Adult Somatic Cells Research Articles

Analysis of Recombinant VP22-OCT4 Fusion Protein Subcellular Location in HepG2 Cell Culture

There are many studies ongoing about the remarkable potential of pluripotent stem cell as the future regenerative therapy. Embryonic stem cell is the only source that has been studied well for it. However, many constraints arise regarding this matter like ethical problems and risk of cell transplantation rejection. Recently, there are many research undergone in exploring new approach through reprogramming somatic cell to become induced pluripotent stem cell (iPSC). OCT4 (Octamer-binding transcription factor 4) is one of the proteins that is responsible for the self-renewal of embryonic stem cell. Therefore, this study aimed to investigate the ability of OCT4 transcription factor, with the help of VP22 (viral protein), to be localized into adult somatic cells (HepG2), which in turn could reprogram it into iPSC. In this study, HepG2 cells are isolated with VP22-OCT4 recombinant fusion protein for 1 hour and 6 hours, which then followed by isolating with mouse monoclonal IgG primary antibody against OCT4 (Ab1) and goat anti-mouse IgG secondary antibody, FITC conjugate (Ab2) respectively for the indirect immunofluorescence staining function. Confocal microscope is utilized to observe the presence of immunofluorescence. The result of the experiment showed a significant difference between the control and the experimental groups for both incubation period (p = 0.005 for 1 hour and p = 0.000 for 6 hours). Moreover, there is also a significance difference between the group of HepG2 cells with VP22-OCT4 incubated for 1 hour and 6 hours (p = 0.044). In conclusion, VP22-OCT4 recombinant protein can be translocated inside the HepG2 cells under 1 hour and 6 hours of incubation periods.

Sequence analysis of cell-free DNA derived from cultured human bone osteosarcoma (143B) cells.

The true importance of cell-free DNA in human biology, together with the potential scale of its clinical utility, is tarnished by a lack of understanding of its composition and origin. In investigating the cell-free DNA present in the growth medium of cultured 143B cells, we previously demonstrated that the majority of cell-free DNA is neither a product of apoptosis nor necrosis. In the present study, we investigated the composition and origin of this cell-free DNA population using next-generation sequencing. We found that the cell-free DNA comprises mainly of repetitive DNA, including α-satellite DNA, mini satellites, and transposons that are currently active or exhibit the capacity to become reactivated. A significant portion of these cell-free DNA fragments originates from specific chromosomes, especially chromosomes 1 and 9. In healthy adult somatic cells, the centromeric and pericentromeric regions of these chromosomes are normally densely methylated. However, in many cancer types, these regions are preferentially hypomethylated. This can lead to double-stranded DNA breaks or it can directly impair the formation of proper kinetochore structures. This type of chromosomal instability is a precursor to the formation of nuclear anomalies, including lagging chromosomes and anaphase bridges. DNA fragments derived from these structures can recruit their own nuclear envelope and form secondary nuclear structures known as micronuclei, which can localize to the nuclear periphery and bud out from the membrane. We postulate that the majority of cell-free DNA present in the growth medium of cultured 143B cells originates from these micronuclei.

Dosage compensation in human pre-implantation embryos: X-chromosome inactivation or dampening?

EMBO Reports (2018) e46294 Eutherian female mammals compensate the dosage of X‐linked gene expression between XY male and XX female, via transcriptional silencing of one of the two X‐chromosomes during embryonic development, a phenomenon known as X‐chromosome inactivation [1]. Studies have shown that like murine adult female somatic cells, the human counterparts also have many genes inactivated in one of the X‐chromosomes. In mouse, there are two forms of X‐inactivation: imprinted and random (Fig 1). Humans on the other hand do not undergo imprinted X‐inactivation [2]. However, X‐chromosome dynamics in human pre‐implantation embryos remains elusive, largely due to the restricted availability of human embryos and technical difficulties. Early experiments on human embryos reported conflicting results [3], [4], [5]. One study showed progressive accumulation of XIST on one of the X‐chromosomes along with the inactivation of X‐linked genes in pre‐implantation female embryos [3]. In contrast, another study reported XIST coating on both X‐chromosomes accompanied by partial exclusion of RNA‐Pol II in most early embryonic cells without the transcriptional silencing of X‐linked genes, indicating incipient X‐inactivation during pre‐implantation development. However, a minor population of cells showed monoallelic Xist expression, and the authors also reported XIST coating of the X‐chromosomes in male embryos [4]. These differences were most likely caused by differences in the sensitivity of the techniques used and/or the low numbers of X‐linked genes studied. In …

Direct Reprogramming of Adult Human Somatic Stem Cells Into Functional Neurons Using Sox2, Ascl1, and Neurog2.

Reprogramming of somatic cells into induced pluripotent stem cells (iPS) or directly into cells from a different lineage, including neurons, has revolutionized research in regenerative medicine in recent years. Mesenchymal stem cells are good candidates for lineage reprogramming and autologous transplantation, since they can be easily isolated from accessible sources in adult humans, such as bone marrow and dental tissues. Here, we demonstrate that expression of the transcription factors (TFs) SRY (sex determining region Y)-box 2 (Sox2), Mammalian achaete-scute homolog 1 (Ascl1), or Neurogenin 2 (Neurog2) is sufficient for reprogramming human umbilical cord mesenchymal stem cells (hUCMSC) into induced neurons (iNs). Furthermore, the combination of Sox2/Ascl1 or Sox2/Neurog2 is sufficient to reprogram up to 50% of transfected hUCMSCs into iNs showing electrical properties of mature neurons and establishing synaptic contacts with co-culture primary neurons. Finally, we show evidence supporting the notion that different combinations of TFs (Sox2/Ascl1 and Sox2/Neurog2) may induce multiple and overlapping neuronal phenotypes in lineage-reprogrammed iNs, suggesting that neuronal fate is determined by a combination of signals involving the TFs used for reprogramming but also the internal state of the converted cell. Altogether, the data presented here contribute to the advancement of techniques aiming at obtaining specific neuronal phenotypes from lineage-converted human somatic cells to treat neurological disorders.

Generation of defined neural populations from pluripotent stem cells.

Effective and efficient generation of human neural stem cells and subsequently functional neural populations from pluripotent stem cells has facilitated advancements in the study of human development and disease modelling. This review will discuss the established protocols for the generation of defined neural populations including regionalized neurons and astrocytes, oligodendrocytes and microglia. Early protocols were established in embryonic stem cells (ESC) but the discovery of induced pluripotent stem cells (iPSC) in 2006 provided a new platform for modelling human disorders of the central nervous system (CNS). The ability to produce patient- and disease-specific iPSC lines has created a new age of disease modelling. Human iPSC may be derived from adult somatic cells and subsequently patterned into numerous distinct cell types. The ability to derive defined and regionalized neural populations from iPSC provides a powerful in vitro model of CNS disorders.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

Epigenetic Regulation of Aldosterone Synthase Gene by Sodium and Angiotensin II.

Background DNA methylation is believed to be maintained in adult somatic cells. Recent findings, however, suggest that all methylation patterns are not stable. We demonstrate that stimulatory signals can change the DNA methylation status around transcription factor binding sites and a transcription start site and activate expression of the aldosterone synthase gene (CYP11B2).Methods and Results DNA methylation of CYP11B2 was analyzed in aldosterone‐producing adenomas, nonfunctioning adrenal adenomas, and adrenal glands and compared with the gene expression levels. CpG dinucleotides in the CYP11B2 promoter were found to be hypormethylated in tissues with high expression, but not in those with low expression, of CYP11B2. Methylation of the CYP11B2 promoter fused to a reporter gene decreased transcriptional activity. Methylation of recognition sequences of transcription factors, including CREB1, NGFIB (NR4A1), and NURR1 (NR4A2) diminished their DNA‐binding activity. A methylated‐CpG‐binding protein MECP2 interacted directly with the methylated CYP11B2 promoter. In rats, low salt intake led to upregulation of CYP11B2 expression and DNA hypomethylation in the adrenal gland. Treatment with angiotensin II type 1 receptor antagonist decreased CYP11B2 expression and led to DNA hypermethylation.Conclusions DNA demethylation may switch the phenotype of CYP11B2 expression from an inactive to an active state and regulate aldosterone biosynthesis.

Identification of Epigenetic Regulators of DUX4-fl for Targeted Therapy of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic de-repression of the disease locus, leading to pathogenic misexpression of the DUX4 gene in skeletal muscle. While the factors and pathways involved in normal repression of the FSHD locus in healthy cells have been well characterized, very little is known about those responsible for the aberrant activation of DUX4-fl in FSHD myocytes. Reasoning that DUX4-fl activators might represent useful targets for small molecule inhibition, we performed a highly targeted, candidate-based screen of epigenetic regulators in primary FSHD myocytes. We confirmed several of the strongest and most specific candidates (ASH1L, BRD2, KDM4C, and SMARCA5) in skeletal myocytes from two other unrelated FSHD1 patients, and we showed that knockdown led to reduced levels of DUX4-fl and DUX4-FL target genes, as well as altered chromatin at the D4Z4 locus. As a second mode of validation, targeting the CRISPR/dCas9-KRAB transcriptional repressor to the promoters of several candidates also led to reduced levels of DUX4-fl. Furthermore, these candidates can be repressed by different methods in skeletal myocytes without major effects on certain critical muscle genes. Our results demonstrate that expression of DUX4-fl is regulated by multiple epigenetic pathways, and they indicate viable, druggable candidates for therapeutic target development.

Optimization of culture conditions for the derivation and propagation of baboon (Papio anubis) induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) offer the possibility of cell replacement therapies using patient-matched cells to treat otherwise intractable diseases and debilitations. To successfully realize this potential, several factors must be optimized including i) selection of the appropriate cell type and numbers to transplant, ii) determination of the means of transplantation and the location into which the transplanted cells should be delivered, and iii) demonstration of the safety and efficacy of the cell replacement protocol to mitigate each targeted disease state. A majority of diseases or debilitations likely to be targeted by cell-based therapeutic approaches represent complex conditions or physiologies manifest predominantly in primates including humans. Nonhuman primates afford the most clinically relevant model system for biomedical studies and testing of cell-based therapies. Baboons have 92% genomic similarity with humans overall and especially significant similarities in their immunogenetic system, rendering this species a particularly valuable model for testing procedures involving cell transplants into living individuals. To maximize the utility of the baboon model, standardized protocols must be developed for the derivation of induced pluripotent stem cells from living adults and the long-term maintenance of these cells in culture. Here we tested four commercially available culture systems (ReproFF, mTeSR1, E8 and Pluristem) for competence to maintain baboon iPSCs in a pluripotent state over multiple passages, and to support the derivation of new lines of baboon iPSCs. Of these four media only Pluristem was able to maintain baboon pluripotency as assessed by morphological characteristics, immunocytochemistry and RT-qPCR. Pluristem also facilitated the derivation of new lines of iPSCs from adult baboon somatic cells, which had previously not been accomplished. We derived multiple iPS cell lines from adult baboon peripheral blood mononuclear cells cultured in Pluristem. These were validated by expression of the pluripotency markers OCT4, NANOG, SOX2, SSEA4 and TRA181, as well as the ability to differentiate into tissues from all three germ layers when injected into immunocompromised mice. These findings further advance the utility of the baboon as an ideal preclinical model system for optimizing iPS cell-based, patient-specific replacement therapies in humans.

The Pleiotropic Effects of the Canonical Wnt Pathway in Early Development and Pluripotency.

The technology to derive embryonic and induced pluripotent stem cells from early embryonic stages and adult somatic cells, respectively, emerged as a powerful resource to enable the establishment of new in vitro models, which recapitulate early developmental processes and disease. Additionally, pluripotent stem cells (PSCs) represent an invaluable source of relevant differentiated cell types with immense potential for regenerative medicine and cell replacement therapies. Pluripotent stem cells support self-renewal, potency and proliferation for extensive periods of culture in vitro. However, the core pathways that rule each of these cellular features specific to PSCs only recently began to be clarified. The Wnt signaling pathway is pivotal during early embryogenesis and is central for the induction and maintenance of the pluripotency of PSCs. Signaling by the Wnt family of ligands is conveyed intracellularly by the stabilization of β-catenin in the cytoplasm and in the nucleus, where it elicits the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of transcription factors. Interestingly, in PSCs, the Wnt/β-catenin–TCF/LEF axis has several unrelated and sometimes opposite cellular functions such as self-renewal, stemness, lineage commitment and cell cycle regulation. In addition, tight control of the Wnt signaling pathway enhances reprogramming of somatic cells to induced pluripotency. Several recent research efforts emphasize the pleiotropic functions of the Wnt signaling pathway in the pluripotent state. Nonetheless, conflicting results and unanswered questions still linger. In this review, we will focus on the diverse functions of the canonical Wnt signaling pathway on the developmental processes preceding embryo implantation, as well as on its roles in pluripotent stem cell biology such as self-renewal and cell cycle regulation and somatic cell reprogramming.

In Vivo Transient and Partial Cell Reprogramming to Pluripotency as a Therapeutic Tool for Neurodegenerative Diseases.

In theory, human diseases in which a specific cell type degenerates, such as neurodegenerative diseases, can be therapeutically addressed by replacement of the lost cells. The classical strategy for cell replacement is exogenous cell transplantation, but now, cell replacement can also be achieved with in situ reprogramming. Indeed, many of these disorders are age-dependent, and "rejuvenating" strategies based on cell epigenetic modifications are a possible approach to counteract disease progression. In this context, transient and/or partial reprogramming of adult somatic cells towards pluripotency can be a promising tool for neuroregeneration. Temporary and controlled in vivo overexpression of Yamanaka reprogramming factors (Oct3/4, Sox2, Klf4, and c-Myc (OSKM)) has been proven feasible in different experimental settings and could be employed to facilitate in situ tissue regeneration; this regeneration can be accomplished either by producing novel stem/precursor cells, without the challenges posed by exogenous cell transplantation, or by changing the epigenetic adult cell signature to the signature of a younger cell. The risk of this procedure resides in the possible lack of perfect control of the process, carrying a potential oncogenic or unexpected cell phenotype hazard. Recent studies have suggested that these limits can be overcome by a tightly controlled cyclic regimen of short-term OSKM expression in vivo that prevents full reprogramming to the pluripotent state and avoids both tumorigenesis and the presence of unwanted undifferentiated cells. On the other hand, this strategy can enhance tissue regeneration for therapeutic purposes in aging-related neurological diseases as well. These data could open the path to further research on the therapeutic potential of in vivo reprogramming in regenerative medicine.

Generation of Functional Dopaminergic Neurons from Reprogramming Fibroblasts by Nonviral-based Mesoporous Silica Nanoparticles

Direct-lineage conversion of the somatic cell by reprogramming, in which mature cells were fully converted into a variety of other cell types bypassing an intermediate pluripotent state, is a promising regenerative medicine approach. Due to the risk of tumorigenesis by viral methods, a non-viral carrier for the delivery of reprogramming factors is very desirable. This study utilized the mesoporous silica nanoparticles (MSNs) as a non-viral delivery system for transduction of the three key factors to achieve conversion of mouse fibroblasts (MFs) into functional dopaminergic neuron-like cells (denoted as fDA-neurons). At the same time, a neurogenesis inducer, ISX-9, was co-delivered with the MSNs to promote the direct conversion of neuron-like cells. Good transfection efficiency of plasmid@MSN allowed repeated dosing to maintain high exogenous gene expression analyzed by qPCR and the changes in neural function markers were monitored. To further validate the dopaminergic function and the electrophysiological properties of fDA-neurons, the results of ELISA assay showed the high levels of secreted-dopamine in the conditional medium and rich Na+/K+-channels were observed in the fDA-neurons on Day 22. The results demonstrated that MSN nanocarrier is effective in delivering the reprogramming factors for the conversion of functional dopaminergic neurons from adult somatic cells.

Cell Transplantation for Spinal Cord Injury: Tumorigenicity of Induced Pluripotent Stem Cell-Derived Neural Stem/Progenitor Cells.

Spinal cord injury (SCI) is an intractable and worldwide difficult medical challenge with limited treatments. Neural stem/progenitor cell (NS/PC) transplantation derived from fetal tissues or embryonic stem cells (ESCs) has demonstrated therapeutic effects via replacement of lost neurons and severed axons and creation of permissive microenvironment to promote repair of spinal cord and axon regeneration but causes ethnical concerns and immunological rejections as well. Thus, the implementation of induced pluripotent stem cells (iPSCs), which can be generated from adult somatic cells and differentiated into NS/PCs, provides an effective alternation in the treatment of SCI. However, as researches further deepen, there is accumulating evidence that the use of iPSC-derived NS/PCs shows mounting concerns of safety, especially the tumorigenicity. This review discusses the tumorigenicity of iPSC-derived NS/PCs focusing on the two different routes of tumorigenicity (teratomas and true tumors) and underlying mechanisms behind them, as well as possible solutions to circumvent them.

Clinical Applications of Induced Pluripotent Stem Cells - Stato Attuale.

In an adult human body, somatic stem cells are present in small amounts in almost all organs with the function of general maintenance and prevention of premature aging. But, these stem cells are not pluripotent and are unable to regenerate large cellular loss caused by infarctions or fractures especially in cells with limited replicative ability such as neurons and cardiomyocytes. These limitations gave rise to the idea of inducing pluripotency to adult somatic cells and thereby restoring their regeneration, replication and plasticity. Though many trials and research were focused on inducing pluripotency, a solid breakthrough was achieved by Yamanaka in 2006. Yamanaka's research identified 4 genes (OCT-4, SOX-2, KLF-4 and c-MYC) as the key requisite for inducing pluripotency in any somatic cells (iPSCs). Our study, reviews the major methods used for inducing pluripotency, differentiation into specific cell types and their application in both cell regeneration and disease modelling. We have also highlighted the current status of iPSCs in clinical applications by analysing the registered clinical trials. We believe that this review will assist the researchers to decide the parameters such as induction method and focus their efforts towards clinical application of iPSCs.

Dedifferentiation of human uterine polyp stem cells into embryo-like cells during inducing pluripotency.

By introduction of Oct4, Sox2, Klf4 and cMyc, human adult somatic cells can be reprogrammed into embryonic stem cell capable of pluripotent differentiation. In several lines of human endometrial polyp- and cervical polyp-mesenchymal stem cells (EPMSCs and CPMSC), we showed introduction of the four transcription factors led to a dedifferentiation of these cells into early embryo-like cells in three days, ranging from one-cell, two-cell, four-cell embryos, and morula to blastocyst. These early embryo-like cells resembled human early embryo derived from in vitro fertilization (IVF) in morphology, and hatching activity. These cells also expressed hypoblast (GATA4) and trophoblast (Cdx2) markers. After culturing the embryo-like cells for one month, the induced pluripotency stem cells (iPSC) could be formed (proved by pluripotency gene expression, by in vitro and in vivo differentiation). C/EBPα expression was also increased in uterine polyps. In contrast, MSCs derived from normal endometrium could not be induced to dedifferentiation to such early embryo-like cells. We conclude that EPMSCs and CPMSCs could be dedifferentiated to early embryo-like cells by the iPSC cocktail. This suggests that polyps of the organ derived from Mullerian duct may harbor epigenetic markers making them vulnerable to reprogramming to the earliest developmental stage. This study provides a simple model to derive early human embryo-like cells by in vitro.

Regenerative medicine using dental tissue derived induced pluripotent stem cell-biomaterials complex

In recent years, many researchers and clinicians found interest in regenerative medicine using induced pluripotent stem cells (iPSCs) with biomaterials due to their pluripotency, which is able to differentiate into any type of cells without human embryo, which of use is ethically controversial. However, there are limitations to make iPSCs from adult somatic cells due to their low stemness and donor site morbidity. Recently, to overcome above drawbacks, dental tissue-derived iPSCs have been highlighted as a type of alternative sources for their high stemness, easy gathering, and their complex (ectomesenchymal) origin, which easily differentiate them to various cell types for nerve, vessel, and other dental tissue regeneration. In other part, utilizing biomaterials for regenerative medicine using cell is recently highlighted because they can modulate cell adhesion, proliferation and (de)differentiation. Therefore, this paper will convey the overview of advantages and drawbacks of dental tissue-derived iPSCs and their future application with biomaterials.

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

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.

Establishment of integration-free induced pluripotent stem cells from human recessive dystrophic epidermolysis bullosa keratinocytes

BackgroundInduced pluripotent stem cell (iPSC) technology enables patient-specific pluripotent stem cells to be derived from adult somatic cells without the use of an embryonic cell source. To date, recessive dystrophic epidermolysis bullosa (RDEB)-specific iPSCs have been generated from patients using integrating retroviral vectors. However, vector integration into the host genome can endanger the biosafety and differentiation propensities of iPSCs. Although various integration-free reprogramming systems have been reported, their utility in reprogramming somatic cells from patients remains largely undetermined. ObjectiveOur study aims to establish safe iPSCs from keratinocytes of RDEB patients using non-integration vector. MethodWe optimized and infected non-integrating Sendai viral vectors to reprogram keratinocytes from healthy volunteers and RDEB patients. ResultsSendai vector infection led to the reproducible generation of genomic modification-free iPSCs from these keratinocytes, which was proved by immunohistochemistry, reverse transcription polymerase chain reaction, methylation assay, teratoma assay and embryoid body formation assay. Furthermore, we confirmed that these iPSCs have the potential to differentiate into dermal fibroblasts and epidermal keratinocytes. ConclusionThis is the first report to prove that the Sendai vector system facilitates the reliable reprogramming of patient keratinocytes into transgene-free iPSCs, providing another pluripotent platform for personalized diagnostic and therapeutic approaches to RDEB.

Molecular Reproduction & Development Volume 84, Issue 12, December 2017

The planarian flatworm Schmidtea mediterranea develops its entire reproductive system post‐embryonically from a population of adult somatic stem cells. Counts et al. (this issue) assessed the requirement of different chaperonin CCT/TRiC subunits during spermatogenesis and somatic integrity using RNA‐interference, which allows for the genetic assessment of specific components during oogenesis and spermatogenesis in this organism. Shown here is a sexually mature planarian (S. mediterranea), imaged by dark‐field microscopy, overlaid on a confocal image of the interior of an individual testis lobe in which the nuclei of cells during various stages of spermatogenesis are revealed.

Multiplexed in vivo homology-directed repair and tumor barcoding enables parallel quantification of Kras variant oncogenicity

Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. However, determining the functional significance of specific alterations remains a major bottleneck in our understanding of the genetic determinants of cancer. Here, we present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice. Using this approach, we introduce a barcoded library of non-synonymous mutations into hotspot codons 12 and 13 of Kras in adult somatic cells to initiate tumors in the lung, pancreas, and muscle. High-throughput sequencing of barcoded KrasHDR alleles from bulk lung and pancreas reveals surprising diversity in Kras variant oncogenicity. Rapid, cost-effective, and quantitative approaches to simultaneously investigate the function of precise genomic alterations in vivo will help uncover novel biological and clinically actionable insights into carcinogenesis.

Reprogramming to pluripotency does not require transition through a primitive streak-like state

Pluripotency can be induced in vitro from adult somatic mammalian cells by enforced expression of defined transcription factors regulating and initiating the pluripotency network. Despite the substantial advances over the last decade to improve the efficiency of direct reprogramming, exact mechanisms underlying the conversion into the pluripotent stem cell state are still vaguely understood. Several studies suggested that induced pluripotency follows reversed embryonic development. For somatic cells of mesodermal and endodermal origin that would require the transition through a Primitive streak-like state, which would necessarily require an Eomesodermin (Eomes) expressing intermediate. We analyzed reprogramming in human and mouse cells of mesodermal as well as ectodermal origin by thorough marker gene analyses in combination with genetic reporters, conditional loss of function and stable fate-labeling for the broad primitive streak marker Eomes. We unambiguously demonstrate that induced pluripotency is not dependent on a transient primitive streak-like stage and thus does not represent reversal of mesendodermal development in vivo.