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One of the biggest roadblocks to using stem cells as the basis for regenerative medicine therapies is the tumorigenicity of stem cells. Unfortunately, the unique abilities of stem cells to self-renew and differentiate into a variety of cell types are also mechanistically linked to their tumorigenic behaviors. Understanding the mechanisms underlying the close relationship between stem cells and cancer cells has therefore become a primary goal in the field. In addition, knowledge gained from investigating the striking parallels between mechanisms orchestrating normal embryogenesis and those that invoke tumorigenesis may well serve as the foundation for developing novel cancer treatments. Emerging discoveries have demonstrated that epigenetic regulatory machinery has important roles in normal stem cell functions, cancer development and cancer stem cell (CSC) identity. These studies provide valuable insights into both the shared and distinct mechanisms by which pluripotency and oncogenicity are established and regulated. In this review, the cancer-related epigenetic mechanisms found in pluripotent stem cells and cancer cells will be discussed, focusing on both the similarities and the differences.

In response to calls for public engagement on human genome editing (HGE), which intensified after the 2018 He Jiankui scandal that resulted in the implantation of genetically modified embryos, we detail an anticipatory approach to the governance of HGE. By soliciting multidisciplinary experts' input on the drivers and uncertainties of HGE development, we developed a set of plausible future scenarios to ascertain publics values-specifically, their hopes and concerns regarding the novel technology and its applications. In turn, we gathered a subset of multidisciplinary experts to propose governance recommendations for HGE that incorporate identified publics' values. These recommendations include: (1) continued participatory public engagement; (2) international harmonization and transparency of multiple governance levers such as professional and scientific societies, funders, and regulators; and (3) development of a formal whistleblower framework., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press.)

Folate supplementation reduces the occurrence of neural tube defects (NTDs), birth defects consisting in the failure of the neural tube to form and close. The mechanisms underlying NTDs and their prevention by folate remain unclear. Here we show that folate receptor 1 (FOLR1) is necessary for the formation of neural tube-like structures in human-cell derived neural organoids. FOLR1 knockdown in neural organoids and in Xenopus laevis embryos leads to NTDs that are rescued by pteroate, a folate precursor that is unable to participate in metabolism. We demonstrate that FOLR1 interacts with and opposes the function of CD2-associated protein, molecule essential for apical endocytosis and turnover of C-cadherin in neural plate cells. In addition, folates increase Ca 2+ transient frequency, suggesting that folate and FOLR1 signal intracellularly to regulate neural plate folding. This study identifies a mechanism of action of folate distinct from its vitamin function during neural tube formation., (© 2024. The Author(s).)

Histone variant H3.3 plays novel roles in development as compared to canonical H3 proteins and is the most commonly mutated histone protein of any kind in human disease. Here we discuss how gene targeting studies of the two H3.3-coding genes H3f3a and H3f3b have provided important insights into H3.3 functions including in gametes as well as brain and lung development. Knockouts have also provided insights into the important roles of H3.3 in maintaining genomic stability and chromatin organization, processes that are also affected when H3.3 is mutated in human diseases such as pediatric tumors and neurodevelopmental syndromes. Overall, H3.3 is a unique histone linking development and disease via epigenomic machinery., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Folate supplementation reduces the occurrence of neural tube defects, one of the most common and serious birth defects, consisting in the failure of the neural tube to form and close early in pregnancy. The mechanisms underlying neural tube defects and folate action during neural tube formation remain unclear. Here we show that folate receptor 1 (FOLR1) is necessary for the formation of neural tube-like structures in human-cell derived neural organoids. Knockdown of FOLR1 in human neural organoids as well as in the Xenopus laevis in vivo model leads to neural tube defects that are rescued by pteroate, a folate precursor that binds to FOLR1 but is unable to participate in metabolic pathways. We demonstrate that FOLR1 interacts with and opposes the function of CD2-associated protein (CD2AP), a molecule that we find is essential for apical endocytosis and the spatiotemporal turnover of the cell adherens junction component C-cadherin in neural plate cells. The counteracting action of FOLR1 on these processes is mediated by regulating CD2AP protein level via a degradation-dependent mechanism. In addition, folate and pteroate increase Ca 2+ transient frequency in the neural plate in a FOLR1-dependent manner, suggesting that folate/FOLR1 signal intracellularly to regulate neural plate folding. This study identifies a mechanism of action of folate distinct from its vitamin function during neural tube formation.

Histone variant H3.3 is encoded by two genes, H3f3a and H3f3b, which can be expressed differentially depending on tissue type. Previous work in our lab has shown that knockout of H3f3b causes some neonatal lethality and infertility in mice, and chromosomal defects in mouse embryonic fibroblasts (MEFs). Studies of H3f3a and H3f3b null mice by others have produced generally similar phenotypes to what we found in our H3f3b nulls, but the relative impacts of the loss of either H3f3a or H3f3b have varied depending on the approach and genetic background. Here we used a knockout-first approach to target the H3f3a gene for inactivation in C57BL6 mice. Homozygous H3f3a targeting produced a lethal phenotype at or before birth. E13.5 null embryos had some potential morphological differences from WT littermates including smaller size and reduced head size. An E18.5 null embryo was smaller than its control littermates with several potential defects including small head and brain size as well as small lungs, which would be consistent with a late gestation lethal phenotype. Despite a reduction in H3.3 and total H3 protein levels, the only histone H3 post-translational modification in the small panel assessed that was significantly altered was the unique H3.3 mark phospho-Serine31, which was consistently increased in null neurospheres. H3f3a null neurospheres also exhibited consistent gene expression changes including in protocadherins. Overall, our findings are consistent with the model that there are differential, cell-type-specific contributions of H3f3a and H3f3b to H3.3 functions in epigenetic and developmental processes., (© 2023 Wiley Periodicals LLC.)

Background: The histone variant H3.3 K27M mutation is a defining characteristic of diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). This histone mutation is responsible for major alterations to histone H3 post-translational modification (PTMs) and subsequent aberrant gene expression. However, much less is known about the effect this mutation has on chromatin structure and function, including open versus closed chromatin regions as well as their transcriptomic consequences., Results: Recently, we developed isogenic CRISPR-edited DIPG cell lines that are wild-type for histone H3.3 that can be compared to their matched K27M lines. Here we show via ATAC-seq analysis that H3.3K27M glioma cells have unique accessible chromatin at regions corresponding to neurogenesis, NOTCH, and neuronal development pathways and associated genes that are overexpressed in H3.3K27M compared to our isogenic wild-type cell line. As to mechanisms, accessible enhancers and super-enhancers corresponding to increased gene expression in H3.3K27M cells were also mapped to genes involved in neurogenesis and NOTCH signaling, suggesting that these pathways are key to DIPG tumor maintenance. Motif analysis implicates specific transcription factors as central to the neuro-oncogenic K27M signaling pathway, in particular, ASCL1 and NEUROD1., Conclusions: Altogether our findings indicate that H3.3K27M causes chromatin to take on a more accessible configuration at key regulatory regions for NOTCH and neurogenesis genes resulting in increased oncogenic gene expression, which is at least partially reversible upon editing K27M back to wild-type., (© 2022. The Author(s).)

It is widely believed that cellular senescence plays a critical role in both aging and cancer, and that senescence is a fundamental, permanent growth arrest that somatic cells cannot avoid. Here we show that Myc plays an important role in self-renewal of esophageal epithelial cells, contributing to their resistance to cellular senescence. Myc is homogeneously expressed in basal cells of the esophageal epithelium and Myc positively regulates their self-renewal by maintaining their undifferentiated state. Indeed, Myc knockout induced a loss of the undifferentiated state of esophageal epithelial cells resulting in cellular senescence while forced MYC expression promoted oncogenic cell proliferation. A superoxide scavenger counteracted Myc knockout-induced senescence, therefore suggesting that a mitochondrial superoxide takes part in inducing senescence. Taken together, these analyses reveal extremely low levels of cellular senescence and senescence-associated phenotypes in the esophageal epithelium, as well as a critical role for Myc in self-renewal of basal cells in this organ. This provides new avenues for studying and understanding the links between stemness and resistance to cellular senescence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hishida, Vazquez-Ferrer, Hishida-Nozaki, Takemoto, Hatanaka, Yoshida, Prieto, Sahu, Takahashi, Reddy, O’Keefe, Rodriguez Esteban, Knoepfler, Nuñez Delicado, Castells, Campistol, Kato, Nakagawa and Izpisua Belmonte.)

Many gene networks are shared between pluripotent stem cells and cancer; a concept exemplified by several DPPA factors such as DPPA2 and DPPA4, which are highly and selectively expressed in stem cells but also found to be reactivated in cancer. Despite their striking expression pattern, for many years the function of DPPA2 and DPPA4 remained a mystery; knockout of Dppa2 and Dppa4 did not affect pluripotency, but caused lung and skeletal defects late in development, long after Dppa2 and Dppa4 expression had been turned off. A number of recent papers have further clarified and defined the roles of these important factors, identifying roles in priming the chromatin and maintaining developmental competency through regulating both H3K4me3 and H3K27me3 at bivalent chromatin domains, and acting to remodel chromatin and facilitate reprogramming of somatic cells to induced pluripotency. These findings highlight an important regulatory role for DPPA2 and DPPA4 at the transitional boundary between pluripotency and differentiation and may have relevance to the functions of DPPA2 and 4 in the context of cancer cells as well., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim: There is a critical need for safe and effective treatments for COVID-19. One possible type of treatment is cellular medicine such as stem cell therapy, but its potential is unclear. Here, our aim was to assess the potential impact of the many cellular medicine trials for COVID-19. Materials & methods: We collected and analyzed data for defined criteria from trial registries. Results: Our data suggest that relatively few of these COVID-19 trials will produce high-level evidence, but that on average they may be somewhat more rigorous than typical cell therapy trials unrelated to COVID-19. Conclusion: Most COVID-19 cellular medicine trials have relatively low potential for rapid, concrete impact. We discuss the findings in the context of the cellular medicine field overall.

In this issue of Cell Stem Cell, Funato et al. (2021) and Bressan et al. (2021) use stem cells as models to define functions of the histone H3.3 G34R mutation in childhood gliomas. Both studies find strong regional specificity to oncohistone activity and implicate specific elements of an aberrantly locked-in neural progenitor transcriptional circuitry., (Copyright © 2021. Published by Elsevier Inc.)

Aim: The industry of unproven stem cell clinics has rapidly mushroomed throughout the USA, posing risks to patients and the research field. In this study, the aim was to better define how this industry changes. Methods: I analyzed a large cohort of US stem cell clinic firms and their distinct clinic locations as defined in 2015-2016 for their status now in 2019. Results: About a quarter of the firms no longer marketed stem cells. Some lacked active websites, while others dropped stem cell services. Even so, the total number of clinics in this group increased because some firms greatly expanded their clinic numbers. Conclusion: Overall, the unproven clinic industry is a moving target requiring ongoing study and regulatory oversight.

The stem cell and regenerative medicine arena has become increasingly complicated in recent years with thousands of people involved. There are as many as a dozen or more main groups of stakeholders, who together may be viewed as one ecosystem that is now rapidly evolving. The nature of the ecosystem and its evolution have major implications for not just those within it, but also for medicine and society at large. Here, I describe this ecosystem and its evolution, as well as the negative impacts within the ecosystem of a constellation of hundreds of unproven for-profit clinics and related businesses. Finally, I propose approaches for how to positively influence and drive the future of the global stem cell ecosystem.

Developmental pluripotency associated factor 4 (Dppa4) is a highly specific marker of pluripotent cells, and is also overexpressed in certain cancers, but its function in either of these contexts is poorly understood. In this study, we use ChIP-Seq to identify Dppa4 binding genome-wide in three distinct cell types: mouse embryonic stem cells (mESC), embryonal carcinoma cells, and 3T3 fibroblasts ectopically expressing Dppa4. We find a core set of Dppa4 binding sites shared across cell types, and also a substantial number of sites unique to each cell type. Across cell types Dppa4 shows a preference for binding to regions with active chromatin signatures, and can influence chromatin modifications at target genes. In 3T3 fibroblasts with enforced Dppa4 expression, Dppa4 represses the cell cycle inhibitor Cdkn2c and activates Ets family transcription factor Etv4, leading to alterations in the cell cycle that likely contribute to the oncogenic phenotype. Dppa4 also directly regulates Etv4 in mESC but represses it in this context, and binds with Oct4 to a set of shared targets that are largely independent of Sox2 and Nanog, indicating that Dppa4 functions independently of the core pluripotency network in stem cells. Together these data provide novel insights into Dppa4 function in both pluripotent and oncogenic contexts., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Regulators are now more often distinguishing between perceived good citizens and "bad actors" in stem cell and regenerative medicine clinical research, resulting in relatively more polar, carrot-and-stick oversight approaches. Here, I discuss why there may be too much carrot and not enough stick by regulators for effective enforcement., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Developmental Pluripotency-Associated-4 (DPPA4) is one of the few core pluripotency genes lacking clearly defined molecular and cellular functions. Here, we used a proteomics screening approach of human embryonic stem cell (hESC) nuclear extract to determine DPPA4 molecular functions through identification of novel cofactors. Unexpectedly, the signaling molecule ERBB3-binding protein 1 (EBP1) was the strongest candidate binding partner for DPPA4 in hESC. EBP1 is a growth factor signaling mediator present in two isoforms, p48 and p42. The two isoforms generally have opposing functions, however their roles in pluripotent cells have not been established. We found that DPPA4 preferentially binds p48 in pluripotent and NTERA-2 cells, but this interaction is largely absent in non-pluripotent cells and is reduced with differentiation. The DPPA4-EBP1 interaction is mediated at least in part in DPPA4 by the highly conserved SAF-A/B, Acinus and PIAS (SAP) domain. Functionally, we found that DPPA4 transcriptional repressive function in reporter assays is significantly increased by specific p48 knockdown, an effect that was abolished with an interaction-deficient DPPA4 ΔSAP mutant. Thus, DPPA4 and EBP1 may cooperate in transcriptional functions through their physical association in a pluripotent cell specific context. Our study identifies EBP1 as a novel pluripotency cofactor and provides insight into potential mechanisms used by DPPA4 in regulating pluripotency through its association with EBP1. Stem Cells 2018;36:671-682., (© 2018 AlphaMed Press.)

Hundreds of businesses in the US currently advertise a wide range of non-US FDA-approved stem cell interventions. Here we present a novel systematic temporal analysis of US companies engaged in direct-to-consumer marketing of putative stem cell treatments. Between 2009 and 2014, the number of new US stem cell businesses with websites grew rapidly, at least doubling on average every year. From 2014 to 2016, approximately 90-100 new stem cell business websites appeared per year. In contrast, from 2012 to the present, regulatory activity in the form of FDA warning letters has been limited. These data point to a problematic disconnect between a rapidly expanding US direct-to-consumer stem cell industry and limited FDA oversight of this marketplace. More consistent, timely and effective FDA actions are urgently needed.

The transcriptional functions of the class I histone deacetylases (HDACs) HDAC1 and HDAC2 are mainly viewed as both repressive and redundant based on murine knockout studies, but they may have additional independent roles and their physiological functions in human cells are not as clearly defined. To address the individual epigenomic functions of HDAC2, here we utilized CRISPR-Cas9 to disrupt HDAC2 in human cells. We find that while HDAC2 null cells exhibited signs of cross-regulation between HDAC1 and HDAC2, specific epigenomic phenotypes were still apparent using RNA-seq and ChIP assays. We identified specific targets of HDAC2 repression, and defined a novel class of genes that are actively expressed in a partially HDAC2-dependent manner. While HDAC2 was required for the recruitment of HDAC1 to repressed HDAC2-gene targets, HDAC2 was dispensable for HDAC1 binding to HDAC2-activated targets, supporting the notion of distinct classes of targets. Both active and repressed classes of gene targets demonstrated enhanced histone acetylation and methylation in HDAC2-null cells. Binding of the HDAC1/2-associated SIN3A corepressor was altered at most HDAC2-targets, but without a clear pattern. Overall, our study defines two classes of HDAC2 targets in human cells, with a dependence of HDAC1 on HDAC2 at one class of targets, and distinguishes unique functions for HDAC2.

Human pluripotent stem cells (hPSC) have great clinical potential through the use of their differentiated progeny, a population in which there is some concern over risks of tumorigenicity or other unwanted cellular behavior due to residual hPSC. Preclinical studies using human stem cells are most often performed within a xenotransplant context. In this study, we sought to measure how undifferentiated hPSC behave following xenotransplant. We directly transplanted undifferentiated human induced pluripotent stem cells (hIPSC) and human embryonic stem cells (hESC) into the adult mouse brain ventricle and analyzed their fates. No tumors or precancerous lesions were present at more than one year after transplantation. This result differed with the tumorigenic capacity we observed after allotransplantation of mouse ESC into the mouse brain. A substantial population of cellular derivatives of undifferentiated hESC and hIPSC engrafted, survived, and migrated within the mouse brain parenchyma. Within brain structures, transplanted cell distribution followed a very specific pattern, suggesting the existence of distinct microenvironments that offer different degrees of permissibility for engraftment. Most of the transplanted hESC and hIPSC that developed into brain cells were NeuN+ neuronal cells, and no astrocytes were detected. Substantial cell and nuclear fusion occurred between host and transplanted cells, a phenomenon influenced by microenvironment. Overall, hIPSC appear to be largely functionally equivalent to hESC in vivo. Altogether, these data bring new insights into the behavior of stem cells without prior differentiation following xenotransplantation into the adult brain.

The growing direct-to-consumer, stem cell clinic industry in the U.S. uses a number of strategies for patient recruitment, including self-styled educational seminars, which may reach thousands of members of the public annually. Here I report on a first-hand experience at such a seminar that I recently attended. Numerous specific medical claims were made at the seminar: no potential for rejection; no side effects, including no pain; proven efficacy for a variety of conditions, including in particular arthritis and pain; and U.S. Food and Drug Administration approval. I discuss the potential impact of these kinds of seminars on the public and on the stem cell field. Stem Cells Translational Medicine 2017;6:14-16., (© 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

The goal of editing the genomes of stem cells to generate model organisms and cell lines for genetic and biological studies has been pursued for decades. There is also exciting potential for future clinical impact in humans. While recent, rapid advances in targeted nuclease technologies have led to unprecedented accessibility and ease of gene editing, biology has benefited from past directed gene modification via homologous recombination, gene traps and other transgenic methodologies. Here we review the history of genome editing in stem cells (including via zinc finger nucleases, transcription activator-like effector nucleases and CRISPR-Cas9), discuss recent developments leading to the implementation of stem cell gene therapies in clinical trials and consider the prospects for future advances in this rapidly evolving field., Competing Interests: Financial & competing interests disclosure This work was support by NIH grant 1R01GM116919-01 to PS Knoepfler and by NCI training fellowship T32CA108459 to KY Chen. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

The phrase "bench-to-bedside" is commonly used to describe the translation of basic discoveries such as those on stem cells to the clinic for therapeutic use in human patients. However, there is a key intermediate step in between the bench and the bedside involving governmental regulatory oversight such as by the Food and Drug Administration (FDA) in the United States (US). Thus, it might be more accurate in most cases to describe the stem cell biological drug development process in this way: from bench to FDA to bedside. The intermediate development and regulatory stage for stem cell-based biological drugs is a multifactorial, continually evolving part of the process of developing a biological drug such as a stem cell-based regenerative medicine product. In some situations, stem cell-related products may not be classified as biological drugs in which case the FDA plays a relatively minor role. However, this middle stage is generally a major element of the process and is often colloquially referred to in an ominous way as "The Valley of Death". This moniker seems appropriate because it is at this point, and in particular in the work that ensues after Phase 1, clinical trials that most drug product development is terminated, often due to lack of funding, diseases being refractory to treatment, or regulatory issues. Not surprisingly, workarounds to deal with or entirely avoid this difficult stage of the process are evolving both inside and outside the domains of official regulatory authorities. In some cases these efforts involve the FDA invoking new mechanisms of accelerating the bench to beside process, but in other cases these new pathways bypass the FDA in part or entirely. Together these rapidly changing stem cell product development and regulatory pathways raise many scientific, ethical, and medical questions. These emerging trends and their potential consequences are reviewed here., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The histone variant H3.3 is involved in diverse biological processes, including development, transcriptional memory and transcriptional reprogramming, as well as diseases, including most notably malignant brain tumors. Recently, we developed a knockout mouse model for the H3f3b gene, one of two genes encoding H3.3. Here, we show that targeted disruption of H3f3b results in a number of phenotypic abnormalities, including a reduction in H3.3 histone levels, leading to male infertility, as well as abnormal sperm and testes morphology. Additionally, null germ cell populations at specific stages in spermatogenesis, in particular spermatocytes and spermatogonia, exhibited increased rates of apoptosis. Disruption of H3f3b also altered histone post-translational modifications and gene expression in the testes, with the most prominent changes occurring at genes involved in spermatogenesis. Finally, H3f3b null testes also exhibited abnormal germ cell chromatin reorganization and reduced protamine incorporation. Taken together, our studies indicate a major role for H3.3 in spermatogenesis through regulation of chromatin dynamics., (© 2014. Published by The Company of Biologists Ltd.)

The transcription factor Miz-1 can either activate or repress gene expression in concert with binding partners including the Myc oncoprotein. The genomic binding of Miz-1 includes both core promoters and more distal sites, but the preferred DNA binding motif of Miz-1 has been unclear. We used a high-throughput in vitro technique, Bind-n-Seq, to identify two Miz-1 consensus DNA binding motif sequences--ATCGGTAATC and ATCGAT (Mizm1 and Mizm2)--bound by full-length Miz-1 and its zinc finger domain, respectively. We validated these sequences directly as high affinity Miz-1 binding motifs. Competition assays using mutant probes indicated that the binding affinity of Miz-1 for Mizm1 and Mizm2 is highly sequence-specific. Miz-1 strongly activates gene expression through the motifs in a Myc-independent manner. MEME-ChIP analysis of Miz-1 ChIP-seq data in two different cell types reveals a long motif with a central core sequence highly similar to the Mizm1 motif identified by Bind-n-Seq, validating the in vivo relevance of the findings. Miz-1 ChIP-seq peaks containing the long motif are predominantly located outside of proximal promoter regions, in contrast to peaks without the motif, which are highly concentrated within 1.5 kb of the nearest transcription start site. Overall, our results indicate that Miz-1 may be directed in vivo to the novel motif sequences we have identified, where it can recruit its specific binding partners to control gene expression and ultimately regulate cell fate.

The stem cell field is at a critical juncture in late 2013. We find ourselves buoyed by building momentum for both transformative basic science discoveries and clinical translation of stem cells. Cellular reprogramming has given the field exciting new avenues as well. The overall prospect of novel stem cell-based therapies becoming a reality for patients in the coming years has never seemed higher. At the same time, we face serious challenges. Some of these challenges, such as stem cell tourism, are familiar to us, although even those are evolving in ways that require adaptability and action by the stem cell field. Other new challenges are also emerging, including an urgent need for formal physician training in stem cells, regulatory compliance balanced with innovation and U.S. Food and Drug Administration reform, and savvy educational outreach. Looking ahead to 2014, both the challenges and opportunities for the stem cell field require a proactive, thoughtful approach to maximize the potential for a positive impact from stem cell advances. In this study, I discuss the key action items for the field as we look ahead to the coming year and beyond.

A host of cancer types exhibit aberrant histone modifications. Recently, distinct and recurrent mutations in a specific histone variant, histone H3.3, have been implicated in a high proportion of malignant pediatric brain cancers. The presence of mutant H3.3 histone disrupts epigenetic posttranslational modifications near genes involved in cancer processes and in brain function. Here, we review possible mechanisms by which mutant H3.3 histones may act to promote tumorigenesis. Furthermore, we discuss how perturbations in normal H3.3 chromatin-related and epigenetic functions may more broadly contribute to the formation of human cancers., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In order to identify novel pluripotency-related oncogenes, an expression screen for oncogenic foci-inducing genes within a retroviral human embryonic stem cell cDNA library was conducted. From this screen, we identified not only known oncogenes but also intriguingly the key pluripotency factor, DPPA4 (developmental pluripotency-associated four) that encodes a DNA binding SAP domain-containing protein. DPPA4 has not been previously identified as an oncogene but is highly expressed in embryonal carcinomas, pluripotent germ cell tumors, and other cancers. DPPA4 is also mutated in some cancers. In direct transformation assays, we validated that DPPA4 is an oncogene in both mouse 3T3 cells and immortalized human dermal fibroblasts. Overexpression of DPPA4 generates oncogenic foci (sarcoma cells) and causes anchorage-independent growth. The in vitro transformed cells also give rise to tumors in immunodeficient mice. Furthermore, functional analyses indicate that both the DNA-binding SAP domain and the histone-binding C-terminal domain are critical for the oncogenic transformation activity of DPPA4. Downregulation of DPPA4 in E14 mouse embryonic stem cells and P19 mouse embryonic carcinoma cells causes decreased cell proliferation in each case. In addition, DPPA4 overexpression induces cell proliferation through genes related to regulation of G1/S transition. Interestingly, we observed similar findings for family member DPPA2. Thus, we have identified a new family of pluripotency-related oncogenes consisting of DPPA2 and DPPA4. Our findings have important implications for stem cell biology and tumorigenesis., (Copyright © 2013 AlphaMed Press.)

In order to elucidate the function of Myc in the maintenance of pluripotency and self-renewal in mouse embryonic stem cells (mESCs), we screened for novel ESC-specific interactors of Myc by mass spectrometry. Undifferentiated embryonic cell transcription factor 1 (Utf1) was identified in the screen as a putative Myc binding protein in mESCs. We found that Myc and Utf1 directly interact. Utf1 is a chromatin-associated factor required for maintaining pluripotency and self-renewal in mESCs. It can also replace c-myc during induced pluripotent stem cell (iPSC) generation with relatively high efficiency, and shares target genes with Myc in mESCs highlighting a potentially redundant functional role between Myc and Utf1. A large region of Utf1 was found to be necessary for direct interaction with N-Myc, while the basic helix-loop-helix leucine zipper domain of N-Myc is necessary for direct interaction with Utf1., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: The histone variant H3.3 plays key roles in regulating chromatin states and transcription. However, the role of endogenous H3.3 in mammalian cells and during development has been less thoroughly investigated. To address this gap, we report the production and phenotypic analysis of mice and cells with targeted disruption of the H3.3-encoding gene, H3f3b., Results: H3f3b knockout (KO) mice exhibit a semilethal phenotype traceable at least in part to defective cell division and chromosome segregation. H3f3b KO cells have widespread ectopic CENP-A protein localization suggesting one possible mechanism for defective chromosome segregation. KO cells have abnormal karyotypes and cell cycle profiles as well. The transcriptome and euchromatin-related epigenome were moderately affected by loss of H3f3b in mouse embryonic fibroblasts (MEFs) with ontology most notably pointing to changes in chromatin regulatory and histone coding genes. Reduced numbers of H3f3b KO mice survive to maturity and almost all survivors from both sexes are infertile., Conclusions: Taken together, our studies suggest that endogenous mammalian histone H3.3 has important roles in regulating chromatin and chromosome functions that in turn are important for cell division, genome integrity, and development.

Stem cell-based regenerative and cellular medicine is an exciting, emerging area of medical practice. While bone marrow transplantation, a stem cell-based therapy, has been a part of medicine for decades, in recent years newer and more diverse forms of stem cell-based therapies are being used to treat a rapidly growing population of patients in the USA as well as worldwide. Nonetheless, to this author's knowledge, there is currently not a single academic medical fellowship training program in the USA that specifically prepares physicians for treating patients with stem cell-based therapies other than bone marrow or hematopoietic stem cell transplantation. An increasing number of physicians untrained in stem cell-based regenerative and cellular medicine are nonetheless transplanting stem cells into hundreds if not thousands of patients for a striking diversity of conditions. Furthermore, as stem cell technology advances, a growing number of physicians with academic affiliations may look to legitimately practice regenerative and cellular medicine. What little training that physicians can currently obtain must be found on an ad hoc basis. This article should act as a call for the development of formal academic medical fellowship programs to train physicians in the practice of cellular and regenerative medicine. The USA is used here as an example of a medical sphere in which it can be argued that such training would be helpful, however such programs would be quite helpful globally.

Myc and N-Myc have widespread impacts on the chromatin state within cells, both in a gene-specific and genome-wide manner. Our laboratory uses functional genomic methods including chromatin immunoprecipitation (ChIP), ChIP-chip, and, more recently, ChIP-seq to analyze the binding and genomic location of Myc. In this chapter, we describe an effective ChIP protocol using specific validated antibodies to Myc and N-Myc. We discuss the application of this protocol to several types of stem and cancer cells, with a focus on aspects of sample preparation prior to library preparation that are critical for successful Myc ChIP assays. Key variables are discussed and include the starting quantity of cells or tissue, lysis and sonication conditions, the quantity and quality of antibody used, and the identification of reliable target genes for ChIP validation.

Induced pluripotent stem cells (iPSCs) have the potential for creating patient-specific regenerative medicine therapies, but the links between pluripotency and tumorigenicity raise important safety concerns. More specifically, the methods employed for the production of iPSCs and oncogenic foci (OF), a form of in vitro produced tumor cells, are surprisingly similar, raising potential concerns about iPSCs. To test the hypotheses that iPSCs and OF are related cell types and, more broadly, that the induction of pluripotency and tumorigenicity are related processes, we produced iPSCs and OF in parallel from common parental fibroblasts. When we compared the transcriptomes of these iPSCs and OF to their parental fibroblasts, similar transcriptional changes were observed in both iPSCs and OF. A significant number of genes repressed during the iPSC formation were also repressed in OF, including a large cohort of differentiation-associated genes. iPSCs and OF shared a limited number of genes that were upregulated relative to parental fibroblasts, but gene ontology analysis pointed toward monosaccharide metabolism as upregulated in both iPSCs and OF. iPSCs and OF were distinct in that only iPSCs activated a host of pluripotency-related genes, while OF activated cellular damage and specific metabolic pathways. We reprogrammed oncogenic foci (ROF) to produce iPSC-like cells, a process dependent on Nanog. However, the ROF had reduced differentiation potential compared to iPSC, suggesting that oncogenic transformation leads to cellular changes that impair complete reprogramming. Taken together, these findings support a model in which OF and iPSCs are related, yet distinct cell types, and in which induced pluripotency and induced tumorigenesis are similar processes.

Recently in Cell, Jia et al. (2012) reported novel Utf1-controlled mechanisms of maintaining pluripotency and self-renewal in embryonic stem cells (ESCs). Utf1 buffers bivalent gene expression by competitive binding with polycomb repressive complex 2 and initiation of mRNA degradation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The production of human induced pluripotent stem cells (hiPSCs) has greatly expanded the realm of possible stem cell-based regenerative medicine therapies and has particularly exciting potential for autologous therapies. However, future therapies based on hiPSCs will first have to address not only similar regulatory issues as those facing human embryonic stem cells with the US FDA and international regulatory agencies, but also hiPSCs have raised unique concerns as well. While the first possible clinical use of hiPSCs remains down the road, as a field it would be wise for us to anticipate potential roadblocks and begin formulating solutions. In this article, I discuss the potential regulatory issues facing hiPSCs and propose some potential changes in the direction of the field in response.

c-Myc is involved in the control of diverse cellular processes and implicated in the maintenance of different tissues including the neural crest. Here, we report that c-Myc is particularly important for pigment cell development and homeostasis. Targeting c-Myc specifically in the melanocyte lineage using the floxed allele of c-Myc and Tyr::Cre transgenic mice results in a congenital gray hair phenotype. The gray coat color is associated with a reduced number of functional melanocytes in the hair bulb and melanocyte stem cells in the hair bulge. Importantly, the gray phenotype does not progress with time, suggesting that maintenance of the melanocyte through the hair cycle does not involve c-Myc function. In embryos, at E13.5, c-Myc-deficient melanocyte precursors are affected in proliferation in concordance with a reduction in numbers, showing that c-Myc is required for the proper melanocyte development. Interestingly, melanocytes from c-Myc-deficient mice display elevated levels of the c-Myc paralog N-Myc. Double deletion of c-Myc and N-Myc results in nearly complete loss of the residual pigmentation, indicating that N-Myc is capable of compensating for c-Myc loss of function in melanocytes., (© 2012 John Wiley & Sons A/S.)

Here we examine the functions of the Myc cofactor and histone acetyltransferase, GCN5/KAT2A, in neural stem and precursor cells (NSC) using a conditional knockout approach driven by nestin-cre. Mice with GCN5-deficient NSC exhibit a 25% reduction in brain mass with a microcephaly phenotype similar to that observed in nestin-cre driven knockouts of c- or N-myc. In addition, the loss of GCN5 inhibits precursor cell proliferation and reduces their populations in vivo, as does loss of N-myc. Gene expression analysis indicates that about one-sixth of genes whose expression is affected by loss of GCN5 are also affected in the same manner by loss of N-myc. These findings strongly support the notion that GCN5 protein is a key N-Myc transcriptional cofactor in NSC, but are also consistent with recruitment of GCN5 by other transcription factors and the use by N-Myc of other histone acetyltransferases. Putative N-Myc/GCN5 coregulated transcriptional pathways include cell metabolism, cell cycle, chromatin, and neuron projection morphogenesis genes. GCN5 is also required for maintenance of histone acetylation both at its putative specific target genes and at Myc targets. Thus, we have defined an important role for GCN5 in NSC and provided evidence that GCN5 is an important Myc transcriptional cofactor in vivo.

Induced pluripotent stem cells are different from embryonic stem cells as shown by epigenetic and genomics analyses. Depending on cell types and culture conditions, such genetic alterations can lead to different metabolic phenotypes which may impact replication rates, membrane properties and cell differentiation. We here applied a comprehensive metabolomics strategy incorporating nanoelectrospray ion trap mass spectrometry (MS), gas chromatography-time of flight MS, and hydrophilic interaction- and reversed phase-liquid chromatography-quadrupole time-of-flight MS to examine the metabolome of induced pluripotent stem cells (iPSCs) compared to parental fibroblasts as well as to reference embryonic stem cells (ESCs). With over 250 identified metabolites and a range of structurally unknown compounds, quantitative and statistical metabolome data were mapped onto a metabolite networks describing the metabolic state of iPSCs relative to other cell types. Overall iPSCs exhibited a striking shift metabolically away from parental fibroblasts and toward ESCs, suggestive of near complete metabolic reprogramming. Differences between pluripotent cell types were not observed in carbohydrate or hydroxyl acid metabolism, pentose phosphate pathway metabolites, or free fatty acids. However, significant differences between iPSCs and ESCs were evident in phosphatidylcholine and phosphatidylethanolamine lipid structures, essential and non-essential amino acids, and metabolites involved in polyamine biosynthesis. Together our findings demonstrate that during cellular reprogramming, the metabolome of fibroblasts is also reprogrammed to take on an ESC-like profile, but there are select unique differences apparent in iPSCs. The identified metabolomics signatures of iPSCs and ESCs may have important implications for functional regulation of maintenance and induction of pluripotency.

Background: A proposed role for Myc in maintaining mouse embryonic stem (ES) cell pluripotency is transcriptional repression of key differentiation-promoting genes, but detail of the mechanism has remained an important open topic., Results: To test the hypothesis that the zinc finger protein Miz-1 plays a central role, in the present work we conducted chromatin immunoprecipitation/microarray (ChIP-chip) analysis of Myc and Miz-1 in human ES cells, finding homeobox (Hox) genes as the most significant functional class of Miz-1 direct targets. Miz-1 differentiation-associated target genes specifically lack acetylated lysine 9 and trimethylated lysine 4 of histone H3 (AcH3K9 and H3K4me3) 9 histone marks, consistent with a repressed transcriptional state. Almost 30% of Miz-1 targets are also bound by Myc and these cobound genes are mostly factors that promote differentiation including Hox genes. Knockdown of Myc increased expression of differentiation genes directly bound by Myc and Miz-1, while a subset of the same genes is downregulated by Miz-1 loss-of-function. Myc and Miz-1 proteins interact with each other and associate with several corepressor factors in ES cells, suggesting a mechanism of repression of differentiation genes., Conclusions: Taken together our data indicate that Miz-1 and Myc maintain human ES cell pluripotency by coordinately suppressing differentiation genes, particularly Hox genes. These data also support a new model of how Myc and Miz-1 function on chromatin.

Induced pluripotent stem cells (iPSCs) hold great promise for autologous cell therapies, but significant roadblocks remain to translating iPSCs to the bedside. For example, concerns about the presumed autologous transplantation potential of iPSCs have been raised by a recent paper demonstrating that iPSC-derived teratomas were rejected by syngeneic hosts. Additionally, the reprogramming process can alter genomic and epigenomic states, so a key goal at this point is to determine the clinical relevance of these changes and minimize those that prove to be deleterious. Finally, thus far few studies have examined the efficacy and tumorigenicity of iPSCs in clinically relevant transplantation scenarios, an essential requirement for the FDA. We discuss potential solutions to these hurdles to provide a roadmap for iPSCs to "jump the dish" and become useful therapies., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Myc family members play crucial roles in regulating cell proliferation, size, and differentiation during organogenesis. Both N-myc and c-myc are expressed throughout inner ear development. To address their function in the mouse inner ear, we generated mice with conditional deletions in either N-myc or c-myc. Loss of c-myc in the inner ear causes no apparent defects, whereas inactivation of N-myc results in reduced growth caused by a lack of proliferation. Reciprocally, the misexpression of N-myc in the inner ear increases proliferation. Morphogenesis of the inner ear in N-myc mouse mutants is severely disturbed, including loss of the lateral canal, fusion of the cochlea with the sacculus and utriculus, and stunted outgrowth of the cochlea. Mutant cochleas are characterized by an increased number of cells exiting the cell cycle that express the cyclin-dependent kinase inhibitor p27(Kip1) and lack cyclin D1, both of which control the postmitotic state of hair cells. Analysis of different molecular markers in N-myc mutant ears reveals the development of a rudimentary organ of Corti containing hair cells and the underlying supporting cells. Differentiated cells, however, fail to form the highly ordered structure characteristic for the organ of Corti but appear as rows or clusters with an excess number of hair cells. The Kölliker's organ, a transient structure neighboring the organ of Corti and a potential source of ectopic hair cells, is absent in the mutant ears. Collectively, our data suggest that N-myc regulates growth, morphogenesis, and pattern formation during the development of the inner ear.

Separate murine knockout (KO) of either c- or N-myc genes in neural stem and precursor cells (NSC) driven by nestin-cre causes microcephaly. The cerebellum is particularly affected in the N-myc KO, leading to a strong reduction in cerebellar granule neural progenitors (CGNP) and mature granule neurons. In humans, mutation of N-myc also causes microcephaly in Feingold Syndrome. We created a double KO (DKO) of c- and N-myc using nestin-cre, which strongly impairs brain growth, particularly that of the cerebellum. Granule neurons were almost absent from the Myc DKO cerebellum, and other cell types were relatively overrepresented, including astroglia, oligodendrocytes, and Purkinje neurons. These findings are indicative of a profound disruption of cell fate of cerebellar stem and precursors. DKO Purkinje neurons were strikingly lacking in normal arborization. Inhibitory neurons were ectopic and exhibited very abnormal GAD67 staining patterns. Also consistent with altered cell fate, the adult DKO cerebellum still retained a residual external germinal layer (EGL). CGNP in the DKO EGL were almost uniformly NeuN and p27KIP1 positive as well as negative for Math1 and BrdU at the peak of normal cerebellar proliferation at P6. The presence of some mitotic CGNP in the absence of S phase cells suggests a possible arrest in M phase. CGNP and NSC metabolism also was affected by loss of Myc as DKO cells exhibited weak nucleolin staining. Together these findings indicate that c- and N-Myc direct cerebellar development by maintaining CGNP and NSC populations through inhibiting differentiation as well as directing rapid cell cycling and active cellular metabolism.

While endogenous Myc (c-myc) and Mycn (N-myc) have been reported to be separately dispensable for murine embryonic stem cell (mESC) function, myc greatly enhances induced pluripotent stem (iPS) cell formation and overexpressed c-myc confers LIF-independence upon mESC. To address the role of myc genes in ESC and in pluripotency generally, we conditionally knocked out both c- and N-myc using myc doubly homozygously floxed mESC lines (cDKO). Both lines of myc cDKO mESC exhibited severely disrupted self-renewal, pluripotency, and survival along with enhanced differentiation. Chimeric embryos injected with DKO mESC most often completely failed to develop or in rare cases survived but with severe defects. The essential nature of myc for self-renewal and pluripotency is at least in part mediated through orchestrating pluripotency-related cell cycle and metabolic programs. This study demonstrates that endogenous myc genes are essential for mESC pluripotency and self-renewal as well as providing the first evidence that myc genes are required for early embryogenesis, suggesting potential mechanisms of myc contribution to iPS cell formation., (Copyright 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

myc genes are associated with a wide variety of human cancers including most types of nervous system tumors. While the mechanisms by which myc overexpression causes tumorigenesis are multifaceted and have yet to be clearly elucidated, they are at least in part related to endogenous myc function in normal cells. Knockout (KO) of either c-myc or N-myc genes in neural stem and precursor cells (NSC) driven by nestin-cre impairs mouse brain growth and mutation of N-myc also causes microcephaly in humans in Feingold Syndrome. To further define myc function in NSC and nervous system development, we created a double KO (DKO) for c- and N-myc using nestin-cre. The DKO mice display profoundly impaired overall brain growth associated with decreased cell cycling and migration of NSC, which are strikingly decreased in number. The DKO brain also exhibits specific changes in gene expression including downregulation of genes involved in protein and nucleotide metabolism, mitosis, and chromatin structure as well as upregulation of genes associated with differentiation. Together these data support a model of nervous system tumorigenesis in which excess myc aberrantly locks in a developmentally active chromatin state characterized by overactive cell cycling, and metabolism as well as blocked differentiation.

Basal progenitors (also called non-surface dividing or intermediate progenitors) have been proposed to regulate the number of neurons during neocortical development through expanding cells committed to a neuronal fate, although the signals that govern this population have remained largely unknown. Here, we show that N-myc mediates the functions of Wnt signaling in promoting neuronal fate commitment and proliferation of neural precursor cells in vitro. Wnt signaling and N-myc also contribute to the production of basal progenitors in vivo. Expression of a stabilized form of beta-catenin, a component of the Wnt signaling pathway, or of N-myc increased the numbers of neocortical basal progenitors, whereas conditional deletion of the N-myc gene reduced these and, as a likely consequence, the number of neocortical neurons. These results reveal that Wnt signaling via N-myc is crucial for the control of neuron number in the developing neocortex.

The activating E2f transcription factors (E2f1, E2f2 and E2f3) induce transcription and are widely viewed as essential positive cell cycle regulators. Indeed, they drive cells out of quiescence, and the 'cancer cell cycle' in Rb1 null cells is E2f-dependent. Absence of activating E2fs in flies or mammalian fibroblasts causes cell cycle arrest, but this block is alleviated by removing repressive E2f or the tumour suppressor p53, respectively. Thus, whether activating E2fs are indispensable for normal division is an area of debate. Activating E2fs are also well known pro-apoptotic factors, providing a defence against oncogenesis, yet E2f1 can limit irradiation-induced apoptosis. In flies this occurs through repression of hid (also called Wrinkled; Smac/Diablo in mammals). However, in mammals the mechanism is unclear because Smac/Diablo is induced, not repressed, by E2f1, and in keratinocytes survival is promoted indirectly through induction of DNA repair targets. Thus, a direct pro-survival function for E2f1-3 and/or its relevance beyond irradiation has not been established. To address E2f1-3 function in normal cells in vivo we focused on the mouse retina, which is a relatively simple central nervous system component that can be manipulated genetically without compromising viability and has provided considerable insight into development and cancer. Here we show that unlike fibroblasts, E2f1-3 null retinal progenitor cells or activated Müller glia can divide. We attribute this effect to functional interchangeability with Mycn. However, loss of activating E2fs caused downregulation of the p53 deacetylase Sirt1, p53 hyperacetylation and elevated apoptosis, establishing a novel E2f-Sirt1-p53 survival axis in vivo. Thus, activating E2fs are not universally required for normal mammalian cell division, but have an unexpected pro-survival role in development.