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Author(s): Alejandro De Los Angeles [1, 2, 3]; Francesco Ferrari [4]; Yuko Fujiwara [1, 2, 3]; Ronald Mathieu [5]; Soohyun Lee [4]; Semin Lee [4]; Ho-Chou Tu [1, 2, 3]; [...]

A key challenge in psychiatry research is the development of high-fidelity model systems that can be experimentally manipulated to explore and test pathophysiological mechanisms of illness. In this respect, the emerging capacity to derive neural cells and circuits from human induced pluripotent stem cells (iPSCs) has generated significant excitement. This review aims to provide a critical appraisal of the potential for iPSCs in illuminating pathophysiological mechanisms in the context of other available technical approaches. We discuss the selection of iPSC phenotypes relevant to psychiatry, the information that researchers can draw on to help guide these decisions, and how researchers choose between the use of 2-dimensional cultures and the use of more complex 3-dimensional model systems. We discuss the strengths and limitations of current models and the challenges and opportunities that they present. Finally, we discuss the potential of iPSC-based model systems for clarifying the mechanisms underlying genetic risk for psychiatry and the steps that will be needed to ensure that robust and reliable conclusions can be drawn. We argue that while iPSC-based models are ideally placed to study fundamental processes occurring within and between neural cells, they are often less well suited for case-control studies, given issues relating to statistical power and the challenges in identifying which cellular phenotypes are meaningful at the level of the whole individual. Our aim is to highlight the importance of considering the hypotheses of a given study to guide decisions about which, if any, iPSC-based system is most appropriate to address it. [ABSTRACT FROM AUTHOR]

Gastrulation is a phase in early mammalian development when the three germ layers are generated and body plan is formed. Although well studied in mice, much less is known about gastrulation in humans. Owing to the lack of access to primary human tissue for study and experimental manipulation, as well as legal and ethical constraints surrounding the use of human embryos, a dissection of the molecular and cellular mechanisms that underlie this process in humans has proven elusive. Nonhuman primates, owing to their relatedness to human species, comprise a tantalizing alternative model system for understanding human biology. Two recent studies have established novel systems to study monkey embryos for 20 days, demonstrating landmark events of early primate embryogenesis with possible relevance to human development. Most strikingly, cells grown in the dish closely resembled cells in in vivo embryos, suggesting that embryo development in a dish might actually be equivalent to that which occurs in vivo. In this piece, the author discusses the tremendous potential of these new methods to unveil insights into mechanisms that mediate primate embryo development. Moreover, repurposing the extended monkey embryo culture methods to create human–monkey embryonic chimeras would aid the development of strategies to create human organs inside livestock species. Finally, the ethical and regulatory issues that emerge from reconsideration of extending time limits for human embryo culture beyond 14 days or primitive streak formation are also briefly considered. [ABSTRACT FROM AUTHOR]

Totipotency refers to single cells' developmental capacity to form an entire organism. Understanding how totipotent stem cells form has implications for chimera generation. In a recent Cell study, Shen et al. (2021) report that inhibition of spliceosomes resets conventional pluripotent stem cells to a cellular state with totipotency features. [ABSTRACT FROM AUTHOR]

Abstract Generating human organs inside interspecies chimeras might one day produce patient-specific organs for clinical applications, but further advances in identifying human chimera-competent pluripotent stem (PS) cells are needed. Moreover, the potential for human PS cells to contribute to the brains in human-animal chimeras raises ethical questions. The use of non-human primate (NHP) chimera-competent PS cells would allow one to test interspecies organogenesis strategies while also bypassing such ethical concerns. Here, we provide the first evidence for a putative chimera-competent pluripotent state in NHPs. Using histone deacetylase (HDAC) and selective kinase inhibition, we converted the PS cells of an Old World monkey, the African Green monkey (aGM), to an ERK-independent cellular state that can be propagated in culture conditions similar to those that sustain chimera-competency in rodent cells. The obtained stem cell lines indefinitely self-renew in MEK inhibitor-containing culture media lacking serum replacement and FGF. Compared to conventional PS cells, the novel stem cells express elevated levels of KLF4, exhibit more intense nuclear staining for TFE3, and manifest increased mitochondrial membrane depolarization. These data are preliminary but indicate that the key to deriving primate chimera-competent PS cells is to shield cells from the activation of ERK, PKC, and WNT signaling. Because of the similarity of aGMs to humans, the more ethically palatable use of NHP cells, and the more similar gestation length between aGMs and large animals such as sheep, the aGM cell lines described herein will serve as a useful tool for evaluating the efficacy and safety of interspecies organogenesis strategies. Future studies will examine chimera-competency and generalizability to human cells. Highlights • Conversion of African green monkey pluripotent stem cells to an ERK-independence using HDAC inhibitor and kinase inhibitors. • Maintenance of African green monkey PS cells in serum-free conditions with MEK, PKC, GSK3, and Tankyrase inhibitors and LIF. • African green monkey ERK-independent pluripotent stem cells possess features associated with a more naïve pluripotent state. • Conversion of African green monkey putative pluripotent stem cells back to a conventional ES cell state. [ABSTRACT FROM AUTHOR]

Rare heterozygous deletions in the neurexin 1 (NRXN1) gene robustly increase an individual's risk of developing neurological and psychiatric disorders. However, the molecular bases by which different mutations result in different clinical presentations, with variable penetrance, are unknown. To better understand the molecular and cellular consequences of heterozygous NRXN1 mutations, Flaherty and colleagues studied how patient mutations influence the NRXN1 isoform repertoire and neuronal phenotypes using induced pluripotent stem (iPS) cells. Advancing from disease association to mechanistic insights, the authors provide insight into how patient mutations might impinge on neuronal function. This research highlights the value of iPS cells for elucidating otherwise elusive links between molecular and neuronal function. In addition, they provide further evidence of the importance of alternative splicing in the pathophysiology of neuropsychiatric diseases. [ABSTRACT FROM AUTHOR]

The search for a better animal model to simulate human disease has been a "holy grail" of biomedical research for decades. Recent identification of different types of pluripotent stem (PS) cells and advances in chimera research might soon permit the generation of interspecies chimeras from closely related species, such as those between humans and other primates. In this study, we suggest that the creation of human–primate chimeras—specifically, the transfer of human stem cells into (non-ape) primate hosts—could not only surpass the limitations of current monkey models of neurological and psychiatric disease but would also raise important ethical considerations concerning the use of monkeys in invasive research. Questions regarding the scientific value and ethical concerns raised by the prospect of human–monkey chimeras are more urgent in light of recent advances in PS cell research and attempts to generate interspecies chimeras between humans and animals. While some jurisdictions prohibit the introduction of human PS cells into monkey preimplantation embryos, other jurisdictions may permit and even encourage such experiments. Therefore, it is useful to consider blastocyst complementation experiments more closely in light of advances that could make these chimeras possible and to consider the ethical and political issues that are raised. [ABSTRACT FROM AUTHOR]

Summary Polycomb Repressive Complex 2 (PRC2) function and DNA methylation (DNAme) are typically correlated with gene repression. Here, we show that PRC2 is required to maintain expression of maternal microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) from the Gtl2 - Rian - Mirg locus, which is essential for full pluripotency of iPSCs. In the absence of PRC2, the entire locus becomes transcriptionally repressed due to gain of DNAme at the intergenic differentially methylated regions (IG-DMRs). Furthermore, we demonstrate that the IG-DMR serves as an enhancer of the maternal Gtl2 - Rian - Mirg locus. Further analysis reveals that PRC2 interacts physically with Dnmt3 methyltransferases and reduces recruitment to and subsequent DNAme at the IG-DMR, thereby allowing for proper expression of the maternal Gtl2 - Rian - Mirg locus. Our observations are consistent with a mechanism through which PRC2 counteracts the action of Dnmt3 methyltransferases at an imprinted locus required for full pluripotency. [ABSTRACT FROM AUTHOR]

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies. [ABSTRACT FROM AUTHOR]

Pluripotency manifests during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Rodent pluripotent stem cells can be considered as two distinct states: naïve and primed. Naïve pluripotent stem cell lines are distinguished from primed cells by self-renewal in response to LIF signaling and MEK/GSK3 inhibition (LIF/2i conditions) and two active X chromosomes in female cells. In rodent cells, the naïve pluripotent state may be accessed through at least three routes: explantation of the inner cell mass, somatic cell reprogramming by ectopic Oct4, Sox2, Klf4, and C-myc, and direct reversion of primed post-implantation-associated epiblast stem cells (EpiSCs). In contrast to their rodent counterparts, human embryonic stem cells and induced pluripotent stem cells more closely resemble rodent primed EpiSCs. A critical question is whether naïve human pluripotent stem cells with bona fide features of both a pluripotent state and naïve-specific features can be obtained. In this review, we outline current understanding of the differences between these pluripotent states in mice, new perspectives on the origins of naïve pluripotency in rodents, and recent attempts to apply the rodent paradigm to capture naïve pluripotency in human cells. Unraveling how to stably induce naïve pluripotency in human cells will influence the full realization of human pluripotent stem cell biology and medicine. [Copyright &y& Elsevier]

Human embryonic germ cells (EGCs) provide a powerful model for identifying molecules involved in the pluripotent state when compared to their progenitors, primordial germ cells (PGCs), and other pluripotent stem cells. Microarray and Principal Component Analysis (PCA) reveals for the first time that human EGCs possess a transcription profile distinct from PGCs and other pluripotent stem cells. Validation with qRT-PCR confirms that human EGCs and PGCs express many pluripotency-associated genes but with quantifiable differences compared to pluripotent embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), and embryonal carcinoma cells (ECCs). Analyses also identified a number of target genes that may be potentially associated with their unique pluripotent states. These include IPO7, MED7, RBM26, HSPD1, and KRAS which were upregulated in EGCs along with other pluripotent stem cells when compared to PGCs. Other potential target genes were also found which may contribute toward a primed ESC-like state. These genes were exclusively up-regulated in ESCs, IPSCs and ECCs including PARP1, CCNE1, CDK6, AURKA, MAD2L1, CCNG1, and CCNB1 which are involved in cell cycle regulation, cellular metabolism and DNA repair and replication. Gene classification analysis also confirmed that the distinguishing feature of EGCs compared to ESCs, ECCs, and IPSCs lies primarily in their genetic contribution to cellular metabolism, cell cycle, and cell adhesion. In contrast, several genes were found upregulated in PGCs which may help distinguish their unipotent state including HBA1, DMRT1, SPANXA1, and EHD2. Together, these findings provide the first glimpse into a unique genomic signature of human germ cells and pluripotent stem cells and provide genes potentially involved in defining different states of germ-line pluripotency.

Cerebral organoids are stem cell-derived, self-organizing three-dimensional cultures. Owing to the remarkable degree to which they recreate the cellular diversity observed in the human brain, they have attracted significant interest as a novel model system for research and drug development, as well as capturing the public imagination. However, many questions remain about the extent to which these cultures recapitulate neurodevelopment and the defining features of the human brain. To clarify the fidelity of human organoid models, Bhaduri and colleagues compared the molecular profile of brain organoid cells with that of primary cells from fetal brain. They observed that, whilst brain organoids broadly recapitulate the cellular profile of human brain, they lack the subtypes of cell classes seen in human brain. In addition, they showed marked expression of cellular stress markers, which could be reversed by transplanting organoid cells into neonatal mouse brain. The authors hypothesise that in vitro culture induces a cellular stress response and that it is this that impairs maturation. Thus, whilst their findings strike a note of caution in the use of organoids as a model for early human brain development, they lay a foundation for improving the accuracy of organoid models in the future. [ABSTRACT FROM AUTHOR]

Pluripotency refers to the potential of single cells to form all cells and tissues of an organism. The observation that pluripotent stem cells can chimerize the embryos of evolutionarily distant species, albeit at very low efficiencies, could with further modifications, facilitate the production of human-animal interspecies chimeras. The generation of human-animal interspecies chimeras, if achieved, will enable practitioners to recapitulate pathologic human tissue formation in vivo and produce patient-specific organs inside livestock species. However, little is known about the nature of chimera-competent cellular states in primates. Here, I discuss recent advances in our understanding of the pluripotency continuum in humans and non-human primates (NHPs). Although undefined differences between humans and NHPs still justify the utility of studying human cells, the complementary use of NHP PS cells could also allow one to conduct pilot studies testing interspecies chimera generation strategies with reduced ethical concerns associated with human interspecies neurological chimerism. However, the availability of standardized, high-quality and validated NHP PS cell lines covering the spectrum of primate pluripotent states is lacking. Therefore, a clearer understanding of the primate pluripotency continuum will facilitate the complementary use of both human and NHP PS cells for testing interspecies organogenesis strategies, with the hope of one day enabling human organ generation inside livestock species. [ABSTRACT FROM AUTHOR]

Pluripotency can be experimentally induced from somatic cells by nuclear transfer, fusion with embryonic stem cells, or ectopic transcription factor induction, but attempts to recapitulate this process by chemical means alone have previously failed. In a recent paper published in Science, Hou et al. pursue a rational, albeit laborious approach to identify cocktails of small molecules whose treatment restores pluripotency in adult somatic cells. [ABSTRACT FROM AUTHOR]

Human cleavage-stage embryos frequently acquire chromosomal aneuploidies during mitosis due to unknown mechanisms. Here, we show that S phase at the 1-cell stage shows replication fork stalling, low fork speed, and DNA synthesis extending into G2 phase. DNA damage foci consistent with collapsed replication forks, DSBs, and incomplete replication form in G2 in an ATR- and MRE11-dependent manner, followed by spontaneous chromosome breakage and segmental aneuploidies. Entry into mitosis with incomplete replication results in chromosome breakage, whole and segmental chromosome errors, micronucleation, chromosome fragmentation, and poor embryo quality. Sites of spontaneous chromosome breakage are concordant with sites of DNA synthesis in G2 phase, locating to gene-poor regions with long neural genes, which are transcriptionally silent at this stage of development. Thus, DNA replication stress in mammalian preimplantation embryos predisposes gene-poor regions to fragility, and in particular in the human embryo, to the formation of aneuploidies, impairing developmental potential., Competing Interests: Declaration of interests D.E. is a member of the Cell editorial board., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Pluripotency refers to the capacity of single cells to form derivatives of the three germ layers-ectoderm, mesoderm, and endoderm. Pluripotency can be captured in vitro as a spectrum of pluripotent stem cell states stabilized in specialized laboratory conditions. The recent discovery that pluripotent stem cells can colonize the embryos of distantly related animal organisms could, with further refinement, enable the generation of chimeric embryos composed of cells of human and animal origin. If achievable, the production of human-animal chimeras will open up new opportunities for regenerative medicine, facilitating human disease modeling and human organ generation inside large animals. However, the generation of human-animal interspecies chimeras is anticipated to require human chimera-competent pluripotent stem cells. Thus, it remains imperative to examine the pluripotency continuum more closely in light of advances that will facilitate the production of human-animal chimeras. This piece will review the current understanding of the pluripotency continuum and interspecies chimeras. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

The production of human organs inside human-animal interspecies chimeras might one day comprise a viable strategy for generating patient-specific organs, but such experiments will require human chimera-competent pluripotent stem (PS) cells. The stabilization of PS cell self-renewal in serum-free medium and ERK blockade might be critical for capturing primate chimera-competent pluripotency. It has recently been shown that shielding primate cells from the activation of ERK, WNT, and PKC signaling is crucial for deriving African green monkey ERK-independent PS cells. Here, I show that this principle is generalizable to human cells. In this chapter, methods are provided to reset conventional human PS cells to ERK-independence using histone deacetylase inhibitors and PGCX media comprised of N2B27 medium supplemented with LIF, PD0325901, Go6983, CHIR99021, and XAV939. The novel stem cells exhibit higher levels of KLF4 and manifest increased mitochondrial membrane depolarization. However, the author observed that not all PS cell lines are amenable to small molecule-mediated resetting. The ERK-independent PS cells described herein will provide a useful resource for testing interspecies organogenesis strategies. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

The search for a better animal model to simulate human disease has been a "holy grail" of biomedical research for decades. Recent identification of different types of pluripotent stem cells (PS cells) and advances in chimera research might soon permit the generation of interspecies chimeras from closely related species, such as those between humans and other primates. Here, we suggest that the creation of human-primate chimeras-specifically, the transfer of human stem cells into (non-ape) primate hosts-could surpass the limitations of current monkey models of neurological and psychiatric disease, but would also raise important ethical considerations concerning the use of monkeys in invasive research. Questions regarding the scientific value and ethical concerns raised by the prospect of human-monkey chimeras are more urgent in light of recent advances in PS cell research and attempts to generate interspecies chimeras between humans and animals. While some jurisdictions prohibit the introduction of human PS cells into monkey preimplantation embryos, other jurisdictions may permit and even encourage such experiments. Therefore, it is useful to consider blastocyst complementation experiments more closely in light of advances that could make these chimeras possible and to consider the ethical and political issues that are raised.

Human pluripotent stem (PS) cells can be isolated from preimplantation embryos or by reprogramming of somatic cells or germline progenitors. Human PS cells are considered the "holy grail" of regenerative medicine because they have the potential to form all cell types of the adult body. Because of their similarity to humans, nonhuman primate (NHP) PS cells are also important models for studying human biology and disease, as well as for developing therapeutic strategies and test bed for cell replacement therapy. This chapter describes adjusted methods for cultivation of PS cells from different primate species, including African green monkey, rhesus monkey, chimpanzee, and human. Supplementation of E8 medium and inhibitors of the Tankyrase and GSK3 kinases to various primate PS cell media reduce line-dependent predisposition for spontaneous differentiation in conventional PS cell cultures. We provide methods for basic characterization of primate PS cell lines, which include immunostaining for pluripotency markers such as OCT4 and TRA-1-60, as well as in vivo teratoma formation assay. We provide methods for generating alternative PS cells including region-selective primed PS cells, two different versions of naïve-like cells, and recently reported extended pluripotent stem (EPS) cells. These derivations are achieved by acclimation of conventional PS cells to target media, episomal reprogramming of somatic cells, or resetting conventional PS cells to a naïve-like state by overexpression of KLF2 and NANOG. We also provide methods for isolation of PS cells from human blastocysts. We describe how to generate interspecies primate-mouse chimeras at the blastocyst and postimplantation embryo stages. Systematic evaluation of the chimeric competency of human and primate PS cells will aid in efforts to overcome species barriers and achieve higher grade chimerism in postimplantation conceptuses that could enable organ-specific enrichment of human xenogeneic PS cell derivatives in large animals such as pigs and sheep.

Humans develop from a unique group of pluripotent cells in early embryos that can produce all cells of the human body. While pluripotency is only transiently manifest in the embryo, scientists have identified conditions that sustain pluripotency indefinitely in the laboratory. Pluripotency is not a monolithic entity, however, but rather comprises a spectrum of different cellular states. Questions regarding the scientific value of examining the continuum of pluripotent stem (PS) cell states have gained increased significance in light of attempts to generate interspecies chimeras between humans and animals. In this chapter, I review our ever-evolving understanding of the continuum of pluripotency. Historically, the discovery of two different PS cell states in mice fostered a general conception of pluripotency comprised of two distinct attractor states: naïve and primed. Naïve pluripotency has been defined by competence to form germline chimeras and governance by unique KLF-based transcription factor (TF) circuitry, whereas primed state is distinguished by an inability to generate chimeras and alternative TF regulation. However, the discovery of many alternative PS cell states challenges the concept of pluripotency as a binary property. Moreover, it remains unclear whether the current molecular criteria used to classify human naïve-like pluripotency also identify human chimera-competent PS cells. Therefore, I examine the pluripotency continuum more closely in light of recent advances in PS cell research and human interspecies chimera research.

Pluripotency refers to the potential of cells to generate all cell types of the embryo proper. Pluripotency spans a spectrum of cellular states. At one polar extreme is naïve pluripotency, which is identified based on the potential to form germline chimeras. At the other polar extreme is primed pluripotency, in which pluripotent cells are primed to differentiate. Mouse naïve PS cells can be derived from preimplantation embryos. Primed epiblast stem (EpiS) cells are typically isolated from epiblasts of early postimplantation mouse embryos. In this chapter, we describe protocols for highly efficient derivation and propagation of murine naïve and primed PS cell lines in serum-free conditions from preimplantation and postimplantation embryos. We describe generation of mouse naïve PS cells using LIF and inhibitors of MEK and GSK3 kinases and of mouse primed PS cells using FGF2 and IWR1 compound which induces the stabilization of Axin proteins.

Stroke is the fifth leading cause of death among Americans each year. Current standard-of-care treatment for stroke deploys intravenous tissue-type plasminogen activator (tPA), mechanical thrombolysis, or delivery of fibrinolytics. Although these therapies have reduced stroke-induced damage, therapeutic options still remain limited. Transplantation of patient-specific neural stem (NS) cells represents a promising strategy for the treatment of stroke. Basic science research has shown that transplanted NS cells can differentiate in the brain of rodent models of stroke and promote behavioral recovery. Clinical trials exploring the feasibility of stem cell treatment for stroke are currently being conducted. However, questions remain regarding the optimal means of delivering NS cells, including cell dose, infusion speed, timing of transplantation, anatomic site, and imaging-assisted monitoring and guidance. Of the different available delivery modalities, intravascular NS delivery after stroke represents one practical approach. In this chapter, I provide methods for intravascular delivery of NS cells in a mouse model of stroke. The techniques involved include cell culture of NS cells, flow cytometry of NS cells, modeling stroke via unilateral common carotid artery occlusion, intra-arterial injection of NS cells into the brain, behavior analyses, and immunohistochemistry. Intra-arterial NS cell therapy has the potential to improve functional recovery after ischemic stroke.

The shortage of human organs for transplantation is a devastating medical problem. One way to expand organ supply is to derive functional organs from patient-specific stem cells. Due to their capacity to grow indefinitely in the laboratory and differentiate into any cell type of the human body, patient-specific pluripotent stem (PS) cells harbor the potential to provide an inexhaustible supply of donor cells for transplantation. However, current efforts to generate functional organs from PS cells have so far been unsuccessful. An alternative and promising strategy is to generate human organs inside large animal species through a technique called interspecies blastocyst complementation. In this method, animals comprised of cells from human and animal species are generated by injecting donor human PS cells into animal host embryos. Critical genes for organ development are knocked out by genome editing, allowing donor human PS cells to populate the vacated niche. In principle, this experimental approach will produce a desired organ of human origin inside a host animal. In this mini-review, we focus on recent advances that may bring the promise of blastocyst complementation to clinical practice. While CRISPR/Cas9 has accelerated the creation of transgenic large animals such as pigs and sheep, we propose that further advances in the generation of chimera-competent human PS cells are needed to achieve interspecies blastocyst complementation. It will also be necessary to define the constituents of the species barrier, which inhibits efficient colonization of host animal embryos with human cells. Interspecies blastocyst complementation is a promising approach to help overcome the organ shortage facing the practice of clinical medicine today.

Self-renewal and pluripotency of embryonic stem cells (ESCs) are established by multiple regulatory pathways operating at several levels. The roles of histone demethylases (HDMs) in these programs are incompletely defined. We conducted a functional RNAi screen for HDMs and identified five potential HDMs essential for mouse ESC identity. In-depth analyses demonstrate that the closely related HDMs Jmjd2b and Jmjd2c are necessary for self-renewal of ESCs and induced pluripotent stem cell generation. Genome-wide occupancy studies reveal that Jmjd2b unique, Jmjd2c unique, and Jmjd2b-Jmjd2c common target sites belong to functionally separable Core, Polycomb repressive complex (PRC), and Myc regulatory modules, respectively. Jmjd2b and Nanog act through an interconnected regulatory loop, whereas Jmjd2c assists PRC2 in transcriptional repression. Thus, two HDMs of the same subclass exhibit distinct and combinatorial functions in control of the ESC state. Such complexity of HDM function reveals an aspect of multilayered transcriptional control., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy, in vitro modeling of human disease, and drug screening. To date, most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively, transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This unit describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector, which includes the coding sequences of human OCT4, SOX2, KLF4, and cMYC linked with picornaviral 2A plasmids. Moreover, after reprogramming has been achieved, this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells., (Curr. Protoc. Stem Cell Biol. 21:4A.5.1-4A.5.16. © 2012 by John Wiley & Sons, Inc.)

Endogenous neural stem cells normally reside in their niche, the subventricular zone, in the uninjured rodent brain. Upon stroke, these cells become more proliferative and migrate away from the subventricular zone into the surrounding parenchyma. It is not known whether this stroke-induced behavior is due to changes in the niche or introduction of attractive cues in the infarct zone, or both. A related question is how transplanted neural stem cells respond to subsequent insults, including whether exogenous stem cells have the plasticity to respond to subsequent injuries after engraftment. We addressed this issue by transplanting neural progenitor cells (NPCs) into the uninjured brain and then subjecting the animal to stroke. We were able to follow the transplanted NPCs in vivo by labeling them with superparamagnetic iron oxide particles and imaging them via high-resolution magnetic resonance imaging (MRI) during engraftment and subsequent to stroke. We find that transplanted NPCs that are latent can be activated in response to stroke and exhibit directional migration into the parenchyma, similar to endogenous neural NPCs, without a niche environment., ((c) 2007 Wiley-Liss, Inc.)

The use of stem cell transplantation to restore neurological function after stroke is being recognized as a potential novel therapy. Before stem cell transplantation can become widely applicable, however, questions remain about the optimal site of delivery and timing of transplantation. In particular, there seems to be increasing evidence that intravascular cell delivery after stroke is a viable alternative to intracerebral transplantation. In this review, the authors focus on the intravascular delivery of stem cells for stroke treatment with an emphasis on timing, transendothelial migration and possible mechanisms leading to neuroprotection, angiogenesis, immunomodulation, and neural plasticity. They also review current concepts of in vivo imaging and tracking of stem cells after stroke.