Defining the regulatory landscape underlying the stemness of human hematopoietic stem cells

The mammalian hematopoietic system is comprised of more than 10 distinct cell types. All are derived from a common progenitor cell, the hematopoietic stem cell (HSC). The HSCs are defined by two main functions; the multilineage differentiation to all terminal blood cell types and their ability to sustain the hematopoietic system through the entire life of an individual through self-renewal. These two features, multilineage differentiation and self-renewal render HSCs both a formidable clinical tool as well as the ideal model for stem cell biology. This work addresses both these features of human HSCs in three experimental approaches. The first one evaluates the efficacy of ex vivo maintenance and expansion of HSCs in the presence of different small chemical molecules and aims at the development of an optimized protocol for ex vivo HSC expansion. HSCs were maintained in the culture under the different conditions and the effect of the small molecules on the immunophenotype of expanded HSCs was investigated. Furthermore, the functional differences as a result of the expansion regimens were studied by assessing the capacity of expanded HSC to repopulate irradiated bone-marrow as well as their multilineage differentiation potential through transplantation experiments in relevant mouse models. Additionally, RNAseq analysis of the differentially expanded populations identifies the transcriptional changes in response to the different expansion protocols tested. Taking into consideration this information, a novel optimized protocol for the ex vivo expansion of human HSC is proposed. The second approach aims at elucidating the epigenetic regulatory landscape during the differentiation of human HSCs into erythrocytes. Dense time-course of chromatin accessibility with DNase I-seq and gene expression using total RNAseq resulted in high resolution maps of the developmentally responsive DNase I Hypersensitive Sites (DHS) and genes during erythropoiesis. Subsequently, the temporal interactions between individual DHS and genes during development are investigated using mathematical modelling, resulting in the identification of epigenetic regulatory modules implicated in lineage commitment and differentiation. Assessment of clonogenic capacity and lineage potential of the differentiating populations along erythropoiesis confirms the existence of discrete functional states that correspond to the regulatory modules identified. Finally, bioinformatic analysis of the single-cell transcriptional dynamics of the populations during erythroid-megakaryocytic development identify distinct populations of progenitor cells and how these diverge during lineage commitment. Finally, the third experimental approach attempts to identify and isolate progenitor populations with defined linage potential. Using single-cell flow-cytometric index sorting, individual hematopoietic progenitor cells are interrogated for both their immunophenotype and their differentiation potential. This resulted in the development of a comprehensive set of cell-surface markers able to discriminate functionally distinct progenitors. Finally, a novel cell-surface marker is identified that can distinguish erythroid progenitors.Στο αιμοποιητικό σύστημα των θηλαστικών, απαντώνται πάνω από 10 διακριτοί διαφοροποιημένοι τύποι κυττάρων και όλοι προέρχονται από έναν προγονικό αιμοποιητικό κυτταρικό τύπο, το αρχέγονο αιμοποιητικό στελεχιαίο κύτταρο (Hematopoietic Stem Cell, HSC). Τα HSCs, πέραν από τη δυνατότητά τους να διαφοροποιούνται προς όλες τις αιμοποιητικές σειρές (πολυγραμμική διαφοροποίηση), είναι αυτά τα οποία συντηρούν ολόκληρο το αιμοποιητικό σύστημα εφ’ όρου ζωής χάρη στην ικανότητά τους να διατηρούν σταθερό τον πληθυσμό τους μέσω της αυτό-ανανέωσης (self-renewal). Αυτά τα δύο λοιπόν χαρακτηριστικά, η αυτό-ανανέωση και η πολυγραμμική διαφοροποίηση είναι αυτά που καθιστούν τα HSCs ένα ισχυρό κλινικό εργαλείο, καθώς και το κατάλληλο μοντέλο για τη βιολογία των στελεχιαίων κυττάρων. Σκοπός της παρούσας εργασίας είναι η διερεύνηση των επιγενετικών, κατά βάση, μηχανισμών που συμμετέχουν στις δύο αυτές λειτουργίες των HSCs του ανθρώπου. Αρχικά, διερευνήθηκαν μέθοδοι ex vivo έκπτυξης των HSCs με την επίδραση μικρών χημικών μορίων (small molecules) και επιδιώχθηκε η ανάπτυξη πρωτοκόλλου που επιτρέπει την βέλτιστη έκπτυξη τους. Μελετήθηακν οι επιδράσεις των μορίων αυτών τόσο στο φαινότυπο των HSCs όσο και στην ικανότητα ενοίκησης ανοσοκατεσταλμένου μυελού των οστών, και διαφοροποίησης με πειράματα ξενο-μεταμόσχευσης σε σχετικό ζωικό μοντέλο. Εν συνεχεία, μελετήθηκαν οι διαφορές στο μεταγραφικό επίπεδο με αλληλούχηση μεταγραφώματος RNAseq και βιοπληροφορική ανάλυση ενώ τέλος ταυτοποιήθηκε ένας συνδυασμός μορίων για την βέλτιστη έκπτυξη των HSCs. Στο δεύτερο σκέλος της εργασίας, διερευνήθηκε η δυναμική του επιγενετικού τοπίου που συμμετέχει στην διαφοροποίηση των HSCs του ανθρώπου προς την ερυθρά σειρά. Αναλύθηκε η προσβασιμότητα χρωματίνης με DNase I-seq και οι μεταγραφικές διαφορές με RNAseq κατά την ex vivo επαγωγή της ερυθροποίησης και δημιουργήθηκαν γενετικοί χάρτες με όλα τα ρυθμιστικά στοιχεία (DNase I Hypersensitive Sites, DHS) και τα γονίδια τα οποία συμμετέχουν κατά την ερυθροποίησης. Εν συνεχεία, με τη χρήση μαθηματικών μοντέλων μελετήθηκαν οι αλληλεπιδράσεις μεταξύ DHS και γονιδίων και πως αυτές μεταβάλλονται κατά τη διαφοροποίηση και ταυτοποιήθηκαν επιγενετικές λειτουργικές δομές που συμμετέχουν στους μηχανισμούς διαφοροποίησης και δέσμευσης στην εκάστοτε κυτταρική σειρά. Πειράματα πολυγραμμικής διαφοροποίησης και κλωνογενούς ικανότητας των κυττάρων κατά την ερυθροποίηση καταδεικνύουν την ύπαρξη διακριτών λειτουργικών σταδίων κατά την διαφοροποίηση, ενδεικτικών των επιγενετικών λειτουργικών δομών που ταυτοποιήθηκαν νωρίτερα. Τέλος, διερευνήθηκαν οι μεταβολές των πληθυσμών των προγονικών κυττάρων στο μεταγραφικό επίπεδο κατά τον διαχωρισμό της ερυθροειδικής και μεγακαρυοκυτταρικής σειράς με ανάλυση του μεταγραφώματος σε μονήρη κύτταρα (single-cell RNAseq) με βιοπληροφορική ανάλυση. Στο τρίτο και τελευταίο σκέλος, έγινε προσπάθεια απομόνωσης των αιμοποιητικών προγονικών κυττάρων με διαφορικό δυναμικό διαφοροποίησης προς τις διάφορες αιμοποιητικές σειρές. Με τη χρήση μεθόδων απομόνωσης μονήρων κυττάρων με κυτταρομετρία ροής και πειραμάτων κλωνογενούς ικανότητας και πολυγραμμικής διαφοροποίησης ταυτοποιήθηκαν προγονικά κύτταρα με συγκεκριμένο διαφοροποιητικό δυναμικό και διακριτό ανοσοφαινότυπο. Τέλος, προτείνεται μια σειρά από δείκτες επιφανείας ικανοί να τα διαχωρίσουν τους διάφορους προγονικούς τύπους βάσει του διαφοροποιητικού τους δυναμικού, ενώ ταυτοποιούνται νέοι δείκτες που ικανοί να διαχωρίσουν τους προγόνους της ερυθράς σειράς

A short phylogenetically informative cpDNA fragment for the identification of Pinus species

The genus Pinus L. consists of ca. 110 ecologically and economically important species extending from the arctic zone to the tropics. Nevertheless, there is little information in the literature on DNA-based methods for the identification of pine species. Here, we identified a new cpDNA fragment (trnV-H/x-h) able to differentiate among pine species and correctly depict the phylogeny within the genus. The fragment was identified based on PCR-RFLP profiles and primers designed based on the sequences of six Pinus species naturally occurring in Greece (Pinus brutia Ten., Pinus halepensis Mill., Pinus leucodermis Antoine, Pinus nigra J.F. Arnold, Pinus pinea L., and Pinus sylvestris L.). We analyzed 90 highly similar pine sequences retrieved from the GenBank to investigate specificity of our marker and the haplotypes found showed to be specific to Pinus and able to differentiate among 39 different species. The phylogenetic tree constructed using these species, correctly depicted the phylogeny of the genus up to the subsection level. These characteristics together with its relatively small size (376-418 bp) make the trnV-H/x-h marker useful for pine identification even in contexts where DNA is degraded, such as in timber tracing, forensic botany and palaeobotanical investigation

Attenuation of cancer proliferation by suppression of glypican-1 and its pleiotropic effects in neoplastic behavior

Glypicans (GPC1-6) are associated with tumorigenic processes and their involvement in neoplastic behavior has been discussed in different cancer types. Here, a cancer-wide GPC expression study, using clinical cancer patient data in The Cancer Genome Atlas, reveals net upregulation of GPC1 and GPC2 in primary solid tumors, whereas GPC3, GPC5 and GPC6 display lowered expression pattern compared to normal tissues. Focusing on GPC1, survival analyses of the clinical cancer patient data reveal statistically significant correlation between high expression of GPC1 and poor prognosis in 10 particular cancer types i.e., bladder urothelial carcinoma, brain lower grade glioma, liver hepatocellular carcinoma, colon adenocarcinoma, kidney renal clear cell carcinoma, lung adenocarcinoma, mesothelioma, ovarian serous cystadenocarcinoma, uterine corpus endometrial carcinoma and uveal melanoma. In vitro studies targeting GPC1 expression by CRISPR/Cas9 or siRNA or treatment with an anti-GPC1 antibody resulted in attenuation of proliferation of cancer cells from bladder carcinoma, glioma and hepatocellular carcinoma patients (T24, U87 and HepG2 cells). Further, overexpression of GPC1 exhibited a significant and negative correlation between GPC1 expression and proliferation of T24 cells. Attempt to reveal the mechanism through which downregulation of GPC1 leads to attenuation of tumor growth using systematic Ingenuity Pathway Analysis indicate that suppression of GPC1 results in ECM-mediated inhibition of specific pro-cancer signaling pathways involving TGF-β and p38 MAPK. Identified differential expression and pleiotropic effects of GPCs in specific cancer types emphasize their potential of as novel diagnostic tools and prognostic factors and open doors for future GPC targeted therapy

Data from: Evolution under dietary restriction increases male reproductive performance without survival cost

Dietary restriction (DR), a reduction in nutrient intake without malnutrition, is the most reproducible way to extend lifespan in a wide range of organisms across the tree of life, yet the evolutionary underpinnings of the DR effect on lifespan are still widely debated. The leading theory suggests that this effect is adaptive and results from reallocation of resources from reproduction to somatic maintenance in order to survive periods of famine in nature. However, such response would cease to be adaptive when DR is chronic and animals are predicted to allocate more resources to reproduction. Nevertheless, chronic DR can also increase the strength of selection resulting in the evolution of more robust genotypes. We evolved Drosophila melanogaster fruitflies on ‘DR’, ‘standard’ and ‘high’ adult diets in replicate populations with overlapping generations. After ~25 generations of experimental evolution, male ‘DR’ flies had higher fitness than males from ‘standard’ and ‘high’ populations. Strikingly, this increase in reproductive success did not come at a cost to survival. Our results suggest that sustained DR selects for more robust male genotypes, which are overall better able to convert resources into energy, which they allocate mostly to reproduction

Evolution under dietary restriction increases male reproductive performance without survival cost

Dietary restriction (DR), a reduction in nutrient intake without malnutrition, is the most reproducible way to extend lifespan in a wide range of organisms across the tree of life, yet the evolutionary underpinnings of the DR effect on lifespan are still widely debated. The leading theory suggests that this effect is adaptive and results from reallocation of resources from reproduction to somatic maintenance, in order to survive periods of famine in nature. However, such response would cease to be adaptive when DR is chronic and animals are selected to allocate more resources to reproduction. Nevertheless, chronic DR can also increase the strength of selection resulting in the evolution of more robust genotypes. We evolved Drosophila melanogaster fruit flies on ‘DR’, ‘standard’ and ‘high’ adult diets in replicate populations with overlapping generations. After approximately 25 generations of experimental evolution, male ‘DR’ flies had higher fitness than males from ‘standard’ and ‘high’ populations. Strikingly, this increase in reproductive success did not come at a cost to survival. Our results suggest that sustained DR selects for more robust male genotypes that are overall better in converting resources into energy, which they allocate mostly to reproduction

Zajitschek et al 2016 Proc B Data

Description of data is provided in the data file

Unraveling hallmark suitability for staging pre- and post-implantation stem cell models

International audienceThe advent of novel 2D and 3D models for human development, including trophoblast stem cells and blastoids, has expanded opportunities for investigating early developmental events, gradually illuminating the enigmatic realm of human development. While these innovations have ushered in new prospects, it has become essential to establish well-defined benchmarks for the cell sources of these models. We aimed to propose a comprehensive characterization of pluripotent and trophoblastic stem cell models by employing a combination of transcriptomic, proteomic, epigenetic, and metabolic approaches. Our findings reveal that extended pluripotent stem cells share many characteristics with primed pluripotent stem cells, with the exception of metabolic activity. Furthermore, our research demonstrates that DNA hypomethylation and high metabolic activity define trophoblast stem cells. These results underscore the necessity of considering multiple hallmarks of pluripotency rather than relying on a single criterion. Multiplying hallmarks alleviate stage-matching bias

Modeling human extraembryonic mesoderm cells using naive pluripotent stem cells

International audienceA hallmark of primate postimplantation embryogenesis is the specification of extraembryonic mesoderm (EXM) before gastrulation, in contrast to rodents where this tissue is formed only after gastrulation. Here, we discover that naive human pluripotent stem cells (hPSCs) are competent to differentiate into EXM cells (EXMCs). EXMCs are specified by inhibition of Nodal signaling and GSK3B, are maintained by mTOR and BMP4 signaling activity, and their transcriptome and epigenome closely resemble that of human and monkey embryo EXM. EXMCs are mesenchymal, can arise from an epiblast intermediate, and are capable of self-renewal. Thus, EXMCs arising via primate-specific specification between implantation and gastrulation can be modeled in vitro. We also find that most of the rare off-target cells within human blastoids formed by triple inhibition (Kagawa et al., 2021) correspond to EXMCs. Our study impacts our ability to model and study the molecular mechanisms of early human embryogenesis and related defects

Integrated multi-omics reveal polycomb repressive complex 2 restricts human trophoblast induction.

Funder: The Marks lab is supported by an NWO-XS grantFunder: M.V. is part of the Oncode Institute, which is partly funded by the Dutch Cancer SocietyFunder: D.W.Z. is part of the Oncode Institute, which is partly funded by the Dutch Cancer SocietyHuman naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human naive pluripotent stem cells are not epigenetically unrestricted, but instead possess chromatin mechanisms that oppose the induction of alternative cell fates