Changes In Fate Research Articles

Symplasmic isolation marks cell fate changes during somatic embryogenesis.

Cell-to-cell signalling is a major mechanism controlling plant morphogenesis. Transport of signalling molecules through plasmodesmata is one way in which plants promote or restrict intercellular signalling over short distances. Plasmodesmata are membrane-lined pores between cells that regulate the intercellular flow of signalling molecules through changes in their size, creating symplasmic fields of connected cells. Here we examine the role of plasmodesmata and symplasmic communication in the establishment of plant cell totipotency, using somatic embryo induction from Arabidopsis explants as a model system. Cell-to-cell communication was evaluated using fluorescent tracers, supplemented with histological and ultrastructural analysis, and correlated with expression of a WOX2 embryo reporter. We showed that embryogenic cells are isolated symplasmically from non-embryogenic cells regardless of the explant type (immature zygotic embryos or seedlings) and inducer system (2,4-dichlorophenoxyacetic acid or the BABY BOOM (BBM) transcription factor), but that the symplasmic domains in different explants differ with respect to the maximum size of molecule capable of moving through the plasmodesmata. Callose deposition in plasmodesmata preceded WOX2 expression in future sites of somatic embryo development, but later was greatly reduced in WOX2-expressing domains. Callose deposition was also associated with a decrease DR5 auxin response in embryogenic tissue. Treatment of explants with the callose biosynthesis inhibitor 2-deoxy-D-glucose supressed somatic embryo formation in all three systems studied, and also blocked the observed decrease in DR5 expression. Together these data suggest that callose deposition at plasmodesmata is required for symplasmic isolation and establishment of cell totipotency in Arabidopsis.

Simultaneous deletion of Prdm1 and Vsx2 enhancers in the retina alters photoreceptor and bipolar cell fate specification, yet differs from deleting both genes.

The transcription factor OTX2 is required for photoreceptor and bipolar cell formation in the retina. It directly activates the transcription factors Prdm1 and Vsx2 through cell type-specific enhancers. PRDM1 and VSX2 work in opposition, such that PRDM1 promotes photoreceptor fate and VSX2 bipolar cell fate. To determine how OTX2+ cell fates are regulated in mice, we deleted Prdm1 and Vsx2 or their cell type-specific enhancers simultaneously using a CRISPR/Cas9 in vivo retina electroporation strategy. Double gene or enhancer targeting effectively removed PRDM1 and VSX2 protein expression. However, double enhancer targeting favored bipolar fate outcomes, whereas double gene targeting favored photoreceptor fate. Both conditions generated excess amacrine cells. Combined, these fate changes suggest that photoreceptors are a default fate outcome in OTX2+ cells and that VSX2 must be present in a narrow temporal window to drive bipolar cell formation. Prdm1 and Vsx2 also appear to redundantly restrict the competence of OTX2+ cells, preventing amacrine cell formation. By taking a combinatorial deletion approach of both coding sequences and enhancers, our work provides new insights into the complex regulatory mechanisms that control cell fate choice.

Топос ДОМ в прозе А. П. Чехова 1890-х годов и ассоциированные с ним мотивы: смирение – бегство – уход

Purpose: The article examines the topos of HOME and following motifs A. P. Chekhov’s late prose. Results: The 1st story, A Woman’s Kingdom, portrays a rich young woman who is bored with her eventless everyday life. Being clever and open-hearted, Anna Akimovna searches for human values, such as empathetic communication, loving family, etc. The fictive time is Christmas Eve and Christmas Day, so genre tradition of Christmas narratives involves miracles connected with status changes (marriage, family reunification, poor relative gets award or gift from their rich uncle, an orphan finds their parents, and so on). Ritual temporality suggests some fate changes, and it makes Anna Akimovna dream about her matrimony matters with the worker Pimenov. At last she realizes that Pimenov is below her socially, and social instinct leads her closer to vapid and stupid such as Lysevich or important administrative official Krylin. Dull days she spends at home compel her to leave her dreams for humility. Thus, home-associated motifs in the story are boredom, weariness, and humility. The second narrative, At Home, is based on a return plot. Vera Kardina, who has received her education courses in Moscow, suffers from primitive customs of her home estate. Eventually, she realizes that she is becoming as rude and narrow-minded as the others inhabitants. Therefore, she decides to escape into an unhappy marriage. Corresponding motifs are the same: boredom and weariness, but the main character’s escape reveals the higher degree of disagreement with her current lifestyle. In the third short story, A Doctor’s Visit, the female character’s disease is explained by her inner anxiety related to her social role. Liza Lyalikova is the heir of a huge factory, but she worries that it makes her unhappy and lonely. Doctor Korolev is sure that the best way for Liza’s recovery is to quit the business. When the characters meet next morning, doctor believes that Liza would leave home. Both characters compare the factory with the devil, the absolute evil force, that forms the main motif of the short story. Here, the HOME topos is reconstructed in symbolic way. Conclusion: We conclude that the topos of home in Chekhov’s late works produces destructive emotions. Main characters of narratives don’t feel safe and calm in their homes. Motif dynamics show the development of characters’ positions from humility to deliverance.

Ascl1 is required to specify a subset of ventromedial hypothalamic neurons.

Despite clear physiological roles, the ventromedial hypothalamus (VMH) developmental programs are poorly understood. Here, we asked whether the proneural gene achaete-scute homolog 1 (Ascl1) contributes to VMH development. Ascl1 transcripts were detected in embryonic day (E) 10.5 to postnatal day 0 VMH neural progenitors. The elimination of Ascl1 reduced the number of VMH neurons at E12.5 and E15.5, particularly within the VMH-central (VMHC) and -dorsomedial (VMHDM) subdomains, and resulted in a VMH cell fate change from glutamatergic to GABAergic. We observed a loss of Neurog3 expression in Ascl1-/- hypothalamic progenitors and an upregulation of Neurog3 when Ascl1 was overexpressed. We also demonstrated a glutamatergic to GABAergic fate switch in Neurog3-null mutant mice, suggesting that Ascl1 might act via Neurog3 to drive VMH cell fate decisions. We also showed a concomitant increase in expression of the central GABAergic fate determinant Dlx1/2 in the Ascl1-null hypothalamus. However, Ascl1 was not sufficient to induce an ectopic VMH fate when overexpressed outside the normal window of competency. Combined, Ascl1 is required but not sufficient to specify the neurotransmitter identity of VMH neurons, acting in a transcriptional cascade with Neurog3.

CD8+ T-cell plasticity regulates vascular regeneration in type-2 diabetes.

In this study, we observe that the ischemic tissues of type-2 diabetic (T2D) patients and mice have significantly more CD8+ T-cells than that of their normoglycemic counterparts, respectively. However, the role of CD8+ T-cells in the pathogenesis of diabetic vascular complication has been less studied.Methods: We employed loss-of-function studies in mouse models using the non-lytic anti-CD8 antibody that blocks tissue infiltration of CD8+ T-cells into the injured tissue. We also performed genome-wide, single-cell RNA-sequencing of CD8+ T-cells to uncover their role in the pathogenesis of diabetic vascular diseases.Results: The vascular density is negatively correlated with the number of CD8+ T-cells in the ischemic tissues of patients and mice after injury. CD8+ T-cells or their supernatant can directly impair human and murine angiogenesis. Compared to normoglycemic mice that can regenerate their blood vessels after injury, T2D mice fail in this regeneration. Treatment with the CD8 checkpoint blocking antibody increases the proliferation and function of endothelial cells in both Leprdb/db mice and diet-induced diabetic Cdh5-Cre;Rosa-YFP lineage-tracing mice after ischemic injury. Furthermore, single-cell transcriptomic profiling reveals that CD8+ T-cells of T2D mice showed a de novo cell fate change from the angiogenic, tissue-resident memory cells towards the effector and effector memory cells after injury. Functional revascularization by CD8 checkpoint blockade is mediated through unleashing such a poised lineage commitment of CD8+ T-cells from T2D mice.Conclusion: Our results reveal that CD8+ T-cell plasticity regulates vascular regeneration; and give clinically relevant insights into the potential development of immunotherapy targeting vascular diseases associated with obesity and diabetes.

Diverse Education Based on Specific Conditions in Rural Areas of China

Connecting the spiritual outlook of the future society, education is the cornerstone of the rejuvenation of a nation. By the year 2018, nearly 600 million people lived in rural areas of China. For being such a large group, their basic necessities of life, and each and every move are closely related to the country’s destiny. So when these two parts get associated, the problem become larger and more difficult. Nowadays we should view the rural education with new eyes. Affected by the deepening of the market economy, the expansion of higher education and the tough job market, rural residents’ views on education are changing and can gradually be divided into two categories: the one is that education is the steering wheel which can lead to the change of fate; the other one is that the education is no longer the only way out. The cost of continuing a child’s education must be seen first. This article holds that in the process of revitalizing rural education, we should take measures according to local conditions basing on rural characteristics, and attach importance to the development of a multi-level and diverse education so that students can see more possibilities besides study and work, which will not only benefit the development of individuals, but also contribute to the progress of the whole society.

Abstract A05: Chromatin dynamics in vivo reveal a complex cell fate transition and master regulators of the development of pancreatic intraepithelial neoplasia

Abstract Pancreatic cancer initiation features a hallmark preneoplastic event wherein the acinar cell acquires a “duct-like” phenotype. Despite this transition in cell fate, little is known about the chromatin specification of pancreatic cell types and the epigenetic dysregulation of normal acinar cells in tumor initiation. To address these questions, we employed a lineage-traced autochthonous mouse model (Mist1-CreERT2 LSL-TdTomato +/- LSL-KrasG12D) to examine systematically perturbed acinar cells that give rise to early PanIN (PanIN 1); in addition, we sorted islet and ductal cell populations and describe the novel generation of Ptf1a-TdTomato mice to reliably sort pancreatic progenitors at e10.5. We observe in our system that Kras activation alone does not disturb acinar cell chromatin nor the histologic appearance of the pancreas. By contrast, caerulein alters chromatin significantly in metaplasia and even in regeneration, with putative enhancers derepressed despite normal histology. In the context of Kras activation and caerulein administration, we find a broad and stable reorganization of chromatin, reflecting cooperativity between oncogenic stress and an inflammatory insult. The chromatin of the reprogrammed acinar cell bears few, if any, ductal, progenitor, or islet features and instead reflects a largely novel cell fate. Detailed analysis of these acinar-derived cells in PanIN1 reveals unexpected features of the newly acquired cell state. First, we find robust induction of epithelial-mesenchymal transition (EMT) gene-proximal enhancer chromatin that is not reflected in the EMT-related transcriptional state. Second, we find evidence of a derepression of a foregut/gastric transcriptional program, not otherwise evident in normal cell types of the adult mouse pancreas. Finally, our genome-wide chromatin accessibility and transcriptome data nominate the pioneer transcription factor Foxa1 as a master regulator of the complex cell fate transition from acinar cell to PanIN. Together, our findings suggest that (1) the acquired acinar cell fate reflects neither true metaplasia nor transdifferentiation nor dedifferentiation events, (2) a gastric/foregut transcript program is unveiled, (3) the process of EMT begins in ADM, and finally (4) that Foxa1 appears to coordinate the change in cell fate. Our data thus demonstrate that histologic appearance does not adequately reflect cellular identity, highlighting the complex spectrum of features acquired in the transition to neoplasia and identifying a putative coordinator of the PanIN cell fate. Citation Format: Rohit Chandwani, Alexa Osterhoudt, Richard Koche, Steven D. Leach. Chromatin dynamics in vivo reveal a complex cell fate transition and master regulators of the development of pancreatic intraepithelial neoplasia [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A05.

Transformation of amino acid tyrosine in chromophoric organic matter solutions: Generation of peroxide and change of bioavailability.

Studying the photochemical transformation mechanism of dissolved free amino acids (DFAA) in chromophoric dissolved organic matter (CDOM) solution facilitates the understanding of DFAA's environmental fate and bioavailability change upon solar irradiation in natural surface waters. Tyrosine oxidation product (Tyr-OH) was synthesized to quantify the primary transformation product (tyrosine peroxide, Tyr-OOH) in CDOM solution. Both reactions between superoxide radical anion (O2-) and tyrosine radical (Tyr) and between singlet oxygen (1O2) and tyrosine (Tyr) yield Tyr-OOH, which is subsequently transformed into Tyr-OH. The reaction between O2- and Tyr not only generated Tyr-OOH but also caused the regeneration of Tyr. O2- and 1O2 contributed 30-44% to Tyr's transformation in CDOM solutions at pH 8.0, in which 1O2 oxidation accounted for 6-11%. The contribution of O2- to Tyr's phototransformation process was the difference between the total contribution of O2- and 1O2 and the individual contribution of 1O2. Compared with the fast assimilation of Tyr, Tyr-OH was stable in natural water under dark incubation, indicating that phototransformation decreased the bioavailability of Tyr.

Abstract A59: CRISPR screen identifies global regulation of H3K36me2 as an epigenomic mechanism underlying epithelial plasticity in pancreatic ductal adenocarcinoma

Abstract Metastasis and chemoresistance, the most lethal consequences of cancer progression, are associated with a form of cellular plasticity known as the epithelial-to-mesenchymal transition (EMT). During this process, epithelial cells disassemble their intercellular junctions and acquire morphologic and motile phenotypes reminiscent of fibroblasts. As a form of cell fate change, EMT is likely driven by alterations to the chromatin landscape. Indeed, there is increasing evidence that epigenetic modifiers act at the promoters of key epithelial and mesenchymal genes. However, since much of this evidence is focused on specific, handpicked loci, the true extent to which EMT-mediated transcriptional rewiring depends on epigenetic factors remains largely unexplored. We therefore performed a targeted CRISPR screen in plastic pancreatic ductal adenocarcinoma (PDA) cell lines to unbiasedly identify epigenetic modifiers critical for this process. More specifically, we found that in the absence of the methyltransferase Nsd2, a writer of H3K36me2, our once plastic cells are fixed in the epithelial state, while in the absence of the demethylase Kdm2a, an eraser of H3K36me2, they remain in the mesenchymal state. These genes were also found to have functional implications for invasion and metastasis in PDA. Since loss of Nsd2 and Kdm2a results in global loss and gain of H3K36me2, respectively, we examined the function of the histone mark itself with a mutant histone that can no longer be methylated at K36 (H3K36M). Expression of H3K36M is sufficient to push plastic cells of various cancer types to an epithelial fate, implying that the modification itself, regardless of which writer or eraser is active, is the key regulator of EMT. Furthermore, we performed histone mass spectrometry and found that even without any genetic perturbations, higher global levels of H3K36me2 are indeed associated with the mesenchymal state. Finally, we mapped H3K36me2 across the genome and found that widespread loss of H3K36me2 leads to dramatic alterations in the enhancer landscape, which in turn correlates with the observed transcriptomic changes. While H3K36me2 is known to play important roles in DNA repair and mRNA splicing, our findings demonstrate a novel role for H3K36me2 as a central epigenetic regulator of EMT and metastasis by regulating enhancer activity genome wide. Just as importantly, despite massive changes in global H3K36me2 levels, the cellular phenotypes affected are remarkably specific to cell identity and differentiation. Collectively, these results implicate a novel mechanism by which global epigenomic changes underpin the transcriptional rewiring associated with epithelial plasticity. Citation Format: Salina Yuan, Ramakrishnan Natesan, Francisco J. Sanchez-Rivera, Jinyang Li, Natarajan V Bhanu, Taiji Yamazoe, Jeffrey H. Lin, Allyson J. Merrell, Stacy K. Thomas, Junwei Shi, Ben A. Garcia, Scott W. Lowe, Irfan A. Asangani, Ben Z. Stanger. CRISPR screen identifies global regulation of H3K36me2 as an epigenomic mechanism underlying epithelial plasticity in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A59.

Submersion of impacting spheres at low Bond and Weber numbers owing to a confined pool

We numerically investigate the mechanism resulting in fate change of a hydrophobic sphere impacting onto a confined pool, that is, at the same impact speed, it does not submerge in a wide pool but does in a narrow pool. We find that the reflection of the impact-induced gravity-capillary waves from the pool boundary is responsible for this phenomenon. In particular, the return of the wave to the symmetry axis may coincide with the rising of the impacting sphere to the water surface, which corresponds to the critical conditions of the fate change. Moreover, for the spheres at the onset of submersion in a wide pool, our analysis suggests that this scenario also accounts for an interesting observation in the numerical simulations. That is, the effective pool size $S_{c}$ , beyond which the submersion of impacting spheres is no longer affected by the pool size $S$ , is mainly dependent on the sphere diameter, no matter whether the surface waves are the capillary or gravity waves. For $S<S_{c}$ , two important pool sizes ( $S_{w,1}$ and $S_{w,2}$ , and $S_{w,2}\geqslant S_{w,1}$ ) are identified at the same impact speed, and the sphere submersion takes place at $S_{w,1}\leqslant S\leqslant S_{w,2}$ . Based on the flow features identified in simulations, a scaling law is proposed to correlate the Weber number and Bond number with $S_{w}$ . The theoretical prediction is shown to agree well with the numerical results.

Reprogramming: identifying the mechanisms that safeguard cell identity.

Development and homeostasis rely upon concerted regulatory pathways to establish the specialized cell types needed for tissue function. Once a cell type is specified, the processes that restrict and maintain cell fate are equally important in ensuring tissue integrity. Over the past decade, several approaches to experimentally reprogram cell fate have emerged. Importantly, efforts to improve and understand these approaches have uncovered novel molecular determinants that reinforce lineage commitment and help resist cell fate changes. In this Review, we summarize recent studies that have provided insights into the various chromatin factors, post-transcriptional processes and features of genomic organization that safeguard cell identity in the context of reprogramming to pluripotency. We also highlight how these factors function in other experimental, physiological and pathological cell fate transitions, including direct lineage conversion, pluripotency-to-totipotency reversion and cancer.

Growth Plate Chondrocytes: Skeletal Development, Growth and Beyond.

Growth plate chondrocytes play central roles in the proper development and growth of endochondral bones. Particularly, a population of chondrocytes in the resting zone expressing parathyroid hormone-related protein (PTHrP) is now recognized as skeletal stem cells, defined by their ability to undergo self-renewal and clonally give rise to columnar chondrocytes in the postnatal growth plate. These chondrocytes also possess the ability to differentiate into a multitude of cell types including osteoblasts and bone marrow stromal cells during skeletal development. Using single-cell transcriptomic approaches and in vivo lineage tracing technology, it is now possible to further elucidate their molecular properties and cellular fate changes. By discovering the fundamental molecular characteristics of these cells, it may be possible to harness their functional characteristics for skeletal growth and regeneration. Here, we discuss our current understanding of the molecular signatures defining growth plate chondrocytes.

Dkk1 Controls Cell-Cell Interaction through Regulation of Non-nuclear β-Catenin Pools.

SummaryDickkopf-1 (Dkk1) is a secreted Wnt antagonist with a well-established role in head induction during development. Numerous studies have emerged implicating Dkk1 in various malignancies and neurodegenerative diseases through an unknown mechanism. Using zebrafish gastrulation as a model for collective cell migration, we unveil such a mechanism, identifying a role for Dkk1 in control of cell connectivity and polarity in vivo, independent of its known function. We find that Dkk1 localizes to adhesion complexes at the plasma membrane and regions of concentrated actomyosin, suggesting a direct involvement in regulation of local cell adhesion. Our results show that Dkk1 represses cell polarization and integrity of cell-cell adhesion, independently of its impact on β-catenin protein degradation. Concurrently, Dkk1 prevents nuclear localization of β-catenin by restricting its distribution to a discrete submembrane pool. We propose that redistribution of cytosolic β-catenin by Dkk1 concomitantly drives repression of cell adhesion and inhibits β-catenin-dependent transcriptional output.

Gibberellin Promotes Sweetpotato Root Vascular Lignification and Reduces Storage-Root Formation.

Sweetpotato yield depends on a change in the developmental fate of adventitious roots into storage-roots. The mechanisms underlying this developmental switch are still unclear. We examined the hypothesis claiming that regulation of root lignification determines storage-root formation. We show that application of the plant hormone gibberellin increased stem elongation and root gibberellin levels, while having inhibitory effects on root system parameters, decreasing lateral root number and length, and significantly reducing storage-root number and diameter. Furthermore, gibberellin enhanced root xylem development, caused increased lignin deposition, and, at the same time, decreased root starch accumulation. In accordance with these developmental effects, gibberellin application upregulated expression levels of sweetpotato orthologues of Arabidopsis vascular development regulators (IbNA075, IbVND7, and IbSND2) and of lignin biosynthesis genes (IbPAL, IbC4H, Ib4CL, IbCCoAOMT, and IbCAD), while downregulating starch biosynthesis genes (IbAGPase and IbGBSS) in the roots. Interestingly, gibberellin downregulated root expression levels of orthologues of the Arabidopsis BREVIPEDICELLUS transcription factor (IbKN2 and IbKN3), regulator of meristem maintenance. The results substantiate our hypothesis and mark gibberellin as an important player in regulation of sweetpotato root development, suggesting that increased fiber formation and lignification inhibit storage-root formation and yield. Taken together, our findings provide insight into the mechanisms underlying sweetpotato storage-root formation and provide a valuable database of genes for further research.

The Ca2+ export pump PMCA clears near-membrane Ca2+ to facilitate store-operated Ca2+ entry and NFAT activation.

Ca2+ signals, which facilitate pluripotent changes in cell fate, reflect the balance between cation entry and export. We found that overexpression of either isoform of the Ca2+-extruding plasma membrane calcium ATPase 4 (PMCA4) pump in Jurkat T cells unexpectedly increased activation of the Ca2+-dependent transcription factor nuclear factor of activated T cells (NFAT). Coexpression of the endoplasmic reticulum-resident Ca2+ sensor stromal interaction molecule 1 (STIM1) with the PMCA4b splice variant further enhanced NFAT activity; however, coexpression with PMCA4a depressed NFAT. No PMCA4 splice variant dependence in STIM1 association was observed, whereas partner of STIM1 (POST) preferentially associated with PMCA4b over PMCA4a, which enhanced, rather than inhibited, PMCA4 function. A comparison of global and near-membrane cytosolic Ca2+ abundances during store-operated Ca2+ entry revealed that PMCA4 markedly depressed near-membrane Ca2+ concentrations, particularly when PMCA4b was coexpressed with STIM1. PMCA4b closely associated with both POST and the store-operated Ca2+ channel Orai1. Furthermore, POST knockdown increased the near-membrane Ca2+ concentration, inhibiting the global cytosolic Ca2+ increase. These observations reveal an unexpected role for POST in coupling PMCA4 to Orai1 to promote Ca2+ entry during T cell activation through Ca2+ disinhibition.

Inactivation of FAM20B causes cell fate changes in annulus fibrosus of mouse intervertebral disc and disc defects via the alterations of TGF-β and MAPK signaling pathways

Intervertebral disc (IVD) disorder is often caused by the defect of annulus fibrosus (AF), especially that of the outer AF. Studies about the mechanisms governing the development of the outer AF are needed for a better understanding of pathogenesis of IVD defects. Glycosaminoglycans (GAGs) are essential components of extracellular matrix (ECM) in AF. FAM20B is a newly identified xylose kinase that catalyzes the biosynthesis of GAGs. In this study, we created Fam20B conditional knockout (cKO) mice in which FAM20B was inactivated in type I collagen-expressing cells, the main type of cells in the outer AF of IVD. The cKO mice showed severe spine deformity and remarkable IVD defects associated with AF malformation. The AF of cKO mice had a lower level of chondroitin sulfate and heparan sulfate, and the outer AF cells lost their normal fibroblast-like morphology and acquired chondrocyte phenotypes, expressing a higher level of Sox 9 and type II collagen along with a reduced level of type I collagen. The level of phospho-Smad 2 and phospho-Smad 3, and that of scleraxis, a downstream target molecule of canonical TGF-β signaling pathway were significantly lower in the AF of cKO mice. The AF in cKO mice also manifested altered levels in the molecules associated with the activations of MAPK pathway; the changes included the increase of phospho-P38 and phospho-ERK and a decrease of phospho-JNK. These results indicate that FAM20B plays an essential role in the development of AF by regulating the TGF-β signaling and MAPK signaling pathways.

Evolving principles underlying neural lineage conversion and their relevance for biomedical translation.

Scientific and technological advances of the past decade have shed light on the mechanisms underlying cell fate acquisition, including its transcriptional and epigenetic regulation during embryonic development. This knowledge has enabled us to purposefully engineer cell fates in vitro by manipulating expression levels of lineage-instructing transcription factors. Here, we review the state of the art in the cell programming field with a focus on the derivation of neural cells. We reflect on what we know about the mechanisms underlying fate changes in general and on the degree of epigenetic remodeling conveyed by the distinct reprogramming and direct conversion strategies available. Moreover, we discuss the implications of residual epigenetic memory for biomedical applications such as disease modeling and neuroregeneration. Finally, we cover recent developments approaching cell fate conversion in the living brain and define questions which need to be addressed before cell programming can become an integral part of translational medicine.

39. CLINICAL INTERPRETATION OF NON-STANDARD PGT-A RESULTS

Introduction Preimplantation genetic testing for chromosome anomalies has been used to improve reproductive outcomes for nearly 20 years. But in some cases, the effectiveness of Preimplantation genetic testing for aneuploidies (PGT-A) may be reduced. Sources of errors during PGT-A are low sensitivity or specificity of either method or interpretation. The threshold value that separates normal result from pathological is not fully formalized for current methods of PGT-A (aCGH, NGS). The decision on the status of the sample is taken by the interpreter (the expert). Can preimplantation genetic testing for aneuploidies reduce IVF efficiency? Yes, if euploid embryo is defined as abnormal or if abnormal embryo is recommended for transfer. To estimate how much the interpretation of the same PGT-A data may differ between the readers we designed the project «Many men, many minds». Materials & Methods We compared the experts’ interpretation with some arbitrary reference. The references are the clinical reports, created in Genetico Laboratory, based on the joined decision after discussion of program data between the clinical geneticist, laboratory geneticist and biologist. Participants of the study were 10 readers, 2 of which are outside Russia. We included 50 samples analyzed by aCGH (Cytochip 24sure, Illumina) and 50 samples analyzed by NGS (Veriseq, Illumina) in our project. The samples for interpretation were not representative of routine samples of Genetico Laboratory (not typical samples were chosen for interpretation, but rather most hard to read ones). The experts received data which include chromosome profiles, QC metrics, and chromosome imbalances which automatically determined by the BlueFuse Multi program (Illumina). We asked experts to provide an answer containing molecular karyotype for each sample and clinical recommendations for embryo transfer. Results Interpretation of PGT-A results depends of expert's “school of thought” – at the same lab opinions tend to be more concordant. And interpretation of chromosome profiles by another expert can lead to a change in the clinical fate of the embryo. It is advisable to discuss PGT-A results between specialists before clinical report making. Interpretation of hard to read data of PGT-A is difficult and the role of the human factor very big. The “low specificity” of an expert may led to samples being recognized as aneuploid, although another expert using the same data will give an estimate of “norm” (or vice versa). Reanalysis of PGT-A data may lead to significant changes in clinical decisions regarding the fate of embryos. And after several months we tested if one interpreter could have two different opinions regarding the same sample. The participants of this additional experiment became 5 experts from initial list of participants. We asked them about the interpretation of 10 samples from the initial list. We did not reveal that these samples were same the experts interpreted several months before. We saw that the same expert may have different opinions on the same sample. Conclusions PGT-A allows to increase IVF efficiency, however, we have room for improvement. It is possible to work on improving the process of data interpretation.

3167 – DEVELOPMENTAL CHANGES IN THE CHROMATIN ACCESSIBILITY OF HEMATOPOIETIC STEM CELLS ARE ASSOCIATED WITH DISTINCT GENETIC PROGRAMS

Hematopoietic stem cells (HSCs) undergo a developmental switch in neonatal mice hallmarked by a decrease in self-renewing divisions and entry into quiescence. Molecular determinants like dynamic changes in the chromatin landscape across the switch hold key insights into developmental changes in cell fate and function but remain poorly understood. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) has emerged as a powerful tool to study chromatin accessibility in rare cells and in combination with genome-wide expression profiling has the potential to resolve cell specific transcriptional networks. To explore the regulation of the switch between fetal liver and adult bone marrow HSC states as well as during the first stages of hematopoietic differentiation, we here asses the changes in chromatin accessibility and genome-wide gene expression patterns in immunophenotypically defined HSCs and lymphoid primed multipotent progenitors (LMPPs). Chromatin accessibility unique to adult bone marrow HSCs were found to be enriched for putative binding sites of the megakaryocyte and erythroid lineage transcription factors Nuclear factor erythroid 2 (NFE2). In parallel, adult HSC gene expression and epigenetic profiles were highly enriched for the megakaryocyte gene signature which is strikingly absent in the E14.5 fetal liver. Taken together, our results identify a previously unrecognized hallmark of the developmental switch of the HSC compartment centered around acquisition of a megakaryocyte bias. These age specific features may have important implications in host response to environmental insults.

Transcription factors in SOX family: Potent regulators for cancer initiation and development in the human body.

Transcription factors (TFs) have a key role in controlling the gene regulatory network that sustains explicit cell states in humans. However, an uncontrolled regulation of these genes potentially results in a wide range of diseases, including cancer. Genes of the SOX family are indeed crucial as deregulation of SOX family TFs can potentially lead to changes in cell fate as well as irregular cell growth. SOX TFs are a conserved group of transcriptional regulators that mediate DNA binding through a highly conserved high-mobility group (HMG) domain. Accumulating evidence demonstrates that cell fate and differentiation in major developmental processes are controlled by SOX TFs. Besides; numerous reports indicate that both up- and down-regulation of SOX TFs may induce cancer progression. In this review, we discuss the involvement of key TFs of SOX family in human cancers.