eSymposia | Tissue Plasticity: Preservation and Alteration of Cellular Identity

Oct 5, 2020 ‐ Oct 7, 2020



Sessions

Loss of TSC1 or TSC2 drives lineage-infidelity and hamartoma formation in a renal organoid model of angiomyolipoma

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Loss of TSC1 or TSC2 drives lineage-infidelity and hamartoma formation in a renal organoid model of angiomyolipoma Renal angiomyolipomas (R-AMLs) are hamartomatous kidney growths which stain positively for markers of adipocytic, vascular, smooth muscle, and melanocytic lineages. These lesions possess loss of function mutations in either TSC1 or TSC2, which are canonical negative regulators of mTORC1 signalling. To date, there exists no in vitro or in vivo model which faithfully recapitulates the architectural and molecular complexity of R-AMLs. Considering these lesions can be detected very early in life- even congenitally- we hypothesized they arise as a consequence of aberrant tissue development. To test this hypothesis, we generated TSC1-/- and TSC2-/- mutants in four human pluripotent stem cell (hPSC) backgrounds using CRISPR/Cas9 genome engineering. Wild type hPSCs differentiated into renal organoids express markers of the glomerulus, proximal and distal tubules, in a topology that resembles human nephron patterning. Remarkably, wild type renal organoids downregulate mTORC1 signalling compared to adjacent undifferentiated cells. In contrast, both TSC1-/- and TSC2-/- hPSCs exhibit substantial lineage infidelity upon differentiation, staining positive for adipocytic and melanocytic markers which are absent in matched wild type controls. Additionally, knockout lines formed nodular growths with disorganized architecture, resembling the hamartomatous organization of R-AML lesions. These lesions exhibit hyperactive mTORC1 signalling, consistent with human pathology. Together, these data suggest three primary findings: loss of TSC1/2 drives lineage infidelity; TSC1/2 may be required for architectural organization of the kidney parenchyma; and a developmental approach to R-AML modelling may best recapitulate the human disease.

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Transflammation: Cell Autonomous Innate Immune Signaling Alters Epigenetic Determinants of Cell Fate

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Transflammation: Cell Autonomous Innate Immune Signaling Alters Epigenetic Determinants of Cell Fate John P. Cooke, Shu Meng, Li Lai, Palas Chanda, Cristina Kriss Houston Methodist Research Institute, Houston TX

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Esculetin supresses EMT phenotype in colon carcinoma cells and induces differentiation in leukemic blast cells accompanying reduced cancer stem cell properties

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Esculetin supresses EMT phenotype in colon carcinoma cells and induces differentiation in leukemic blast cells accompanying reduced cancer stem cell properties Esculetin supresses EMT phenotype in colon carcinoma cells and induces differentiation in leukemic blast cells accompanying reduced cancer stem cell properties Ankit Mathur, Daman Saluja Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India The intra-tumoural heterogeneity partially attributed to clonal evolution of transforming tumour cells results in a complex sub-clonal hierarchical organization and therapy resistance. The phenotypic trans-differentiation of the cancer cells can be best explained by Epithelial-Mesenchymal Transition (EMT) in solid tumours as well as accumulated immature blast cells endowed with cancer stem cell (CSC) properties in Acute Myeloid Leukemia (AML). We have previously demonstrated anti-proliferative, anti-leukemic, and anti-oxidative effects of natural compound (Esculetin) on AML and pancreatic cancer cells. The current study supports the rationale of using esculetin to force transdifferentiating cells to undergo a less aggressive counterpart. The Trans-differentiating potential of esculetin was assessed on two in vitro cellular models using AML cells (Kasumi-1cell line) with t(8;21/AML-ETO) translocation as well as human colon carcinoma cell line (HCT116) with p53 and p73 knockdowns. Three HCT116 cell strains with sequential knockdown of p53 and p73 genes progressively exhibited altered morphology indicative of acquisition of mesenchymal property. EMT marker expressions and Wnt activation coupled with anchorage independent growth, anoikis resistance, enhanced cell mobility, and saturation density confirmed progressive transformation accompanying EMT. Esculetin (100μM), exerted cytostatic effect with reduced aggressiveness and reversion of EMT phenotypes in HCT116 strains. Interestingly, morphological alterations associated with neutrophilic differentiation as well as corresponding acquisition of myeloid lineage markers indicate terminal differentiation potential of esculetin in leukemic blasts. The study highlights the possibility that the cells that escaped the early apoptosis program were stimulated to undergo differentiation upon esculetin treatment. Esculetin also showed potential to revert the CSC marker expressions consistent with reduced functional CSC properties and suppression of Wnt associated genes in colon cancer as well as leukemic cells. Thus, the study may provide significant therapeutic innervations of esculetin as a differentiating agent in both solid cancers as well as in leukemia.

Speaker(s):
  • Ankit Mathur, PhD, Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi

Changes in the Pelvic Floor Muscle Stem Cell Phenotype During Pregnancy

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Changes in the Pelvic Floor Muscle Stem Cell Phenotype During Pregnancy Francesca Boscolo, PhD1; Alessandra Sacco, PhD2; Marianna Alperin1, MD MS. 1Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Diego. 2Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute. Pelvic floor disorders (PFDs) are a major public health issue given their high prevalence, negative impact on quality of life of millions of women, and related economic burden. Maternal childbirth injury to the pelvic floor supportive structures, including pelvic floor muscles (PFMs), confers the greatest hazard for subsequent PFDs. Computational models of human parturition demonstrate that PFMs’ elongation up to 300% of resting muscle length is necessary to achieve fetal delivery. Thus, muscle injury would be expected to occur in most, if not all, vaginal deliveries; however, the majority of women do not exhibit PFM injuries after childbirth. We hypothesized that structural and functional adaptations, acquired by the PFMs during pregnancy, may account for the ability of these muscles to withstand “supraphysiologic” strains during parturition without injury. Using the validated rat model, we have previously shown that during pregnancy PFMs undergo fiber elongation via sarcomerogenesis, or serial addition of muscle functional units, known as sarcomeres. This protective adaptation enables PFMs to withstand deformations during parturition without excessive sarcomere hyperelongation, in turn, decreasing PFMs’ susceptibility to mechanical birth injury. Muscle stem cells (MuSC), are responsible for maintenance of muscle homeostasis, response to altered physiological conditions, such as increased load, and regeneration in case of injury. To exert their function, MuSCs, quiescent in unperturbed muscles, become activated, proliferate and differentiate. In this study, we tested the hypothesis that MuSCs are involved in pregnancy-induced adaptations of the rat PFMs. To determine phenotypic changes associated with MuSCs during pregnancy, we first assessed in vivo cells proliferative ability at different time points across the rat gestational period: non-pregnant (NP), mid-pregnant (MP; D11 out of 23-day gestation), and late-pregnant (LP; D21). Through EdU incorporation assay together with Pax7 (MuSC marker) immunohistochemistry, we observed a significant increase in MuSC proliferation in MP animals. LP MuSCs showed reduced proliferation compared to NP and MP cells. These results suggest that MuSCs become activated during the first half of pregnancy, and then return to quiescence by the end of the gestational period. To determine the fate of the activated cells, we assessed the proportion of MuSCs positive for Myogenin (differentiation marker). In MP rats, Myogenin+ MuSCs were significantly increased compared to NP and LP time points. These results were further validated through qRT-PCR performed on freshly isolated cells. To determine whether the observed changes were cell autonomous, we isolated MuSCs from rat PFMs and assessed their ability to divide in vitro through time-lapse microscopy. Consistent with our in vivo data, MuSCs from MP animals entered the cell cycle faster compared to MuSCs procured from NP and LP rats, indicating the increased activation. To determine whether LP MuSCs re-entry into quiescence is comparable to NP quiescent state, we performed RNA sequencing analysis and observed a significant decrease in cell growth, cell proliferation, and cell differentiation pathways in LP cells. To validate these results, we assessed MuSC differentiation in vitro. LP MuSCs exhibited significant decrease in differentiation ability compared to NP cells. Altogether these results suggest that MuSC quiescent state is not comparable in NP and LP animals, and that the observed difference is cell autonomous. In summary, the physiological changes associated with pregnancy have a strong effect on the MuSC autonomous behavior. During the first half of pregnancy, MuSCs become activated, returning to quiescence during the second half of pregnancy. These changes in MuSCs behavior are consistent with pregnancy-induced adaptation timeline, where fiber elongation starts during the first half of pregnancy, aided by activated MuSCs, to be completed by the end of the rat gestation. In late pregnancy, MuSCs return to quiescence. Altogether these results strongly suggest that MuSCs are involved in fiber elongation via sarcomerogenesis during gestation. Thus, PFM resident stem cells contribute to the generation of protective antepartum adaptations that mitigate susceptibility to muscle birth injury. Future directions focus on determining the specific physiological stimuli that govern PFM stem cell changes during pregnancy.

Speaker(s):

Helix-loop-helix transcription factor Ascl4 induces myogenic program in pluripotent stem cells

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Helix-loop-helix transcription factor Ascl4 induces myogenic program in pluripotent stem cells Yusaku Kodakaa, Shuichi Watanabe, Hikaru Ito, Mayank Verma, Tomohide Takaya, Yoko Asakura, Michael Kyba, Atsushi Asakura Stem Cell institute, bPaul & Sheila Wellstone Muscular Dystrophy Center, cDepartment of Neurology, dDepartment of Pediatrics, University of Minnesota Medical School, Minneapolis, MN The basic helix-loop-helix (bHLH) transcription factors play central roles in developmental processes including cell fate specification such as MyoD family for myogenesis and Achaete-scute complex-like 1 (Ascl1) for neurogenesis. Here, we found that all Ascl family (Ascl1-5) has the ability to induce myogenic program when overexpressed in embryonic stem cells (ESCs). Among them, we noticed that Ascl4 expression is detected in dermomyotome, a place for myogenic progenitor cells during mouse embryogenesis. In both ESCs and induced Pluripotent Stem Cells (iPSCs), overexpression of Ascl4 efficiently induces MyoD/Pax7-positive myogenic cells followed by myosin heavy chain positive terminally differentiated myocytes when Ascl4 expression was withdrawn. Integrative analysis of RNA-seq and ChIP-seq data revealed that Ascl4 is able to induce MyoD expression through the binding of novel MyoD enhancer region termed embryonic enhancer region (EER) located at 60 kb upstream of its start site. Together, these findings imply that Ascl4 may mediate activation of MyoD as a key regulator of somitic myogenesis.

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Divergent roles of HMGCS2 in intestinal stemness and colonic tumorigenesis

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Divergent roles of HMGCS2 in intestinal stemness and colonic tumorigenesis Chia-Wei Cheng1*, Omer H. Yilmaz2* 1Department of Genetics and Development at Columbia University; 2Koch Institute at MIT *Corresponding author Little is known how an endogenous metabolite functions as a cell-fate determinant and by which to influence tumor development. We previously reported that the ketone body-producing enzyme HMGCS2 (HMG-CoA synthase 2), distinguishes self-renewing Lgr5+ intestinal stem cells (ISCs) from differentiated cell types in the small intestine. ISCs rely on the HMGCS2-derived ketone body βOHB to regulate stem cell self-renewal and lineage commitment through HDAC inhibition and Notch signaling. Here, we investigate the role of HMGCS2 and the ketone body signaling in colon and colon cancer. In humans and mice, bimodal HMGCS2 expression distinguishes between proximal and distal colon. In humans, the anatomically relevant HMGCS2 expression pattern is established during development and maintained in adulthood but becomes less evident in tumors. Notably, TCGA data for colon adenocarcinoma indicates a poor overall survival in patients with HMGCS2low tumors. In mice, proximal and distal colonic crypts display distinct organoid forming capacities and cellular lineage compositions, with HMGCS2 exclusively expressed in the absorptive lineage. Loss of HMGCS2 in the APChet tumor mouse model reduces tumor lesions in the small intestine while exacerbating tumor progression in the distal colon, a novel phenotype previously thought to be an unsolved mouse versus human conundrum. These findings highlight the divergent roles of Hmgcs2 in intestinal stemness and colonic tumorigenesis and raise the possibility that dysregulation of metabolic cell-fate determinant may destabilize the lineage commitment and contribute to oncogenic transformation.

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Mechanisms driving in vivo transdifferentiation

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Mechanisms driving in vivo transdifferentiation The extent to which differentiated cells, while remaining in their native microenvironment, can be reprogrammed to assume a different identity will reveal fundamental insight into cellular plasticity and impact regenerative medicine. To investigate in vivo cell lineage potential, we leveraged the zebrafish as a practical vertebrate platform to determine factors and mechanisms necessary to induce differentiated cells of one germ layer to adopt the lineage of another. We found that ectopic co-expression of Sox32 and Oct4 in several non-endoderm lineages, including skeletal muscle cells, can specifically trigger an early endoderm genetic program. Endoderm-induced muscle cells rapidly lose muscle gene expression and morphology, while specifically gaining endoderm organ lineage markers via a mechanism resembling normal development. Further, muscle lineage conversion is independent of a dedifferentiation or a pluripotency mechanism, suggesting that reprogramming occurs via direct transdifferentiation. However, inhibition of the skeletal muscle master regulator Myod and activation of the cell recycling process autophagy are necessary for, and can enhance, the earliest events in muscle cell reprogramming, revealing that active repression of muscle identity is critical for the initiation of lineage conversion. Importantly, examination of other models of direct lineage conversion, such as induced neurons, cardiomyocytes, and pancreatic endocrine cells, reveal that autophagy is fundamental for transdifferentiation. Our work demonstrates that within a vertebrate animal, differentiated cells can be induced to directly adopt the identity of a completely unrelated cell lineage, while remaining in a distinct microenvironment, suggesting that differentiated cells in vivo may be more amenable to lineage conversion than previously appreciated. Furthermore, our mechanistic studies suggest stimulating the loss of a cell’s identity, both its transcriptome and proteome, to be a novel strategy for increasing the efficiency and efficacy of induced transdifferentiation.

Speaker(s):
  • Duc S. Dong, PhD, Sanford Burnham Prebys Medical Discovery Institute

Vascular smooth muscle-derived adipocyte progenitors are the cellular origin of cold-induced brown adipocytes

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Vascular smooth muscle-derived adipocyte progenitors are the cellular origin of cold-induced brown adipocytes The adipose tissue plays a central role in the regulation of energy homeostasis and is therefore a major contributor to the pathophysiology of obesity and metabolic diseases. Brown adipose tissue is specialized in thermogenic energy expenditure and is an attractive target for anti-obesity therapies. Prolonged cold exposure increases BAT mass and activity partially through de novo recruitment of brown adipocytes as well as the coordinated expansion of other cells within the adipose niche to enable maximal thermogenic activity. However, the source of cold-induced brown adipogenesis and the molecular mechanism regulating BAT expansion is not known. To delineate the cellular remodeling of the thermogenic niche and identify the cellular origin of brown adipocytes, we used single-cell RNA sequencing. We identified a previously unknown population of adipocyte progenitors derived from the vascular smooth muscle lineage (VSM-APC) as the source of brown adipocytes recruited in cold. VSM-APCs specifically express the temperature-sensitive ion channel transient receptor potential cation channel subfamily V member 1 (Trpv1) in brown and white adipose tissue. Lineage tracing studies using the Trpv1Cre Rosa26mTmG mouse model confirmed that the Trpv1pos VSM-APCs are a distinct population of adipocyte progenitors that contribute to brown and white adipocyte pools in vivo and in vitro. Intriguingly, cold exposure promotes the proliferation and differentiation of Trpv1pos progenitors into highly thermogenic brown adipocytes. Together, this work illustrated the landscape of the thermogenic adipose niche remodeling at the single cell resolution and established a new cellular origin of thermogenic adipocytes. This new model for the development of BAT could be critical in designing strategies to increase the number of brown adipocytes in humans. Farnaz Shamsi1,5, Matthew D. Lynes1,5, Mary Piper2, Li-Lun Ho3, Tian Lian Huang1, Yu-Hua Tseng1,4 1 Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Harvard Medical School, Boston, MA, USA 2 Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA 3 Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA, USA 4 Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA 5 Equal Contributions

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The IL33/ST2 axis in Celiac Disease

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

The IL33/ST2 axis in Celiac Disease Federico Perez1, Carolina Ruera1, Emanuel Miculan1, Paula Carasi1, Karen Dubois-Camacho2, Laura Garbi3, Luciana Guzman3, Marcela A. Hermoso2, Fernando Chirdo1 1Instituto de Estudios Inmunológicos y Fisiopatológicos UNLP-CONICET, Argentina. 2Innate Immunity Laboratory, Immunology Program, Biomedical Sciences Institute, Universidad de Chile, Chile. 3Gastroenterology Unit of San Martin Hospital of La Plata and Sor Maria Ludovica, Children Hospital of La Plata. Argentina. IL-33 is cytokine able to promote regulatory T cells, reparation processes and type 2 immune responses in the gut. These functions can be inhibited by the presence of IFN???? and IL-23. Furthermore, IL-33 functions are regulated by a soluble form of its cellular receptor (ST2L), named sST2 (Molofsky et al. 2015). Celiac Disease (CD) is characterized by an immune response dominated by IFN????+ Th1 cells responsive to gluten, and enterocyte death by cytotoxic CD8+ T cells (Meresse et al. 2006). In this context, we have found that IL-33 and sST2 are increased in sera of CD patients. Also, CD8+ T cells are differentially increased in active CD patients. Additionally, CD patients have an increase in the ST2L as well as, in sST2 in CD duodenum. Thus, it is possible that IL-33 could target ST2L+ cells. Western blot analysis showed an increase in IL-33 mature fragments of about 18-20 kDa in protein extracts of duodenal samples of CD patients. These fragments are thought to be produced by granulocytes derived enzymes in the extracellular medium, and have much greater bioactivity on target cells (Lefrançais et al. 2012; 2014). Inflammatory cell death is considered to be one of the main sources of free IL-33 in inflamed tissues. Accordingly, we have found that Cleaved Caspase 1 and active IL1β, as markers of pyroptosis, are increased in CD patients. Therefore, IL-33 may act as a pro-cytotoxic alarmin released by pyroptotic cells in CD patients. In turn free IL-33 may boost programmed cell death mechanisms on stressed cells by targeting Cytotoxic ST2L+ CD8+T cells. Also, we considered that its wound-healing and regulatory properties may be inhibited by IFN???? and IL-23, which are released in response to gluten (Nilsen et al. 1998; Harris, et al. 2008). This work was funded by grant PICT 2017 0880 from the Agencia Nacional de Promoción Científica y Tecnológica from Ministerio de Ciencia, Argentine.

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TNFAIP8 controls intestinal stell cell homeostasis and paligenosis by regulating microbiome-induced PI3K/Akt/β-catenin signaling

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

TNFAIP8 controls intestinal stell cell homeostasis and paligenosis by regulating microbiome-induced PI3K/Akt/β-catenin signaling Jason R Goldsmith1, Nina Spitofsky1, Ali Zamani1, Ryan Hood1, Amanda Boggs1, Xinyuan Li1, Mingyue Li1, Elizabeth Reiner1,2, Arshad Ayyaz3, Zienab Etwebi1, Ling Lu1, Javier Rivera Guzman1,4, Mayassa J. Bou-Dargham1, Terry Cathoupolis1, Hakon Hakonarson5,6, Honghong Sun1, Jeffrey L. Wrana3,7, Michael V. Gonzalez5,6, Youhai H Chen1,# 1Dept of Pathology and Lab Medicine, PSOM, UPenn; 2Univ. of Pikeville-Kentucky School of Osteopathic Medicine; 3Centre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital; 4Dept of Biology, Univ. of Maryland Baltimore College; 5Center for Applied Genomics, The Children’s Hospital of Philadelphia; 6Dept of Pediatrics, PSOM, UPenn; 7Dept of Molecular Genetics, Univ. of Toronto Epithelial cells and their stem cell niche are regenerated after injury by de-differentiated adult cells in a process named paligenosis. In the intestine, paligenosis is YAP-dependent and gives rise to Sca1+/Clu+ revival stem cells. However, the molecular mechanisms that regulate this process are unknown. Here we show that the protein TNFAIP8 (aka TIPE0) is a critical regulator of intestinal paligenosis and functions through the inhibition of basal microbiome-dependent PI3K/Akt signaling. Loss of TIPE0 results in hyperactivation of the Akt pathway, leading to resistance to ischemic injury. However, when the epithelium was disrupted through chemical means, the intestine was unable to regenerate. The loss of TIPE0 resulted in a baseline shift to more partially differentiated enterocytes, with inappropriate baseline activation of the Sca-1+/Clu+ regenerative program, but a lack of appropriate YAP/Sca-1/Clu induction after injury. Subsequent cellular signaling analysis demonstrated that PI3K/Akt signaling was enhanced upon loss of TIPE0 and that this was responsible both for the resistance to injury and lack of regenerative function. TIPE0 was found to be a critical regulator of PI3K/Akt signaling, inhibiting commensal microbial stimulation by extracting PIP2 from the plasma membrane and inhibiting PIP3 accumulation. In summary, TIPE0 is needed for intestinal homeostasis, and loss results in hyperactivation of PI3K/Akt-mediated signaling, leading to altered differentiation as well as an inability to respond injury, with a gut that fails to regenerate but also resists some injuries.

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