Cellular Reprogramming

The gastrointestinal (GI) epithelium is a highly regenerative tissue with the potential to provide a renewable source of insulin + cells using cellular reprogramming. Here, I describe the antral stomach as a previously unrecognized source highly amenable to conversion into functional insulin-secreting cells. Native antral endocrine cells share a surprising degree of transcriptional similarity with pancreatic ! cells. Expression of !-cell reprogramming factors (Ngn3, Pdx1, and Mafa) in vivo converts antral cells efficiently into insulin + cells with close molecular and functional resemblance to endogenous ! cells. My data further indicate that Cdx2, an intestine-specific transcription factor, acts as a molecular barrier for !-cell conversion. Induced GI insulin + cells can suppress hyperglycemia over at least 6 months, and they regenerate rapidly after ablation from the native stem-cell compartment. Transplantation of bioengineered stomach mini-organs also produced insulin + cells and suppressed hyperglycemia. These studies demonstrate the potential of developing engineered stomach tissue as a renewable source of functional ! cells for glycemic control.

Advances in regenerative nanomedicine raise a host of ethical, legal, and social questions that healthcare providers and scientists will need to consider. These questions and concerns include definitions, appropriate applications, dual use, potential risks, regulations, and access. In this chapter, we provide an overview of the questions and concerns and recommend proactive consideration and solutions.

Here we use high-throughput RT-PCR technology to take a snapshot of splicing changes in the full spectrum of high-and low-expressed genes during induction of fibroblasts, from several donors, into iPSCs and their subsequent redifferentiation. We uncover a programme of concerted alternative splicing changes involved in late mesoderm differentiation and controlled by key splicing regulators MBNL1 and RBFOX2. These critical splicing adjustments arise early in vertebrate evolution and remain fixed in at least 10 genes (including PLOD2, CLSTN1, ATP2A1, PALM, ITGA6, KIF13A, FMNL3, PPIP5K1, MARK2 and FNIP1), implying that vertebrates require alternative splicing to fully implement the instructions of transcriptional control networks.

Nuclear reprogramming, the conversion of the epigenome of a differentiated cell to one that is similar to the undifferentiated embryonic state, can be facilitated by several methods, such as nuclear transfer, cell fusion, use of embryonic stem cell extracts, and more recently, by the introduction of exogenous transcription factors. Amongst these various strategies, somatic cell nuclear transfer (SCNT) is, by far, the most effective method of nuclear reprogramming. The majority of SCNT studies have been carried out using enucleated mature oocytes, as reprogramming is efficient and can be completed within hours following the introduction of the somatic cell nuclei into the recipient oocyte. Fertilized eggs, on the other hand, were regarded as poor recipients for nuclear transfer, as previous studies showed that embryonic blastomeres transferred into enucleated zygotes were unable to develop to blastocysts. However, more recent studies have demonstrated that the method of enucleation and the cell cycle phase of the embryos can impact the success of somatic cell reprogramming when zygotes were used as nuclear recipients. It is, therefore, timely to revisit and further explore the nuclear reprogramming capacity of zygotes as recipients for SCNT. Assessment of the various factors that influence the reprogramming capacity of zygotes in SCNT also provide hints of the mechanistic nature of nuclear reprogramming.

The environment can have a long-lasting influence on an individual's physiology and behavior. While some environmental conditions can be beneficial and result in adaptive responses, others can lead to pathological behaviors. Many studies have demonstrated that changes induced by the environment are expressed not only by the individuals directly exposed, but also by the offspring sometimes across multiple generations. Epigenetic alterations have been proposed as underlying mechanisms for such transmissible effects. Here, we review the most relevant literature on these changes and the developmental stages they affect the most. We discuss current evidence for transgenerational effects of prenatal and postnatal factors on bodily functions and behavioral responses, and the potential epigenetic mechanisms involved. We also discuss the need for a careful evaluation of the evolutionary importance with respect to health and disease, and possible directions for future research in the field.

Like breadcrumbs in the forest, cotranscriptionally acquired histone methylation acts as a memory of prior transcription. Because it can be retained through cell divisions, transcriptional memory allows cells to coordinate complex transcriptional programs during development. However, if not reprogrammed properly during cell fate transitions, it can also disrupt cellular identity. In this review, we discuss the consequences of failure to reprogram histone methylation during three crucial epigenetic reprogramming windows: maternal reprogramming at fertilization, embryonic stem cell (ESC) differentiation, and the continuous maintenance of cell identity in differentiated cells. In addition, we discuss how following the wrong breadcrumb trail of transcriptional memory provides a framework for understanding how heterozygous loss-of-function mutations in histone-modifying enzymes may cause severe neurodevelopmental disorders.

The discovery of human embryonic stem cells (hESCs) raised promises for a universal resource for cell based therapies in regenerative medicine. Recently, fast-paced progress has been made towards the generation of pluripotent stem cells (PSCs) amenable for clinical applications, culminating in reprogramming of adult somatic cells to autologous PSCs that can be indefinitely expanded in vitro. However, besides the efficient generation of bona fide, clinically safe PSCs (e.g., without the use of oncoproteins and gene transfer based on viruses inserting randomly into the genome), a major challenge in the field remains how to efficiently differentiate PSCs to specific lineages and how to select cells that will function normally upon transplantation in adults. In this review, we analyse the in vitro differentiation potential of PSCs to the hematopoietic lineage by discussing blood cell types that can be currently obtained, limitations in derivation of adult-type HSCs and prospects for clinical application of PSCs-derived blood cells.

Epigenetic reprogramming of germ cells involves the genome-wide erasure and subsequent reestablishment of DNA methylation, along with reprogramming of histone modification profiles and the eventual incorporation of histone variants. These linked processes appear to be key for the establishment of the correct epigenetic regulation of this cell lineage. Mouse studies indicate that DNA demethylation may be initiated at E (embryonic day) 8 with rapid and substantial erasure occurring between E11.5 and E12.5. This is accompanied by a reduction in H3K9 dimethylation and an increase in H3K27 trimethylation. DNA remethylation subsequently occurs in late gestation in male germ cells and postnatally in female germ cells. This reprogramming occurs throughout the genome, with the exception of specific sequences. The conservation of this process across species remains largely undetermined, and, with recent discoveries of new DNA modifications, there is still much to be explored.

M aintenance of cellular identity relies on the expression of cell type-specific transcription factors and the underlying state of the epigenome. During somatic cell reprogramming , lineage-committed cells can be converted into iPSCs on expression of critical transcriptional regulators of embryonic stem cells; namely, OCT4, SOX2, KLF4 and MYC (OSKM) 1. This process results in a complete change in cellular identity, and involves extensive transcriptional and epigenome-wide changes 2. The state of an epigenome is determined by the activities of chromatin writer, reader and eraser proteins. Therefore, chromatin regulators have emerged as important factors of cell identity and can act as facilita-tors or barriers to reprogramming 3. Inhibition of a number of major chromatin-related pathways can facilitate reprogramming, such as DNA methylation 4 , histone deacetylation 5 , histone H3 lysine 9 methylation 6,7 , CAF-1 complex 8 and NCoR/SMRT corepressors 9. These factors safeguard cellular identity mainly by preventing the activation of pluripotency genes. However, chromatin factors also safeguard cellular identity by perpetuating active somatic-specific gene transcription programs or by preventing the silencing of such genes on OSKM expression. Recent work has indicated that loss of the 'active' histone H3 lysine 27 acet-ylation (H3K27) mark from the enhancers of somatic-specific genes is an early step in reprogramming 10. An important remaining question is which chromatin factors counteract the OSKM-mediated silencing of lineage-specific gene expression in reprogramming. We had previously identified DOT1L-mediated histone H3 lysine 79 methylation as one of the key barriers to reprogramming that counteracts the silencing of lineage-specific genes 6. DOT1L inhibition could also replace KLF4 and MYC in human iPSC generation. In subsequent studies, inhibition of DOT1L activity has been shown to increase reprogramming efficiency in a wide range of systems 11,12. Importantly, DOT1L inhibitors facilitate the derivation of chemically induced pluripotent stem cells (ciPSCs) from mouse somatic cells 13. However, the generation of ciPSCs from human cells has not been achieved so far, suggesting that additional barriers exist for human cell reprogramming. The CBP and EP300 proteins are closely related histone acetyl-transferases (HATs) that act as transcriptional coactivators 14. CBP and EP300 (also known as KAT3A and KAT3B, respectively) are large, multi-domain proteins, which in addition to their catalytic HAT domain, contain bromodomains that bind acetylated histones and are required for chromatin binding 15-17. Localization of CBP/ EP300 in the genome has been used to identify cell type-specific enhancers in mice and humans 18,19. These coactivators also occupy super-enhancer regions, which have an important role in maintaining cell identity 20,21. Both coactivators are required for early embry-onic development and proper differentiation of a wide range of cell types 22. However, the role of these two major coactivators in reprogramming remains largely unknown. In this study, we carried out a chromatin-focused chemical screen to identify epigenetic regulators that can collaborate with DOT1L inhibition in reprogramming. We discover that CBP/EP300 bromodomain inhibition enhances reprogramming to pluripotency by facilitating the silencing of the somatic gene expression program. Results A chromatin-focused chemical screen for reprogramming. We carried out a reprogramming screen in the presence of inhibitors Silencing of the somatic cell type-specific genes is a critical yet poorly understood step in reprogramming. To uncover pathways that maintain cell identity, we performed a reprogramming screen using inhibitors of chromatin factors. Here, we identify acetyl-lysine competitive inhibitors targeting the bromodomains of coactivators CREB (cyclic-AMP response element binding protein) binding protein (CBP) and E1A binding protein of 300 kDa (EP300) as potent enhancers of reprogramming. These inhibitors accelerate reprogramming, are critical during its early stages and, when combined with DOT1L inhibition, enable efficient derivation of human induced pluripotent stem cells (iPSCs) with OCT4 and SOX2. In contrast, catalytic inhibition of CBP/EP300 prevents iPSC formation, suggesting distinct functions for different coactivator domains in reprogramming. CBP/EP300 bromodomain inhibition decreases somatic-specific gene expression, histone H3 lysine 27 acetylation (H3K27Ac) and chromatin accessibility at target promoters and enhancers. The master mesenchymal transcription factor PRRX1 is one such functionally important target of CBP/EP300 bromodomain inhibition. Collectively, these results show that CBP/EP300 bromodomains sustain cell-type-specific gene expression and maintain cell identity. NATURE ChEMiCAl BiOlOGy | VOL 15 | MAY 2019 | 519-528 | www.nature.com/naturechemicalbiology 519

Adult stem cells (SCs) are essential for tissue maintenance and regeneration yet are susceptible to senescence during aging. We demonstrate the importance of the amount of the oxidized form of cellular nicotinamide adenine dinucleotide (NAD(+)) and its impact on mitochondrial activity as a pivotal switch to modulate muscle SC (MuSC) senescence. Treatment with the NAD(+) precursor nicotinamide riboside (NR) induced the mitochondrial unfolded protein response (UPR(mt)) and synthesis of prohibitin proteins, and this rejuvenated MuSCs in aged mice. NR also prevented MuSC senescence in the Mdx mouse model of muscular dystrophy. We furthermore demonstrate that NR delays senescence of neural SCs (NSCs) and melanocyte SCs (McSCs), and increased mouse lifespan. Strategies that conserve cellular NAD(+) may reprogram dysfunctional SCs and improve lifespan in mammals.

Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson's disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons. In particular, transplantation of embryonic-stem-cell-derived dopaminergic neurons has been shown to be efficient in restoring motor symptoms in conditions of dopamine deficiency. However, the use of pluripotent-derived cells might lead to the development of tumours if not properly controlled. Here we identified a minimal set of three transcription factors--Mash1 (also known as Ascl1), Nurr1 (also known as Nr4a2) and Lmx1a--that are able to generate directly functional dopaminergic neurons from mouse and human fibroblasts without reverting to a progenitor cell stage. Induced dopaminergic (iDA) cells release dopamine and show spontaneous electrical activity organized in regular spikes consistent with the pacemaker activity featured by brain dopaminergic neurons. The three factors were able to elicit dopaminergic neuronal conversion in prenatal and adult fibroblasts from healthy donors and Parkinson's disease patients. Direct generation of iDA cells from somatic cells might have significant implications for understanding critical processes for neuronal development, in vitro disease modelling and cell replacement therapies.

Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient-and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells, capable of germline transmission, from mouse somatic cells by transduction of four defined transcription factors. Here, we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. Furthermore, these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts.

Familial hypertrophic cardiomyopathy (HCM) is one the most common heart disorders, with gene mutations in the cardiac sarcomere. Studying HCM with patient-specific induced pluripotent stem-cell (iPSC)-derived cardiomyocytes (CMs) would benefit the understanding of HCM mechanism, as well as the development of personalized therapeutic strategies. To investigate the molecular mechanism underlying the abnormal CM functions in HCM, we derived iPSCs from an HCM patient with a single missense mutation (Arginine442Glycine) in the MYH7 gene. CMs were next enriched from HCM and healthy iPSCs, followed with whole transcriptome sequencing and pathway enrichment analysis. A widespread increase of genes responsible for 'Cell Proliferation' was observed in HCM iPSC-CMs when compared with control iPSC-CMs. Additionally, HCM iPSC-CMs exhibited disorganized sarcomeres and electrophysiological irregularities. Furthermore, disease phenotypes of HCM iPSC-CMs were attenuated with pharmaceutical t...

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for ...

Reprogramming somatic cells to induced pluripotent stem (iPS) cells has been accomplished by expressing pluripotency factors and oncogenes 1-8 , but the low frequency and tendency to induce malignant transformation 9 compromise the clinical utility of this powerful approach. We address both issues by investigating the mechanisms limiting reprogramming efficiency in somatic cells.

Practical clinical applications for current induced pluripotent stem cell (iPSC) technologies are hindered by very low generation efficiencies. Here, we demonstrate that newborn human (h) and mouse (m) extra-embryonic amnion (AM) and yolk-sac (YS) cells, in which endogenous KLF4 ⁄ Klf4, c-MYC ⁄ c-Myc and RONIN ⁄ Ronin are expressed, can be reprogrammed to hiPSCs and miPSCs with efficiencies for AM cells of 0.02% and 0.1%, respectively. Both hiPSC and miPSCs are indistinguishable from embryonic stem cells in colony morphology, expression of pluripotency markers, global gene expression profile, DNA methylation status of OCT4 and NANOG, teratoma formation and, in the case of miPSCs, generation of germline transmissible chimeric mice. As copious amounts of human AM cells can be collected without invasion, and stored long term by conventional means without requirement for in vitro culture, they represent an ideal source for cell banking and subsequent 'on demand' generation of hiPSCs for personal regenerative and pharmaceutical applications.

Large animal models provide useful data for preclinical research including regenerative medicine. However whereas the derivation of tissue specific stem cells has been successful. pluripotent stem cells so far have been difficult to obtain in these species. A possible alternative could be direct reprogramming but this has only been described in mouse and human. We have recently described an alternative method for reprogramming human somatic cells based on a brief demethylation step immediately followed by an induction protocol. Aim of the present paper was to determine whether this method is applicable to pig in the attempt to achieve cell reprogramming in a large animal model for the first time. Pig dermal fibroblasts were exposed to DNA methyltransferase inhibitor 5-aza-cytidine (5-aza-CR) for 18 h. After a brief recovery period, fibroblast were subjected to a three-step protocol for the induction of endocrine pancreatic differentiation that was completed after 42 days. During the process pig fibroblast rapidly lost their typical elongated form and gradually became organized in a reticular pattern that evolved into distinct cell aggregates. After a brief expression of some pluripotency genes, cells expression pattern mimicked the transition from primitive endoderm to endocrine pancreas. Not only converted cells expressed insulin but were able to release it in response to a physiological glucose challenge in vitro. Finally they were able to protect recipient mice against streptozotocin-induced diabetes. This work shows, that the conversion of a somatic cell into another, even if belonging to a different germ layer, is possible also in pig.

Background: Inactivation of one X chromosome is established early in female mammalian development and can be reversed in vivo and in vitro when pluripotency factors are re-expressed. The extent of reactivation along the inactive X chromosome (Xi) and the determinants of locus susceptibility are, however, poorly understood. Here we use cell fusion-mediated pluripotent reprograming to study human Xi reactivation and allele-specific single nucleotide polymorphisms (SNPs) to identify reactivated loci. Results: We show that a subset of human Xi genes is rapidly reactivated upon re-expression of the pluripotency network. These genes lie within the most evolutionary recent segments of the human X chromosome that are depleted of LINE1 and enriched for SINE elements, predicted to impair XIST spreading. Interestingly, this cadre of genes displays stochastic Xi expression in human fibroblasts ahead of reprograming. This stochastic variability is evident between clones, by RNA-sequencing, and at the single-cell level, by RNA-FISH, and is not attributable to differences in repressive histone H3K9me3 or H3K27me3 levels. Treatment with the DNA demethylating agent 5-deoxy-azacytidine does not increase Xi expression ahead of reprograming, but instead reveals a second cadre of genes that only become susceptible to reactivation upon induction of pluripotency. Conclusions: Collectively, these data not only underscore the multiple pathways that contribute to maintaining silencing along the human Xi chromosome but also suggest that transcriptional stochasticity among human cells could be useful for predicting and engineering epigenetic strategies to achieve locus-specific or domain-specific human Xi gene reactivation.

The discovery of induced pluripotent stem cells (iPSCs) has the potential to revolutionize the field of regenerative medicine. In the past few years, iPSCs have been the subject of intensive research towards their application in disease modeling and drug screening. In the future, these cells may be applied in cell therapy to replace or regenerate tissues by autologous transplantation. However, two major hurdles need to be resolved in order to reach the later goal: the low reprogramming efficiency and the safety risks,

Introduction: Advances in the field of stem cells have led to novel avenues for generating induced pluripotent stem cells (iPSCs) from differentiated somatic cells. iPSCs are typically obtained by the introduction of four factors-OCT4, SOX2, KLF4, and cMYC-via integrating vectors. Here, we report the feasibility of a novel reprogramming process based on vectors derived from the non-integrating vaccine strain of measles virus (MV). Methods: We produced a one-cycle MV vector by substituting the viral attachment protein gene with the green fluorescent protein (GFP) gene. This vector was further engineered to encode for OCT4 in an additional transcription unit.

The oocyte is the only cell of the body that can reprogram transplanted somatic nuclei and sets the gold standard for all reprogramming methods. Therefore, an in-depth characterization of its proteome holds promise to advance our understanding of reprogramming and germ cell biology. To date, limitations on oocyte numbers and proteomic technology have impeded this task, and the search for reprogramming factors has been conducted in embryonic stem (ES) cells instead. Here, we present the proteome of metaphase II mouse oocytes to a depth of 3699 proteins, which substantially extends the number of proteins identified until now in mouse oocytes and is comparable by size to the proteome of undifferentiated mouse ES cells. Twenty-eight oocyte proteins, also detected in ES cells, match the criteria of our multilevel approach to screen for reprogramming factors, namely nuclear localization, chromatin modification, and catalytic activity. Our oocyte proteome catalog thus advances the definition of the "reprogrammome", the set of molecules-proteins, RNAs, lipids, and small molecules-that enable reprogramming.

Introduction: Stem cells are cells with the ability to grow and differentiate into more than 200 cell types. Sources of data: We review here the characteristics and potential of human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs) and adult stem cells (ASCs). Areas of agreement: The differentiation ability of all stem cell types could be stimulated to obtain specialized cells that represent renewable sources of functional cells useful for cell-based therapy. Areas of controversy: The proof of functional differentiated cells needs to be investigated in more detail using both in vitro and in vivo assays including animal disease models and clinical studies. Growing points: Much progress has been made in the ASCs-based therapies. Meanwhile hESCs and iPSCs have dramatically emerged as novel approaches to understand pathogenesis of different diseases. Areas timely for developing research: A number of new strategies become very important in regenerative medicine. However, we discuss the limitations of stem cells and latest development in the reprogramming research.

In C. elegans, the H3K36 methyltransferase, MES-4, helps establish germ cell fate by maintaining H3K36me2/3 at germline genes between generations. Previously, we showed that the H3K4me2 demethylase, SPR-5, and the H3K9 methyltransferase, MET-2, reprogram histone methylation at fertilization to prevent the ectopic expression of germline genes in somatic tissues. Together, this indicates that SPR-5 and MET-2 maternal reprogramming may antagonize MES-4 to establish germline versus soma. Here, we show that spr-5; met-2 mutant progeny have a severe developmental delay that is associated with the ectopic maintenance of H3K36me2/3 at MES-4 targeted germline genes in somatic tissues, and the ectopic expression of these genes. We further show that the developmental delay and the ectopic expression are dependent upon MES-4. Thus, we propose that SPR-5, MET-2, and MES-4 balance inherited histone methylation to establish germline versus soma. Without this balance, the inappropriate transcription of germline genes in somatic tissues causes developmental delay.

, but the use of iPSCs is hindered by the use of viral delivery systems. Chemical-induced reprogramming offers a novel approach to generating iPSCs without any viral vector-based genetic modification. Previous reports showed that several small molecules could replace some of the reprogramming factors although at least two transcription factors, Oct4 and Klf4, are still required to generate iPSCs from mouse embryonic fibroblasts. Here, we identify a specific chemical combination, which is sufficient to permit reprogramming from mouse embryonic and adult fibroblasts in the presence of a single transcription factor, Oct4, within 20 days, replacing Sox2, Klf4 and c-Myc. The iPSCs generated using this treatment resembled mouse embryonic stem cells in terms of global gene expression profile, epigenetic status and pluripotency both in vitro and in vivo. We also found that 8 days of Oct4 induction was sufficient to enable Oct4-induced reprogramming in the presence of the small molecules, which suggests that reprogramming was initiated within the first 8 days and was independent of continuous exogenous Oct4 expression. These discoveries will aid in the future generation of iPSCs without genetic modification, as well as elucidating the molecular mechanisms that underlie the reprogramming process.

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine.

| For decades, Waddington's concept of the 'epigenetic landscape' has served as an educative hierarchical model to illustrate the progressive restriction of cell differentiation potential during normal development. While still being highly valuable in the context of normal development, the Waddington model falls short of accommodating recent breakthroughs in cell programming. The advent of induced pluripotent stem (iPS) cells and advances in direct cell fate conversion (also known as transdifferentiation) suggest that somatic and pluripotent cell fates can be interconverted without transiting through distinct hierarchies. We propose a non-hierarchical 'epigenetic disc' model to explain such cell fate transitions, which provides an alternative landscape for modelling cell programming and reprogramming.

The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous 'secondary' somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25-to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming.

Werner syndrome (WS) patients exhibit premature aging predominantly in mesenchyme-derived tissues, but not in neural lineages, a consequence of telomere dysfunction and accelerated senescence. The cause of this lineage-specific aging remains unknown. Here, we document that reprogramming of WS fibroblasts to pluripotency elongated telomere length and prevented telomere dysfunction. To obtain mechanistic insight into the origin of tissue-specific aging, we differentiated iPSCs to mesenchymal stem cells (MSCs) and neural stem/progenitor cells (NPCs). We observed recurrence of premature senescence associated with accelerated telomere attrition and defective synthesis of the lagging strand telomeres in MSCs, but not in NPCs. We postulate this ''aging'' discrepancy is regulated by telomerase. Expression of hTERT or p53 knockdown ameliorated the accelerated aging phenotypein MSC, whereas inhibition of telomerase sensitized NPCs to DNA damage. Our findings unveil a role for telomerase in the protection of accelerated aging in a specific lineage of stem cells.

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.