Characterization of Alternative Splicing in High-Risk Wilms' Tumors.

The significant heterogeneity of Wilms' tumors between different patients is thought to arise from genetic and epigenetic distortions that occur during various stages of fetal kidney development in a way that is poorly understood. To address this, we characterized the heterogeneity of alternative mRNA splicing in Wilms' tumors using a publicly available RNAseq dataset of high-risk Wilms' tumors and normal kidney samples. Through Pareto task inference and cell deconvolution, we found that the tumors and normal kidney samples are organized according to progressive stages of kidney development within a triangle-shaped region in latent space, whose vertices, or "archetypes", resemble the cap mesenchyme, the nephrogenic stroma, and epithelial tubular structures of the fetal kidney. We identified a set of genes that are alternatively spliced between tumors located in different regions of latent space and found that many of these genes are associated with the epithelial-to-mesenchymal transition (EMT) and muscle development. Using motif enrichment analysis, we identified putative splicing regulators, some of which are associated with kidney development. Our findings provide new insights into the etiology of Wilms' tumors and suggest that specific splicing mechanisms in early stages of development may contribute to tumor development in different patients.

Characterization of Continuous Transcriptional Heterogeneity in High-Risk Blastemal-Type Wilms' Tumors Using Unsupervised Machine Learning.

Wilms' tumors are pediatric malignancies that are thought to arise from faulty kidney development. They contain a wide range of poorly differentiated cell states resembling various distorted developmental stages of the fetal kidney, and as a result, differ between patients in a continuous manner that is not well understood. Here, we used three computational approaches to characterize this continuous heterogeneity in high-risk blastemal-type Wilms' tumors. Using Pareto task inference, we show that the tumors form a triangle-shaped continuum in latent space that is bounded by three tumor archetypes with "stromal", "blastemal", and "epithelial" characteristics, which resemble the un-induced mesenchyme, the cap mesenchyme, and early epithelial structures of the fetal kidney. By fitting a generative probabilistic "grade of membership" model, we show that each tumor can be represented as a unique mixture of three hidden "topics" with blastemal, stromal, and epithelial characteristics. Likewise, cellular deconvolution allows us to represent each tumor in the continuum as a unique combination of fetal kidney-like cell states. These results highlight the relationship between Wilms' tumors and kidney development, and we anticipate that they will pave the way for more quantitative strategies for tumor stratification and classification.

Characterization of alternative mRNA splicing in cultured cell populations representing progressive stages of human fetal kidney development.

Nephrons are the functional units of the kidney. During kidney development, cells from the cap mesenchyme-a transient kidney-specific progenitor state-undergo a mesenchymal to epithelial transition (MET) and subsequently differentiate into the various epithelial cell types that create the tubular structures of the nephron. Faults in this transition can lead to a pediatric malignancy of the kidney called Wilms' tumor that mimics normal kidney development. While human kidney development has been characterized at the gene expression level, a comprehensive characterization of alternative splicing is lacking. Therefore, in this study, we performed RNA sequencing on cell populations representing early, intermediate, and late developmental stages of the human fetal kidney, as well as three blastemal-predominant Wilms' tumor patient-derived xenografts. Using this newly generated RNAseq data, we identified a set of transcripts that are alternatively spliced between the different developmental stages. Moreover, we found that cells from the earliest developmental stage have a mesenchymal splice-isoform profile that is similar to that of blastemal-predominant Wilms' tumor xenografts. RNA binding motif enrichment analysis suggests that the mRNA binding proteins ESRP1, ESRP2, RBFOX2, and QKI regulate alternative mRNA splicing during human kidney development. These findings illuminate new molecular mechanisms involved in human kidney development and pediatric kidney cancer.

Generation and characterization of iPSC lines from two nuclear envelopathy patients with a homozygous nonsense mutation in the TOR1AIP1 gene.

LAP1 is an inner nuclear membrane protein encoded by TOR1AIP1. A homozygous c.961C > T loss of function mutation in TOR1AIP1 that affects both isoforms of LAP1 was recently described. This mutation leads to the development of a severe multisystemic nuclear envelopathy syndrome. Here we describe the generation and characterization of two human induced pluripotent stem cell (hiPSC) lines derived from skin fibroblasts of two patients carrying the homozygous c.961C > T mutation. These novel lines can be used as a powerful tool to investigate the molecular mechanism by which LAP1 deficiency leads to the development of this severe hereditary disorder.

Apoptosis induction by the stem cell factor LIN28A.

BACKGROUND INFORMATION: Lin28A and its paralog Lin28B are RNA binding proteins expressed in stem and progenitor cells, regulating the balance between their proliferation and differentiation. In-vivo and in-vitro experiments have shown that overexpression of these genes leads to abnormal cell proliferation, which results in many cases in cell transformation and tumor formation. RESULTS: Here we show, for the first time, that Lin28A overexpression can also lead to the opposite effect, i.e. apoptosis induction. We further demonstrate that this effect is specific to Lin28A but not to Lin28B and that it is mediated via the Let-7 independent pathway in a complex mechanism that involves at least several proteins. CONCLUSIONS AND SIGNIFICANCE: This unexpected observation suggests that cell fate regulation by Lin28 is dependent on a specific cellular/genetic context. Unraveling the cellular and molecular mechanisms underlying this Lin28A overexpression effect may pave the way for novel tumor therapeutic strategies, as Lin28 is commonly expressed in many types of tumors but not in most normal adult cells.

Human Pluripotent Stem Cell Fate Regulation by SMARCB1.

Epigenetic regulation by the SWI/SNF complex is essential for normal self-renewal capacity and pluripotency of human pluripotent stem cells (hPSCs). It has been shown that different subunits of the complex have a distinct role in this regulation. Specifically, the SMARCB1 subunit has been shown to regulate the activity of enhancers in diverse types of cells, including hPSCs. Here, we report the establishment of conditional hPSC lines, enabling control of SMARCB1 expression from complete loss of function to significant overexpression. Using this system, we show that any deviation from normal SMARCB1 expression leads to cell differentiation. We further found that SMARCB1 expression is not required for differentiation of hPSCs into progenitor cells, but rather for later stages of differentiation. Finally, we identify SMARCB1 as a critical player in regulation of cell-cell and cell-ECM interactions in hPSCs and show that this regulation is mediated at least in part by the WNT pathway.

Single-Cell RNA Sequencing Reveals mRNA Splice Isoform Switching during Kidney Development.

BACKGROUND: During mammalian kidney development, nephron progenitors undergo a mesenchymal-to-epithelial transition and eventually differentiate into the various tubular segments of the nephron. Recently, Drop-seq single-cell RNA sequencing technology for measuring gene expression from thousands of individual cells identified the different cell types in the developing kidney. However, that analysis did not include the additional layer of heterogeneity that alternative mRNA splicing creates. METHODS: Full transcript length single-cell RNA sequencing characterized the transcriptomes of 544 individual cells from mouse embryonic kidneys. RESULTS: Gene expression levels measured with full transcript length single-cell RNA sequencing identified each cell type. Further analysis comprehensively characterized splice isoform switching during the transition between mesenchymal and epithelial cellular states, which is a key transitional process in kidney development. The study also identified several putative splicing regulators, including the genes Esrp1/2 and Rbfox1/2. CONCLUSIONS: Discovery of the sets of genes that are alternatively spliced as the fetal kidney mesenchyme differentiates into tubular epithelium will improve our understanding of the molecular mechanisms that drive kidney development.

Heterochronic regulation of lung development via the Lin28-Let-7 pathway.

The heterochronic gene Lin28 regulates diverse developmental processes. It was shown previously that global Lin28A overexpression during mouse embryogenesis results in perinatal lethality. However, the reason for this early lethality has not been elucidated. Here, we showed that Lin28A overexpression prevents normal lung development via the inhibition of the Let-7 micro RNAs, thus causing the perinatal lethality. We further found that Lin28A overexpression in lung mesenchymal cells, but not epithelial cells, is sufficient to recapitulate the lung phenotype. Moreover, we defined the specific time window wherein Lin28A expression exerts its effect. Deep characterization of the transgenic lungs suggests that the Lin28A-Let-7 pathway delays the transition from one developmental stage to another but does not completely abrogate the differentiation capacity of the lung progenitor cells. Finally, we suggested that the effect of Lin28A-Let-7 on embryonic lung development is mediated at least in part through the TGF-ß1-signaling pathway. Altogether, these findings define for the first time the Lin28-Let-7 pathway as a critical heterochronic regulator of lung development.-Komarovsky Gulman, N., Armon, L., Shalit, T., Urbach, A. Heterochronic regulation of lung development via the Lin28-Let-7 pathway.

The Lin28/let-7 Pathway Regulates the Mammalian Caudal Body Axis Elongation Program.

The heterochronic genes Lin28a/b and let-7 regulate invertebrate development, but their functions in patterning the mammalian body plan remain unexplored. Here, we describe how Lin28/let-7 influence caudal vertebrae number during body axis formation. We found that FoxD1-driven overexpression of Lin28a strikingly increased caudal vertebrae number and tail bud cell proliferation, whereas its knockout did the opposite. Lin28a overexpression downregulated the neural marker Sox2, causing a pro-mesodermal phenotype with a decreased proportion of neural tissue relative to nascent mesoderm. Manipulating Lin28a and let-7 led to opposite effects, and manipulating Lin28a's paralog, LIN28B caused similar yet distinct phenotypes. These findings suggest that Lin28/let-7 play a role in the regulation of tail length through heterochrony of the body plan. We propose that the Lin28/let-7 pathway controls the pool of caudal progenitors during tail development, promoting their self-renewal and balancing neural versus mesodermal cell fate decisions.

Geometry of Gene Expression Space of Wilms' Tumors From Human Patients.

Wilms' tumor is a pediatric malignancy that is thought to originate from faulty kidney development during the embryonic stage. However, there is a large variation between tumors from different patients in both histology and gene expression that is not well characterized. Here we use a meta-analysis of published microarray datasets to show that Favorable Histology Wilms' Tumors (FHWT's) fill a triangle-shaped continuum in gene expression space of which the vertices represent three idealized "archetypes". We show that these archetypes have predominantly renal blastemal, stromal, and epithelial characteristics and that they correlate well with the three major lineages of the developing embryonic kidney. Moreover, we show that advanced stage tumors shift towards the renal blastemal archetype. These results illustrate the potential of this methodology for characterizing the cellular composition of Wilms' tumors and for assessing disease progression.

Peroxisome proliferator-activated receptor gamma (PPARγ) is central to the initiation and propagation of human angiomyolipoma, suggesting its potential as a therapeutic target

Angiomyolipoma (AML), the most common benign renal tumor, can result in severe morbidity from hemorrhage and renal failure. While mTORC1 activation is involved in its growth, mTORC1 inhibitors fail to eradicate AML, highlighting the need for new therapies. Moreover, the identity of the AML cell of origin is obscure. AML research, however, is hampered by the lack of in vivo models. Here, we establish a human AML-xenograft (Xn) model in mice, recapitulating AML at the histological and molecular levels. Microarray analysis demonstrated tumor growth in vivo to involve robust PPARγ-pathway activation. Similarly, immunostaining revealed strong PPARγ expression in human AML specimens. Accordingly, we demonstrate that while PPARγ agonism accelerates AML growth, PPARγ antagonism is inhibitory, strongly suppressing AML proliferation and tumor-initiating capacity, via a TGFB-mediated inhibition of PDGFB and CTGF. Finally, we show striking similarity between AML cell lines and mesenchymal stem cells (MSCs) in terms of antigen and gene expression and differentiation potential. Altogether, we establish the first in vivo human AML model, which provides evidence that AML may originate in a PPARγ-activated renal MSC lineage that is skewed toward adipocytes and smooth muscle and away from osteoblasts, and uncover PPARγ as a regulator of AML growth, which could serve as an attractive therapeutic target.

LIN28: A Stem Cell Factor with a Key Role in Pediatric Tumor Formation.

Differentiation and development are normally unidirectional processes in which progenitor/stem cells differentiate into more mature cells. Transformation of adult cells into cancer cells is accompanied in many cases by dedifferentiation of the adult cell, while differentiation failure of progenitor cells can result in the formation of unique type of cancers called pediatric cancer. LIN28A and its paralog LIN28B are pluripotent genes that are expressed mainly in stem/progenitor cells. Since the first identification of LIN28 in mammals, numerous studies demonstrated the general oncogenic features of these genes. In this review, we emphasize the unique role of LIN28 in pediatric tumor formation. We show, based on comprehensive literature screen and analysis of published microarray data, that LIN28 expression in pediatric tumors is even more common than in adult tumors, and discuss the possibility that in the case of pediatric cancers, LIN28 acts by preventing normal development/differentiation rather than by transformation of mature cells into cancer cells. Overall, this review highlights the role of LIN28 as a bridge point between embryonic development, stem cell biology, and cancer.

Lin28 sustains early renal progenitors and induces Wilms tumor.

Wilms Tumor, the most common pediatric kidney cancer, evolves from the failure of terminal differentiation of the embryonic kidney. Here we show that overexpression of the heterochronic regulator Lin28 during kidney development in mice markedly expands nephrogenic progenitors by blocking their final wave of differentiation, ultimately resulting in a pathology highly reminiscent of Wilms tumor. Using lineage-specific promoters to target Lin28 to specific cell types, we observed Wilms tumor only when Lin28 is aberrantly expressed in multiple derivatives of the intermediate mesoderm, implicating the cell of origin as a multipotential renal progenitor. We show that withdrawal of Lin28 expression reverts tumorigenesis and markedly expands the numbers of glomerulus-like structures and that tumor formation is suppressed by enforced expression of Let-7 microRNA. Finally, we demonstrate overexpression of the LIN28B paralog in a significant percentage of human Wilms tumor. Our data thus implicate the Lin28/Let-7 pathway in kidney development and tumorigenesis.

Comparing ESC and iPSC-Based Models for Human Genetic Disorders.

Traditionally, human disorders were studied using animal models or somatic cells taken from patients. Such studies enabled the analysis of the molecular mechanisms of numerous disorders, and led to the discovery of new treatments. Yet, these systems are limited or even irrelevant in modeling multiple genetic diseases. The isolation of human embryonic stem cells (ESCs) from diseased blastocysts, the derivation of induced pluripotent stem cells (iPSCs) from patients' somatic cells, and the new technologies for genome editing of pluripotent stem cells have opened a new window of opportunities in the field of disease modeling, and enabled studying diseases that couldn't be modeled in the past. Importantly, despite the high similarity between ESCs and iPSCs, there are several fundamental differences between these cells, which have important implications regarding disease modeling. In this review we compare ESC-based models to iPSC-based models, and highlight the advantages and disadvantages of each system. We further suggest a roadmap for how to choose the optimal strategy to model each specific disorder.

The Lin28/let-7 axis regulates glucose metabolism.

The let-7 tumor suppressor microRNAs are known for their regulation of oncogenes, while the RNA-binding proteins Lin28a/b promote malignancy by inhibiting let-7 biogenesis. We have uncovered unexpected roles for the Lin28/let-7 pathway in regulating metabolism. When overexpressed in mice, both Lin28a and LIN28B promote an insulin-sensitized state that resists high-fat-diet induced diabetes. Conversely, muscle-specific loss of Lin28a or overexpression of let-7 results in insulin resistance and impaired glucose tolerance. These phenomena occur, in part, through the let-7-mediated repression of multiple components of the insulin-PI3K-mTOR pathway, including IGF1R, INSR, and IRS2. In addition, the mTOR inhibitor, rapamycin, abrogates Lin28a-mediated insulin sensitivity and enhanced glucose uptake. Moreover, let-7 targets are enriched for genes containing SNPs associated with type 2 diabetes and control of fasting glucose in human genome-wide association studies. These data establish the Lin28/let-7 pathway as a central regulator of mammalian glucose metabolism.

Generation of induced pluripotent stem cells from human blood.

Human dermal fibroblasts obtained by skin biopsy can be reprogrammed directly to pluripotency by the ectopic expression of defined transcription factors. Here, we describe the derivation of induced pluripotent stem cells from CD34+ mobilized human peripheral blood cells using retroviral transduction of OCT4/SOX2/KLF4/MYC. Blood-derived human induced pluripotent stem cells are indistinguishable from human embryonic stem cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, DNA methylation status at pluripotent cell-specific genes, and the capacity to differentiate in vitro and in teratomas. The ability to reprogram cells from human blood will allow the generation of patient-specific stem cells for diseases in which the disease-causing somatic mutations are restricted to cells of the hematopoietic lineage.

Studying early lethality of 45,XO (Turner's syndrome) embryos using human embryonic stem cells.

Turner's syndrome (caused by monosomy of chromosome X) is one of the most common chromosomal abnormalities in females. Although 3% of all pregnancies start with XO embryos, 99% of these pregnancies terminate spontaneously during the first trimester. The common genetic explanation for the early lethality of monosomy X embryos, as well as the phenotype of surviving individuals is haploinsufficiency of pseudoautosomal genes on the X chromosome. Another possible mechanism is null expression of imprinted genes on the X chromosome due to the loss of the expressed allele. In contrast to humans, XO mice are viable, and fertile. Thus, neither cells from patients nor mouse models can be used in order to study the cause of early lethality in XO embryos. Human embryonic stem cells (HESCs) can differentiate in culture into cells from the three embryonic germ layers as well as into extraembryonic cells. These cells have been shown to have great value in modeling human developmental genetic disorders. In order to study the reasons for the early lethality of 45,XO embryos we have isolated HESCs that have spontaneously lost one of their sex chromosomes. To examine the possibility that imprinted genes on the X chromosome play a role in the phenotype of XO embryos, we have identified genes that were no longer expressed in the mutant cells. None of these genes showed a monoallelic expression in XX cells, implying that imprinting is not playing a major role in the phenotype of XO embryos. To suggest an explanation for the embryonic lethality caused by monosomy X, we have differentiated the XO HESCs in vitro an in vivo. DNA microarray analysis of the differentiated cells enabled us to compare the expression of tissue specific genes in XO and XX cells. The tissue that showed the most significant differences between the clones was the placenta. Many placental genes are expressed at much higher levels in XX cells in compare to XO cells. Thus, we suggest that abnormal placental differentiation as a result of haploinsufficiency of X-linked pseudoautosomal genes causes the early lethality in XO human embryos.