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The future of safe cell-based therapy rests on overcoming teratoma/ tumor formation, in particular when using human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Because the presence of a few remaining undifferentiated hPSCs can cause undesirable teratomas after transplantation, complete removal of these cells with no/minimal damage to differentiated cells is a prerequisite for clinical application of hPSC-based therapy. Having identified a unique hESC signature of pro-and antiapoptotic gene expression profile, we hypothesized that targeting hPSC-specific antiapoptotic factor(s) (i.e., survivin or Bcl10) represents an efficient strategy to selectively eliminate pluripotent cells with teratoma potential. Here we report the successful identification of small molecules that can effectively inhibit these antiapoptotic factors, leading to selective and efficient removal of pluripotent stem cells through apoptotic cell death. In particular, a single treatment of hESC-derived mixed population with chemical inhibitors of survivin (e.g., quercetin or YM155) induced selective and complete cell death of undifferentiated hPSCs. In contrast, differentiated cell types (e.g., dopamine neurons and smooth-muscle cells) derived from hPSCs survived well and maintained their functionality. We found that quercetin-induced selective cell death is caused by mitochondrial accumulation of p53 and is sufficient to prevent teratoma formation after transplantation of hESC-or hiPSC-derived cells. Taken together, these results provide the "proof of concept" that small-molecule targeting of hPSC-specific antiapoptotic pathway(s) is a viable strategy to prevent tumor formation by selectively eliminating remaining undifferentiated pluripotent cells for safe hPSC-based therapy.

Somaclonal variation is manifested as cytological abnormalities, frequent qualitative and quantitative phenotypic mutation, sequence change, and gene activation and silencing. Activation of quiescent transposable elements and retrotransposons indicate that epigenetic changes occur through the culture process. Epigenetic activation of DNA elements further suggests that epigenetic changes may also be involved in cytogenetic instability through modification of heterochromatin, and as a basis of phenotypic variation through the modulation of gene function. The observation that DNA methylation patterns are highly variable among regenerated plants and their progeny provides evidence that DNA modifications are less stable in culture than in seed-grown plants. Future research will determine the relative importance of epigenetic versus sequence or chromosome variation in conditioning somaclonal variation in plants.

Intracellular Vitamin C (VC) is maintained at high levels in the developing brain by the activity of sodium-dependent VC transporter 2 (Svct2), suggesting specific VC functions in brain development. A role of VC as a cofactor for Fe(II)-2-oxoglutarate-dependent dioxygenases has recently been suggested. We show that VC supplementation in neural stem cell (NSC) cultures derived from embryonic midbrains greatly enhanced differentiation towards midbrain-type DA (mDA) neurons, the neuronal subtype associated with Parkinson’s disease. VC induced gain of 5-hydroxymethylcytosine (5hmC) and loss of H3K27m3 in DA phenotype gene promoters, which are catalyzed by Tet1 and Jmjd3, respectively. Consequently VC enhanced DA phenotype gene transcriptions in the progenitors by Nurr1, a transcription factor critical for mDA neuron development, to be more accessible to the gene promoters. Further mechanism studies including Tet1 and Jmjd3 knockdown/inhibition experiments revealed that both the 5hmC and H3K27m3 changes, specifically in the progenitor cells, are indispensible for the VC-mediated mDA neuron differentiation. We finally show that in Svct2 knockout mouse embryos, mDA neuron formation in the developing midbrain decreased along with the 5hmC/ H3k27m3 changes. These findings together indicate an epigenetic role of VC in midbrain DA neuron development.

Understanding how dopamine (DA) phenotypes are acquired in midbrain DA (mDA) neuron development is important for bioassays and cell replacement therapy for mDA neuron-associated disorders. Here, we demonstrate a feed-forward mechanism of mDA neuron development involving Nurr1 and Foxa2. Nurr1 acts as a transcription factor for DA phenotype gene expression. However, Nurr1-mediated DA gene expression was inactivated by forming a protein complex with CoREST, and then recruiting histone deacetylase 1 (Hdac1), an enzyme catalyzing histone deacetylation, to DA gene promoters. Coexpression of Nurr1 and Foxa2 was established in mDA neuron precursor cells by a positive cross-regulatory loop. In the presence of Foxa2, the Nurr1-CoREST interaction was diminished (by competitive formation of the Nurr1-Foxa2 activator complex), and CoRESTHdac1 proteins were less enriched in DA gene promoters. Consequently, histone 3 acetylation (H3Ac), which is responsible for open chromatin structures, was strikingly increased at DA phenotype gene promoters. These data establish the interplay of Nurr1 and Foxa2 as the crucial determinant for DA phenotype acquisition during mDA neuron development.KEY WORDS: Foxa2, Nurr1, Midbrain dopamine neuron, Development, Neural precursor cell, Epigenetic control, CoREST, Hdac, Mouse INTRODUCTIONMidbrain dopamine (mDA) neurons play important roles in voluntary movement, emotion and reward-based behaviors. Dysfunction or degeneration of this neuronal subtype is related to major neuropsychiatric disorders such as Parkinson's disease (PD), schizophrenia and drug addiction. Owing to the pathophysiological implications, mDA neurons are the most extensively studied cells. A molecular understanding of mDA neuron development is of high clinical interest as replacing this cell population in diseased brains is considered to be one of the most promising therapeutic approaches for PD (Deierborg et al., 2008;Morizane et al., 2008). In addition, developmental information can be exploited to establish optimal bioassays for mDA neuron-related disorders. mDA neurons arise from floor plate cells at the ventral midline of the embryonic midbrain (Bonilla et al., 2008;Ono et al., 2007). Sonic hedgehog (Shh), secreted initially by the notochord and later by floor plate cells, induces expression of forkhead family of winged-helix transcription factor 2 (Foxa2; also known as hepatocyte nuclear factor 3 beta), in the midbrain floor plate cells [mouse embryonic day (E) 8.5] (Ang et al., 1993;Monaghan et al., 1993;Placzek, 1995;Sasaki and Hogan, 1994;Sasaki et al., 1997). Foxa2 acts as a master regulator to induce expression of developmental factors specifying mDA neuron precursors such as Nurr1, Pitx3, Lmx1a, Msx1, neurogenin 2 and Mash1 (Ascl1 -Mouse Genome Informatics) (Ang, 2009;Ferri et al., 2007;Kittappa et al., 2007;Lee et al., 2010;Metzakopian et al., 2012). The early inductive role of Foxa2 is probably achieved by cooperation with the Wnt-Lmx1a/b regulatory loop from the isthmic organizer (Chung et al., 2009;Naka...

Understanding midbrain dopamine (DA) neuron differentiation is of importance, because of physiological and clinical implications of this neuronal subtype. We show that prolonged membrane depolarization induced by KCl treatment promotes DA neuron differentiation from neural precursor cells (NPCs) derived from embryonic ventral midbrain (VM). Interestingly, the depolarization-induced increase of DA neuron yields was not abolished by L-type calcium channel blockers, along with no depolarizationmediated change of intracellular calcium level in the VMderived NPCs (VM-NPCs), suggesting that the depolarization effect is due to a calcium-independent mechanism. Experiments with labeled DA neuron progenitors indicate that membrane depolarization acts at the differentiation fate determination stage and promotes the expression of DA phenotype genes (tyrosine hydroxylase [TH] and DA transporter [DAT]). Recruitment of Nurr1, a transcription factor crucial for midbrain DA neuron development, to the promoter of TH gene was enhanced by depolarization, along with increases of histone 3 acetylation (H3Ac) and trimethylation of histone3 on lysine 4 (H3K4m3), and decreases of H3K9m3 and H3K27m3 in the consensus Nurr1 binding regions of TH promoter. Depolarization stimuli on differentiating VM-NPCs also induced dissociation of methyl CpG binding protein 2 and related repressor complex molecules (repressor element-1 silencing transcription factor corepressor and histone deacetylase 1) from the CpG sites of TH and DAT promoters. Based on these findings, we suggest that membrane depolarization promotes DA neuron differentiation by opening chromatin structures surrounding DA phenotype genes and inhibiting the binding of corepressors, thus allowing transcriptional activators such as Nurr1 to access DA neuron differentiation gene promoter regions.

Use of the physiological mechanisms promoting midbrain DA (mDA) neuron survival seems an appropriate option for developing treatments for Parkinson's disease (PD). mDA neurons are specifically marked by expression of the transcription factors Nurr1 and Foxa2. We show herein that Nurr1 and Foxa2 interact to protect mDA neurons against various toxic insults, but their expression is lost during aging and degenerative processes. In addition to their proposed cell-autonomous actions in mDA neurons, forced expression of these factors in neighboring glia synergistically protects degenerating mDA neurons in a paracrine mode. As a consequence of these bimodal actions, adeno-associated virus (AAV)-mediated gene delivery of Nurr1 and Foxa2 in a PD mouse model markedly protected mDA neurons and motor behaviors associated with nigrostriatal DA neurotransmission. The effects of the combined gene delivery were dramatic, highly reproducible, and sustained for at least 1 year, suggesting that expression of these factors is a promising approach in PD therapy.

Plants regenerated from tissue culture often display somaclonal variation, that is, somatic and often meiotically heritable phenotypic variation that can result from both genetic and epigenetic modifications. To better understand the molecular basis of somaclonal variation, we have characterized four unique tissue culture-derived epialleles of the pericarp color1 (p1) gene of maize (Zea mays L.). The progenitor p1 allele, P1-wr, is composed of multiple head-to-tail tandemly arranged copies of the complete gene unit and specifies brick-red phlobaphene pigmentation in the cob glumes. The novel epialleles identified in progeny plants regenerated from tissue culture showed partial to complete loss of p1 function indicated by pink or colorless cob glumes. Loss of pigmentation was correlated with nearly complete loss of p1 steady-state transcripts. DNA gel-blot analysis and genomic bisulfite sequencing showed that silencing of the epialleles was associated with hypermethylation of a region in the second intron of P1-wr. Presence of Unstable factor for orange1 (Ufo1), an unlinked epigenetic modifier of p1, restored the cob glume pigmentation in the silenced alleles, and such reactivation was accompanied by hypomethylation of the p1 sequence. This observation confirmed that silencing of the epialleles is indeed due to epigenetic modifications and that the p1 epialleles were capable of functioning in the presence of the correct transacting factors. While the low-copy regions of the genome generally undergo hypomethylation during tissue culture, our study shows that the tandemly repeated genes are also prone to hypermethylation and epigenetic silencing.