging evidence supports that epigenetic reprogramming indeed occurs during sexual reproduction in plants and that it has a major role in maintaining genome integrity and a potential contribution to epiallelic variation.

In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells

PubMed Central

Komuta, Yukari; Ishii, Toshiyuki; Kaneda, Makoto; Ueda, Yasuji; Miyamoto, Kiyoko; Toyoda, Masashi; Umezawa, Akihiro

2016-01-01

ABSTRACT Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs) have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration. PMID:27170256

Decoding the regulatory landscape of melanoma reveals TEADS as regulators of the invasive cell state

PubMed Central

Verfaillie, Annelien; Imrichova, Hana; Atak, Zeynep Kalender; Dewaele, Michael; Rambow, Florian; Hulselmans, Gert; Christiaens, Valerie; Svetlichnyy, Dmitry; Luciani, Flavie; Van den Mooter, Laura; Claerhout, Sofie; Fiers, Mark; Journe, Fabrice; Ghanem, Ghanem-Elias; Herrmann, Carl; Halder, Georg; Marine, Jean-Christophe; Aerts, Stein

2015-01-01

Transcriptional reprogramming of proliferative melanoma cells into a phenotypically distinct invasive cell subpopulation is a critical event at the origin of metastatic spreading. Here we generate transcriptome, open chromatin and histone modification maps of melanoma cultures; and integrate this data with existing transcriptome and DNA methylation profiles from tumour biopsies to gain insight into the mechanisms underlying this key reprogramming event. This shows thousands of genomic regulatory regions underlying the proliferative and invasive states, identifying SOX10/MITF and AP-1/TEAD as regulators, respectively. Knockdown of TEADs shows a previously unrecognized role in the invasive gene network and establishes a causative link between these transcription factors, cell invasion and sensitivity to MAPK inhibitors. Using regulatory landscapes and in silico analysis, we show that transcriptional reprogramming underlies the distinct cellular states present in melanoma. Furthermore, it reveals an essential role for the TEADs, linking it to clinically relevant mechanisms such as invasion and resistance. PMID:25865119

Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors

PubMed Central

Doulatov, Sergei; Vo, Linda T.; Macari, Elizabeth R.; Wahlster, Lara; Kinney, Melissa A.; Taylor, Alison M.; Barragan, Jessica; Gupta, Manav; McGrath, Katherine; Lee, Hsiang-Ying; Humphries, Jessica M.; DeVine, Alex; Narla, Anupama; Alter, Blanche P.; Beggs, Alan H.; Agarwal, Suneet; Ebert, Benjamin L.; Gazda, Hanna T.; Lodish, Harvey F.; Sieff, Colin A.; Schlaeger, Thorsten M.; Zon, Leonard I.; Daley, George Q.

2017-01-01

Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA. PMID:28179501

Excision of a viral reprogramming cassette by delivery of synthetic Cre mRNA

PubMed Central

Loh, Yuin-Han; Yang, Jimmy Chen; De Los Angeles, Alejandro; Guo, Chunguang; Cherry, Anne; Rossi, Derrick J.; Park, In-Hyun; Daley, George Q.

2012-01-01

The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy, in vitro modeling of human disease, and drug screening. To date, most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively, transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This report describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector, which includes the coding sequences of human OCT4, SOX2, KLF4, and cMYC linked with picornaviral 2A plasmids. Moreover, after reprogramming has been achieved, this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells. PMID:22605648

PRC2 Represses Hormone-Induced Somatic Embryogenesis in Vegetative Tissue of Arabidopsis thaliana

PubMed Central

Mozgová, Iva

2017-01-01

Many plant cells can be reprogrammed into a pluripotent state that allows ectopic organ development. Inducing totipotent states to stimulate somatic embryo (SE) development is, however, challenging due to insufficient understanding of molecular barriers that prevent somatic cell dedifferentiation. Here we show that Polycomb repressive complex 2 (PRC2)-activity imposes a barrier to hormone-mediated transcriptional reprogramming towards somatic embryogenesis in vegetative tissue of Arabidopsis thaliana. We identify factors that enable SE development in PRC2-depleted shoot and root tissue and demonstrate that the establishment of embryogenic potential is marked by ectopic co-activation of crucial developmental regulators that specify shoot, root and embryo identity. Using inducible activation of PRC2 in PRC2-depleted cells, we demonstrate that transient reduction of PRC2 activity is sufficient for SE formation. We suggest that modulation of PRC2 activity in plant vegetative tissue combined with targeted activation of developmental pathways will open possibilities for novel approaches to cell reprogramming. PMID:28095419

Mammalian genes induce partially reprogrammed pluripotent stem cells in non-mammalian vertebrate and invertebrate species

PubMed Central

Rosselló, Ricardo Antonio; Chen, Chun-Chun; Dai, Rui; Howard, Jason T; Hochgeschwender, Ute; Jarvis, Erich D

2013-01-01

Cells are fundamental units of life, but little is known about evolution of cell states. Induced pluripotent stem cells (iPSCs) are once differentiated cells that have been re-programmed to an embryonic stem cell-like state, providing a powerful platform for biology and medicine. However, they have been limited to a few mammalian species. Here we found that a set of four mammalian transcription factor genes used to generate iPSCs in mouse and humans can induce a partially reprogrammed pluripotent stem cell (PRPSCs) state in vertebrate and invertebrate model organisms, in mammals, birds, fish, and fly, which span 550 million years from a common ancestor. These findings are one of the first to show cross-lineage stem cell-like induction, and to generate pluripotent-like cells for several of these species with in vivo chimeras. We suggest that the stem-cell state may be highly conserved across a wide phylogenetic range. DOI: http://dx.doi.org/10.7554/eLife.00036.001 PMID:24015354

DNA Methylation in Embryo Development: Epigenetic Impact of ART (Assisted Reproductive Technologies).

PubMed

Canovas, Sebastian; Ross, Pablo J; Kelsey, Gavin; Coy, Pilar

2017-11-01

DNA methylation can be considered a component of epigenetic memory with a critical role during embryo development, and which undergoes dramatic reprogramming after fertilization. Though it has been a focus of research for many years, the reprogramming mechanism is still not fully understood. Recent results suggest that absence of maintenance at DNA replication is a major factor, and that there is an unexpected role for TET3-mediated oxidation of 5mC to 5hmC in guarding against de novo methylation. Base-resolution and genome-wide profiling methods are enabling more comprehensive assessments of the extent to which ART might impair DNA methylation reprogramming, and which sequence elements are most vulnerable. Indeed, as we also review here, studies showing the effect of culture media, ovarian stimulation or embryo transfer on the methylation pattern of embryos emphasize the need to face ART-associated defects and search for strategies to mitigate adverse effects on the health of ART-derived children. © 2017 WILEY Periodicals, Inc.

Cellular reprogramming dynamics follow a simple 1D reaction coordinate

NASA Astrophysics Data System (ADS)

Teja Pusuluri, Sai; Lang, Alex H.; Mehta, Pankaj; Castillo, Horacio E.

2018-01-01

Cellular reprogramming, the conversion of one cell type to another, induces global changes in gene expression involving thousands of genes, and understanding how cells globally alter their gene expression profile during reprogramming is an ongoing problem. Here we reanalyze time-course data on cellular reprogramming from differentiated cell types to induced pluripotent stem cells (iPSCs) and show that gene expression dynamics during reprogramming follow a simple 1D reaction coordinate. This reaction coordinate is independent of both the time it takes to reach the iPSC state as well as the details of the experimental protocol used. Using Monte-Carlo simulations, we show that such a reaction coordinate emerges from epigenetic landscape models where cellular reprogramming is viewed as a ‘barrier-crossing’ process between cell fates. Overall, our analysis and model suggest that gene expression dynamics during reprogramming follow a canonical trajectory consistent with the idea of an ‘optimal path’ in gene expression space for reprogramming.

Inhibition of TGFβ signaling increases direct conversion of fibroblasts to induced cardiomyocytes.

PubMed

Ifkovits, Jamie L; Addis, Russell C; Epstein, Jonathan A; Gearhart, John D

2014-01-01

Recent studies have been successful at utilizing ectopic expression of transcription factors to generate induced cardiomyocytes (iCMs) from fibroblasts, albeit at a low frequency in vitro. This work investigates the influence of small molecules that have been previously reported to improve differentiation to cardiomyocytes as well as reprogramming to iPSCs in conjunction with ectopic expression of the transcription factors Hand2, Nkx2.5, Gata4, Mef2C, and Tbx5 on the conversion to functional iCMs. We utilized a reporter system in which the calcium indicator GCaMP is driven by the cardiac Troponin T promoter to quantify iCM yield. The TGFβ inhibitor, SB431542 (SB), was identified as a small molecule capable of increasing the conversion of both mouse embryonic fibroblasts and adult cardiac fibroblasts to iCMs up to ∼5 fold. Further characterization revealed that inhibition of TGFβ by SB early in the reprogramming process led to the greatest increase in conversion of fibroblasts to iCMs in a dose-responsive manner. Global transcriptional analysis at Day 3 post-induction of the transcription factors revealed an increased expression of genes associated with the development of cardiac muscle in the presence of SB compared to the vehicle control. Incorporation of SB in the reprogramming process increases the efficiency of iCM generation, one of the major goals necessary to enable the use of iCMs for discovery-based applications and for the clinic.

Inhibition of TGFβ Signaling Increases Direct Conversion of Fibroblasts to Induced Cardiomyocytes

PubMed Central

Ifkovits, Jamie L.; Addis, Russell C.; Epstein, Jonathan A.; Gearhart, John D.

2014-01-01

Recent studies have been successful at utilizing ectopic expression of transcription factors to generate induced cardiomyocytes (iCMs) from fibroblasts, albeit at a low frequency in vitro. This work investigates the influence of small molecules that have been previously reported to improve differentiation to cardiomyocytes as well as reprogramming to iPSCs in conjunction with ectopic expression of the transcription factors Hand2, Nkx2.5, Gata4, Mef2C, and Tbx5 on the conversion to functional iCMs. We utilized a reporter system in which the calcium indicator GCaMP is driven by the cardiac Troponin T promoter to quantify iCM yield. The TGFβ inhibitor, SB431542 (SB), was identified as a small molecule capable of increasing the conversion of both mouse embryonic fibroblasts and adult cardiac fibroblasts to iCMs up to ∼5 fold. Further characterization revealed that inhibition of TGFβ by SB early in the reprogramming process led to the greatest increase in conversion of fibroblasts to iCMs in a dose-responsive manner. Global transcriptional analysis at Day 3 post-induction of the transcription factors revealed an increased expression of genes associated with the development of cardiac muscle in the presence of SB compared to the vehicle control. Incorporation of SB in the reprogramming process increases the efficiency of iCM generation, one of the major goals necessary to enable the use of iCMs for discovery-based applications and for the clinic. PMID:24586958

Polycistronic lentiviral vector for "hit and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells.

PubMed

Chang, Chia-Wei; Lai, Yi-Shin; Pawlik, Kevin M; Liu, Kaimao; Sun, Chiao-Wang; Li, Chao; Schoeb, Trenton R; Townes, Tim M

2009-05-01

We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts using a single, polycistronic lentiviral vector encoding the reprogramming factors Oct4, Sox2, and Klf4. Porcine teschovirus-1 2A sequences that trigger ribosome skipping were inserted between human cDNAs for these factors, and the polycistron was subcloned downstream of the elongation factor 1 alpha promoter in a self-inactivating (SIN) lentiviral vector containing a loxP site in the truncated 3' long terminal repeat (LTR). Adult skin fibroblasts from a humanized mouse model of sickle cell disease were transduced with this single lentiviral vector, and iPS cell colonies were picked within 30 days. These cells expressed endogenous Oct4, Sox2, Nanog, alkaline phosphatase, stage-specific embryonic antigen-1, and other markers of pluripotency. The iPS cells produced teratomas containing tissue derived from all three germ layers after injection into immunocompromised mice and formed high-level chimeras after injection into murine blastocysts. iPS cell lines with as few as three lentiviral insertions were obtained. Expression of Cre recombinase in these iPS cells resulted in deletion of the lentiviral vector, and sequencing of insertion sites demonstrated that remnant 291-bp SIN LTRs containing a single loxP site did not interrupt coding sequences, promoters, or known regulatory elements. These results suggest that a single, polycistronic "hit and run" vector can safely and effectively reprogram adult dermal fibroblasts into iPS cells.

Cellular programming and reprogramming: sculpting cell fate for the production of dopamine neurons for cell therapy.

PubMed

Aguila, Julio C; Hedlund, Eva; Sanchez-Pernaute, Rosario

2012-01-01

Pluripotent stem cells are regarded as a promising cell source to obtain human dopamine neurons in sufficient amounts and purity for cell replacement therapy. Importantly, the success of clinical applications depends on our ability to steer pluripotent stem cells towards the right neuronal identity. In Parkinson disease, the loss of dopamine neurons is more pronounced in the ventrolateral population that projects to the sensorimotor striatum. Because synapses are highly specific, only neurons with this precise identity will contribute, upon transplantation, to the synaptic reconstruction of the dorsal striatum. Thus, understanding the developmental cell program of the mesostriatal dopamine neurons is critical for the identification of the extrinsic signals and cell-intrinsic factors that instruct and, ultimately, determine cell identity. Here, we review how extrinsic signals and transcription factors act together during development to shape midbrain cell fates. Further, we discuss how these same factors can be applied in vitro to induce, select, and reprogram cells to the mesostriatal dopamine fate.

A practical and efficient cellular substrate for the generation of induced pluripotent stem cells from adults: blood-derived endothelial progenitor cells.

PubMed

Geti, Imbisaat; Ormiston, Mark L; Rouhani, Foad; Toshner, Mark; Movassagh, Mehregan; Nichols, Jennifer; Mansfield, William; Southwood, Mark; Bradley, Allan; Rana, Amer Ahmed; Vallier, Ludovic; Morrell, Nicholas W

2012-12-01

Induced pluripotent stem cells (iPSCs) have the potential to generate patient-specific tissues for disease modeling and regenerative medicine applications. However, before iPSC technology can progress to the translational phase, several obstacles must be overcome. These include uncertainty regarding the ideal somatic cell type for reprogramming, the low kinetics and efficiency of reprogramming, and karyotype discrepancies between iPSCs and their somatic precursors. Here we describe the use of late-outgrowth endothelial progenitor cells (L-EPCs), which possess several favorable characteristics, as a cellular substrate for the generation of iPSCs. We have developed a protocol that allows the reliable isolation of L-EPCs from peripheral blood mononuclear cell preparations, including frozen samples. As a proof-of-principle for clinical applications we generated EPC-iPSCs from both healthy individuals and patients with heritable and idiopathic forms of pulmonary arterial hypertension. L-EPCs grew clonally; were highly proliferative, passageable, and bankable; and displayed higher reprogramming kinetics and efficiencies compared with dermal fibroblasts. Unlike fibroblasts, the high efficiency of L-EPC reprogramming allowed for the reliable generation of iPSCs in a 96-well format, which is compatible with high-throughput platforms. Array comparative genome hybridization analysis of L-EPCs versus donor-matched circulating monocytes demonstrated that L-EPCs have normal karyotypes compared with their subject's reference genome. In addition, >80% of EPC-iPSC lines tested did not acquire any copy number variations during reprogramming compared with their parent L-EPC line. This work identifies L-EPCs as a practical and efficient cellular substrate for iPSC generation, with the potential to address many of the factors currently limiting the translation of this technology.

p63 Silencing induces reprogramming of cardiac fibroblasts into cardiomyocyte-like cells.

PubMed

Patel, Vivekkumar; Singh, Vivek P; Pinnamaneni, Jaya Pratap; Sanagasetti, Deepthi; Olive, Jacqueline; Mathison, Megumi; Cooney, Austin; Flores, Elsa R; Crystal, Ronald G; Yang, Jianchang; Rosengart, Todd K

2018-04-13

Reprogramming of fibroblasts into induced cardiomyocytes represents a potential new therapy for heart failure. We hypothesized that inactivation of p63, a p53 gene family member, may help overcome human cell resistance to reprogramming. p63 Knockout ( -/- ) and knockdown murine embryonic fibroblasts (MEFs), p63 -/- adult murine cardiac fibroblasts, and human cardiac fibroblasts were assessed for cardiomyocyte-specific feature changes, with or without treatment by the cardiac transcription factors Hand2-Myocardin (HM). Flow cytometry revealed that a significantly greater number of p63 -/- MEFs expressed the cardiac-specific marker cardiac troponin T (cTnT) in culture compared with wild-type (WT) cells (38% ± 11% vs 0.9% ± 0.9%, P < .05). HM treatment of p63 -/- MEFs increased cTnT expression to 74% ± 3% of cells but did not induce cTnT expression in wild-type murine embryonic fibroblasts. shRNA-mediated p63 knockdown likewise yielded a 20-fold increase in cTnT microRNA expression compared with untreated MEFs. Adult murine cardiac fibroblasts demonstrated a 200-fold increase in cTnT gene expression after inducible p63 knockout and expressed sarcomeric α-actinin as well as cTnT. These p63 -/- adult cardiac fibroblasts exhibited calcium transients and electrically stimulated contractions when co-cultured with neonatal rat cardiomyocytes and treated with HM. Increased expression of cTnT and other marker genes was also observed in p63 knockdown human cardiac fibroblasts procured from patients undergoing procedures for heart failure. Downregulation of p63 facilitates direct cardiac cellular reprogramming and may help overcome the resistance of human cells to reprogramming. Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

CD44 Is a Negative Cell Surface Marker for Pluripotent Stem Cell Identification during Human Fibroblast Reprogramming

PubMed Central

Vaz, Candida; Tanavde, Vivek; Lakshmipathy, Uma

2014-01-01

Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones. PMID:24416407

The AP-1 transcription factor FOSL1 causes melanocyte reprogramming and transformation.

PubMed

Maurus, K; Hufnagel, A; Geiger, F; Graf, S; Berking, C; Heinemann, A; Paschen, A; Kneitz, S; Stigloher, C; Geissinger, E; Otto, C; Bosserhoff, A; Schartl, M; Meierjohann, S

2017-09-07

The MAPK pathway is activated in the majority of melanomas and is the target of therapeutic approaches. Under normal conditions, it initiates the so-called immediate early response, which encompasses the transient transcription of several genes belonging to the AP-1 transcription factor family. Under pathological conditions, such as continuous MAPK pathway overactivation due to oncogenic alterations occurring in melanoma, these genes are constitutively expressed. The consequences of a permanent expression of these genes are largely unknown. Here, we show that FOSL1 is the main immediate early AP-1 member induced by melanoma oncogenes. We first examined its role in established melanoma cells. We found that FOSL1 is involved in melanoma cell migration as well as cell proliferation and anoikis-independent growth, which is mediated by the gene product of its target gene HMGA1, encoding a multipotent chromatin modifier. As FOSL1 expression is increased in patient melanoma samples compared to nevi, we investigated the effect of enhanced FOSL1 expression on melanocytes. Intriguingly, we found that FOSL1 acts oncogenic and transforms melanocytes, enabling subcutaneous tumor growth in vivo. During the process of transformation, FOSL1 reprogrammed the melanocytes and downregulated MITF in a HMGA1-dependent manner. At the same time, AXL was upregulated, leading to a shift in the MITF/AXL balance. Furthermore, FOSL1 re-enforced pro-tumorigenic transcription factors MYC, E2F3 and AP-1. Together, this led to the enhancement of several growth-promoting processes, such as ribosome biogenesis, cellular detachment and pyrimidine metabolism. Overall, we demonstrate that FOSL1 is a novel reprogramming factor for melanocytes with potent tumor transformation potential.

The grapevine expression atlas reveals a deep transcriptome shift driving the entire plant into a maturation program.

PubMed

Fasoli, Marianna; Dal Santo, Silvia; Zenoni, Sara; Tornielli, Giovanni Battista; Farina, Lorenzo; Zamboni, Anita; Porceddu, Andrea; Venturini, Luca; Bicego, Manuele; Murino, Vittorio; Ferrarini, Alberto; Delledonne, Massimo; Pezzotti, Mario

2012-09-01

We developed a genome-wide transcriptomic atlas of grapevine (Vitis vinifera) based on 54 samples representing green and woody tissues and organs at different developmental stages as well as specialized tissues such as pollen and senescent leaves. Together, these samples expressed ∼91% of the predicted grapevine genes. Pollen and senescent leaves had unique transcriptomes reflecting their specialized functions and physiological status. However, microarray and RNA-seq analysis grouped all the other samples into two major classes based on maturity rather than organ identity, namely, the vegetative/green and mature/woody categories. This division represents a fundamental transcriptomic reprogramming during the maturation process and was highlighted by three statistical approaches identifying the transcriptional relationships among samples (correlation analysis), putative biomarkers (O2PLS-DA approach), and sets of strongly and consistently expressed genes that define groups (topics) of similar samples (biclustering analysis). Gene coexpression analysis indicated that the mature/woody developmental program results from the reiterative coactivation of pathways that are largely inactive in vegetative/green tissues, often involving the coregulation of clusters of neighboring genes and global regulation based on codon preference. This global transcriptomic reprogramming during maturation has not been observed in herbaceous annual species and may be a defining characteristic of perennial woody plants.

Efficient Generation of iPS Cells from Skeletal Muscle Stem Cells

PubMed Central

Tan, Kah Yong; Eminli, Sarah; Hettmer, Simone; Hochedlinger, Konrad; Wagers, Amy J.

2011-01-01

Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that “epigenetic memory” significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle. PMID:22028872

A case of cellular alchemy: lineage reprogramming and its potential in regenerative medicine

PubMed Central

Asuelime, Grace E.; Shi, Yanhong

2012-01-01

The field of regenerative medicine is rapidly gaining momentum as an increasing number of reports emerge concerning the induced conversions observed in cellular fate reprogramming. While in recent years, much attention has been focused on the conversion of fate-committed somatic cells to an embryonic-like or pluripotent state, there are still many limitations associated with the applications of induced pluripotent stem cell reprogramming, including relatively low reprogramming efficiency, the times required for the reprogramming event to take place, the epigenetic instability, and the tumorigenicity associated with the pluripotent state. On the other hand, lineage reprogramming involves the conversion from one mature cell type to another without undergoing conversion to an unstable intermediate. It provides an alternative approach in regenerative medicine that has a relatively lower risk of tumorigenesis and increased efficiency within specific cellular contexts. While lineage reprogramming provides exciting potential, there is still much to be assessed before this technology is ready to be applied in a clinical setting. PMID:22371436

A rare human syndrome provides genetic evidence that WNT signaling is required for reprogramming of fibroblasts to induced pluripotent stem cells

PubMed Central

Ross, Jason; Busch, Julia; Mintz, Ellen; Ng, Damian; Stanley, Alexandra; Brafman, David; Sutton, V. Reid; Van den Veyver, Ignatia; Willert, Karl

2015-01-01

SUMMARY WNT signaling promotes the reprogramming of somatic cells to an induced pluripotent state. We provide genetic evidence that WNT signaling is a requisite step during the induction of pluripotency. Fibroblasts from individuals with Focal Dermal Hypoplasia (FDH), a rare genetic syndrome caused by mutations in the essential WNT processing enzyme PORCN, fail to reprogram using standard methods. This blockade in reprogramming is overcome by ectopic WNT signaling and by PORCN overexpression, thus demonstrating that WNT signaling is essential for reprogramming. The rescue of reprogramming is critically dependent on the level of WNT signaling: steady baseline activation of the WNT pathway yields karyotypically normal iPS cells, whereas daily stimulation with Wnt3a produces FDH-iPS cells with severely abnormal karyotypes. Therefore, although WNT signaling is required for cellular reprogramming, inappropriate activation of WNT signaling induces chromosomal instability, highlighting the precarious nature of ectopic WNT activation, and its tight relationship with oncogenic transformation. PMID:25464842

Using low-risk factors to generate non-integrated human induced pluripotent stem cells from urine-derived cells.

PubMed

Wang, Linli; Chen, Yuehua; Guan, Chunyan; Zhao, Zhiju; Li, Qiang; Yang, Jianguo; Mo, Jian; Wang, Bin; Wu, Wei; Yang, Xiaohui; Song, Libing; Li, Jun

2017-11-02

Because the lack of an induced pluripotent stem cell (iPSC) induction system with optimal safety and efficiency limits the application of these cells, development of such a system is important. To create such an induction system, we screened a variety of reprogrammed plasmid combinations and multiple compounds and then verified the system's feasibility using urine cells from different individuals. We also compared large-scale iPSC chromosomal variations and expression of genes associated with genomic stability between this system and the traditional episomal system using karyotype and quantitative reverse transcription polymerase chain reaction analyses. We developed a high-efficiency episomal system, the 6F/BM1-4C system, lacking tumorigenic factors for human urine-derived cell (hUC) reprogramming. This system includes six low-risk factors (6F), Oct4, Glis1, Klf4, Sox2, L-Myc, and the miR-302 cluster. Transfected hUCs were treated with four compounds (4C), inhibitor of lysine-demethylase1, methyl ethyl ketone, glycogen synthase kinase 3 beta, and histone deacetylase, within a short time period. Comparative analysis revealed significantly decreased chromosomal variation in iPSCs and significantly increased Sirt1 expression compared with iPSCs induced using the traditional episomal system. The 6F/BM1-4C system effectively induces reprogramming of urine cells in samples obtained from different individuals. iPSCs induced using the 6F/BM1-4C system are more stable at the cytogenetic level and have potential value for clinical application.

Epigenetic reprogramming - is deamination key to active DNA demethylation?

PubMed Central

Teperek-Tkacz, Marta; Pasque, Vincent; Gentsch, George; Ferguson-Smith, Anne C.

2013-01-01

DNA demethylation processes are important for reproduction being central in epigenetic reprogramming during embryonic and germ cell development. While the enzymes methylating DNA have been known for many years, identification of factors capable of mediating active DNA demethylation has been challenging. Recent findings suggest that cytidine deaminases may be key players in active DNA demethylation. One of the most investigated candidates is AID (activation-induced cytidine deaminase) best known for its role in generating secondary antibody diversity in B cells. We evaluate evidence for cytidine deaminases in DNA demethylation pathways in vertebrates and discuss possible models for their targeting and activity regulation. These findings are also considered alongside alternative demethylation pathways involving hydroxymethylation. PMID:21911441

Reprogramming of enteroendocrine K cells to pancreatic β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Esder; Ryu, Gyeong Ryul; Moon, Sung-Dae

2014-01-17

Highlights: •K cells were selected from STC-1 cells, a heterogeneous enteroendocrine cell line. •K cells did not express Nkx6.1 and Neurogenin3. •Combined expression of Nkx6.1 and Neurogenin3 reprogrammed K cells to β-cells. •Reprogramming of K cells to β-cells was not complete. -- Abstract: Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic β-cells in a process called cell reprogramming. Enteroendocrine cells and β-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in β-cells. Thus, K cells could be reprogrammed to β-cellsmore » under certain conditions. However, there is no clear evidence on whether these cells convert to β-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1{sup +}-K cells) was established. Induction of Neurogenin3 expression in Nkx6.1{sup +}-K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1{sup +}-K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture.« less

Optimizing the method for generation of integration-free induced pluripotent stem cells from human peripheral blood.

PubMed

Gu, Haihui; Huang, Xia; Xu, Jing; Song, Lili; Liu, Shuping; Zhang, Xiao-Bing; Yuan, Weiping; Li, Yanxin

2018-06-15

Generation of induced pluripotent stem cells (iPSCs) from human peripheral blood provides a convenient and low-invasive way to obtain patient-specific iPSCs. The episomal vector is one of the best approaches for reprogramming somatic cells to pluripotent status because of its simplicity and affordability. However, the efficiency of episomal vector reprogramming of adult peripheral blood cells is relatively low compared with cord blood and bone marrow cells. In the present study, integration-free human iPSCs derived from peripheral blood were established via episomal technology. We optimized mononuclear cell isolation and cultivation, episomal vector promoters, and a combination of transcriptional factors to improve reprogramming efficiency. Here, we improved the generation efficiency of integration-free iPSCs from human peripheral blood mononuclear cells by optimizing the method of isolating mononuclear cells from peripheral blood, by modifying the integration of culture medium, and by adjusting the duration of culture time and the combination of different episomal vectors. With this optimized protocol, a valuable asset for banking patient-specific iPSCs has been established.

System-Wide Hypersensitive Response-Associated Transcriptome and Metabolome Reprogramming in Tomato1[W][OA

PubMed Central

Etalo, Desalegn W.; Stulemeijer, Iris J.E.; Peter van Esse, H.; de Vos, Ric C.H.; Bouwmeester, Harro J.; Joosten, Matthieu H.A.J.

2013-01-01

The hypersensitive response (HR) is considered to be the hallmark of the resistance response of plants to pathogens. To study HR-associated transcriptome and metabolome reprogramming in tomato (Solanum lycopersicum), we used plants that express both a resistance gene to Cladosporium fulvum and the matching avirulence gene of this pathogen. In these plants, massive reprogramming occurred, and we found that the HR and associated processes are highly energy demanding. Ubiquitin-dependent protein degradation, hydrolysis of sugars, and lipid catabolism are used as alternative sources of amino acids, energy, and carbon skeletons, respectively. We observed strong accumulation of secondary metabolites, such as hydroxycinnamic acid amides. Coregulated expression of WRKY transcription factors and genes known to be involved in the HR, in addition to a strong enrichment of the W-box WRKY-binding motif in the promoter sequences of the coregulated genes, point to WRKYs as the most prominent orchestrators of the HR. Our study has revealed several novel HR-related genes, and reverse genetics tools will allow us to understand the role of each individual component in the HR. PMID:23719893

Generation of Arbas Cashmere Goat Induced Pluripotent Stem Cells Through Fibroblast Reprogramming.

PubMed

Tai, Dapeng; Liu, Pengxia; Gao, Jing; Jin, Muzi; Xu, Teng; Zuo, Yongchun; Liang, Hao; Liu, Dongjun

2015-08-01

Various factors affect the process of obtaining stable Arbas cashmere goat embryonic stem cells (ESCs), for example, the difficulty in isolating cells at the appropriate stage of embryonic development, the in vitro culture environment, and passage methods. With the emergence of induced pluripotent stem cell (iPSC) technology, it has become possible to use specific genes to induce somatic cell differentiation in PSCs. We transferred OCT4, SOX2, c-MYC, and KLF4 into Arbas cashmere goat fetal fibroblasts, then induced and cultured them using a drug-inducible system to obtain Arbas goat iPSCs that morphologically resembled mouse iPSCs. After identification, the obtained goat iPSCs expressed ESC markers, had a normal karyotype, could differentiate into embryoid bodies in vitro, and could differentiate into three germ layer cell types and form teratomas in vivo. We used microarray gene expression profile analysis to elucidate the reprogramming process. Our results provide the experimental basis for establishing cashmere goat iPSC lines and for future in-depth studies on molecular mechanism of cashmere goat somatic cell reprogramming.

Biological pacemaker created by minimally invasive somatic reprogramming in pigs with complete heart block

PubMed Central

Hu, Yu-Feng; Dawkins, James Frederick; Cho, Hee Cheol; Marbán, Eduardo; Cingolani, Eugenio

2016-01-01

Somatic reprogramming by reexpression of the embryonic transcription factor T-box 18 (TBX18) converts cardiomyocytes into pacemaker cells. We hypothesized that this could be a viable therapeutic avenue for pacemaker-dependent patients afflicted with device-related complications, and therefore tested whether adenoviral TBX18 gene transfer could create biological pacemaker activity in vivo in a large-animal model of complete heart block. Biological pacemaker activity, originating from the intramyocardial injection site, was evident in TBX18-transduced animals starting at day 2 and persisted for the duration of the study (14 days) with minimal backup electronic pacemaker use. Relative to controls transduced with a reporter gene, TBX18-transduced animals exhibited enhanced autonomic responses and physiologically superior chronotropic support of physical activity. Induced sinoatrial node cells could be identified by their distinctive morphology at the site of injection in TBX18-transduced animals, but not in controls. No local or systemic safety concerns arose. Thus, minimally invasive TBX18 gene transfer creates physiologically relevant pacemaker activity in complete heart block, providing evidence for therapeutic somatic reprogramming in a clinically relevant disease model. PMID:25031269

Coordination of m6A mRNA methylation and gene transcription by ZFP217 regulates pluripotency and reprogramming

PubMed Central

Aguilo, Francesca; Zhang, Fan; Sancho, Ana; Fidalgo, Miguel; Di Cecilia, Serena; Vashisht, Ajay; Lee, Dung-Fang; Chen, Chih-Hung; Rengasamy, Madhumitha; Andino, Blanca; Jahouh, Farid; Roman, Angel; Krig, Sheryl R.; Wang, Rong; Zhang, Weijia; Wohlschlegel, James A.; Wang, Jianlong; Walsh, Martin J.

2015-01-01

SUMMARY Epigenetic and epitranscriptomic networks have important functions in maintaining pluripotency of embryonic stem cells (ESCs) and somatic cell reprogramming. However the mechanisms integrating the actions of these distinct networks are only partially understood. Here, we show that the chromatin-associated zinc finger protein 217 (ZFP217) coordinates epigenetic and epitranscriptomic regulation. ZFP217 interacts with several epigenetic regulators, activates transcription of key pluripotency genes, and modulates N6-methyladenosine (m6A) deposition on their transcripts by sequestering the enzyme m6A methyltransferase-like 3 (METTL3). Consistently, Zfp217 depletion compromises ESC self-renewal and somatic cell reprogramming, globally increases m6A RNA levels, and enhances m6A modification of Nanog, Sox2, Klf4, and c-Myc mRNAs, promoting their degradation. ZFP217 binds its own target gene mRNAs, which are also METTL3-associated, and is enriched at promoters of m6A-modified transcripts. Collectively, these findings shed light on how a transcription factor can tightly couple gene transcription to m6A RNA modification to insure ESC identity. PMID:26526723

Efficient method to create integration-free, virus-free, Myc and Lin28-free human induced pluripotent stem cells from adherent cells.

PubMed

Kamath, Anant; Ternes, Sara; McGowan, Stephen; English, Anthony; Mallampalli, Rama; Moy, Alan B

2017-08-01

Nonviral induced pluripotent stem cell (IPSC) reprogramming is not efficient without the oncogenes, Myc and Lin28 . We describe a robust Myc and Lin28 -free IPSC reprogramming approach using reprogramming molecules. IPSC colony formation was compared in the presence and absence of Myc and Lin28 by the mixture of reprogramming molecules and episomal vectors. While more colonies were observed in cultures transfected with the aforementioned oncogenes, the Myc and Lin28 -free method achieved the same reprogramming efficiency as reports that used these oncogenes. Further, all colonies were fully reprogrammed based on expression of SSEA4, even in the absence of Myc and Lin28 . This approach satisfies an important regulatory pathway for developing IPSC cell therapies with lower clinical risk.

Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks.

PubMed

Liu, Kun-Hsiang; Niu, Yajie; Konishi, Mineko; Wu, Yue; Du, Hao; Sun Chung, Hoo; Li, Lei; Boudsocq, Marie; McCormack, Matthew; Maekawa, Shugo; Ishida, Tetsuya; Zhang, Chao; Shokat, Kevan; Yanagisawa, Shuichi; Sheen, Jen

2017-05-18

Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report unique Ca 2+ signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca 2+ -sensor protein kinases (CPKs) as master regulators that orchestrate primary nitrate responses. A chemical switch with the engineered mutant CPK10(M141G) circumvents embryo lethality and enables conditional analyses of cpk10 cpk30 cpk32 triple mutants to define comprehensive nitrate-associated regulatory and developmental programs. Nitrate-coupled CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors to specify the reprogramming of gene sets for downstream transcription factors, transporters, nitrogen assimilation, carbon/nitrogen metabolism, redox, signalling, hormones and proliferation. Conditional cpk10 cpk30 cpk32 and nlp7 mutants similarly impair nitrate-stimulated system-wide shoot growth and root establishment. The nutrient-coupled Ca 2+ signalling network integrates transcriptome and cellular metabolism with shoot-root coordination and developmental plasticity in shaping organ biomass and architecture.

The endoparasitoid, Cotesia vestalis, regulates host physiology by reprogramming the neuropeptide transcriptional network

USDA-ARS?s Scientific Manuscript database

Endoparasitoids develop inside another insect; success depends on regulating host immunity and development by maternal factors injected into hosts during oviposition, including venom, polydnaviruses and teratocytes. Although prior results provide insights into parasitism-induced immunosuppression, l...

Characterization of porcine partially reprogrammed iPSCs from adipose-derived stem cells.

PubMed

Wei, Chao; Li, Xia; Zhang, Pengfei; Zhang, Yu; Liu, Tong; Jiang, Shaoshuai; Han, Fei; Zhang, Yunhai

2015-05-01

Partially reprogrammed induced pluripotent stem cells (PiPSCs) have great potential for investigating reprogramming mechanisms and represent an alternative potential material for making genetically modified animals and regenerative medicine. To date, PiPSCs have scarcely been reported in detail when compared with mice and humans. In this study, we obtained PiPSCs from porcine adipose-derived stem cells (pADSCs) by ectopic expression of human transcription factors (OCT4, SOX2, c-MYC, and KLF4) in feeder-free condition. The morphology and proliferation activity of porcine PiPSCs (pPiPSCs) were similar to those of porcine fully reprogrammed iPSCs (pFiPSCs); furthermore, pPiPSCs expressed higher levels of the typical surface molecules (CD29) found in pADSCs. However, pPiPSCs were negative for key proteins (NANOG) connected with stemness and possessed lower differentiation ability in vivo and in vitro. When differentiation-inhibiting factors were withdrawn, pPiPSCs-derived cells (pPiPSC-DCs) showed similar features to pADSCs in many aspects, including proliferation, differentiation, and immunosuppression. When both types of cells were used to produce cloned embryos, we found that the blastocyst formation rate of 19DC (one of the pPiPSC-DC cell lines)-derived cloned embryos was obviously higher than that of others. The total cell number of 19DC-derived blastocysts was significantly higher than the 30DC (one pFiPSC-DC cell line)-derived blastocysts. In all, through limited differentiation ability, the proliferation activity of pPiPSCs is similar to that of pFiPSCs, and pPiPSCs can retain several of the features of pADSCs, which are beneficial to cell therapy. Furthermore, the differentiation of pPiPSCs is more favorable for producing high-quality reconstructed embryos. © 2015 Society for Reproduction and Fertility.

Advances in Reprogramming-Based Study of Neurologic Disorders

PubMed Central

Baldwin, Kristin K.

2015-01-01

The technology to convert adult human non-neural cells into neural lineages, through induced pluripotent stem cells (iPSCs), somatic cell nuclear transfer, and direct lineage reprogramming or transdifferentiation has progressed tremendously in recent years. Reprogramming-based approaches aimed at manipulating cellular identity have enormous potential for disease modeling, high-throughput drug screening, cell therapy, and personalized medicine. Human iPSC (hiPSC)-based cellular disease models have provided proof of principle evidence of the validity of this system. However, several challenges remain before patient-specific neurons produced by reprogramming can provide reliable insights into disease mechanisms or be efficiently applied to drug discovery and transplantation therapy. This review will first discuss limitations of currently available reprogramming-based methods in faithfully and reproducibly recapitulating disease pathology. Specifically, we will address issues such as culture heterogeneity, interline and inter-individual variability, and limitations of two-dimensional differentiation paradigms. Second, we will assess recent progress and the future prospects of reprogramming-based neurologic disease modeling. This includes three-dimensional disease modeling, advances in reprogramming technology, prescreening of hiPSCs and creating isogenic disease models using gene editing. PMID:25749371

Algorithm for cellular reprogramming.

PubMed

Ronquist, Scott; Patterson, Geoff; Muir, Lindsey A; Lindsly, Stephen; Chen, Haiming; Brown, Markus; Wicha, Max S; Bloch, Anthony; Brockett, Roger; Rajapakse, Indika

2017-11-07

The day we understand the time evolution of subcellular events at a level of detail comparable to physical systems governed by Newton's laws of motion seems far away. Even so, quantitative approaches to cellular dynamics add to our understanding of cell biology. With data-guided frameworks we can develop better predictions about, and methods for, control over specific biological processes and system-wide cell behavior. Here we describe an approach for optimizing the use of transcription factors (TFs) in cellular reprogramming, based on a device commonly used in optimal control. We construct an approximate model for the natural evolution of a cell-cycle-synchronized population of human fibroblasts, based on data obtained by sampling the expression of 22,083 genes at several time points during the cell cycle. To arrive at a model of moderate complexity, we cluster gene expression based on division of the genome into topologically associating domains (TADs) and then model the dynamics of TAD expression levels. Based on this dynamical model and additional data, such as known TF binding sites and activity, we develop a methodology for identifying the top TF candidates for a specific cellular reprogramming task. Our data-guided methodology identifies a number of TFs previously validated for reprogramming and/or natural differentiation and predicts some potentially useful combinations of TFs. Our findings highlight the immense potential of dynamical models, mathematics, and data-guided methodologies for improving strategies for control over biological processes. Copyright © 2017 the Author(s). Published by PNAS.

Inhibition of TGF-β signaling in combination with TLR7 ligation re-programs a tumoricidal phenotype in tumor-associated macrophages.

PubMed

Peng, Jiao; Tsang, Julia Yuen Shan; Li, Daxu; Niu, Na; Ho, Derek Hoi Hang; Lau, Kwok Fai; Lui, Vincent Chi Hang; Lamb, Jonathan Robert; Chen, Yan; Tam, Paul Kwong Hang

2013-05-01

Inadequate immunity that occurs in a tumor environment is in part due to the presence of M2-type tumor-associated macrophages (TAMs). TGF-β has a multi-functional role in tumor development including modulating the biological activity of both the tumor and TAMs. In this study, using an in vitro TAM/tumor cell co-culture system ligation of TLR7, which is expressed on TAMs but not the tumor cells, in the presence of TGF-β receptor I inhibitor re-programmed the phenotype of the TAMs. In part they adopted the phenotype characteristic of M1-type macrophages, namely they had increased tumoricidal activity and elevated expression of iNOS, CD80 and MHC class II, while TGF-β secretion was reduced. The reprogrammed phenotype was accompanied by enhanced NF-κB nuclear translocation. The pro-angiogenesis factor VEGF was down-regulated and in vivo the number of CD31-positive tumor capillaries was also reduced. Furthermore, in vivo we observed that TLR7 ligation/TGF-β receptor I inhibition increased tumor apoptosis and elevated the number of CD4+, CD8+, and CD19+ cells as well as neutrophils infiltrating the tumor. Our data demonstrate that selective TLR stimulation with TGF-β inhibition can reprogram TAMs towards an M1-like phenotype and thereby provides new perspectives in cancer therapy. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Cell-autonomous regulation of Mu-opioid receptor recycling by substance P.

PubMed

Bowman, Shanna L; Soohoo, Amanda L; Shiwarski, Daniel J; Schulz, Stefan; Pradhan, Amynah A; Puthenveedu, Manojkumar A

2015-03-24

How neurons coordinate and reprogram multiple neurotransmitter signals is an area of broad interest. Here, we show that substance P (SP), a neuropeptide associated with inflammatory pain, reprograms opioid receptor recycling and signaling. SP, through activation of the neurokinin 1 (NK1R) receptor, increases the post-endocytic recycling of the mu-opioid receptor (MOR) in trigeminal ganglion (TG) neurons in an agonist-selective manner. SP-mediated protein kinase C (PKC) activation is both required and sufficient for increasing recycling of exogenous and endogenous MOR in TG neurons. The target of this cross-regulation is MOR itself, given that mutation of either of two PKC phosphorylation sites on MOR abolishes the SP-induced increase in recycling and resensitization. Furthermore, SP enhances the resensitization of fentanyl-induced, but not morphine-induced, antinociception in mice. Our results define a physiological pathway that cross-regulates opioid receptor recycling via direct modification of MOR and suggest a mode of homeostatic interaction between the pain and analgesic systems.

Transfection in perfused microfluidic cell culture devices: A case study.

PubMed

Raimes, William; Rubi, Mathieu; Super, Alexandre; Marques, Marco P C; Veraitch, Farlan; Szita, Nicolas

2017-08-01

Automated microfluidic devices are a promising route towards a point-of-care autologous cell therapy. The initial steps of induced pluripotent stem cell (iPSC) derivation involve transfection and long term cell culture. Integration of these steps would help reduce the cost and footprint of micro-scale devices with applications in cell reprogramming or gene correction. Current examples of transfection integration focus on maximising efficiency rather than viable long-term culture. Here we look for whole process compatibility by integrating automated transfection with a perfused microfluidic device designed for homogeneous culture conditions. The injection process was characterised using fluorescein to establish a LabVIEW-based routine for user-defined automation. Proof-of-concept is demonstrated by chemically transfecting a GFP plasmid into mouse embryonic stem cells (mESCs). Cells transfected in the device showed an improvement in efficiency (34%, n = 3) compared with standard protocols (17.2%, n = 3). This represents a first step towards microfluidic processing systems for cell reprogramming or gene therapy.

Cell-Autonomous Regulation of Mu-Opioid Receptor Recycling by Substance P

PubMed Central

Bowman, Shanna L.; Soohoo, Amanda L.; Shiwarski, Daniel J.; Schulz, Stefan; Pradhan, Amynah A.; Puthenveedu, Manojkumar A.

2015-01-01

SUMMARY How neurons coordinate and reprogram multiple neurotransmitter signals is an area of broad interest. Here, we show that substance P (SP), a neuropep-tide associated with inflammatory pain, reprograms opioid receptor recycling and signaling. SP, through activation of the neurokinin 1 (NK1R) receptor, increases the post-endocytic recycling of the muopioid receptor (MOR) in trigeminal ganglion (TG) neurons in an agonist-selective manner. SP-mediated protein kinase C (PKC) activation is both required and sufficient for increasing recycling of exogenous and endogenous MOR in TG neurons. The target of this cross-regulation is MOR itself, given that mutation of either of two PKC phosphorylation sites on MOR abolishes the SP-induced increase in recycling and resensitization. Furthermore, SP enhances the resensitization of fentanyl-induced, but not morphine-induced, antinociception in mice. Our results define a physiological pathway that cross-regulates opioid receptor recycling via direct modification of MOR and suggest a mode of homeo-static interaction between the pain and analgesic systems. PMID:25801029

Highly Expandable Human iPS Cell-Derived Neural Progenitor Cells (NPC) and Neurons for Central Nervous System Disease Modeling and High-Throughput Screening.

PubMed

Cheng, Chialin; Fass, Daniel M; Folz-Donahue, Kat; MacDonald, Marcy E; Haggarty, Stephen J

2017-01-11

Reprogramming of human somatic cells into induced pluripotent stem (iPS) cells has greatly expanded the set of research tools available to investigate the molecular and cellular mechanisms underlying central nervous system (CNS) disorders. Realizing the promise of iPS cell technology for the identification of novel therapeutic targets and for high-throughput drug screening requires implementation of methods for the large-scale production of defined CNS cell types. Here we describe a protocol for generating stable, highly expandable, iPS cell-derived CNS neural progenitor cells (NPC) using multi-dimensional fluorescence activated cell sorting (FACS) to purify NPC defined by cell surface markers. In addition, we describe a rapid, efficient, and reproducible method for generating excitatory cortical-like neurons from these NPC through inducible expression of the pro-neural transcription factor Neurogenin 2 (iNgn2-NPC). Finally, we describe methodology for the use of iNgn2-NPC for probing human neuroplasticity and mechanisms underlying CNS disorders using high-content, single-cell-level automated microscopy assays. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

HOXB7 overexpression in lung cancer is a hallmark of acquired stem-like phenotype.

PubMed

Monterisi, Simona; Lo Riso, Pietro; Russo, Karin; Bertalot, Giovanni; Vecchi, Manuela; Testa, Giuseppe; Di Fiore, Pier Paolo; Bianchi, Fabrizio

2018-03-26

HOXB7 is a homeodomain (HOX) transcription factor involved in regional body patterning of invertebrates and vertebrates. We previously identified HOXB7 within a ten-gene prognostic signature for lung adenocarcinoma, where increased expression of HOXB7 was associated with poor prognosis. This raises the question of how HOXB7 overexpression can influence the metastatic behavior of lung adenocarcinoma. Here, we analyzed publicly available microarray and RNA-seq lung cancer expression datasets and found that HOXB7-overexpressing tumors are enriched in gene signatures characterizing adult and embryonic stem cells (SC), and induced pluripotent stem cells (iPSC). Experimentally, we found that HOXB7 upregulates several canonical SC/iPSC markers and sustains the expansion of a subpopulation of cells with SC characteristics, through modulation of LIN28B, an emerging cancer gene and pluripotency factor, which we discovered to be a direct target of HOXB7. We validated this new circuit by showing that HOXB7 enhances reprogramming to iPSC with comparable efficiency to LIN28B or its target c-MYC, which is a canonical reprogramming factor.

Fip1 regulates mRNA alternative polyadenylation to promote stem cell self-renewal

PubMed Central

Lackford, Brad; Yao, Chengguo; Charles, Georgette M; Weng, Lingjie; Zheng, Xiaofeng; Choi, Eun-A; Xie, Xiaohui; Wan, Ji; Xing, Yi; Freudenberg, Johannes M; Yang, Pengyi; Jothi, Raja; Hu, Guang; Shi, Yongsheng

2014-01-01

mRNA alternative polyadenylation (APA) plays a critical role in post-transcriptional gene control and is highly regulated during development and disease. However, the regulatory mechanisms and functional consequences of APA remain poorly understood. Here, we show that an mRNA 3′ processing factor, Fip1, is essential for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Fip1 promotes stem cell maintenance, in part, by activating the ESC-specific APA profiles to ensure the optimal expression of a specific set of genes, including critical self-renewal factors. Fip1 expression and the Fip1-dependent APA program change during ESC differentiation and are restored to an ESC-like state during somatic reprogramming. Mechanistically, we provide evidence that the specificity of Fip1-mediated APA regulation depends on multiple factors, including Fip1-RNA interactions and the distance between APA sites. Together, our data highlight the role for post-transcriptional control in stem cell self-renewal, provide mechanistic insight on APA regulation in development, and establish an important function for APA in cell fate specification. PMID:24596251

Browning of human adipocytes requires KLF11 and reprogramming of PPARγ superenhancers.

PubMed

Loft, Anne; Forss, Isabel; Siersbæk, Majken Storm; Schmidt, Søren Fisker; Larsen, Ann-Sofie Bøgh; Madsen, Jesper Grud Skat; Pisani, Didier F; Nielsen, Ronni; Aagaard, Mads Malik; Mathison, Angela; Neville, Matt J; Urrutia, Raul; Karpe, Fredrik; Amri, Ez-Zoubir; Mandrup, Susanne

2015-01-01

Long-term exposure to peroxisome proliferator-activated receptor γ (PPARγ) agonists such as rosiglitazone induces browning of rodent and human adipocytes; however, the transcriptional mechanisms governing this phenotypic switch in adipocytes are largely unknown. Here we show that rosiglitazone-induced browning of human adipocytes activates a comprehensive gene program that leads to increased mitochondrial oxidative capacity. Once induced, this gene program and oxidative capacity are maintained independently of rosiglitazone, suggesting that additional browning factors are activated. Browning triggers reprogramming of PPARγ binding, leading to the formation of PPARγ "superenhancers" that are selective for brown-in-white (brite) adipocytes. These are highly associated with key brite-selective genes. Based on such an association, we identified an evolutionarily conserved metabolic regulator, Kruppel-like factor 11 (KLF11), as a novel browning transcription factor in human adipocytes that is required for rosiglitazone-induced browning, including the increase in mitochondrial oxidative capacity. KLF11 is directly induced by PPARγ and appears to cooperate with PPARγ in a feed-forward manner to activate and maintain the brite-selective gene program. © 2015 Loft et al.; Published by Cold Spring Harbor Laboratory Press.

Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

PubMed Central

Li, Dong; Secher, Jan O.; Mashayekhi, Kaveh; Nielsen, Troels T.; Hyttel, Poul; Freude, Kristine K.

2017-01-01

ABSTRACT Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore, reprogramming of SSEA-1+ sorted pEFs led to higher reprogramming efficiency. Subsequent transcriptome profiling of the SSEA-1+ vs. the SSEA-1neg cell fraction revealed highly comparable gene signatures. However several genes that were found to be upregulated in the SSEA-1+ cells were similarly expressed in mesenchymal stem cells (MSCs). We therefore termed these cells SSEA-1 Expressing Enhanced Reprogramming (SEER) cells. Interestingly, SEER cells were more effective at differentiating into osteocytes and chondrocytes in vitro. We conclude that SEER cells are more amenable for reprogramming and that the expression of mesenchymal stem cell genes is advantageous in the reprogramming process. This data provides evidence supporting the elite theory and helps to delineate which cell types and specific genes are important for reprogramming in the pig. PMID:28426281

A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells

PubMed Central

Bhutani, Nidhi; Decker, Matthew N.; Brady, Jennifer J.; Bussat, Rose T.; Burns, David M.; Corbel, Stephane Y.; Blau, Helen M.

2013-01-01

Mechanistic insights into the reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) are limited, particularly for early acting molecular regulators. Here we use an acute loss of function approach to demonstrate that activation-induced deaminase (AID) activity is necessary for the initiation of reprogramming to iPSCs. While AID is well known for antibody diversification, it has also recently been shown to have a role in active DNA demethylation in reprogramming toward pluripotency and development. These findings suggested a potential role for AID in iPSC generation, yet, iPSC yield from AID-knockout mouse fibroblasts was similar to that of wild-type (WT) fibroblasts. We reasoned that an acute loss of AID function might reveal effects masked by compensatory mechanisms during development, as reported for other proteins. Accordingly, we induced an acute reduction (>50%) in AID levels using 4 different shRNAs and determined that reprogramming to iPSCs was significantly impaired by 79 ± 7%. The deaminase activity of AID was critical, as coexpression of WT but not a catalytic mutant AID rescued reprogramming. Notably, AID was required only during a 72-h time window at the onset of iPSC reprogramming. Our findings show a critical role for AID activity in the initiation of reprogramming to iPSCs.—Bhutani, N., Decker, M. N., Brady, J. J., Bussat, R. T., Burns, D. M., Corbel, S. Y., Blau, H. M. A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells. PMID:23212122

Engineered proteins with PUF scaffold to manipulate RNA metabolism

PubMed Central

Wang, Yang; Wang, Zefeng; Tanaka Hall, Traci M.

2013-01-01

Pumilio/fem-3 mRNA binding factor (FBF) proteins are characterized by a sequence-specific RNA-binding domain. This unique single-stranded RNA recognition module, whose sequence specificity can be reprogrammed, has been fused with functional modules to engineer protein factors with various functions. Here we summarize the advancement in developing RNA regulatory tools and opportunities for the future. PMID:23731364

Posttranscriptional (Re)programming of Cell Fate: Examples in Stem Cells, Progenitor, and Differentiated Cells.

PubMed

Kanellopoulou, Chrysi; Muljo, Stefan A

2018-01-01

How a single genome can give rise to many different transcriptomes and thus all the different cell lineages in the human body is a fundamental question in biology. While signaling pathways, transcription factors, and chromatin architecture, to name a few determinants, have been established to play critical roles, recently, there is a growing appreciation of the roles of non-coding RNAs and RNA-binding proteins in controlling cell fates posttranscriptionally. Thus, it is vital that these emerging players are also integrated into models of gene regulatory networks that underlie programs of cellular differentiation. Sometimes, we can leverage knowledge about such posttranscriptional circuits to reprogram patterns of gene expression in meaningful ways. Here, we review three examples from our work.

Developmental Programming of Renal Function and Re-Programming Approaches

PubMed Central

Nüsken, Eva; Dötsch, Jörg; Weber, Lutz T.; Nüsken, Kai-Dietrich

2018-01-01

Chronic kidney disease affects more than 10% of the population. Programming studies have examined the interrelationship between environmental factors in early life and differences in morbidity and mortality between individuals. A number of important principles has been identified, namely permanent structural modifications of organs and cells, long-lasting adjustments of endocrine regulatory circuits, as well as altered gene transcription. Risk factors include intrauterine deficiencies by disturbed placental function or maternal malnutrition, prematurity, intrauterine and postnatal stress, intrauterine and postnatal overnutrition, as well as dietary dysbalances in postnatal life. This mini-review discusses critical developmental periods and long-term sequelae of renal programming in humans and presents studies examining the underlying mechanisms as well as interventional approaches to “re-program” renal susceptibility toward disease. Clinical manifestations of programmed kidney disease include arterial hypertension, proteinuria, aggravation of inflammatory glomerular disease, and loss of kidney function. Nephron number, regulation of the renin–angiotensin–aldosterone system, renal sodium transport, vasomotor and endothelial function, myogenic response, and tubuloglomerular feedback have been identified as being vulnerable to environmental factors. Oxidative stress levels, metabolic pathways, including insulin, leptin, steroids, and arachidonic acid, DNA methylation, and histone configuration may be significantly altered by adverse environmental conditions. Studies on re-programming interventions focused on dietary or anti-oxidative approaches so far. Further studies that broaden our understanding of renal programming mechanisms are needed to ultimately develop preventive strategies. Targeted re-programming interventions in animal models focusing on known mechanisms will contribute to new concepts which finally will have to be translated to human application. Early nutritional concepts with specific modifications in macro- or micronutrients are among the most promising approaches to improve future renal health. PMID:29535992

In situ reprogramming to transdifferentiate fibroblasts into cardiomyocytes using adenoviral vectors: Implications for clinical myocardial regeneration.

PubMed

Mathison, Megumi; Singh, Vivek P; Chiuchiolo, Maria J; Sanagasetti, Deepthi; Mao, Yun; Patel, Vivekkumar B; Yang, Jianchang; Kaminsky, Stephen M; Crystal, Ronald G; Rosengart, Todd K

2017-02-01

The reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells improves ventricular function in myocardial infarction models. Only integrating persistent expression vectors have thus far been used to induce reprogramming, potentially limiting its clinical applicability. We therefore tested the reprogramming potential of nonintegrating, acute expression adenoviral (Ad) vectors. Ad or lentivirus vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) were validated in vitro. Sprague-Dawley rats then underwent coronary ligation and Ad-mediated administration of vascular endothelial growth factor to generate infarct prevascularization. Three weeks later, animals received Ad or lentivirus encoding G, M, or T (AdGMT or LentiGMT) or an equivalent dose of a null vector (n = 11, 10, and 10, respectively). Outcomes were analyzed by echocardiography, magnetic resonance imaging, and histology. Ad and lentivirus vectors provided equivalent G, M, and T expression in vitro. AdGMT and LentiGMT both likewise induced expression of the cardiomyocyte marker cardiac troponin T in approximately 6% of cardiac fibroblasts versus <1% cardiac troponin T expression in AdNull (adenoviral vector that does not encode a transgene)-treated cells. Infarcted myocardium that had been treated with AdGMT likewise demonstrated greater density of cells expressing the cardiomyocyte marker beta myosin heavy chain 7 compared with AdNull-treated animals. Echocardiography demonstrated that AdGMT and LentiGMT both increased ejection fraction compared with AdNull (AdGMT: 21% ± 3%, LentiGMT: 14% ± 5%, AdNull: -0.4% ± 2%; P < .05). Ad vectors are at least as effective as lentiviral vectors in inducing cardiac fibroblast transdifferentiation into induced cardiomyocyte-like cells and improving cardiac function in postinfarct rat hearts. Short-term expression Ad vectors may represent an important means to induce cardiac cellular reprogramming in humans. Copyright © 2016 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

Extraocular muscle regeneration in zebrafish requires late signals from Insulin-like growth factors.

PubMed

Saera-Vila, Alfonso; Louie, Ke'ale W; Sha, Cuilee; Kelly, Ryan M; Kish, Phillip E; Kahana, Alon

2018-01-01

Insulin-like growth factors (Igfs) are key regulators of key biological processes such as embryonic development, growth, and tissue repair and regeneration. The role of Igf in myogenesis is well documented and, in zebrafish, promotes fin and heart regeneration. However, the mechanism of action of Igf in muscle repair and regeneration is not well understood. Using adult zebrafish extraocular muscle (EOM) regeneration as an experimental model, we show that Igf1 receptor blockage using either chemical inhibitors (BMS754807 and NVP-AEW541) or translation-blocking morpholino oligonucleotides (MOs) reduced EOM regeneration. Zebrafish EOMs regeneration depends on myocyte dedifferentiation, which is driven by early epigenetic reprogramming and requires autophagy activation and cell cycle reentry. Inhibition of Igf signaling had no effect on either autophagy activation or cell proliferation, indicating that Igf signaling was not involved in the early reprogramming steps of regeneration. Instead, blocking Igf signaling produced hypercellularity of regenerating EOMs and diminished myosin expression, resulting in lack of mature differentiated muscle fibers even many days after injury, indicating that Igf was involved in late re-differentiation steps. Although it is considered the main mediator of myogenic Igf actions, Akt activation decreased in regenerating EOMs, suggesting that alternative signaling pathways mediate Igf activity in muscle regeneration. In conclusion, Igf signaling is critical for re-differentiation of reprogrammed myoblasts during late steps of zebrafish EOM regeneration, suggesting a regulatory mechanism for determining regenerated muscle size and timing of differentiation, and a potential target for regenerative therapy.

Direct Reprogramming of Spiral Ganglion Non-neuronal Cells into Neurons: Toward Ameliorating Sensorineural Hearing Loss by Gene Therapy

PubMed Central

Noda, Teppei; Meas, Steven J.; Nogami, Jumpei; Amemiya, Yutaka; Uchi, Ryutaro; Ohkawa, Yasuyuki; Nishimura, Koji; Dabdoub, Alain

2018-01-01

Primary auditory neurons (PANs) play a critical role in hearing by transmitting sound information from the inner ear to the brain. Their progressive degeneration is associated with excessive noise, disease and aging. The loss of PANs leads to permanent hearing impairment since they are incapable of regenerating. Spiral ganglion non-neuronal cells (SGNNCs), comprised mainly of glia, are resident within the modiolus and continue to survive after PAN loss. These attributes make SGNNCs an excellent target for replacing damaged PANs through cellular reprogramming. We used the neurogenic pioneer transcription factor Ascl1 and the auditory neuron differentiation factor NeuroD1 to reprogram SGNNCs into induced neurons (iNs). The overexpression of both Ascl1 and NeuroD1 in vitro generated iNs at high efficiency. Transcriptome analyses revealed that iNs displayed a transcriptome profile resembling that of endogenous PANs, including expression of several key markers of neuronal identity: Tubb3, Map2, Prph, Snap25, and Prox1. Pathway analyses indicated that essential pathways in neuronal growth and maturation were activated in cells upon neuronal induction. Furthermore, iNs extended projections toward cochlear hair cells and cochlear nucleus neurons when cultured with each respective tissue. Taken together, our study demonstrates that PAN-like neurons can be generated from endogenous SGNNCs. This work suggests that gene therapy can be a viable strategy to treat sensorineural hearing loss caused by degeneration of PANs. PMID:29492404

Reprogramming retinal neurons and standardized quantification of their differentiation in 3-dimensional retinal cultures

PubMed Central

Hiler, Daniel J.; Barabas, Marie E.; Griffiths, Lyra M.; Dyer, Michael A.

2017-01-01

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. Other protocols to reprogram postmitotic neurons have required the inactivation of the p53 tumor suppressor. We describe a method that does not require p53 inactivation and induces reprogramming in cells purified from the retinae of reprogrammable mice in aggregates with wild-type retinal cells. After the first 10 days of reprogramming, the aggregates are then dispersed and plated on irradiated feeder cells to propagate and isolate individual iPSC clones. The reprogramming efficiency of different neuronal populations at any stage of development can be quantitated using this protocol. Reprogramming retinal neurons with this protocol will take 56 days, and these retina-derived iPSCs can undergo retinal differentiation to produce retinae in 34 days. In addition, we describe a quantitative assessment of retinal differentiation from these neuron-derived iPSCs called STEM-RET. The procedure quantitates eye field specification, optic cup formation, and retinal differentiation in 3-dimensional cultures using molecular, cellular and morphological criteria. An advanced level of cell culture experience is required to carry out this protocol. PMID:27658012

Cell reprogramming modelled as transitions in a hierarchy of cell cycles

NASA Astrophysics Data System (ADS)

Hannam, Ryan; Annibale, Alessia; Kühn, Reimer

2017-10-01

We construct a model of cell reprogramming (the conversion of fully differentiated cells to a state of pluripotency, known as induced pluripotent stem cells, or iPSCs) which builds on key elements of cell biology viz. cell cycles and cell lineages. Although reprogramming has been demonstrated experimentally, much of the underlying processes governing cell fate decisions remain unknown. This work aims to bridge this gap by modelling cell types as a set of hierarchically related dynamical attractors representing cell cycles. Stages of the cell cycle are characterised by the configuration of gene expression levels, and reprogramming corresponds to triggering transitions between such configurations. Two mechanisms were found for reprogramming in a two level hierarchy: cycle specific perturbations and a noise induced switching. The former corresponds to a directed perturbation that induces a transition into a cycle-state of a different cell type in the potency hierarchy (mainly a stem cell) whilst the latter is a priori undirected and could be induced, e.g. by a (stochastic) change in the cellular environment. These reprogramming protocols were found to be effective in large regimes of the parameter space and make specific predictions concerning reprogramming dynamics which are broadly in line with experimental findings.

Current Concept and Update of the Macrophage Plasticity Concept: Intracellular Mechanisms of Reprogramming and M3 Macrophage “Switch” Phenotype

PubMed Central

Malyshev, Igor; Malyshev, Yuri

2015-01-01

Macrophages play a key role in immunity. In this review, we consider the traditional notion of macrophage plasticity, data that do not fit into existing concepts, and a hypothesis for existence of a new switch macrophage phenotype. Depending on the microenvironment, macrophages can reprogram their phenotype toward the proinflammatory M1 phenotype or toward the anti-inflammatory M2 phenotype. Macrophage reprogramming involves well-coordinated changes in activities of signalling and posttranslational mechanisms. Macrophage reprogramming is provided by JNK-, PI3K/Akt-, Notch-, JAK/STAT-, TGF-β-, TLR/NF-κB-, and hypoxia-dependent pathways. Posttranscriptional regulation is based on micro-mRNA. We have hypothesized that, in addition to the M1 and M2 phenotypes, an M3 switch phenotype exists. This switch phenotype responds to proinflammatory stimuli with reprogramming towards the anti-inflammatory M2 phenotype or, contrarily, it responds to anti-inflammatory stimuli with reprogramming towards the proinflammatory M1 phenotype. We have found signs of such a switch phenotype in lung diseases. Understanding the mechanisms of macrophage reprogramming will assist in the selection of new therapeutic targets for correction of impaired immunity. PMID:26366410

Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alberio, Ramiro; Johnson, Andrew D.; Stick, Reimer

2005-07-01

The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requiresmore » permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells.« less

Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes.

PubMed

Tan, Xiaobing; Dai, Qingli; Guo, Tao; Xu, Jingshu; Dai, Qingyuan

2018-01-22

Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy. Copyright © 2017. Published by Elsevier Inc.

Epigenomic Reprogramming of the Developing Reproductive Tract and Disease Susceptibility in Adulthood

PubMed Central

Walker, Cheryl Lyn

2014-01-01

During development, epigenetic programs are “installed” on the genome that direct differentiation and normal tissue and organ function in adulthood. Consequently, development is also a period of susceptibility to reprogramming of the epigenome. Developmental reprogramming occurs when an adverse stimulus or insult interrupts the proper “install” of epigenetic programs during development, reprogramming normal physiological responses in such a way as to promote disease later in life. Some of the best examples of developmental reprogramming involve the reproductive tract, where early life exposures to environmental estrogens can increase susceptibility to benign and malignant tumors in adulthood including leiomyoma (fibroids), endometrial and prostate cancer. Although specific mechanism(s) by which environmental estrogens reprogram the developing epigenome were unknown, both DNA and histone methylation were considered likely targets for epigenetic reprogramming. We have now identified a mechanism by which developmental exposures to environmental estrogens reprogram the epigenome by inducing inappropriate activation of nongenomic estrogen receptor (ER) signaling. Activation of non-genomic ER signaling via the PI3K pathway activates the kinase AKT/PKB in the developing reproductive tract, which phosphorylates the histone lysine methyltransferase (HKMT) EZH2, the key “installer” of epigenetic histone H3 lysine 27 trimethylation (H3K27me3). AKT phosphorylation inactivates EZH2, decreasing levels of H3K27 methylation, a repressive mark that inhibits gene expression, in the developing uterus. As a result of this developmental reprogramming, many estrogen-responsive genes become hypersensitive to estrogen in adulthood, exhibiting elevated expression throughout the estrus cycle, and resulting in a “hyper-estrogenized” phenotype in the adult uterus that promotes development of hormone dependent tumors. PMID:21656660

Sox11 Expression Promotes Regeneration of Some Retinal Ganglion Cell Types but Kills Others.

PubMed

Norsworthy, Michael W; Bei, Fengfeng; Kawaguchi, Riki; Wang, Qing; Tran, Nicholas M; Li, Yi; Brommer, Benedikt; Zhang, Yiming; Wang, Chen; Sanes, Joshua R; Coppola, Giovanni; He, Zhigang

2017-06-21

At least 30 types of retinal ganglion cells (RGCs) send distinct messages through the optic nerve to the brain. Available strategies of promoting axon regeneration act on only some of these types. Here we tested the hypothesis that overexpressing developmentally important transcription factors in adult RGCs could reprogram them to a "youthful" growth-competent state and promote regeneration of other types. From a screen of transcription factors, we identified Sox11 as one that could induce substantial axon regeneration. Transcriptome profiling indicated that Sox11 activates genes involved in cytoskeletal remodeling and axon growth. Remarkably, α-RGCs, which preferentially regenerate following treatments such as Pten deletion, were killed by Sox11 overexpression. Thus, Sox11 promotes regeneration of non-α-RGCs, which are refractory to Pten deletion-induced regeneration. We conclude that Sox11 can reprogram adult RGCs to a growth-competent state, suggesting that different growth-promoting interventions promote regeneration in distinct neuronal types. Copyright © 2017 Elsevier Inc. All rights reserved.

Metabolic reprogramming during TGFβ1-induced epithelial-to-mesenchymal transition

PubMed Central

Jiang, Lei; Xiao, Ling; Sugiura, Hidekazu; Huang, Xiumei; Ali, Aktar; Kuro-o, Makoto; Deberardinis, Ralph J.; Boothman, David A.

2014-01-01

Metastatic progression, including extravasation and micro-metastatic outgrowth, is the main cause of cancer patient death. Recent studies suggest that cancer cells reprogram their metabolism to support increased proliferation through increased glycolysis and biosynthetic activities, including lipogenesis pathways. However, metabolic changes during metastatic progression, including alterations in regulatory gene expression, remain undefined. We show that transforming growth factor beta 1 (TGFβ1) induced Epithelial-to-Mesenchymal Transition (EMT) is accompanied by coordinately reduced enzyme expression required to convert glucose into fatty acids, and concomitant enhanced respiration. Over-expressed Snail1, a transcription factor mediating TGFβ1-induced EMT, was sufficient to suppress carbohydrate-responsive-element-binding protein (ChREBP, a master lipogenic regulator), and fatty acid synthase (FASN), its effector lipogenic gene. Stable FASN knock-down was sufficient to induce EMT, stimulate migration and extravasation in vitro. FASN silencing enhanced lung metastasis and death in vivo. These data suggest that a metabolic transition that suppresses lipogenesis and favors energy production is an essential component of TGFβ1-induced EMT and metastasis. PMID:25284588

Spin glass model for dynamics of cell reprogramming

NASA Astrophysics Data System (ADS)

Pusuluri, Sai Teja; Lang, Alex H.; Mehta, Pankaj; Castillo, Horacio E.

2015-03-01

Recent experiments show that differentiated cells can be reprogrammed to become pluripotent stem cells. The possible cell fates can be modeled as attractors in a dynamical system, the ``epigenetic landscape.'' Both cellular differentiation and reprogramming can be described in the landscape picture as motion from one attractor to another attractor. We perform Monte Carlo simulations in a simple model of the landscape. This model is based on spin glass theory and it can be used to construct a simulated epigenetic landscape starting from the experimental genomic data. We re-analyse data from several cell reprogramming experiments and compare with our simulation results. We find that the model can reproduce some of the main features of the dynamics of cell reprogramming.

Efficient Direct Lineage Reprogramming of Fibroblasts into Induced Cardiomyocytes Using Nanotopographical Cues.

PubMed

Yoo, Junsang; Chang, Yujung; Kim, Hongwon; Baek, Soonbong; Choi, Hwan; Jeong, Gun-Jae; Shin, Jaein; Kim, Hongnam; Kim, Byung-Soo; Kim, Jongpil

2017-03-01

Induced cardiomyocytes (iCMs) generated via direct lineage reprogramming offer a novel therapeutic target for the study and treatment of cardiac diseases. However, the efficiency of iCM generation is significantly low for therapeutic applications. Here, we show an efficient direct conversion of somatic fibroblasts into iCMs using nanotopographic cues. Compared with flat substrates, the direct conversion of fibroblasts into iCMs on nanopatterned substrates resulted in a dramatic increase in the reprogramming efficiency and maturation of iCM phenotypes. Additionally, enhanced reprogramming by substrate nanotopography was due to changes in the activation of focal adhesion kinase and specific histone modifications. Taken together, these results suggest that nanotopographic cues can serve as an efficient stimulant for direct lineage reprogramming into iCMs.

Current reprogramming systems in regenerative medicine: from somatic cells to induced pluripotent stem cells.

PubMed

Hu, Chenxia; Li, Lanjuan

2016-01-01

Induced pluripotent stem cells (iPSCs) paved the way for research fields including cell therapy, drug screening, disease modeling and the mechanism of embryonic development. Although iPSC technology has been improved by various delivery systems, direct transduction and small molecule regulation, low reprogramming efficiency and genomic modification steps still inhibit its clinical use. Improvements in current vectors and the exploration of novel vectors are required to balance efficiency and genomic modification for reprogramming. Herein, we set out a comprehensive analysis of current reprogramming systems for the generation of iPSCs from somatic cells. By clarifying advantages and disadvantages of the current reprogramming systems, we are striding toward an effective route to generate clinical grade iPSCs.

The Wnt/β-catenin signaling pathway tips the balance between apoptosis and reprograming of cell fusion hybrids.

PubMed

Lluis, Frederic; Pedone, Elisa; Pepe, Stefano; Cosma, Maria Pia

2010-11-01

Cell-cell fusion contributes to cell differentiation and developmental processes. We have previously showed that activation of Wnt/β-catenin enhances somatic cell reprograming after polyethylene glycol (PEG)-mediated fusion. Here, we show that neural stem cells and ESCs can fuse spontaneously in cocultures, although with very low efficiency (about 2%), as the hybrids undergo apoptosis. In contrast, when Wnt/β-catenin signaling is activated in ESCs and leads to accumulation of low amounts of β-catenin in the nucleus, activated ESCs can reprogram somatic cells with very high efficiency after spontaneous fusion. Furthermore, we also show that different levels of β-catenin accumulation in the ESC nuclei can modulate cell proliferation, although in our experimental setting, cell proliferation does not modulate the reprograming efficiency per se. Overall, the present study provides evidence that spontaneous fusion occurs, while the survival of the reprogramed clones is strictly dependent on induction of a Wnt-mediated reprograming pathway. Copyright © 2010 AlphaMed Press.

Neutrophils induce macrophage anti-inflammatory reprogramming by suppressing NF-κB activation.

PubMed

Marwick, John A; Mills, Ross; Kay, Oliver; Michail, Kyriakos; Stephen, Jillian; Rossi, Adriano G; Dransfield, Ian; Hirani, Nikhil

2018-06-04

Apoptotic cells modulate the function of macrophages to control and resolve inflammation. Here, we show that neutrophils induce a rapid and sustained suppression of NF-κB signalling in the macrophage through a unique regulatory relationship which is independent of apoptosis. The reduction of macrophage NF-κB activation occurs through a blockade in transforming growth factor β-activated kinase 1 (TAK1) and IKKβ activation. As a consequence, NF-κB (p65) phosphorylation is reduced, its translocation to the nucleus is inhibited and NF-κB-mediated inflammatory cytokine transcription is suppressed. Gene Set Enrichment Analysis reveals that this suppression of NF-κB activation is not restricted to post-translational modifications of the canonical NF-κB pathway, but is also imprinted at the transcriptional level. Thus neutrophils exert a sustained anti-inflammatory phenotypic reprogramming of the macrophage, which is reflected by the sustained reduction in the release of pro- but not anti- inflammatory cytokines from the macrophage. Together, our findings identify a novel apoptosis-independent mechanism by which neutrophils regulate the mediator profile and reprogramming of monocytes/macrophages, representing an important nodal point for inflammatory control.

Pluripotent stem cells and reprogrammed cells in farm animals.

PubMed

Nowak-Imialek, Monika; Kues, Wilfried; Carnwath, Joseph W; Niemann, Heiner

2011-08-01

Pluripotent cells are unique because of their ability to differentiate into the cell lineages forming the entire organism. True pluripotent stem cells with germ line contribution have been reported for mice and rats. Human pluripotent cells share numerous features of pluripotentiality, but confirmation of their in vivo capacity for germ line contribution is impossible due to ethical and legal restrictions. Progress toward derivation of embryonic stem cells from domestic species has been made, but the derived cells were not able to produce germ line chimeras and thus are termed embryonic stem-like cells. However, domestic animals, in particular the domestic pig (Sus scrofa), are excellent large animals models, in which the clinical potential of stem cell therapies can be studied. Reprogramming technologies for somatic cells, including somatic cell nuclear transfer, cell fusion, in vitro culture in the presence of cell extracts, in vitro conversion of adult unipotent spermatogonial stem cells into germ line derived pluripotent stem cells, and transduction with reprogramming factors have been developed with the goal of obtaining pluripotent, germ line competent stem cells from domestic animals. This review summarizes the present state of the art in the derivation and maintenance of pluripotent stem cells in domestic animals.

Metabolic changes in tumor cells and tumor-associated macrophages: A mutual relationship.

PubMed

Netea-Maier, Romana T; Smit, Johannes W A; Netea, Mihai G

2018-01-28

In order to adapt to the reduced availability of nutrients and oxygen in the tumor microenvironment and the increased requirements of energy and building blocks necessary for maintaining their high proliferation rate, malignant cells undergo metabolic changes that result in an increased production of lactate, nitric oxide, reactive oxygen species, prostaglandins and other byproducts of arachidonic acid metabolism that influence both the composition of the inflammatory microenvironment and the function of the tumor-associated macrophages (TAMs). In response to cues present in the TME, among which products of altered tumor cell metabolism, TAMs are also required to reprogram their metabolism, with activation of glycolysis, fatty acid synthesis and altered nitrogen cycle metabolism. These changes result in functional reprogramming of TAMs which includes changes in the production of cytokines and angiogenetic factors, and contribute to the tumor progression and metastasis. Understanding the metabolic changes governing the intricate relationship between the tumor cells and the TAMs represents an essential step towards developing novel therapeutic approaches targeting the metabolic reprogramming of the immune cells to potentiate their tumoricidal potential and to circumvent therapy resistance. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Reserve stem cells: Reprogramming of differentiated cells fuels repair, metaplasia, and neoplasia in the adult gastrointestinal tract

PubMed Central

Mills, Jason C.; Sansom, Owen J.

2016-01-01

It has long been known that differentiated cells can switch fates, especially in vitro, but only recently has there been a critical mass of publications describing the mechanisms adult, post-mitotic cells use in vivo to reverse their differentiation state. We propose that this sort of cellular reprogramming is a fundamental cellular process akin to apoptosis or mitosis. Because reprogramming can invoke regenerative cells from mature cells, it is critical to the longterm maintenance of tissues like the pancreas, which encounter large insults during adulthood but lack constitutively active adult stem cells to repair the damage. However, even in tissues with adult stem cells, like stomach and intestine, reprogramming may allow mature cells to serve as reserve (“quiescent”) stem cells when normal stem cells are compromised. We propose that the potential downside to reprogramming is that it increases risk for cancers that occur late in adulthood. Mature, long-lived cells may have years of exposure to mutagens. Mutations that affect the physiological function of differentiated, post-mitotic cells may lead to apoptosis, but mutations in genes that govern proliferation might not be selected against. Hence, reprogramming with reentry into the cell cycle might unmask those mutations, causing an irreversible progenitor-like, proliferative state. We review recent evidence showing that reprogramming fuels irreversible metaplastic and precancerous proliferations in stomach and pancreas. Finally, we illustrate how we think reprogrammed differentiated cells are likely candidates as cells of origin for cancers of the intestine. PMID:26175494

Forward Programming of Cardiac Stem Cells by Homogeneous Transduction with MYOCD plus TBX5

PubMed Central

Belian, Elisa; Noseda, Michela; Abreu Paiva, Marta S.; Leja, Thomas; Sampson, Robert; Schneider, Michael D.

2015-01-01

Adult cardiac stem cells (CSCs) express many endogenous cardiogenic transcription factors including members of the Gata, Hand, Mef2, and T-box family. Unlike its DNA-binding targets, Myocardin (Myocd)—a co-activator not only for serum response factor, but also for Gata4 and Tbx5—is not expressed in CSCs. We hypothesised that its absence was a limiting factor for reprogramming. Here, we sought to investigate the susceptibility of adult mouse Sca1+ side population CSCs to reprogramming by supplementing the triad of GATA4, MEF2C, and TBX5 (GMT), and more specifically by testing the effect of the missing co-activator, Myocd. Exogenous factors were expressed via doxycycline-inducible lentiviral vectors in various combinations. High throughput quantitative RT-PCR was used to test expression of 29 cardiac lineage markers two weeks post-induction. GMT induced more than half the analysed cardiac transcripts. However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2. Adding MYOCD to GMT affected only slightly the breadth and level of gene induction, but, importantly, triggered expression of all three proteins examined (α-cardiac actin, atrial natriuretic peptide, sarcomeric myosin heavy chains). MYOCD + TBX was the most effective pairwise combination in this system. In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized. In summary: (1) GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2) Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3) Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy. Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate. PMID:26047103

Forward Programming of Cardiac Stem Cells by Homogeneous Transduction with MYOCD plus TBX5.

PubMed

Belian, Elisa; Noseda, Michela; Abreu Paiva, Marta S; Leja, Thomas; Sampson, Robert; Schneider, Michael D

2015-01-01

Adult cardiac stem cells (CSCs) express many endogenous cardiogenic transcription factors including members of the Gata, Hand, Mef2, and T-box family. Unlike its DNA-binding targets, Myocardin (Myocd)-a co-activator not only for serum response factor, but also for Gata4 and Tbx5-is not expressed in CSCs. We hypothesised that its absence was a limiting factor for reprogramming. Here, we sought to investigate the susceptibility of adult mouse Sca1+ side population CSCs to reprogramming by supplementing the triad of GATA4, MEF2C, and TBX5 (GMT), and more specifically by testing the effect of the missing co-activator, Myocd. Exogenous factors were expressed via doxycycline-inducible lentiviral vectors in various combinations. High throughput quantitative RT-PCR was used to test expression of 29 cardiac lineage markers two weeks post-induction. GMT induced more than half the analysed cardiac transcripts. However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2. Adding MYOCD to GMT affected only slightly the breadth and level of gene induction, but, importantly, triggered expression of all three proteins examined (α-cardiac actin, atrial natriuretic peptide, sarcomeric myosin heavy chains). MYOCD + TBX was the most effective pairwise combination in this system. In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized. (1) GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2) Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3) Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy. Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate.

Actin stress in cell reprogramming

PubMed Central

Guo, Jun; Wang, Yuexiu; Sachs, Frederick; Meng, Fanjie

2014-01-01

Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin–cpstFRET–actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals. PMID:25422450

Inhibitor of differentiation 1 transcription factor promotes metabolic reprogramming in hepatocellular carcinoma cells

PubMed Central

Sharma, Bal Krishan; Kolhe, Ravindra; Black, Stephen M.; Keller, Jonathan R.; Mivechi, Nahid F.; Satyanarayana, Ande

2016-01-01

Reprograming of metabolism is one of the central hallmarks of cancer. The majority of cancer cells depend on high rates of glycolysis and glutaminolysis for their growth and survival. A number of oncogenes and tumor suppressors have been connected to the regulation of altered glucose and glutamine metabolism in cancer cells. For example, the oncogene c-Myc plays vital roles in cancer cell metabolic adaptation by directly regulating various genes that participate in aerobic glycolysis and glutaminolysis. Inhibitor of differentiation 1 (Id1) is a helix-loop-helix transcription factor that plays important roles in cell proliferation, differentiation, and cell fate determination. Overexpression of Id1 causes intestinal adenomas and thymic lymphomas in mice, suggesting that Id1 could function as an oncogene. Despite it being an oncogene, whether Id1 plays any prominent role in cancer cell metabolic reprograming is unknown. Here, we demonstrate that Id1 is strongly expressed in human and mouse liver tumors and in hepatocellular carcinoma (HCC) cell lines, whereas its expression is very low or undetectable in normal liver tissues. In HCC cells, Id1 expression is regulated by the MAPK/ERK pathway at the transcriptional level. Knockdown of Id1 suppressed aerobic glycolysis and glutaminolysis, suggesting that Id1 promotes a metabolic shift toward aerobic glycolysis. At the molecular level, Id1 mediates its metabolic effects by regulating the expression levels of c-Myc. Knockdown of Id1 resulted in down-regulation (∼75%) of c-Myc, whereas overexpression of Id1 strongly induced (3-fold) c-Myc levels. Interestingly, knockdown of c-Myc resulted in down-regulation (∼60%) of Id1, suggesting a positive feedback-loop regulatory mechanism between Id1 and c-Myc. Under anaerobic conditions, both Id1 and c-Myc are down-regulated (50–70%), and overexpression of oxygen-insensitive hypoxia-inducible factor 1α (Hif1α) or its downstream target Mxi1 resulted in a significant reduction of c-Myc and Id1 (∼70%), suggesting that Hif1α suppresses Id1 and c-Myc under anaerobic conditions via Mxi1. Together, our findings indicate a prominent novel role for Id1 in liver cancer cell metabolic adaptation.—Sharma, B. K., Kolhe, R., Black, S. M., Keller, J. R., Mivechi, N. F., Satyanarayana, A. Inhibitor of differentiation 1 transcription factor promotes metabolic reprogramming in hepatocellular carcinoma cells. PMID:26330493

Cloning animals by somatic cell nuclear transfer – biological factors

PubMed Central

Tian, X Cindy; Kubota, Chikara; Enright, Brian; Yang, Xiangzhong

2003-01-01

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other specie, this review will be focused on somatic cell cloning of cattle. PMID:14614770

Cloning animals by somatic cell nuclear transfer--biological factors.

PubMed

Tian, X Cindy; Kubota, Chikara; Enright, Brian; Yang, Xiangzhong

2003-11-13

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other species, this review will be focused on somatic cell cloning of cattle.

iPSCs-based anti-aging therapies: Recent discoveries and future challenges.

PubMed

Pareja-Galeano, Helios; Sanchis-Gomar, Fabián; Pérez, Laura M; Emanuele, Enzo; Lucia, Alejandro; Gálvez, Beatriz G; Gallardo, María Esther

2016-05-01

The main biological hallmarks of the aging process include stem cell exhaustion and cellular senescence. Consequently, research efforts to treat age-related diseases as well as anti-aging therapies in general have recently focused on potential 'reprogramming' regenerative therapies. These new approaches are based on induced pluripotent stem cells (iPSCs), including potential in vivo reprogramming for tissue repair. Another possibility is targeting pathways of cellular senescence, e.g., through modulation of p16INK4a signaling and especially inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Here, we reviewed and discussed these recent developments together with their possible usefulness for future treatments against sarcopenia, a major age-related condition. Copyright © 2016 Elsevier B.V. All rights reserved.

Did Convergent Protein Evolution Enable Phytoplasmas to Generate 'Zombie Plants'?

PubMed

Rümpler, Florian; Gramzow, Lydia; Theißen, Günter; Melzer, Rainer

2015-12-01

Phytoplasmas are pathogenic bacteria that reprogram plant development such that leaf-like structures instead of floral organs develop. Infected plants are sterile and mainly serve to propagate phytoplasmas and thus have been termed 'zombie plants'. The developmental reprogramming relies on specific interactions of the phytoplasma protein SAP54 with a small subset of MADS-domain transcription factors. Here, we propose that SAP54 folds into a structure that is similar to that of the K-domain, a protein-protein interaction domain of MADS-domain proteins. We suggest that undergoing convergent structural and sequence evolution, SAP54 evolved to mimic the K-domain. Given the high specificity of resulting developmental alterations, phytoplasmas might be used to study flower development in genetically intractable plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

SREBF1 Activity is Regulated by an AR/mTOR Nuclear Axis in Prostate Cancer.

PubMed

Audet-Walsh, Etienne; Vernier, Mathieu; Yee, Tracey; Laflamme, Chloe E; Li, Susan; Chen, Yonghong; Giguere, Vincent

2018-05-21

Reprogramming of cellular metabolism is an important feature of prostate cancer (PCa), including altered lipid metabolism. Recently, it was observed that the nuclear fraction of mTOR is essential for the androgen-mediated metabolic reprogramming of PCa cells. Herein, it is demonstrated that the androgen receptor (AR) and mTOR bind to regulatory regions of sterol regulatory element binding transcription factor 1 (SREBF1) to control its expression, while dual activation of these signaling pathways also promotes SREBF1 cleavage and its translocation to the nucleus. Consequently, SREBF1 recruitment to regulatory regions of its target genes is induced upon treatment with the synthetic androgen R1881, an effect abrogated upon inhibition of the mTOR signaling pathway. In turn, pharmacological and genetic inhibition of SREBF1 activity impairs the androgen-mediated induction of the key lipogenic genes fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD1). Consistent with these observations, the expression of SREBF1, FASN and SCD1 is significantly correlated in human PCa tumor clinical specimens. Functionally, blockade of SREBF1 activity reduces the androgen-driven lipid accumulation. Interestingly, decreased triglyceride accumulation observed upon SREBF1 inhibition is paralleled by an increase in mitochondrial respiration, indicating a potential rewiring of citrate metabolism in PCa cells. Altogether, these data define an AR/mTOR nuclear axis, in the context of PCa, as a novel pathway regulating SREBF1 activity and citrate metabolism. The finding that an AR/mTOR complex promotes SREBP expression and activity enhances our understanding of the metabolic adaptation necessary for prostate cancer cell growth and suggests novel therapeutic approaches to target metabolic vulnerabilities in tumors. Copyright ©2018, American Association for Cancer Research.

Diet-Induced Obesity Reprograms the Inflammatory Response of the Murine Lung to Inhaled Endotoxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tilton, Susan C.; Waters, Katrina M.; Karin, Norman J.

The co-occurrence of environmental factors is common in complex human diseases and, as such, understanding the molecular responses involved is essential to determine risk and susceptibility to disease. We have investigated the key biological pathways that define susceptibility for pulmonary infection during obesity in diet-induced obese (DIO) and regular weight (RW) C57BL/6 mice exposed to inhaled lipopolysaccharide (LPS). LPS induced a strong inflammatory response in all mice as indicated by elevated cell counts of macrophages and neutrophils and levels of proinflammatory cytokines (MDC, MIP-1γ, IL-12, RANTES) in the bronchoalveolar lavage fluid. Additionally, DIO mice exhibited 50% greater macrophage cell counts,more » but decreased levels of the cytokines, IL-6, TARC, TNF-α, and VEGF relative to RW mice. Microarray analysis of lung tissue showed over half of the LPS-induced expression in DIO mice consisted of genes unique for obese mice, suggesting that obesity reprograms how the lung responds to subsequent insult. In particular, we found that obese animals exposed to LPS have gene signatures showing increased inflammatory and oxidative stress response and decreased antioxidant capacity compared with RW. Because signaling pathways for these responses can be common to various sources of environmentally induced lung damage, we further identified biomarkers that are indicative of specific toxicant exposure by comparing gene signatures after LPS exposure to those from a parallel study with cigarette smoke. These data show obesity may increase sensitivity to further insult and that co-occurrence of environmental stressors result in complex biosignatures that are not predicted from analysis of individual exposures.« less

Extended Self-Renewal and Accelerated Reprogramming in the Absence of Kdm5b

PubMed Central

Hu, Gangqing; Yu, Zu-Xi; Liu, Chengyu

2013-01-01

Embryonic stem (ES) cell pluripotency is thought to be regulated in part by H3K4 methylation. However, it is unclear how H3K4 demethylation contributes to ES cell function and participates in induced pluripotent stem (iPS) cell reprogramming. Here, we show that KDM5B, which demethylates H3K4, is important for ES cell differentiation and presents a barrier to the reprogramming process. Depletion of Kdm5b leads to an extension in the self-renewal of ES cells in the absence of LIF. Transcriptome analysis revealed the persistent expression of pluripotency genes and underexpression of developmental genes during differentiation in the absence of Kdm5b, suggesting that KDM5B plays a key role in cellular fate changes. We also observed accelerated reprogramming of differentiated cells in the absence of Kdm5b, demonstrating that KDM5B is a barrier to the reprogramming process. Expression analysis revealed that mesenchymal master regulators associated with the epithelial-to-mesenchymal transition (EMT) are downregulated during reprogramming in the absence of Kdm5b. Moreover, global analysis of H3K4me3/2 revealed that enhancers of fibroblast genes are rapidly deactivated in the absence of Kdm5b, and genes associated with EMT lose H3K4me3/2 during the early reprogramming process. These findings provide functional insight into the role for KDM5B in regulating ES cell differentiation and as a barrier to the reprogramming process. PMID:24100015

Overcoming the hurdles for a reproducible generation of human functionally mature reprogrammed neurons.

PubMed

Broccoli, Vania; Rubio, Alicia; Taverna, Stefano; Yekhlef, Latefa

2015-06-01

The advent of cell reprogramming technologies has widely disclosed the possibility to have direct access to human neurons for experimental and biomedical applications. Human pluripotent stem cells can be instructed in vitro to generate specific neuronal cell types as well as different glial cells. Moreover, new approaches of direct neuronal cell reprogramming can strongly accelerate the generation of different neuronal lineages. However, genetic heterogeneity, reprogramming fidelity, and time in culture of the starting cells can still significantly bias their differentiation efficiency and quality of the neuronal progenies. In addition, reprogrammed human neurons exhibit a very slow pace in gaining a full spectrum of functional properties including physiological levels of membrane excitability, sustained and prolonged action potential firing, mature synaptic currents and synaptic plasticity. This delay poses serious limitations for their significance as biological experimental model and screening platform. We will discuss new approaches of neuronal cell differentiation and reprogramming as well as methods to accelerate the maturation and functional activity of the converted human neurons. © 2015 by the Society for Experimental Biology and Medicine.

Single-Cell RNA-Seq Reveals Dynamic Early Embryonic-like Programs during Chemical Reprogramming.

PubMed

Zhao, Ting; Fu, Yao; Zhu, Jialiang; Liu, Yifang; Zhang, Qian; Yi, Zexuan; Chen, Shi; Jiao, Zhonggang; Xu, Xiaochan; Xu, Junquan; Duo, Shuguang; Bai, Yun; Tang, Chao; Li, Cheng; Deng, Hongkui

2018-06-12

Chemical reprogramming provides a powerful platform for exploring the molecular dynamics that lead to pluripotency. Although previous studies have uncovered an intermediate extraembryonic endoderm (XEN)-like state during this process, the molecular underpinnings of pluripotency acquisition remain largely undefined. Here, we profile 36,199 single-cell transcriptomes at multiple time points throughout a highly efficient chemical reprogramming system using RNA-sequencing and reconstruct their progression trajectories. Through identifying sequential molecular events, we reveal that the dynamic early embryonic-like programs are key aspects of successful reprogramming from XEN-like state to pluripotency, including the concomitant transcriptomic signatures of two-cell (2C) embryonic-like and early pluripotency programs and the epigenetic signature of notable genome-wide DNA demethylation. Moreover, via enhancing the 2C-like program by fine-tuning chemical treatment, the reprogramming process is remarkably accelerated. Collectively, our findings offer a high-resolution dissection of cell fate dynamics during chemical reprogramming and shed light on mechanistic insights into the nature of induced pluripotency. Copyright © 2018 Elsevier Inc. All rights reserved.

The Histone Acetyltransferase MOF Promotes Induces Generation of Pluripotent Stem Cells.

PubMed

Mu, Xupeng; Yan, Shaohua; Fu, Changhao; Wei, Anhui

2015-08-01

Histone modification plays an important role in maintaining pluripotency and self-renewal of embryonic stem cells (ESCs). The histone acetyltransferase MOF is a key regulator of ESCs; however, the role of MOF in the process of reprogramming back to induced pluripotent stem cells (iPSCs) remains unclear. In this study, we investigated the function of MOF on the generation of iPSCs. We show that iPSCs contain high levels of MOF mRNA, and the expression level of MOF protein is dramatically upregulated following reprogramming. Most importantly, overexpression of MOF improves reprogramming efficiency and facilitates the formation of iPSCs, whereas small hairpin RNA (shRNA)-mediated knockdown of MOF impairs iPSCs generation during reprogramming. Further investigation reveals that MOF interacts with the H3K4 methyltransferase Wdr5 to promote endogenous Oct4 expression during the reprogramming process. Knockdown of MOF reduces H4K16ac and H3K4me3 modification at the Oct4 promoter. In conclusion, our data indicate that MOF is an important epigenetic regulator that is critical for efficient reprogramming.

Knockdown Brm and Baf170, components of chromatin remodeling complex, facilitates reprogramming of somatic cells

USDA-ARS?s Scientific Manuscript database

The SWI/SNF (SWItch/Sucrose NonFermentable or BAF, Brg/Brahma-associated factors) complexes are epigenetic modifiers of chromatin structure and undergo progressive changes in subunit composition during cellular differentiation. For example, in embryonic stem cells (ESCs) esBAF contains Brg1 and Baf...

RNA-Seq analysis of stuA mutants in Fusarium verticillioides indicates dramatic genomic wide transcriptional reprogramming

USDA-ARS?s Scientific Manuscript database

StuA, first discovered in Aspergillus nidulans and a member of the APSES class of transcription factors, regulates several essential developmental stages in fungi such as virulence, sporulation and toxin production in phytopathogenic fungi. Fusarium verticillioides (Fv), a maize phytopathogen, produ...

SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence

PubMed Central

Winiecka-Klimek, Marta; Smolarz, Maciej; Walczak, Maciej P.; Zieba, Jolanta; Hulas-Bigoszewska, Krystyna; Kmieciak, Blazej; Piaskowski, Sylwester; Rieske, Piotr; Grzela, Dawid P.; Stoczynska-Fidelus, Ewelina

2015-01-01

Tumorigenic potential of induced pluripotent stem cells (iPSCs) infiltrating population of induced neural stem cells (iNSCs) generated from iPSCs may limit their medical applications. To overcome such a difficulty, direct reprogramming of adult somatic cells into iNSCs was proposed. The aim of this study was the systematic comparison of induced neural cells (iNc) obtained with different methods—direct reprogramming of human adult fibroblasts with either SOX2 (SiNSc-like) or SOX2 and c-MYC (SMiNSc-like) and induced pluripotent stem cells differentiation to ebiNSc—in terms of gene expression profile, differentiation potential as well as proliferation properties. Immunocytochemistry and real-time PCR analyses were used to evaluate gene expression profile and differentiation potential of various iNc types. Bromodeoxyuridine (BrdU) incorporation and senescence-associated beta-galactosidase (SA-β-gal) assays were used to estimate proliferation potential. All three types of iNc were capable of neuronal differentiation; however, astrocytic differentiation was possible only in case of ebiNSc. Contrary to ebiNSc generation, the direct reprogramming was rarely a propitious process, despite 100% transduction efficiency. The potency of direct iNSCs-like cells generation was lower as compared to iNSCs obtained by iPSCs differentiation, and only slightly improved when c-MYC was added. Directly reprogrammed iNSCs-like cells were lacking the ability to differentiate into astrocytic cells and characterized by poor efficiency of neuronal cells formation. Such features indicated that these cells could not be fully reprogrammed, as confirmed mainly with senescence detection. Importantly, SiNSc-like and SMiNSc-like cells were unable to achieve the long-term survival and became senescent, which limits their possible therapeutic applicability. Our results suggest that iNSCs-like cells, generated in the direct reprogramming attempts, were either not fully reprogrammed or reprogrammed only into neuronal progenitors, mainly because of the inaccuracies of currently available protocols. PMID:26535892

Androgen Receptor Deregulation Drives Bromodomain-Mediated Chromatin Alterations in Prostate Cancer.

PubMed

Urbanucci, Alfonso; Barfeld, Stefan J; Kytölä, Ville; Itkonen, Harri M; Coleman, Ilsa M; Vodák, Daniel; Sjöblom, Liisa; Sheng, Xia; Tolonen, Teemu; Minner, Sarah; Burdelski, Christoph; Kivinummi, Kati K; Kohvakka, Annika; Kregel, Steven; Takhar, Mandeep; Alshalalfa, Mohammed; Davicioni, Elai; Erho, Nicholas; Lloyd, Paul; Karnes, R Jeffrey; Ross, Ashley E; Schaeffer, Edward M; Vander Griend, Donald J; Knapp, Stefan; Corey, Eva; Feng, Felix Y; Nelson, Peter S; Saatcioglu, Fahri; Knudsen, Karen E; Tammela, Teuvo L J; Sauter, Guido; Schlomm, Thorsten; Nykter, Matti; Visakorpi, Tapio; Mills, Ian G

2017-06-06

Global changes in chromatin accessibility may drive cancer progression by reprogramming transcription factor (TF) binding. In addition, histone acetylation readers such as bromodomain-containing protein 4 (BRD4) have been shown to associate with these TFs and contribute to aggressive cancers including prostate cancer (PC). Here, we show that chromatin accessibility defines castration-resistant prostate cancer (CRPC). We show that the deregulation of androgen receptor (AR) expression is a driver of chromatin relaxation and that AR/androgen-regulated bromodomain-containing proteins (BRDs) mediate this effect. We also report that BRDs are overexpressed in CRPCs and that ATAD2 and BRD2 have prognostic value. Finally, we developed gene stratification signature (BROMO-10) for bromodomain response and PC prognostication, to inform current and future trials with drugs targeting these processes. Our findings provide a compelling rational for combination therapy targeting bromodomains in selected patients in which BRD-mediated TF binding is enhanced or modified as cancer progresses. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Comprehensive analysis of the dynamic structure of nuclear localization signals.

PubMed

Yamagishi, Ryosuke; Okuyama, Takahide; Oba, Shuntaro; Shimada, Jiro; Chaen, Shigeru; Kaneko, Hiroki

2015-12-01

Most transcription and epigenetic factors in eukaryotic cells have nuclear localization signals (NLSs) and are transported to the nucleus by nuclear transport proteins. Understanding the features of NLSs and the mechanisms of nuclear transport might help understand gene expression regulation, somatic cell reprogramming, thus leading to the treatment of diseases associated with abnormal gene expression. Although many studies analyzed the amino acid sequence of NLSs, few studies investigated their three-dimensional structure. Therefore, we conducted a statistical investigation of the dynamic structure of NLSs by extracting the conformation of these sequences from proteins examined by X-ray crystallography and using a quantity defined as conformational determination rate (a ratio between the number of amino acids determining the conformation and the number of all amino acids included in a certain region). We found that determining the conformation of NLSs is more difficult than determining the conformation of other regions and that NLSs may tend to form more heteropolymers than monomers. Therefore, these findings strongly suggest that NLSs are intrinsically disordered regions.

Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells

PubMed Central

Lee, Jonghyeob; Sugiyama, Takuya; Liu, Yinghua; Wang, Jing; Gu, Xueying; Lei, Ji; Markmann, James F; Miyazaki, Satsuki; Miyazaki, Jun-ichi; Szot, Gregory L; Bottino, Rita; Kim, Seung K

2013-01-01

Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001 PMID:24252877

Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells.

PubMed

Lee, Jonghyeob; Sugiyama, Takuya; Liu, Yinghua; Wang, Jing; Gu, Xueying; Lei, Ji; Markmann, James F; Miyazaki, Satsuki; Miyazaki, Jun-Ichi; Szot, Gregory L; Bottino, Rita; Kim, Seung K

2013-11-19

Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001.

Dynamic epigenetic regulation of glioblastoma tumorigenicity through LSD1 modulation of MYC expression

PubMed Central

Kozono, David; Li, Jie; Nitta, Masayuki; Sampetrean, Oltea; Gonda, David; Kushwaha, Deepa S.; Merzon, Dmitry; Ramakrishnan, Valya; Zhu, Shan; Zhu, Kaya; Matsui, Hiroko; Harismendy, Olivier; Hua, Wei; Mao, Ying; Kwon, Chang-Hyuk; Saya, Hideyuki; Nakano, Ichiro; Pizzo, Donald P.; VandenBerg, Scott R.; Chen, Clark C.

2015-01-01

The available evidence suggests that the lethality of glioblastoma is driven by small subpopulations of cells that self-renew and exhibit tumorigenicity. It remains unclear whether tumorigenicity exists as a static property of a few cells or as a dynamically acquired property. We used tumor-sphere and xenograft formation as assays for tumorigenicity and examined subclones isolated from established and primary glioblastoma lines. Our results indicate that glioblastoma tumorigenicity is largely deterministic, yet the property can be acquired spontaneously at low frequencies. Further, these dynamic transitions are governed by epigenetic reprogramming through the lysine-specific demethylase 1 (LSD1). LSD depletion increases trimethylation of histone 3 lysine 4 at the avian myelocytomatosis viral oncogene homolog (MYC) locus, which elevates MYC expression. MYC, in turn, regulates oligodendrocyte lineage transcription factor 2 (OLIG2), SRY (sex determining region Y)-box 2 (SOX2), and POU class 3 homeobox 2 (POU3F2), a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Our model suggests epigenetic regulation of key transcription factors governs transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development. PMID:26159421

Capsicum annuum transcription factor WRKYa positively regulates defense response upon TMV infection and is a substrate of CaMK1 and CaMK2.

PubMed

Huh, Sung Un; Lee, Gil-Je; Jung, Ji Hoon; Kim, Yunsik; Kim, Young Jin; Paek, Kyung-Hee

2015-01-23

Plants are constantly exposed to pathogens and environmental stresses. To minimize damage caused by these potentially harmful factors, plants respond by massive transcriptional reprogramming of various stress-related genes via major transcription factor families. One of the transcription factor families, WRKY, plays an important role in diverse stress response of plants and is often useful to generate genetically engineered crop plants. In this study, we carried out functional characterization of CaWRKYa encoding group I WRKY member, which is induced during hypersensitive response (HR) in hot pepper (Capsicum annuum) upon Tobacco mosaic virus (TMV) infection. CaWRKYa was involved in L-mediated resistance via transcriptional reprogramming of pathogenesis-related (PR) gene expression and affected HR upon TMV-P0 infection. CaWRKYa acts as a positive regulator of this defense system and could bind to the W-box of diverse PR genes promoters. Furthermore, we found Capsicum annuum mitogen-activated protein kinase 1 (CaMK1) and 2 (CaMK2) interacted with CaWRKYa and phosphorylated the SP clusters but not the MAPK docking (D)-domain of CaWRKYa. Thus, these results demonstrated that CaWRKYa was regulated by CaMK1 and CaMK2 at the posttranslational level in hot pepper.

Generating induced pluripotent stem cell derived endothelial cells and induced endothelial cells for cardiovascular disease modelling and therapeutic angiogenesis.

PubMed

Clayton, Z E; Sadeghipour, S; Patel, S

2015-10-15

Standard therapy for atherosclerotic coronary and peripheral arterial disease is insufficient in a significant number of patients because extensive disease often precludes effective revascularization. Stem cell therapy holds promise as a supplementary treatment for these patients, as pre-clinical and clinical research has shown transplanted cells can promote angiogenesis via direct and paracrine mechanisms. Induced pluripotent stem cells (iPSCs) are a novel cell type obtained by reprogramming somatic cells using exogenous transcription factor cocktails, which have been introduced to somatic cells via viral or plasmid constructs, modified mRNA or small molecules. IPSCs are now being used in disease modelling and drug testing and are undergoing their first clinical trial, but despite recent advances, the inefficiency of the reprogramming process remains a major limitation, as does the lack of consensus regarding the optimum transcription factor combination and delivery method and the uncertainty surrounding the genetic and epigenetic stability of iPSCs. IPSCs have been successfully differentiated into vascular endothelial cells (iPSC-ECs) and, more recently, induced endothelial cells (iECs) have also been generated by direct differentiation, which bypasses the pluripotent intermediate. IPSC-ECs and iECs demonstrate endothelial functionality in vitro and have been shown to promote neovessel growth and enhance blood flow recovery in animal models of myocardial infarction and peripheral arterial disease. Challenges remain in optimising the efficiency, safety and fidelity of the reprogramming and endothelial differentiation processes and establishing protocols for large-scale production of clinical-grade, patient-derived cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

BMP-induced reprogramming of the neural retina into retinal pigment epithelium requires Wnt signalling

PubMed Central

Steinfeld, Jörg; Steinfeld, Ichie; Bausch, Alexander; Coronato, Nicola; Hampel, Meggi-Lee; Depner, Heike; Layer, Paul G.

2017-01-01

ABSTRACT In vertebrates, the retinal pigment epithelium (RPE) and photoreceptors of the neural retina (NR) comprise a functional unit required for vision. During vertebrate eye development, a conversion of the RPE into NR can be induced by growth factors in vivo at optic cup stages, but the reverse process, the conversion of NR tissue into RPE, has not been reported. Here, we show that bone morphogenetic protein (BMP) signalling can reprogram the NR into RPE at optic cup stages in chick. Shortly after BMP application, expression of Microphthalmia-associated transcription factor (Mitf) is induced in the NR and selective cell death on the basal side of the NR induces an RPE-like morphology. The newly induced RPE differentiates and expresses Melanosomalmatrix protein 115 (Mmp115) and RPE65. BMP-induced Wnt2b expression is observed in regions of the NR that become pigmented. Loss of function studies show that conversion of the NR into RPE requires both BMP and Wnt signalling. Simultaneous to the appearance of ectopic RPE tissue, BMP application reprogrammed the proximal RPE into multi-layered retinal tissue. The newly induced NR expresses visual segment homeobox-containing gene (Vsx2), and the ganglion and photoreceptor cell markers Brn3α and Visinin are detected. Our results show that high BMP concentrations are required to induce the conversion of NR into RPE, while low BMP concentrations can still induce transdifferentiation of the RPE into NR. This knowledge may contribute to the development of efficient standardized protocols for RPE and NR generation for cell replacement therapies. PMID:28546339

Quantifying Cell Fate Decisions for Differentiation and Reprogramming of a Human Stem Cell Network: Landscape and Biological Paths

PubMed Central

Li, Chunhe; Wang, Jin

2013-01-01

Cellular reprogramming has been recently intensively studied experimentally. We developed a global potential landscape and kinetic path framework to explore a human stem cell developmental network composed of 52 genes. We uncovered the underlying landscape for the stem cell network with two basins of attractions representing stem and differentiated cell states, quantified and exhibited the high dimensional biological paths for the differentiation and reprogramming process, connecting the stem cell state and differentiated cell state. Both the landscape and non-equilibrium curl flux determine the dynamics of cell differentiation jointly. Flux leads the kinetic paths to be deviated from the steepest descent gradient path, and the corresponding differentiation and reprogramming paths are irreversible. Quantification of paths allows us to find out how the differentiation and reprogramming occur and which important states they go through. We show the developmental process proceeds as moving from the stem cell basin of attraction to the differentiation basin of attraction. The landscape topography characterized by the barrier heights and transition rates quantitatively determine the global stability and kinetic speed of cell fate decision process for development. Through the global sensitivity analysis, we provided some specific predictions for the effects of key genes and regulation connections on the cellular differentiation or reprogramming process. Key links from sensitivity analysis and biological paths can be used to guide the differentiation designs or reprogramming tactics. PMID:23935477

Brief Report: Human Acute Myeloid Leukemia Reprogramming to Pluripotency Is a Rare Event and Selects for Patient Hematopoietic Cells Devoid of Leukemic Mutations.

PubMed

Lee, Jong-Hee; Salci, Kyle R; Reid, Jennifer C; Orlando, Luca; Tanasijevic, Borko; Shapovalova, Zoya; Bhatia, Mickie

2017-09-01

Induced pluripotent stem cell reprogramming has provided critical insights into disease processes by modeling the genetics and related clinical pathophysiology. Human cancer represents highly diverse genetics, as well as inter- and intra-patient heterogeneity, where cellular model systems capable of capturing this disease complexity would be invaluable. Acute myeloid leukemia (AML) represents one of most heterogeneous cancers and has been divided into genetic subtypes correlated with unique risk stratification over the decades. Here, we report our efforts to induce pluripotency from the heterogeneous population of human patients that represents this disease in the clinic. Using robust optimized reprogramming methods, we demonstrate that reprogramming of AML cells harboring leukemic genomic aberrations is a rare event with the exception of those with de novo mixed-lineage leukemia (MLL) mutations that can be reprogrammed and model drug responses in vitro. Our findings indicate that unlike hematopoietic cells devoid of genomic aberrations, AML cells harboring driver mutations are refractory to reprogramming. Expression of MLL fusion proteins in AML cells did not contribute to induced reprogramming success, which continued to select for patient derived cells devoid of AML patient-specific aberrations. Our study reveals that unanticipated blockades to achieving pluripotency reside within the majority of transformed AML patient cells. Stem Cells 2017;35:2095-2102. © 2017 AlphaMed Press.

The long noncoding RNA Chaer defines an epigenetic checkpoint in cardiac hypertrophy.

PubMed

Wang, Zhihua; Zhang, Xiao-Jing; Ji, Yan-Xiao; Zhang, Peng; Deng, Ke-Qiong; Gong, Jun; Ren, Shuxun; Wang, Xinghua; Chen, Iris; Wang, He; Gao, Chen; Yokota, Tomohiro; Ang, Yen Sin; Li, Shen; Cass, Ashley; Vondriska, Thomas M; Li, Guangping; Deb, Arjun; Srivastava, Deepak; Yang, Huang-Tian; Xiao, Xinshu; Li, Hongliang; Wang, Yibin

2016-10-01

Epigenetic reprogramming is a critical process of pathological gene induction during cardiac hypertrophy and remodeling, but the underlying regulatory mechanisms remain to be elucidated. Here we identified a heart-enriched long noncoding (lnc)RNA, named cardiac-hypertrophy-associated epigenetic regulator (Chaer), which is necessary for the development of cardiac hypertrophy. Mechanistically, Chaer directly interacts with the catalytic subunit of polycomb repressor complex 2 (PRC2). This interaction, which is mediated by a 66-mer motif in Chaer, interferes with PRC2 targeting to genomic loci, thereby inhibiting histone H3 lysine 27 methylation at the promoter regions of genes involved in cardiac hypertrophy. The interaction between Chaer and PRC2 is transiently induced after hormone or stress stimulation in a process involving mammalian target of rapamycin complex 1, and this interaction is a prerequisite for epigenetic reprogramming and induction of genes involved in hypertrophy. Inhibition of Chaer expression in the heart before, but not after, the onset of pressure overload substantially attenuates cardiac hypertrophy and dysfunction. Our study reveals that stress-induced pathological gene activation in the heart requires a previously uncharacterized lncRNA-dependent epigenetic checkpoint.

Partners in crime: VEGF and IL-4 conscript tumour-promoting macrophages.

PubMed

De Palma, Michele

2012-05-01

Tumour-associated macrophages (TAMs) foster tumour progression by several mechanisms, including the promotion of angiogenesis, tissue remodelling, and immunosuppression. Such pro-tumoural activities are thought to be executed by TAM subtypes that harbour features of alternatively activated (or M2-polarized) macrophages. However, the molecular signals in tumours that induce recruitment and differentiation of M2-like macrophages are not fully defined. In this issue of The Journal of Pathology, Linde et al investigate the role of the tumour-derived cytokines, VEGF and IL-4, in the recruitment and polarization of macrophages in a mouse model of skin cancer. The authors report that while VEGF-A recruits monocytes from the peripheral circulation, IL-4 induces their differentiation into tumour-promoting, M2-like macrophages. IL-4 signalling blockade was sufficient to reprogram TAMs away from the M2-like phenotype and inhibited tumour angiogenesis and growth. This study attests to the potential of reprogramming TAMs to abate their pro-angiogenic and pro-tumoural functions in tumours. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Five classic articles in somatic cell reprogramming.

PubMed

Park, In-Hyun

2010-09-01

Research on somatic cell reprogramming has progressed significantly over the past few decades, from nuclear transfer into frogs' eggs in 1952 to the derivation of human-induced pluripotent stem (iPS) cells in the present day. In this article, I review five landmark papers that have laid the foundation for current efforts to apply somatic cell reprogramming in the clinic.

Metabolic changes associated with the long winter fast dominate the liver proteome in 13-lined ground squirrels

PubMed Central

Hindle, Allyson G.; Grabek, Katharine R.; Epperson, L. Elaine; Karimpour-Fard, Anis

2014-01-01

Small-bodied hibernators partition the year between active homeothermy and hibernating heterothermy accompanied by fasting. To define molecular events underlying hibernation that are both dependent and independent of fasting, we analyzed the liver proteome among two active and four hibernation states in 13-lined ground squirrels. We also examined fall animals transitioning between fed homeothermy and fasting heterothermy. Significantly enriched pathways differing between activity and hibernation were biased toward metabolic enzymes, concordant with the fuel shifts accompanying fasting physiology. Although metabolic reprogramming to support fasting dominated these data, arousing (rewarming) animals had the most distinct proteome among the hibernation states. Instead of a dominant metabolic enzyme signature, torpor-arousal cycles featured differences in plasma proteins and intracellular membrane traffic and its regulation. Phosphorylated NSFL1C, a membrane regulator, exhibited this torpor-arousal cycle pattern; its role in autophagosome formation may promote utilization of local substrates upon metabolic reactivation in arousal. Fall animals transitioning to hibernation lagged in their proteomic adjustment, indicating that the liver is more responsive than preparatory to the metabolic reprogramming of hibernation. Specifically, torpor use had little impact on the fall liver proteome, consistent with a dominant role of nutritional status. In contrast to our prediction of reprogramming the transition between activity and hibernation by gene expression and then within-hibernation transitions by posttranslational modification (PTM), we found extremely limited evidence of reversible PTMs within torpor-arousal cycles. Rather, acetylation contributed to seasonal differences, being highest in winter (specifically in torpor), consistent with fasting physiology and decreased abundance of the mitochondrial deacetylase, SIRT3. PMID:24642758

The therapeutic potential of cell identity reprogramming for the treatment of aging-related neurodegenerative disorders

PubMed Central

Smith, Derek K.; He, Miao; Zhang, Chun-Li; Zheng, Jialin C.

2018-01-01

Neural cell identity reprogramming strategies aim to treat age-related neurodegenerative disorders with newly induced neurons that regenerate neural architecture and functional circuits in vivo. The isolation and neural differentiation of pluripotent embryonic stem cells provided the first in vitro models of human neurodegenerative disease. Investigation into the molecular mechanisms underlying stem cell pluripotency revealed that somatic cells could be reprogrammed to induced pluripotent stem cells (iPSCs) and these cells could be used to model Alzheimer disease, amyotrophic lateral sclerosis, Huntington disease, and Parkinson disease. Additional neural precursor and direct transdifferentiation strategies further enabled the induction of diverse neural linages and neuron subtypes both in vitro and in vivo. In this review, we highlight neural induction strategies that utilize stem cells, iPSCs, and lineage reprogramming to model or treat age-related neurodegenerative diseases, as well as, the clinical challenges related to neural transplantation and in vivo reprogramming strategies. PMID:26844759

Cardiac Biomarkers: a Focus on Cardiac Regeneration

PubMed Central

Forough, Reza; Scarcello, Catherine; Perkins, Matthew

2011-01-01

Historically, biomarkers have been used in two major ways to maintain and improve better health status: first, for diagnostic purposes, and second, as specific targets to treat various diseases. A new era in treatment and even cure for the some diseases using reprograming of somatic cells is about to be born. In this approach, scientists are successfully taking human skin cells (previously considered terminally-differentiated cells) and re-programming them into functional cardiac myocytes and other cell types in vitro. A cell reprograming approach for treatment of cardiovascular diseases will revolutionize the field of medicine and significantly expand the human lifetime. Availability of a comprehensive catalogue for cardiac biomarkers is necessary for developing cell reprograming modalities to treat cardiac diseases, as well as for determining the progress of reprogrammed cells as they become cardiac cells. In this review, we present a comprehensive survey of the cardiac biomarkers currently known. PMID:23074366

Advances in generation of functional β-cells from induced pluripotent stem cells as a cure for diabetes mellitus.

PubMed

Kalra, Kunal; Chandrabose, Srijaya Thekkeparambil; Ramasamy, Thamil Selvee; Kasim, Noor Hayaty Binti Abu

2018-06-04

Diabetes mellitus is one of the leading cause for death worldwide. Loss and functional failure of pancreatic β-cells, the parenchyma cells in the islets of Langerhans onsets and progresses diabetes mellitus. The increasing incidence of this metabolic disorder necessitates efficient strategies to produce functional β-cells for treating diabetes mellitus. Human induced pluripotent stem cells (hiPSC), holds potential for treating diabetes owning to their self-renewal capacity and ability to differentiate into β-cells. iPSC technology also provides unlimited starting material to generate differentiated cells for regenerative applications. Progress has also been made in establishing in-vitro culture protocols to yield definitive endoderm, pancreatic endoderm progenitor cells and β-cells via different reprogramming strategies and growth factor supplementation. However, these generated β-cells are still immature, lack functional characteristics and exhibit lower capability in reversing the diseases conditions. Current methods employed to generate mature and functional β-cells include; use of small and large molecules to enhance the reprogramming and differentiation efficiency, 3D culture systems to improve the functional properties and heterogeneity of differentiated cells. This review details recent advancements in the generation of mature β-cells by reprogramming stem cells into iPSCs that is further programmed to β-cells. It also provides deeper insight of current reprogramming protocols and their efficacy, focusing on the underlying mechanism of chemical based approach to generate iPSCs. Furthermore, we have highlighted the recent differentiation strategies both in-vitro and in-vivo to date and the future prospects in generation of mature β-cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Effect of the time interval between fusion and activation on epigenetic reprogramming and development of bovine somatic cell nuclear transfer embryos.

PubMed

Liu, Jun; Wang, Yongsheng; Su, Jianmin; Wang, Lijun; Li, Ruizhe; Li, Qian; Wu, Yongyan; Hua, Song; Quan, Fusheng; Guo, Zekun; Zhang, Yong

2013-04-01

Previous studies have shown that the time interval between fusion and activation (FA interval) play an important role in nuclear remodeling and in vitro development of somatic cell nuclear transfer (SCNT) embryos. However, the effects of FA interval on the epigenetic reprogramming and in vivo developmental competence of SCNT embryos remain unknown. In the present study, the effects of different FA intervals (0 h, 2 h, and 4 h) on the epigenetic reprogramming and developmental competence of bovine SCNT embryos were assessed. The results demonstrated that H3 lysine 9 (H3K9ac) levels decreased rapidly after fusion in all three groups. H3K9ac was practically undetectable 2 h after fusion in the 2-h and 4-h FA interval groups. However, H3K9ac was still evidently detectable in the 0-h FA interval group. The H3K9ac levels increased 10 h after fusion in all three groups, but were higher in the 2-h and 4-h FA interval groups than that in the 0-h FA interval group. The methylation levels of the satellite I region in day-7 blastocysts derived from the 2-h or 4-h FA interval groups was similar to that of in vitro fertilization blastocysts and is significantly lower than that of the 0-h FA interval group. SCNT embryos derived from 2-h FA interval group showed higher developmental competence than those from the 0-h and 4-h FA interval groups in terms of cleavage rate, blastocyst formation rate, apoptosis index, and pregnancy and calving rates. Hence, the FA interval is an important factor influencing the epigenetic reprogramming and developmental competence of bovine SCNT embryos.

Heme oxygenase-1 affects generation and spontaneous cardiac differentiation of induced pluripotent stem cells.

PubMed

Stepniewski, Jacek; Pacholczak, Tomasz; Skrzypczyk, Aniela; Ciesla, Maciej; Szade, Agata; Szade, Krzysztof; Bidanel, Romain; Langrzyk, Agnieszka; Grochowski, Radoslaw; Vandermeeren, Felix; Kachamakova-Trojanowska, Neli; Jez, Mateusz; Drabik, Grazyna; Nakanishi, Mahito; Jozkowicz, Alicja; Dulak, Jozef

2018-02-01

Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO-1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1 -/- fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1 +/+ cells. Reversely, pharmacological enhancement of HO-1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53-regulated miR-34a and 14-3-3σ was observed in HO-1-deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO-1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1 +/+ and Hmox1 -/- iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO-1-deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas-9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2-lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2 +/+ and Nfe2l2 -/- iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO-1 expression can further optimize generation and cardiac differentiation of iPSCs. © 2018 IUBMB Life, 70(2):129-142, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

Current practice patterns and knowledge among gynecologic surgeons of InterStim® programming after implantation.

PubMed

Hobson, Deslyn T G; Gaskins, Jeremy T; Frazier, LaTisha; Francis, Sean L; Kinman, Casey L; Meriwether, Kate V

2017-10-03

The objective of this study was to describe surgeons' current practices in InterStim® programming after initial implantation and their knowledge of programming parameters. We hypothesized that surgeons performing their own reprogramming would have increased knowledge. We administered a written survey to attendees at the Society of Gynecologic Surgeons Scientific Meeting and analyzed those on which surgeons indicated they offer InterStim® care. The survey queried surgeon characteristics, experience with InterStim® implantation and programming, and clinical opinions regarding reprogramming and tested six knowledge-based questions about programming parameters. Correct response to all six questions was the primary outcome. One hundred and thirty-five of 407 (33%) attendees returned the survey, of which 99 met inclusion criteria. Most respondents (88 of 99; 89%) were between 36 and 60 years, 27 (73%) were women, 76 (77%) practiced in a university setting, and 76 (77%) were trained in Female Pelvic Medicine and Reconstructive Surgery (FPMRS). Surgeons who had InterStim® programming training were more likely to perform their own programming [15/46 (32%) vs 6/47 (13%), p = 0.03]. Most answered all knowledge-based questions correctly (62/90, 69%); no surgeon characteristics were significantly associated with this outcome. Most surgeons cited patient comfort (71/80, 89%) and symptom relief (64/80, 80%) as important factors when reprogramming, but no prevalent themes emerged on how and why surgeons change certain programming parameters. Surgeons who had formal InterStim® programming training are more likely to perform programming themselves. No surgeon characteristic was associated with improved programming knowledge. We found that surgeons prioritize patient comfort and symptoms when deciding to reprogram.

Marine Corps Budgetary Reprogramming Effectiveness

DTIC Science & Technology

2015-03-01

infrastructure (Appropriations Act of Congress, 2008). The environmental restoration is a transfer account controlled by the DOD. Usually in the case of...at an average just over 11 percent and the Marine Corps encircle the backend of the DOD portion of reprogramming with the Marine Corps reprogramming...blue force tracker (BFT), radio systems, high mobility multipurpose wheeled vehicle (HMMWV), medium tactical vehicle replacement (MTVR), and

Xenopatients 2.0: reprogramming the epigenetic landscapes of patient-derived cancer genomes.

PubMed

Menendez, Javier A; Alarcón, Tomás; Corominas-Faja, Bruna; Cuyàs, Elisabet; López-Bonet, Eugeni; Martin, Angel G; Vellon, Luciano

2014-01-01

In the science-fiction thriller film Minority Report, a specialized police department called "PreCrime" apprehends criminals identified in advance based on foreknowledge provided by 3 genetically altered humans called "PreCogs". We propose that Yamanaka stem cell technology can be similarly used to (epi)genetically reprogram tumor cells obtained directly from cancer patients and create self-evolving personalized translational platforms to foresee the evolutionary trajectory of individual tumors. This strategy yields a large stem cell population and captures the cancer genome of an affected individual, i.e., the PreCog-induced pluripotent stem (iPS) cancer cells, which are immediately available for experimental manipulation, including pharmacological screening for personalized "stemotoxic" cancer drugs. The PreCog-iPS cancer cells will re-differentiate upon orthotopic injection into the corresponding target tissues of immunodeficient mice (i.e., the PreCrime-iPS mouse avatars), and this in vivo model will run through specific cancer stages to directly explore their biological properties for drug screening, diagnosis, and personalized treatment in individual patients. The PreCog/PreCrime-iPS approach can perform sets of comparisons to directly observe changes in the cancer-iPS cell line vs. a normal iPS cell line derived from the same human genetic background. Genome editing of PreCog-iPS cells could create translational platforms to directly investigate the link between genomic expression changes and cellular malignization that is largely free from genetic and epigenetic noise and provide proof-of-principle evidence for cutting-edge "chromosome therapies" aimed against cancer aneuploidy. We might infer the epigenetic marks that correct the tumorigenic nature of the reprogrammed cancer cell population and normalize the malignant phenotype in vivo. Genetically engineered models of conditionally reprogrammable mice to transiently express the Yamanaka stemness factors following the activation of phenotypic copies of specific cancer diseases might crucially evaluate a "reprogramming cure" for cancer. A new era of xenopatients 2.0 generated via nuclear reprogramming of the epigenetic landscapes of patient-derived cancer genomes might revolutionize the current personalized translational platforms in cancer research.

Totipotency, Pluripotency and Nuclear Reprogramming

NASA Astrophysics Data System (ADS)

Mitalipov, Shoukhrat; Wolf, Don

Mammalian development commences with the totipotent zygote which is capable of developing into all the specialized cells that make up the adult animal. As development unfolds, cells of the early embryo proliferate and differentiate into the first two lineages, the pluripotent inner cell mass and the trophectoderm. Pluripotent cells can be isolated, adapted and propagated indefinitely in vitro in an undifferentiated state as embryonic stem cells (ESCs). ESCs retain their ability to differentiate into cells representing the three major germ layers: endoderm, mesoderm or ectoderm or any of the 200+ cell types present in the adult body. Since many human diseases result from defects in a single cell type, pluripotent human ESCs represent an unlimited source of any cell or tissue type for replacement therapy thus providing a possible cure for many devastating conditions. Pluripotent cells resembling ESCs can also be derived experimentally by the nuclear reprogramming of somatic cells. Reprogrammed somatic cells may have an even more important role in cell replacement therapies since the patient's own somatic cells can be used for reprogramming thereby eliminating immune based rejection of transplanted cells. In this review, we summarize two major approaches to reprogramming: (1) somatic cell nuclear transfer and (2) direct reprogramming using genetic manipulations.

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids.

PubMed

Meraviglia, Viviana; Zanon, Alessandra; Lavdas, Alexandros A; Schwienbacher, Christine; Silipigni, Rosamaria; Di Segni, Marina; Chen, Huei-Sheng Vincent; Pramstaller, Peter P; Hicks, Andrew A; Rossini, Alessandra

2015-06-05

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation. In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Barriers for Deriving Transgene-Free Pig iPS Cells with Episomal Vectors.

PubMed

Du, Xuguang; Feng, Tao; Yu, Dawei; Wu, Yuanyuan; Zou, Huiying; Ma, Shuangyu; Feng, Chong; Huang, Yongye; Ouyang, Hongsheng; Hu, Xiaoxiang; Pan, Dengke; Li, Ning; Wu, Sen

2015-11-01

To date no authentic embryonic stem cell (ESC) line or germline-competent-induced pluripotent stem cell (iPSC) line has been established for large animals. Despite this fact, there is an impression in the field that large animal ESCs or iPSCs are as good as mouse counterparts. Clarification of this issue is important for a healthy advancement of the stem cell field. Elucidation of the causes of this failure in obtaining high quality iPSCs/ESCs may offer essential clues for eventual establishment of authentic ESCs for large animals including humans. To this end, we first generated porcine iPSCs using nonintegrating replicating episomal plasmids. Although these porcine iPSCs met most pluripotency criteria, they could neither generate cloned piglets through nuclear transfer, nor contribute to later stage chimeras through morula injections or aggregations. We found that the reprogramming genes in iPSCs could not be removed even under negative selection, indicating they are required to maintain self-renewal. The persistent expression of these genes in porcine iPSCs in turn caused differentiation defects in vivo. Therefore, incomplete reprogramming manifested by a reliance on sustained expression of exogenous-reprogramming factors appears to be the main reason for the inability of porcine iPSCs to form iPSC-derived piglets. © 2015 AlphaMed Press.

Metabolic Reprogramming in Thyroid Carcinoma

PubMed Central

Coelho, Raquel Guimaraes; Fortunato, Rodrigo S.; Carvalho, Denise P.

2018-01-01

Among all the adaptations of cancer cells, their ability to change metabolism from the oxidative to the glycolytic phenotype is a hallmark called the Warburg effect. Studies on tumor metabolism show that improved glycolysis and glutaminolysis are necessary to maintain rapid cell proliferation, tumor progression, and resistance to cell death. Thyroid neoplasms are common endocrine tumors that are more prevalent in women and elderly individuals. The incidence of thyroid cancer has increased in the Past decades, and recent findings describing the metabolic profiles of thyroid tumors have emerged. Currently, several drugs are in development or clinical trials that target the altered metabolic pathways of tumors are undergoing. We present a review of the metabolic reprogramming in cancerous thyroid tissues with a focus on the factors that promote enhanced glycolysis and the possible identification of promising metabolic targets in thyroid cancer. PMID:29629339

Regeneration of fat cells from myofibroblasts during wound healing.

PubMed

Plikus, Maksim V; Guerrero-Juarez, Christian F; Ito, Mayumi; Li, Yun Rose; Dedhia, Priya H; Zheng, Ying; Shao, Mengle; Gay, Denise L; Ramos, Raul; Hsi, Tsai-Ching; Oh, Ji Won; Wang, Xiaojie; Ramirez, Amanda; Konopelski, Sara E; Elzein, Arijh; Wang, Anne; Supapannachart, Rarinthip June; Lee, Hye-Lim; Lim, Chae Ho; Nace, Arben; Guo, Amy; Treffeisen, Elsa; Andl, Thomas; Ramirez, Ricardo N; Murad, Rabi; Offermanns, Stefan; Metzger, Daniel; Chambon, Pierre; Widgerow, Alan D; Tuan, Tai-Lan; Mortazavi, Ali; Gupta, Rana K; Hamilton, Bruce A; Millar, Sarah E; Seale, Patrick; Pear, Warren S; Lazar, Mitchell A; Cotsarelis, George

2017-02-17

Although regeneration through the reprogramming of one cell lineage to another occurs in fish and amphibians, it has not been observed in mammals. We discovered in the mouse that during wound healing, adipocytes regenerate from myofibroblasts, a cell type thought to be differentiated and nonadipogenic. Myofibroblast reprogramming required neogenic hair follicles, which triggered bone morphogenetic protein (BMP) signaling and then activation of adipocyte transcription factors expressed during development. Overexpression of the BMP antagonist Noggin in hair follicles or deletion of the BMP receptor in myofibroblasts prevented adipocyte formation. Adipocytes formed from human keloid fibroblasts either when treated with BMP or when placed with human hair follicles in vitro . Thus, we identify the myofibroblast as a plastic cell type that may be manipulated to treat scars in humans. Copyright © 2017, American Association for the Advancement of Science.

SOX2 O-GlcNAcylation alters its protein-protein interactions and genomic occupancy to modulate gene expression in pluripotent cells

PubMed Central

Myers, Samuel A; Peddada, Sailaja; Chatterjee, Nilanjana; Friedrich, Tara; Tomoda, Kiichrio; Krings, Gregor; Thomas, Sean; Maynard, Jason; Broeker, Michael; Thomson, Matthew; Pollard, Katherine; Yamanaka, Shinya; Burlingame, Alma L; Panning, Barbara

2016-01-01

The transcription factor SOX2 is central in establishing and maintaining pluripotency. The processes that modulate SOX2 activity to promote pluripotency are not well understood. Here, we show SOX2 is O-GlcNAc modified in its transactivation domain during reprogramming and in mouse embryonic stem cells (mESCs). Upon induction of differentiation SOX2 O-GlcNAcylation at serine 248 is decreased. Replacing wild type with an O-GlcNAc-deficient SOX2 (S248A) increases reprogramming efficiency. ESCs with O-GlcNAc-deficient SOX2 exhibit alterations in gene expression. This change correlates with altered protein-protein interactions and genomic occupancy of the O-GlcNAc-deficient SOX2 compared to wild type. In addition, SOX2 O-GlcNAcylation impairs the SOX2-PARP1 interaction, which has been shown to regulate ESC self-renewal. These findings show that SOX2 activity is modulated by O-GlcNAc, and provide a novel regulatory mechanism for this crucial pluripotency transcription factor. DOI: http://dx.doi.org/10.7554/eLife.10647.001 PMID:26949256

Allelic exchange of pheromones and their receptors reprograms sexual identity in Cryptococcus neoformans.

PubMed

Stanton, Brynne C; Giles, Steven S; Staudt, Mark W; Kruzel, Emilia K; Hull, Christina M

2010-02-26

Cell type specification is a fundamental process that all cells must carry out to ensure appropriate behaviors in response to environmental stimuli. In fungi, cell identity is critical for defining "sexes" known as mating types and is controlled by components of mating type (MAT) loci. MAT-encoded genes function to define sexes via two distinct paradigms: 1) by controlling transcription of components common to both sexes, or 2) by expressing specially encoded factors (pheromones and their receptors) that differ between mating types. The human fungal pathogen Cryptococcus neoformans has two mating types (a and alpha) that are specified by an extremely unusual MAT locus. The complex architecture of this locus makes it impossible to predict which paradigm governs mating type. To identify the mechanism by which the C. neoformans sexes are determined, we created strains in which the pheromone and pheromone receptor from one mating type (a) replaced the pheromone and pheromone receptor of the other (alpha). We discovered that these "alpha(a)" cells effectively adopt a new mating type (that of a cells); they sense and respond to alpha factor, they elicit a mating response from alpha cells, and they fuse with alpha cells. In addition, alpha(a) cells lose the alpha cell type-specific response to pheromone and do not form germ tubes, instead remaining spherical like a cells. Finally, we discovered that exogenous expression of the diploid/dikaryon-specific transcription factor Sxi2a could then promote complete sexual development in crosses between alpha and alpha(a) strains. These data reveal that cell identity in C. neoformans is controlled fully by three kinds of MAT-encoded proteins: pheromones, pheromone receptors, and homeodomain proteins. Our findings establish the mechanisms for maintenance of distinct cell types and subsequent developmental behaviors in this unusual human fungal pathogen.

Mitochondrial-Associated Cell Death Mechanisms Are Reset to an Embryonic-Like State in Aged Donor-Derived iPS Cells Harboring Chromosomal Aberrations

PubMed Central

Prigione, Alessandro; Hossini, Amir M.; Lichtner, Björn; Serin, Akdes; Fauler, Beatrix; Megges, Matthias; Lurz, Rudi; Lehrach, Hans; Zouboulis, Christos C.

2011-01-01

Somatic cells reprogrammed into induced pluripotent stem cells (iPSCs) acquire features of human embryonic stem cells (hESCs) and thus represent a promising source for cellular therapy of debilitating diseases, such as age-related disorders. However, reprogrammed cell lines have been found to harbor various genomic alterations. In addition, we recently discovered that the mitochondrial DNA of human fibroblasts also undergoes random mutational events upon reprogramming. Aged somatic cells might possess high susceptibility to nuclear and mitochondrial genome instability. Hence, concerns over the oncogenic potential of reprogrammed cells due to the lack of genomic integrity may hinder the applicability of iPSC-based therapies for age-associated conditions. Here, we investigated whether aged reprogrammed cells harboring chromosomal abnormalities show resistance to apoptotic cell death or mitochondrial-associated oxidative stress, both hallmarks of cancer transformation. Four iPSC lines were generated from dermal fibroblasts derived from an 84-year-old woman, representing the oldest human donor so far reprogrammed to pluripotency. Despite the presence of karyotype aberrations, all aged-iPSCs were able to differentiate into neurons, re-establish telomerase activity, and reconfigure mitochondrial ultra-structure and functionality to a hESC-like state. Importantly, aged-iPSCs exhibited high sensitivity to drug-induced apoptosis and low levels of oxidative stress and DNA damage, in a similar fashion as iPSCs derived from young donors and hESCs. Thus, the occurrence of chromosomal abnormalities within aged reprogrammed cells might not be sufficient to over-ride the cellular surveillance machinery and induce malignant transformation through the alteration of mitochondrial-associated cell death. Taken together, we unveiled that cellular reprogramming is capable of reversing aging-related features in somatic cells from a very old subject, despite the presence of genomic alterations. Nevertheless, we believe it will be essential to develop reprogramming protocols capable of safeguarding the integrity of the genome of aged somatic cells, before employing iPSC-based therapy for age-associated disorders. PMID:22110631

Stem Cell Differentiation Stage Factors and Their Role in Triggering Symmetry Breaking Processes during Cancer Development: A Quantum Field Theory Model for Reprogramming Cancer Cells to Healthy Phenotypes.

PubMed

Biava, Pier Mario; Burigana, Fabio; Germano, Roberto; Kurian, Philip; Verzegnassi, Claudio; Vitiello, Giuseppe

2017-09-20

A long history of research has pursued the use of embryonic factors isolated during cell differentiation processes for the express purpose of transforming cancer cells back to healthy phenotypes. Recent results have clarified that the substances present at different stages of cell differentiation-which we call stem cell differentiation stage factors (SCDSFs)-are proteins with low molecular weight and nucleic acids that regulate genomic expression. The present review summarizes how these substances, taken at different stages of cellular maturation, are able to retard proliferation of many human tumor cell lines and thereby reprogram cancer cells to healthy phenotypes. The model presented here is a quantum field theory (QFT) model in which SCDSFs are able to trigger symmetry breaking processes during cancer development. These symmetry breaking processes, which lie at the root of many phenomena in elementary particle physics and condensed matter physics, govern the phase transitions of totipotent cells to higher degrees of diversity and order, resulting in cell differentiation. In cancers, which share many genomic and metabolic similarities with embryonic stem cells, stimulated re-differentiation often signifies the phenotypic reversion back to health and non-proliferation. In addition to acting on key components of the cellular cycle, SCDSFs are able to reprogram cancer cells by delicately influencing the cancer microenvironment, modulating the electrochemistry and thus the collective electrodynamic behaviors between dipole networks in biomacromolecules and the interstitial water field. Coherent effects in biological water, which are derived from a dissipative QFT framework, may offer new diagnostic and therapeutic targets at a systemic level, before tumor instantiation occurs in specific tissues or organs. Thus, by including the environment as an essential component of our model, we may push the prevailing paradigm of mutation-driven oncogenesis toward a closer description of reality. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

CD24 tracks divergent pluripotent states in mouse and human cells

PubMed Central

Shakiba, Nika; White, Carl A.; Lipsitz, Yonatan Y.; Yachie-Kinoshita, Ayako; Tonge, Peter D; Hussein, Samer M. I.; Puri, Mira C.; Elbaz, Judith; Morrissey-Scoot, James; Li, Mira; Munoz, Javier; Benevento, Marco; Rogers, Ian M.; Hanna, Jacob H.; Heck, Albert J. R.; Wollscheid, Bernd; Nagy, Andras; Zandstra, Peter W

2015-01-01

Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths. Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells. Here we use cell surface proteomics to interrogate mouse cell reprogramming dynamics and discover CD24 as a marker that tracks the emergence of reprogramming-responsive cells, while enabling the analysis and enrichment of transgene-dependent (F-class) and -independent (traditional) induced pluripotent stem cells (iPSCs) at later stages. Furthermore, CD24 can be used to delineate epiblast stem cells (EpiSCs) from embryonic stem cells (ESCs) in mouse pluripotent culture. Importantly, regulated CD24 expression is conserved in human pluripotent stem cells (PSCs), tracking the conversion of human ESCs to more naive-like PSC states. Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture. PMID:26076835

AMP-Activated Protein Kinase as a Reprogramming Strategy for Hypertension and Kidney Disease of Developmental Origin.

PubMed

Tain, You-Lin; Hsu, Chien-Ning

2018-06-12

Suboptimal early-life conditions affect the developing kidney, resulting in long-term programming effects, namely renal programming. Adverse renal programming increases the risk for developing hypertension and kidney disease in adulthood. Conversely, reprogramming is a strategy aimed at reversing the programming processes in early life. AMP-activated protein kinase (AMPK) plays a key role in normal renal physiology and the pathogenesis of hypertension and kidney disease. This review discusses the regulation of AMPK in the kidney and provides hypothetical mechanisms linking AMPK to renal programming. This will be followed by studies targeting AMPK activators like metformin, resveratrol, thiazolidinediones, and polyphenols as reprogramming strategies to prevent hypertension and kidney disease. Further studies that broaden our understanding of AMPK isoform- and tissue-specific effects on renal programming are needed to ultimately develop reprogramming strategies. Despite the fact that animal models have provided interesting results with regard to reprogramming strategies targeting AMPK signaling to protect against hypertension and kidney disease with developmental origins, these results await further clinical translation.

X Chromosome of female cells shows dynamic changes in status during human somatic cell reprogramming.

PubMed

Kim, Kun-Yong; Hysolli, Eriona; Tanaka, Yoshiaki; Wang, Brandon; Jung, Yong-Wook; Pan, Xinghua; Weissman, Sherman Morton; Park, In-Hyun

2014-06-03

Induced pluripotent stem cells (iPSCs) acquire embryonic stem cell (ESC)-like epigenetic states, including the X chromosome. Previous studies reported that human iPSCs retain the inactive X chromosome of parental cells, or acquire two active X chromosomes through reprogramming. Most studies investigated the X chromosome states in established human iPSC clones after completion of reprogramming. Thus, it is still not fully understood when and how the X chromosome reactivation occurs during reprogramming. Here, we report a dynamic change in the X chromosome state throughout reprogramming, with an initial robust reactivation of the inactive X chromosome followed by an inactivation upon generation of nascent iPSC clones. iPSCs with two active X chromosomes or an eroded X chromosome arise in passaging iPSCs. These data provide important insights into the plasticity of the X chromosome of human female iPSCs and will be crucial for the future application of such cells in cell therapy and X-linked disease modeling.

NF-κB activation impairs somatic cell reprogramming in ageing.

PubMed

Soria-Valles, Clara; Osorio, Fernando G; Gutiérrez-Fernández, Ana; De Los Angeles, Alejandro; Bueno, Clara; Menéndez, Pablo; Martín-Subero, José I; Daley, George Q; Freije, José M P; López-Otín, Carlos

2015-08-01

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.

Local epigenetic reprograming induced by G-quadruplex ligands

PubMed Central

Recolin, Bénédicte; Campbell, Beth C.; Maiter, Ahmed; Sale, Julian E.; Balasubramanian, Shankar

2017-01-01

DNA and histone modifications regulate transcriptional activity and thus represent valuable targets to reprogram the activity of genes. Current epigenetic therapies target the machinery that regulates these modifications, leading to global transcriptional reprogramming with the potential for extensive undesired effects. Epigenetic information can also be modified as a consequence of disrupting processive DNA replication. Here we demonstrate that impeding replication by small molecule-mediated stabilisation of G-quadruplex nucleic acid secondary structures triggers local epigenetic plasticity. We report the use of the BU-1 locus of chicken DT40 cells to screen for small molecules able to induce G-quadruplex-dependent transcriptional reprogramming. Further characterisation of the top hit compound revealed its ability to induce a dose-dependent inactivation of BU-1 expression in two steps, first loss of H3K4me3 and subsequently DNA cytosine methylation, changes that were heritable across cell divisions even after the compound was removed. Targeting DNA secondary structures thus represents a potentially new approach for locus-specific epigenetic reprogramming. PMID:29064488

Reprogramming cellular identity for regenerative medicine

PubMed Central

Cherry, Anne B.C.; Daley, George Q.

2012-01-01

The choreographed development of over 200 distinct differentiated cell types from a single zygote is a complex and poorly understood process. Whereas development leads unidirectionally towards more restricted cell fates, recent work in cellular reprogramming has proven that striking conversions of one cellular identity into another can be engineered, promising countless applications in biomedical research and paving the way for modeling disease with patient-derived stem cells. To date, there has been little discussion of which disease models are likely to be most informative. We here review evidence demonstrating that because environmental influences and epigenetic signatures are largely erased during reprogramming, patient-specific models of diseases with strong genetic bases and high penetrance are likely to prove most informative in the near term. However, manipulating in vitro culture conditions may ultimately enable cell-based models to recapitulate gene-environment interactions. Here, we discuss the implications of the new reprogramming paradigm in biomedicine and outline how reprogramming of cell identities is enhancing our understanding of cell differentiation and prospects for cellular therapies and in vivo regeneration. PMID:22424223

The therapeutic potential of cell identity reprogramming for the treatment of aging-related neurodegenerative disorders.

PubMed

Smith, Derek K; He, Miao; Zhang, Chun-Li; Zheng, Jialin C

2017-10-01

Neural cell identity reprogramming strategies aim to treat age-related neurodegenerative disorders with newly induced neurons that regenerate neural architecture and functional circuits in vivo. The isolation and neural differentiation of pluripotent embryonic stem cells provided the first in vitro models of human neurodegenerative disease. Investigation into the molecular mechanisms underlying stem cell pluripotency revealed that somatic cells could be reprogrammed to induced pluripotent stem cells (iPSCs) and these cells could be used to model Alzheimer disease, amyotrophic lateral sclerosis, Huntington disease, and Parkinson disease. Additional neural precursor and direct transdifferentiation strategies further enabled the induction of diverse neural linages and neuron subtypes both in vitro and in vivo. In this review, we highlight neural induction strategies that utilize stem cells, iPSCs, and lineage reprogramming to model or treat age-related neurodegenerative diseases, as well as, the clinical challenges related to neural transplantation and in vivo reprogramming strategies. Copyright © 2016. Published by Elsevier Ltd.

Stress-triggered atavistic reprogramming (STAR) addiction: driving force behind head and neck cancer?

PubMed Central

Masuda, Muneyuki; Wakasaki, Takahiro; Toh, Satoshi

2016-01-01

Recent results of the Cancer Genome Atlas on head and neck squamous cell carcinoma (HNSCC) revealed that HNSCC lacked predominant gain-of-function mutations in oncogenes, whereas an essential role for epigenetics in oncogenesis has become apparent. In parallel, it has gained general acceptance that cancer is considered as complex adaptive system, which evolves responding environmental selective pressures. This somatic evolution appears to proceed concurrently with the acquisition of an atavistic pluripotent state (i.e., “stemness”), which is inducible by intrinsic epigenetic reprogramming program as demonstrated by induced pluripotent stem (iPS) cells. This Nobel prize-winning discovery has markedly accelerated and expanded cancer stem cell research from the point of epigenetic reprogramming. Taken together, we hypothesize that stress-triggered atavistic reprogramming (STAR) may be the major driving force of HNSCC evolution. In this perspective, we discuss the possible mechanisms of STAR in HNSCC, focusing on recent topics of epigenetic reprogramming in developmental and cancer cell biology. PMID:27429838

Identification and Successful Negotiation of a Metabolic Checkpoint in Direct Neuronal Reprogramming.

PubMed

Gascón, Sergio; Murenu, Elisa; Masserdotti, Giacomo; Ortega, Felipe; Russo, Gianluca L; Petrik, David; Deshpande, Aditi; Heinrich, Christophe; Karow, Marisa; Robertson, Stephen P; Schroeder, Timm; Beckers, Johannes; Irmler, Martin; Berndt, Carsten; Angeli, José P Friedmann; Conrad, Marcus; Berninger, Benedikt; Götz, Magdalena

2016-03-03

Despite the widespread interest in direct neuronal reprogramming, the mechanisms underpinning fate conversion remain largely unknown. Our study revealed a critical time point after which cells either successfully convert into neurons or succumb to cell death. Co-transduction with Bcl-2 greatly improved negotiation of this critical point by faster neuronal differentiation. Surprisingly, mutants with reduced or no affinity for Bax demonstrated that Bcl-2 exerts this effect by an apoptosis-independent mechanism. Consistent with a caspase-independent role, ferroptosis inhibitors potently increased neuronal reprogramming by inhibiting lipid peroxidation occurring during fate conversion. Genome-wide expression analysis confirmed that treatments promoting neuronal reprogramming elicit an anti-oxidative stress response. Importantly, co-expression of Bcl-2 and anti-oxidative treatments leads to an unprecedented improvement in glial-to-neuron conversion after traumatic brain injury in vivo, underscoring the relevance of these pathways in cellular reprograming irrespective of cell type in vitro and in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Local epigenetic reprogramming induced by G-quadruplex ligands

NASA Astrophysics Data System (ADS)

Guilbaud, Guillaume; Murat, Pierre; Recolin, Bénédicte; Campbell, Beth C.; Maiter, Ahmed; Sale, Julian E.; Balasubramanian, Shankar

2017-11-01

DNA and histone modifications regulate transcriptional activity and thus represent valuable targets to reprogram the activity of genes. Current epigenetic therapies target the machinery that regulates these modifications, leading to global transcriptional reprogramming with the potential for extensive undesired effects. Epigenetic information can also be modified as a consequence of disrupting processive DNA replication. Here, we demonstrate that impeding replication by small-molecule-mediated stabilization of G-quadruplex nucleic acid secondary structures triggers local epigenetic plasticity. We report the use of the BU-1 locus of chicken DT40 cells to screen for small molecules able to induce G-quadruplex-dependent transcriptional reprogramming. Further characterization of the top hit compound revealed its ability to induce a dose-dependent inactivation of BU-1 expression in two steps: the loss of H3K4me3 and then subsequent DNA cytosine methylation, changes that were heritable across cell divisions even after the compound was removed. Targeting DNA secondary structures thus represents a potentially new approach for locus-specific epigenetic reprogramming.

Reprogramming of non-genomic estrogen signaling by the stemness factor SOX2 enhances the tumor-initiating capacity of breast cancer cells.

PubMed

Vazquez-Martin, Alejandro; Cufí, Sílvia; López-Bonet, Eugeni; Corominas-Faja, Bruna; Cuyàs, Elisabet; Vellon, Luciano; Iglesias, Juan Manuel; Leis, Olatz; Martín, Angel G; Menendez, Javier A

2013-11-15

The restoration of pluripotency circuits by the reactivation of endogenous stemness factors, such as SOX2, may provide a new paradigm in cancer development. The tumoral stem cell reprogramming hypothesis, i.e., the ability of stemness factors to redirect normal and differentiated tumor cells toward a less-differentiated and stem-like state, adds new layers of complexity to cancer biology, because the effects of such reprogramming may remain dormant until engaged later in response to (epi)genetic and/or (micro)environmental events. To test this hypothesis, we utilized an in vitro model of a SOX2-overexpressing cancer stem cell (CSC)-like cellular state that was recently developed in our laboratory by employing Yamanaka's nuclear reprogramming technology in the estrogen receptor α (ERα)-positive MCF-7 breast cancer cell line. Despite the acquisition of distinct molecular features that were compatible with a breast CSC-like cellular state, such as strong aldehyde dehydrogenase activity, as detected by ALDEFLUOR, and overexpression of the SSEA-4 and CD44 breast CSC markers, the tumor growth-initiating ability of SOX2-overexpressing CSC-like MCF-7 cells solely occurred in female nude mice supplemented with estradiol when compared with MCF-7 parental cells. Ser118 phosphorylation of estrogen receptor α (ERα), which is a pivotal integrator of the genomic and nongenomic E 2/ERα signaling pathways, drastically accumulated in nuclear speckles in the interphase nuclei of SOX2-driven CSC-like cell populations. Moreover, SOX2-positive CSC-like cells accumulated significantly higher numbers of actively dividing cells, and the highest levels of phospho-Ser118-ERα occurred when chromosomes lined up on a metaphase plate. The previously unrecognized link between E 2/ERα signaling and SOX2-driven stem cell circuitry may significantly impact our current understanding of breast cancer initiation and progression, i.e., SOX2 can promote non-genomic E 2 signaling that leads to nuclear phospho-Ser118-ERα, which ultimately exacerbates genomic ER signaling in response to E 2. Because E 2 stimulation has been recently shown to enhance breast tumor-initiating cell survival by downregulating miR-140, which targets SOX2, the establishment of a bidirectional cross-talk interaction between the stem cell self-renewal regulator, SOX2, and the local and systemic ability of E 2 to increase breast CSC activity may have profound implications for the development of new CSC-directed strategies for breast cancer prevention and therapy.

Temporally distinct transcriptional regulation of myocyte dedifferentiation and Myofiber growth during muscle regeneration.

PubMed

Louie, Ke'ale W; Saera-Vila, Alfonso; Kish, Phillip E; Colacino, Justin A; Kahana, Alon

2017-11-09

Tissue regeneration requires a series of steps, beginning with generation of the necessary cell mass, followed by cell migration into damaged area, and ending with differentiation and integration with surrounding tissues. Temporal regulation of these steps lies at the heart of the regenerative process, yet its basis is not well understood. The ability of zebrafish to dedifferentiate mature "post-mitotic" myocytes into proliferating myoblasts that in turn regenerate lost muscle tissue provides an opportunity to probe the molecular mechanisms of regeneration. Following subtotal excision of adult zebrafish lateral rectus muscle, dedifferentiating residual myocytes were collected at two time points prior to cell cycle reentry and compared to uninjured muscles using RNA-seq. Functional annotation (GAGE or K-means clustering followed by GO enrichment) revealed a coordinated response encompassing epigenetic regulation of transcription, RNA processing, and DNA replication and repair, along with protein degradation and translation that would rewire the cellular proteome and metabolome. Selected candidate genes were phenotypically validated in vivo by morpholino knockdown. Rapidly induced gene products, such as the Polycomb group factors Ezh2 and Suz12a, were necessary for both efficient dedifferentiation (i.e. cell reprogramming leading to cell cycle reentry) and complete anatomic regeneration. In contrast, the late activated gene fibronectin was important for efficient anatomic muscle regeneration but not for the early step of myocyte cell cycle reentry. Reprogramming of a "post-mitotic" myocyte into a dedifferentiated myoblast requires a complex coordinated effort that reshapes the cellular proteome and rewires metabolic pathways mediated by heritable yet nuanced epigenetic alterations and molecular switches, including transcription factors and non-coding RNAs. Our studies show that temporal regulation of gene expression is programmatically linked to distinct steps in the regeneration process, with immediate early expression driving dedifferentiation and reprogramming, and later expression facilitating anatomical regeneration.

In vivo cardiac cellular reprogramming efficacy is enhanced by angiogenic preconditioning of the infarcted myocardium with vascular endothelial growth factor.

PubMed

Mathison, Megumi; Gersch, Robert P; Nasser, Ahmed; Lilo, Sarit; Korman, Mallory; Fourman, Mitchell; Hackett, Neil; Shroyer, Kenneth; Yang, Jianchang; Ma, Yupo; Crystal, Ronald G; Rosengart, Todd K

2012-12-01

In situ cellular reprogramming offers the possibility of regenerating functional cardiomyocytes directly from scar fibroblasts, obviating the challenges of cell implantation. We hypothesized that pretreating scar with gene transfer of the angiogenic vascular endothelial growth factor (VEGF) would enhance the efficacy of this strategy. Gata4, Mef2c, and Tbx5 (GMT) administration via lentiviral transduction was demonstrated to transdifferentiate rat fibroblasts into (induced) cardiomyocytes in vitro by cardiomyocyte marker studies. Fisher 344 rats underwent coronary ligation and intramyocardial administration of an adenovirus encoding all 3 major isoforms of VEGF (AdVEGF-All6A(+)) or an AdNull control vector (n=12/group). Lentivirus encoding GMT or a GFP control was administered to each animal 3 weeks later, followed by histologic and echocardiographic analyses. GMT administration reduced the extent of fibrosis by half compared with GFP controls (12 ± 2% vs 24 ± 3%, P<0.01) and reduced the number of myofibroblasts detected in the infarct zone by 4-fold. GMT-treated animals also demonstrated greater density of cardiomyocyte-specific marker beta myosin heavy chain 7(+) cells compared with animals receiving GFP with or without VEGF (P<0.01). Ejection fraction was significantly improved after GMT vs GFP administration (12 ± 3% vs -7 ± 3%, P<0.01). Eight (73%) GFP animals but no GMT animals demonstrated decreased ejection fraction during this interval (P<0.01). Also, improvement in ejection fraction was 4-fold greater in GMT/VEGF vs GMT/null animals (17 ± 2% vs 4 ± 1%, P<0.05). VEGF administration to infarcted myocardium enhances the efficacy of GMT-mediated cellular reprogramming in improving myocardial function and reducing the extent of myocardial fibrosis compared with the use of GMT or VEGF alone.

Epigenetic reprogramming in mammalian species after SCNT-based cloning.

PubMed

Niemann, Heiner

2016-07-01

The birth of "Dolly," the first mammal cloned from an adult mammary epithelial cell, abolished the decades-old scientific dogma implying that a terminally differentiated cell cannot be reprogrammed into a pluripotent embryonic state. The most dramatic epigenetic reprogramming occurs in SCNT when the expression profile of a differentiated cell is abolished and a new embryo-specific expression profile, involving 10,000 to 12,000 genes, and thus, most genes of the entire genome is established, which drives embryonic and fetal development. The initial release from somatic cell epigenetic constraints is followed by establishment of post-zygotic expression patterns, X-chromosome inactivation, and adjustment of telomere length. Somatic cell nuclear transfer may be associated with a variety of pathologic changes of the fetal and placental phenotype in a proportion of cloned offspring, specifically in ruminants, that are thought to be caused by aberrant epigenetic reprogramming. Improvements in our understanding of this dramatic epigenetic reprogramming event will be instrumental in realizing the great potential of SCNT for basic research and for important agricultural and biomedical applications. Here, current knowledge on epigenetic reprogramming after use of SCNT in livestock is reviewed, with emphasis on gene-specific and global DNA methylation, imprinting, X-chromosome inactivation, and telomere length restoration in early development. Copyright © 2016 Elsevier Inc. All rights reserved.

Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange

PubMed Central

Kelton, William; Waindok, Ann Cathrin; Pesch, Theresa; Pogson, Mark; Ford, Kyle; Parola, Cristina; Reddy, Sai T.

2017-01-01

The development of programmable nucleases has enabled the application of new genome engineering strategies for cellular immunotherapy. While targeted nucleases have mostly been used to knock-out or knock-in genes in immune cells, the scarless exchange of entire immunogenomic alleles would be of great interest. In particular, reprogramming the polymorphic MHC locus could enable the creation of matched donors for allogeneic cellular transplantation. Here we show a proof-of-concept for reprogramming MHC-specificity by performing CRISPR-Cas9-assisted cassette exchange. Using murine antigen presenting cell lines (RAW264.7 macrophages), we demonstrate that the generation of Cas9-induced double-stranded breaks flanking the native MHC-I H2-Kd locus led to exchange of an orthogonal H2-Kb allele. MHC surface expression allowed for easy selection of reprogrammed cells by flow cytometry, thus obviating the need for additional selection markers. MHC-reprogrammed cells were fully functional as they could present H2-Kd-restricted peptide and activate cognate T cells. Finally, we investigated the role of various donor template formats on exchange efficiency, discovering that templates that underwent in situ linearization resulted in the highest MHC-reprogramming efficiency. These findings highlight a potential new approach for the correcting of MHC mismatches in cellular transplantation. PMID:28374766

Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase

PubMed Central

2015-01-01

The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity. PMID:24517311

Histone Deacetylase Inhibitors in Cell Pluripotency, Differentiation, and Reprogramming

PubMed Central

Kretsovali, Androniki; Hadjimichael, Christiana; Charmpilas, Nikolaos

2012-01-01

Histone deacetylase inhibitors (HDACi) are small molecules that have important and pleiotropic effects on cell homeostasis. Under distinct developmental conditions, they can promote either self-renewal or differentiation of embryonic stem cells. In addition, they can promote directed differentiation of embryonic and tissue-specific stem cells along the neuronal, cardiomyocytic, and hepatic lineages. They have been used to facilitate embryo development following somatic cell nuclear transfer and induced pluripotent stem cell derivation by ectopic expression of pluripotency factors. In the latter method, these molecules not only increase effectiveness, but can also render the induction independent of the oncogenes c-Myc and Klf4. Here we review the molecular pathways that are involved in the functions of HDAC inhibitors on stem cell differentiation and reprogramming of somatic cells into pluripotency. Deciphering the mechanisms of HDAC inhibitor actions is very important to enable their exploitation for efficient and simple tissue regeneration therapies. PMID:22550500

A chemical approach to myocardial protection and regeneration.

PubMed

Piccoli, Marco; Cirillo, Federica; Tettamanti, Guido; Anastasia, Luigi

2016-04-28

The possibility of generating induced pluripotent stem cells from mouse embryonic fibroblasts and human adult fibroblasts has introduced new perspectives for possible therapeutic strategies to repair damaged hearts. However, obtaining large numbers of adult stem cells is still an ongoing challenge, and the safety of genetic reprogramming with lenti- or retro-viruses has several drawbacks not easy to be addressed. Furthermore, the majority of adult stem cell-based clinical trials for heart regeneration have had generally poor and controversial results. Nonetheless, it is now clear that the injected cells activate the growth and differentiation of progenitor cells that are already present in the heart. This is achieved by the release of signalling factors and/or exosomes carrying them. Along this line, chemistry may play a major role in developing new strategies for activating resident stem cells to regenerate the heart. In particular, this review focuses on small molecule approaches for cell reprogramming, cell differentiation, and activation of cell protection.

A sequential EMT-MET mechanism drives the differentiation of human embryonic stem cells towards hepatocytes.

PubMed

Li, Qiuhong; Hutchins, Andrew P; Chen, Yong; Li, Shengbiao; Shan, Yongli; Liao, Baojian; Zheng, Dejin; Shi, Xi; Li, Yinxiong; Chan, Wai-Yee; Pan, Guangjin; Wei, Shicheng; Shu, Xiaodong; Pei, Duanqing

2017-05-03

Reprogramming has been shown to involve EMT-MET; however, its role in cell differentiation is unclear. We report here that in vitro differentiation of hESCs to hepatic lineage undergoes a sequential EMT-MET with an obligatory intermediate mesenchymal phase. Gene expression analysis reveals that Activin A-induced formation of definitive endoderm (DE) accompanies a synchronous EMT mediated by autocrine TGFβ signalling followed by a MET process. Pharmacological inhibition of TGFβ signalling blocks the EMT as well as DE formation. We then identify SNAI1 as the key EMT transcriptional factor required for the specification of DE. Genetic ablation of SNAI1 in hESCs does not affect the maintenance of pluripotency or neural differentiation, but completely disrupts the formation of DE. These results reveal a critical mesenchymal phase during the acquisition of DE, highlighting a role for sequential EMT-METs in both differentiation and reprogramming.

[TSA improve transgenic porcine cloned embryo development and transgene expression].

PubMed

Kong, Qing-Ran; Zhu, Jiang; Huang, Bo; Huan, Yan-Jun; Wang, Feng; Shi, Yong-Qian; Liu, Zhong-Feng; Wu, Mei-Ling; Liu, Zhong-Hua

2011-07-01

Uncompleted epigenetic reprogramming is attributed to the low efficiency of producing transgenic cloned animals. Histone modification associated with epigenetics can directly influence the embryo development and transgene expression. Trichostatin A (TSA), as an inhibitor of histone deacetylase, can change the status of histone acetylation, improve somatic cell reprogramming, and enhance cloning efficiency. TSA prevents the chromatin structure from being condensed, so that transcription factor could binds to DNA sequence easily and enhance transgene expression. Our study established the optimal TSA treatment on porcine donor cells and cloned embryos, 250 nmol/L, 24 h and 40 nmol/L, 24 h, respectively. Furthermore, we found that both the cloned embryo and the donor cell treated by TSA resulted in the highest development efficiency. Meanwhile, TSA can improve transgene expression in donor cell and cloned embryo. In summary, TSA can significantly improve porcine reconstructed embryo development and transgene expression.

Induced pluripotent stem cells: advances to applications

PubMed Central

Nelson, Timothy J; Martinez-Fernandez, Almudena; Yamada, Satsuki; Ikeda, Yasuhiro; Perez-Terzic, Carmen; Terzic, Andre

2010-01-01

Induced pluripotent stem cell (iPS) technology has enriched the armamentarium of regenerative medicine by introducing autologous pluripotent progenitor pools bioengineered from ordinary somatic tissue. Through nuclear reprogramming, patient-specific iPS cells have been derived and validated. Optimizing iPS-based methodology will ensure robust applications across discovery science, offering opportunities for the development of personalized diagnostics and targeted therapeutics. Here, we highlight the process of nuclear reprogramming of somatic tissues that, when forced to ectopically express stemness factors, are converted into bona fide pluripotent stem cells. Bioengineered stem cells acquire the genuine ability to generate replacement tissues for a wide-spectrum of diseased conditions, and have so far demonstrated therapeutic benefit upon transplantation in model systems of sickle cell anemia, Parkinson’s disease, hemophilia A, and ischemic heart disease. The field of regenerative medicine is therefore primed to adopt and incorporate iPS cell-based advancements as a next generation stem cell platforms. PMID:21165156

Robotics in the Construction Industry

DTIC Science & Technology

1990-06-01

accomplished through reprogramming and the attachment of different end effectors. 2.1.2.3 Manipulator I This is the mechanism for moving objects in the...other 3 types of robots), limited repeatability (ability to "hit" the same point in space time after time without reprogramming or adjustment by the... Reprogramming for a different 3 sequence of steps is generally difficult and time- consuming, as the stops must be relocated and I calibrated for the new sequence

Reprogramming of Somatic Cells Towards Pluripotency by Cell Fusion.

PubMed

Malinowski, Andrzej R; Fisher, Amanda G

2016-01-01

Pluripotent reprogramming can be dominantly induced in a somatic nucleus upon fusion with a pluripotent cell such as embryonic stem (ES) cell. Cell fusion between ES cells and somatic cells results in the formation of heterokaryons, in which the somatic nuclei begin to acquire features of the pluripotent partner. The generation of interspecies heterokaryons between mouse ES- and human somatic cells allows an experimenter to distinguish the nuclear events occurring specifically within the reprogrammed nucleus. Therefore, cell fusion provides a simple and rapid approach to look at the early nuclear events underlying pluripotent reprogramming. Here, we describe a polyethylene glycol (PEG)-mediated cell fusion protocol to generate interspecies heterokaryons and intraspecies hybrids between ES cells and B lymphocytes or fibroblasts.

Environmentally Induced Transgenerational Epigenetic Reprogramming of Primordial Germ Cells and the Subsequent Germ Line

PubMed Central

Skinner, Michael K.; Haque, Carlos Guerrero-Bosagna M.; Nilsson, Eric; Bhandari, Ramji; McCarrey, John R.

2013-01-01

A number of environmental factors (e.g. toxicants) have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. Transgenerational inheritance requires the germline transmission of altered epigenetic information between generations in the absence of direct environmental exposures. The primary periods for epigenetic programming of the germ line are those associated with primordial germ cell development and subsequent fetal germline development. The current study examined the actions of an agricultural fungicide vinclozolin on gestating female (F0 generation) progeny in regards to the primordial germ cell (PGC) epigenetic reprogramming of the F3 generation (i.e. great-grandchildren). The F3 generation germline transcriptome and epigenome (DNA methylation) were altered transgenerationally. Interestingly, disruptions in DNA methylation patterns and altered transcriptomes were distinct between germ cells at the onset of gonadal sex determination at embryonic day 13 (E13) and after cord formation in the testis at embryonic day 16 (E16). A larger number of DNA methylation abnormalities (epimutations) and transcriptional alterations were observed in the E13 germ cells than in the E16 germ cells. These observations indicate that altered transgenerational epigenetic reprogramming and function of the male germline is a component of vinclozolin induced epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance are provided. PMID:23869203

Chemicals as the Sole Transformers of Cell Fate.

PubMed

Ebrahimi, Behnam

2016-05-30

Forced expression of lineage-specific transcription factors in somatic cells can result in the generation of different cell types in a process named direct reprogramming, bypassing the pluripotent state. However, the introduction of transgenes limits the therapeutic applications of the produced cells. Numerous small-molecules have been introduced in the field of stem cell biology capable of governing self-renewal, reprogramming, transdifferentiation and regeneration. These chemical compounds are versatile tools for cell fate conversion toward desired outcomes. Cell fate conversion using small-molecules alone (chemical reprogramming) has superiority over arduous traditional genetic techniques in several aspects. For instance, rapid, transient, and reversible effects in activation and inhibition of functions of specific proteins are of the profits of small-molecules. They are cost-effective, have a long half-life, diversity on structure and function, and allow for temporal and flexible regulation of signaling pathways. Additionally, their effects could be adjusted by fine-tuning concentrations and combinations of different small-molecules. Therefore, chemicals are powerful tools in cell fate conversion and study of stem cell and chemical biology in vitro and in vivo. Moreover, transgene-free and chemical-only transdifferentiation approaches provide alternative strategies for the generation of various cell types, disease modeling, drug screening, and regenerative medicine. The current review gives an overview of the recent findings concerning transdifferentiation by only small-molecules without the use of transgenes.

Human Urinary Epithelial Cells as a Source of Engraftable Hepatocyte-Like Cells Using Stem Cell Technology.

PubMed

Sauer, Vanessa; Tchaikovskaya, Tatyana; Wang, Xia; Li, Yanfeng; Zhang, Wei; Tar, Krisztina; Polgar, Zsuzsanna; Ding, Jianqiang; Guha, Chandan; Fox, Ira J; Roy-Chowdhury, Namita; Roy-Chowdhury, Jayanta

2016-12-13

Although several types of somatic cells have been reprogrammed into induced pluripotent stem cells (iPSCs) and then differentiated to hepatocyte-like cells (iHeps), the method for generating such cells from renal tubular epithelial cells shed in human urine and transplanting them into animal livers has not been described systematically. We report reprogramming of human urinary epithelial cells into iPSCs and subsequent hepatic differentiation, followed by a detailed characterization of the newly generated iHeps. The epithelial cells were reprogrammed into iPSCs by delivering the pluripotency factors OCT3/4, SOX2, KLF4, and MYC using methods that do not involve transgene integration, such as nucleofection of episomal (oriP/EBNA-1) plasmids or infection with recombinant Sendai viruses. After characterization of stable iPSC lines, a three-step differentiation toward hepatocytes was performed. The iHeps expressed a large number of hepatocyte-preferred genes, including nuclear receptors that regulate genes involved in cholesterol homeostasis, bile acid transport, and detoxification. MicroRNA profile of the iHeps largely paralleled that of primary human hepatocytes. The iHeps engrafted into the livers of Scid mice transgenic for mutant human SERPINA1 after intrasplenic injection. Thus, urine is a readily available source for generating human iHeps that could be potentially useful for disease modeling, pharmacological development, and regenerative medicine.

Metabolic Reprogramming by 3-Iodothyronamine (T1AM): A New Perspective to Reverse Obesity through Co-Regulation of Sirtuin 4 and 6 Expression.

PubMed

Assadi-Porter, Fariba M; Reiland, Hannah; Sabatini, Martina; Lorenzini, Leonardo; Carnicelli, Vittoria; Rogowski, Micheal; Selen Alpergin, Ebru S; Tonelli, Marco; Ghelardoni, Sandra; Saba, Alessandro; Zucchi, Riccardo; Chiellini, Grazia

2018-05-22

Obesity is a complex disease associated with environmental and genetic factors. 3-Iodothyronamine (T1AM) has revealed great potential as an effective weight loss drug. We used metabolomics and associated transcriptional gene and protein expression analysis to investigate the tissue specific metabolic reprogramming effects of subchronic T1AM treatment at two pharmacological daily doses (10 and 25 mg/kg) on targeted metabolic pathways. Multi-analytical results indicated that T1AM at 25 mg/kg can act as a novel master regulator of both glucose and lipid metabolism in mice through sirtuin-mediated pathways. In liver, we observed an increased gene and protein expression of Sirt6 (a master gene regulator of glucose) and Gck (glucose kinase) and a decreased expression of Sirt4 (a negative regulator of fatty acids oxidation (FAO)), whereas in white adipose tissue only Sirt6 was increased. Metabolomics analysis supported physiological changes at both doses with most increases in FAO, glycolysis indicators and the mitochondrial substrate, at the highest dose of T1AM. Together our results suggest that T1AM acts through sirtuin-mediated pathways to metabolically reprogram fatty acid and glucose metabolism possibly through small molecules signaling. Our novel mechanistic findings indicate that T1AM has a great potential as a drug for the treatment of obesity and possibly diabetes.

CSF1/CSF1R Blockade Reprograms Tumor-Infiltrating Macrophages and Improves Response to T Cell Checkpoint Immunotherapy in Pancreatic Cancer Models

PubMed Central

Zhu, Yu; Knolhoff, Brett L.; Meyer, Melissa A.; Nywening, Timothy M.; West, Brian L.; Luo, Jingqin; Wang-Gillam, Andrea; Goedegebuure, S Peter; Linehan, David C.; DeNardo, David G.

2014-01-01

Cancer immunotherapy generally offers limited clinical benefit without coordinated strategies to mitigate the immunosuppressive nature of the tumor microenvironment. Critical drivers of immune escape in the tumor microenvironment include tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC), which not only mediate immune suppression but also promote metastatic dissemination and impart resistance to cytotoxic therapies. Thus, strategies to ablate the effects of these myeloid cell populations may offer great therapeutic potential. In this report, we demonstrate in a mouse model of pancreatic ductal adenocarcinoma (PDAC) that inhibiting signaling by the myeloid growth factor receptor CSF1R can functionally reprogram macrophage responses that enhance antigen presentation and productive anti-tumor T cell responses. Investigations of this response revealed that CSF1R blockade also upregulated T cell checkpoint molecules, including PDL1 and CTLA4, thereby restraining beneficial therapeutic effects. We found that PD1 and CTLA4 antagonists showed limited efficacy as single agents to restrain PDAC growth, but that that combining these agents with CSF1R blockade potently elicited tumor regressions, even in larger established tumors. Taken together, our findings provide a rationale to reprogram immunosuppressive myeloid cell populations in the tumor microenvironment under conditions that can significantly empower the therapeutic effects of checkpoint-based immunotherapeutics. PMID:25082815

CSF1/CSF1R blockade reprograms tumor-infiltrating macrophages and improves response to T-cell checkpoint immunotherapy in pancreatic cancer models.

PubMed

Zhu, Yu; Knolhoff, Brett L; Meyer, Melissa A; Nywening, Timothy M; West, Brian L; Luo, Jingqin; Wang-Gillam, Andrea; Goedegebuure, S Peter; Linehan, David C; DeNardo, David G

2014-09-15

Cancer immunotherapy generally offers limited clinical benefit without coordinated strategies to mitigate the immunosuppressive nature of the tumor microenvironment. Critical drivers of immune escape in the tumor microenvironment include tumor-associated macrophages and myeloid-derived suppressor cells, which not only mediate immune suppression, but also promote metastatic dissemination and impart resistance to cytotoxic therapies. Thus, strategies to ablate the effects of these myeloid cell populations may offer great therapeutic potential. In this report, we demonstrate in a mouse model of pancreatic ductal adenocarcinoma (PDAC) that inhibiting signaling by the myeloid growth factor receptor CSF1R can functionally reprogram macrophage responses that enhance antigen presentation and productive antitumor T-cell responses. Investigations of this response revealed that CSF1R blockade also upregulated T-cell checkpoint molecules, including PDL1 and CTLA4, thereby restraining beneficial therapeutic effects. We found that PD1 and CTLA4 antagonists showed limited efficacy as single agents to restrain PDAC growth, but that combining these agents with CSF1R blockade potently elicited tumor regressions, even in larger established tumors. Taken together, our findings provide a rationale to reprogram immunosuppressive myeloid cell populations in the tumor microenvironment under conditions that can significantly empower the therapeutic effects of checkpoint-based immunotherapeutics. ©2014 American Association for Cancer Research.

A Multistate Toggle Switch Defines Fungal Cell Fates and Is Regulated by Synergistic Genetic Cues

PubMed Central

Anderson, Matthew Z.; Porman, Allison M.; Wang, Na; Mancera, Eugenio; Bennett, Richard J.

2016-01-01

Heritable epigenetic changes underlie the ability of cells to differentiate into distinct cell types. Here, we demonstrate that the fungal pathogen Candida tropicalis exhibits multipotency, undergoing stochastic and reversible switching between three cellular states. The three cell states exhibit unique cellular morphologies, growth rates, and global gene expression profiles. Genetic analysis identified six transcription factors that play key roles in regulating cell differentiation. In particular, we show that forced expression of Wor1 or Efg1 transcription factors can be used to manipulate transitions between all three cell states. A model for tristability is proposed in which Wor1 and Efg1 are self-activating but mutually antagonistic transcription factors, thereby forming a symmetrical self-activating toggle switch. We explicitly test this model and show that ectopic expression of WOR1 can induce white-to-hybrid-to-opaque switching, whereas ectopic expression of EFG1 drives switching in the opposite direction, from opaque-to-hybrid-to-white cell states. We also address the stability of induced cell states and demonstrate that stable differentiation events require ectopic gene expression in combination with chromatin-based cues. These studies therefore experimentally test a model of multistate stability and demonstrate that transcriptional circuits act synergistically with chromatin-based changes to drive cell state transitions. We also establish close mechanistic parallels between phenotypic switching in unicellular fungi and cell fate decisions during stem cell reprogramming. PMID:27711197

In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

PubMed

Mitani, Yasuyuki; Vagnozzi, Ronald J; Millay, Douglas P

2017-01-01

Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming. © FASEB.

In vivo myomaker-mediated heterologous fusion and nuclear reprogramming

PubMed Central

Mitani, Yasuyuki; Vagnozzi, Ronald J.; Millay, Douglas P.

2017-01-01

Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non–muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle–specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.—Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming. PMID:27825107

Binary colloidal crystals (BCCs) as a feeder-free system to generate human induced pluripotent stem cells (hiPSCs)

PubMed Central

Wang, Peng-Yuan; Hung, Sandy Shen-Chi; Thissen, Helmut; Kingshott, Peter; Wong, Raymond Ching-Bong

2016-01-01

Human induced pluripotent stem cells (hiPSCs) are capable of differentiating into any cell type and provide significant advances to cell therapy and regenerative medicine. However, the current protocol for hiPSC generation is relatively inefficient and often results in many partially reprogrammed colonies, which increases the cost and reduces the applicability of hiPSCs. Biophysical stimulation, in particular from tuning cell-surface interactions, can trigger specific cellular responses that could in turn promote the reprogramming process. In this study, human fibroblasts were reprogrammed into hiPSCs using a feeder-free system and episomal vectors using novel substrates based on binary colloidal crystals (BCCs). BCCs are made from two different spherical particle materials (Si and PMMA) ranging in size from nanometers to micrometers that self-assemble into hexagonal close-packed arrays. Our results show that the BCCs, particularly those made from a crystal of 2 μm Si and 0.11 μm PMMA particles (2SiPM) facilitate the reprogramming process and increase the proportion of fully reprogrammed hiPSC colonies, even without a vitronectin coating. Subsequent isolation of clonal hiPSC lines demonstrates that they express pluripotent markers (OCT4 and TRA-1-60). This proof-of-concept study demonstrates that cell reprogramming can be improved on substrates where surface properties are tailored to the application. PMID:27833126

Privileged Communication Embryonic Development Following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation

PubMed Central

Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A.; Shen, Li; Inoue, Azusa; Zhang, Yi

2014-01-01

SUMMARY Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by IVF but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells, and its removal by ectopic expression of the H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency. PMID:25417163

Direct reprogramming of human bone marrow stromal cells into functional renal cells using cell-free extracts.

PubMed

Papadimou, Evangelia; Morigi, Marina; Iatropoulos, Paraskevas; Xinaris, Christodoulos; Tomasoni, Susanna; Benedetti, Valentina; Longaretti, Lorena; Rota, Cinzia; Todeschini, Marta; Rizzo, Paola; Introna, Martino; Grazia de Simoni, Maria; Remuzzi, Giuseppe; Goligorsky, Michael S; Benigni, Ariela

2015-04-14

The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs), also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes-formation of "domes" and tubule-like structures-and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Somatic Nucleus Reprogramming Is Significantly Improved by m-Carboxycinnamic Acid Bishydroxamide, a Histone Deacetylase Inhibitor*

PubMed Central

Dai, Xiangpeng; Hao, Jie; Hou, Xiao-jun; Hai, Tang; Fan, Yong; Yu, Yang; Jouneau, Alice; Wang, Liu; Zhou, Qi

2010-01-01

Somatic cell nuclear transfer (SCNT) has shown tremendous potential for understanding the mechanisms of reprogramming and creating applications in the realms of agriculture, therapeutics, and regenerative medicine, although the efficiency of reprogramming is still low. Somatic nucleus reprogramming is triggered in the short time after transfer into recipient cytoplasm, and therefore, this period is regarded as a key stage for optimizing SCNT. Here we report that CBHA, a histone deacetylase inhibitor, modifies the acetylation status of somatic nuclei and increases the developmental potential of mouse cloned embryos to reach pre- and post-implantation stages. Furthermore, the cloned embryos treated by CBHA displayed higher efficiency in the derivation of nuclear transfer embryonic stem cell lines by promoting outgrowths. More importantly, CBHA increased blastocyst quality compared with trichostatin A, another prevalent histone deacetylase inhibitor reported previously. Use of CBHA should improve the productivity of SCNT for a variety of research and clinical applications, and comparisons of cells with different levels of pluripotency and treated with CBHA versus trichostatin A will facilitate studies of the mechanisms of reprogramming. PMID:20566633

Engineering Poly(ethylene glycol) Materials to Promote Cardiogenesis

NASA Astrophysics Data System (ADS)

Smith, Amanda Walker

Heart failure is one of the leading causes of death worldwide, and the current costs of treatment put a significant economic burden on our societies. After an infarction, fibrotic tissue begins to form as part of the heart failure cascade. Current options to slow this process include a wide range of pharmaceutical agents, and ultimately the patient may require a heart transplant. Innovative treatment approaches are needed to bring down costs and improve quality of life. The possibility of regenerating or replacing damaged tissue with healthy cardiomyocytes is generating considerable excitement, but there are still many obstacles to overcome. First, while cell injections into the myocardium have demonstrated slight improvements in cardiac function, the actual engraftment of transplanted cells is very low. It is anticipated that improving engraftment will boost outcomes. Second, cellular differentiation and reprogramming protocols have not yet produced cells that are identical to adult cardiomyocytes, and immunogenicity continues to be a problem despite the advent of autologously derived induced pluripotent stem cells. This dissertation will explore biomaterials approaches to addressing these two obstacles. Tissue engineering scaffolds may improve cell engraftment by providing bioactive factors, preventing cell anoikis, and reducing cell washout by blood flow. Poly(ethylene glycol) (PEG) is often used as a coating to reduce implant rejection because it is highly resistant to protein adsorption. Because fibrosis of a material in contact with the myocardium could cause arrhythmias, PEG materials are highly relevant for cardiac tissue engineering applications. In Chapter 2, we describe a novel method for crosslinking PEG microspheres around cells to form a scaffold for tissue engineering. We then demonstrate that HL-1 cardiomyocyte viability and phenotype are retained throughout the fabrication process and during the first 7 weeks of culture. In the third chapter of the dissertation, we demonstrate that the use of PEG cell culture substrates can improve efficiency of direct reprogramming from fibroblasts to cardiomyocytes for cell transplantation. Standard tissue culture plastic adsorbs proteins from the cell media, increasing experimental variability via non-specific signaling. Because of its protein resistant properties, PEG provides cells with highly specific signals. In addition to improving the efficiency, we found that presentation of RGD peptides stimulated proliferation during reprogramming. Combined, the improvements enabled us to approximately double the number of cardiomyocytes produced by the protocol. In Chapter 4, we explore the effects of 3D culture on the direct reprogramming protocol described in Chapter 3. We demonstrate that the variables involved in 3D culture, including scaffold material, diffusion, cellular remodeling, and scaffold topography, have significant effects on reprogramming efficiency. This chapter provides the groundwork for future studies developing 3D microenvironments for efficient and scalable reprogramming to cardiomyocytes.

Reprogramming the Metastatic Microenvironment to Combat Disease Recurrence

DTIC Science & Technology

2016-10-01

the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this...lead to pathological bone loss, which can stimulate tumor cell outgrowth. In addition to contributing to morbidity, this ‘vicious cycle’ also...surveillance and antigen presentation affect metastatic relapse in the bone. In so doing, we discovered that inhibiting colony stimulating factor-1

Dissecting Embryonic Stem Cell Self-Renewal and Differentiation Commitment from Quantitative Models.

PubMed

Hu, Rong; Dai, Xianhua; Dai, Zhiming; Xiang, Qian; Cai, Yanning

2016-10-01

To model quantitatively embryonic stem cell (ESC) self-renewal and differentiation by computational approaches, we developed a unified mathematical model for gene expression involved in cell fate choices. Our quantitative model comprised ESC master regulators and lineage-specific pivotal genes. It took the factors of multiple pathways as input and computed expression as a function of intrinsic transcription factors, extrinsic cues, epigenetic modifications, and antagonism between ESC master regulators and lineage-specific pivotal genes. In the model, the differential equations of expression of genes involved in cell fate choices from regulation relationship were established according to the transcription and degradation rates. We applied this model to the Murine ESC self-renewal and differentiation commitment and found that it modeled the expression patterns with good accuracy. Our model analysis revealed that Murine ESC was an attractor state in culture and differentiation was predominantly caused by antagonism between ESC master regulators and lineage-specific pivotal genes. Moreover, antagonism among lineages played a critical role in lineage reprogramming. Our results also uncovered that the ordered expression alteration of ESC master regulators over time had a central role in ESC differentiation fates. Our computational framework was generally applicable to most cell-type maintenance and lineage reprogramming.

Therapeutic cloning and cellular reprogramming.

PubMed

Rodriguez, Ramon M; Ross, Pablo J; Cibelli, Jose B

2012-01-01

Embryonic stem cells are capable of differentiating into any cell-type present in an adult organism, and constitute a renewable source of tissue for regenerative therapies. The transplant of allogenic stem cells is challenging due to the risk of immune rejection. Nevertheless, somatic cell reprogramming techniques allow the generation of isogenic embryonic stem cells, genetically identical to the patient. In this chapter we will discuss the cellular reprogramming techniques in the context of regenerative therapy and the biological and technical barriers that they will need to overcome before clinical use.

Budget Execution Exercise for Use in the Financial Management in the Armed Forces Course at the Naval Postgraduate School.

DTIC Science & Technology

1985-12-01

provide additional funding immediately; the program had to be initiated within existing budget constraints by reprogramming funds at the local level. Some...comptroller could recall only one instance during the past two fiscal years wherein COMNAVAIRLANT rejected a request for reprogramming in excess of $50...lists. The first contains items which must be fully funded in the course of the year, even if reprogramming authority must . be requested from the sponsor

Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks

PubMed Central

Liu, Kun-hsiang; Niu, Yajie; Konishi, Mineko; Wu, Yue; Du, Hao; Sun Chung, Hoo; Li, Lei; Boudsocq, Marie; McCormack, Matthew; Maekawa, Shugo; Ishida, Tetsuya; Zhang, Chao; Shokat, Kevan; Yanagisawa, Shuichi; Sheen, Jen

2018-01-01

Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report novel Ca2+ signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca2+-sensor protein kinases (CPKs) as master regulators orchestrating primary nitrate responses. A chemical switch with the engineered CPK10(M141G) kinase enables conditional analyses of cpk10,30,32 to define comprehensive nitrate-associated regulatory and developmental programs, circumventing embryo lethality. Nitrate-CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors (TFs) to specify reprogramming of gene sets for downstream TFs, transporters, N-assimilation, C/N-metabolism, redox, signalling, hormones, and proliferation. Conditional cpk10,30,32 and nlp7 similarly impair nitrate-stimulated system-wide shoot growth and root establishment. The nutrient-coupled Ca2+ signalling network integrates transcriptome and cellular metabolism with shoot-root coordination and developmental plasticity in shaping organ biomass and architecture. PMID:28489820

Extensive epigenetic reprogramming during the life cycle of Marchantia polymorpha.

PubMed

Schmid, Marc W; Giraldo-Fonseca, Alejandro; Rövekamp, Moritz; Smetanin, Dmitry; Bowman, John L; Grossniklaus, Ueli

2018-01-25

In plants, the existence and possible role of epigenetic reprogramming has been questioned because of the occurrence of stably inherited epialleles. Evidence suggests that epigenetic reprogramming does occur during land plant reproduction, but there is little consensus on the generality and extent of epigenetic reprogramming in plants. We studied DNA methylation dynamics during the life cycle of the liverwort Marchantia polymorpha. We isolated thalli and meristems from male and female gametophytes, archegonia, antherozoids, as well as sporophytes at early and late developmental stages, and compared their DNA methylation profiles. Of all cytosines tested for differential DNA methylation, 42% vary significantly in their methylation pattern throughout the life cycle. However, the differences are limited to few comparisons between specific stages of the life cycle and suggest four major epigenetic states specific to sporophytes, vegetative gametophytes, antherozoids, and archegonia. Further analyses indicated clear differences in the mechanisms underlying reprogramming in the gametophytic and sporophytic generations, which are paralleled by differences in the expression of genes involved in DNA methylation. Differentially methylated cytosines with a gain in methylation in antherozoids and archegonia are enriched in the CG and CHG contexts, as well as in gene bodies and gene flanking regions. In contrast, gain of DNA methylation during sporophyte development is mostly limited to the CHH context, LTR retrotransposons, DNA transposons, and repeats. We conclude that epigenetic reprogramming occurs at least twice during the life cycle of M. polymorpha and that the underlying mechanisms are likely different between the two events.

Developmental Programming of Adult Disease: Reprogramming by Melatonin?

PubMed

Tain, You-Lin; Huang, Li-Tung; Hsu, Chien-Ning

2017-02-16

Adult-onset chronic non-communicable diseases (NCDs) can originate from early life through so-called the "developmental origins of health and disease" (DOHaD) or "developmental programming". The DOHaD concept offers the "reprogramming" strategy to shift the treatment from adulthood to early life, before clinical disease is apparent. Melatonin, an endogenous indoleamine produced by the pineal gland, has pleiotropic bioactivities those are beneficial in a variety of human diseases. Emerging evidence support that melatonin is closely inter-related to other proposed mechanisms contributing to the developmental programming of a variety of chronic NCDs. Recent animal studies have begun to unravel the multifunctional roles of melatonin in many experimental models of developmental programming. Even though some progress has been made in research on melatonin as a reprogramming strategy to prevent DOHaD-related NCDs, future human studies should aim at filling the translational gap between animal models and clinical trials. Here, we review several key themes on the reprogramming effects of melatonin in DOHaD research. We have particularly focused on the following areas: mechanisms of developmental programming; the interrelationship between melatonin and mechanisms underlying developmental programming; pathophysiological roles of melatonin in pregnancy and fetal development; and insight provided by animal models to support melatonin as a reprogramming therapy. Rates of NCDs are increasing faster than anticipated all over the world. Hence, there is an urgent need to understand reprogramming mechanisms of melatonin and to translate experimental research into clinical practice for halting a growing list of DOHaD-related NCDs.

In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes

PubMed Central

Qian, Li; Huang, Yu; Spencer, C. Ian; Foley, Amy; Vedantham, Vasanth; Liu, Lei; Conway, Simon J.; Fu, Ji-dong; Srivastava, Deepak

2012-01-01

SUMMARY The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported that cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here, we use genetic lineage-tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became bi-nucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast activating peptide, Thymosin β4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes. PMID:22522929

Clinically relevant HIF-1α-dependent metabolic reprogramming in oropharyngeal squamous cell carcinomas includes coordinated activation of CAIX and the miR-210/ISCU signaling axis, but not MCT1 and MCT4 upregulation

PubMed Central

Sáenz-de-Santa-María, Inés; Bernardo-Castiñeira, Cristóbal; Secades, Pablo; Bernaldo-de-Quirós, Sandra; Rodrigo, Juan Pablo; Astudillo, Aurora; Chiara, María-Dolores

2017-01-01

Metabolic reprogramming is a very heterogeneous phenomenon in cancer. It mostly consists on increased glycolysis, lactic acid formation and extracellular acidification. These events have been associated to increased activity of the hypoxia inducible factor, HIF-1α. This study aimed at defining the metabolic program activated by HIF-1α in oropharyngeal squamous cell carcinomas (SCC) and assessing its clinical impact. Global gene/miRNA expression was analyzed in SCC-derived cells exposed to hypoxia. Expression of HIF-1α, the carbonic anhydrase CAIX, and the lactate/H+ transporters MCT1 and MCT4 were analyzed by immunohistochemistry in 246 SCCs. Cell-based analysis revealed that HIF-1α-driven metabolic program includes over-expression of glycolytic enzymes and the microRNA miR-210 coupled to down-regulation of its target, the iron-sulfur cluster assembly protein, ISCU. pH-regulator program entailed over-expression of CAIX, but not MCT1 or MCT4. Accordingly, significant overlapping exists between over-expression of HIF-1α and CAIX, but not HIF-1α and MCT1 or MCT4, in tumor cells. Increased miR-210 and concomitant decreased ISCU RNA levels were found in ~40% of tumors and this was significantly associated with HIF-1α and CAIX, but not MCT1 or MCT4, over-expression. HIF-1α and/or CAIX over-expression was associated with high recurrence rate and low overall survival of surgically treated patients. By contrast, clinically significant correlations were not found in tumors with MCT1 or MCT4 over-expression. This is the first study that provides in vivo evidences of coordinated activation of HIF-1α, CAIX, miR-210 and ISCU in carcinoma and association with poor prognosis, a finding with important implications for the development of metabolic-targeting therapies against hypoxia. PMID:28099149

Clinically relevant HIF-1α-dependent metabolic reprogramming in oropharyngeal squamous cell carcinomas includes coordinated activation of CAIX and the miR-210/ISCU signaling axis, but not MCT1 and MCT4 upregulation.

PubMed

Sáenz-de-Santa-María, Inés; Bernardo-Castiñeira, Cristóbal; Secades, Pablo; Bernaldo-de-Quirós, Sandra; Rodrigo, Juan Pablo; Astudillo, Aurora; Chiara, María-Dolores

2017-02-21

Metabolic reprogramming is a very heterogeneous phenomenon in cancer. It mostly consists on increased glycolysis, lactic acid formation and extracellular acidification. These events have been associated to increased activity of the hypoxia inducible factor, HIF-1α. This study aimed at defining the metabolic program activated by HIF-1α in oropharyngeal squamous cell carcinomas (SCC) and assessing its clinical impact. Global gene/miRNA expression was analyzed in SCC-derived cells exposed to hypoxia. Expression of HIF-1α, the carbonic anhydrase CAIX, and the lactate/H+ transporters MCT1 and MCT4 were analyzed by immunohistochemistry in 246 SCCs. Cell-based analysis revealed that HIF-1α-driven metabolic program includes over-expression of glycolytic enzymes and the microRNA miR-210 coupled to down-regulation of its target, the iron-sulfur cluster assembly protein, ISCU. pH-regulator program entailed over-expression of CAIX, but not MCT1 or MCT4. Accordingly, significant overlapping exists between over-expression of HIF-1α and CAIX, but not HIF-1α and MCT1 or MCT4, in tumor cells. Increased miR-210 and concomitant decreased ISCU RNA levels were found in ~40% of tumors and this was significantly associated with HIF-1α and CAIX, but not MCT1 or MCT4, over-expression. HIF-1α and/or CAIX over-expression was associated with high recurrence rate and low overall survival of surgically treated patients. By contrast, clinically significant correlations were not found in tumors with MCT1 or MCT4 over-expression. This is the first study that provides in vivo evidences of coordinated activation of HIF-1α, CAIX, miR-210 and ISCU in carcinoma and association with poor prognosis, a finding with important implications for the development of metabolic-targeting therapies against hypoxia.

Cancer: A Problem of Developmental Biology; Scientific Evidence for Reprogramming and Differentiation Therapy.

PubMed

Sell, Stewart; Nicolini, Andrea; Ferrari, Paola; Biava, Pier M

2016-01-01

Current medical literature acknowledges that embryonic micro-environment is able to suppress tumor development. Administering carcinogenic substances during organogenesis in fact leads to embryonic malformations, but not to offspring tumor growth. Once organogenesis has ended, administration of carcinogenic substances causes a rise in offspring tumor development. These data indicate that cancer can be considered a deviation in normal development, which can be regulated by factors of the embryonic microenvironment. Furthermore, it has been demonstrated that teratoma differentiates into normal tissues once it is implanted in the embryo. Recently, it has been shown that implanting a melanoma in Zebrafish embryo did not result in a tumor development; however, it did in the adult specimen. This demonstrates that cancer cells can differentiate into normal tissues when implanted in the embryo. In addition, it was demonstrated that other tumors can revert into a normal phenotype and/or differentiate into normal tissue when implanted in the embryo. These studies led some authors to define cancer as a problem of developmental biology and to predict the present concept of "cancer stem cells theory". In this review, we record the most important researches about the reprogramming and differentiation treatments of cancer cells to better clarify how the substances taken from developing embryo or other biological substances can induce differentiation of malignant cells. Lastly, a model of cancer has been proposed here, conceived by one of us, which is consistent with the reality, as demonstrated by a great number of researches. This model integrates the theory of the "maturation arrest" of cancer cells as conceived by B. Pierce with the theory which describes cancer as a process of deterministic chaos determined by genetic and/or epigenetic alterations in differentiated cells, which leads a normal cell to become cancerous. All the researches here described demonstrated that cancer can be considered a problem of developmental biology and that one of the most important hallmarks of cancer is the loss of differentiation as already described by us in other articles.

Diet-induced obesity reprograms the inflammatory response of the murine lung to inhaled endotoxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tilton, Susan C., E-mail: susan.tilton@pnnl.gov; Waters, Katrina M.; Karin, Norman J.

The co-occurrence of environmental factors is common in complex human diseases and, as such, understanding the molecular responses involved is essential to determine risk and susceptibility to disease. We have investigated the key biological pathways that define susceptibility for pulmonary infection during obesity in diet-induced obese (DIO) and regular weight (RW) C57BL/6 mice exposed to inhaled lipopolysaccharide (LPS). LPS induced a strong inflammatory response in all mice as indicated by elevated cell counts of macrophages and neutrophils and levels of proinflammatory cytokines (MDC, MIP-1γ, IL-12, RANTES) in the bronchoalveolar lavage fluid. Additionally, DIO mice exhibited 50% greater macrophage cell counts,more » but decreased levels of the cytokines, IL-6, TARC, TNF-α, and VEGF relative to RW mice. Microarray analysis of lung tissue showed over half of the LPS-induced expression in DIO mice consisted of genes unique for obese mice, suggesting that obesity reprograms how the lung responds to subsequent insult. In particular, we found that obese animals exposed to LPS have gene signatures showing increased inflammatory and oxidative stress response and decreased antioxidant capacity compared with RW. Because signaling pathways for these responses can be common to various sources of environmentally induced lung damage, we further identified biomarkers that are indicative of specific toxicant exposure by comparing gene signatures after LPS exposure to those from a parallel study with cigarette smoke. These data show obesity may increase sensitivity to further insult and that co-occurrence of environmental stressors result in complex biosignatures that are not predicted from analysis of individual exposures. - Highlights: ► Obesity modulates inflammatory markers in BAL fluid after LPS exposure. ► Obese animals have a unique transcriptional signature in lung after LPS exposure. ► Obesity elevates inflammatory stress and reduces antioxidant capacity in the lung. ► Toxicant-specific biomarkers predict exposure independent of systemic inflammation.« less

Fundamentals of cancer metabolism

PubMed Central

DeBerardinis, Ralph J.; Chandel, Navdeep S.

2016-01-01

Tumors reprogram pathways of nutrient acquisition and metabolism to meet the bioenergetic, biosynthetic, and redox demands of malignant cells. These reprogrammed activities are now recognized as hallmarks of cancer, and recent work has uncovered remarkable flexibility in the specific pathways activated by tumor cells to support these key functions. In this perspective, we provide a conceptual framework to understand how and why metabolic reprogramming occurs in tumor cells, and the mechanisms linking altered metabolism to tumorigenesis and metastasis. Understanding these concepts will progressively support the development of new strategies to treat human cancer. PMID:27386546

Generation of a human iPSC line from a patient with Leigh syndrome.

PubMed

Galera, Teresa; Zurita, Francisco; González-Páramos, Cristina; Moreno-Izquierdo, Ana; Fraga, Mario F; Fernández, Agustin F; Garesse, Rafael; Gallardo, M Esther

2016-01-01

Human iPSC line LND554SV.3 was generated from heteroplasmic fibroblasts of a patient with Leigh syndrome carrying a mutation in the MT-ND5 gene (m.13513GNA; p.D393N). Reprogramming factors Oct3/4, Sox2, Klf4,and cMyc were delivered using a non-integrative methodology that involves the use of Sendai virus.

Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming

PubMed Central

McClellan, Michael J.; Wood, C. David; Ojeniyi, Opeoluwa; Cooper, Tim J.; Kanhere, Aditi; Arvey, Aaron; Webb, Helen M.; Palermo, Richard D.; Harth-Hertle, Marie L.; Kempkes, Bettina; Jenner, Richard G.; West, Michelle J.

2013-01-01

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors. PMID:24068937

In Vivo Reprogramming for CNS Repair: Regenerating Neurons from Endogenous Glial Cells

PubMed Central

Li, Hedong; Chen, Gong

2017-01-01

Neuroregeneration in the central nervous system (CNS) has proven to be difficult despite decades of research. The old dogma that CNS neurons cannot be regenerated in the adult mammalian brain has been overturned; however, endogenous adult neurogenesis appears to be insufficient for brain repair. Stem cell therapy once held promise for generating large quantities of neurons in the CNS, but immunorejection and long-term functional integration remain major hurdles. In this perspective, we discuss the use of in vivo reprogramming as an emerging technology to regenerate functional neurons from endogenous glial cells inside the brain and spinal cord. Besides the CNS, in vivo reprogramming has been demonstrated successfully in the pancreas, heart and liver, and may be adopted in other organs. Although challenges remain for translating this technology into clinical therapies, we anticipate that in vivo reprogramming may revolutionize regenerative medicine by using a patient’s own internal cells for tissue repair. PMID:27537482

Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency

PubMed Central

Cacchiarelli, Davide; Trapnell, Cole; Ziller, Michael J.; Soumillon, Magali; Cesana, Marcella; Karnik, Rahul; Donaghey, Julie; Smith, Zachary D.; Ratanasirintrawoot, Sutheera; Zhang, Xiaolan; Ho Sui, Shannan J.; Wu, Zhaoting; Akopian, Veronika; Gifford, Casey A.; Doench, John; Rinn, John L.; Daley, George Q.; Meissner, Alexander; Lander, Eric S.; Mikkelsen, Tarjei S.

2015-01-01

Summary Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered reactivation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PMID:26186193

A comparison of non-integrating reprogramming methods

PubMed Central

Schlaeger, Thorsten M; Daheron, Laurence; Brickler, Thomas R; Entwisle, Samuel; Chan, Karrie; Cianci, Amelia; DeVine, Alexander; Ettenger, Andrew; Fitzgerald, Kelly; Godfrey, Michelle; Gupta, Dipti; McPherson, Jade; Malwadkar, Prerana; Gupta, Manav; Bell, Blair; Doi, Akiko; Jung, Namyoung; Li, Xin; Lynes, Maureen S; Brookes, Emily; Cherry, Anne B C; Demirbas, Didem; Tsankov, Alexander M; Zon, Leonard I; Rubin, Lee L; Feinberg, Andrew P; Meissner, Alexander; Cowan, Chad A; Daley, George Q

2015-01-01

Human induced pluripotent stem cells (hiPSCs1–3) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV)4, episomal (Epi)5 and mRNA transfection mRNA6 methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation. PMID:25437882

Design principles for nuclease-deficient CRISPR-based transcriptional regulators.

PubMed

Jensen, Michael K

2018-06-01

The engineering of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins continues to expand the toolkit available for genome editing, reprogramming gene regulation, genome visualisation and epigenetic studies of living organisms. In this review, the emerging design principles on the use of nuclease-deficient CRISPR-based reprogramming of gene expression will be presented. The review will focus on the designs implemented in yeast both at the level of CRISPR proteins and guide RNA (gRNA), but will lend due credits to the seminal studies performed in other species where relevant. In addition to design principles, this review also highlights applications benefitting from the use of CRISPR-mediated transcriptional regulation and discusses the future directions to further expand the toolkit for nuclease-deficient reprogramming of genomes. As such, this review should be of general interest for experimentalists to get familiarised with the parameters underlying the power of reprogramming genomic functions by use of nuclease-deficient CRISPR technologies.

The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

PubMed

Araki, Ryoko; Mizutani, Eiji; Hoki, Yuko; Sunayama, Misato; Wakayama, Sayaka; Nagatomo, Hiroaki; Kasama, Yasuji; Nakamura, Miki; Wakayama, Teruhiko; Abe, Masumi

2017-05-01

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196. © 2017 AlphaMed Press.

Coactivator SRC-2–dependent metabolic reprogramming mediates prostate cancer survival and metastasis

PubMed Central

Dasgupta, Subhamoy; Putluri, Nagireddy; Long, Weiwen; Zhang, Bin; Wang, Jianghua; Kaushik, Akash K.; Arnold, James M.; Bhowmik, Salil K.; Stashi, Erin; Brennan, Christine A.; Rajapakshe, Kimal; Coarfa, Cristian; Mitsiades, Nicholas; Ittmann, Michael M.; Chinnaiyan, Arul M.; Sreekumar, Arun; O’Malley, Bert W.

2015-01-01

Metabolic pathway reprogramming is a hallmark of cancer cell growth and survival and supports the anabolic and energetic demands of these rapidly dividing cells. The underlying regulators of the tumor metabolic program are not completely understood; however, these factors have potential as cancer therapy targets. Here, we determined that upregulation of the oncogenic transcriptional coregulator steroid receptor coactivator 2 (SRC-2), also known as NCOA2, drives glutamine-dependent de novo lipogenesis, which supports tumor cell survival and eventual metastasis. SRC-2 was highly elevated in a variety of tumors, especially in prostate cancer, in which SRC-2 was amplified and overexpressed in 37% of the metastatic tumors evaluated. In prostate cancer cells, SRC-2 stimulated reductive carboxylation of α-ketoglutarate to generate citrate via retrograde TCA cycling, promoting lipogenesis and reprogramming of glutamine metabolism. Glutamine-mediated nutrient signaling activated SRC-2 via mTORC1-dependent phosphorylation, which then triggered downstream transcriptional responses by coactivating SREBP-1, which subsequently enhanced lipogenic enzyme expression. Metabolic profiling of human prostate tumors identified a massive increase in the SRC-2–driven metabolic signature in metastatic tumors compared with that seen in localized tumors, further implicating SRC-2 as a prominent metabolic coordinator of cancer metastasis. Moreover, SRC-2 inhibition in murine models severely attenuated the survival, growth, and metastasis of prostate cancer. Together, these results suggest that the SRC-2 pathway has potential as a therapeutic target for prostate cancer. PMID:25664849

Specific Cell (Re-)Programming: Approaches and Perspectives.

PubMed

Hausburg, Frauke; Jung, Julia Jeannine; David, Robert

2018-01-01

Many disorders are manifested by dysfunction of key cell types or their disturbed integration in complex organs. Thereby, adult organ systems often bear restricted self-renewal potential and are incapable of achieving functional regeneration. This underlies the need for novel strategies in the field of cell (re-)programming-based regenerative medicine as well as for drug development in vitro. The regenerative field has been hampered by restricted availability of adult stem cells and the potentially hazardous features of pluripotent embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Moreover, ethical concerns and legal restrictions regarding the generation and use of ESCs still exist. The establishment of direct reprogramming protocols for various therapeutically valuable somatic cell types has overcome some of these limitations. Meanwhile, new perspectives for safe and efficient generation of different specified somatic cell types have emerged from numerous approaches relying on exogenous expression of lineage-specific transcription factors, coding and noncoding RNAs, and chemical compounds.It should be of highest priority to develop protocols for the production of mature and physiologically functional cells with properties ideally matching those of their endogenous counterparts. Their availability can bring together basic research, drug screening, safety testing, and ultimately clinical trials. Here, we highlight the remarkable successes in cellular (re-)programming, which have greatly advanced the field of regenerative medicine in recent years. In particular, we review recent progress on the generation of cardiomyocyte subtypes, with a focus on cardiac pacemaker cells. Graphical Abstract.

Investigation of Rett syndrome using pluripotent stem cells.

PubMed

Dajani, Rana; Koo, Sung-Eun; Sullivan, Gareth J; Park, In-Hyun

2013-11-01

Rett syndrome (RTT) is one of most prevalent female neurodevelopmental disorders. De novo mutations in X-linked MECP2 are mostly responsible for RTT. Since the identification of MeCP2 as the underlying cause of RTT, murine models have contributed to understanding the pathophysiology of RTT and function of MeCP2. Reprogramming is a procedure to produce induced pluripotent stem cells (iPSCs) by overexpression of four transcription factors. iPSCs obtain similar features as embryonic stem cells and are capable of self-renewing and differentiating into cells of all three layers. iPSCs have been utilized in modeling human diseases in vitro. Neurons differentiated from RTT-iPSCs showed the recapitulation of RTT phenotypes. Despite the early success, genetic and epigenetic instability upon reprogramming and ensuing maintenance of iPSCs raise concerns in using RTT-iPSCs as an accurate in vitro model. Here, we update the current iPSC-based RTT modeling, and its concerns and challenges. © 2013 Wiley Periodicals, Inc.

Protein kinase D1 drives pancreatic acinar cell reprogramming and progression to intraepithelial neoplasia

NASA Astrophysics Data System (ADS)

Liou, Geou-Yarh; Döppler, Heike; Braun, Ursula B.; Panayiotou, Richard; Scotti Buzhardt, Michele; Radisky, Derek C.; Crawford, Howard C.; Fields, Alan P.; Murray, Nicole R.; Wang, Q. Jane; Leitges, Michael; Storz, Peter

2015-02-01

The transdifferentiation of pancreatic acinar cells to a ductal phenotype (acinar-to-ductal metaplasia, ADM) occurs after injury or inflammation of the pancreas and is a reversible process. However, in the presence of activating Kras mutations or persistent epidermal growth factor receptor (EGF-R) signalling, cells that underwent ADM can progress to pancreatic intraepithelial neoplasia (PanIN) and eventually pancreatic cancer. In transgenic animal models, ADM and PanINs are initiated by high-affinity ligands for EGF-R or activating Kras mutations, but the underlying signalling mechanisms are not well understood. Here, using a conditional knockout approach, we show that protein kinase D1 (PKD1) is sufficient to drive the reprogramming process to a ductal phenotype and progression to PanINs. Moreover, using 3D explant culture of primary pancreatic acinar cells, we show that PKD1 acts downstream of TGFα and Kras, to mediate formation of ductal structures through activation of the Notch pathway.

Salmonella enterica serovar-specific transcriptional reprogramming of infected cells.

PubMed

Hannemann, Sebastian; Galán, Jorge E

2017-07-01

Despite their high degree of genomic similarity, different Salmonella enterica serovars are often associated with very different clinical presentations. In humans, for example, the typhoidal S. enterica serovar Typhi causes typhoid fever, a life-threatening systemic disease. In contrast, the non-typhoidal S. enterica serovar Typhimurium causes self-limiting gastroenteritis. The molecular bases for these different clinical presentations are incompletely understood. The ability to re-program gene expression in host cells is an essential virulence factor for typhoidal and non-typhoidal S. enterica serovars. Here, we have compared the transcriptional profile of cultured epithelial cells infected with S. Typhimurium or S. Typhi. We found that both serovars stimulated distinct transcriptional responses in infected cells that are associated with the stimulation of specific signal transduction pathways. These specific responses were associated with the presence of a distinct repertoire of type III secretion effector proteins. These observations provide major insight into the molecular bases for potential differences in the pathogenic mechanisms of typhoidal and non-typhoidal S. enterica serovars.

Spatiotemporal dynamics of OCT4 protein localization during preimplantation development in mice.

PubMed

Fukuda, Atsushi; Mitani, Atsushi; Miyashita, Toshiyuki; Kobayashi, Hisato; Umezawa, Akihiro; Akutsu, Hidenori

2016-11-01

Spatiotemporal expression of transcription factors is crucial for genomic reprogramming. Pou5f1 (Oct4) is an essential transcription factor for reprogramming. A recent study reported that OCT4A, which is crucial for establishment and maintenance of pluripotent cells, is expressed in oocytes, but maternal OCT4A is dispensable for totipotency induction. Whereas another study reported that OCT4B, which is not related to pluripotency, is predominantly expressed instead of OCT4A during early preimplantation phases in mice. To determine the expression states of OCT4 in murine preimplantation embryos, we conducted in-depth expression and functional analyses. We found that pluripotency-related OCT4 mainly localizes to the cytoplasm in early preimplantation phases, with no major nuclear localization until the 8-16-cell stage despite high expression in both oocytes and early embryos. RNA-sequencing analysis using oocytes and early preimplantation embryos could not identify the splice variants creating alternative forms of OCT4 protein. Forced expression of OCT4 in zygotes by the injection of polyadenylated mRNA clearly showed nuclear localization of OCT4 protein around 3-5-fold greater than physiological levels and impaired developmental competency in a dose-dependent manner. Embryos with modest overexpression of OCT4 could develop to the 16-cell stage; however, more than 50% of the embryos were arrested at this stage, similar to the results for OCT4 depletion. In contrast, extensive overexpression of OCT4 resulted in complete arrest at the 2-cell stage accompanied by downregulation of zygotically activated genes and repetitive elements related to the totipotent state. These results demonstrated that OCT4 protein localization was spatiotemporally altered during preimplantation development, and strict control of Oct4 protein levels was essential for proper totipotential reprogramming. © 2016 Society for Reproduction and Fertility.

Evaluation of porcine stem cell competence for somatic cell nuclear transfer and production of cloned animals.

PubMed

Secher, Jan O; Liu, Ying; Petkov, Stoyan; Luo, Yonglun; Li, Dong; Hall, Vanessa J; Schmidt, Mette; Callesen, Henrik; Bentzon, Jacob F; Sørensen, Charlotte B; Freude, Kristine K; Hyttel, Poul

2017-03-01

Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells. Copyright © 2017 Elsevier B.V. All rights reserved.

In Vivo Cardiac Cellular Reprogramming Efficacy Is Enhanced by Angiogenic Preconditioning of the Infarcted Myocardium With Vascular Endothelial Growth Factor

PubMed Central

Mathison, Megumi; P. Gersch, Robert; Nasser, Ahmed; Lilo, Sarit; Korman, Mallory; Fourman, Mitchell; Hackett, Neil; Shroyer, Kenneth; Yang, Jianchang; Ma, Yupo; Crystal, Ronald G.; Rosengart, Todd K.

2012-01-01

Background In situ cellular reprogramming offers the possibility of regenerating functional cardiomyocytes directly from scar fibroblasts, obviating the challenges of cell implantation. We hypothesized that pretreating scar with gene transfer of the angiogenic vascular endothelial growth factor (VEGF) would enhance the efficacy of this strategy. Methods and Results Gata4, Mef2c, and Tbx5 (GMT) administration via lentiviral transduction was demonstrated to transdifferentiate rat fibroblasts into (induced) cardiomyocytes in vitro by cardiomyocyte marker studies. Fisher 344 rats underwent coronary ligation and intramyocardial administration of an adenovirus encoding all 3 major isoforms of VEGF (AdVEGF‐All6A+) or an AdNull control vector (n=12/group). Lentivirus encoding GMT or a GFP control was administered to each animal 3 weeks later, followed by histologic and echocardiographic analyses. GMT administration reduced the extent of fibrosis by half compared with GFP controls (12±2% vs 24±3%, P<0.01) and reduced the number of myofibroblasts detected in the infarct zone by 4‐fold. GMT‐treated animals also demonstrated greater density of cardiomyocyte‐specific marker beta myosin heavy chain 7+ cells compared with animals receiving GFP with or without VEGF (P<0.01). Ejection fraction was significantly improved after GMT vs GFP administration (12±3% vs −7±3%, P<0.01). Eight (73%) GFP animals but no GMT animals demonstrated decreased ejection fraction during this interval (P<0.01). Also, improvement in ejection fraction was 4‐fold greater in GMT/VEGF vs GMT/null animals (17±2% vs 4±1%, P<0.05). Conclusions VEGF administration to infarcted myocardium enhances the efficacy of GMT‐mediated cellular reprogramming in improving myocardial function and reducing the extent of myocardial fibrosis compared with the use of GMT or VEGF alone. PMID:23316332

Mammalian designer cells: Engineering principles and biomedical applications.

PubMed

Xie, Mingqi; Fussenegger, Martin

2015-07-01

Biotechnology is a widely interdisciplinary field focusing on the use of living cells or organisms to solve established problems in medicine, food production and agriculture. Synthetic biology, the science of engineering complex biological systems that do not exist in nature, continues to provide the biotechnology industry with tools, technologies and intellectual property leading to improved cellular performance. One key aspect of synthetic biology is the engineering of deliberately reprogrammed designer cells whose behavior can be controlled over time and space. This review discusses the most commonly used techniques to engineer mammalian designer cells; while control elements acting on the transcriptional and translational levels of target gene expression determine the kinetic and dynamic profiles, coupling them to a variety of extracellular stimuli permits their remote control with user-defined trigger signals. Designer mammalian cells with novel or improved biological functions not only directly improve the production efficiency during biopharmaceutical manufacturing but also open the door for cell-based treatment strategies in molecular and translational medicine. In the future, the rational combination of multiple sets of designer cells could permit the construction and regulation of higher-order systems with increased complexity, thereby enabling the molecular reprogramming of tissues, organisms or even populations with highest precision. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fast-ball sports experts depend on an inhibitory strategy to reprogram their movement timing.

PubMed

Nakamoto, Hiroki; Ikudome, Sachi; Yotani, Kengo; Maruyama, Atsuo; Mori, Shiro

2013-07-01

The purpose of our study was to clarify whether an inhibitory strategy is used for reprogramming of movement timing by experts in fast-ball sports when they correct their movement timing due to unexpected environmental changes. We evaluated the influence of disruption of inhibitory function of the right inferior frontal gyrus (rIFG) on reprogramming of movement timing of experts and non-experts in fast-ball sports. The task was to manually press a button to coincide with the arrival of a moving target. The target moved at a constant velocity, and its velocity was suddenly either increased or decreased in some trials. The task was performed either with or without transcranial magnetic stimulation (TMS), which was delivered to the region of the rIFG. Under velocity change conditions without TMS, the experts showed significantly smaller timing errors and a higher rate of reprogramming of movement timing than the non-experts. Moreover, TMS application during the task significantly diminished the expert group's performance, but not the control group, particularly in the condition where the target velocity decreases. These results suggest that experts use an inhibitory strategy for reprogramming of movement timing. In addition, the rIFG inhibitory function contributes to the superior movement correction of experts in fast-ball sports.

Plant chromatin warms up in Madrid

PubMed Central

Jarillo, José A; Gaudin, Valerie; Hennig, Lars; Köhler, Claudia; Piñeiro, Manuel

2014-01-01

The 3rd European Workshop on Plant Chromatin (EWPC) was held on August 2013 in Madrid, Spain. A number of different topics on plant chromatin were presented during the meeting, including new factors mediating Polycomb Group protein function in plants, chromatin-mediated reprogramming in plant developmental transitions, the role of histone variants, and newly identified chromatin remodeling factors. The function of interactions between chromatin and transcription factors in the modulation of gene expression, the role of chromatin dynamics in the control of nuclear processes and the influence of environmental factors on chromatin organization were also reported. In this report, we highlight some of the new insights emerging in this growing area of research, presented at the 3rd EWPC. PMID:24504145

Overcoming Endocrine Resistance by Targeting ER/FoxA1/IL 8 Axis

DTIC Science & Technology

2016-10-01

INTRODUCTION Approximately 75% of breast cancers express the hormone estrogen receptor α (ER). As a critical determinant in estrogen response and oncogenic...factor of estrogen receptor α (ER)–chromatin binding and function, yet its aberration in endocrine-resistant (Endo-R) breast cancer is unknown. Here, we...positive tumors. FOXA1 | estrogen receptor | breast cancer | transcriptional reprogramming | endocrine resistance About 75% of breast cancers express

Human Factors Research in Aircrew Performance and Training: 1990 Annual Summary Report

DTIC Science & Technology

1991-06-01

TRS) . . . . . . . . . 97 DEVELOPMENT OF A METHODOLOGY FOR MEASURING BOTH CONSCIOUS AND SUBCONSCIOUS ASPECTS OF AIRCREW COORDINATION IN ARMY HELICOPTER...Perkin-Elmer mini- computer and FORTRAN programming language. The model was later reprogrammed using the TOSS software and an IBM personal computer. The...will be conducted by the UAFDL. 101 44 DEVELOPMENT OF A METHODOLOGY FOR MEASURING BOTH CONSCIOUS .AND SUBCONSCIOUS ASPECTS OF AIRCREW COORDINATION IN

Cloning Mice.

PubMed

Ogura, Atsuo

2017-08-01

Viable and fertile mice can be generated by somatic nuclear transfer into enucleated oocytes, presumably because the transplanted somatic cell genome becomes reprogrammed by factors in the oocyte. The first somatic cloned offspring of mice were obtained by directly injecting donor nuclei into recipient enucleated oocytes. When this method is used (the so-called Honolulu method of somatic cell nuclear transfer [SCNT]), the donor nuclei readily and completely condense within the enucleated metaphase II-arrested oocytes, which contain high levels of M-phase-promoting factor (MPF). It is believed that the condensation of the donor chromosomes promotes complete reprogramming of the donor genome within the mouse oocytes. Another key to the success of mouse cloning is the use of blunt micropipettes attached to a piezo impact-driving micromanipulation device. This system saves a significant amount of time during the micromanipulation of oocytes and thus minimizes the loss of oocyte viability in vitro. For example, a group of 20 oocytes can be enucleated within 10 min by an experienced operator. This protocol is composed of seven parts: (1) preparing micropipettes, (2) setting up the enucleation and injection micropipettes, (3) collecting and enucleating oocytes, (4) preparing nucleus donor cells, (5) injecting donor nuclei, (6) activating embryos and culturing, and (7) transferring cloned embryos. © 2017 Cold Spring Harbor Laboratory Press.

Reprogramming of Melanoma Tumor-Infiltrating Lymphocytes to Induced Pluripotent Stem Cells

PubMed Central

Saito, Hidehito; Okita, Keisuke; Fusaki, Noemi; Sabel, Michael S.; Chang, Alfred E.; Ito, Fumito

2016-01-01

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for autologous cell therapies. One of the possible applications of iPSCs is to use them as a cell source for producing autologous lymphocytes for cell-based therapy against cancer. Tumor-infiltrating lymphocytes (TILs) that express programmed cell death protein-1 (PD-1) are tumor-reactive T cells, and adoptive cell therapy with autologous TILs has been found to achieve durable complete response in selected patients with metastatic melanoma. Here, we describe the derivation of human iPSCs from melanoma TILs expressing high level of PD-1 by Sendai virus-mediated transduction of the four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC. TIL-derived iPSCs display embryonic stem cell-like morphology, have normal karyotype, express stem cell-specific surface antigens and pluripotency-associated transcription factors, and have the capacity to differentiate in vitro and in vivo. A wide variety of T cell receptor gene rearrangement patterns in TIL-derived iPSCs confirmed the heterogeneity of T cells infiltrating melanomas. The ability to reprogram TILs containing patient-specific tumor-reactive repertoire might allow the generation of patient- and tumor-specific polyclonal T cells for cancer immunotherapy. PMID:27057178

Transcriptional control of the autophagy-lysosome system in pancreatic cancer

PubMed Central

Perera, Rushika M.; Stoykova, Svetlana; Nicolay, Brandon N.; Ross, Kenneth N.; Fitamant, Julien; Boukhali, Myriam; Lengrand, Justine; Deshpande, Vikram; Selig, Martin K.; Ferrone, Cristina R.; Settleman, Jeff; Stephanopoulos, Gregory; Dyson, Nicholas J.; Zoncu, Roberto; Ramaswamy, Sridhar; Haas, Wilhelm; Bardeesy, Nabeel

2016-01-01

Activation of cellular stress response pathways to maintain metabolic homeostasis is emerging as a critical growth and survival mechanism in many cancers1. The pathogenesis of pancreatic ductal adenocarcinoma (PDA) requires high levels of autophagy2–4, a conserved self-degradative process5. However, the regulatory circuits that activate autophagy and reprogram PDA cell metabolism are unknown. We now show that autophagy induction in PDA occurs as part of a broader transcriptional program that coordinates activation of lysosome biogenesis and function, and nutrient scavenging, mediated by the MiT/TFE family transcription factors. In PDA cells, the MiT/TFE proteins6 – MITF, TFE3 and TFEB – are decoupled from regulatory mechanisms that control their cytoplasmic retention. Increased nuclear import in turn drives the expression of a coherent network of genes that induce high levels of lysosomal catabolic function essential for PDA growth. Unbiased global metabolite profiling reveals that MiT/TFE-dependent autophagy-lysosomal activation is specifically required to maintain intracellular amino acid (AA) pools. These results identify the MiT/TFE transcription factors as master regulators of metabolic reprogramming in pancreatic cancer and demonstrate activation of clearance pathways converging on the lysosome as a novel hallmark of aggressive malignancy. PMID:26168401

Thinking outside the liver: Induced pluripotent stem cells for hepatic applications

PubMed Central

Subba Rao, Mekala; Sasikala, Mitnala; Reddy, D Nageshwar

2013-01-01

The discovery of induced pluripotent stem cells (iPSCs) unraveled a mystery in stem cell research, after identification of four re-programming factors for generating pluripotent stem cells without the need of embryos. This breakthrough in generating iPSCs from somatic cells has overcome the ethical issues and immune rejection involved in the use of human embryonic stem cells. Hence, iPSCs form a great potential source for developing disease models, drug toxicity screening and cell-based therapies. These cells have the potential to differentiate into desired cell types, including hepatocytes, under in vitro as well as under in vivo conditions given the proper microenvironment. iPSC-derived hepatocytes could be useful as an unlimited source, which can be utilized in disease modeling, drug toxicity testing and producing autologous cell therapies that would avoid immune rejection and enable correction of gene defects prior to cell transplantation. In this review, we discuss the induction methods, role of reprogramming factors, and characterization of iPSCs, along with hepatocyte differentiation from iPSCs and potential applications. Further, we discuss the location and detection of liver stem cells and their role in liver regeneration. Although tumor formation and genetic mutations are a cause of concern, iPSCs still form a promising source for clinical applications. PMID:23801830

Thinking outside the liver: induced pluripotent stem cells for hepatic applications.

PubMed

Subba Rao, Mekala; Sasikala, Mitnala; Nageshwar Reddy, D

2013-06-14

The discovery of induced pluripotent stem cells (iPSCs) unraveled a mystery in stem cell research, after identification of four re-programming factors for generating pluripotent stem cells without the need of embryos. This breakthrough in generating iPSCs from somatic cells has overcome the ethical issues and immune rejection involved in the use of human embryonic stem cells. Hence, iPSCs form a great potential source for developing disease models, drug toxicity screening and cell-based therapies. These cells have the potential to differentiate into desired cell types, including hepatocytes, under in vitro as well as under in vivo conditions given the proper microenvironment. iPSC-derived hepatocytes could be useful as an unlimited source, which can be utilized in disease modeling, drug toxicity testing and producing autologous cell therapies that would avoid immune rejection and enable correction of gene defects prior to cell transplantation. In this review, we discuss the induction methods, role of reprogramming factors, and characterization of iPSCs, along with hepatocyte differentiation from iPSCs and potential applications. Further, we discuss the location and detection of liver stem cells and their role in liver regeneration. Although tumor formation and genetic mutations are a cause of concern, iPSCs still form a promising source for clinical applications.

Cell Fusion Reprogramming Leads to a Specific Hepatic Expression Pattern during Mouse Bone Marrow Derived Hepatocyte Formation In Vivo

PubMed Central

Arza, Elvira; Alvarez-Barrientos, Alberto; Fabregat, Isabel; Garcia-Bravo, Maria; Meza, Nestor W.; Segovia, Jose C.

2012-01-01

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-β1 (TGF-β1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation. PMID:22457803

Heart Development, Diseases, and Regeneration　- New Approaches From Innervation, Fibroblasts, and Reprogramming.

PubMed

Ieda, Masaki

2016-09-23

It is well known that cardiac function is tightly controlled by neural activity; however, the molecular mechanism of cardiac innervation during development and the relationship with heart disease remain undetermined. My work has revealed the molecular networks that govern cardiac innervation and its critical roles in heart diseases such as silent myocardial ischemia and arrhythmias. Cardiomyocytes proliferate during embryonic development, but lose their proliferative capacity after birth. Cardiac fibroblasts are a major source of cells during fibrosis and induce cardiac hypertrophy after myocardial injury in the adult heart. Despite the importance of fibroblasts in the adult heart, the role of fibroblasts in embryonic heart development was previously not determined. I demonstrated that cardiac fibroblasts play important roles in myocardial growth and cardiomyocyte proliferation during embryonic development, and I identified key paracrine factors and signaling pathways. In contrast to embryonic cardiomyocytes, adult cardiomyocytes have little regenerative capacity, leading to heart failure and high mortality rates after myocardial infarction. Leveraging the knowledge of developmental biology, I identified cardiac reprogramming factors that can directly convert resident cardiac fibroblasts into cardiomyocytes for heart regeneration. These findings greatly improved our understanding of heart development and diseases, and provide a new strategy for heart regenerative therapy. (Circ J 2016; 80: 2081-2088).

Shaping Gene Expression by Landscaping Chromatin Architecture: Lessons from a Master.

PubMed

Sartorelli, Vittorio; Puri, Pier Lorenzo

2018-05-19

Since its discovery as a skeletal muscle-specific transcription factor able to reprogram somatic cells into differentiated myofibers, MyoD has provided an instructive model to understand how transcription factors regulate gene expression. Reciprocally, studies of other transcriptional regulators have provided testable hypotheses to further understand how MyoD activates transcription. Using MyoD as a reference, in this review, we discuss the similarities and differences in the regulatory mechanisms employed by tissue-specific transcription factors to access DNA and regulate gene expression by cooperatively shaping the chromatin landscape within the context of cellular differentiation. Copyright © 2018 Elsevier Inc. All rights reserved.

Krüppel-like factors in mammalian stem cells and development

PubMed Central

Bialkowska, Agnieszka B.; Yang, Vincent W.

2017-01-01

Krüppel-like factors (KLFs) are a family of zinc-finger transcription factors that are found in many species. Recent studies have shown that KLFs play a fundamental role in regulating diverse biological processes such as cell proliferation, differentiation, development and regeneration. Of note, several KLFs are also crucial for maintaining pluripotency and, hence, have been linked to reprogramming and regenerative medicine approaches. Here, we review the crucial functions of KLFs in mammalian embryogenesis, stem cell biology and regeneration, as revealed by studies of animal models. We also highlight how KLFs have been implicated in human diseases and outline potential avenues for future research. PMID:28246209

Environmental Estrogens Differentially Engage the Histone Methyltransferase EZH2 to Increase Risk of Uterine Tumorigenesis

PubMed Central

Greathouse, K. Leigh; Bredfeldt, Tiffany; Everitt, Jeffrey I.; Lin, Kevin; Berry, Tia; Kannan, Kurunthachalam; Mittelstadt, Megan L.; Ho, Shuk-mei; Walker, Cheryl L.

2013-01-01

Environmental exposures during sensitive windows of development can reprogram normal physiological responses and alter disease susceptibility later in life in a process known as developmental reprogramming. For example, exposure to the xenoestrogen diethylstilbestrol (DES) during reproductive tract development can reprogram estrogen-responsive gene expression in the myometrium, resulting in hyper-responsiveness to hormone in the adult uterus and promotion of hormone-dependent uterine leiomyoma. We show here that the environmental estrogens genistein (GEN), a soy phytoestrogen, and the plasticizer bisphenol A (BPA), differ in their pattern of developmental reprogramming and promotion of tumorigenesis (leiomyomas) in the uterus. While both GEN and BPA induce genomic estrogen receptor (ER) signaling in the developing uterus, only GEN induced PI3K/AKT non-genomic ER signaling to the histone methyltransferase Enhancer of Zeste homolog 2 (EZH2). As a result, this “pre-genomic” signaling phosphorylates and represses EZH2, and reduces levels of H3K27 repressive mark in chromatin. Furthermore, only GEN caused estrogen-responsive genes in the adult myometrium to become hyper-responsive to hormone; estrogen-responsive genes were repressed in BPA exposed uteri. Importantly, this pattern of EZH2 engagement to decrease versus increase H3K27 methylation correlated with the effect of these xenoestrogens on tumorigenesis. Developmental reprogramming by GEN promoted development of uterine leiomyomas, increasing tumor incidence and multiplicity, while BPA did not. These data demonstrate that environmental estrogens have distinct non-genomic effects in the developing uterus that determines their ability to engage the epigenetic regulator EZH2, decrease levels of the repressive epigenetic histone H3K27 methyl mark in chromatin during developmental reprogramming, and promote uterine tumorigenesis. PMID:22504913

Reprogramming of human cancer cells to pluripotency for models of cancer progression

PubMed Central

Kim, Jungsun; Zaret, Kenneth S

2015-01-01

The ability to study live cells as they progress through the stages of cancer provides the opportunity to discover dynamic networks underlying pathology, markers of early stages, and ways to assess therapeutics. Genetically engineered animal models of cancer, where it is possible to study the consequences of temporal-specific induction of oncogenes or deletion of tumor suppressors, have yielded major insights into cancer progression. Yet differences exist between animal and human cancers, such as in markers of progression and response to therapeutics. Thus, there is a need for human cell models of cancer progression. Most human cell models of cancer are based on tumor cell lines and xenografts of primary tumor cells that resemble the advanced tumor state, from which the cells were derived, and thus do not recapitulate disease progression. Yet a subset of cancer types have been reprogrammed to pluripotency or near-pluripotency by blastocyst injection, by somatic cell nuclear transfer and by induced pluripotent stem cell (iPS) technology. The reprogrammed cancer cells show that pluripotency can transiently dominate over the cancer phenotype. Diverse studies show that reprogrammed cancer cells can, in some cases, exhibit early-stage phenotypes reflective of only partial expression of the cancer genome. In one case, reprogrammed human pancreatic cancer cells have been shown to recapitulate stages of cancer progression, from early to late stages, thus providing a model for studying pancreatic cancer development in human cells where previously such could only be discerned from mouse models. We discuss these findings, the challenges in developing such models and their current limitations, and ways that iPS reprogramming may be enhanced to develop human cell models of cancer progression. PMID:25712212

The Phosphatidylinositol 3,5-Bisphosphate (PI(3,5)P2)-dependent Tup1 Conversion (PIPTC) Regulates Metabolic Reprogramming from Glycolysis to Gluconeogenesis*

PubMed Central

Han, Bong-Kwan; Emr, Scott D.

2013-01-01

Glucose/carbon metabolism is a fundamental cellular process in living cells. In response to varying environments, eukaryotic cells reprogram their glucose/carbon metabolism between aerobic or anaerobic glycolysis, oxidative phosphorylation, and/or gluconeogenesis. The distinct type of glucose/carbon metabolism that a cell carries out has significant effects on the cell's proliferation and differentiation. However, it is poorly understood how the reprogramming of glucose/carbon metabolism is regulated. Here, we report a novel endosomal PI(3,5)P2 lipid-dependent regulatory mechanism that is required for metabolic reprogramming from glycolysis to gluconeogenesis in Saccharomyces cerevisiae. Certain gluconeogenesis genes, such as FBP1 (encoding fructose-1,6-bisphosphatase 1) and ICL1 (encoding isocitrate lyase 1) are under control of the Mig1 repressor and Cyc8-Tup1 corepressor complex. We previously identified the PI(3,5)P2-dependent Tup1 conversion (PIPTC), a mechanism to convert Cyc8-Tup1 corepressor to Cti6-Cyc8-Tup1 coactivator. We demonstrate that the PIPTC plays a critical role for transcriptional activation of FBP1 and ICL1. Furthermore, without the PIPTC, the Cat8 and Sip4 transcriptional activators cannot be efficiently recruited to the promoters of FBP1 and ICL1, suggesting a key role for the PIPTC in remodulating the chromatin architecture at the promoters. Our findings expand our understanding of the regulatory mechanisms for metabolic reprogramming in eukaryotes to include key regulation steps outside the nucleus. Given that Tup1 and the metabolic enzymes that control PI(3,5)P2 are highly conserved among eukaryotes, our findings may provide important insights toward understanding glucose/carbon metabolic reprogramming in other eukaryotes, including humans. PMID:23733183

The phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)-dependent Tup1 conversion (PIPTC) regulates metabolic reprogramming from glycolysis to gluconeogenesis.

PubMed

Han, Bong-Kwan; Emr, Scott D

2013-07-12

Glucose/carbon metabolism is a fundamental cellular process in living cells. In response to varying environments, eukaryotic cells reprogram their glucose/carbon metabolism between aerobic or anaerobic glycolysis, oxidative phosphorylation, and/or gluconeogenesis. The distinct type of glucose/carbon metabolism that a cell carries out has significant effects on the cell's proliferation and differentiation. However, it is poorly understood how the reprogramming of glucose/carbon metabolism is regulated. Here, we report a novel endosomal PI(3,5)P2 lipid-dependent regulatory mechanism that is required for metabolic reprogramming from glycolysis to gluconeogenesis in Saccharomyces cerevisiae. Certain gluconeogenesis genes, such as FBP1 (encoding fructose-1,6-bisphosphatase 1) and ICL1 (encoding isocitrate lyase 1) are under control of the Mig1 repressor and Cyc8-Tup1 corepressor complex. We previously identified the PI(3,5)P2-dependent Tup1 conversion (PIPTC), a mechanism to convert Cyc8-Tup1 corepressor to Cti6-Cyc8-Tup1 coactivator. We demonstrate that the PIPTC plays a critical role for transcriptional activation of FBP1 and ICL1. Furthermore, without the PIPTC, the Cat8 and Sip4 transcriptional activators cannot be efficiently recruited to the promoters of FBP1 and ICL1, suggesting a key role for the PIPTC in remodulating the chromatin architecture at the promoters. Our findings expand our understanding of the regulatory mechanisms for metabolic reprogramming in eukaryotes to include key regulation steps outside the nucleus. Given that Tup1 and the metabolic enzymes that control PI(3,5)P2 are highly conserved among eukaryotes, our findings may provide important insights toward understanding glucose/carbon metabolic reprogramming in other eukaryotes, including humans.

Focal adhesion kinase-promoted tumor glucose metabolism is associated with a shift of mitochondrial respiration to glycolysis.

PubMed

Zhang, J; Gao, Q; Zhou, Y; Dier, U; Hempel, N; Hochwald, S N

2016-04-14

Cancer cells often gains a growth advantage by taking up glucose at a high rate and undergoing aerobic glycolysis through intrinsic cellular factors that reprogram glucose metabolism. Focal adhesion kinase (FAK), a key transmitter of growth factor and anchorage stimulation, is aberrantly overexpressed or activated in most solid tumors, including pancreatic ductal adenocarcinomas (PDACs). We determined whether FAK can act as an intrinsic driver to promote aerobic glycolysis and tumorigenesis. FAK inhibition decreases and overexpression increases intracellular glucose levels during unfavorable conditions, including growth factor deficiency and cell detachment. Amplex glucose assay, fluorescence and carbon-13 tracing studies demonstrate that FAK promotes glucose consumption and glucose-to-lactate conversion. Extracellular flux analysis indicates that FAK enhances glycolysis and decreases mitochondrial respiration. FAK increases key glycolytic proteins, including enolase, pyruvate kinase M2 (PKM2), lactate dehydrogenase and monocarboxylate transporter. Furthermore, active/tyrosine-phosphorylated FAK directly binds to PKM2 and promotes PKM2-mediated glycolysis. On the other hand, FAK-decreased levels of mitochondrial complex I can result in reduced oxidative phosphorylation (OXPHOS). Attenuation of FAK-enhanced glycolysis re-sensitizes cancer cells to growth factor withdrawal, decreases cell viability and reduces growth of tumor xenografts. These observations, for the first time, establish a vital role of FAK in cancer glucose metabolism through alterations in the OXPHOS-to-glycolysis balance. Broadly targeting the common phenotype of aerobic glycolysis and more specifically FAK-reprogrammed glucose metabolism will disrupt the bioenergetic and biosynthetic supply for uncontrolled growth of tumors, particularly glycolytic PDAC.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhadurihauck, Anjuli; Li, Lei; Li, Qianqian

Adoptive cell transfer therapy (ACT) is one of the most promising immunotherapies against cancer, using tumor-infiltrating lymphocytes (TILs) expanded in vitro. Tumor-infiltrating cytotoxic T lymphocytes (TICTLs) play a prominent role in cancer control. TILs terminally differentiate in response to immunosuppressive environments within tumors, and thus are slow to expand and challenging to maintain both in vitro and in patients. To reverse this exhaustion, we utilize a nuclear protein delivery system that exposes TICTLs to the SOX2, Oct-4, and NANOG (SON) proteins. Unlike activated naïve CTLs (effector CTLs), TICTLs respond favorably to SON treatment, exhibiting steady proliferation and extended survivability independent of cytokinemore » and antigen stimulation. Though TICTLs treated with SON (STICTLs) still express T cell receptors as well as other critical downstream components, they are unresponsive to antigen challenge, suggesting that SON treatment regresses TICTLs into a state similar to that of an early double negative T cell. Our findings indicate the TICTL response to SON proteins is unique when compared to effector CTLs, suggesting TICTLs may be sensitive to regulation by other lineage-specific transcription factors and opening a promising new avenue into cancer immunotherapy. To our knowledge, this is the first report on lineage reprogramming of TILs using protein stem cell transcription factors delivered directly to the nucleus. -- Highlights: •TICTLs are sensitive to reprogramming by proteins of stem cell transcription factors, but effector CTLs were not. •TICTLs are regressed back to an early double negative T cell stage. •TCR signaling is deregulated by these transcription factors.« less

Nanog is an essential factor for induction of pluripotency in somatic cells from endangered felids.

PubMed

Verma, Rajneesh; Liu, Jun; Holland, Michael Kenneth; Temple-Smith, Peter; Williamson, Mark; Verma, Paul John

2013-02-01

Nanog has an important role in pluripotency induction in bovines and snow leopards. To examine whether it was required for wild felids globally, we examined the induction of pluripotency in felids from Asia (Bengal tiger, Panthera tigris), Africa (serval, Leptailurus serval), and the Americas (jaguar, Panthera onca). Dermal fibroblasts were transduced with genes encoding the human transcription factors OCT4, SOX2, KLF4, and cMYC with or without NANOG. Both four- and five-factor induction resulted in colony formation at day 3 in all three species tested; however, we were not able to maintain colonies that were generated without NANOG beyond passage (P) 7. Five-factor induced pluripotent stem cell (iPSC) colonies from wild cats were expanded in vitro on feeder layers and were positive for alkaline phosphatase and protein expression of OCT-4, NANOG, and stage-specific embryonic antigen-4 at P4 and P14. Reverse-transcription polymerase chain reaction confirmed that all five human transgenes were transcribed at P4; however, OCT4, SOX2, and NANOG transgenes were silenced by P14. Endogenous OCT4 and NANOG transcripts were detected at P4 and P14 in all cell lines confirming successful reprogramming. At P14, the iPSCs from all three species remained euploid and differentiated in vivo and in vitro into derivatives of the three germ layers. This study describes an effective method for inducing pluripotency in three endangered wild cats from across the globe and confirms Nanog as an essential factor in the reprogramming event. Efficient production of iPSC from endangered felids creates a unique opportunity for species preservation through gamete production, nuclear transfer, embryo complementation, and future novel technologies.

Nanog Is an Essential Factor for Induction of Pluripotency in Somatic Cells from Endangered Felids

PubMed Central

Verma, Rajneesh; Liu, Jun; Holland, Michael Kenneth; Temple-Smith, Peter; Williamson, Mark

2013-01-01

Abstract Nanog has an important role in pluripotency induction in bovines and snow leopards. To examine whether it was required for wild felids globally, we examined the induction of pluripotency in felids from Asia (Bengal tiger, Panthera tigris), Africa (serval, Leptailurus serval), and the Americas (jaguar, Panthera onca). Dermal fibroblasts were transduced with genes encoding the human transcription factors OCT4, SOX2, KLF4, and cMYC with or without NANOG. Both four- and five-factor induction resulted in colony formation at day 3 in all three species tested; however, we were not able to maintain colonies that were generated without NANOG beyond passage (P) 7. Five-factor induced pluripotent stem cell (iPSC) colonies from wild cats were expanded in vitro on feeder layers and were positive for alkaline phosphatase and protein expression of OCT-4, NANOG, and stage-specific embryonic antigen-4 at P4 and P14. Reverse-transcription polymerase chain reaction confirmed that all five human transgenes were transcribed at P4; however, OCT4, SOX2, and NANOG transgenes were silenced by P14. Endogenous OCT4 and NANOG transcripts were detected at P4 and P14 in all cell lines confirming successful reprogramming. At P14, the iPSCs from all three species remained euploid and differentiated in vivo and in vitro into derivatives of the three germ layers. This study describes an effective method for inducing pluripotency in three endangered wild cats from across the globe and confirms Nanog as an essential factor in the reprogramming event. Efficient production of iPSC from endangered felids creates a unique opportunity for species preservation through gamete production, nuclear transfer, embryo complementation, and future novel technologies. PMID:23514873

Altered gene products involved in the malignant reprogramming of cancer stem/progenitor cells and multitargeted therapies

PubMed Central

Mimeault, Murielle; Batra, Surinder K.

2013-01-01

Recent studies in the field of cancer stem cells have revealed that the alterations in key gene products involved in the epithelial-mesenchymal transition (EMT) program, altered metabolic pathways such as enhanced glycolysis, lipogenesis and/or autophagy and treatment resistance may occur in cancer stem/progenitor cells and their progenies during cancer progression. Particularly, the sustained activation of diverse developmental cascades such as hedgehog, epidermal growth factor receptor (EGFR), Wnt/β-catenin, Notch, transforming growth factor-β (TGF-β)/TGF-βR receptors and/or stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) can play critical functions for high self-renewal potential, survival, invasion and metastases of cancer stem/progenitor cells and their progenies. It has also been observed that cancer cells may be reprogrammed to re-express different pluripotency-associated stem cell-like markers such as Myc, Oct-3/4, Nanog and Sox-2 along the EMT process and under stressful and hypoxic conditions. Moreover, the enhanced expression and/or activities of some drug resistance-associated molecules such as Bcl-2, Akt/molecular target of rapamycin (mTOR), nuclear factor-kappaB (NF-κB), hypoxia-inducible factors (HIFs), macrophage inhibitory cytokine-1 (MIC-1) and ATP-binding cassette (ABC) multidrug transporters frequently occur in cancer cells during cancer progression and metastases. These molecular events may cooperate for the survival and acquisition of a more aggressive and migratory behavior by cancer stem/progenitor cells and their progenies during cancer transition to metastatic and recurrent disease states. Of therapeutic interest, these altered gene products may also be exploited as molecular biomarkers and therapeutic targets to develop novel multitargeted strategies for improving current cancer therapies and preventing disease relapse. PMID:23994756

Fatty acid metabolic reprogramming via mTOR-mediated inductions of PPARγ directs early activation of T cells

PubMed Central

Angela, Mulki; Endo, Yusuke; Asou, Hikari K.; Yamamoto, Takeshi; Tumes, Damon J.; Tokuyama, Hirotake; Yokote, Koutaro; Nakayama, Toshinori

2016-01-01

To fulfil the bioenergetic requirements for increased cell size and clonal expansion, activated T cells reprogramme their metabolic signatures from energetically quiescent to activated. However, the molecular mechanisms and essential components controlling metabolic reprogramming in T cells are not well understood. Here, we show that the mTORC1–PPARγ pathway is crucial for the fatty acid uptake programme in activated CD4+ T cells. This pathway is required for full activation and rapid proliferation of naive and memory CD4+ T cells. PPARγ directly binds and induces genes associated with fatty acid uptake in CD4+ T cells in both mice and humans. The PPARγ-dependent fatty acid uptake programme is critical for metabolic reprogramming. Thus, we provide important mechanistic insights into the metabolic reprogramming mechanisms that govern the expression of key enzymes, fatty acid metabolism and the acquisition of an activated phenotype during CD4+ T cell activation. PMID:27901044

Design principles for nuclease-deficient CRISPR-based transcriptional regulators

PubMed Central

Jensen, Michael K

2018-01-01

Abstract The engineering of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins continues to expand the toolkit available for genome editing, reprogramming gene regulation, genome visualisation and epigenetic studies of living organisms. In this review, the emerging design principles on the use of nuclease-deficient CRISPR-based reprogramming of gene expression will be presented. The review will focus on the designs implemented in yeast both at the level of CRISPR proteins and guide RNA (gRNA), but will lend due credits to the seminal studies performed in other species where relevant. In addition to design principles, this review also highlights applications benefitting from the use of CRISPR-mediated transcriptional regulation and discusses the future directions to further expand the toolkit for nuclease-deficient reprogramming of genomes. As such, this review should be of general interest for experimentalists to get familiarised with the parameters underlying the power of reprogramming genomic functions by use of nuclease-deficient CRISPR technologies. PMID:29726937

Reprogramming Glia Into Neurons in the Peripheral Auditory System as a Solution for Sensorineural Hearing Loss: Lessons From the Central Nervous System

PubMed Central

Meas, Steven J.; Zhang, Chun-Li; Dabdoub, Alain

2018-01-01

Disabling hearing loss affects over 5% of the world’s population and impacts the lives of individuals from all age groups. Within the next three decades, the worldwide incidence of hearing impairment is expected to double. Since a leading cause of hearing loss is the degeneration of primary auditory neurons (PANs), the sensory neurons of the auditory system that receive input from mechanosensory hair cells in the cochlea, it may be possible to restore hearing by regenerating PANs. A direct reprogramming approach can be used to convert the resident spiral ganglion glial cells into induced neurons to restore hearing. This review summarizes recent advances in reprogramming glia in the CNS to suggest future steps for regenerating the peripheral auditory system. In the coming years, direct reprogramming of spiral ganglion glial cells has the potential to become one of the leading biological strategies to treat hearing impairment. PMID:29593497

Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis.

PubMed

Mao, Guohong; Meng, Xiangzong; Liu, Yidong; Zheng, Zuyu; Chen, Zhixiang; Zhang, Shuqun

2011-04-01

Plant sensing of invading pathogens triggers massive metabolic reprogramming, including the induction of secondary antimicrobial compounds known as phytoalexins. We recently reported that MPK3 and MPK6, two pathogen-responsive mitogen-activated protein kinases, play essential roles in the induction of camalexin, the major phytoalexin in Arabidopsis thaliana. In search of the transcription factors downstream of MPK3/MPK6, we found that WRKY33 is required for MPK3/MPK6-induced camalexin biosynthesis. In wrky33 mutants, both gain-of-function MPK3/MPK6- and pathogen-induced camalexin production are compromised, which is associated with the loss of camalexin biosynthetic gene activation. WRKY33 is a pathogen-inducible transcription factor, whose expression is regulated by the MPK3/MPK6 cascade. Chromatin immunoprecipitation assays reveal that WRKY33 binds to its own promoter in vivo, suggesting a potential positive feedback regulatory loop. Furthermore, WRKY33 is a substrate of MPK3/MPK6. Mutation of MPK3/MPK6 phosphorylation sites in WRKY33 compromises its ability to complement the camalexin induction in the wrky33 mutant. Using a phospho-protein mobility shift assay, we demonstrate that WRKY33 is phosphorylated by MPK3/MPK6 in vivo in response to Botrytis cinerea infection. Based on these data, we conclude that WRKY33 functions downstream of MPK3/MPK6 in reprogramming the expression of camalexin biosynthetic genes, which drives the metabolic flow to camalexin production in Arabidopsis challenged by pathogens.

MicroRNA in Metabolic Re-Programming and Their Role in Tumorigenesis

PubMed Central

Tomasetti, Marco; Amati, Monica; Santarelli, Lory; Neuzil, Jiri

2016-01-01

The process of metabolic re-programing is linked to the activation of oncogenes and/or suppression of tumour suppressor genes, which are regulated by microRNAs (miRNAs). The interplay between oncogenic transformation-driven metabolic re-programming and modulation of aberrant miRNAs further established their critical role in the initiation, promotion and progression of cancer by creating a tumorigenesis-prone microenvironment, thus orchestrating processes of evasion to apoptosis, angiogenesis and invasion/migration, as well metastasis. Given the involvement of miRNAs in tumour development and their global deregulation, they may be perceived as biomarkers in cancer of therapeutic relevance. PMID:27213336

Superenhancer reprogramming drives a B-cell–epithelial transition and high-risk leukemia

PubMed Central

Hu, Yeguang; Zhang, Zhihong; Kashiwagi, Mariko; Yoshida, Toshimi; Joshi, Ila; Jena, Nilamani; Somasundaram, Rajesh; Emmanuel, Akinola Olumide; Sigvardsson, Mikael; Fitamant, Julien; El-Bardeesy, Nabeel; Gounari, Fotini; Van Etten, Richard A.; Georgopoulos, Katia

2016-01-01

IKAROS is required for the differentiation of highly proliferative pre-B-cell precursors, and loss of IKAROS function indicates poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL). Here we show that IKAROS regulates this developmental stage by positive and negative regulation of superenhancers with distinct lineage affiliations. IKAROS defines superenhancers at pre-B-cell differentiation genes together with B-cell master regulators such as PAX5, EBF1, and IRF4 but is required for a highly permissive chromatin environment, a function that cannot be compensated for by the other transcription factors. IKAROS is also highly enriched at inactive enhancers of genes normally expressed in stem–epithelial cells. Upon IKAROS loss, expression of pre-B-cell differentiation genes is attenuated, while a group of extralineage transcription factors that are directly repressed by IKAROS and depend on EBF1 relocalization at their enhancers for expression is induced. LHX2, LMO2, and TEAD–YAP1, normally kept separate from native B-cell transcription regulators by IKAROS, now cooperate directly with them in a de novo superenhancer network with its own feed-forward transcriptional reinforcement. Induction of de novo superenhancers antagonizes Polycomb repression and superimposes aberrant stem–epithelial cell properties in a B-cell precursor. This dual mechanism of IKAROS regulation promotes differentiation while safeguarding against a hybrid stem–epithelial–B-cell phenotype that underlies high-risk B-ALL. PMID:27664237

Proper reprogramming of imprinted and non-imprinted genes in cloned cattle gametogenesis.

PubMed

Kaneda, Masahiro; Watanabe, Shinya; Akagi, Satoshi; Inaba, Yasushi; Geshi, Masaya; Nagai, Takashi

2017-11-01

Epigenetic abnormalities in cloned animals are caused by incomplete reprogramming of the donor nucleus during the nuclear transfer step (first reprogramming). However, during the second reprogramming step that occurs only in the germline cells, epigenetic errors not corrected during the first step are repaired. Consequently, epigenetic abnormalities in the somatic cells of cloned animals should be erased in their spermatozoa or oocytes. This is supported by the fact that offspring from cloned animals do not exhibit defects at birth or during postnatal development. To test this hypothesis in cloned cattle, we compared the DNA methylation level of two imprinted genes (H19 and PEG3) and three non-imprinted genes (XIST, OCT4 and NANOG) and two repetitive elements (Satellite I and Satellite II) in blood and sperm DNAs from cloned and non-cloned bulls. We found no differences between cloned and non-cloned bulls. We also analyzed the DNA methylation levels of four repetitive elements (Satellite I, Satellite II, Alpha-satellite and Art2) in oocytes recovered from cloned and non-cloned cows. Again, no significant differences were observed between clones and non-clones. These results suggested that imprinted and non-imprinted genes and repetitive elements were properly reprogramed during gametogenesis in cloned cattle; therefore, they contributed to the soundness of cloned cattle offspring. © 2017 Japanese Society of Animal Science.

DNA double-strand break response in stem cells: mechanisms to maintain genomic integrity.

PubMed

Nagaria, Pratik; Robert, Carine; Rassool, Feyruz V

2013-02-01

Embryonic stem cells (ESCs) represent the point of origin of all cells in a given organism and must protect their genomes from both endogenous and exogenous genotoxic stress. DNA double-strand breaks (DSBs) are one of the most lethal forms of damage, and failure to adequately repair DSBs would not only compromise the ability of SCs to self-renew and differentiate, but will also lead to genomic instability and disease. Herein, we describe the mechanisms by which ESCs respond to DSB-inducing agents such as reactive oxygen species (ROS) and ionizing radiation, compared to somatic cells. We will also discuss whether the DSB response is fully reprogrammed in induced pluripotent stem cells (iPSCs) and the role of the DNA damage response (DDR) in the reprogramming of these cells. ESCs have distinct mechanisms to protect themselves against DSBs and oxidative stress compared to somatic cells. The response to damage and stress is crucial for the maintenance of self-renewal and differentiation capacity in SCs. iPSCs appear to reprogram some of the responses to genotoxic stress. However, it remains to be determined if iPSCs also retain some DDR characteristics of the somatic cells of origin. The mechanisms regulating the genomic integrity in ESCs and iPSCs are critical for its safe use in regenerative medicine and may shed light on the pathways and factors that maintain genomic stability, preventing diseases such as cancer. This article is part of a Special Issue entitled Biochemistry of Stem Cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Reversible Reprogramming of Circulating Memory T Follicular Helper Cell Function during Chronic HIV Infection

PubMed Central

Cubas, Rafael; van Grevenynghe, Julien; Wills, Saintedym; Kardava, Lela; Santich, Brian H.; Buckner, Clarisa M.; Muir, Roshell; Tardif, Virginie; Nichols, Carmen; Procopio, Francesco; He, Zhong; Metcalf, Talibah; Ghneim, Khader; Locci, Michela; Ancuta, Petronella; Routy, Jean-Pierre; Trautmann, Lydie; Li, Yuxing; McDermott, Adrian B.; Koup, Rick A.; Petrovas, Constantinos; Migueles, Steven A.; Connors, Mark; Tomaras, Georgia D.; Moir, Susan; Crotty, Shane

2015-01-01

Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2–responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART. PMID:26546609

Xenopatients 2.0

PubMed Central

Menendez, Javier A; Alarcón, Tomás; Corominas-Faja, Bruna; Cuyàs, Elisabet; López-Bonet, Eugeni; Martin, Ángel G; Vellon, Luciano

2014-01-01

In the science-fiction thriller film Minority Report, a specialized police department called “PreCrime” apprehends criminals identified in advance based on foreknowledge provided by 3 genetically altered humans called “PreCogs”. We propose that Yamanaka stem cell technology can be similarly used to (epi)genetically reprogram tumor cells obtained directly from cancer patients and create self-evolving personalized translational platforms to foresee the evolutionary trajectory of individual tumors. This strategy yields a large stem cell population and captures the cancer genome of an affected individual, i.e., the PreCog-induced pluripotent stem (iPS) cancer cells, which are immediately available for experimental manipulation, including pharmacological screening for personalized “stemotoxic” cancer drugs. The PreCog-iPS cancer cells will re-differentiate upon orthotopic injection into the corresponding target tissues of immunodeficient mice (i.e., the PreCrime-iPS mouse avatars), and this in vivo model will run through specific cancer stages to directly explore their biological properties for drug screening, diagnosis, and personalized treatment in individual patients. The PreCog/PreCrime-iPS approach can perform sets of comparisons to directly observe changes in the cancer-iPS cell line vs. a normal iPS cell line derived from the same human genetic background. Genome editing of PreCog-iPS cells could create translational platforms to directly investigate the link between genomic expression changes and cellular malignization that is largely free from genetic and epigenetic noise and provide proof-of-principle evidence for cutting-edge “chromosome therapies” aimed against cancer aneuploidy. We might infer the epigenetic marks that correct the tumorigenic nature of the reprogrammed cancer cell population and normalize the malignant phenotype in vivo. Genetically engineered models of conditionally reprogrammable mice to transiently express the Yamanaka stemness factors following the activation of phenotypic copies of specific cancer diseases might crucially evaluate a “reprogramming cure” for cancer. A new era of xenopatients 2.0 generated via nuclear reprogramming of the epigenetic landscapes of patient-derived cancer genomes might revolutionize the current personalized translational platforms in cancer research. PMID:24406535

Boundary formulations for sensitivity analysis without matrix derivatives

NASA Technical Reports Server (NTRS)

Kane, J. H.; Guru Prasad, K.

1993-01-01

A new hybrid approach to continuum structural shape sensitivity analysis employing boundary element analysis (BEA) is presented. The approach uses iterative reanalysis to obviate the need to factor perturbed matrices in the determination of surface displacement and traction sensitivities via a univariate perturbation/finite difference (UPFD) step. The UPFD approach makes it possible to immediately reuse existing subroutines for computation of BEA matrix coefficients in the design sensitivity analysis process. The reanalysis technique computes economical response of univariately perturbed models without factoring perturbed matrices. The approach provides substantial computational economy without the burden of a large-scale reprogramming effort.

Authentication in Reprogramming of Sensor Networks for Mote Class Adversaries

DTIC Science & Technology

2006-01-01

based approach. In this paper, we propose a symmetric key-based protocol for authenticating the reprogramming process. Our protocol is based on the ... secret instantiation algorithm, which requires only O(log n) keys to be maintained at each sensor. We integrate this algorithm with the existing

Blood pressure reprogramming adapter assists signal recording

NASA Technical Reports Server (NTRS)

Vick, H. A.

1967-01-01

Blood pressure reprogramming adapter separates the two components of a blood pressure signal, a dc pressure signal and an ac Korotkoff sounds signal, so that the Korotkoff sounds are recorded on one channel as received while the dc pressure signal is converted to FM and recorded on a second channel.

Reprogramming Bacteria to Seek and Destroy Small Molecules (JGI Seventh Annual User Meeting 2012: Genomics of Energy and Environment)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gallivan, Justin

2012-03-21

Justin Gallivan, of Emory University presents a talk titled "Reprogramming Bacteria to Seek and Destroy Small Molecules" at the JGI User 7th Annual Genomics of Energy & Environment Meeting on March 21, 2012 in Walnut Creek, CA.

Reprogramming Bacteria to Seek and Destroy Small Molecules (JGI Seventh Annual User Meeting 2012: Genomics of Energy and Environment)

ScienceCinema

Gallivan, Justin

2018-05-01

Justin Gallivan, of Emory University presents a talk titled "Reprogramming Bacteria to Seek and Destroy Small Molecules" at the JGI User 7th Annual Genomics of Energy & Environment Meeting on March 21, 2012 in Walnut Creek, CA.

Stem cell therapies for age-related macular degeneration: the past, present, and future.

PubMed

Dang, Yalong; Zhang, Chun; Zhu, Yu

2015-01-01

In the developed world, age-related macular degeneration (AMD) is one of the major causes of irreversible blindness in the elderly. Although management of neovascular AMD (wet AMD) has dramatically progressed, there is still no effective treatment for nonneovascular AMD (dry AMD), which is characterized by retinal pigment epithelial (RPE) cell death (or dysfunction) and microenvironmental disruption in the retina. Therefore, RPE replacement and microenvironmental regulation represent viable treatments for dry AMD. Recent advances in cell biology have demonstrated that RPE cells can be easily generated from several cell types (pluripotent stem cells, multipotent stem cells, or even somatic cells) by spontaneous differentiation, coculturing, defined factors or cell reprogramming, respectively. Additionally, in vivo studies also showed that the restoration of visual function could be obtained by transplanting functional RPE cells into the subretinal space of recipient. More importantly, clinical trials approved by the US government have shown promising prospects in RPE transplantation. However, key issues such as implantation techniques, immune rejection, and xeno-free techniques are still needed to be further investigated. This review will summarize recent advances in cell transplantation for dry AMD. The obstacles and prospects in this field will also be discussed.

Post-translational regulation of WRKY transcription factors in plant immunity.

PubMed

Ishihama, Nobuaki; Yoshioka, Hirofumi

2012-08-01

Plants have evolved immune system to protect themselves against invading pathogens. Recent research has illustrated that signaling networks, after perception of diverse pathogen-derived signals, facilitate transcriptional reprogramming through mitogen-activated protein kinase (MAPK) cascades. WRKY proteins, which comprise a large family of plant transcription factors, are key players in plant immune responses. WRKY transcription factors participate in the control of defense-related genes either as positive or as negative regulators, and essentially are regulated at the transcriptional level. Emerging evidence emphasizes that group I WRKY transcription factors, which contain a conserved motif in the N-terminal region, are also activated by MAPK-dependent phosphorylation, underlining their importance in plant immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Aging and reprogramming: a two-way street

PubMed Central

Mahmoudi, Salah; Brunet, Anne

2012-01-01

Aging is accompanied by the functional decline of cells, tissues, and organs, as well as a striking increase in a wide range of diseases. The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) opens new avenues for the aging field and has important applications for therapeutic treatments of age-related diseases. Here we review emerging studies on how aging and age-related pathways influence iPSC generation and property. We discuss the exciting possibility that reverting to a pluripotent stem cell stage erases several deficits associated with aging and will provide new strategies for rejuvenation. Finally, we argue that reprogramming provides a unique opportunity to model aging and perhaps exceptional longevity. PMID:23146768

Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors.

PubMed

Bar-Nur, Ori; Gerli, Mattia F M; Di Stefano, Bruno; Almada, Albert E; Galvin, Amy; Coffey, Amy; Huebner, Aaron J; Feige, Peter; Verheul, Cassandra; Cheung, Priscilla; Payzin-Dogru, Duygu; Paisant, Sylvain; Anselmo, Anthony; Sadreyev, Ruslan I; Ott, Harald C; Tajbakhsh, Shahragim; Rudnicki, Michael A; Wagers, Amy J; Hochedlinger, Konrad

2018-05-08

Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7 + cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Introduction to thematic minireview series: Development of human therapeutics based on induced pluripotent stem cell (iPSC) technology.

PubMed

Rao, Mahendra; Gottesfeld, Joel M

2014-02-21

With the advent of human induced pluripotent stem cell (hiPSC) technology, it is now possible to derive patient-specific cell lines that are of great potential in both basic research and the development of new therapeutics for human diseases. Not only do hiPSCs offer unprecedented opportunities to study cellular differentiation and model human diseases, but the differentiated cell types obtained from iPSCs may become therapeutics themselves. These cells can also be used in the screening of therapeutics and in toxicology assays for potential liabilities of therapeutic agents. The remarkable achievement of transcription factor reprogramming to generate iPSCs was recognized by the award of the Nobel Prize in Medicine to Shinya Yamanaka in 2012, just 6 years after the first publication of reprogramming methods to generate hiPSCs (Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007) Cell 131, 861-872). This minireview series highlights both the promises and challenges of using iPSC technology for disease modeling, drug screening, and the development of stem cell therapeutics.

Generation of induced pluripotent stem cells with high efficiency from human embryonic renal cortical cells.

PubMed

Yao, Ling; Chen, Ruifang; Wang, Pu; Zhang, Qi; Tang, Hailiang; Sun, Huaping

2016-01-01

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) emerges as a prospective therapeutic angle in regenerative medicine and a tool for drug screening. Although increasing numbers of iPSCs from different sources have been generated, there has been limited progress in yield of iPSC. Here, we show that four Yamanaka factors Oct4, Sox2, Klf4 and c-Myc can convert human embryonic renal cortical cells (hERCCs) to pluripotent stem cells with a roughly 40-fold higher reprogramming efficiency compared with that of adult human dermal fibroblasts. These iPSCs show pluripotency in vitro and in vivo, as evidenced by expression of pluripotency associated genes, differentiation into three embryonic germ layers by teratoma tests, as well as neuronal fate specification by embryoid body formation. Moreover, the four exogenous genes are effectively silenced in these iPSCs. This study highlights the use of hERCCs to generate highly functional human iPSCs which may aid the study of genetic kidney diseases and accelerate the development of cell-based regenerative therapy.

Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells.

PubMed

Chang, Chan-Jung; Mitra, Koyel; Koya, Mariko; Velho, Michelle; Desprat, Romain; Lenz, Jack; Bouhassira, Eric E

2011-01-01

We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.

The function of the Mediator complex in plant immunity.

PubMed

An, Chuanfu; Mou, Zhonglin

2013-03-01

Upon pathogen infection, plants undergo dramatic transcriptome reprogramming to shift from normal growth and development to immune response. During this rapid process, the multiprotein Mediator complex has been recognized as an important player to fine-tune gene-specific and pathway-specific transcriptional reprogramming by acting as an adaptor/coregulator between sequence-specific transcription factor and RNA polymerase II (RNAPII). Here, we review current understanding of the role of five functionally characterized Mediator subunits (MED8, MED15, MED16, MED21 and MED25) in plant immunity. All these Mediator subunits positively regulate resistance against leaf-infecting biotrophic bacteria or necrotrophic fungi. While MED21 appears to regulate defense against fungal pathogens via relaying signals from upstream regulators and chromatin modification to RNAPII, the other four Mediator subunits locate at different positions of the defense network to convey phytohormone signal(s). Fully understanding the role of Mediator in plant immunity needs to characterize more Mediator subunits in both Arabidopsis and other plant species. Identification of interacting proteins of Mediator subunits will further help to reveal their specific regulatory mechanisms in plant immunity.

Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming.

PubMed

Rubio, Alicia; Luoni, Mirko; Giannelli, Serena G; Radice, Isabella; Iannielli, Angelo; Cancellieri, Cinzia; Di Berardino, Claudia; Regalia, Giulia; Lazzari, Giovanna; Menegon, Andrea; Taverna, Stefano; Broccoli, Vania

2016-11-18

The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular, this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells), hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here, we described a new platform which enables, rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular, we devised a protocol that, combining the expression of the CRISPR components with neurogenic factors, generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy, fast and that does not require the generation of stable isogenic clones, practice that is time consuming and for some genes not feasible.

Impairment of DNA Methylation Maintenance Is the Main Cause of Global Demethylation in Naive Embryonic Stem Cells.

PubMed

von Meyenn, Ferdinand; Iurlaro, Mario; Habibi, Ehsan; Liu, Ning Qing; Salehzadeh-Yazdi, Ali; Santos, Fátima; Petrini, Edoardo; Milagre, Inês; Yu, Miao; Xie, Zhenqing; Kroeze, Leonie I; Nesterova, Tatyana B; Jansen, Joop H; Xie, Hehuang; He, Chuan; Reik, Wolf; Stunnenberg, Hendrik G

2016-06-16

Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

Obesity, male infertility, and the sperm epigenome.

PubMed

Craig, James R; Jenkins, Timothy G; Carrell, Douglas T; Hotaling, James M

2017-04-01

Obesity is a growing epidemic and a common problem among reproductive-age men that can both cause and exacerbate male-factor infertility by means of endocrine abnormalities, associated comorbidities, and direct effects on the fidelity and throughput of spermatogenesis. Robust epidemiologic, clinical, genetic, epigenetic, and nonhuman animal data support these findings. Recent works in the burgeoning field of epigenetics has demonstrated that paternal obesity can affect offspring metabolic and reproductive phenotypes by means of epigenetic reprogramming of spermatogonial stem cells. Understanding the impact of this reprogramming is critical to a comprehensive view of the impact of obesity on subsequent generations. Furthermore, and perhaps more importantly, conveying the impact of these lifestyle changes on future progeny can serve as a powerful tool for obese men to modify their behavior. Reproductive urologists and endocrinologists must learn to assimilate these new findings to better counsel men about the importance of paternal preconception health, a topic recently being championed by the Centers for Disease Control and Prevention. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

An Orchestrated Intron Retention Program in Meiosis Controls Timely Usage of Transcripts during Germ Cell Differentiation.

PubMed

Naro, Chiara; Jolly, Ariane; Di Persio, Sara; Bielli, Pamela; Setterblad, Niclas; Alberdi, Antonio J; Vicini, Elena; Geremia, Raffaele; De la Grange, Pierre; Sette, Claudio

2017-04-10

Global transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Experts in Fast-Ball Sports Reduce Anticipation Timing Cost by Developing Inhibitory Control

ERIC Educational Resources Information Center

Nakamoto, Hiroki; Mori, Shiro

2012-01-01

The present study was conducted to examine the relationship between expertise in movement correction and rate of movement reprogramming within limited time periods, and to clarify the specific cognitive processes regarding superior reprogramming ability in experts. Event-related potentials (ERPs) were recorded in baseball experts (n = 7) and…

Reprogramming Enhancers to Drive Metastasis.

PubMed

Mostoslavsky, Raul; Bardeesy, Nabeel

2017-08-24

Acquired molecular changes can promote the spreading of primary tumor cells to distant tissues. In this issue of Cell, Roe et al. show that metastatic progression of pancreatic cancer involves large-scale enhancer reprogramming by Foxa1, which activates transcriptional program specifying early endodermal stem cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Metabolic Reprogramming and Oncogenesis: One Hallmark, Many Organelles.

PubMed

Costa, A S H; Frezza, C

2017-01-01

The process of tumorigenesis can be described by a series of molecular features, among which alteration of cellular metabolism has recently emerged. This metabolic rewiring fulfills the energy and biosynthetic demands of fast proliferating cancer cells and amplifies their metabolic repertoire to survive and proliferate in the poorly oxygenated and nutrient-deprived tumor microenvironment. During the last decade, the complex reprogramming of cancer cell metabolism has been widely investigated, revealing cancer-specific metabolic alterations. These include dysregulation of glucose and glutamine metabolism, alterations of lipid synthesis and oxidation, and a complex rewiring of mitochondrial function. However, mitochondria are not the only metabolically active organelles within the cell, and other organelles, including lysosomes, peroxisomes, and endoplasmic reticulum, harbor components of the metabolic network. Of note, dysregulation of the function of these organelles is increasingly recognized in cancer cells. However, to what extent these organelles contribute to the metabolic reprogramming of cancer is not fully understood. In this review, we describe the main metabolic functions of these organelles and provide insights into how they communicate to orchestrate a coordinated metabolic reprogramming during transformation. © 2017 Elsevier Inc. All rights reserved.

Biochemical basis for pharmacological intervention as a reprogramming strategy against hypertension and kidney disease of developmental origin.

PubMed

Tain, You-Lin; Chan, Samuel H H; Chan, Julie Y H

2018-07-01

The concept of "developmental origins of health and disease" (DOHaD) stipulates that both hypertension and kidney disease may take origin from early-life insults. The DOHaD concept also offers reprogramming strategies aiming at shifting therapeutic interventions from adulthood to early life, even before clinical symptoms are evident. Based on those two concepts, this review will present the evidence for the existence of, and the programming mechanisms in, kidney developmental programming that may lead to hypertension and kidney disease. This will be followed by potential pharmacological interventions that may serve as a reprogramming strategy to counter the rising epidemic of hypertension and kidney disease. We point out that before patients could benefit from this strategy, the most pressing issue is for the growing body of evidence from animal studies in support of pharmacological intervention as a reprogramming strategy to long-term protect against hypertension and kidney disease of developmental origins to be validated clinically and the critical window, drug dose, dosing regimen, and therapeutic duration identified. Copyright © 2018 Elsevier Inc. All rights reserved.

BCG Vaccination Protects against Experimental Viral Infection in Humans through the Induction of Cytokines Associated with Trained Immunity.

PubMed

Arts, Rob J W; Moorlag, Simone J C F M; Novakovic, Boris; Li, Yang; Wang, Shuang-Yin; Oosting, Marije; Kumar, Vinod; Xavier, Ramnik J; Wijmenga, Cisca; Joosten, Leo A B; Reusken, Chantal B E M; Benn, Christine S; Aaby, Peter; Koopmans, Marion P; Stunnenberg, Hendrik G; van Crevel, Reinout; Netea, Mihai G

2018-01-10

The tuberculosis vaccine bacillus Calmette-Guérin (BCG) has heterologous beneficial effects against non-related infections. The basis of these effects has been poorly explored in humans. In a randomized placebo-controlled human challenge study, we found that BCG vaccination induced genome-wide epigenetic reprograming of monocytes and protected against experimental infection with an attenuated yellow fever virus vaccine strain. Epigenetic reprogramming was accompanied by functional changes indicative of trained immunity. Reduction of viremia was highly correlated with the upregulation of IL-1β, a heterologous cytokine associated with the induction of trained immunity, but not with the specific IFNγ response. The importance of IL-1β for the induction of trained immunity was validated through genetic, epigenetic, and immunological studies. In conclusion, BCG induces epigenetic reprogramming in human monocytes in vivo, followed by functional reprogramming and protection against non-related viral infections, with a key role for IL-1β as a mediator of trained immunity responses. Copyright © 2017 Elsevier Inc. All rights reserved.

Direct Reprogramming of Fibroblasts via a Chemically Induced XEN-like State.

PubMed

Li, Xiang; Liu, Defang; Ma, Yantao; Du, Xiaomin; Jing, Junzhan; Wang, Lipeng; Xie, Bingqing; Sun, Da; Sun, Shaoqiang; Jin, Xueqin; Zhang, Xu; Zhao, Ting; Guan, Jingyang; Yi, Zexuan; Lai, Weifeng; Zheng, Ping; Huang, Zhuo; Chang, Yanzhong; Chai, Zhen; Xu, Jun; Deng, Hongkui

2017-08-03

Direct lineage reprogramming, including with small molecules, has emerged as a promising approach for generating desired cell types. We recently found that during chemical induction of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, cells pass through an extra-embryonic endoderm (XEN)-like state. Here, we show that these chemically induced XEN-like cells can also be induced to directly reprogram into functional neurons, bypassing the pluripotent state. The induced neurons possess neuron-specific expression profiles, form functional synapses in culture, and further mature after transplantation into the adult mouse brain. Using similar principles, we were also able to induce hepatocyte-like cells from the XEN-like cells. Cells in the induced XEN-like state were readily expandable over at least 20 passages and retained genome stability and lineage specification potential. Our study therefore establishes a multifunctional route for chemical lineage reprogramming and may provide a platform for generating a diverse range of cell types via application of this expandable XEN-like state. Copyright © 2017 Elsevier Inc. All rights reserved.

Abnormalities in human pluripotent cells due to reprogramming mechanisms

PubMed Central

Ma, Hong; Morey, Robert; O’Neil, Ryan C.; He, Yupeng; Daughtry, Brittany; Schultz, Matthew D.; Hariharan, Manoj; Nery, Joseph R.; Castanon, Rosa; Sabatini, Karen; Thiagarajan, Rathi D.; Tachibana, Masahito; Kang, Eunju; Tippner-Hedges, Rebecca; Ahmed, Riffat; Gutierrez, Nuria Marti; Van Dyken, Crystal; Polat, Alim; Sugawara, Atsushi; Sparman, Michelle; Gokhale, Sumita; Amato, Paula; Wolf, Don P.; Ecker, Joseph R.; Laurent, Louise C.; Mitalipov, Shoukhrat

2016-01-01

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the ‘gold standard’, they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies. PMID:25008523

Identification of secretaglobin Scgb2a1 as a target for developmental reprogramming by BPA in the rat prostate.

PubMed

Wong, Rebecca Lee Yean; Wang, Quan; Treviño, Lindsey S; Bosland, Maarten C; Chen, Jing; Medvedovic, Mario; Prins, Gail S; Kannan, Kurunthachalam; Ho, Shuk-Mei; Walker, Cheryl Lyn

2015-01-01

Secretoglobins are a superfamily of secreted proteins thought to participate in inflammation, tissue repair, and tumorigenesis. Secretoglobin family 2A member 1 (Scgb2a1) is a component of prostatein, a major androgen-binding protein secreted by the rat prostate. Using a rat model for developmental reprogramming of susceptibility to prostate carcinogenesis, we identified, by RNA-seq, that Scgb2a1 is significantly upregulated (>100-fold) in the prostate of adult rats neonatally exposed to bisphenol A (BPA), with increased gene expression confirmed by quantitative RT-PCR and chromatin immunoprecipitation for histone H3 lysine 9 acetylation. Bisulfite analysis of both CpG islands located within 10 kb of the Scgb2a1 promoter identified significant hypomethylation of the CpG island upstream of the transcription start site of this gene in the reprogrammed prostate. These data suggest that expression of Scgb2a1 in the adult prostate could be epigenetically reprogrammed by BPA exposure during prostate development, with potential implications for cancer risk and response to chemotherapeutics associated with prostatein binding.

Human X chromosome inactivation and reactivation: implications for cell reprogramming and disease.

PubMed

Cantone, Irene; Fisher, Amanda G

2017-11-05

X-chromosome inactivation (XCI) is an exemplar of epigenetic regulation that is set up as pluripotent cells differentiate. Once established, XCI is stably propagated, but can be reversed in vivo or by pluripotent reprogramming in vitro Although reprogramming provides a useful model for inactive X (Xi) reactivation in mouse, the relative instability and heterogeneity of human embryonic stem (ES) cells and induced pluripotent stem cells hampers comparable progress in human. Here we review studies aimed at reactivating the human Xi using different reprogramming strategies. We outline our recent results using mouse ES cells to reprogramme female human fibroblasts by cell-cell fusion. We show that pluripotent reprogramming induces widespread and rapid chromatin remodelling in which the human Xi loses XIST and H3K27m3 enrichment and selected Xi genes become reactivated, ahead of mitotic division. Using RNA sequencing to map the extent of human Xi reactivation, and chromatin-modifying drugs to potentiate reactivation, we outline how this approach could be used to better design strategies to re-express human X-linked loci. As cell fusion induces the expression of human pluripotency genes that represent both the 'primed' and 'naive' states, this approach may also offer a fresh opportunity to segregate human pluripotent states with distinct Xi expression profiles, using single-cell-based approaches.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'. © 2017 The Author(s).

Transgene-free iPSCs generated from small volume peripheral blood nonmobilized CD34+ cells

PubMed Central

Merling, Randall K.; Sweeney, Colin L.; Choi, Uimook; De Ravin, Suk See; Myers, Timothy G.; Otaizo-Carrasquero, Francisco; Pan, Jason; Linton, Gilda; Chen, Lifeng; Koontz, Sherry; Theobald, Narda L.; Malech, Harry L.

2013-01-01

A variety of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), but CD34+ hematopoietic stem cells (HSCs) present in nonmobilized peripheral blood (PB) would be a convenient target. We report a method for deriving iPSC from PB HSCs using immunobead purification and 2- to 4-day culture to enrich CD34+ HSCs to 80% ± 9%, followed by reprogramming with loxP-flanked polycistronic (human Oct4, Klf4, Sox2, and c-Myc) STEMCCA-loxP lentivector, or with Sendai vectors. Colonies arising with STEMCCA-loxP were invariably TRA-1-60+, yielding 5.3 ± 2.8 iPSC colonies per 20 mL PB (n = 17), where most colonies had single-copy STEMCCA-loxP easily excised by transient Cre expression. Colonies arising with Sendai were variably reprogrammed (10%-80% TRA-1-60+), with variable yield (6 to >500 TRA-1-60+ iPSC colonies per 10 mL blood; n = 6). Resultant iPSC clones expressed pluripotent cell markers and generated teratomas. Genomic methylation patterns of STEMCCA-loxP–reprogrammed clones closely matched embryonic stem cells. Furthermore, we showed that iPSCs are derived from the nonmobilized CD34+ HSCs enriched from PB rather than from any lymphocyte or monocyte contaminants because they lack somatic rearrangements typical of T or B lymphocytes and because purified CD14+ monocytes do not yield iPSC colonies under these reprogramming conditions. PMID:23386128

RNA-Generated and Gene-Edited Induced Pluripotent Stem Cells for Disease Modeling and Therapy.

PubMed

Kehler, James; Greco, Marianna; Martino, Valentina; Pachiappan, Manickam; Yokoe, Hiroko; Chen, Alice; Yang, Miranda; Auerbach, Jonathan; Jessee, Joel; Gotte, Martin; Milanesi, Luciano; Albertini, Alberto; Bellipanni, Gianfranco; Zucchi, Ileana; Reinbold, Rolland A; Giordano, Antonio

2017-06-01

Cellular reprogramming by epigenomic remodeling of chromatin holds great promise in the field of human regenerative medicine. As an example, human-induced Pluripotent Stem Cells (iPSCs) obtained by reprograming of patient somatic cells are sufficiently similar to embryonic stem cells (ESCs) and can generate all cell types of the human body. Clinical use of iPSCs is dependent on methods that do not utilize genome altering transgenic technologies that are potentially unsafe and ethically unacceptable. Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogramming may prove to be more suitable for clinical applications and provide stable starting cell lines for gene-editing, isolation, and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene-editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. We present optimized RNA-based laboratory procedure for making and editing iPSCs. In the near-term these two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic, and translational research. J. Cell. Physiol. 232: 1262-1269, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Violacein Induces Death of Resistant Leukaemia Cells via Kinome Reprogramming, Endoplasmic Reticulum Stress and Golgi Apparatus Collapse

PubMed Central

Queiroz, Karla C. S.; Milani, Renato; Ruela-de-Sousa, Roberta R.; Fuhler, Gwenny M.; Justo, Giselle Z.; Zambuzzi, Willian F.; Duran, Nelson; Diks, Sander H.; Spek, C. Arnold; Ferreira, Carmen V.; Peppelenbosch, Maikel P.

2012-01-01

It is now generally recognised that different modes of programmed cell death (PCD) are intimately linked to the cancerous process. However, the mechanism of PCD involved in cancer chemoprevention is much less clear and may be different between types of chemopreventive agents and tumour cell types involved. Therefore, from a pharmacological view, it is crucial during the earlier steps of drug development to define the cellular specificity of the candidate as well as its capacity to bypass dysfunctional tumoral signalling pathways providing insensitivity to death stimuli. Studying the cytotoxic effects of violacein, an antibiotic dihydro-indolone synthesised by an Amazon river Chromobacterium, we observed that death induced in CD34+/c-Kit+/P-glycoprotein+/MRP1+ TF1 leukaemia progenitor cells is not mediated by apoptosis and/or autophagy, since biomarkers of both types of cell death were not significantly affected by this compound. To clarify the working mechanism of violacein, we performed kinome profiling using peptide arrays to yield comprehensive descriptions of cellular kinase activities. Pro-death activity of violacein is actually carried out by inhibition of calpain and DAPK1 and activation of PKA, AKT and PDK, followed by structural changes caused by endoplasmic reticulum stress and Golgi apparatus collapse, leading to cellular demise. Our results demonstrate that violacein induces kinome reprogramming, overcoming death signaling dysfunctions of intrinsically resistant human leukaemia cells. PMID:23071514

Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

DOE PAGES

Hayashi, Yohei; Caboni, Laura; Das, Debanu; ...

2015-03-30

NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutantsmore » based on the protein–DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings indicate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering.« less

Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

PubMed Central

Hayashi, Yohei; Caboni, Laura; Das, Debanu; Yumoto, Fumiaki; Clayton, Thomas; Deller, Marc C.; Nguyen, Phuong; Farr, Carol L.; Chiu, Hsiu-Ju; Miller, Mitchell D.; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Tomoda, Kiichiro; Conklin, Bruce R.; Wilson, Ian A.; Yamanaka, Shinya; Fletterick, Robert J.

2015-01-01

NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutants based on the protein–DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings demonstrate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering. PMID:25825768

Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayashi, Yohei; Caboni, Laura; Das, Debanu

NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutantsmore » based on the protein–DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings indicate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering.« less

Keith's MAGIC: Cloning and the Cell Cycle.

PubMed

Wells, D N

2013-10-01

Abstract Professor Keith Campbell's critical contribution to the discovery that a somatic cell from an adult animal can be fully reprogrammed by oocyte factors to form a cloned individual following nuclear transfer (NT)(Wilmut et al., 1997 ) overturned a dogma concerning the reversibility of cell fate that many scientists had considered to be biologically impossible. This seminal experiment proved the totipotency of adult somatic nuclei and finally confirmed that adult cells could differentiate without irreversible changes to the genetic material.

WIND1 Promotes Shoot Regeneration through Transcriptional Activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis[OPEN

PubMed Central

Ohnuma, Mariko; Kurata, Tetsuya; Nakata, Masaru; Ohme-Takagi, Masaru

2017-01-01

Many plant species display remarkable developmental plasticity and regenerate new organs after injury. Local signals produced by wounding are thought to trigger organ regeneration but molecular mechanisms underlying this control remain largely unknown. We previously identified an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) as a central regulator of wound-induced cellular reprogramming in plants. In this study, we demonstrate that WIND1 promotes callus formation and shoot regeneration by upregulating the expression of the ENHANCER OF SHOOT REGENERATION1 (ESR1) gene, which encodes another AP2/ERF transcription factor in Arabidopsis thaliana. The esr1 mutants are defective in callus formation and shoot regeneration; conversely, its overexpression promotes both of these processes, indicating that ESR1 functions as a critical driver of cellular reprogramming. Our data show that WIND1 directly binds the vascular system-specific and wound-responsive cis-element-like motifs within the ESR1 promoter and activates its expression. The expression of ESR1 is strongly reduced in WIND1-SRDX dominant repressors, and ectopic overexpression of ESR1 bypasses defects in callus formation and shoot regeneration in WIND1-SRDX plants, supporting the notion that ESR1 acts downstream of WIND1. Together, our findings uncover a key molecular pathway that links wound signaling to shoot regeneration in plants. PMID:28011694

Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers

PubMed Central

Gao, Xuefei; Tsang, Jason C.H.; Gaba, Fortis; Wu, Donghai; Lu, Liming; Liu, Pentao

2014-01-01

The transcription activator–like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (Cas9) utlilize distinct molecular mechanisms in targeting site recognition. The two proteins can be modified to carry additional functional domains to regulate expression of genomic loci in mammalian cells. In this study, we have compared the two systems in activation and suppression of the Oct4 and Nanog loci by targeting their enhancers. Although both are able to efficiently activate the luciferase reporters, the CRISPR/dCas9 system is much less potent in activating the endogenous loci and in the application of reprogramming somatic cells to iPS cells. Nevertheless, repression by CRISPR/dCas9 is comparable to or even better than TALE repressors. We demonstrated that dCas9 protein binding results in significant physical interference to binding of native transcription factors at enhancer, less efficient active histone markers induction or recruitment of activating complexes in gene activation. This study thus highlighted the merits and drawbacks of transcription regulation by each system. A combined approach of TALEs and CRISPR/dCas9 should provide an optimized solution to regulate genomic loci and to study genetic elements such as enhancers in biological processes including somatic cell reprogramming and guided differentiation. PMID:25223790

Trained immunity: a program of innate immune memory in health and disease

PubMed Central

Netea, Mihai G.; Joosten, Leo A.B.; Latz, Eicke; Mills, Kingston H.G.; Natoli, Gioacchino; Stunnenberg, Hendrik G.; O’Neill, Luke A.J.; Xavier, Ramnik J.

2016-01-01

The general view that only adaptive immunity can build immunological memory has recently been challenged. In organisms lacking adaptive immunity as well as in mammals, the innate immune system can mount resistance to reinfection, a phenomenon termed trained immunity or innate immune memory. Trained immunity is orchestrated by epigenetic reprogramming, broadly defined as sustained changes in gene expression and cell physiology that do not involve permanent genetic changes such as mutations and recombination, which are essential for adaptive immunity. The discovery of trained immunity may open the door for novel vaccine approaches, for new therapeutic strategies for the treatment of immune deficiency states, and for modulation of exaggerated inflammation in autoinflammatory diseases. PMID:27102489

Mitochondrial Pyruvate Carrier Function and Cancer Metabolism

PubMed Central

Rauckhorst, Adam J.

2016-01-01

Metabolic reprograming in cancer supports the increased biosynthesis required for unchecked proliferation. Increased glucose utilization is a defining feature of many cancers that is accompanied by altered pyruvate partitioning and mitochondrial metabolism. Cancer cells also require mitochondrial tricarboxylic acid cycle activity and electron transport chain function for biosynthetic competency and proliferation. Recent evidence demonstrates that mitochondrial pyruvate carrier (MPC) function is abnormal in some cancers and that increasing MPC activity may decrease cancer proliferation. Here we examine recent findings on MPC function and cancer metabolism. Special emphasis is placed on the compartmentalization of pyruvate metabolism and the alternative routes of metabolism that maintain the cellular biosynthetic pools required for unrestrained proliferation in cancer. PMID:27269731

A programmable microfluidic static droplet array for droplet generation, transportation, fusion, storage, and retrieval.

PubMed

Jin, Si Hyung; Jeong, Heon-Ho; Lee, Byungjin; Lee, Sung Sik; Lee, Chang-Soo

2015-01-01

We present a programmable microfluidic static droplet array (SDA) device that can perform user-defined multistep combinatorial protocols. It combines the passive storage of aqueous droplets without any external control with integrated microvalves for discrete sample dispensing and dispersion-free unit operation. The addressable picoliter-volume reaction is systematically achieved by consecutively merging programmable sequences of reagent droplets. The SDA device is remarkably reusable and able to perform identical enzyme kinetic experiments at least 30 times via automated cross-contamination-free removal of droplets from individual hydrodynamic traps. Taking all these features together, this programmable and reusable universal SDA device will be a general microfluidic platform that can be reprogrammed for multiple applications.

Manufacturing Cell Therapies Using Engineered Biomaterials.

PubMed

Abdeen, Amr A; Saha, Krishanu

2017-10-01

Emerging manufacturing processes to generate regenerative advanced therapies can involve extensive genomic and/or epigenomic manipulation of autologous or allogeneic cells. These cell engineering processes need to be carefully controlled and standardized to maximize safety and efficacy in clinical trials. Engineered biomaterials with smart and tunable properties offer an intriguing tool to provide or deliver cues to retain stemness, direct differentiation, promote reprogramming, manipulate the genome, or select functional phenotypes. This review discusses the use of engineered biomaterials to control human cell manufacturing. Future work exploiting engineered biomaterials has the potential to generate manufacturing processes that produce standardized cells with well-defined critical quality attributes appropriate for clinical testing. Copyright © 2017 Elsevier Ltd. All rights reserved.

Binary Cell Fate Decisions and Fate Transformation in the Drosophila Larval Eye

PubMed Central

Rister, Jens; Ng, June; Celik, Arzu; Sprecher, Simon G.

2013-01-01

The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs) make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5) or the green-sensitive Rhodopsin 6 (Rh6). Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. We show that interplay of two transcription factors Senseless (Sens) and Hazy control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. Our findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner. PMID:24385925

Binary cell fate decisions and fate transformation in the Drosophila larval eye.

PubMed

Mishra, Abhishek Kumar; Tsachaki, Maria; Rister, Jens; Ng, June; Celik, Arzu; Sprecher, Simon G

2013-01-01

The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs) make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5) or the green-sensitive Rhodopsin 6 (Rh6). Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. We show that interplay of two transcription factors Senseless (Sens) and Hazy control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. Our findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner.

Computer Network Operations Methodology

DTIC Science & Technology

2004-03-01

means of their computer information systems. Disrupt - This type of attack focuses on disrupting as “attackers might surreptitiously reprogram enemy...by reprogramming the computers that control distribution within the power grid. A disruption attack introduces disorder and inhibits the effective...between commanders. The use of methodologies is widespread and done subconsciously to assist individuals in decision making. The processes that

Nutrient deprivation-related OXPHOS/glycolysis interconversion via HIF-1α/C-MYC pathway in U251 cells.

PubMed

Liu, Zhongjian; Sun, Yang; Tan, Shirui; Liu, Liang; Hu, Suqiong; Huo, Hongyu; Li, Meizhang; Cui, Qinghua; Yu, Min

2016-05-01

Although the Warburg effect is a dominant metabolic phenotype observed in cancers, the metabolic changes and adaptation occurring in tumors have been demonstrated to extend beyond the Warburg effect and thus considered a secondary effect to the transformation process of carcinogenesis, including nutritional deficiencies. However, the role of nutritional deficiencies in this metabolic reprogramming (e. g., oxidative phosphorylation (OXPHOS)/glycolysis interconversion) is not completely known yet. Here, we showed that under regular culture condition, the proliferation of U251 cells, but not other tumor cell lines, preferentially performed the Warburg effect and was remarkably inhibited by oxamic acid which can inhibit the activity of lactate dehydrogenase (LDH); whereas under serum starvation, glycolysis was depressed, tricarboxylic acid cycle (TCA) was enhanced, and the activity of OXPHOS was reinforced to maintain cellular ATP content in a high level, but interestingly, we observed a decreased expression of reactive oxygen species (ROS). Moreover, the upregulated activity of mitochondrial complex I was confirmed by Western blots and showed that the mitochondrial-related protein, NDUFA9, NDUFB8, ND1, and VDAC1 were remarkably increased after serum starved. Mechanistically, nutritional deficiencies could reduce hypoxia-inducible factor α (HIF-1α) protein expression to increase C-MYC protein level, which in turn increased nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) transcription to enhance the activity of OXPHOS, suggesting that metabolic reprogramming by the changes of microenvironment during the carcinogenesis can provide some novel therapeutic clues to traditional cancer treatments.

Transgenerational response to stress in plants and its application for breeding.

PubMed

Bilichak, Andriy; Kovalchuk, Igor

2016-03-01

A growing number of reports indicate that plants possess the ability to maintain a memory of stress exposure throughout their ontogenesis and even transmit it faithfully to the following generation. Some of the features of transgenerational memory include elevated genome instability, a higher tolerance to stress experienced by parents, and a cross-tolerance. Although the underlying molecular mechanisms of this phenomenon are not clear, a likely contributing factor is the absence of full-scale reprogramming of the epigenetic landscape during gametogenesis; therefore, epigenetic marks can occasionally escape the reprogramming process and can be passed on to the progeny. To date, it is not entirely clear which part of the epigenetic landscape is more likely to escape the reprogramming events, and whether such a process is random or directed and sequence specific. The identification of specific epigenetic marks associated with specific stressors would allow generation of stress-tolerant plants through the recently discovered techniques for precision epigenome engineering. The engineered DNA-binding domains (e.g. ZF, TALE, and dCas9) fused to particular chromatin modifiers would make it possible to target epigenetic modifications to the selected loci, probably allowing stress tolerance to be achieved in the progeny. This approach, termed epigenetic breeding, along with other methods has great potential to be used for both the assessment of the propagation of epigenetic marks across generations and trait improvement in plants. In this communication, we provide a short overview of recent reports demonstrating a transgenerational response to stress in plants, and discuss the underlying potential molecular mechanisms of this phenomenon and its use for plant biotechnology applications. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Accelerated geroncogenesis in hereditary breast-ovarian cancer syndrome

PubMed Central

Menendez, Javier A.; Folguera-Blasco, Núria; Cuyàs, Elisabet; Fernández-Arroyo, Salvador; Joven, Jorge; Alarcón, Tomás

2016-01-01

The geroncogenesis hypothesis postulates that the decline in metabolic cellular health that occurs naturally with aging drives a “field effect” predisposing normal tissues for cancer development. We propose that mutations in the cancer susceptibility genes BRCA1/2 might trigger “accelerated geroncogenesis” in breast and ovarian epithelia. By speeding up the rate at which the metabolic threshold becomes “permissive” with survival and expansion of genomically unstable pre-tumoral epithelial cells, BRCA haploinsufficiency-driven metabolic reprogramming would operate as a bona fide oncogenic event enabling malignant transformation and tumor formation in BRCA carriers. The metabolic facet of BRCA1 one-hit might involve tissue-specific alterations in acetyl-CoA, α-ketoglutarate, NAD+, FAD, or S-adenosylmethionine, critical factors for de/methylation or de/acetylation dynamics in the nuclear epigenome. This in turn might induce faulty epigenetic reprogramming at the “install phase” that directs cell-specific differentiation of breast/ovarian epithelial cells, which can ultimately determine the penetrance of BRCA defects during developmental windows of susceptibility. This model offers a framework to study whether metabolic drugs that prevent or revert metabolic reprogramming induced by BRCA haploinsufficiency might displace the “geroncogenic risk” of BRCA carriers to the age typical for those without the mutation. The identification of the key nodes that directly communicate changes in cellular metabolism to the chromatin in BRCA haploinsufficient cells may allow the epigenetic targeting of genomic instability using exclusively metabolic means. The validation of accelerated geroncogenesis as an inherited “one-hit” metabolic “field effect” might offer new strategies to therapeutically revisit the apparently irreversible genetic-hereditary fate of women with hereditary breast-ovarian cancer syndrome. PMID:26943589

The effect of chemically modified alginates on macrophage phenotype and biomolecule transport.

PubMed

Bygd, Hannah C; Bratlie, Kaitlin M

2016-07-01

Macrophage (MΦ) reprogramming has received significant attention in applications such as cancer therapeutics and tissue engineering where the host immune response to biomaterials is crucial in determining the success or failure of an implanted device. Polymeric systems can potentially be used to redirect infiltrating M1 MΦs toward a proangiogenic phenotype. This work exploits the concept of MΦ reprogramming in the engineering of materials for improving the longevity of tissue engineering scaffolds. We have investigated the effect of 13 different chemical modifications of alginate on MΦ phenotype. Markers of the M1 response-tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase-and the M2 response-arginase-were measured and used to determine the ability of the materials to alter MΦ phenotype. It was found that some modifications were able to reduce the pro-inflammatory response of M1 MΦs, others appeared to amplify the M2 phenotype, and the results for two materials suggested they were able to reprogram a MΦ population from M1 to M2. These findings were supplemented by studies done to examine the permselectivity of the materials. Diffusion of TNF-α was completely prevented through some of these materials, while up to 84% was found to diffuse through others. The diffusion of insulin through the materials was statistically consistent. These results suggest that the modification of these materials might alter mass transport in beneficial ways. The ability to control polarization of MΦ phenotypes with immunoprotective materials has the potential to augment the success of tissue engineering scaffolds. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1707-1719, 2016. © 2016 Wiley Periodicals, Inc.

Prenatal Nutritional Deficiency Reprogrammed Postnatal Gene Expression in Mammal Brains: Implications for Schizophrenia

PubMed Central

Xu, Jiawei; He, Guang; Zhu, Jingde; Zhou, Xinyao; St Clair, David; Wang, Teng; Xiang, Yuqian; Zhao, Qingzhu; Xing, Qinghe; Liu, Yun; Wang, Lei; Li, Qiaoli

2015-01-01

Background: Epidemiological studies have identified prenatal exposure to famine as a risk factor for schizophrenia, and animal models of prenatal malnutrition display structural and functional brain abnormalities implicated in schizophrenia. Methods: The offspring of the RLP50 rat, a recently developed animal model of prenatal famine malnutrition exposure, was used to investigate the changes of gene expression and epigenetic modifications in the brain regions. Microarray gene expression analysis was carried out in the prefrontal cortex and the hippocampus from 8 RLP50 offspring rats and 8 controls. MBD-seq was used to test the changes in DNA methylation in hippocampus depending on prenatal malnutrition exposure. Results: In the prefrontal cortex, offspring of RLP50 exhibit differences in neurotransmitters and olfactory-associated gene expression. In the hippocampus, the differentially-expressed genes are related to synaptic function and transcription regulation. DNA methylome profiling of the hippocampus also shows widespread but systematic epigenetic changes; in most cases (87%) this involves hypermethylation. Remarkably, genes encoded for the plasma membrane are significantly enriched for changes in both gene expression and DNA methylome profiling screens (p = 2.37×10–9 and 5.36×10–9, respectively). Interestingly, Mecp2 and Slc2a1, two genes associated with cognitive impairment, show significant down-regulation, and Slc2a1 is hypermethylated in the hippocampus of the RLP50 offspring. Conclusions: Collectively, our results indicate that prenatal exposure to malnutrition leads to the reprogramming of postnatal brain gene expression and that the epigenetic modifications contribute to the reprogramming. The process may impair learning and memory ability and result in higher susceptibility to schizophrenia. PMID:25522397

Reprogramming of single-cell derived mesenchymal stem cells into hair cell-like cells

PubMed Central

Lin, Zhaoyu; Perez, Philip; Sun, Zhenyu; Liu, Jan-Jan; Shin, June Ho; Hyrc, Krzysztof L.; Samways, Damien; Egan, Terry; Holley, Matthew C.; Bao, Jianxin

2012-01-01

Hypothesis Adult mesenchymal stem cells (MSCs) can be converted into hair cell-like cells by transdetermination. Background Given the fundamental role sensory hair cells play in sound detection and the irreversibility of their loss in mammals, much research has focused on developing methods to generate new hair cells as a means of treating permanent hearing loss. Although MSCs can differentiate into multiple cell lineages, no efficient means of reprogramming them into sensory hair cells exists. Earlier work has shown that the transcription factor Atoh1 is necessary for early development of hair cells, but it is not clear whether Atoh1 can be used to convert MSCs into hair cells. Methods Clonal MSC cell lines were established and reprogrammed into hair cell-like cells by a combination of protein transfer, adenoviral based gene transfer and co-culture with neurons. During transdetermination, inner ear molecular markers were analyzed by RT-PCR, and cell structures were examined by immunocytochemistry. Results Atoh1 overexpression in MSCs failed to convert MSCs into hair cell-like cells, suggesting that the ability of Atoh1 to induce hair cell differentiation is context dependent. Because Atoh1 overexpression successfully transforms VOT-E36 cells into hair cell-like cells, we modified the cell context of MSCs by performing a total protein transfer from VOT-E36 cells prior to overexpressing Atoh1. The modified MSCs were transformed into hair cell-like cells and attracted contacts from spiral ganglion neurons in a co-culture model. Conclusion We established a new procedure, consisting of VOT-E36 protein transfer, Atoh1 overexpression, and co-culture with spiral ganglion neurons, which can transform MSCs into hair cell-like cells. PMID:23111404