Unexpected redundancy of Gpr56 and Gpr97 during hematopoietic cell development and differentiation

Abstract Integrated molecular signals regulate cell fate decisions in the embryonic aortic endothelium to drive hematopoietic stem cell (HSC) generation during development. The G-protein–coupled receptor 56 (Gpr56, also called Adgrg1) is the most highly upregulated receptor gene in cells that take on hematopoietic fate and is expressed by adult bone marrow HSCs. Despite the requirement for Gpr56 in hematopoietic stem/progenitor cell (HS/PC) generation in zebrafish embryos and the highly upregulated expression of GPR56 in treatment-resistant leukemic patients, its function in normal mammalian hematopoiesis remains unclear. Here, we examine the role of Gpr56 in HS/PC development in Gpr56 conditional knockout (cKO) mouse embryos and Gpr knockout (KO) embryonic stem cell (ESC) hematopoietic differentiation cultures. Our results show a bias toward myeloid differentiation of Gpr56 cKO fetal liver HSCs and an increased definitive myeloid progenitor cell frequency in Gpr56KO ESC differentiation cultures. Surprisingly, we find that mouse Gpr97 can rescue Gpr56 morphant zebrafish hematopoietic generation, and that Gpr97 expression is upregulated in mouse Gpr56 deletion models. When both Gpr56 and Gpr97 are deleted in ESCs, no or few hematopoietic PCs (HPCs) are generated upon ESC differentiation. Together, our results reveal novel and redundant functions for these 2 G-protein coupled receptors in normal mammalian hematopoietic cell development and differentiation.

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Unexpected redundancy of Gpr56 and Gpr97 during hematopoietic cell development and differentiation

10.1101/2020.09.18.303230 ◽

2020 ◽

Author(s):

A. Maglitto ◽

S.A. Mariani ◽

E. de Pater ◽

C. Rodriguez-Seoane ◽

C.S. Vink ◽

...

Keyword(s):

Stem Cell ◽

G Protein ◽

Progenitor Cell ◽

Embryonic Stem ◽

Cell Development ◽

Hematopoietic Cell ◽

Conditional Knockout ◽

Hematopoietic Stem ◽

Development And Differentiation ◽

G Protein Coupled

AbstractIntegrated molecular signals regulate cell fate during embryonic hematopoietic stem cell (HSC) generation. The G-protein coupled receptor 56 (Gpr56) is the most highly-upregulated receptor gene in cells that take on hematopoietic fate and it is expressed by adult bone marrow HSCs. Although Gpr56 is required for hematopoietic stem/progenitor cell (HS/PC) generation in zebrafish embryos, its function in mammalian hematopoiesis remains unclear. Here we examine the role of Gpr56 in HS/PC development in Gpr56 conditional knockout (cKO) mouse embryos and Gpr knockout (KO) embryonic stem cell (ESC) hematopoietic differentiation cultures. Our results show a myeloid bias of Gpr56 cKO fetal liver HSCs and an increased definitive myeloid progenitor cell frequency in Gpr56KO ESC differentiation cultures. Surprisingly, we find that mouse Gpr97 rescues Gpr56 morphant zebrafish hematopoietic generation, and that Gpr97 expression is upregulated in mouse Gpr56 deletion models. When both Gpr56 and Gpr97 are deleted in ESCs, no/few HS/PCs are generated upon ESC differentiation. Together, our results reveal novel and redundant functions for these two G-protein coupled receptors in normal mammalian hematopoietic cell development and differentiation.

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Lineage Instructive Function of C/EBPα in Multipotent Hematopoietic Progenitor Cells Revealed in a Novel Cebpa-Cre Knock-in Model

Blood ◽

10.1182/blood.v112.11.2458.2458 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2458-2458

Author(s):

Albert Wolfler ◽

Astrid A Danen-van Oorschot ◽

Jurgen Haanstra ◽

Marijke Valkhof ◽

Paulette van Strien ◽

...

Keyword(s):

Progenitor Cells ◽

Cell Fate ◽

Cell Development ◽

Hematopoietic Cell ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

Multipotent Progenitors ◽

Reported Data

Abstract Transcription factors control the lineage specification and differentiation of hematopoietic progenitor cells. They are expressed in a cell type-restricted pattern and activate lineage specific genetic programs. Recent studies have demonstrated that expression of GATA-1 or PU.1 in multipotent lin−Sca-1+c-Kit+ (LSK) cells specifies them to develop into myeloerythroid progenitors or lymphomyeloid progenitors, respectively. In contrast, C/EBPα, a transcription factor indispensable for the production of granulocytes and macrophages, is thought to predominantly act at a later stage of hematopoietic commitment, by governing the transition from common myeloid progenitors (CMPs) into granulocytic/monocytic progenitors (GMPs). To study whether C/EBPα may already exert a lineage instructive function at an earlier stage of hematopoietic cell development, i.e., at the level of multipotent LSK cells, we generated a knock-in mouse model expressing Cre recombinase under the regulation of the cebpa promoter and crossed C/EBPαcre/+ mice with R26 YFP reporter mice. This model faithfully demonstrates high levels of C/EBPα expression in myeloid cells and enabled us to trace cebpa-driven Cre/YFP expression in single LSK cells and their progeny by flow cytometry and colony cultures. On average cebpa-driven YFP expression was found in 17% (range 10–25%) of the total LSK fraction (n=12 mice). Within the CD150+CD48− CD34− subset of LSK cells, which contains the most primitive hematopoietic stem cells (HSC), 3–8% of the cells expressed YFP, indicating that cebpa is lowly expressed in bona fide HSC. This low level of expression appears insufficient for lineage determination, since the same levels of YFP expression (1–10%) were found in peripheral T and B cells. Within the CD34+ fraction of LSK cells, a population enriched for multipotent progenitors, 19% (range 14%–28%) of the cells expressed YFP. Identical distributions of YFP+ cells among the different LSK subsets were found in fetal livers of day 14.5 embryos, suggesting a comparable regulation of cebpa expression in fetal and adult cells. Similar to the reported data for GATA-1 and PU.1, cebpa-expressing LSK cells were predominantly found in the Sca-1low fraction. When cultured in a multilineage cytokine cocktail, YFP+ LSK cells gave predominantly rise to GM colonies (73% of all colonies; range 65–85%), whereas YFP− cells formed multiple types of colonies including mixed, megakaryocytic and erythroid colonies. The predominant outgrowth of YFP+ LSK cells to GM lineages was further supported in GM-CSF-supplemented colony assays, which gave rise to cloning efficiencies of 26% for YFP+ and 4% for YFP− LSK cells, respectively. In conclusion, our results show that C/EBPα starts to exert its instructive function towards GM cell development already within the LSK population, at the level of the multipotent progenitors. This has important ramifications for our understanding of the role of C/EBPα in early hematopoietic cell fate decisions.

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The Biological and Clinical Relevance of G Protein-Coupled Receptors to the Outcomes of Hematopoietic Stem Cell Transplantation: A Systematized Review

International Journal of Molecular Sciences ◽

10.3390/ijms20163889 ◽

2019 ◽

Vol 20 (16) ◽

pp. 3889 ◽

Cited By ~ 1

Author(s):

Hadrien Golay ◽

Simona Jurkovic Mlakar ◽

Vid Mlakar ◽

Tiago Nava ◽

Marc Ansari

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

G Protein ◽

Cell Transplantation ◽

Hematopoietic Stem Cell ◽

G Protein Coupled Receptors ◽

Current Status ◽

Hematopoietic Stem ◽

G Protein Coupled

Hematopoietic stem cell transplantation (HSCT) remains the only curative treatment for several malignant and non-malignant diseases at the cost of serious treatment-related toxicities (TRTs). Recent research on extending the benefits of HSCT to more patients and indications has focused on limiting TRTs and improving immunological effects following proper mobilization and engraftment. Increasing numbers of studies report associations between HSCT outcomes and the expression or the manipulation of G protein-coupled receptors (GPCRs). This large family of cell surface receptors is involved in various human diseases. With ever-better knowledge of their crystal structures and signaling dynamics, GPCRs are already the targets for one third of the current therapeutic arsenal. The present paper assesses the current status of animal and human research on GPCRs in the context of selected HSCT outcomes via a systematized survey and analysis of the literature.

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The combination of valproic acid and lithium delays hematopoietic stem/progenitor cell differentiation

Blood ◽

10.1182/blood-2011-08-375386 ◽

2012 ◽

Vol 119 (13) ◽

pp. 3050-3059 ◽

Cited By ~ 26

Author(s):

Marta A. Walasek ◽

Leonid Bystrykh ◽

Vincent van den Boom ◽

Sandra Olthof ◽

Albertina Ausema ◽

...

Keyword(s):

Valproic Acid ◽

Stem Cell ◽

Small Molecules ◽

Cell Fate ◽

Progenitor Cell ◽

Synergistic Effects ◽

Cell Phenotype ◽

Hematopoietic Stem ◽

Stem Cell Fate ◽

Cell Fate Decisions

Abstract Despite increasing knowledge on the regulation of hematopoietic stem/progenitor cell (HSPC) self-renewal and differentiation, in vitro control of stem cell fate decisions has been difficult. The ability to inhibit HSPC commitment in culture may be of benefit to cell therapy protocols. Small molecules can serve as tools to manipulate cell fate decisions. Here, we tested 2 small molecules, valproic acid (VPA) and lithium (Li), to inhibit differentiation. HSPCs exposed to VPA and Li during differentiation-inducing culture preserved an immature cell phenotype, provided radioprotection to lethally irradiated recipients, and enhanced in vivo repopulating potential. Anti-differentiation effects of VPA and Li were observed also at the level of committed progenitors, where VPA re-activated replating activity of common myeloid progenitor and granulocyte macrophage progenitor cells. Furthermore, VPA and Li synergistically preserved expression of stem cell–related genes and repressed genes involved in differentiation. Target genes were collectively co-regulated during normal hematopoietic differentiation. In addition, transcription factor networks were identified as possible primary regulators. Our results show that the combination of VPA and Li potently delays differentiation at the biologic and molecular levels and provide evidence to suggest that combinatorial screening of chemical compounds may uncover possible additive/synergistic effects to modulate stem cell fate decisions.

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130 EXPRESSION AND LOCALIZATION OF µ OPIOID RECEPTOR IN PORCINE PRE-IMPLANTATION EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab130 ◽

2010 ◽

Vol 22 (1) ◽

pp. 224 ◽

Cited By ~ 1

Author(s):

R. Minoia ◽

T. Q. Dang-Nguyen ◽

K. Matsukawa ◽

M. Kaneda ◽

M. E. Dell'Aquila ◽

...

Keyword(s):

Stem Cell ◽

G Protein ◽

Cell Fate ◽

Opioid Receptors ◽

Embryonic Stem ◽

Endogenous Opioids ◽

The Body ◽

Cell Fate Decisions ◽

G Protein Coupled

Embryonic stem cells can become any tissue in the body, excluding a placenta. Growth factors, hormones, and neurotransmitters have been implicated in the regulation of their fate. Because various neural precursors express functional neurotransmitter receptors, as G-protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. Moreover, a high level of endogenous opioids linked to G-protein-coupled receptor above all μ opioid receptors (MOR) has been shown to interfere with normal calcium metabolism and with the activity of the mitogen-activated protein kinase (MAPK). Thus it is very important to understand the possible influence of opioid activities in the regulation of stem cell fate. In this study we investigated the presence of MOR on porcine in vitro-produced embryos at one-cell, 4-cell, morula, and blastocyst stages by immunostaining. The COC were collected by aspiration, cultured in NCSU-37 medium supplemented with hormones for 20 to 22 h, and then in maturation medium without hormones for 24 h. After this time, COC were inseminated with frozen-thawed epididymal spermatozoa at the concentration of 10 × 5 sperm cells mL-1 for 3 h. After removal of cumulus cells, putative zygotes were cultured in IVC Pyr-Lac medium for the first 2 days and in IVC Glu medium until Day 6 (the day of IVF was defined as Day 0). Embryos at different stages were collected at 12, 36, 120, and 144 h post fertilization, and kept in 4% (v/v) paraformaldehyde until examination. All samples were washed and incubated for 30 min in PBS-1%BSA. Controls were incubated in PBS-1% BSA for 90 min, whereas embryos were incubated with a 1 : 2500 dilution of the primary rabbit antibody against the third extracellular loop of MOR. Prior to examination, all samples were washed in PBS and incubated with a FITC-conjugated anti rabbit IgG-secondary antibody diluted 1:200 in Evans Blue/PBS1x. Samples were visualized by laser scanning confocal microscope (Nikon). The immunofluorescence localize, by intense brilliant green, the presence of MOR on blastomers of all stage embryos examined, whereas the embryos of negative control did not show any fluorescent region or spotted coloring. Our results support specific implication of the opioid receptors in developmental process of porcine embryos. Their presence suggests a possible role of MOR in embryonic development. Thus it can be speculated that there is a role for MOR in controlling key events of the stem cell life. However, these primary results must be confirmed by the demonstration of protein expression (by Western blot) of MOR in the embryos and deeply studied to understand the exact functional role of MOR in them at this level. JSPS short-term scholarship.

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The Spliceosomal Component Sf3b1 Is Essential for Hematopoietic Differentiation

Blood ◽

10.1182/blood.v126.23.3582.3582 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3582-3582

Author(s):

Adriana De La Garza-Sauceda ◽

Rosannah C. Cameron ◽

Sara Nik ◽

Michelle Gulfo ◽

Sara G. Payne ◽

...

Keyword(s):

Flow Cytometry ◽

Endothelial Cells ◽

Cell Differentiation ◽

Progenitor Cell ◽

Cell Development ◽

Hematopoietic Cell ◽

Lateral Plate Mesoderm ◽

Lateral Plate ◽

Hematopoietic Stem ◽

Primitive Erythropoiesis

Abstract Myelodysplastic syndrome (MDS) is a disorder arising from hematopoietic stem and progenitor cell (HSPC) dysfunction resulting in ineffective hematopoiesis. A multitude of recurrent somatic mutations in spliceosomal components were recently identified in MDS that likely contribute to the pathogenesis of the disease. The lack of in vivo models to study cell-type specific effects of spliceosomal mutations limits our understanding of why such mutations lead to hematopoietic abnormalities. Using a zebrafish with a loss-of-function mutation in sf3b1 (sf3b1hi3394), an essential member of the spliceosome, we demonstrate hematopoietic cell differentiation and hematopoietic stem and progenitor cell (HSPC) specification are processes sensitive to spliceosomal malfunction. Primitive erythropoiesis initiates normally in sf3b1 mutants as evidenced by expression of scl in the posterior lateral plate mesoderm at 14 hours post fertilization (hpf) as well as gata1 and beta-globin at 24 hpf. Flow cytometry quantification of gata1:gfp positive erythrocytes showed sf3b1 mutants have 25% more cells at 24 hpf, but greater than 3-fold fewer cells at 36 and 48 hpf, time points when wild type erythroblasts are expanding and differentiating. At 48 hpf, we also observed decreased levels of o-dianisidine positive erythrocytes, low numbers of morphologically mature erythroblasts, and higher levels of immature erythroblasts in sf3b1 mutants. Similarly, we observed normal initiation of primitive myelopoiesis marked by stem cell leukemia (scl) expression in the anterior lateral plate mesoderm at 14 hpf, but diminished expression of more differentiated markers, l-plastin and myeloperoxidase at 24 and 28 hpf in sf3b1 mutants. Quantification of lysozyme C:dsred positivemyeloid cells using flow cytometry also showed 24-fold fewer mature myeloid cells in sf3b1 mutants at 36 hpf. Our data on primitive erythropoiesis and myelopoiesis indicate sf3b1 is required for hematopoietic cell differentiation. Additionally, sf3b1 mutants have diminished expression of the definitive HSPC marker runx1 within the aorta at 28 hpf. In contrast, we observed normal expression of the pan-endothelial marker kinase insert domain receptor-like (kdrl) and aorta-specific markers notch1b and notch3 at 24 hpf. Flow cytometry quantification of kdrl:gfp endothelial cells at 24 hpf shows no difference in the frequency of endothelial cells in sf3b1 mutants. Moreover, we observed fewer cmyb:gfp; kdrl:dsred double positive HSPCs along the dorsal aorta. The data suggest that decreased HSPC formation in sf3b1 mutants is due to a failure in hemogenic induction. From these studies, we show sf3b1 is required at specific stages of hematopoietic cell development. These results provide novel insight into the role of splicing in blood cell development, and can afford a deeper understanding of the mechanism of splicing regulation on the origins of MDS. Disclosures No relevant conflicts of interest to declare.

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