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Variability between human pluripotent stem cell (hPSC) lines remains a challenge and opportunity in biomedicine. In this study, hPSC lines from multiple donors were differentiated toward neuroectoderm and mesendoderm lineages. We revealed dynamic transcriptomic patterns that delineate the emergence of these lineages, which were conserved across lines, along with individual line-specific transcriptional signatures that were invariant throughout differentiation. These transcriptomic signatures predicted an antagonism between SOX21-driven forebrain fates and retinoic acid-induced hindbrain fates. Replicate lines and paired adult tissue demonstrated the stability of these line-specific transcriptomic traits. We show that this transcriptomic variation in lineage bias had both genetic and epigenetic origins, aligned with the anterior-to-posterior structure of early mammalian development, and was present across a large collection of hPSC lines. These findings contribute to developing systematic analyses of PSCs to define the origin and consequences of variation in the early events orchestrating individual human development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Better understanding of the progression of neural stem cells (NSCs) in the developing cerebral cortex is important for modeling neurogenesis and defining the pathogenesis of neuropsychiatric disorders. Here, we use RNA sequencing, cell imaging, and lineage tracing of mouse and human in vitro NSCs and monkey brain sections to model the generation of cortical neuronal fates. We show that conserved signaling mechanisms regulate the acute transition from proliferative NSCs to committed glutamatergic excitatory neurons. As human telencephalic NSCs develop from pluripotency in vitro, they transition through organizer states that spatially pattern the cortex before generating glutamatergic precursor fates. NSCs derived from multiple human pluripotent lines vary in these early patterning states, leading differentially to dorsal or ventral telencephalic fates. This work furthers systematic analyses of the earliest patterning events that generate the major neuronal trajectories of the human telencephalon., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Summary: Non-negative Matrix Factorization (NMF) algorithms associate gene expression with biological processes (e.g. time-course dynamics or disease subtypes). Compared with univariate associations, the relative weights of NMF solutions can obscure biomarkers. Therefore, we developed a novel patternMarkers statistic to extract genes for biological validation and enhanced visualization of NMF results. Finding novel and unbiased gene markers with patternMarkers requires whole-genome data. Therefore, we also developed Genome-Wide CoGAPS Analysis in Parallel Sets (GWCoGAPS), the first robust whole genome Bayesian NMF using the sparse, MCMC algorithm, CoGAPS. Additionally, a manual version of the GWCoGAPS algorithm contains analytic and visualization tools including patternMatcher, a Shiny web application. The decomposition in the manual pipeline can be replaced with any NMF algorithm, for further generalization of the software. Using these tools, we find granular brain-region and cell-type specific signatures with corresponding biomarkers in GTEx data, illustrating GWCoGAPS and patternMarkers ascertainment of data-driven biomarkers from whole-genome data., Availability and Implementation: PatternMarkers & GWCoGAPS are in the CoGAPS Bioconductor package (3.5) under the GPL license., Contact: gsteinobrien@jhmi.edu or ccolantu@jhmi.edu or ejfertig@jhmi.edu., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

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.

Differentiating pluripotent cells from fibroblast progenitors is a potentially transformative tool in personalized medicine. We previously identified relatively greater success culturing dura-derived fibroblasts than scalp-derived fibroblasts from postmortem tissue. We hypothesized that these differences in culture success were related to epigenetic differences between the cultured fibroblasts by sampling location, and therefore generated genome-wide DNA methylation and transcriptome data on 11 intrinsically matched pairs of dural and scalp fibroblasts from donors across the lifespan (infant to 85 years). While these cultured fibroblasts were several generations removed from the primary tissue and morphologically indistinguishable, we found widespread epigenetic differences by sampling location at the single CpG (N = 101,989), region (N = 697), "block" (N = 243), and global spatial scales suggesting a strong epigenetic memory of original fibroblast location. Furthermore, many of these epigenetic differences manifested in the transcriptome, particularly at the region-level. We further identified 7,265 CpGs and 11 regions showing significant epigenetic memory related to the age of the donor, as well as an overall increased epigenetic variability, preferentially in scalp-derived fibroblasts-83% of loci were more variable in scalp, hypothesized to result from cumulative exposure to environmental stimuli in the primary tissue. By integrating publicly available DNA methylation datasets on individual cell populations in blood and brain, we identified significantly increased inter-individual variability in our scalp- and other skin-derived fibroblasts on a similar scale as epigenetic differences between different lineages of blood cells. Lastly, these epigenetic differences did not appear to be driven by somatic mutation--while we identified 64 probable de-novo variants across the 11 subjects, there was no association between mutation burden and age of the donor (p = 0.71). These results depict a strong component of epigenetic memory in cell culture from primary tissue, even after several generations of daughter cells, related to cell state and donor age.

Loss of insulin-producing pancreatic islet β-cells is a hallmark of type 1 diabetes. Several experimental paradigms demonstrate that these cells can, in principle, be regenerated from multiple endogenous sources using signaling pathways that are also used during pancreas development. A thorough understanding of these pathways will provide improved opportunities for therapeutic intervention. It is now appreciated that signaling pathways should not be seen as "on" or "off" but that the degree of activity may result in wildly different cellular outcomes. In addition to the degree of operation of a signaling pathway, noncanonical branches also play important roles. Thus, a pathway, once considered as "off" or "low" may actually be highly operational but may be using noncanonical branches. Such branches are only now revealing themselves as new tools to assay them are being generated. A formidable source of noncanonical signal transduction concepts is neural stem cells because these cells appear to have acquired unusual signaling interpretations to allow them to maintain their unique dual properties (self-renewal and multipotency). We discuss how such findings from the neural field can provide a blueprint for the identification of new molecular mechanisms regulating pancreatic biology, with a focus on Notch, Hes/Hey, and hedgehog pathways., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

The endocrine system involves communication among different tissues in distinct organs, including the pancreas and components of the Hypothalamic-Pituitary-Adrenal Axis. The molecular mechanisms underlying these complex interactions are a subject of intense study as they may hold clues for the progression and treatment of a variety of metabolic and degenerative diseases. A plethora of signaling pathways, activated by hormones and other endocrine factors have been implicated in this communication. Recent advances in the stem cell field introduce a new level of complexity: adult progenitor cells appear to utilize distinct signaling pathways than the more mature cells in the tissue they co-reside. It is therefore important to elucidate the signal transduction requirements of adult progenitor cells in addition to those of mature cells. Recent evidence suggests that a common non-canonical signaling pathway regulates adult progenitors in several different tissues, rendering it as a potentially valuable starting point to explore their biology. The STAT3-Ser/Hes3 Signaling Axis was first identified as a major regulator of neural stem cells and, subsequently, cancer stem cells. In the endocrine/neuroendocrine system, this pathway operates on several levels, regulating other types of plastic cells: (a) it regulates pancreatic islet cell function and insulin release; (b) insulin in turn activates the pathway in broadly distributed neural progenitors and possibly also hypothalamic tanycytes, cells with important roles in the control of the adrenal gland; (c) adrenal progenitors themselves operate this pathway. The STAT3-Ser/Hes3 Signaling Axis therefore deserves additional research in the context of endocrinology., (© Georg Thieme Verlag KG Stuttgart · New York.)

As a group, we met to discuss the current challenges for creating meaningful patient-specific in vitro models to study brain disorders. Although the convergence of findings between laboratories and patient cohorts provided us confidence and optimism that hiPSC-based platforms will inform future drug discovery efforts, a number of critical technical challenges remain. This opinion piece outlines our collective views on the current state of hiPSC-based disease modeling and discusses what we see to be the critical objectives that must be addressed collectively as a field., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Genomic data production is at its highest level and continues to increase, making available novel primary data and existing public data to researchers for exploration. Here we explore the consequences of "batch" correction for biological discovery in two publicly available expression datasets. We consider this to include the estimation of and adjustment for wide-spread systematic heterogeneity in genomic measurements that is unrelated to the effects under study, whether it be technical or biological in nature., Methods: We present three illustrative data analyses using surrogate variable analysis (SVA) and describe how to perform artifact discovery in light of natural heterogeneity within biological groups, secondary biological questions of interest, and non-linear treatment effects in a dataset profiling differentiating pluripotent cells (GSE32923) and another from human brain tissue (GSE30272)., Results: Careful specification of biological effects of interest is very important to factor-based approaches like SVA. We demonstrate greatly sharpened global and gene-specific differential expression across treatment groups in stem cell systems. Similarly, we demonstrate how to preserve major non-linear effects of age across the lifespan in the brain dataset. However, the gains in precisely defining known effects of interest come at the cost of much other information in the "cleaned" data, including sex, common copy number effects and sample or cell line-specific molecular behavior., Conclusions: Our analyses indicate that data "cleaning" can be an important component of high-throughput genomic data analysis when interrogating explicitly defined effects in the context of data affected by robust technical artifacts. However, caution should be exercised to avoid removing biological signal of interest. It is also important to note that open data exploration is not possible after such supervised "cleaning", because effects beyond those stipulated by the researcher may have been removed. With the goal of making these statistical algorithms more powerful and transparent to researchers in the biological sciences, we provide exploratory plots and accompanying R code for identifying and guiding "cleaning" process (https://github.com/andrewejaffe/StemCellSVA). The impact of these methods is significant enough that we have made newly processed data available for the brain data set at http://braincloud.jhmi.edu/plots/ and GSE30272.

Unlabelled: Interest is great in the new molecular concepts that explain, at the level of signal transduction, the process of reprogramming. Usually, transcription factors with developmental importance are used, but these approaches give limited information on the signaling networks involved, which could reveal new therapeutic opportunities. Recent findings involving reprogramming by genetic means and soluble factors with well-studied downstream signaling mechanisms, including signal transducer and activator of transcription 3 (STAT3) and hairy and enhancer of split 3 (Hes3), shed new light into the molecular mechanisms that might be involved. We examine the appropriateness of common culture systems and their ability to reveal unusual (noncanonical) signal transduction pathways that actually operate in vivo. We then discuss such novel pathways and their importance in various plastic cell types, culminating in their emerging roles in reprogramming mechanisms. We also discuss a number of reprogramming paradigms (mouse induced pluripotent stem cells, direct conversion to neural stem cells, and in vivo conversion of acinar cells to β-like cells). Specifically for acinar-to-β-cell reprogramming paradigms, we discuss the common view of the underlying mechanism (involving the Janus kinase-STAT pathway that leads to STAT3-tyrosine phosphorylation) and present alternative interpretations that implicate STAT3-serine phosphorylation alone or serine and tyrosine phosphorylation occurring in sequential order. The implications for drug design and therapy are important given that different phosphorylation sites on STAT3 intercept different signaling pathways. We introduce a new molecular perspective in the field of reprogramming with broad implications in basic, biotechnological, and translational research., Significance: Reprogramming is a powerful approach to change cell identity, with implications in both basic and applied biology. Most efforts involve the forced expression of key transcription factors, but recently, success has been reported with manipulating signal transduction pathways that might intercept them. It is important to start connecting the function of the classic reprogramming genes to signaling pathways that also mediate reprogramming, unifying the sciences of signal transduction, stem cell biology, and epigenetics. Neural stem cell studies have revealed the operation of noncanonical signaling pathways that are now appreciated to also operate during reprogramming, offering new mechanistic explanations., (©AlphaMed Press.)

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., (©AlphaMed Press.)

Naive mouse embryonic stem cells (mESCs) and primed epiblast stem cells (mEpiSCs) represent successive snapshots of pluripotency during embryogenesis. Using transcriptomic and epigenomic mapping we show that a small fraction of transcripts are differentially expressed between mESCs and mEpiSCs and that these genes show expected changes in chromatin at their promoters and enhancers. Unexpectedly, the cis-regulatory circuitry of genes that are expressed at identical levels between these cell states also differs dramatically. In mESCs, these genes are associated with dominant proximal enhancers and dormant distal enhancers, which we term seed enhancers. In mEpiSCs, the naive-dominant enhancers are lost, and the seed enhancers take up primary transcriptional control. Seed enhancers have increased sequence conservation and show preferential usage in downstream somatic tissues, often expanding into super enhancers. We propose that seed enhancers ensure proper enhancer utilization and transcriptional fidelity as mammalian cells transition from naive pluripotency to a somatic regulatory program., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Published by Elsevier B.V.)

Following their birth in the adult hippocampal dentate gyrus, newborn progenitor cells migrate into the granule cell layer where they differentiate, mature, and functionally integrate into existing circuitry. The hypothesis that adult hippocampal neurogenesis is physiologically important has gained traction, but the precise role of newborn neurons in hippocampal function remains unclear. We investigated whether loss of new neurons impacts dendrite morphology and glutamate levels in area CA3 of the hippocampus by utilizing a human GFAP promoter-driven thymidine kinase genetic mouse model to conditionally suppress adult neurogenesis. We found that chronic ablation of new neurons induces remodeling in CA3 pyramidal cells and increases stress-induced release of the neurotransmitter glutamate. The ability of persistent impairment of adult neurogenesis to influence hippocampal dendrite morphology and excitatory amino acid neurotransmission has important implications for elucidating newborn neuron function, and in particular, understanding the role of these cells in stress-related excitoxicity.

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., (© 2013 AlphaMed Press.)

Finding a cell that reprograms in a nonstochastic manner without genetic manipulation has proven elusive. In this issue, Guo et al. report the identification of a cell defined by an ultrafast cycle whose progeny reprogram in a synchronous and rapid manner., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Signal transducer and activator of transcription 3 (STAT3) dramatically increases during the first post-natal week, and supports the survival of mature hippocampal neurons. Recently, we reported that chronic elevation of excitability leads to a loss of STAT3 signal, inducing vulnerability in neurons. The loss of STAT3 signal was due to impaired Erk1/2 activation. While overnight elevation of activity attenuated STAT3 signal, brief low-frequency stimuli, which induce long-term depression, have been shown to activate STAT3. Here we investigated how STAT3 responds to depolarization in mature neurons. A brief depolarization results in the transient activation of STAT3: it induces calcium influx through L-type voltage-gated calcium channels, which triggers activation of Src family kinases. Src family kinases are required for phosphorylation of STAT3 at Tyr-705 and Ser-727. PTyr-705 is Janus kinase (JAK)-dependent, while PSer-727 is dependent on Akt, the Ser/Thr kinase. Both PTyr-705 and PSer-727 are necessary for nuclear translocation of STAT3 in these neurons. Chronic elevation of spontaneous activity by an A-type potassium blocker, 4-aminopyridine (4-AP), also induced the transient phosphorylation of STAT3, which after 4 h fell to basal levels despite the presence of 4-AP. These results suggest that phasic and chronic neuronal activation induce distinct molecular pathways, resulting in opposing regulation of STAT3 signal., (© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Neural stem cells (NSCs) are pluripotent precursors with the ability to proliferate and differentiate into 3 neural cell lineages, neurons, astrocytes and oligodendrocytes. Elucidation of the mechanisms underlying these biologic processes is essential for understanding both physiologic and pathologic neural development and regeneration after injury. Nuclear hormone receptors (NRs) and their transcriptional coregulators also play crucial roles in neural development, functions and fate. To identify key NRs and their transcriptional regulators in NSC differentiation, we examined mRNA expression of 49 NRs and many of their coregulators during differentiation (0-5 days) of mouse embryonic NSCs induced by withdrawal of fibroblast growth factor-2 (FGF2). 37 out of 49 NRs were expressed in NSCs before induction of differentiation, while receptors known to play major roles in neural development, such as THRα, RXRs, RORs, TRs, and COUP-TFs, were highly expressed. CAR, which plays important roles in xenobiotic metabolism, was also highly expressed. FGF2 withdrawal induced mRNA expression of RORγ, RXRγ, and MR by over 20-fold. Most of the transcriptional coregulators examined were expressed basally and throughout differentiation without major changes, while FGF2 withdrawal strongly induced mRNA expression of several histone deacetylases (HDACs), including HDAC11. Dexamethasone and aldosterone, respectively a synthetic glucocorticoid and natural mineralocorticoid, increased NSC numbers and induced differentiation into neurons and astrocytes. These results indicate that the NRs and their coregulators are present and/or change their expression during NSC differentiation, suggesting that they may influence development of the central nervous system in the absence or presence of their ligands., (© Georg Thieme Verlag KG Stuttgart · New York.)

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., (Published by Elsevier B.V.)

Tumors exhibit complex organization and contain a variety of cell populations. The realization that the regenerative properties of a tumor may be largely confined to a cell subpopulation (cancer stem cell) is driving a new era of anti-cancer research. Cancer stem cells from Glioblastoma Multiforme tumors express markers that are also expressed in non-cancerous neural stem cells, including nestin and Sox2. We previously showed that the transcription factor Hes3 is a marker of neural stem cells, and that its expression is inhibited by JAK activity. Here we show that Hes3 is also expressed in cultures from glioblastoma multiforme which express neural stem cell markers, can differentiate into neurons and glia, and can recapitulate the tumor of origin when transplanted into immunocompromised mice. Similar to observations in neural stem cells, JAK inhibits Hes3 expression. Hes3 RNA interference reduces the number of cultured glioblastoma cells suggesting a novel therapeutic strategy.

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., (Published by Elsevier B.V.)

Chronically altered levels of network activity lead to changes in the morphology and functions of neurons. However, little is known of how changes in neuronal activity alter the intracellular signaling pathways mediating neuronal survival. Here, we use primary cultures of rat hippocampal neurons to show that elevated neuronal activity impairs phosphorylation of the serine/threonine kinase, Erk1/2, and the activation of signal transducer and activator of transcription 3 (STAT3) by phosphorylation of serine 727. Chronically stimulated neurons go through apoptosis when they fail to activate another serine/threonine kinase, Akt. Gain- and loss-of-function experiments show that STAT3 plays the key role directly downstream from Erk1/2 as the alternative survival pathway. Elevated neuronal activity resulted in increased expression of a tumor suppressor, p53, and its target gene, Bax. These changes are observed in Kv4.2 knock-out mouse hippocampal neurons, which are also sensitive to the blockade of TrkB signaling, confirming that the alteration occurs in vivo. Thus, this study provides new insight into a mechanism by which chronic elevation of activity may cause neurodegeneration.

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., (Copyright © 2012 AlphaMed Press.)

Sox2 is an important transcriptional regulator in embryonic and adult stem cells. Recently, Sox2 was identified as an oncogene in many endodermal cancers, including colon cancer. There is great interest in how Sox2 cooperates with other transcription factors to regulate stem cell renewal, differentiation, and reprogramming. However, we still lack a general understanding of Sox2 transcriptional action. To determine transcriptional partners of Sox2 in adult cells, we generated mice where gene expression could be induced by an externally applied stimulus. We analyzed the consequences in the intestine where cell turnover is rapid. Sox2 expression, but not Oct4, specifically increased the numbers of stem cells and repressed Cdx2, a master regulator of endodermal identity. In vivo studies demonstrated that Sox21, another member of the SoxB gene family, was a specific, immediate, and cell-autonomous target of Sox2 in intestinal stem cells. In vitro experiments showed that Sox21 was sufficient to repress Cdx2 in colon cancer cells and in pluripotent stem cells. Sox21 was also specifically induced by Sox2 in fibroblasts and inhibition of Sox21 blocked reprogramming to the pluripotent state. These results show that transcriptional induction of Sox21 is a rapid and general mediator of the effects of Sox2 on cell identity in a wide range of cell types., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The number of neurons in the adult rodent brain is strongly influenced by events in early postnatal life that eliminate approximately half of the neurons. Recently, we reported that neurotrophins induced survival of neonatal rat hippocampal neurons by promoting neural activity and activation of the Ser/Thr kinase, Akt. The survival of neurons also depended on integrin signaling, but a role for the extracellular matrix (ECM) in this mechanism was yet to be explored. Here, we show that levels of the matrix metalloproteinase-9 (MMP9) decrease, and the level of the ECM protein laminin increases in rat hippocampus during the period of neuronal death. Hippocampi from MMP9 null mice showed higher levels of laminin expression than wild type at P1 and no further increase at P10. In vitro, the matrix metalloproteinase inhibitor FN-439 promoted survival of neurons in a laminin-integrin β1-dependent manner. Blocking laminin signaling attenuated activation of Akt by depolarization. In vivo, injecting FN-439 into the neonatal hippocampus increased the level of laminin and promoted neuronal survival through an integrin-dependent mechanism. These results show signals from the ECM are not simply permissive but rather actively regulated, and they interact with neuronal activity to control the number of hippocampal neurons. This work is the first to report a role for MMP9 in regulating neuronal survival through the developmental process that establishes the functional brain.

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 β-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., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The adult hippocampus is involved in learning and memory. As a consequence, it is a brain region of remarkable plasticity. This plasticity exhibits itself both as cellular changes and neurogenesis. For neurogenesis to occur, a population of local stem cells and progenitor cells is maintained in the adult brain and these are able to proliferate and differentiate into neurons which contribute to the hippocampal circuitry. There is much interest in understanding the role of immature cells in the hippocampus, in relation to learning and memory. Methods and mechanisms that increase the numbers of these cells will be valuable in this research field. We show here that single injections of soluble factors into the lateral ventricle of adult rats and mice induces the rapid (within one week) increase in the number of putative stem cells/progenitor cells in the hippocampus. The established progenitor marker Sox2 together with the more recently established marker Hes3, were used to quantify the manipulation of the Sox2/Hes3 double-positive cell population. We report that in both adult rodent species, Sox2+/Hes3+ cell numbers can be increased within one week. The most prominent increase was observed in the hilus of the dentate gyrus. This study presents a fast, pharmacological method to manipulate the numbers of endogenous putative stem cells/progenitor cells. This method may be easily modified to alter the degree of activation (e.g. by the use of osmotic pumps for delivery, or by repeat injections through implanted cannulas), in order to be best adapted to different paradigms of research (neurodegenerative disease, neuroprotection, learning, memory, plasticity, etc).

Malignant gliomas contain a population of self-renewing tumorigenic stem-like cells; however, it remains unclear how these glioma stem cells (GSCs) self-renew or generate cellular diversity at the single-cell level. Asymmetric cell division is a proposed mechanism to maintain cancer stem cells, yet the modes of cell division that GSCs utilize remain undetermined. Here, we used single-cell analyses to evaluate the cell division behavior of GSCs. Lineage-tracing analysis revealed that the majority of GSCs were generated through expansive symmetric cell division and not through asymmetric cell division. The majority of differentiated progeny was generated through symmetric pro-commitment divisions under expansion conditions and in the absence of growth factors, occurred mainly through asymmetric cell divisions. Mitotic pair analysis detected asymmetric CD133 segregation and not any other GSC marker in a fraction of mitoses, some of which were associated with Numb asymmetry. Under growth factor withdrawal conditions, the proportion of asymmetric CD133 divisions increased, congruent with the increase in asymmetric cell divisions observed in the lineage-tracing studies. Using single-cell-based observation, we provide definitive evidence that GSCs are capable of different modes of cell division and that the generation of cellular diversity occurs mainly through symmetric cell division, not through asymmetric cell division.

It is important to determine the mechanisms controlling the number of neurons in the nervous system. Previously, we reported that neuronal activity plays a central role in controlling neuron number in the neonatal hippocampus of rodents. Neuronal survival requires sustained activation of the serine-threonine kinase Akt, which is initiated by neurotrophins and continued for several hours by neuronal activity and integrin signaling. Here, we focus on the CA3 region to show that neuronal apoptosis requires p53. As in wild-type animals, neuronal death occurs in the first postnatal week and ends by postnatal day (P)10 in p53(-/-) mice. During this period, the CA3 region of p53(-/-) mice contains significantly lower numbers of apoptotic cells, and at the end of the death period, it contains more neurons than the wild type. At P10, the p53(-/-) CA3 region contains a novel subpopulation of neurons with small soma size. These neurons show normal levels of tropomyosin receptor kinase receptor activation, but lower levels of activated Akt than the neurons with somata of normal size. These results suggest that p53 is the key downstream regulator of the novel survival-signaling pathway that regulates the number of CA3 neurons in the first 10 days of postnatal life., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

The nervous system develops through a program that first produces neurons in excess and then eliminates as many as half in a specific period of early postnatal life. Neurotrophins are widely thought to regulate neuronal survival, but this role has not been clearly defined in the CNS. Here we show that neurotrophins promote survival of young neurons by promoting spontaneous activity. Survival of hippocampal neurons in neonatal rat requires spontaneous activity that depends on the excitatory action of GABA. Neurotrophins facilitate recruitment of cultured neurons into active networks, and it is this activity, combined with integrin receptor signaling, that controls neuronal survival. In vivo, neurotrophins require integrin signaling to control neuron number. These data are the first to link the early excitatory action of GABA to the developmental death period and to assign an essential role for activity in neurotrophin-mediated survival that establishes appropriate networks.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.

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.

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.

Hypoxia contributes to the progression of a variety of cancers by activating adaptive transcriptional programs that promote cell survival, motility and tumor angiogenesis. Although the importance of hypoxia and subsequent hypoxia-inducible factor-1alpha (HIF-1alpha) activation in tumor angiogenesis is well known, their role in the regulation of glioma-derived stem cells is unclear. In this study, we show that hypoxia (1% oxygen) promotes the self-renewal capacity of CD133-positive human glioma-derived cancer stem cells (CSCs). Propagation of the glioma-derived CSCs in a hypoxic environment also led to the expansion of cells bearing CXCR4 (CD184), CD44(low) and A2B5 surface markers. The enhanced self-renewal activity of the CD133-positive CSCs in hypoxia was preceded by upregulation of HIF-1alpha. Knockdown of HIF-1alpha abrogated the hypoxia-mediated CD133-positive CSC expansion. Inhibition of the phosphatidylinositol 3-kinase(PI3K)-Akt or ERK1/2 pathway reduced the hypoxia-driven CD133 expansion, suggesting that these signaling cascades may modulate the hypoxic response. Finally, CSCs propagated at hypoxia robustly retained the undifferentiated phenotype, whereas CSCs cultured at normoxia did not. These results suggest that response to hypoxia by CSCs involves the activation of HIF-1alpha to enhance the self-renewal activity of CD133-positive cells and to inhibit the induction of CSC differentiation. This study illustrates the importance of the tumor microenvironment in determining cellular behavior.

In Parkinson's disease, multiple cell types in many brain regions are afflicted. As a consequence, a therapeutic strategy that activates a general neuroprotective response may be valuable. We have previously shown that Notch ligands support neural precursor cells in vitro and in vivo. Here we show that neural precursors express the angiopoietin receptor Tie2 and that injections of angiopoietin2 activate precursors in the adult brain. Signaling downstream of Tie2 and the Notch receptor regulate blood vessel formation. In the adult brain, angiopoietin2 and the Notch ligand Dll4 activate neural precursors with opposing effects on the density of blood vessels. A model of Parkinson's disease was used to show that angiopoietin2 and Dll4 rescue injured dopamine neurons with motor behavioral improvement. A combination of growth factors with little impact on the vasculature retains the ability to stimulate neural precursors and protect dopamine neurons. The cellular and pharmacological basis of the neuroprotective effects achieved by these single treatments merits further analysis.