Ana Sevilla

Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome

The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth and deafness), an autosomal-dominant developmental disorder belonging to a relatively prevalent class of inherited RAS–mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype.

Nanog-dependent feedback loops regulate murine embryonic stem cell heterogeneity

A number of key regulators of mouse embryonic stem (ES) cell identity, including the transcription factor Nanog, show strong expression fluctuations at the single-cell level. The molecular basis for these fluctuations is unknown. Here we used a genetic complementation strategy to investigate expression changes during transient periods of Nanog downregulation. Employing an integrated approach that includes high-throughput single-cell transcriptional profiling and mathematical modelling, we found that early molecular changes subsequent to Nanog loss are stochastic and reversible. However, analysis also revealed that Nanog loss severely compromises the self-sustaining feedback structure of the ES cell regulatory network. Consequently, these nascent changes soon become consolidated to committed fate decisions in the prolonged absence of Nanog. Consistent with this, we found that exogenous regulation of Nanog-dependent feedback control mechanisms produced a more homogeneous ES cell population. Taken together our results indicate that Nanog-dependent feedback loops have a role in controlling both ES cell fate decisions and population variability.

Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease, as well as an emerging source of tissue for regenerative medicine. The preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes limiting the scale and reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming enabling automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. We demonstrate that automated reprogramming and the pooled selection of polyclonal pluripotent cells results in high-quality, stable iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single-colony subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines.

Prdm16 is a physiologic regulator of hematopoietic stem cells

Fetal liver and adult bone marrow hematopoietic stem cells (HSCs) renew or differentiate into committed progenitors to generate all blood cells. PRDM16 is involved in human leukemic translocations and is expressed highly in some karyotypically normal acute myeloblastic leukemias. As many genes involved in leukemogenic fusions play a role in normal hematopoiesis, we analyzed the role of Prdm16 in the biology of HSCs using Prdm16-deficient mice. We show here that, within the hematopoietic system, Prdm16 is expressed very selectively in the earliest stem and progenitor compartments, and, consistent with this expression pattern, is critical for the establishment and maintenance of the HSC pool during development and after transplantation. Prdm16 deletion enhances apoptosis and cycling of HSCs. Expression analysis revealed that Prdm16 regulates a remarkable number of genes that, based on knockout models, both enhance and suppress HSC function, and affect quiescence, cell cycling, renewal, differentiation, and apoptosis to various extents. These data suggest that Prdm16 may be a critical node in a network that contains negative and positive feedback loops and integrates HSC renewal, quiescence, apoptosis, and differentiation.

Construction and validation of a regulatory network for pluripotency and self-renewal of mouse embryonic stem cells.

A 30-node signed and directed network responsible for self-renewal and pluripotency of mouse embryonic stem cells (mESCs) was extracted from several ChIP-Seq and knockdown followed by expression prior studies. The underlying regulatory logic among network components was then learned using the initial network topology and single cell gene expression measurements from mESCs cultured in serum/LIF or serum-free 2i/LIF conditions. Comparing the learned network regulatory logic derived from cells cultured in serum/LIF vs. 2i/LIF revealed differential roles for Nanog, Oct4/Pou5f1, Sox2, Esrrb and Tcf3. Overall, gene expression in the serum/LIF condition was more variable than in the 2i/LIF but mostly consistent across the two conditions. Expression levels for most genes in single cells were bimodal across the entire population and this motivated a Boolean modeling approach. In silico predictions derived from removal of nodes from the Boolean dynamical model were validated with experimental single and combinatorial RNA interference (RNAi) knockdowns of selected network components. Quantitative post-RNAi expression level measurements of remaining network components showed good agreement with the in silico predictions. Computational removal of nodes from the Boolean network model was also used to predict lineage specification outcomes. In summary, data integration, modeling, and targeted experiments were used to improve our understanding of the regulatory topology that controls mESC fate decisions as well as to develop robust directed lineage specification protocols.

SETD6 monomethylates H2AZ on lysine 7 and is required for the maintenance of embryonic stem cell self-renewal

The histone H2A variant H2AZ is an essential chromatin signaling factor. Herein, we report that H2AZ is monomethylated at lysine 7 (H2AZK7me1) by the lysine methyltransferase SETD6. We observed that methylation of H2AZ increased noticeably upon cellular differentiation of mouse embryonic stem cells (mESCs). H2AZK7me1 and the repressive H3K27me3 mark were found near the transcriptional start sites of differentiation marker genes, but were removed upon retinoic acid-induced cellular differentiation. The depletion of Setd6 in mESCs led to cellular differentiation, compromised self-renewal, and poor clonogenicity. These findings demonstrate that mESCs require Setd6 for self-renewal and portray H2AZK7me1 as a marker of cellular differentiation.

Combining competition assays with genetic complementation strategies to dissect mouse embryonic stem cell self-renewal and pluripotency

Substantial scientific interest has been dedicated recently to the crucial factors that control the pluripotent state of stem cells. To gain a comprehensive understanding of the molecular mechanisms regulating mouse embryonic stem cell (mESC) self-renewal and lineage differentiation, we have developed a robust method for studying the role of a particular gene in these processes. This protocol describes detailed procedures for the design and generation of the complementation rescue system and its application in dissecting the network of pluripotency-associated factors, using mESCs as a model. Specifically, three main procedures are described: (i) screening pluripotency-associated factors by competition assay; (ii) setting up an inducible complementation rescue system; and (iii) dynamically studying the pluripotency network response to target depletion. Completion of the competition assay and complementation rescue system takes 35 and 30 d, respectively, and an additional 16 d to study the dynamic molecular effects of a gene of interest in the pluripotency network.

Myeloid Dysregulation in a Human Induced Pluripotent Stem Cell Model of PTPN11-Associated Juvenile Myelomonocytic Leukemia

Somatic PTPN11 mutations cause juvenile myelomonocytic leukemia (JMML). Germline PTPN11 defects cause Noonan syndrome (NS), and specific inherited mutations cause NS/JMML. Here, we report that hematopoietic cells differentiated from human induced pluripotent stem cells (hiPSCs) harboring NS/JMML-causing PTPN11 mutations recapitulated JMML features. hiPSC-derived NS/JMML myeloid cells exhibited increased signaling through STAT5 and upregulation of miR-223 and miR-15a. Similarly, miR-223 and miR-15a were upregulated in 11/19 JMML bone marrow mononuclear cells harboring PTPN11 mutations, but not those without PTPN11 defects. Reducing miR-223s function in NS/JMML hiPSCs normalized myelogenesis. MicroRNA target gene expression levels were reduced in hiPSC-derived myeloid cells as well as in JMML cells with PTPN11 mutations. Thus, studying an inherited human cancer syndrome with hiPSCs illuminated early oncogenesis prior to the accumulation of secondary genomic alterations, enabling us to discover microRNA dysregulation, establishing a genotype-phenotype association for JMML and providing therapeutic targets.

Post-translational modifications of the histone variant H2AZ.

Gene expression is regulated by complex coordinated processes, including chromatin remodeling, post-translational modifications of histones, and incorporation of non-allelic histone variants. The histone variant H2AZ constitutes only a few percents of the total H2A cellular pool (Ball et al., 1983; West and Bonner, 1980). However, H2AZ is essential in several multi-cellular organisms (Faast et al., 2001; Ren and Gorovsky, 2001; Ridgway et al., 2004; van Daal and Elgin, 1992) and required for normal proliferation in Schizosaccharomyces pombe and Saccharomyces cerevisiae (Carr et al., 1994; Santisteban et al., 2000). In mammalian cells, H2AZ is involved in embryonic stem (ES) cell biology (Faast et al., 2001; Creyghton et al., 2008). Recent reports define the importance of H2AZ and H2AZ-modifying enzymes in self-renewal of ES cells (Binda et al., 2013; Hu et al., 2013; Li et al., 2012). In addition, H2AZ is post-translationally modified by acetylation, SUMOylation, ubiquitination, and methylation of lysines (Binda et al., 2013). Herein, we summarize our perspective on the emergent role of H2AZ in the biology of ES cells, while bringing a particular emphasis on the functions of its post-translational modifications.

Patterning pluripotency in embryonic stem cells.

Developmental gradients of morphogens and the formation of boundaries guide the choices between self‐renewal and differentiation in stem cells. Still, surprisingly little is known about gene expression signatures of differentiating stem cells at the boundaries between regions. We thus combined inducible gene expression with a microfluidic technology to pattern gene expression in murine embryonic stem cells. Regional depletion of the Nanog transcriptional regulator was achieved through the exposure of cells to microfluidic gradients of morphogens. In this way, we established pluripotency‐differentiation boundaries between Nanog expressing cells (pluripotency zone) and Nanog suppressed cells (early differentiation zone) within the same cell population, with a gradient of Nanog expression across the individual cell colonies, to serve as a mimic of the developmental process. Using this system, we identified strong interactions between Nanog and its target genes by constructing a network with Nanog as the root and the measured levels of gene expression in each region. Gene expression patterns at the pluripotency‐differentiation boundaries recreated in vitro were similar to those in the developing blastocyst. This approach to the study of cellular commitment at the boundaries between gene expression domains, a phenomenon critical for understanding of early development, has potential to benefit fundamental research of stem cells and their application in regenerative medicine. Stem Cells 2013;31:1806‐1815