Designing signaling environments to steer transcriptional diversity in neural progenitor cell populations

Stem cell populations within developing embryos are diverse, composed of many different subpopulations of cells with varying developmental potential. The structure of stem cell populations in cell culture remains poorly understood and presents a barrier to differentiating stem cells for therapeutic applications. In this paper we develop a framework for controlling the architecture of stem cell populations in cell culture using high-throughput single cell mRNA-seq and computational analysis. We find that the transcriptional diversity of neural stem cell populations collapses in cell culture. Cell populations are depleted of committed neuron progenitor cells and become dominated by a single pre-astrocytic cell population. By analyzing the response of neural stem cell populations to forty distinct signaling conditions, we demonstrate that signaling environments can restructure cell populations by modulating the relative abundance of pre-astrocyte and pre-neuron subpopulations according to a simple linear code. One specific combination of BMP4, EGF, and FGF2 ligands switches the default population balance such that 70% of cells correspond to the committed neurons. Our work demonstrates that single-cell RNA-seq can be applied to modulate the diversity of in vitro stem cell populations providing a new strategy for population-level stem cell control

The Caenorhabditis elegans Female State: Decoupling the Transcriptomic Effects of Aging and Sperm-Status

Understanding genome and gene function in a whole organism requires us to fully comprehend the life cycle and the physiology of the organism in question. Caenorhabditis elegans XX animals are hermaphrodites that exhaust their sperm after 3 d of egg-laying. Even though C. elegans can live for many days after cessation of egg-laying, the molecular physiology of this state has not been as intensely studied as other parts of the life cycle, despite documented changes in behavior and metabolism. To study the effects of sperm depletion and aging of C. elegans during the first 6 d of adulthood, we measured the transcriptomes of first-day adult hermaphrodites and sixth-day sperm-depleted adults, and, at the same time points, mutant fog-2(lf) worms that have a feminized germline phenotype. We found that we could separate the effects of biological aging from sperm depletion. For a large subset of genes, young adult fog-2(lf) animals had the same gene expression changes as sperm-depleted sixth-day wild-type hermaphrodites, and these genes did not change expression when fog-2(lf) females reached the sixth day of adulthood. Taken together, this indicates that changing sperm status causes a change in the internal state of the worm, which we call the female-like state. Our data provide a high-quality picture of the changes that happen in global gene expression throughout the period of early aging in the worm

Designing signaling environments to steer transcriptional diversity in neural progenitor cell populations

Stem cell populations within developing embryos are diverse, composed of many different subpopulations of cells with varying developmental potential. The structure of stem cell populations in cell culture remains poorly understood and presents a barrier to differentiating stem cells for therapeutic applications. In this paper we develop a framework for controlling the architecture of stem cell populations in cell culture using high-throughput single cell mRNA-seq and computational analysis. We find that the transcriptional diversity of neural stem cell populations collapses in cell culture. Cell populations are depleted of committed neuron progenitor cells and become dominated by a single pre-astrocytic cell population. By analyzing the response of neural stem cell populations to forty distinct signaling conditions, we demonstrate that signaling environments can restructure cell populations by modulating the relative abundance of pre-astrocyte and pre-neuron subpopulations according to a simple linear code. One specific combination of BMP4, EGF, and FGF2 ligands switches the default population balance such that 70% of cells correspond to the committed neurons. Our work demonstrates that single-cell RNA-seq can be applied to modulate the diversity of in vitro stem cell populations providing a new strategy for population-level stem cell control

Dissecting heterogeneous cell populations across drug and disease conditions with PopAlign

Single-cell measurement techniques can now probe gene expression in heterogeneous cell populations from the human body across a range of environmental and physiological conditions. However, new mathematical and computational methods are required to represent and analyze gene expression changes that occur in complex mixtures of single cells as they respond to signals, drugs, or disease states. Here, we introduce a mathematical modeling platform, PopAlign, that automatically identifies subpopulations of cells within a heterogeneous mixture, and tracks gene expression and cell abundance changes across subpopulations by constructing and comparing probabilistic models. We apply PopAlign to analyze the impact of 42 different immunomodulatory compounds on a heterogeneous population of donor-derived human immune cells as well as patient-specific disease signatures in multiple myeloma. PopAlign scales to comparisons involving tens to hundreds of samples, enabling large-scale studies of natural and engineered cell populations as they respond to drugs, signals or physiological change

The Caenorhabditis elegans Female State: Decoupling the Transcriptomic Effects of Aging and Sperm-Status

Understanding genome and gene function in a whole organism requires us to fully comprehend the life cycle and the physiology of the organism in question. Caenorhabditis elegans XX animals are hermaphrodites that exhaust their sperm after 3 d of egg-laying. Even though C. elegans can live for many days after cessation of egg-laying, the molecular physiology of this state has not been as intensely studied as other parts of the life cycle, despite documented changes in behavior and metabolism. To study the effects of sperm depletion and aging of C. elegans during the first 6 d of adulthood, we measured the transcriptomes of first-day adult hermaphrodites and sixth-day sperm-depleted adults, and, at the same time points, mutant fog-2(lf) worms that have a feminized germline phenotype. We found that we could separate the effects of biological aging from sperm depletion. For a large subset of genes, young adult fog-2(lf) animals had the same gene expression changes as sperm-depleted sixth-day wild-type hermaphrodites, and these genes did not change expression when fog-2(lf) females reached the sixth day of adulthood. Taken together, this indicates that changing sperm status causes a change in the internal state of the worm, which we call the female-like state. Our data provide a high-quality picture of the changes that happen in global gene expression throughout the period of early aging in the worm

The Caenorhabditis elegans Female-Like State: Decoupling the Transcriptomic Effects of Aging and Sperm Status

Understanding genome and gene function in a whole organism requires us to fully comprehend the life cycle and the physiology of the organism in question. Caenorhabditis elegans XX animals are hermaphrodites that exhaust their sperm after 3 d of egg-laying. Even though C. elegans can live for many days after cessation of egg-laying, the molecular physiology of this state has not been as intensely studied as other parts of the life cycle, despite documented changes in behavior and metabolism. To study the effects of sperm depletion and aging of C. elegans during the first 6 d of adulthood, we measured the transcriptomes of first-day adult hermaphrodites and sixth-day sperm-depleted adults, and, at the same time points, mutant fog-2(lf) worms that have a feminized germline phenotype. We found that we could separate the effects of biological aging from sperm depletion. For a large subset of genes, young adult fog-2(lf) animals had the same gene expression changes as sperm-depleted sixth-day wild-type hermaphrodites, and these genes did not change expression when fog-2(lf) females reached the sixth day of adulthood. Taken together, this indicates that changing sperm status causes a change in the internal state of the worm, which we call the female-like state. Our data provide a high-quality picture of the changes that happen in global gene expression throughout the period of early aging in the worm

Vitrification with Dimethyl Sulfoxide Induces Transcriptomic Alteration of Gene and Transposable Element Expression in Immature Human Oocytes

Despite substantial advancements in the field of cryobiology, oocyte and embryo cryopreservation still compromise developmental competence. Furthermore, dimethyl sulfoxide (DMSO), one of the most commonly used cryoprotectants, has been found to exert potent effects on the epigenetic landscape of cultured human cells, as well as mouse oocytes and embryos. Little is known about its impact on human oocytes. Additionally, few studies investigate the effects of DMSO on transposable elements (TE), the control of which is essential for the maintenance of genomic instability. The objective of this study was to investigate the impact of vitrification with DMSO-containing cryoprotectant on the transcriptome, including on TEs, of human oocytes. Twenty-four oocytes at the GV stage were donated by four healthy women undergoing elective oocyte cryopreservation. Oocytes were paired such that half from each patient were vitrified with DMSO-containing cryoprotectant (Vitrified Cohort), while the other half were snap frozen in phosphate buffer, unexposed to DMSO (Non-Vitrified Cohort). All oocytes underwent RNA sequencing via a method with high fidelity for single cell analysis, and which allows for the analysis of TE expression through Switching Mechanism at the 5′-end of the RNA Transcript sequencing 2 (SMARTseq2), followed by functional enrichment analysis. Of the 27,837 genes identified by SMARTseq2, 7331 (26.3%) were differentially expressed (p PIWIL2, DNMT3A, and DNMT3B, and negatively correlated with age. These findings suggest that the current standard process of oocyte vitrification, involving DMSO-containing cryoprotectant, induces significant transcriptome changes, including those involving TEs

An ordered pattern of Ana2 phosphorylation by Plk4 is required for centriole assembly

Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 is then phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STil/ANa2 (STAN) domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N terminus of Ana2, which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4-Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.Division of Intramural Research at the National Heart, Lung, and Blood Institute [1ZIA HL006104]; National Cancer Institute [P30 CA23074]; National Institute of General Medical Sciences [R01GFM110166]; National Science Foundation [MCB1158151]; Phoenix Friends6 month embargo; published online: 1 March 2018This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Dacomitinib (PF-00299804), a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor, demonstrates remarkable activity against HER2-amplified uterine serous endometrial cancer in vitro

Uterine serous carcinoma (USC) is an aggressive subtype of endometrial cancer that carries an extremely poor prognosis. Up to 35% of USC may overexpress the epidermal growth factor receptor-2 (HER2/neu) at strong (i.e., 3+) level by immunohistochemistry (IHC) or harbor HER2/neu gene amplification by fluorescence in situ hybridization (FISH). In this study, we assessed the sensitivity of a panel of USC cell lines with and without HER2/neu gene amplification to dacomitinib (PF-00299804), an irreversible pan-human epidermal growth factor receptor tyrosine kinase inhibitor. Eight primary cell lines (i.e., four harboring HER2/neu gene amplification by FISH and four FISH-cell lines), all demonstrating similar in vitro growth rates, were evaluated in viability/proliferation assays. The effect of dacomitinib on cell growth, cell cycle distribution, and signaling was determined using flow cytometry-based assays. Dacomitinib caused a significantly stronger growth inhibition in HER2/neu FISH+ USC cell lines when compared to FISH- USC (dacomitinib half maximal inhibitory concentration (IC50) mean+/-SEM= 0.02803+/-0.003355 mu M in FISH+ versus 1.498+/-0.2209 mu M in FISH- tumors, P<0.0001). Dacomitinib growth inhibition was associated with a significant and dose-dependent decline in phosphorylated HER2/neu and S6 transcription factor and a dose-dependent and time-dependent cell cycle arrest in G0/G1 in FISH+ USC. Dacomitinib is remarkably effective against chemotherapy-resistant HER2/neu gene-amplified USC. Clinical studies with dacomitinib in HER2/neu FISH+ USC patients resistant to standard salvage chemotherapy are warranted