Notch signaling regulates motor neuron differentiation of human embryonic stem cells.

In the pMN domain of the spinal cord, Notch signaling regulates the balance between motor neuron differentiation and maintenance of the progenitor state for later oligodendrocyte differentiation. Here, we sought to study the role of Notch signaling in regulation of the switch from the pMN progenitor state to differentiated motor neurons in a human model system. Human embryonic stem cells (hESCs) were directed to differentiate to pMN-like progenitor cells by the inductive action of retinoic acid and a Shh agonist, purmorphamine. We found that the expression of the Notch signaling effector Hes5 was induced in hESC-derived pMN-like progenitors and remained highly expressed when they were cultured under conditions favoring motor neuron differentiation. Inhibition of Notch signaling by a γ-secretase inhibitor in the differentiating pMN-like progenitor cells decreased Hes5 expression and enhanced the differentiation toward motor neurons. Conversely, over-expression of Hes5 in pMN-like progenitor cells during the differentiation interfered with retinoic acid- and purmorphamine-induced motor neuron differentiation and inhibited the emergence of motor neurons. Inhibition of Notch signaling had a permissive rather than an inductive effect on motor neuron differentiation. Our results indicate that Notch signaling has a regulatory role in the switch from the pMN progenitor to the differentiated motor neuron state. Inhibition of Notch signaling can be harnessed to enhance the differentiation of hESCs toward motor neurons.

Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer.

Many genes associated with CpG islands undergo de novo methylation in cancer. Studies have suggested that the pattern of this modification may be partially determined by an instructive mechanism that recognizes specifically marked regions of the genome. Using chromatin immunoprecipitation analysis, here we show that genes methylated in cancer cells are specifically packaged with nucleosomes containing histone H3 trimethylated on Lys27. This chromatin mark is established on these unmethylated CpG island genes early in development and then maintained in differentiated cell types by the presence of an EZH2-containing Polycomb complex. In cancer cells, as opposed to normal cells, the presence of this complex brings about the recruitment of DNA methyl transferases, leading to de novo methylation. These results suggest that tumor-specific targeting of de novo methylation is pre-programmed by an established epigenetic system that normally has a role in marking embryonic genes for repression.

Continuous Immune-Modulatory Effects of Human Olig2+ Precursor Cells Attenuating a Chronic-Active Model of Multiple Sclerosis.

Neuroglial precursor cells (NPC) possess immune-modulatory properties by which they prevent immune-mediated injury in experimental autoimmune encephalomyelitis (EAE). It is unclear whether cell transplantation in a clinical-relevant setup induces ongoing therapeutic effects in a chronic-active model of progressive multiple sclerosis (MS). We examined whether human embryonic stem cell (hESC)-derived NPCs inhibit progressive EAE in Biozzi AB/H mice, manifesting with chronic-active neuroinflammation and demyelinated plaques. hESC-derived NPCs were propagated for 6-8 weeks as spheres enriched for Olig2+ cells to switch from neuronal to glial commitment and to enrich for oligodendrocyte progenitor cells. NPC were transplanted intracerebroventricularly at 30 days post-EAE induction, after the acute relapse. We evaluated effects of cell transplantation on clinical parameters, neuroinflammation, myelination, and axonal loss. Transplanted animals exhibited a significantly milder disease, reduced neuroinflammation, reduced demyelination, and reduced axonal loss as compared to control EAE mice. Toluidine-blue semi-thin staining showed a bystander neuroprotective effect of human precursor cells preventing the loss of myelinated fibers in superficial layer of the cervical dorsal funiculus. Human Olig2+ cells were detected along spinal cord meninges after 65 days of follow-up. In co-cultures in vitro, Olig2+ human precursors inhibited Concanavalin A-induced murine T cell activation and proliferation. To conclude, glial-committed human NPC induce ongoing immune-regulatory and neuroprotective effects, following transplantation into mice with a clinical-relevant model of chronic-active MS and during established disease, entering the chronic phase. These properties highlight the therapeutic potential of human NPC transplantation in chronic MS and their delivery via the cerebrospinal fluid.

Systemically transplanted mesenchymal stem cells induce vascular-like structure formation in a rat model of vaginal injury.

The beneficial effect of mesenchymal stem cells (MSCs) on wound healing is mostly attributed to a trophic effect that promotes angiogenesis. Whether MSCs can contribute to the formation of new blood vessels by direct differentiation is still controversial. Pelvic floor dysfunction (PFD) is a group of disorders that negatively affect the quality of women's lives. Traditional vaginal surgical repair provides disappointing anatomical outcome. Stem cell transplantation may be used to supplement surgery and improve its outcome. Here we aimed to examine the engraftment, survival, differentiation and angiogenic effect of transplanted MSCs in a vaginal injury rat model. MSCs were obtained from the bone marrow of Sprague Drawley (SD) rats, expanded and characterized in vitro. The MSCs expressed CD90 and CD29, did not express CD45, CD34, CD11b and CD31 and could differentiate into osteogenic, chondrogenic and adipogenic lineages. Cells were labeled with either PKH-26 or GFP and transplanted systemically or locally to female SD rats, just after a standardized vaginal incision was made. Engraftment after local transplantation was less efficient at all-time points compared to systemic administration. In the systemically transplanted animal group, MSCs migrated to the injury site and were present in the healed vagina for at least 30 days. Both systemic and local MSCs transplantation promoted host angiogenesis. Systemically transplanted MSCs created new vascular-like structures by direct differentiation into endothelium. These findings pave the way to further studies of the potential role of MSCs transplantation in improving surgical outcome in women with PFD.

Hyperinsulinism of infancy: novel ABCC8 and KCNJ11 mutations and evidence for additional locus heterogeneity.

Hyperinsulinism of infancy is a genetically heterogeneous disease characterized by dysregulation of insulin secretion resulting in severe hypoglycemia. To date, mutations in five different genes, the sulfonylurea receptor (SUR1, ABCC8), the inward rectifying potassium channel (K(IR)6.2, KCNJ11), glucokinase (GCK), glutamate dehydrogenase (GLUD1), and short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD), have been implicated. Previous reports suggest that, in 40% of patients, no mutation can be identified in any of these genes, suggesting additional locus heterogeneity. However, previous studies did not screen all five genes using direct sequencing, the most sensitive technique available for mutation detection. We selected 15 hyperinsulinism of infancy patients and systematically sequenced the promoter and all coding exons and intron/exon boundaries of ABCC8 and KCNJ11. If no mutation was identified, the coding sequence and intron/exon boundaries of GCK, GLUD1, and SCHAD were sequenced. Seven novel mutations were found in the ABCC8 coding region, one mutation was found in the KCNJ11 coding region, and one novel mutation was found in each of the two promoter regions screened. Functional studies on beta-cells from six patients showed abnormal ATP-sensitive K+ channel function in five of the patients; the sixth had normal channel activity, and no mutations were found. Photolabeling studies using a reconstituted system showed that all missense mutations altered intracellular trafficking. Each of the promoter mutations decreased expression of a reporter gene by about 60% in a heterologous expression system. In four patients (27%), no mutations were identified. Thus, further genetic heterogeneity is suggested in this disorder. These patients represent a cohort that can be used for searching for mutations in other candidate genes.

Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells.

Dysfunction and loss of retinal pigment epithelium (RPE) leads to degeneration of photoreceptors in age-related macular degeneration and subtypes of retinitis pigmentosa. Human embryonic stem cells (hESCs) may serve as an unlimited source of RPE cells for transplantation in these blinding conditions. Here we show the directed differentiation of hESCs toward an RPE fate under defined culture conditions. We demonstrate that nicotinamide promotes the differentiation of hESCs to neural and subsequently to RPE fate. In the presence of nicotinamide, factors from the TGF-beta superfamily, which presumably pattern RPE development during embryogenesis, further direct RPE differentiation. The hESC-derived pigmented cells exhibit the morphology, marker expression, and function of authentic RPE and rescue retinal structure and function after transplantation to an animal model of retinal degeneration caused by RPE dysfunction. These results are an important step toward the future use of hESCs to replenish RPE in blinding diseases.

MicroRNA expression patterns and function in endodermal differentiation of human embryonic stem cells.

BACKGROUND/AIMS: microRNAs (miRNAs) are small noncoding RNAs that regulate cognate mRNAs post-transcriptionally. Human embryonic stem cells (hESC), which exhibit the characteristics of pluripotency and self-renewal, may serve as a model to study the role of miRNAs in early human development. We aimed to determine whether endodermally-differentiated hESC demonstrate a unique miRNA expression pattern, and whether overexpression of endoderm-specific miRNA may affect hESC differentiation. METHODS: miRNA expression was profiled in undifferentiated and NaButyrate-induced differentiated hESC of two lines, using microarray and quantitative RT-PCR. Then, the effect of lentiviral-based overexpression of liver-specific miR-122 on hESC differentiation was analyzed, using genomewide gene microarrays. RESULTS: The miRNA profiling revealed expression of three novel miRNAs in undifferentiated and differentiated hESC. Upon NaButyrate induction, two of the most upregulated miRNAs common to both cell lines were miR-24 and miR-10a, whose target genes have been shown to inhibit endodermal differentiation. Furthermore, induction of several liver-enriched miRNAs, including miR-122 and miR-192, was observed in parallel to induction of endodermal gene expression. Stable overexpression of miR-122 in hESC was unable to direct spontaneous differentiation towards a clear endodermal fate, but rather, delayed general differentiation of these cells. CONCLUSIONS: Our results demonstrate that expression of specific miRNAs correlates with that of specific genes upon differentiation, and highlight the potential role of miRNAs in endodermal differentiation of hESC.

Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway.

The Wnt pathway controls numerous developmental processes via the beta-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of beta-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic beta-catenin associates with APC and axin and is continuously phosphorylated by GSK-3beta, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3beta from phosphorylating beta-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. Here we study the regulation of beta-catenin phosphorylation and degradation by the Wnt pathway. Using mass spectrometry and phosphopeptide-specific antibodies, we show that a complex of axin and casein kinase I (CKI) induces beta-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate beta-catenin in vitro at S45; CKI inhibition suppresses this phosphorylation in vivo. CKI phosphorylation creates a priming site for GSK-3beta and is both necessary and sufficient to initiate the beta-catenin phosphorylation-degradation cascade. Wnt3A signaling and Dvl overexpression suppress S45 phosphorylation, thereby precluding the initiation of the cascade. Thus, a single, CKI-dependent phosphorylation event serves as a molecular switch for the Wnt pathway.

Pseudosubstrate regulation of the SCF(beta-TrCP) ubiquitin ligase by hnRNP-U.

beta-TrCP/E3RS (E3RS) is the F-box protein that functions as the receptor subunit of the SCF(beta-TrCP) ubiquitin ligase (E3). Surprisingly, although its two recognized substrates, IkappaB(alpha) and beta-catenin, are present in the cytoplasm, we have found that E3RS is located predominantly in the nucleus. Here we report the isolation of the major E3RS-associated protein, hnRNP-U, an abundant nuclear phosphoprotein. This protein occupies E3RS in a specific and stoichiometric manner, stabilizes the E3 component, and is likely responsible for its nuclear localization. hnRNP-U binding was abolished by competition with a pIkappaB(alpha) peptide, or by a specific point mutation in the E3RS WD region, indicating an E3-substrate-type interaction. However, unlike pI(kappa)Balpha, which is targeted by SCF(beta-TrCP) for degradation, the E3-bound hnRNP-U is stable and is, therefore, a pseudosubstrate. Consequently, hnRNP-U engages a highly neddylated active SCF(beta-TrCP), which dissociates in the presence of a high-affinity substrate, resulting in ubiquitination of the latter. Our study points to a novel regulatory mechanism, which secures the localization, stability, substrate binding threshold, and efficacy of a specific protein-ubiquitin ligase.