Lodovica Borghese

High-resolution DNA analysis of human embryonic stem cell lines reveals culture-induced copy number changes and loss of heterozygosity

Prolonged culture of human embryonic stem cells (hESCs) can lead to adaptation and the acquisition of chromosomal abnormalities, underscoring the need for rigorous genetic analysis of these cells. Here we report the highest-resolution study of hESCs to date using an Affymetrix SNP 6.0 array containing 906,600 probes for single nucleotide polymorphisms (SNPs) and 946,000 probes for copy number variations (CNVs). Analysis of 17 different hESC lines maintained in different laboratories identified 843 CNVs of 50 kb–3 Mb in size. We identified, on average, 24% of the loss of heterozygosity (LOH) sites and 66% of the CNVs changed in culture between early and late passages of the same lines. Thirty percent of the genes detected within CNV sites had altered expression compared to samples with normal copy number states, of which >44% were functionally linked to cancer. Furthermore, LOH of the q arm of chromosome 16, which has not been observed previously in hESCs, was detected.

Inhibition of Notch Signaling in Human Embryonic Stem Cell-Derived Neural Stem Cells Delays G1/S Phase Transition and Accelerates Neuronal Differentiation In Vitro and In Vivo

The controlled in vitro differentiation of human embryonic stem cells (hESCs) and other pluripotent stem cells provides interesting prospects for generating large numbers of human neurons for a variety of biomedical applications. A major bottleneck associated with this approach is the long time required for hESC‐derived neural cells to give rise to mature neuronal progeny. In the developing vertebrate nervous system, Notch signaling represents a key regulator of neural stem cell (NSC) maintenance. Here, we set out to explore whether this signaling pathway can be exploited to modulate the differentiation of hESC‐derived NSCs (hESNSCs). We assessed the expression of Notch pathway components in hESNSCs and demonstrate that Notch signaling is active under self‐renewing culture conditions. Inhibition of Notch activity by the γ‐secretase inhibitor N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT) in hESNSCs affects the expression of human homologues of known targets of Notch and of several cell cycle regulators. Furthermore, DAPT‐mediated Notch inhibition delays G1/S‐phase transition and commits hESNSCs to neurogenesis. Combined with growth factor withdrawal, inhibition of Notch signaling results in a marked acceleration of differentiation, thereby shortening the time required for the generation of electrophysiologically active hESNSC‐derived neurons. This effect can be exploited for neural cell transplantation, where transient Notch inhibition before grafting suffices to promote the onset of neuronal differentiation of hESNSCs in the host tissue. Thus, interference with Notch signaling provides a tool for controlling human NSC differentiation both in vitro and in vivo. STEM CELLS 2010;28:955–964

Cancer Genes Hypermethylated in Human Embryonic Stem Cells

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.

MicroRNA-Based Promotion of Human Neuronal Differentiation and Subtype Specification

MicroRNAs are key regulators of neural cell proliferation, differentiation and fate choice. Due to the limited access to human primary neural tissue, the role of microRNAs in human neuronal differentiation remains largely unknown. Here, we use a population of long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) derived from human embryonic stem cells to study the expression and function of microRNAs at early stages of human neural stem cell differentiation and neuronal lineage decision. Based on microRNA expression profiling followed by gain- and loss-of-function analyses in lt-NES cells and their neuronal progeny, we demonstrate that miR-153, miR-324-5p/3p and miR-181a/a* contribute to the shift of lt-NES cells from self-renewal to neuronal differentiation. We further show that miR-125b and miR-181a specifically promote the generation of neurons of dopaminergic fate, whereas miR-181a* inhibits the development of this neurotransmitter subtype. Our data demonstrate that time-controlled modulation of specific microRNA activities not only regulates human neural stem cell self-renewal and differentiation but also contributes to the development of defined neuronal subtypes.

Pluripotent Stem Cell-Derived Somatic Stem Cells as Tool to Study the Role of MicroRNAs in Early Human Neural Development

The in vitro differentiation of human pluripotent stem cells represents a convenient approach to generate large numbers of neural cells for basic and translational research. We recently described the derivation of homogeneous populations of long-term self-renewing neuroepithelial-like stem cells from human pluripotent stem cells (lt-NES® cells). These cells constitute a suitable source of neural stem cells for in vitro modelling of early human neural development. Recent evidence demonstrates that microRNAs are important regulators of stem cells and nervous system development. Studies in several model organisms suggest that microRNAs contribute to different stages of neurogenesis - from progenitor self-renewal to survival and function of differentiated neurons. However, the understanding of the impact of microRNA-based regulation in human neural development is still at its dawn. Here, we give an overview on the current state of microRNA biology in stem cells and neural development and examine the role of the neural-associated miR-124, miR- 125b and miR-9/9* in human lt-NES® cells. We show that overexpression of miR-124, as well as overexpression of miR-125b, impair lt-NES® cell self-renewal and induce differentiation into neurons. Overexpression of the miR-9/9* locus also impairs self-renewal of lt-NES® cells and supports their commitment to neuronal differentiation. A detailed examination revealed that overexpression of miR-9 promotes differentiation, while overexpression of miR-9* affects both proliferation and differentiation of lt-NES® cells. This work provides insights into the regulation of early human neuroepithelial cells by microRNAs and highlights the potential of controlling differentiation of human stem cells by modulating the expression of selected microRNAs.

Reciprocal Regulation between Bifunctional miR-9/9∗ and its Transcriptional Modulator Notch in Human Neural Stem Cell Self-Renewal and Differentiation

Summary Tight regulation of the balance between self-renewal and differentiation of neural stem cells is crucial to assure proper neural development. In this context, Notch signaling is a well-known promoter of stemness. In contrast, the bifunctional brain-enriched microRNA miR-9/9∗ has been implicated in promoting neuronal differentiation. Therefore, we set out to explore the role of both regulators in human neural stem cells. We found that miR-9/9∗ decreases Notch activity by targeting NOTCH2 and HES1, resulting in an enhanced differentiation. Vice versa, expression levels of miR-9/9∗ depend on the activation status of Notch signaling. While Notch inhibits differentiation of neural stem cells, it also induces miR-9/9∗ via recruitment of the Notch intracellular domain (NICD)/RBPj transcriptional complex to the miR-9/9∗_2 genomic locus. Thus, our data reveal a mutual interaction between bifunctional miR-9/9∗ and the Notch signaling cascade, calibrating the delicate balance between self-renewal and differentiation of human neural stem cells.

Non-Genetic Modulation of Notch Activity by Artificial Delivery of Notch Intracellular Domain into Neural Stem Cells

Stem cells have become a major focus of scientific interest as a potential source of somatic cell types for biomedical applications. Understanding and controlling the elicitors and mechanisms in differentiation of pluripotent stem cell-derived somatic cell types remains a key challenge. The major types of molecular processes that control cellular differentiation involve evolutionary conserved cell signaling pathways. Notch receptors participate in a wide variety of biological processes, including cell fate decisions of stem cells. This study explores the potential of protein transduction to directly deliver recombinant Notch-1 intracellular domain (NICD) into mammalian cells in order to accomplish transgene-free Notch activation. We engineered a cell-permeant version of NICD and explored its function on mouse and human neural stem cells. We show that NICD transduction modulates known direct and indirect Notch target genes and antagonizes the DAPT-mediated inhibition of Notch signaling on the transcriptional level. Moreover, NICD enhances cell proliferation accompanied by increased cyclin D1 and decreased p27 protein levels. In the absence of growth factors NICD strongly impairs neuronal differentiation while being insufficient to keep cells in a proliferative state. Furthermore, our studies depict NICD protein transduction as a novel tool for a time and dose-dependent non-genetic modulation of Notch signaling to decipher its cellular functions.

Correction: MicroRNA-Based Promotion of Human Neuronal Differentiation and Subtype Specification

[This corrects the article DOI: 10.1371/journal.pone.0059011.].