scholarly journals Διερεύνηση και ανάλυση της επιγενετικής ρύθμισης των λειτουργιών των αιμοποιητικών στελεχιαίων κυττάρων του ανθρώπου

2019 ◽  
Author(s):  
Γρηγόριος Γεωργολόπουλος
Keyword(s):  
Stem Cell ◽  
Small Molecules ◽  
Single Cell ◽  
Hematopoietic Stem Cell ◽  
Ex Vivo ◽  
Dnase I ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites

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.

Enhanced Human Hematopoietic Stem Cell Self-Renewal Enabled by Controlling Feedback Signaling From Lineage Committed Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1274-1274
Author(s):  
Elizabeth Csaszar ◽  
Daniel Kirouac ◽  
Mei Yu ◽  
Caryn Ito ◽  
Peter W. Zandstra
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Population ◽  
Ex Vivo ◽  
Stem Cell Population ◽  
Fed Batch ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Numbers

Abstract Abstract 1274 Clinical outcomes of hematopoietic stem cell (HSC) transplantation are correlated with infused progenitor cell dose. Limited cell numbers in a typical umbilical cord blood (UCB) unit restricts the therapeutic potential of UCB and motivates ex vivo expansion of these cells. Strategies to grow HSCs have relied on the supplement of molecules acting directly on the stem cell population; however, in all cases, sustained HSC growth is limited by the concurrent growth of more mature cells and their endogenously produced inhibitory signaling factors. Despite increasing evidence for the important role of intercellular (between cell) communication networks, the identity and impact of non-stem cell autonomous feedback signaling remains poorly understood. Simultaneous kinetic tracking of more than 30 secreted factors produced during UCB culture, including TGF-b1, MIP-1b, and MCP-1, in combination with computational simulations of cell population dynamics, enabled us to develop a global control strategy predicted to reduce inhibitory paracrine signaling and, consequently, increase HSC self-renewal. By maintaining endogenously produced ligands at specified levels using a tuneable fed-batch (automated media dilution) strategy, we achieved significant improvements in expansions of total cell numbers (∼180-fold), CD34+ cells (∼80-fold), and NOD/SCID/IL-2Rgc-null (NSG) repopulating cells (∼11-fold, detected at limiting dilution). The fed-batch strategy has been integrated into an automated bioreactor, allowing for the generation of a clinically-relevant cell product after 12 days of culture, with minimal user manipulation. As this strategy targets the HSC environment and not the stem cells directly, it has the ability to act in combination with other expansion strategies to produce synergistic results. Unexpectedly, supplementation of the soluble protein, TAT-HOXB4, to the system, yielded the expected boost in progenitor expansion only in “sub-optimal” control conditions but not in the fed-batch system. Hypothesizing that the efficacy of HOXB4 may be dependent on the skewing of supportive vs. non-supportive cell populations, and the consequent impact of paracrine ligand production, we performed kinetic tracking of 20 hematopoietic cell types during several supportive (fed-batch, HOXB4 supplemented, Notch ligand Delta1 supplemented) vs. non-supportive (control) cultures. Meta analysis of these data revealed a non-autonomous link between HOXB4, increased megakaryocyte production, and stem cell proliferation, as well as between Notch delta-1 ligand, decreased myeloid cell production, and a decrease in the growth inhibition of stem cells. These predictions have been experimentally validated using co-cultures of sorted purified HSCs and CD41+ megakaryocykes and CD14+ monocytes. Our results identify complex connections between mature cell lineages and stem cell fate decisions and we expect to report a direct link between cell-cell interactions emerging from culture manipulations and the resulting impact on HSC self-renewal. Collectively, these studies support a dominant role for non-stem cell autonomous feedback signaling in the regulation of HSC self-renewal. Overcoming cell non-autonomous inhibition of HSC self-renewal has allowed for novel strategies to enhance HSC numbers ex vivo, thereby facilitating the production of clinically relevant quantities of stem and progenitor cells and enabling more effective strategies to treat hematologic disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Enforcing Post-Transcriptional Circuitries to Achieve Human Hematopoietic Stem Cell Expansion

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-46-SCI-46
Author(s):  
Kristin Hope
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Transcriptional Control ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Self Renewal ◽  
Control Of Gene Expression ◽  
Hematopoietic Stem

Abstract The balance between hematopoietic stem cell (HSC) differentiation and self-renewal is central to clinical regenerative paradigms. Unravelling the precise molecular mechanisms that govern HSC fate choices will thus have far reaching consequences for the development of effective therapies for hematopoietic and immunological disorders. There is an emerging recognition that beyond transcription, HSC homeostasis is subject to post-transcriptional control by RNA-binding proteins (RBPs) that ensure precise control of gene expression by modulating mRNA splicing, polyadenylation, localization, degradation or translation. RBPs can synchronously regulate the fates of operationally similar RNAs, in what have been termed RNA regulons. We have used a variety of functional approaches, in combination with unbiased genome- and proteome-scale, methods to define the tenets that govern this regulation and to determine key downstream circuitries of stem cell-regulating RBPs whose targeting could provide the basis for novel regenerative treatments. Through loss-of-function efforts, we have identified the RBP, MSI2, as a required factor for human HSC maintenance. By contrast, at supraphysiological levels, MSI2 has a profound positive effect on human HSC self-renewal decisions. Using a combination of global profiling, including mapping MSI2's targets through cross-linking immunoprecipitation (CLIP)-seq, we show that MSI2 achieves its ex vivo self-renewal-promoting effects by directing a co-ordinated post-transcriptional repression of key targets within the aryl hydrocarbon receptor (AHR) pathway. We are currently exploring the "rules" by which MSI2 influences its downstream effects on target RNAs and how it functions, in combination with other protein interactors, to instill a putative RBP regulatory code in HSCs. HSCs exhibit highly unique epigenomes, transcriptomes and proteomes and it is this distinctive molecular landscape that provides the canvas upon which MSI2, and indeed any other HSC-specific RBP exert their post-transcriptional influence over stem cell function. As such, to decipher the bona fide RNA networks that RBPs function upon in HSCs and to understand how they influence this network to enforce self-renewal, we are working towards performing systematic studies of RBP regulons in these cells specifically. In turn these approaches are elucidating a host of RBPs and post-transcriptional control mechanisms previously unappreciated for their role in HSC control. When modulated appropriately, we can successfully tailor these post-transcriptional regulons to enforce desired HSC outputs ex vivo. In summary, approaches to elucidate key HSC-regulatory RBPs and their protein and RNA interactomes provide valuable insights into a layer of HSC control previously not well understood, and one that can be capitalized on to achieve successful HSC expansion. Disclosures No relevant conflicts of interest to declare.

scholarly journals M1 and M2 macrophages differentially regulate hematopoietic stem cell self-renewal and ex vivo expansion

2018 ◽  
Vol 2 (8) ◽  
pp. 859-870 ◽  
Author(s):  
Yi Luo ◽  
Lijian Shao ◽  
Jianhui Chang ◽  
Wei Feng ◽  
Y. Lucy Liu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Differential Expression ◽  
Hematopoietic Stem Cell ◽  
Ex Vivo ◽  
M2 Macrophages ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Key Points ◽  
M1 And M2 Macrophages

Key Points M2-MΦs promote and M1-MΦs inhibit HSC self-renewal via differential expression of Arg1 and NOS2, respectively. Coculture of hUCB CD34+ cells with M2-MΦs resulted in a significant expansion of CD34+ cells and SCID–mice repopulating cells.

Hematopoietic Stem Cell Expansion by HOXB4 Is Greatly Enhanced in p21 Deficient Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1688-1688 ◽  
Author(s):  
Noriko Miyake ◽  
Ann C.M. Brun ◽  
Mattias Magnusson ◽  
David T. Scadden ◽  
Stefan Karlsson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, enforced expression of HOXB4 is a potent stimulus for murine hematopoietic stem cell (HSC) self-renewal. Murine HSCs engineered to overexpress HoxB4 expand significantly more than control cells in vivo and ex vivo while maintaining a normal differentiation program. HSCs are regulated by the cell proliferation machinery that is intrinsically controlled by cyclin-dependent kinase inhibitors such as p21Cip1/Waf1(p21) and p27Kip1 (p27). The p21 protein restricts cell cycling of the hematopoietic stem cell pool and maintains hematopoietic stem cell quiescence. In order to ask whether enhanced proliferation due to HOXB4 overexpression is mediated through suppression of p21 we overexpressed HOXB4 in hematopoietic cells from p21−/− mice. First, we investigated whether human HOXB4 enhances in vitro expansion of BM cells from p21−/− mice compared to p21+/+ mice. 5FU treated BM cells from p21−/− or p21+/+ mice were pre-stimulated with SCF, IL-6, IL-3 for 2 days followed by transduction using retroviral vector expressing HOXB4 together with GFP (MIGB4) or the control GFP vector (MIG). The cells were maintained in suspension cultures for 13 days and analyzed for GFP positive cells by flow-cytometry. Compared to MIG transduced BM cells from p21+/+ mice (MIG/p21+), the numbers of GFP positive cells were increased 1.1-fold in MIG/p21−, 3.2-fold in MIGB4/p21+ and 10.0-fold in MIGB4/p21− respectively (n=5). CFU assays were performed after 13 days of culture. The numbers of CFU were increased 4.8-fold in MIG/p21−, 19.5-fold in MIG/p21+ and 33.9 -fold in MIGB4/p21− respectively. Next, we evaluated level of HSCs expansion by bone marrow repopulation assays. After 12-days of culture, 1.5 x 105 MIGB4 or MIG-transduced cells (Ly5.2) were transplanted into lethally irradiated mice in combination with 8 x 105 fresh Ly5.1 bone marrow cells. Sixteen weeks after transplantation, no Ly5.2 cells could be detected in MIG/p21+ or MIG/p21− transplanted mice (n=6). In contrast, Ly5.2 positive cells were detected in both MIGB4/p21+/+ and MIGB4/p21−/− cells. The % of Ly5.2 positive cells in MIGB4/p21− transplanted mice was 9.9-fold higher than MIGB4/p21+ transplanted mice. (38.4 % vs 3.9 %, P<0.02, n=5). These Ly5.2 positive cells differentiated into all lineages, as determined by proportions of Mac-1, B-220, CD3 and Ter119 positive populations. Currently, we are enumerating the expansion of HOXB4 transduced HSCs in p21 deficient BM cells using the CRU assay. Our findings suggest that HOXB4 increases the self-renewal of hematopoietic stem cells by a mechanism that is independent of p21. In addition, the findings demonstrate that deficiency of p21 in combination with enforced expression of HOXB4 can be used to rapidly and effectively expand hematopoietic stem cells.

2002 - EX VIVO ACTIVATION OF HEAT SHOCK FACTOR 1 (HSF1) PROMOTES SUSTAINED HEMATOPOIETIC STEM CELL SELF-RENEWAL

2019 ◽  
Vol 76 ◽  
pp. S42-S43
Author(s):  
Robert Signer ◽  
Miriama Kruta ◽  
Mary Jean Sunshine ◽  
Yunpeng Fu ◽  
Lorena Hidalgo San Jose
Keyword(s):  
Stem Cell ◽  
Heat Shock ◽  
Hematopoietic Stem Cell ◽  
Ex Vivo ◽  
Heat Shock Factor ◽  
Heat Shock Factor 1 ◽  
Self Renewal ◽  
Hematopoietic Stem

scholarly journals Loss of the Rho GTPase activating protein p190-B enhances hematopoietic stem cell engraftment potential

Blood ◽  
2009 ◽  
Vol 114 (17) ◽  
pp. 3557-3566 ◽  
Author(s):  
Haiming Xu ◽  
Satyam Eleswarapu ◽  
Hartmut Geiger ◽  
Kathleen Szczur ◽  
Deidre Daria ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Rho Gtpase ◽  
Ex Vivo ◽  
Negative Regulator ◽  
Transcriptional Analysis ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cell (HSC) engraftment is a multistep process involving HSC homing to bone marrow, self-renewal, proliferation, and differentiation to mature blood cells. Here, we show that loss of p190-B RhoGTPase activating protein, a negative regulator of Rho GTPases, results in enhanced long-term engraftment during serial transplantation. This effect is associated with maintenance of functional HSC-enriched cells. Furthermore, loss of p190-B led to marked improvement of HSC in vivo repopulation capacity during ex vivo culture without altering proliferation and multilineage differentiation of HSC and progeny. Transcriptional analysis revealed that p190-B deficiency represses the up-regulation of p16Ink4a in HSCs in primary and secondary transplantation recipients, providing a possible mechanism of p190-B–mediated HSC functions. Our study defines p190-B as a critical transducer element of HSC self-renewal activity and long-term engraftment, thus suggesting that p190-B is a target for HSC-based therapies requiring maintenance of engraftment phenotype.

scholarly journals Single-cell approaches identify the molecular network driving malignant hematopoietic stem cell self-renewal

Blood ◽  
2018 ◽  
Vol 132 (8) ◽  
pp. 791-803 ◽  
Author(s):  
Mairi S. Shepherd ◽  
Juan Li ◽  
Nicola K. Wilson ◽  
Caroline A. Oedekoven ◽  
Jiangbing Li ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Hematopoietic Stem Cell ◽  
Myeloproliferative Neoplasms ◽  
Molecular Network ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points

Key Points Single-cell approaches identify regulators of malignant HSC self-renewal. Identification of novel roles for Bmi1, Pbx1, and Meis1 in myeloproliferative neoplasms.

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