Bdnf deficiency in the neonatal hippocampus contributes to global dna hypomethylation and adult behavioral changes.

Schizophrenia is a neurodevelopmental psychiatric disorder, encompassing genetic and environmental risk factors. For several decades, investigators have been implementing the use of lesions of the neonatal rodent hippocampus to model schizophrenia, resulting in a broad spectrum of adult schizophrenia-related behavioral changes. Despite the extensive use of these proposed animal models of schizophrenia, the mechanisms by which these lesions result in schizophrenia-like behavioral alterations remain unclear. Here we provide in vivo evidence that transient pharmacological inactivation of the hippocampus via tetrodotoxin microinjections or a genetic reduction in brain derived neurotrophic factor (BDNF) protein levels (BDNF+/- rats) lead to global DNA hypomethylation, disrupted maturation of the neuronal nucleus and aberrant acoustic startle response in the adult rat. The similarity between the effects of the two treatments strongly indicate that BDNF signaling is involved in effects obtained after the TTX microinjections. These findings may shed light on the cellular mechanisms underlying the phenotypical features of neonatal transient inhibition of the hippocampus as a preclinical model of schizophrenia and suggest that BDNF signaling represents a target pathway for development of novel treatment therapies.

Dissecting transcriptomic signatures of neuronal differentiation and maturation using iPSCs.

Human induced pluripotent stem cells (hiPSCs) are a powerful model of neural differentiation and maturation. We present a hiPSC transcriptomics resource on corticogenesis from 5 iPSC donor and 13 subclonal lines across 9 time points over 5 broad conditions: self-renewal, early neuronal differentiation, neural precursor cells (NPCs), assembled rosettes, and differentiated neuronal cells. We identify widespread changes in the expression of both individual features and global patterns of transcription. We next demonstrate that co-culturing human NPCs with rodent astrocytes results in mutually synergistic maturation, and that cell type-specific expression data can be extracted using only sequencing read alignments without cell sorting. We lastly adapt a previously generated RNA deconvolution approach to single-cell expression data to estimate the relative neuronal maturity of iPSC-derived neuronal cultures and human brain tissue. Using many public datasets, we demonstrate neuronal cultures are maturationally heterogeneous but contain subsets of neurons more mature than previously observed.

A Role for Sox2 in the Adult Cerebellum.

The cerebellum, a derivative of the hindbrain, plays a crucial role in balance and posture as well as in higher cognitive and locomotive processes. Cerebellar development is initiated during the segmental phase of hindbrain formation. Here, we describe the phenotype, of a single surviving adult conditional mouse mutant mouse, in which Sox2 function is ablated in embryonic radial glial cells by means of hGFAP-CRE. The single Sox2RGINV/mosaic adult mutant mouse displays motor disability, microsomia, reduced Central Nervous System (CNS) size and cerebellar defects associated with human genetically related congenital abnormalities.

Novel inhibitors of As(III) S-adenosylmethionine methyltransferase (AS3MT) identified by virtual screening.

Due to increased interest in As(III) S-adenosylmethionine methyltransferase (AS3MT), a search for chemical probes that can help elucidate function was initiated. A homology model was built based on related enzymes, and virtual screening produced 426 potential hits. Evaluation of these compounds in a functional enzymatic assay revealed several modest inhibitors including an O-substituted 2-amino-3-cyano indole scaffold. Two iterations of near neighbor searches revealed compound 5 as a potent inhibitor of AS3MT with good selectivity over representative methyltransferases DOT1L and NSD2 as well as a representative set of diverse receptors. Compound 5 should prove to be a useful tool to investigate the role of AS3MT and a potential starting point for further optimization.

Pluripotent Stem Cell Platforms for Drug Discovery.

Use of human pluripotent stem cells (hPSCs) and their differentiated derivatives have led to recent proof-of-principle drug discoveries, defining a pathway to the implementation of hPSC-based drug discovery (hPDD). Current hPDD strategies, however, have inevitable conceptual biases and technological limitations, including the dimensionality of cell-culture methods, cell maturity and functionality, experimental variability, and data reproducibility. In this review, we dissect representative hPDD systems via analysis of hPSC-based 2D-monolayers, 3D culture, and organoids. We discuss mechanisms of drug discovery and drug repurposing, and roles of membrane drug transporters in tissue maturation and hPDD using the example of drugs that target various mutations of CFTR, the cystic fibrosis transmembrane conductance regulator gene, in patients with cystic fibrosis.

Concise Review: Conceptualizing Paralogous Stem-Cell Niches and Unfolding Bone Marrow Progenitor Cell Identities.

Lineage commitment and differentiation of skeletal stem cells/bone marrow stromal cells (SSCs/BMSCs, often called bone marrow-derived "mesenchymal stem/stromal" cells) offer an important opportunity to study skeletal and hematopoietic diseases, and for tissue engineering and regenerative medicine. Currently, many studies in this field have relied on cell lineage tracing methods in mouse models, which have provided a significant advancement in our knowledge of skeletal and hematopoietic stem-cell niches in bone marrow (BM). However, there is a lack of agreement in numerous fundamental areas, including origins of various BM stem-cell niches, cell identities, and their physiological roles in the BM. In order to resolve these issues, we propose a new hypothesis of "paralogous" stem-cell niches (PSNs); that is, progressively altered parallel niches within an individual species throughout the life span of the organism. A putative PSN code seems to be plausible based on analysis of transcriptional signatures in two representative genes that encode Nes-GFP and leptin receptors, which are frequently used to monitor SSC lineage development in BM. Furthermore, we suggest a dynamic paralogous BM niche (PBMN) model that elucidates the coupling and uncoupling mechanisms between BM stem-cell niches and their zones of active regeneration during different developmental stages. Elucidation of these PBMNs would enable us to resolve the existing controversies, thus paving the way to achieving precision regenerative medicine and pharmaceutical applications based on these BM cell resources. Stem Cells 2018;36:11-21.

A "Hit and Run" Approach to Inducible Direct Reprogramming of Astrocytes to Neural Stem Cells.

Temporal and spatial control of gene expression can be achieved using an inducible system as a fundamental tool for regulated transcription in basic, applied and eventually in clinical research. We describe a novel "hit and run" inducible direct reprogramming approach. In a single step, 2 days post-transfection, transiently transfected Sox2(FLAG) under the Leu3p-αIPM inducible control (iSox2) triggers the activation of endogenous Sox2, redirecting primary astrocytes into abundant distinct nestin-positive radial glia cells. This technique introduces a unique novel tool for safe, rapid and efficient reprogramming amendable to regenerative medicine.

Directed differentiation of human induced pluripotent stem cells toward bone and cartilage: in vitro versus in vivo assays.

The ability to differentiate induced pluripotent stem cells (iPSCs) into committed skeletal progenitors could allow for an unlimited autologous supply of such cells for therapeutic uses; therefore, we attempted to create novel bone-forming cells from human iPSCs using lines from two distinct tissue sources and methods of differentiation that we previously devised for osteogenic differentiation of human embryonic stem cells, and as suggested by other publications. The resulting cells were assayed using in vitro methods, and the results were compared with those obtained from in vivo transplantation assays. Our results show that true bone was formed in vivo by derivatives of several iPSC lines, but that the successful cell lines and differentiation methodologies were not predicted by the results of the in vitro assays. In addition, bone was formed equally well from iPSCs originating from skin or bone marrow stromal cells (also known as bone marrow-derived mesenchymal stem cells), suggesting that the iPSCs did not retain a "memory" of their previous life. Furthermore, one of the iPSC-derived cell lines formed verifiable cartilage in vivo, which likewise was not predicted by in vitro assays.

Developmental insights from early mammalian embryos and core signaling pathways that influence human pluripotent cell growth and differentiation.

Human pluripotent stem cells (hPSCs) have two potentially attractive applications: cell replacement-based therapies and drug discovery. Both require the efficient generation of large quantities of clinical-grade stem cells that are free from harmful genomic alterations. The currently employed colony-type culture methods often result in low cell yields, unavoidably heterogeneous cell populations, and substantial chromosomal abnormalities. Here, we shed light on the structural relationship between hPSC colonies/embryoid bodies and early-stage embryos in order to optimize current culture methods based on the insights from developmental biology. We further highlight core signaling pathways that underlie multiple epithelial-to-mesenchymal transitions (EMTs), cellular heterogeneity, and chromosomal instability in hPSCs. We also analyze emerging methods such as non-colony type monolayer (NCM) and suspension culture, which provide alternative growth models for hPSC expansion and differentiation. Furthermore, based on the influence of cell-cell interactions and signaling pathways, we propose concepts, strategies, and solutions for production of clinical-grade hPSCs, stem cell precursors, and miniorganoids, which are pivotal steps needed for future clinical applications.

Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics.

Human pluripotent stem cells (hPSCs) provide powerful resources for application in regenerative medicine and pharmaceutical development. In the past decade, various methods have been developed for large-scale hPSC culture that rely on combined use of multiple growth components, including media containing various growth factors, extracellular matrices, 3D environmental cues, and modes of multicellular association. In this Protocol Review, we dissect these growth components by comparing cell culture methods and identifying the benefits and pitfalls associated with each one. We further provide criteria, considerations, and suggestions to achieve optimal cell growth for hPSC expansion, differentiation, and use in future therapeutic applications.

FGF2 and insulin signaling converge to regulate cyclin D expression in multipotent neural stem cells.

The ex vivo expansion of stem cells is making major contribution to biomedical research. The multipotent nature of neural precursors acutely isolated from the developing central nervous system has been established in a series of studies. Understanding the mechanisms regulating cell expansion in tissue culture would support their expanded use either in cell therapies or to define disease mechanisms. Basic fibroblast growth factor (FGF2) and insulin, ligands for tyrosine kinase receptors, are sufficient to sustain neural stem cells (NSCs) in culture. Interestingly, real-time imaging shows that these cells become multipotent every time they are passaged. Here, we analyze the role of FGF2 and insulin in the brief period when multipotent cells are present. FGF2 signaling results in the phosphorylation of Erk1/2, and activation of c-Fos and c-Jun that lead to elevated cyclin D mRNA levels. Insulin signals through the PI3k/Akt pathway to regulate cyclins at the post-transcriptional level. This precise Boolean regulation extends our understanding of the proliferation of multipotent NSCs and provides a basis for further analysis of proliferation control in the cell states defined by real-time mapping of the cell lineages that form the central nervous system.

StemCellDB: the human pluripotent stem cell database at the National Institutes of Health.

Much of the excitement generated by induced pluripotent stem cell technology is concerned with the possibility of disease modeling as well as the potential for personalized cell therapy. However, to pursue this it is important to understand the 'normal' pluripotent state including its inherent variability. We have performed various molecular profiling assays for 21 hESC lines and 8 hiPSC lines to generate a comprehensive snapshot of the undifferentiated state of pluripotent stem cells. Analysis of the gene expression data revealed no iPSC-specific gene expression pattern in accordance with previous reports. We further compared cells, differentiated as embryoid bodies in 2 media proposed to initiate differentiation towards separate cell fates, as well as 20 adult tissues. From this analysis we have generated a gene list which defines pluripotency and establishes a baseline for the pluripotent state. Finally, we provide lists of genes enriched under both differentiation conditions which show the proposed bias toward independent cell fates.

Regulation and expression of the ATP-binding cassette transporter ABCG2 in human embryonic stem cells.

The expression and function of several multidrug transporters (including ABCB1 and ABCG2) have been studied in human cancer cells and in mouse and human adult stem cells. However, the expression of ABCG2 in human embryonic stem cells (hESCs) remains unclear. Limited and contradictory results in the literature from two research groups have raised questions regarding its expression and function. In this study, we used quantitative real-time PCR, Northern blots, whole genome RNA sequencing, Western blots, and immunofluorescence microscopy to study ABCG2 expression in hESCs. We found that full-length ABCG2 mRNA transcripts are expressed in undifferentiated hESC lines. However, ABCG2 protein was undetectable even under embryoid body differentiation or cytotoxic drug induction. Moreover, surface ABCG2 protein was coexpressed with the differentiation marker stage-specific embryonic antigen-1 of hESCs, following constant BMP-4 signaling at days 4 and 6. This expression was tightly correlated with the downregulation of two microRNAs (miRNAs) (i.e., hsa-miR-519c and hsa-miR-520h). Transfection of miRNA mimics and inhibitors of these two miRNAs confirmed their direct involvement in the regulation ABCG2 translation. Our findings clarify the controversy regarding the expression of the ABCG2 gene and also provide new insights into translational control of the expression of membrane transporter mRNAs by miRNAs in hESCs.

Non-colony type monolayer culture of human embryonic stem cells.

Regenerative medicine, relying on human embryonic stem cell (hESC) technology, opens promising new avenues for therapy of many severe diseases. However, this approach is restricted by limited production of the desired cells due to the refractory properties of hESC growth in vitro. It is further hindered by insufficient control of cellular stress, growth rates, and heterogeneous cellular states under current culture conditions. In this study, we report a novel cell culture method based on a non-colony type monolayer (NCM) growth. Human ESCs under NCM remain pluripotent as determined by teratoma assays and sustain the potential to differentiate into three germ layers. This NCM culture has been shown to homogenize cellular states, precisely control growth rates, significantly increase cell production, and enhance hESC recovery from cryopreservation without compromising chromosomal integrity. This culture system is simple, robust, scalable, and suitable for high-throughput screening and drug discovery.

Direct and indirect contribution of human embryonic stem cell-derived hepatocyte-like cells to liver repair in mice.

BACKGROUND & AIMS: Many studies of embryonic stem cells have investigated direct cell replacement of damaged tissues, but little is known about how donor cell-derived signals affect host tissue regeneration. We investigated the direct and indirect roles of human embryonic stem cell-derived cells in liver repair in mice. METHODS: To promote the initial differentiation of human embryonic stem cells into mesendoderm, we activated the ß-catenin signaling pathway with lithium; cells were then further differentiated into hepatocyte-like cells. The differentiated cells were purified by indocyanine green staining and laser microdissection and characterized by immunostaining, polymerase chain reaction, biochemical function, electron microscopy, and transplantation analyses. To investigate indirect effects of these cells, secreted proteins (secretomes) were analyzed by a label-free quantitative mass spectrometry. Carbon tetrachloride was used to induce acute liver injury in mice; cells or secreted proteins were administered by intrasplenic or intraperitoneal injection, respectively. RESULTS: The differentiated hepatocyte-like cells had multiple features of normal hepatocytes, engrafted efficiently into mice, and continued to have hepatic features; they promoted proliferation of host hepatocytes and revascularization of injured host liver tissues. Proteomic analysis identified proteins secreted from these cells that might promote host tissue repair. Injection of the secreted proteins into injured livers of mice promoted significant amounts of tissue regeneration without cell grafts. CONCLUSIONS: Hepatocyte-like cells derived from human embryonic stem cells contribute to recovery of injured liver tissues in mice, not only by cell replacement but also by delivering trophic factors that support endogenous liver regeneration.

Isolation of epiblast stem cells from preimplantation mouse embryos.

Pluripotent stem cells provide a platform to interrogate control elements that function to generate all cell types of the body. Despite their utility for modeling development and disease, the relationship of mouse and human pluripotent stem cell states to one another remains largely undefined. We have shown that mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) are distinct, pluripotent states isolated from pre- and post-implantation embryos respectively. Human ES cells are different than mouse ES cells and share defining features with EpiSCs, yet are derived from pre-implantation human embryos. Here we show that EpiSCs can be routinely derived from pre-implantation mouse embryos. The preimplantation-derived EpiSCs exhibit molecular features and functional properties consistent with bona fide EpiSCs. These results provide a simple method for isolating EpiSCs and offer direct insight into the intrinsic and extrinsic mechanisms that regulate the acquisition of distinct pluripotent states.

Cholera toxin regulates a signaling pathway critical for the expansion of neural stem cell cultures from the fetal and adult rodent brains.

BACKGROUND: New mechanisms that regulate neural stem cell (NSC) expansion will contribute to improved assay systems and the emerging regenerative approach that targets endogenous stem cells. Expanding knowledge on the control of stem cell self renewal will also lead to new approaches for targeting the stem cell population of cancers. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that Cholera toxin regulates two recently characterized NSC markers, the Tie2 receptor and the transcription factor Hes3, and promotes the expansion of NSCs in culture. Cholera toxin increases immunoreactivity for the Tie2 receptor and rapidly induces the nuclear localization of Hes3. This is followed by powerful cultured NSC expansion and induction of proliferation both in the presence and absence of mitogen. CONCLUSIONS/SIGNIFICANCE: Our data suggest a new cell biological mechanism that regulates the self renewal and differentiation properties of stem cells, providing a new logic to manipulate NSCs in the context of regenerative disease and cancer.

Epiblast stem cells contribute new insight into pluripotency and gastrulation.

Gastrulation is the defining feature of metazoan development where it serves to apportion seemingly equivalent, pluripotent cells to specific fates. The three embryonic germ layers generated during gastrulation from the pluripotent epiblast including ectoderm, mesoderm, and definitive endoderm, contain the progenitors required to build all of the tissues of the developing organism. As a result, there is great interest in understanding the events that coordinate gastrulation. Because developing embryos in placental mammals are relatively inaccessible, stem cells are widely used for experimental and biochemical interrogation of these processes. Epiblast stem cells (EpiSCs) are grown from the post-implantation epiblast, which is the most proximal pluripotent tissue to the early somatic and germ cell precursors. Because EpiSCs can be propagated indefinitely in vitro as a stable state that recapitulates the properties of the post-implantation epiblast, they are uniquely positioned to provide novel insight into the developmental window where somatic and germ cell lineages are first established. Here we discuss the nature of EpiSCs and their significance in understanding gastrulation and cell specification in relationship to other pluripotent cell culture models.

Angiogenic factors stimulate growth of adult neural stem cells.

BACKGROUND: The ability to grow a uniform cell type from the adult central nervous system (CNS) is valuable for developing cell therapies and new strategies for drug discovery. The adult mammalian brain is a source of neural stem cells (NSC) found in both neurogenic and non-neurogenic zones but difficulties in culturing these hinders their use as research tools. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that NSCs can be efficiently grown in adherent cell cultures when angiogenic signals are included in the medium. These signals include both anti-angiogenic factors (the soluble form of the Notch receptor ligand, Dll4) and pro-angiogenic factors (the Tie-2 receptor ligand, Angiopoietin 2). These treatments support the self renewal state of cultured NSCs and expression of the transcription factor Hes3, which also identifies the cancer stem cell population in human tumors. In an organotypic slice model, angiogenic factors maintain vascular structure and increase the density of dopamine neuron processes. CONCLUSIONS/SIGNIFICANCE: We demonstrate new properties of adult NSCs and a method to generate efficient adult NSC cultures from various central nervous system areas. These findings will help establish cellular models relevant to cancer and regeneration.