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1.  Concise Review: Reprogramming, Behind the Scenes: Noncanonical Neural Stem Cell Signaling Pathways Reveal New, Unseen Regulators of Tissue Plasticity With Therapeutic Implications 
Stem Cells Translational Medicine  2015;4(11):1251-1257.
This study examined the appropriateness of common culture systems and their ability to reveal unusual (noncanonical) signal transduction pathways that actually operate in vivo. Novel pathways and their importance in various plastic cell types, culminating in their emerging roles in reprogramming mechanisms, and a number of reprogramming paradigms are also discussed. A new molecular perspective in the field of reprogramming with broad implications in basic, biotechnological, and translational research is introduced.
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.
doi:10.5966/sctm.2015-0105
PMCID: PMC4622411  PMID: 26371344
Cellular reprogramming; Cellular transdifferentiation; Induced pluripotent stem cells; Signal transduction; STAT3 transcription factor; Hes3 protein; Pancreatic islets
3.  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 Sox2FLAG 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.
doi:10.3389/fphys.2016.00127
PMCID: PMC4828628  PMID: 27148066
FLP; tetracycline; doxycycline; CREERT2; embryonic stem cells; induced pluripotent stem cells; neural progenitor cells; tissue regeneration
4.  Efficient serum-free derivation of oligodendrocyte precursors from neural stem cell-enriched cultures 
Stem cells (Dayton, Ohio)  2009;27(1):116-125.
Oligodendrocytes derived in the laboratory from stem cells have been proposed as a treatment for acute and chronic injury to the central nervous system. Platelet-derived growth factor-receptor alpha (PDGFRα) signaling is known to regulate oligodendrocyte precursor cell numbers both during development and adulthood. Here, we analyze the effects of PDGFRα signaling on central nervous system (CNS) stem cell-enriched cultures. We find that AC133 selection for CNS progenitors acutely isolated from the fetal cortex enriches for PDGF-AA responsive cells. PDGF-AA treatment of FGF2-expanded CNS stem cell-enriched cultures increases nestin+ cell number, viability, proliferation, and glycolytic rate. We show that a brief exposure to PDGF-AA rapidly and efficiently permits the derivation of O4+ oligodendrocyte-lineage cells from CNS stem cell-enriched cultures. The derivation of oligodendrocyte-lineage cells demonstrated here may support the effective use of stem cells in understanding fate choice mechanisms and the development of new therapies targeting this cell type.
doi:10.1634/stemcells.2007-0205
PMCID: PMC4772902  PMID: 18403757
oligodendrocytes; multipotent stem cells; platelet-derived growth factor; glycolysis; extracellular signal-regulated MAP kinases; phosphatidylinositol 3-kinase
5.  Strong Components of Epigenetic Memory in Cultured Human Fibroblasts Related to Site of Origin and Donor Age 
PLoS Genetics  2016;12(2):e1005819.
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.
Author Summary
Regenerative medicine specialists have been using a type of cell commonly found in the skin called the fibroblast because it is easily obtained from skin samples, grows well in culture, and can be manipulated in the laboratory to de-differentiate into a primordial state known as the induced pluripotent stem cell. These primitive stem cells can then be transformed into mature tissues, such as liver or pancreas cells. Here we show that fibroblasts, coming from different locations in the same individual, vary significantly in epigenetic marks called DNA methylation, which are involved in the regulation of gene expression. In addition to location-specific patterns of DNA methylation, we also find that fibroblasts from different anatomical locations respond differently in epigenetic patterns related to aging. As the field of regenerative medicine advances, our study demonstrates that deciding upon the source of fibroblasts from an individual to generate new tissues and organs may be an important consideration.
doi:10.1371/journal.pgen.1005819
PMCID: PMC4767228  PMID: 26913521
6.  Creating Patient-Specific Neural Cells for the In Vitro Study of Brain Disorders 
Stem Cell Reports  2015;5(6):933-945.
Summary
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.
Highlights
•A key limitation of the field is difficulty in accurately defining cell state•Next step will be building complexity by achieving network and circuit structures•Epigenetic factors and somatic mosaicism in iPS cells may contribute to disease•A critical advance will be improving scalability and reproducibility of assays
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.
doi:10.1016/j.stemcr.2015.10.011
PMCID: PMC4881284  PMID: 26610635
7.  Practical impacts of genomic data “cleaning” on biological discovery using surrogate variable analysis 
BMC Bioinformatics  2015;16:372.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s12859-015-0808-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12859-015-0808-5
PMCID: PMC4636836  PMID: 26545828
Batch correction; Gene expression; Surrogate variable analysis
8.  Epigenomic comparison reveals activation of ‘seed’ enhancers during transition from naive to primed pluripotency 
Cell stem cell  2014;14(6):854-863.
SUMMARY
Naïve 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 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 naïve-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 naïve pluripotency to a somatic regulatory program.
doi:10.1016/j.stem.2014.05.005
PMCID: PMC4149284  PMID: 24905169
9.  Complementary roles for histone deacetylases 1, 2, and 3 in differentiation of pluripotent stem cells 
In eukaryotic cells, covalent modifications to core histones contribute to the establishment and maintenance of cellular phenotype via regulation of gene expression. Histone acetyltransferases (HATs) cooperate with histone deacetylases (HDACs) to establish and maintain specific patterns of histone acetylation. HDAC inhibitors can cause pluripotent stem cells to cease proliferating and enter terminal differentiation pathways in culture. To better define the roles of individual HDACs in stem cell differentiation, we have constructed “dominant-negative” stem cell lines expressing mutant, Flag-tagged HDACs with reduced enzymatic activity. Replacement of a single residue (His → Ala) in the catalytic center reduced the activity of HDACs 1 and 2 by 80%, and abolished HDAC3 activity; the mutant HDACs were expressed at similar levels and in the same multiprotein complexes as wild-type HDACs. Hexamethylene bisacetamide-induced MEL cell differentiation was potentiated by the individual mutant HDACs, but only to 2%, versus 60% for an HDAC inhibitor, sodium butyrate, suggesting that inhibition of multiple HDACs is required for full potentiation. Cultured E14.5 cortical stem cells differentiate to neurons, astrocytes, and oligodendrocytes upon withdrawal of basic fibroblast growth factor. Transduction of stem cells with mutant HDACs 1, 2, or 3 shifted cell fate choice toward oligodendrocytes. Mutant HDAC2 also increased differentiation to astrocytes, while mutant HDAC1 reduced differentiation to neurons by 50%. These results indicate that HDAC activity inhibits differentiation to oligodendrocytes, and that HDAC2 activity specifically inhibits differentiation to astrocytes, while HDAC1 activity is required for differentiation to neurons.
doi:10.1111/j.1432-0436.2007.00232.x
PMCID: PMC4428170  PMID: 18021260
HDAC; dominant negative; transduction; cell fate; MEL; neural stem cell
10.  Atrophy of pyramidal neurons and increased stress-induced glutamate levels in CA3 following chronic suppression of adult neurogenesis 
Brain structure & function  2013;219(3):1139-1148.
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 new-born 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.
doi:10.1007/s00429-013-0532-8
PMCID: PMC3795860  PMID: 23483239
Neurogenesis; Dentate gyrus; Excitotoxicity; Atrophy; Dendrite; CA3
11.  Developmental insights from early mammalian embryos and core signaling pathways that influence human pluripotent cell growth and differentiation 
Stem cell research  2014;12(3):610-621.
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.
doi:10.1016/j.scr.2014.02.002
PMCID: PMC4075444  PMID: 24603366
Embryonic stem cells; induced pluripotent stem cells; cell culture; suspension culture; non-colony type monolayer; single cell; embryoid body; epithelial-to-mesenchymal transition; heterogeneity; chromosomal instability
12.  Human Pluripotent Stem Cell Culture: Considerations for Maintenance, Expansion, and Therapeutics 
Cell stem cell  2014;14(1):13-26.
Summary
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, three-dimensional environmental (3D) cues and modes of multicellular association. In this 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.
doi:10.1016/j.stem.2013.12.005
PMCID: PMC3915741  PMID: 24388173
Human embryonic stem cells; induced pluripotent stem cells; cell culture; expansion; differentiation; regenerative medicine
13.  Directed Differentiation of Human Induced Pluripotent Stem Cells Toward Bone and Cartilage: In Vitro Versus In Vivo Assays 
The ability to generate large numbers of bone- and cartilage-forming cells from induced pluripotent stem cells (iPSCs) would mark a major advance in tissue engineering. A number of protocols exist, but the overall quality and consistency of this type of differentiation are under-reported. In this study, the authors analyzed differentiated iPSCs in vitro and in vivo by stringent criteria, and found that in vitro analysis does not predict in vivo differentiation.
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.
doi:10.5966/sctm.2013-0154
PMCID: PMC4073820  PMID: 24855277
Induced pluripotent stem cells; Bone; Osteoblast; Chondrogenesis; Transplantation
14.  StemCellDB: The Human Pluripotent Stem Cell Database at the National Institutes of Health 
Stem cell research  2012;10(1):57-66.
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.
doi:10.1016/j.scr.2012.09.002
PMCID: PMC3590800  PMID: 23117585
15.  Non-colony type monolayer culture of human embryonic stem cells 
Stem cell research  2012;9(3):237-248.
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.
doi:10.1016/j.scr.2012.06.003
PMCID: PMC3490040  PMID: 22910561
16.  Regulation and Expression of the ATP-binding Cassette Transporter ABCG2 in Human Embryonic Stem Cells 
Stem cells (Dayton, Ohio)  2012;30(10):2175-2187.
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 SSEA-1 of hESCs, following constant BMP-4 signaling at days 4 and 6. This expression was tightly correlated with the down-regulation of two microRNAs (i.e., hsa-miR-519c and hsa-miR-520h). Transfection of microRNA mimics and inhibitors of these two microRNAs 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 microRNAs in hESCs.
doi:10.1002/stem.1195
PMCID: PMC3480739  PMID: 22887864
human embryonic stem cells; ATP-binding cassette; ABCG2; BMP-4; differentiation
17.  Loss of STAT3 signaling during elevated activity causes vulnerability in hippocampal neurons 
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 knockout 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 to a mechanism by which chronic elevation of activity may cause neurodegeneration.
doi:10.1523/JNEUROSCI.2940-12.2012
PMCID: PMC3504487  PMID: 23115188
18.  In the newborn hippocampus, neurotrophin-dependent survival requires spontaneous activity and integrin signaling 
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 post-natal life. Neurotrophins are widely thought to regulate neuronal survival but this role has not been clearly defined in the central nervous system. 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 γ-aminobutyric acid (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.
doi:10.1523/JNEUROSCI.0202-11.2011
PMCID: PMC3500851  PMID: 21613492
19.  Toward xeno-free culture of human embryonic stem cells 
The culture of human embryonic stem cells (hESCs) is limited, both technically and with respect to clinical potential, by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. The concern over xenogeneic contaminants from the mouse feeder cells may restrict transplantation to humans and the variability in MEFs from batch-to-batch and laboratory-to-laboratory may contribute to some of the variability in experimental results. Finally, use of any feeder layer increases the work load and subsequently limits the large-scale culture of human ES cells. Thus, the development of feeder-free cultures will allow more reproducible culture conditions, facilitate scale-up and potentiate the clinical use of cells differentiated from hESC cultures. In this review, we describe various methods tested to culture cells in the absence of MEF feeder layers and other advances in eliminating xenogeneic products from the culture system.
doi:10.1016/j.biocel.2005.12.014
PMCID: PMC3449300  PMID: 16469522
Human embryonic stem cells; MEF; Feeder-free cell culture; Matrigel
20.  Isolation of epiblast stem cells from pre-implantation mouse embryos 
Cell stem cell  2011;8(3):318-325.
SUMMARY
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 pre-implantation-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.
doi:10.1016/j.stem.2011.01.016
PMCID: PMC3073125  PMID: 21362571
epiblast stem cells; pluripotency; embryonic stem cells; blastocyst
21.  Genetic variation in FGF20 modulates hippocampal biology 
We explored the effect of Single Nucleotide Polymorphisms (SNPs) in the Fibroblast Growth Factor 20 gene (FGF20) associated with risk for Parkinson’s disease (PD) on brain structure and function in a large sample of healthy young-adult human subjects and also in elderly subjects to look at the interaction between genetic variations and age (N = 237, 116 men, 18–87 years). We analyzed high resolution anatomical magnetic resonance images using voxel-based morphometry, a quantitative neuroanatomical technique. We also measured FGF20 mRNA expression in post-mortem human brain tissue to determine the molecular correlates of these SNPs (N = 108, 72 men, 18–74 years). We found that the T allele carriers of rs12720208 in the 3’ UTR had relatively larger hippocampal volume (p = 0.0059), diminished verbal episodic memory (p = 0.048) and showed steeper decreases of hippocampal volume with normal ageing (p = 0.026). In post-mortem brain, T allele carriers had greater expression of hippocampal FGF20 mRNA (p = 0.037), consistent with a previously characterized microRNA mechanism. The C allele matches a predicted miR-433 microRNA binding domain, whereas the T allele disrupts it, resulting in higher FGF20 protein translation. The strong FGF20 genetic effects in hippocampus are presumably mediated by activation of the FGF receptor 1 (FGFR1), which is expressed in mammalian brain most abundantly in the hippocampus. These associations, from mRNA expression to brain morphology to cognition and an interaction with ageing, confirm a role of FGF20 in human brain structure and function during development and aging.
doi:10.1523/JNEUROSCI.5773-09.2010
PMCID: PMC2909689  PMID: 20427658
FGF20; Genetic; Voxel-Based Morphometry; Neuroimaging; MRI; Hippocampus
22.  Cholera Toxin Regulates a Signaling Pathway Critical for the Expansion of Neural Stem Cell Cultures from the Fetal and Adult Rodent Brains 
PLoS ONE  2010;5(5):e10841.
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.
doi:10.1371/journal.pone.0010841
PMCID: PMC2877108  PMID: 20520777
23.  Angiogenic Factors Stimulate Growth of Adult Neural Stem Cells 
PLoS ONE  2010;5(2):e9414.
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 [1], [2], [3], [4], [5], [6].
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.
doi:10.1371/journal.pone.0009414
PMCID: PMC2829079  PMID: 20195471
24.  Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells 
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.
doi:10.1007/s11626-010-9297-z
PMCID: PMC2855804  PMID: 20186512
Human embryonic stem cell; Cell culture; Defined cell culture media; Comparative study
25.  A novel blastocyst-derived stem cell line reveals an active role for growth factor signaling in the induction of stem cell pluripotency 
Cell  2008;135(3):449-461.
SUMMARY
Pluripotent stem cell lines can be derived from blastocyst embryos, which yield embryonic stem cell lines (ES cells), as well as the post-implantation epiblast, which gives rise to epiblast stem cell lines (EpiSCs). Remarkably, ES cells and EpiSCs display profound differences in the combination of growth factors that maintain their pluripotent state. Molecular and functional differences between these two stem cell types demonstrate that the tissue of origin and/or the growth factor milieu may be important determinants of the stem cell identity. We explored how developmental stage of the tissue of origin and culture growth factor conditions affect the stem cell pluripotent state. Our findings reveal that novel stem cell lines can be generated from blastocyst embryos with unique functional and molecular properties. We demonstrate that the culture growth factor environment and cell-cell interaction play a critical role in defining several unique and stable stem cell ground states.
doi:10.1016/j.cell.2008.08.035
PMCID: PMC2767270  PMID: 18984157

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