Human Molecular Genetics

Human Molecular Genetics 2008 17(R1):R67-R75; doi:10.1093/hmg/ddn065

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Multiple layers of molecular controls modulate self-renewal and neuronal lineage specification of embryonic stem cells

Gene W. Yeo^1,2,, Nicole Coufal^1,, Stefan Aigner¹, Beate Winner¹, Jonathan A. Scolnick¹, Maria C.N. Marchetto¹, Alysson R. Muotri¹, Christian Carson³ and Fred H. Gage^1,*

¹ Laboratory of Genetics ² Crick-Jacobs Center for Computational and Theoretical Biology, Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA ³ BD Biosciences, 11077 North Torrey Pines Road, La Jolla, CA 92037, USA

^* To whom correspondence should be addressed. Email: gage{at}salk.edu

Received February 13, 2008; Revised February 13, 2008; Accepted February 28, 2008

	ABSTRACT

TOP ABSTRACT THE IMPORTANCE OF STEM... CURRENT MODELS FOR NEURONAL... EPIGENETICS OF STEM CELL... MICRORNAS MODULATE PROLIFERATION... GENE EXPRESSION AND STEM... POSTTRANSLATIONAL CHANGES IN... PERSPECTIVES FUNDING REFERENCES

 
Elucidating the molecular changes that arise during neural differentiation and fate specification is crucial for building accurate in vitro models of neurodegenerative diseases using human embryonic stem cells (hESCs). Here we review the importance of hESCs and derived progenitors in treating and modeling neurological diseases, and summarize the current efforts for the differentiation of hESCs into neural progenitors and defined neurons. We recapitulate the recent findings and discuss open questions on aspects of molecular control of gene expression by chromatin modification and methylation, posttranscriptional regulation by alternative splicing and the action of microRNAs, and protein modification. An integrative view of the different levels will hopefully provide much needed insight into understanding stem cell biology.

	THE IMPORTANCE OF STEM CELLS IN NEUROLOGICAL DISEASES

TOP ABSTRACT THE IMPORTANCE OF STEM... CURRENT MODELS FOR NEURONAL... EPIGENETICS OF STEM CELL... MICRORNAS MODULATE PROLIFERATION... GENE EXPRESSION AND STEM... POSTTRANSLATIONAL CHANGES IN... PERSPECTIVES FUNDING REFERENCES

 
Human neurological diseases are complex and often difficult to model in vitro, and rodent models are frequently genetically irrelevant. The hallmarks of several neurodegenerative disorders, such as Parkinson’s disease (PD), Huntington’s disease (HD) and Alzheimer’s disease (AD), are slow progressive losses of specific neuronal populations in the brain. So far only symptomatic therapeutic approaches are available, making these diseases potential candidates for restorative therapeutic approaches. Therefore, major research efforts have focused on cell transplantation of hESCs, derived neural progenitors and/or neural stem cells (NSCs) to restore depleted diseased cells.

For instance, motor deficits in PD are due to the progressive degeneration of the dopaminergic neurons (DAs) of the substantia nigra pars compacta resulting in decreased dopamine release into the striatum. Transplantation strategies aiming toward rectifying the striatal dopamine deficit conducted in PD patients provided the proof of principle that transplanted neurons can elicit beneficial effects. However, difficulty in obtaining cells suitable for transplantation reduces the utility of the transplantation approach. More recently, there has been an intense effort to use human embryonic stem cell (hESC)-derived neural progenitor cells (NPCs) for transplantation. These cells can be grown in large numbers from a single source to help control for variations in source tissue. It may also be possible to differentiate the cells in vitro in such a way as to avoid the problems of fetal cell transplants (e.g. uncontrolled dopamine release, ethical concerns). However, many barriers still need to be overcome before hESC-derived neural progenitors can be considered safe for transplantation into humans. For example, Roy et al. showed that hESC-derived neural progenitors may be tumorigenic in vivo (1). In fact, hESC-derived neurons will be more beneficial as an in vitro model system in which diseases such as PD can be studied.

Induced pluripotent stem cells (iPSCs) offer a potentially new direction for the study and treatment of neurodegenerative diseases. Several groups recently demonstrated that overexpression of a combination of several genes, including OCT4, SOX2, and some combination of Klf4, Lin28 and c-Myc in human fibroblasts leads to the creation of pluripotent embryonic stem-like cells that can contribute to all cell lineages (2,3). In principle, Human iPSCs can be generated from an individual affected by a neurodegenerative disease, allowing researchers to produce neurons in vitro that contain the same genetic makeup as the patient. These patient-specific neurons will provide a useful resource for understanding the genetic-basis for sporadic cases of neurodegeneration.

	CURRENT MODELS FOR NEURONAL DIFFERENTIATION OF HESCS

TOP ABSTRACT THE IMPORTANCE OF STEM... CURRENT MODELS FOR NEURONAL... EPIGENETICS OF STEM CELL... MICRORNAS MODULATE PROLIFERATION... GENE EXPRESSION AND STEM... POSTTRANSLATIONAL CHANGES IN... PERSPECTIVES FUNDING REFERENCES

 
Accurate differentiation of hESCs (or iPSCs) into NSCs will be crucial for in vitro modeling and treatment. Figure 1 summarizes the current and future efforts in neural specification and enrichment from hESCs. There are three major approaches for differentiating hESCs into NSCs: co-culturing hESCs with a stromal feeder layer such as PA6 (4) and MS5 (5); directly differentiating hESC colonies in culture by addition of the bone morphogenetic protein (BMP) antagonist Noggin (6,7); and isolating NSC-containing neuroectoderm from embryoid bodies (EBs) (8). Researchers have developed methods to promote the neuroectoderm population in EBs including incubating EBs in a defined neural promoting media (8), in conditioned media from stromal HepG2 cells (9) or in the presence of Noggin (7,10–12). With the exception of complete neural differentiation by co-culture on PA6 cells, the majority of these methods converge at the formation of neural rosettes (4). Rosettes consist of radial arrangements of columnar cells that express many markers also found in the developing neural tube. Rosettes can be manually dissected and plated to form NSCs, which can be propagated as neuropheres (8) or as adherent cultures (12) for multiple passages before further differentiation into neurons and glia. Moreover, engraftment of hESC-derived NSCs into rodents can result in functional neuronal integration (8).

View larger version (34K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1. Derivation and differentiation of human embryonic stem cells to a neuronal lineage. Traditionally, hESCs are derived by culturing the inner cell mass of a blastocyst, although recent evidence suggests overexpression of subset of key transcription factors can also result in hES-type cells. Neuronal differentiation of hESCs is achieved through either co-culture with mouse PA6 cells or embryoid body (EB) formation resulting in neural rosettes which are manually dissected. Neuronal precursor cells derived from rosettes can be enriched through sorting, used for transplantation studies, or further differentiated in vitro.

 
Directed differentiation of hESCs into specific functional neuronal subtypes is more complex than specification of NSCs alone and requires mimicking the in vivo development of each unique neuronal subtype. Many laboratories are focusing on generating two specific neuronal subtypes, DAs, which could be useful for furthering the understanding and treatment of PD, and motor neurons expressing choline acetyltransferase, which could be used in the study and treatment of amylotrophic lateral sclerosis (also known as Lou Gehrig’s disease) and spinal cord injury. Both differentiation schemes employ defined signaling molecules to specifically pattern the cells. Sonic Hedgehog (SHH) and FGF8 (13) or Noggin (14,15) have been used to pattern cells toward the dopaminergic subtype, and a recent study employed the co-culture of an immortalized human fetal midbrain astrocyte cell line, in addition to signaling molecules to significantly enhance the differentiation (1). Similarly, SHH and retinoic acid (RA) have been used to promote motor neuron differentiation (16,17). Importantly, many groups have demonstrated that hESC-derived DA neurons and motor neurons are functional both in vitro and in vivo. Differentiated DA neurons have been shown to fire action potentials and release dopamine upon depolarization in vitro (13) and have been shown to survive transplantation and engraft in rodent models of PD (1,15,18). Differentiated motor neurons exhibit spontaneous action potentials and make contacts with co-cultured myotubes in vitro (17), and transplantation experiments of motor neuron progeny into developing chick embryo and adult rat spinal cord have resulted in robust engraftments (16). In addition to these two neural subtypes, the field will most certainly broaden its repertoire of differentiation methods to include the plethora of neural subtypes that exist. Already, advances have been reported in the differentiation of peripheral sensory neurons (19) and neural crest fates (16).

The future of hESC or iPSC neuronal differentiation lies in improving the relative abundance of the desired neuronal subtype within the total cell population and by removing nonspecific contaminating cell types and remaining proliferative cells from postmitotic neuronal cultures, thereby obtaining near-pure populations for transplantation experiments. These goals have been successfully pursued by fluorescent-activated cell sorting of the cell population, using subtype-specific promoters driving fluorescent reporter proteins and by antibody staining for subtype-specific cell surface markers. For instance, by utilizing an HB9 promoter green fluorescent protein (GFP) construct, Soundararajan et al. (20) enriched for putative motor neuron precursors. They subsequently showed that these HB9-positive cells engrafted into the chick neural tube and exhibited proper migration to the medial motor column, projected axons to the appropriate target muscles and showed synaptic activity that resembled that of endogenous motor neurons. A synapsin promoter-driving GFP has also been used to successfully isolate neurons from a diverse cell population (21). Similarly, the research community is just beginning to appreciate the utility of the cell surface marker profile of different NPC populations and subtype-specific cells. For example, cell surface markers such as CD133, 5E12, CD34, CD45 and CD24/lo have been employed to enrich for neurosphere-forming cells from fetal brain (22). Similarly, SSEA-4 and prominin-1/CD133 have been shown to demarcate fetal NSCs from human brain (23,24).

An important consequence of furthering the purity and differentiation specificity of hESCs has been the opportunity to systematically study in a genome-wide fashion the molecular pathways governing self-renewal and lineage specification and differentiation.

	EPIGENETICS OF STEM CELL BIOLOGY: CHROMATIN AND METHYLATION

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Aside from the key transcription factors, such as Oct4, Nanog and Sox2, which are necessary and sufficient for specifying self-renewal in hESCs (25), epigenetic changes mediated by other factors also play important roles in maintaining a pluripotent state. For example, Polycomb group (PcG) proteins are transcriptional repressors that modify chromatin structure and have been implicated in hESC pluripotency (26). PcG proteins can act in two distinct complexes, PRC1 and PRC2, to repress gene expression. To identify target genes in murine embryonic stem cells, Boyer et al. performed genome-wide location analysis using antibodies against core components of PRC1 (phc1 and Rnf2) and PRC2 (Suz12 and Eed) (27). From these studies, the view emerged that PRC2 initiates transcriptional repression by inhibiting initiation, whereas the PRC1 complex maintains the repressive conditions. Mutations in the components of PRCs eliminate the pluripotent ability of mES cells, leading to embryonic lethality (28,29).

Attesting to the importance of PRC1 and PRC2 in controlling gene expression in mESCs, 90% of genomic sequences detected in chromatin immunoprecipitation (ChIP) experiments using antibodies against PRC1 and PRC2 components were within 1 kb of a transcription start site, making these complexes likely regulators of these genes. PRC1 and PRC2 components overlap in a subset of these genes, many of which encode transcription factors with important roles in a variety of developmental processes. Also, all these co-occupied genes are highly enriched for histone H3 trimethylated at lysine 27 (H3K27me3), a marker strongly associated with transcriptionally repressed chromatin (30). Since H3K27 trimethylation is catalyzed by PcG proteins (26), it is likely that this mark is deposited at these promoters by recruited PcG proteins. Upon differentiation of mESCs, 93% of PcG target transcripts were preferentially upregulated. Specifically, the differentiation of mESCs cells toward NPCs increased the expression of neuron-specific genes, whereas the loss of H3K27me3 was concomitant with an increase in RNA polymerase II occupancy and H3K4me3, a histone modification associated with active transcription that is created by trithorax-group (trxG) proteins (26).

These data suggest that PcG proteins have specialized roles in silencing genes at the embryonic stage that correlate with differentiation and loss of pluripotency but maintain the potential to become activated upon lineage commitment (31,32). In this new model, pluripotent cells can keep important tissue-specific regulator genes poised for expression by an opposing histone modification. The fact that PcG proteins can repress genes that are poised for activation indicates that the dynamic role of PcG complexes seems to be different from other irreversible epigenetic mechanisms, such as DNA methylation. Oct4, Sox2 and Nanog, the three key pluripotency transcription factors in hESCs (33), can bind many genes identified as PcG targets. Moreover, PcG proteins can target a similar set of developmental regulators in hESCs (34). These data suggest that at least for a subset of genes, PcG proteins can act as transcriptional repressors by collaborating with a specific set of transcriptional factors.

Recently, Mikkelsen et al. (35) used ChIP for specific histone modifications coupled with high-throughput sequencing technology to generate genome-wide maps of chromatin state from mESCs and lineage-committed cells. The authors used H3K27me3 and H3K4me3 antibodies to study 17, 762 promoter regions, discriminating between CpG-rich promoters (associated with both ubiquitously expressed housekeeping genes, and genes with more complex expression patterns), CpG-poor promoters (generally associated with highly tissue-specific genes) and promoters with intermediate CpG content. In mESCs cells, CpG-rich promoters are associated with active chromatin, as judged by the presence of the H3K4me3 mark. However, around 22% of these promoters are bivalent, exhibiting histone markers of both active (H3K4me3) and repressed (H3K27me3) chromatin. These genes show low transcriptional levels, suggesting that the repressive effect of PcG activity can be dominant over the TrxG activity. Most promoters marked with H3K4me3 alone retain this mark upon neural differentiation while about half of the promoters with bivalent marks resolve to H3K4me3, concomitant with an increase in gene expression. Conversely, CpG-poor promoters behave quite differently. In mESCs, only 6.5% have significant H3K4me3 marks and virtually none showed enrichment for H3K27me3. In neuronally derived cells, most of these promoters lost the H3K4me3 mark, whereas 1.5% gained the mark. In both situations mESCs and neuron-derived cells—the expression levels of the associated genes—strongly correlated with the presence or absence of H3K4me3. Altogether, these findings suggest that CpG-poor promoters may be selectively activated by cell-type or tissue-specific factors. In fact, it was shown that in embryonic stem cells, windows of unmethylated CpG dinucleotides and possible interacting factors mark enhancers for tissue-specific genes (36).

While these studies have been performed in mESCs, it is likely that hESCs are highly dependent on similar modes of epigenetic regulation. Not only can genome-wide maps of chromatin-state provide a rich source of information about different stages of development, they also reveal stem cell- and lineage-specific epigenomic signatures that will ultimately reveal how a single genome produces such a diversity of cell types.

	MICRORNAS MODULATE PROLIFERATION AND DIFFERENTIATION

TOP ABSTRACT THE IMPORTANCE OF STEM... CURRENT MODELS FOR NEURONAL... EPIGENETICS OF STEM CELL... MICRORNAS MODULATE PROLIFERATION... GENE EXPRESSION AND STEM... POSTTRANSLATIONAL CHANGES IN... PERSPECTIVES FUNDING REFERENCES

 
Small non-coding RNAs may act as an additional layer controlling the gene expression patterns required for maintaining the pluripotent state of stem cells on the one hand and promoting lineage-specific differentiation on the other. MicroRNAs represent the most abundant class of functional small RNAs in mammalian cells (37) and are also the best understood. These 19- to 23-nucleotide RNAs are transcribed embedded in long primary transcripts, from which they are released in a multi-step processing pathway (38). Upon incorporation of the mature microRNAs into the effector complex, they negatively regulate gene expression by recognizing mRNAs through partial sequence complimentarity within mRNA 3' untranslated regions, thereby targeting these transcripts for degradation, destabilization and/or repression of productive translation (39).

Experimental evidence indicates that a single microRNA can directly target at least 100 distinct mRNA species (40,41). Progress in determining the functional targets of microRNAs has largely been driven by improvements in the design and delivery of microRNA mimics and antagonists to cultured cells and in live animals (42–44). For instance, the microRNA paralogs miR-125a and miR-125b are dramatically upregulated during RA-induced neuronal differentiation of P19 mouse embryonic carcinoma cells, and they have been shown to directly target and downregulate the product of the lin-28 gene, a regulator of developmental timing (45). The brain-enriched microRNA miR-124a targets the laminin gamma 1 and integrin beta 1 mRNAs, and ectopic expression of miR-124a in chick embryo causes defects in the basal lamina of the developing neural tube (46). Similarly, another brain-enriched microRNA, miR-134, has been shown to be involved in control of spine development via downregulating expression of the protein kinase Limk1 (47). These examples illustrate the profound yet specific effects that microRNAs exert on central biological processes.

Several lines of evidence from mouse embryonic stem cell studies indicate that the microRNA pathway is critical both for stem cell self-renewal and differentiation in general and for neurogenesis in particular. Although mESCs deleted for either of the two RNase enzymes essential for microRNA maturation are viable and do not lose their capacity to generate embryonic stem cell colonies, the knock-out cells display proliferation defects (48–50). Despite the differences in embryonic stem cell maintenance regulation between mouse and human (51), it is likely that stem cell maintenance in humans is similarly dependent on microRNAs. This view is supported by the fact that many stem cell enriched microRNAs that have been found by cloning in mouse (52) and human (53) embryonic stem cells are highly related to each other in sequence, suggesting that they have similar mRNA targets. In humans, this hESC-enriched microRNA family consists of miR-93, miR-302a-d, miR-371, miR-372, miR-373 and miR-520a-h. Since miR-302a-d are also found expressed highly in embryonic carcinoma cells (53), only miR-371, miR-372 and miR-373 appear specific to normal hESCs cells. While no functional studies of these microRNAs have been conducted in hESCs cells, the results from primary human somatic and cancer cells point to a role for miR-372 and miR-373 in allowing cells to proliferate by promoting the bypass of the G1/S cell cycle checkpoint (54). A similar role for microRNA-dependent regulation has been observed for stem cell division in Drosophila (55), attesting to the evolutionary conservation of their role in stem cell maintenance.

In mESCs, several individual microRNAs have recently been shown to be able to promote and direct neurogenesis at all stages. For example, over-expression of mir-134, which is highly expressed in the adult CNS, boosts mESC differentiation toward the ectodermal lineage, even in the presence of the stem cell maintenance factor LIF (56). Interestingly, miR-134 may exert this effect in part by directly downregulating the Oct4 activators Nanog and LRH1 (56). Modulation of the relative levels of functional miR-124a and miR-9—both brain-enriched microRNAs—during differentiation of mESCs-derived neural progenitors alters the ratio of cells of the glial versus neuronal lineages, perhaps by acting via the STAT3 pathway (57). Lastly, miR-133b, a midbrain-enriched microRNA, negatively regulates the maturation and function of mES-derived DAs (42). It appears that miR-133b directly targets the transcription factor Pitx3, an activator of tyrosine hydroxylase, the enzyme catalyzing the rate-limiting step in dopamine (42). In agreement with the notion that microRNAs may regulate neuronal subtype specification, recent microRNA profiling of hESC lines revealed cell line-specific intrinsic biases toward particular neuronal lineages, which were accompanied by distinct microRNA expression patterns (58,59).

Thus, although we are still awaiting experimental proof, it seems reasonable to hypothesize that the manipulation of cellular levels of specific microRNAs may, in the near future, become a viable option to improve upon—and perhaps even substitute for—current differentiation protocols for hESCs. Such an approach would likely entail the dosed delivery of agonists and inhibitors of sets of microRNAs at several stages during the differentiation procedure.

	GENE EXPRESSION AND STEM CELL TRANSCRIPTOME DIVERSITY

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Genome-wide expression profiling provides a basis to examine changes of genes and pathways in response to hESC differentiation. Several groups have utilized high-throughput profiling methods such as microarrays, expressed sequence tags (ESTs), serial analysis of gene expression, massively parallel signature sequencing profiling (MPSS) and RT-PCR methods to compare transcriptional profiles in different hESC lines and to study expression changes during the differentiation of hESCs to various lineages (60–64). By studying ESTs, Brandenberger et al. (63) identified 32 000 unique transcripts as being expressed in undifferentiated hESCs, among them 500 were significantly upregulated, and 150 were downregulated. Wright et al. (65) identified ‘expressed’ and ‘not expressed’ genes in NPCs isolated from the human embryonic cortex; Cai et al. (66) used the MPSS technique to analyze expression of fetal NPCs in comparison to hESCs and astrocyte precursors; Maisel et al. (67) used Affymetrix Gene Chip arrays to compare adult and fetal NPCs propagated as neurospheres. Using SAGE and MPSS, Richards et al. (68) and Miura et al. (62) identified 190 or 50 genes that were upregulated in hESCs compared with differentiated cells. Some genes were often found in different expression datasets, which included Oct4, Sox2 and Nanog, and were considered to be molecular signatures of ESCs.

Apart from transcriptional changes, co- or posttranscriptional mRNA regulation also occurs through alternative splicing (AS), whereby more than one transcript isoform of a gene is produced. In the case of stem cells, AS generates different transcripts at different stages of differentiation and frequently contributes to the regulation of gene expression by generating tissue-specific mRNA and protein isoforms (69–72). Underscoring the importance of AS in gene regulation, recent studies using splicing-sensitive microarrays suggested that up to 75% of human genes undergo AS (73). Hence, it is not surprising that AS plays important roles in regulating neuronal gene expression and function (74,75).

A number of regulatory proteins are likely involved in regulating AS. These proteins are in the spliceosome particles, the SR protein family, the heterogeneous nuclear ribonucleoproteins, as well as auxiliary splicing factors, typically RNA-binding proteins (76). These splicing factors bind to short, degenerate cis-regulatory elements located in the exonic and intronic regions of AS exons regulate splicing choices, and disruptions in these elements lead to genetic diseases (77). While almost nothing is known about which splicing factors are important for AS regulation in human or mouse ESCs, several factors have been implicated in being important in regulating neuronal differentiation and maturation. Recently, three publications demonstrate that the polypyrimidine tract-binding protein (PTB/PTBP1) is critical in keeping non-neuronal cells from differentiating into neurons (78–80). PTB is known as a splicing repressor of neuron-specific exon usage in a myriad of pre-mRNAs (81). The three studies showed that knocking down PTB protein is sufficient to trigger neuronal-specific AS in non-neuronal cells. Interestingly, neurons express a paralog of PTB, nPTB (PTBP2), which acts as a weaker splicing repressor compared with PTB; and PTB and nPTB are expressed in a mutually exclusive fashion. PTB directly represses nPTB expression in non-neuronal cells by preventing exon 10 inclusion in nPTB, which introduces a premature translation termination codon, thereby degrading nPTB mRNA via the nonsense-mediated pathway. To answer why and how PTB is excluded from neurons, Makeyev et al. (79) show that a neuronal-enriched microRNA (mir-124) directly targets the 3' untranslated region of PTB, silences PTB expression, and does not induce but promotes neuronal differentiation of mouse P19 cells. It is an open question whether these same regulatory networks pertain to hESCs, a non-neuronal cell and differentiated NSCs.

The splicing field is currently in a cataloguing phase of attempting to identify all isoforms in various tissues and cell lines. To our knowledge, AS has not been implicated in stem cell biology, until recently—a newly identified isoform of FGF4, FGF4si counters the growth-promoting effects of FGF4 (82). Interestingly, while FGF4 ceases to be expressed in late differentiated cells, FGF4si does not. It is still unclear which splicing factor regulates the generation of the FGFsi isoform. To systematically identify AS isoforms, two approaches have been taken, namely ESTs and microarrays. Pritsker et al. (83) identified alternatively spliced isoforms of thousands of genes, using ESTs derived from embryonic and hematopoietic stem cells. Genes identified as producing splice variants showed significant enrichment for those encoding components of signaling pathways, as well those involved in stem cell self-renewal and differentiation. Using splicing-sensitive microarrays, Yeo et al. identified and characterized AS events that distinguish pluripotent hESCs from NPCs, in order to pave the way for future candidate gene approaches to study the impact of AS in hESCs and NPCs. REAP, a regression-based method for analyzing exon array data, was introduced and was applied to discover AS events in hESCs, compared with NPCs derived from hESC (12). REAP detected more than 1000 AS events. Interestingly, the study showed that only a minority of AS events was common between various hESC-to-NPC comparisons. A possible explanation is that the cell lines were not only genetically different but were also exposed to different isolation and culture conditions. It is also possible that posttranscriptional changes such as AS may be more variable despite the cells being at acknowledged ‘end-points’ defined by a limited set of immunohistochemical markers. However, this suggests that utilizing differences in AS as a molecular signature of various degrees of differentiation may be far more sensitive and accurate than simply gene expression. These insights clearly highlight the previously underappreciated complexity in gene regulation by AS in stem cell and neuronal differentiation.

	POSTTRANSLATIONAL CHANGES IN STEM CELL DIFFERENTIATION

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Although patterns of gene expression and protein activity are crucial for biological processes, many of the interesting regulatory steps, particularly involved in cell proliferation, migration and differentiation, are likely to depend on proteins’ posttranslational modifications rather than expression. However, there is a dearth of literature on the importance of posttranslational in stem cell biology. A recent example, Zhang et al. (84) showed that Oct-4, which plays an important role in maintaining self-renewal in embryonic stem cells, can be modulated posttranslationally by SUMO. This underscores the need for proteomics in regulating the posttranslational modifications regulating embryonic stem cell biology. For example, protein phosphorylation can alter the interactions between the phosphorylated protein and the rest of the proteome. Phosphoproteome analysis performed on mESCs (85) found that many chromatin remodeling proteins are differentially regulated by phosphorylation in embryonic stem cells compared with differentiated cells. Because the activity of kinases and phophatases can be regulated by small molecules, identifying the phosphoproteome will present an excellent opportunity to identify small molecule chemical regulators of pluripotency or directed differentiation of hESCs.

	PERSPECTIVES

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The diversity of regulatory processing at multiple layers during embryonic stem cell differentiation suggests many opportunities for intervention to promote self-renewal or directed neural differentiation. Figure 2 presents a summary of the layers discussed in this review. For example, small molecules have been shown to affect the epigenetic regulation of gene expression (DNA methylation and histone modifications) which affects stem cell proliferation and differentiation (86–88). RNA interference (RNAi) can also be utilized to silence genes and reveal their potential roles in proliferation and differentiation (89–91). Endogenous microRNAs may also be utilized to control proliferation and differentiation into specific lineages. For example, mir-133b has been shown to regulate the maturation and function of midbrain DAs in mammals (42). However, our overview of the recent advances in these aspects of molecular control of stem cell biology reveals more questions than answers, and illuminates significant gaps in our understanding, and much room for future work. We end with two broad future goals for the fields: (i) determining how epigenetic signatures relate to transcriptional changes in protein-coding and miRNA-coding genes will be important for understanding stem cell biology (92); (ii) with the diversity of neural subtypes that can be generated from hESCs, it is a matter of time before we see more crossings between AS, microRNAs and posttranslational medications as modes of biological richness.

View larger version (42K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 2. Molecular regulation of stem cells. Regulation of biological processes in hESCs occurs at multiple levels such as epigenetic changes (methylation and acetylation), regulation of alternative splicing and through post-translational modifications such as phosphorylation, through the action of microRNAs which can lead to the translational repression and/or degradation of target mRNAs.

 

	FUNDING

TOP ABSTRACT THE IMPORTANCE OF STEM... CURRENT MODELS FOR NEURONAL... EPIGENETICS OF STEM CELL... MICRORNAS MODULATE PROLIFERATION... GENE EXPRESSION AND STEM... POSTTRANSLATIONAL CHANGES IN... PERSPECTIVES FUNDING REFERENCES

 
G.W.Y. is a Junior Fellow at the Crick-Jacobs Center of Computational and Theoretical Biology. B.W. is a Feodor-Lynnen fellow of the Alexander von Humboldt Foundation. S.A. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-1859-05). A.R.M. is supported by the Rett Syndrome Research Foundation. M.C.N.M. is supported by the George E. Hewitt Foundation for Medical Research. J.A.S. is supported by the California Institute of Regenerative Medicine. N.C. is supported by the Lookout Fund. F.H.G. is supported by the Lookout Fund and the National Institutes of Health; National Institute on Aging and National Institute of Neurological Disease and Stroke.

	Acknowledgements

 
The authors would like to thank Jamie Simon for figure preparation.

Conflict of Interest statement. None declared.

	FOOTNOTES

 
These authors contributed equally to this work.

	REFERENCES

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