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Type 2 alveolar epithelial cells (AT2s), facultative progenitor cells of the lung alveolus, play a vital role in the biology of the distal lung. In vitro model systems that incorporate human cells, recapitulate the biology of primary AT2s, and interface with the outside environment could serve as useful tools to elucidate functional characteristics of AT2s in homeostasis and disease. We and others recently adapted human induced pluripotent stem cell-derived AT2s (iAT2s) for air-liquid interface (ALI) culture. Here, we comprehensively characterize the effects of ALI culture on iAT2s and benchmark their transcriptional profile relative to both freshly sorted and cultured primary human fetal and adult AT2s. We find that iAT2s cultured at ALI maintain an AT2 phenotype while upregulating expression of transcripts associated with AT2 maturation. We then leverage this platform to assay the effects of exposure to clinically significant, inhaled toxicants including cigarette smoke and electronic cigarette vapor.

Naive human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional "primed" hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Here, we describe the results from a high-throughput screen using ∼3,000 well-annotated compounds to identify essential signaling requirements for naive human pluripotency. We report that MEK1/2 inhibitors can be replaced during maintenance of naive human pluripotency by inhibitors targeting either upstream (FGFR, RAF) or downstream (ERK1/2) kinases. Naive hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naive resetting in combination with PKC, ROCK, and TNKS inhibitors and activin A. This work demonstrates that induction and maintenance of naive human pluripotency are governed by distinct signaling requirements., Competing Interests: Declaration of interests R.J. is a co-founder of Fate Therapeutics, Fulcrum Therapeutics, and Omega Therapeutics and an advisor to Dewpoint Therapeutics. T.W.T., S.A.K., and R.J. are co-inventors on a patent application related to naive hPSCs., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

How pluripotent stem cells differentiate into the main germ layers is a key question of developmental biology. Here, we show that the chromatin-related factor Whsc1 (also known as Nsd2 and MMSET) has a dual role in pluripotency exit and germ layer specification of embryonic stem cells. On induction of differentiation, a proportion of Whsc1-depleted embryonic stem cells remain entrapped in a pluripotent state and fail to form mesendoderm, although they are still capable of generating neuroectoderm. These functions of Whsc1 are independent of its methyltransferase activity. Whsc1 binds to enhancers of the mesendodermal regulators Gata4, T (Brachyury), Gata6 and Foxa2, together with Brd4, and activates the expression of these genes. Depleting each of these regulators also delays pluripotency exit, suggesting that they mediate the effects observed with Whsc1. Our data indicate that Whsc1 links silencing of the pluripotency regulatory network with activation of mesendoderm lineages.

Type 1 diabetes is characterized by autoimmune destruction of β cells located in pancreatic islets. However, tractable in vivo models of human pancreatic β cells have been limited. Here, we generated xenogeneic human pancreatic β-like cells in the mouse pancreas by orthotopic transplantation of stem cell-derived β (SC-β) cells into the pancreas of neonatal mice. The engrafted β-like cells expressed β cell transcription factors and markers associated with functional maturity. Engrafted human cells recruited mouse endothelial cells, suggesting functional integration. Human insulin was detected in the blood circulation of transplanted mice for months after transplantation and increased upon glucose stimulation. In addition to β-like cells, human cells expressing markers for other endocrine pancreas cell types, acinar cells, and pancreatic ductal cells were identified in the pancreata of transplanted mice, indicating that this approach allows studying other human pancreatic cell types in the mouse pancreas. Our results demonstrate that orthotopic transplantation of human SC-β cells into neonatal mice is an experimental platform that allows the generation of mice with human pancreatic β-like cells in the endogenous niche., Competing Interests: Conflict of interest statement: R.J. is a cofounder and advisor of Fate Therapeutics, Fulcrum Therapeutics, and Omega Therapeutics. D.A.M. is the founder of Semma Therapeutics.

Pluripotent stem cells have broad utility in biomedical research and their molecular regulation has thus garnered substantial interest. While the principles that establish and regulate pluripotency have been well defined in the mouse, it has been difficult to extrapolate these insights to the human system due to species-specific differences and the distinct developmental identities of mouse versus human embryonic stem cells. In this Review, we examine genome-wide approaches to elucidate the regulatory principles of pluripotency in human embryos and stem cells, and highlight where differences exist in the regulation of pluripotency in mice and humans. We review recent insights into the nature of human pluripotent cells in vivo , obtained by the deep sequencing of pre-implantation embryos. We also present an integrated overview of the principal layers of global gene regulation in human pluripotent stem cells. Finally, we discuss the transcriptional and epigenomic remodeling events associated with cell fate transitions into and out of human pluripotency., Competing Interests: Competing interestsR.J. is co-founder of Fate Therapeutics, Fulcrum Therapeutics and Omega Therapeutics., (© 2017. Published by The Company of Biologists Ltd.)

Naive human embryonic stem cells (hESCs) can be derived from primed hESCs or directly from blastocysts, but their X chromosome state has remained unresolved. Here, we show that the inactive X chromosome (X i ) of primed hESCs was reactivated in naive culture conditions. Like cells of the blastocyst, the resulting naive cells contained two active X chromosomes with XIST expression and chromosome-wide transcriptional dampening and initiated XIST-mediated X inactivation upon differentiation. Both establishment of and exit from the naive state (differentiation) happened via an XIST-negative X a X a intermediate. Together, these findings identify a cell culture system for functionally exploring the two X chromosome dosage compensation processes in early human development: X dampening and X inactivation. However, remaining differences between naive hESCs and embryonic cells related to mono-allelic XIST expression and non-random X inactivation highlight the need for further culture improvement. As the naive state resets X i abnormalities seen in primed hESCs, it may provide cells better suited for downstream applications., (Published by Elsevier Inc.)

In vitro differentiation of human pluripotent stem cells provides a systematic platform to investigate the physiological development and function of the human nervous system, as well as the etiology and consequence when these processes go awry. Recent development in three-dimensional (3D) organotypic culture systems allows modeling of the complex structure formation of the human CNS, and the intricate interactions between various resident neuronal and glial cell types. Combined with an ever-expanding genome editing and regulation toolkit such as CRISPR/Cas9, it is now a possibility to study human neurological disease in the relevant molecular, cellular and anatomical context. In this article, we review recent progress in 3D neural culture and the implications for disease modeling., (Copyright © 2016. Published by Elsevier Ltd.)

Recent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development. We also show that induction of the naive state is accompanied by genome-wide DNA hypomethylation, which is reversible except at imprinted genes, and that the X chromosome status resembles that of the human preimplantation embryo. However, we did not see efficient incorporation of naive human cells into mouse embryos. Overall, the different naive conditions we tested showed varied relationships to human embryonic states based on molecular criteria, providing a backdrop for future analysis of naive human pluripotency., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, we describe the 3D chromosome regulatory landscape of human naive and primed embryonic stem cells. To devise this map, we identified transcriptional enhancers and insulators in these cells and placed them within the context of cohesin-associated CTCF-CTCF loops using cohesin ChIA-PET data. The CTCF-CTCF loops we identified form a chromosomal framework of insulated neighborhoods, which in turn form topologically associating domains (TADs) that are largely preserved during the transition between the naive and primed states. Regulatory changes in enhancer-promoter interactions occur within insulated neighborhoods during cell state transition. The CTCF anchor regions we identified are conserved across species, influence gene expression, and are a frequent site of mutations in cancer cells, underscoring their functional importance in cellular regulation. These 3D regulatory maps of human pluripotent cells therefore provide a foundation for future interrogation of the relationships between chromosome structure and gene control in development and disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Stem cells self-renew and generate specialized progeny through differentiation, but vary in the range of cells and tissues they generate, a property called developmental potency. Pluripotent stem cells produce all cells of an organism, while multipotent or unipotent stem cells regenerate only specific lineages or tissues. Defining stem-cell potency relies upon functional assays and diagnostic transcriptional, epigenetic and metabolic states. Here we describe functional and molecular hallmarks of pluripotent stem cells, propose a checklist for their evaluation, and illustrate how forensic genomics can validate their provenance.

Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question of whether an earlier, "naive" state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles that of mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Deciphering the mechanisms of epigenetic reprogramming provides fundamental insights into cell fate decisions, which in turn reveal strategies to make the reprogramming process increasingly efficient. Here we review recent advances in epigenetic reprogramming to pluripotency with a focus on the principal molecular regulators. We examine the trajectories connecting somatic and pluripotent cells, genetic and chemical methodologies for inducing pluripotency, the role of endogenous master transcription factors in establishing the pluripotent state, and functional interactions between reprogramming factors and epigenetic regulators. Defining the crosstalk among the diverse molecular actors implicated in cellular reprogramming presents a major challenge for future inquiry., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many applications are limited due to the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. Here, using an endogenous LGR5-GFP reporter, we derived adult stem cells from hPSCs that gave rise to functional human intestinal tissue comprising all major cell types of the intestine. Histological and functional analyses revealed that such human organoid cultures could be derived with high purity and with a composition and morphology similar to those of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. This adult stem cell system provides a platform for studying human intestinal disease in vitro using genetically engineered hPSCs.

The homeodomain transcription factor Nanog is a central part of the core pluripotency transcriptional network and plays a critical role in embryonic stem cell (ESC) self-renewal. Several reports have suggested that Nanog expression is allelically regulated and that transient downregulation of Nanog in a subset of pluripotent cells predisposes them toward differentiation. Using single-cell gene expression analyses combined with different reporters for the two alleles of Nanog, we show that Nanog is biallelically expressed in ESCs independently of culture condition. We also show that the overall variation in endogenous Nanog expression in ESCs is very similar to that of several other pluripotency markers. Our analysis suggests that reporter-based studies of gene expression in pluripotent cells can be significantly influenced by the gene-targeting strategy and genetic background employed., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

Despite recent critical insights into the pluripotent state of embryonic stem cells (ESCs), there is little agreement over the inaugural and subsequent steps leading to its generation [1-4]. Here we show that inner cell mass (ICM)-generated cells expressing Blimp1, a key transcriptional repressor of the somatic program during germ cell specification [5, 6], emerge on day 2 of blastocyst culture. Single-cell gene expression profiling indicated that many of these Blimp1-positive cells coexpress other genes typically associated with early germ cell specification. When genetically traced in vitro, these cells acquired properties normally associated with primordial germ cells. Importantly, fate-mapping experiments revealed that ESCs commonly arise from Blimp1-positive precursors; indeed, prospective sorting of such cells from ICM outgrowths increased the rate of ESC derivation more than 9-fold. Finally, using genetic ablation or distinct small molecules [7, 8], we show that epiblast cells can become ESCs without first acquiring Blimp1 positivity. Our findings suggest that the germ cell-like state is facultative for the stabilization of pluripotency in vitro. Thus, the association of Blimp1 expression with ESC development furthers understanding of how the pluripotent state of these cells is established in vitro and suggests a means to enhance the generation of new stem cell lines from blastocysts., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The Tet family of enzymes (Tet1/2/3) converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Mouse embryonic stem cells (mESCs) highly express Tet1 and have an elevated level of 5hmC. Tet1 has been implicated in ESC maintenance and lineage specification in vitro but its precise function in development is not well defined. To establish the role of Tet1 in pluripotency and development, we have generated Tet1 mutant mESCs and mice. Tet1(-/-) ESCs have reduced levels of 5hmC and subtle changes in global gene expression, and are pluripotent and support development of live-born mice in tetraploid complementation assay, but display skewed differentiation toward trophectoderm in vitro. Tet1 mutant mice are viable, fertile, and grossly normal, though some mutant mice have a slightly smaller body size at birth. Our data suggest that Tet1 loss leading to a partial reduction in 5hmC levels does not affect pluripotency in ESCs and is compatible with embryonic and postnatal development., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease, as well as a promising source for cell replacement therapies. One crucial limitation has been the inability to perform experiments under genetically defined conditions. This is particularly relevant for late age onset disorders in which in vitro phenotypes are predicted to be subtle and susceptible to significant effects of genetic background variations. By combining zinc finger nuclease (ZFN)-mediated genome editing and iPSC technology, we provide a generally applicable solution to this problem, generating sets of isogenic disease and control human pluripotent stem cells that differ exclusively at either of two susceptibility variants for Parkinson's disease by modifying the underlying point mutations in the α-synuclein gene. The robust capability to genetically correct disease-causing point mutations in patient-derived hiPSCs represents significant progress for basic biomedical research and an advance toward hiPSC-based cell replacement therapies., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Induced pluripotent stem cells (iPSCs) are generated from somatic cells by the transduction of defined transcription factors, and this process involves dynamic changes in DNA methylation. While the reprogramming of somatic cells is accompanied by demethylation of pluripotency genes, the functional importance of de novo DNA methylation has not been clarified. Here, using loss-of-function studies, we generated iPSCs from fibroblasts that were deficient in de novo DNA methylation mediated by Dnmt3a and Dnmt3b. These iPSCs reactivated pluripotency genes, underwent self-renewal, and showed restricted developmental potential that was rescued upon reintroduction of Dnmt3a and Dnmt3b. We conclude that de novo DNA methylation by Dnmt3a and Dnmt3b is dispensable for nuclear reprogramming of somatic cells.

Direct reprogramming of somatic cells to induced pluripotent stem cells by ectopic expression of defined transcription factors has raised fundamental questions regarding the epigenetic stability of the differentiated cell state. In addition, evidence has accumulated that distinct states of pluripotency can interconvert through the modulation of both cell-intrinsic and exogenous factors. To fully realize the potential of in vitro reprogrammed cells, we need to understand the molecular and epigenetic determinants that convert one cell type into another. Here we review recent advances in this rapidly moving field and emphasize unresolved and controversial questions., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Recent advances in deriving induced pluripotent stem (iPS) cells from patients offer new possibilities for biomedical research and clinical applications, as these cells could be used for autologous transplantation. We differentiated iPS cells from patients with Parkinson's disease (PD) into dopaminergic (DA) neurons and show that these DA neurons can be transplanted without signs of neurodegeneration into the adult rodent striatum. The PD patient iPS (PDiPS) cell-derived DA neurons survived at high numbers, showed arborization, and mediated functional effects in an animal model of PD as determined by reduction of amphetamine- and apomorphine-induced rotational asymmetry, but only a few DA neurons projected into the host striatum at 16 wk after transplantation. We next applied FACS for the neural cell adhesion molecule NCAM on differentiated PDiPS cells before transplantation, which resulted in surviving DA neurons with functional effects on amphetamine-induced rotational asymmetry in a 6-OHDA animal model of PD. Morphologically, we found that PDiPS cell-derived non-DA neurons send axons along white matter tracts into specific close and remote gray matter target areas in the adult brain. Such findings establish the transplantation of human PDiPS cell-derived neurons as a long-term in vivo method to analyze potential disease-related changes in a physiological context. Our data also demonstrate proof of principle of survival and functional effects of PDiPS cell-derived DA neurons in an animal model of PD and encourage further development of differentiation protocols to enhance growth and function of implanted PDiPS cell-derived DA neurons in regard to potential therapeutic applications.

Multipotent adult germ-line stem cells (maGSCs) and induced pluripotent stem cells (iPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, effects of the immune system on these cells have not been investigated. We have compared the susceptibility of maGSC lines to IL-2-activated natural killer (NK) cells with embryonic stem cell (ESC) lines, iPSCs, and F9 teratocarcinoma cells. The killing of pluripotent cell lines by syngeneic, allogeneic, and xenogeneic killer cells ranged between 48 and 265% in chromium release assays when compared to YAC-1 cells, which served as highly susceptible reference cells. With the exception of 2 maGSC lines, they expressed ligands for the activating NK receptor NKG2D that belong to the RAE-1 family, and killing could be inhibited by soluble NKG2D, demonstrating a functional role of these molecules. Furthermore, ligands of the activating receptor DNAM-1 were frequently expressed. The susceptibility to NK cells might constitute a common feature of pluripotent cells. It could result in rejection after transplantation, as suggested by a reduced teratoma growth after NK cell activation in vivo, but it might also offer a strategy to deplete contaminating pluripotent cells before grafting of differentiated cells.

Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have very different biological properties, and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse-derived epiblast stem cells (EpiSCs). Here we rewire the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by ectopic induction of Oct4, Klf4, and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3beta (GSK3beta) and mitogen-activated protein kinase (ERK1/2) pathway. Forskolin, a protein kinase A pathway agonist which can induce Klf4 and Klf2 expression, transiently substitutes for the requirement for ectopic transgene expression. In contrast to conventional human ESCs, these epigenetically converted cells have growth properties, an X-chromosome activation state (XaXa), a gene expression profile, and a signaling pathway dependence that are highly similar to those of mouse ESCs. Finally, the same growth conditions allow the derivation of human induced pluripotent stem (iPS) cells with similar properties as mouse iPS cells. The generation of validated "naïve" human ESCs will allow the molecular dissection of a previously undefined pluripotent state in humans and may open up new opportunities for patient-specific, disease-relevant research.

Mouse embryonic stem cells (mESCs) have the ability to differentiate into any cell type and can generate chimeric mice when transplanted into a host blastocyst. This remarkable potential, together with the development of robust gene targeting strategies in mESCs, were essential for establishing the mouse as the most widely used model organism in biomedical research. Recent advances have allowed the isolation of human embryonic stem cells and the derivation of induced pluripotent stem cells. Genetic tools similar to those proven routine in the mouse system are needed to realize the full potential of human pluripotent cells as disease models and putative therapeutics. Gene targeting in human cells, however, has proven to be more difficult, more time-consuming, and less robust than in mESCs. In this chapter, we discuss the strategies that have been used to allow specific genetic modifications in human pluripotent cells. We focus on the novel application of custom-engineered zinc-finger nucleases for gene targeting, which has promise to become a robust tool for efficient genetic manipulation of human pluripotent cells.

Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc transcription factors, but only a minority of donor somatic cells can be reprogrammed to pluripotency. Here we demonstrate that reprogramming by these transcription factors is a continuous stochastic process where almost all mouse donor cells eventually give rise to iPS cells on continued growth and transcription factor expression. Additional inhibition of the p53/p21 pathway or overexpression of Lin28 increased the cell division rate and resulted in an accelerated kinetics of iPS cell formation that was directly proportional to the increase in cell proliferation. In contrast, Nanog overexpression accelerated reprogramming in a predominantly cell-division-rate-independent manner. Quantitative analyses define distinct cell-division-rate-dependent and -independent modes for accelerating the stochastic course of reprogramming, and suggest that the number of cell divisions is a key parameter driving epigenetic reprogramming to pluripotency.

Several types of terminally differentiated somatic cells can be reprogrammed into a pluripotent state by ectopic expression of Klf4, Oct3/4, Sox2, and c-Myc. Such induced pluripotent stem (iPS) cells have great potential to serve as an autologous source of cells for tissue repair. In the process of developing iPS-cell-based therapies, the major goal is to determine whether differentiated cells derived from iPS cells, such as cardiomyocytes (CMs), have the same functional properties as their physiological in vivo counterparts. Therefore, we differentiated murine iPS cells to CMs in vitro and characterized them by RT-PCR, immunocytochemistry, and electrophysiology. As key markers of cardiac lineages, transcripts for Nkx2.5, alphaMHC, Mlc2v, and cTnT could be identified. Immunocytochemical stainings revealed the presence of organized sarcomeric actinin but the absence of mature atrial natriuretic factor. We examined characteristics and developmental changes of action potentials, as well as functional hormonal regulation and sensitivity to channel blockers. In addition, we determined expression patterns and functionality of cardiac-specific voltage-gated Na+, Ca2+, and K+ channels at early and late differentiation stages and compared them with CMs derived from murine embryonic stem cells (ESCs) as well as with fetal CMs. We conclude that iPS cells give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed cardiac ion channels; CMs generated from iPS cells have a ventricular phenotype; and cardiac development of iPS cells is delayed compared with maturation of native fetal CMs and of ESC-derived CMs. This difference may reflect the incomplete reprogramming of iPS cells and should be critically considered in further studies to clarify the suitability of the iPS model for regenerative medicine of heart disorders.

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.

Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the postimplantation epiblast and display a restricted developmental potential. Here we characterize pluripotent states in the nonobese diabetic (NOD) mouse strain, which prior to this study was considered "nonpermissive" for ESC derivation. We find that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming. The NOD ESCs and iPSCs appear to be "metastable," as they acquire an alternative EpiSC-like identity after removal of the exogenous factors, while their reintroduction converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by exogenous factors.

Ectopic expression of defined transcription factors can reprogram somatic cells to induced pluripotent stem (iPS) cells, but the utility of iPS cells is hampered by the use of viral delivery systems. Small molecules offer an alternative to replace virally transduced transcription factors with chemical signaling cues responsible for reprogramming. In this report we describe a small-molecule screening platform applied to identify compounds that functionally replace the reprogramming factor Klf4. A series of small-molecule scaffolds were identified that activate Nanog expression in mouse fibroblasts transduced with a subset of reprogramming factors lacking Klf4. Application of one such molecule, kenpaullone, in lieu of Klf4 gave rise to iPS cells that are indistinguishable from murine embryonic stem cells. This experimental platform can be used to screen large chemical libraries in search of novel compounds to replace the reprogramming factors that induce pluripotency. Ultimately, such compounds may provide mechanistic insight into the reprogramming process.

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.

Directed reprogramming of somatic cells by defined factors provides a novel method for the generation of patient-specific stem cells with the potential to bypass both the practical and ethical concerns associated with somatic cell nuclear transfer (SCNT) and human embryonic stem (hES) cells. Although the generation of induced pluripotent stem (iPS) cells has proven a robust technology in mouse and human, a major impediment to the use of iPS cells for therapeutic purposes has been the viral-based delivery of the reprogramming factors because multiple proviral integrations pose the danger of insertional mutagenesis. Here we report a novel approach to reduce the number of viruses necessary to reprogram somatic cells by delivering reprogramming factors in a single virus using 2A "self-cleaving" peptides, which support efficient polycistronic expression from a single promoter. We find that up to four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can be expressed from a single virus to generate iPS cells in both embryonic and adult somatic mouse cells and we show that a single proviral copy is sufficient to generate iPS cells from mouse embryonic fibroblasts. In addition we have generated human induced pluripotent stem (hiPS) cell lines from human keratinocytes, demonstrating that a single polycistronic virus can reprogram human somatic cells.

Aims: Induced pluripotent stem (iPS) cells have a developmental potential similar to that of blastocyst-derived embryonic stem (ES) cells and may serve as an autologous source of cells for tissue repair, in vitro disease modelling and toxicity assays. Here we aimed at generating iPS cell-derived cardiomyocytes (CMs) and comparing their molecular and functional characteristics with CMs derived from native murine ES cells., Methods and Results: Beating cardiomyocytes were generated using a mass culture system from murine N10 and O9 iPS cells as well as R1 and D3 ES cells. Transcripts of the mesoderm specification factor T-brachyury and non-atrial cardiac specific genes were expressed in differentiating iPS EBs. Using immunocytochemistry to determine the expression and intracellular organisation of cardiac specific structural proteins we demonstrate strong similarity between iPS-CMs and ES-CMs. In line with a previous study electrophysiological analyses showed that hormonal response to beta-adrenergic and muscarinic receptor stimulation was intact. Action potential (AP) recordings suggested that most iPS-CMs measured up to day 23 of differentiation are of ventricular-like type. Application of lidocaine, Cs+, SEA0400 and verapamil+ nifedipine to plated iPS-EBs during multi-electrode array (MEA) measurements of extracellular field potentials and intracellular sharp electrode recordings of APs revealed the presence of I(Na), I(f), I(NCX), and I(CaL), respectively, and suggested their involvement in cardiac pacemaking, with I(CaL) being of major importance. Furthermore, iPS-CMs developed and conferred force to avitalized ventricular tissue that was responsive to beta-adrenergic stimulation., Conclusions: Our data demonstrate that the cardiogenic potential of iPS cells is comparable to that of ES cells and that iPS-CMs possess all fundamental functional elements of a typical cardiac cell, including spontaneous beating, hormonal regulation, cardiac ion channel expression and contractility. Therefore, iPS-CMs can be regarded as a potentially valuable source of cells for in vitro studies and cellular cardiomyoplasty., (2009 S. Karger AG, Basel.)

Expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc in mesodermal and endodermal derivatives, including fibroblasts, lymphocytes, liver, stomach, and beta cells, generates induced pluripotent stem (iPS) cells. It remains unknown, however, whether cell types of the ectodermal lineage are equally amenable to reprogramming into iPS cells by the same combination of factors. To test this, we have isolated genetically marked neural progenitor cells (NPCs) from neonatal mouse brains and infected them with viral vectors expressing Oct4, Sox2, Klf4, and c-Myc. Infected NPCs gave rise to iPS cells that expressed markers of embryonic stem cells, showed demethylation of pluripotency genes, formed teratomas, and contributed to viable chimeras. In contrast to other somatic cell types, NPCs expressed high levels of endogenous Sox2 and thus did not require viral Sox2 expression for reprogramming into iPS cells. Our data show that in addition to mesoderm- and endoderm-derived cell types, neural progenitor cells of the ectodermal lineage can be reprogrammed into iPS cells, suggesting that in vitro reprogramming is a universal process. These results also imply that the combination of factors necessary for reprogramming is dependent on cellular context. Disclosure of potential conflicts of interest is found at the end of this article.

Current approaches to reprogram human somatic cells to pluripotent iPSCs utilize viral transduction of different combinations of transcription factors. These protocols are highly inefficient because only a small fraction of cells carry the appropriate number and stoichiometry of proviral insertions to initiate the reprogramming process. Here we have generated genetically homogeneous "secondary" somatic cells, which carry the reprogramming factors as defined doxycycline (DOX)-inducible transgenes. These cells were obtained by infecting fibroblasts with DOX-inducible lentiviruses, isolating "primary" iPSCs in the presence of the drug, and finally differentiating to "secondary" fibroblasts. When "secondary" fibroblast lines were cultured in the presence of DOX without further viral infection, up to 2% of the cells were reprogrammed to pluripotent "secondary" human iPSCs. This system will facilitate the characterization of the reprogramming process and provides a unique platform for genetic or chemical screens to enhance reprogramming or replace individual factors.

DNA methylation is essential for normal development and has been implicated in many pathologies including cancer. Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that 'weak' CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, in a pattern reminiscent of that reported in some primary tumours. More generally, the results establish reduced representation bisulphite sequencing as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine.

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.

Somatic cells can be reprogrammed to a pluripotent state through the ectopic expression of defined transcription factors. Understanding the mechanism and kinetics of this transformation may shed light on the nature of developmental potency and suggest strategies with improved efficiency or safety. Here we report an integrative genomic analysis of reprogramming of mouse fibroblasts and B lymphocytes. Lineage-committed cells show a complex response to the ectopic expression involving induction of genes downstream of individual reprogramming factors. Fully reprogrammed cells show gene expression and epigenetic states that are highly similar to embryonic stem cells. In contrast, stable partially reprogrammed cell lines show reactivation of a distinctive subset of stem-cell-related genes, incomplete repression of lineage-specifying transcription factors, and DNA hypermethylation at pluripotency-related loci. These observations suggest that some cells may become trapped in partially reprogrammed states owing to incomplete repression of transcription factors, and that DNA de-methylation is an inefficient step in the transition to pluripotency. We demonstrate that RNA inhibition of transcription factors can facilitate reprogramming, and that treatment with DNA methyltransferase inhibitors can improve the overall efficiency of the reprogramming process.

Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency.

Since its discovery as a critical regulator of pluripotency in embryonic stem (ES) cells and the inner cells mass of the developing blastocyst, the Pou domain-containing transcription factor Oct4 has become a proxy for "stemness" in numerous studies of somatic stem cells as its presence is often taken as evidence of pluripotency in these cells. Recent studies, however, have demonstrated that not only is Oct4 dispensable for maintaining potency in somatic stem cell compartments, but also that the methods applied to detect Oct4 and the interpretation of the resulting data may be flawed. Here we contrast pathways known to govern pluripotency in embryonic stem cells with those in adult stem cells and critically discuss the concept of pluripotency in adult stem cells of the mammalian soma.

Reprogramming of somatic cells to a pluripotent embryonic stem cell-like state has been achieved by nuclear transplantation of a somatic nucleus into an enucleated egg and most recently by introducing defined transcription factors into somatic cells. Nuclear reprogramming is of great medical interest, as it has the potential to generate a source of patient-specific cells. Here, we review strategies to reprogram somatic cells to a pluripotent embryonic state and discuss our understanding of the molecular mechanisms of reprogramming based on recent insights into the regulatory circuitry of the pluripotent state.

Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs, we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first, followed by stage-specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene, marking fully reprogrammed cells, was only observed late in the process. Importantly, the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step toward understanding some of the molecular events governing epigenetic reprogramming.

It has recently been demonstrated that mouse and human fibroblasts can be reprogrammed into an embryonic stem cell-like state by introducing combinations of four transcription factors. However, the therapeutic potential of such induced pluripotent stem (iPS) cells remained undefined. By using a humanized sickle cell anemia mouse model, we show that mice can be rescued after transplantation with hematopoietic progenitors obtained in vitro from autologous iPS cells. This was achieved after correction of the human sickle hemoglobin allele by gene-specific targeting. Our results provide proof of principle for using transcription factor-induced reprogramming combined with gene and cell therapy for disease treatment in mice. The problems associated with using retroviruses and oncogenes for reprogramming need to be resolved before iPS cells can be considered for human therapy.

In vitro reprogramming of somatic cells into a pluripotent embryonic stem cell-like state has been achieved through retroviral transduction of murine fibroblasts with Oct4, Sox2, c-myc and Klf4. In these experiments, the rare 'induced pluripotent stem' (iPS) cells were isolated by stringent selection for activation of a neomycin-resistance gene inserted into the endogenous Oct4 (also known as Pou5f1) or Nanog loci. Direct isolation of pluripotent cells from cultured somatic cells is of potential therapeutic interest, but translation to human systems would be hindered by the requirement for transgenic donors in the present iPS isolation protocol. Here we demonstrate that reprogrammed pluripotent cells can be isolated from genetically unmodified somatic donor cells solely based upon morphological criteria.

We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells. By obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse embryonic stem cells, neural progenitor cells and embryonic fibroblasts. We find that lysine 4 and lysine 27 trimethylation effectively discriminates genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential. Lysine 36 trimethylation marks primary coding and non-coding transcripts, facilitating gene annotation. Trimethylation of lysine 9 and lysine 20 is detected at satellite, telomeric and active long-terminal repeats, and can spread into proximal unique sequences. Lysine 4 and lysine 9 trimethylation marks imprinting control regions. Finally, we show that chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations.

Nuclear transplantation can reprogramme a somatic genome back into an embryonic epigenetic state, and the reprogrammed nucleus can create a cloned animal or produce pluripotent embryonic stem cells. One potential use of the nuclear cloning approach is the derivation of 'customized' embryonic stem (ES) cells for patient-specific cell treatment, but technical and ethical considerations impede the therapeutic application of this technology. Reprogramming of fibroblasts to a pluripotent state can be induced in vitro through ectopic expression of the four transcription factors Oct4 (also called Oct3/4 or Pou5f1), Sox2, c-Myc and Klf4. Here we show that DNA methylation, gene expression and chromatin state of such induced reprogrammed stem cells are similar to those of ES cells. Notably, the cells-derived from mouse fibroblasts-can form viable chimaeras, can contribute to the germ line and can generate live late-term embryos when injected into tetraploid blastocysts. Our results show that the biological potency and epigenetic state of in-vitro-reprogrammed induced pluripotent stem cells are indistinguishable from those of ES cells.

Transcriptional regulators and epigenetic modifiers play crucial roles throughout development to ensure that proper gene expression patterns are established and maintained in any given cell type. Recent genome-wide studies have begun to unravel how genetic and epigenetic factors maintain the undifferentiated state of embryonic stem cells while allowing these cells to remain poised to differentiate into somatic cells in response to developmental cues. These studies provide a conceptual framework for understanding pluripotency and lineage-specification at the molecular level.

The cloning of mammals from differentiated donor cells has refuted the old dogma that development is an irreversible process. It has demonstrated that the oocyte can reprogramme an adult nucleus into an embryonic state that can direct development of a new organism. The prospect of deriving patient-specific embryonic stem cells by nuclear transfer underscores the potential use of this technology in regenerative medicine. The future challenge will be to study alternatives to nuclear transfer in order to recapitulate reprogramming in a Petri dish without the use of oocytes.