scholarly journals EpoR-tdTomato-Cre mice enable identification of EpoR expression in subsets of tissue macrophages and hematopoietic cells

Blood ◽  
2021 ◽  
Author(s):  
Huan Zhang ◽  
Shihui Wang ◽  
Donghao Liu ◽  
Chengjie Gao ◽  
Yongshuai Han ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Erythropoietin Receptor ◽  
Specific Gene ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Functional Studies ◽  
Egfp Signal ◽  
Epor Expression

The erythropoietin receptor (EpoR) has been traditionally thought as an erythroid specific gene. Notably, accumulating evidence suggests that EpoR is expressed well beyond erythroid cells. However, the expression of EpoR in non-erythroid cells has been controversial. In the present study we generated EpoR-tdTomato-Cre mice and used them to examine the expression of EpoR in tissue macrophages and hematopoietic cells. We show that in marked contrast to the previously available EpoR-eGFPcre mice in which very weak eGFP signal was detected in erythroid cells, tdTomato was readily detectable in both fetal liver (FL) and bone marrow (BM) erythroid cells at all developmental stages and exhibited dynamic changes during erythropoiesis. Consistent with our recent finding that erythroblastic island (EBI) macrophages are characterized by the expression of EpoR, tdTomato was readily detected in both FL and BM EBI macrophages. Moreover, tdTomato was also detected in subsets of hematopoietic stem cells, progenitors, megakaryocytes and B cells in BM as well as in spleen red pulp macrophages and liver Kupffer cells. The expression of EpoR was further demonstrated by the EpoR-tdTomato-Cre mediated excision of the floxed STOP sequence. Importantly, EPO injection selectively promoted proliferation of the EpoR-expressing cells and induced erythroid lineage bias during hematopoiesis. Our findings imply broad roles of EPO/EpoR in hematopoiesis which warrant further investigation. The EpoR-tdTomato-Cre mouse line provides a powerful tool to facilitate future studies on EpoR expression and regulation in various non-hematopoietic cells and to conditionally manipulate gene expression in EpoR-expressing cells for functional studies.

Abstract 255: The Kidney and Heart are Hemogenic Organs During Embryonic Life.

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Yan Hu ◽  
Minghong Li ◽  
R. Ariel Gomez ◽  
Maria Luisa S Sequeira Lopez
Keyword(s):  
General Circulation ◽  
Heart Function ◽  
Erythropoietin Receptor ◽  
Erythroid Cells ◽  
Scientific Controversy ◽  
Hematopoietic Stem ◽  
Adult Mice ◽  
Circulating Cells ◽  
Receptor Locus ◽  
Embryonic Life

During early embryonic life, the heart starts to beat before an effective circulation is established, and the kidney starts to form its vasculature before it connects to the general circulation. We and others have shown a close lineage relationship between endothelial cells (ECs) and hematopoietic cells. In fact, during embryonic development erythroblasts bud from the endothelium of developing vessels, a process we termed hemovasculogenesis. Those studies suggested the possibility that embryonic organs may have hemogenic potential. To test this hypothesis, we performed lineage studies and colony forming unit (CFC) assays to trace the fate of hematopoietic stem cells (HSCs), erythroid cells, and ECs in heart and kidney from embryonic mice. Using ER-GFPcre mice that express both GFP and cre under control of the erythropoietin receptor locus in the erythroid cells, we identified hematopoietic progenitors (Hb+Nanog+) within nascent vessels in the early embryonic kidney and heart. Using EC-SCL-Cre-ERT transgenic mice that specifically express tamoxifen inducible Cre in EC progenitors, we found both circulating and non-circulating cells from the EC lineage in the early embryonic heart and kidney. CFC assays using HSC-SCL-Cre-ERT; mTmG mice (which express GFP in the cells from the HSC lineage upon tamoxifen induction) showed that both the embryonic kidney and heart possess HSCs. Further, transplantation studies of pre-vascular embryonic kidneys from EC-SCL-Cre-ERT;R26R mice under the kidney capsule of WT adult mice showed blood cells derived from the embryonic kidney suggesting that the embryonic kidney also possesses HSCs that originate in situ. These studies indicate that the embryonic kidney and heart function as hematopoietic organs during early embryogenesis. In addition to solve an important scientific controversy in our understanding of lineage/fate relationships in the developing embryo, these findings are relevant for tissue repair/regeneration and may help explain why under pathological circumstances, hematopoiesis occurs in extramedullary organs.

Expansion of mouse hematopoietic stem/progenitor cells in three-dimensional cocultures on growth-suppressed stromal cell layer

2019 ◽  
Vol 42 (7) ◽  
pp. 374-379 ◽  
Author(s):  
Hirotoshi Miyoshi ◽  
Chiaki Sato ◽  
Yuichiro Shimizu ◽  
Misa Morita
Keyword(s):  
Progenitor Cells ◽  
Mitomycin C ◽  
Stromal Cells ◽  
Fetal Liver ◽  
Three Dimensional ◽  
Hematopoietic Cells ◽  
Liver Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

With the aim of establishing an effective method to expand hematopoietic stem/progenitor cells for application in hematopoietic stem cell transplantation, we performed ex vivo expansion of hematopoietic stem/progenitor cells derived from mouse fetal liver cells in three-dimensional cocultures with stromal cells. In these cocultures, stromal cells were first cultured within three-dimensional scaffolds to form stromal layers and then fetal liver cells containing hematopoietic cells were seeded on these scaffolds to expand the hematopoietic cells over the 2 weeks of coculture in a serum-containing medium without the addition of cytokines. Prior to coculture, stromal cell growth was suppressed by treatment with the DNA synthesis inhibitor mitomycin C, and its effect on hematopoietic stem/progenitor cell expansion was compared with that in control cocultures in which fetal liver cells were cocultured with three-dimensional freeze-thawed stromal cells. After coculture with mitomycin C-treated stromal cells, we achieved a several-fold expansion of the primitive hematopoietic cells (c-kit+hematopoietic progenitor cells >7.8-fold, and CD34+hematopoietic stem/progenitor cells >3.5-fold). Compared with control cocultures, expansion of hematopoietic stem/progenitor cells tended to be lower, although that of hematopoietic progenitor cells was comparable. Thus, our results suggest that three-dimensional freeze-thawed stromal cells have higher potential to expand hematopoietic stem/progenitor cells compared with mitomycin C-treated stromal cells.

scholarly journals The sialomucin CD34 is expressed on hematopoietic cells and blood vessels during murine development

Blood ◽  
1995 ◽  
Vol 85 (1) ◽  
pp. 96-105 ◽  
Author(s):  
PE Young ◽  
S Baumhueter ◽  
LA Lasky
Keyword(s):  
Blood Vessels ◽  
Matrix Protein ◽  
Vascular System ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Embryoid Bodies ◽  
Extracellular Matrix Protein ◽  
Hematopoietic Stem ◽  
Endothelial Growth Factor ◽  
High Degree

The processes of angiogenesis and hematopoiesis require a high degree of coordination during embryogenesis. Whereas much is understood about the development of the vascular system in avian embryos, little information has been attained in mammals, predominantly because there are no specific markers for either blood vessels or hematopoietic cells in any developing mammalian system. We have recently shown that murine CD34 (mCD34) is expressed on the vascular endothelium in all organs and tissues of the adult mouse as well as on a small percentage of presumably hematopoietic stem cells in the bone marrow and fetal liver. Here we show that mCD34 is also expressed on the endothelium of blood vessels and on a subset of hematopoietic-like cells throughout murine development. mCD34 is first observed on the yolk sac endothelium of day 7.5 embryos and on a subset of hematopoietic cells within these yolk sacs. mCD34 expression is maintained on vessels and hematopoietic cells in all organs and tissues throughout embryogenesis. In addition, mCD34 is localized on growth conelike filopodial processes that appear at the budding edge of newly sprouted capillaries. Double staining of capillaries for mCD34 and laminin shows that these growth conelike processes seem to be free of laminin, whereas the formed capillaries seem to be coated with this extracellular matrix protein. Analysis of vessels in developing brain shows that these filopodial processes seem to be directed toward the ventricular epithelium, a previously described site of vascular endothelial growth factor synthesis. Finally, we show that the vascular structures of developing murine embryoid bodies also express mCD34. These data suggest that mCD34 is a useful marker for the analysis of the development of the blood vascular system in murine embryos.

A Kinetic Model for the Homing and Migration of Prenatally Transplanted Marrow

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3251-3257 ◽  
Author(s):  
Aimen F. Shaaban ◽  
Heung Bae Kim ◽  
Ross Milner ◽  
Alan W. Flake
Keyword(s):  
Stem Cell ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Donor Cell ◽  
Cell Phenotype ◽  
Functional Assay ◽  
Cell Frequency ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
Kinetic Profile

Abstract Currently little is known about the mechanisms regulating the homing and the early engraftment of prenatally transplanted hematopoietic cells due to the lack of a relevant functional assay. In this study, we have defined a reproducible kinetic profile of the homing and the early engraftment events in a murine model of prenatal stem cell transplantation. Light density mononuclear cells (LDMCs) from adult C57Pep3b and SJL/J marrow were transplanted by intraperitoneal (IP) injection into C57BL/6 fetuses (106 LDMCs/fetus) at 14 days of gestation. The fetuses were sacrificed at early time points (1.5 to 96 hours) after transplantation. Recipient fetal liver and cord blood were analyzed for donor cell frequency and donor cell phenotype by dual color flow cytometry. Pertinent findings included the following: (1) a triphasic kinetic profile exists after in utero hematopoietic stem cell (HSC) transplantation (homing of circulating donor cells, rapid reduction of donor cell frequency, and donor cell competitive equilibration); (2) homing to the fetal liver is nonselective and reflects the phenotypic profile of the donor population; and (3) the kinetics after the prenatal transplantation of congenic or fully allogeneic cells are identical. This model will facilitate a systematic analysis of the mechanisms that regulate the homing of prenatally transplanted hematopoietic cells.

Kruppel-Like Factor 4 Upregulates p21 and Downregulates Proliferation of Human and Mouse HSPCs, but Is Not Essential for Mouse HSPC Repopulation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1317-1317
Author(s):  
Jonathan K. Alder ◽  
Robert W. Georgantas ◽  
Richard L. Hildreth ◽  
Xiaobing Yu ◽  
Curt I. Civin
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Fetal Liver ◽  
Family Members ◽  
Hematopoietic Cells ◽  
Normal Numbers ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Klf4 Expression ◽  
And Function

Abstract Several Kruppel-like factor family members, including KLF1, KLF2, KLF3, and KLF6 have pivotal roles in hematopoiesis. Experiments in zebrafish have suggested that KLF4 may play a similar role. Here we found that enforced expression of KLF4 in hematopoietic cells induced cell cycle arrest without triggering apoptosis. Based on the high levels of expression of KLF4 in mouse and human hematopoietic stem-progenitor cells (HSPCs), we hypothesized and demonstrated that KLF4 regulates proliferation of these cells through regulation of p21cip1/waf1 (p21). Nevertheless, KLF4−/− mouse fetal liver cells had normal numbers of all mature lineages and provided radioprotection, similar to wild type (wt) controls. Furthermore, in long-term competitive repopulation assays, KLF4−/− mouse HSPCs demonstrated hematopoietic potency equivalent to wt. We found that KLF2 is expressed at higher levels than KLF4 in mouse HSPCs and is a more potent activator of p21, suggesting that KLF2 (and/or other KLF family members) may play a compensatory role in KLF4−/− HSPCs. Thus, although is not essential for their normal development and function, KLF4 expression is sufficient to induce p21-mediated cell cycle arrest in hematopoietic cells.

SCL/TAL1 Regulates Erythropoietin Receptor Expression.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1195-1195
Author(s):  
Heather M. Rogers ◽  
Xiaobing Yu ◽  
Constance Tom Noguchi
Keyword(s):  
Erythroid Differentiation ◽  
Erythropoietin Receptor ◽  
Receptor Expression ◽  
Binding Motif ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Mild Anemia ◽  
Adult Mice ◽  
High Level

Abstract The basic-helix-loop-helix transcription factor SCL/TAL1, is required for erythropoiesis during development, and conditional deletion in adult hematopoiesis results in hematopoietic stem cells with a competitive repopulation disadvantage and defective erythropoiesis in vitro. However, adult mice with a conditional SCL/TAL1 deletion survive with mild anemia, suggesting defective erythroid proliferation and indicating that SCL/TAL1 is important, but not essential in mature red blood cell production. We find that during erythroid differentiation of primary human hematopoietic CD34+ cells, SCL/TAL1 expression peaks at day 8–10 following erythropoietin (EPO) stimulation, concomitant with peak expression of GATA-1 and EKLF. Treatment with SCL/TAL1 antisense oligonucleotides during erythroid differentiation markedly decreases erythroid differentiation as indicated by decreased expression of GATA-1 and both b- and g-globin expression, along with the absence of the characteristic decrease in GATA-2. Microarray analysis of erythroid cells overexpressing SCL/TAL1 indicate increased gene expression for b- and g-globin, and other genes related to erythropoiesis including EPO receptor (EPO-R), and these results are confirmed in stable cell lines with increasing SCL/TAL1 expression. Examination of EPO-R transcription regulation indicates that E-boxes in the 5′ UTR can bind SCL/TAL1 in vitro and, in addition to the GATA-1 binding motif, provide transcription activity in reporter gene assays. These data indicate that in addition to the importance of SCL/TAL1 DNA binding for proliferation of BFU-E and expression of glycophorin A and protein 4.2, SCL/TAL1 is also necessary for high level expression of EPO-R. Reduction in EPO-R expression likely contributes to the anemia associated with the conditional adult deletion of SCL/TAL1 and to the proliferative defect of erythroid cells observed in vitro. Early expression of SCL/TAL1 in hematopoietic cells may activate expression of EPO-R prior to EPO stimulation of erythropoiesis and induction of GATA-1.

Constitutive TGF-β1 Deficiency Induces a Defect in Hematopoietic Stem/Progenitor Cell Compartment in Fetus and Neonate.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1397-1397
Author(s):  
Claude Capron ◽  
Catherine Lacout ◽  
Yann Lecluse ◽  
Valérie Jalbert ◽  
Elisabeth Cramer Bordé ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Type I ◽  
Activated T Cells ◽  
Hematopoietic Stem ◽  
Tgf Β1

Abstract TGF-β1 is a cytokine with pleiotropic effects. It has been considered that TGF-β1plays a major role on hematopoietic stem cells (HSC) based on in vitro experiment. Achieving in vivo experiments proved to be difficult because constitutive TGF-β1 knock-out (KO) in mice leads to lethality during the first 4 weeks of life from a wasting syndrome related to tissue infiltration by activated T cells and macrophages. For this reason, hematopoiesis of TGF-β1−/− mice has not been studied in details. In contrast the role of TGF-β1 has been recently extensively studied in conditional TGF-β type I receptor (TβRI) KO mice. No clear effect was observed on HSC functions, suggesting that TGF-β1 was not a key physiological regulator of hematopoiesis in the adult. However, these experiments have some limitations. They do not exclude a putative role for TGF-β1 during fetal hematopoiesis and they do not specifically address the role of TGF-β1 on hematopoiesis because KO of TGF-β receptor leads to signaling arrest for all TGF-βs. In addition, other receptors may be involved in TGF-β1 signaling. For these reasons, we have investigated the hematopoiesis of constitutive TGF-β1 KO mice with a mixed Sv129 × CF-1 genetic background allowing the birth of a high proportion of homozygotes. In 2 week-old neonate mice, we have shown a decrease of bone marrow (BM) and spleen progenitors and a decrease of immature progenitors colony forming unit of the spleen (CFU-s). Moreover this was associated with a loss in reconstitutive activity of TGF-β1−/− HSC from BM. However, although asymptomatic, these mice had an excess of activated lymphocytes and an augmentation of Sca-1 antigen on hematopoietic cells suggesting an excess of γ-interferon release. Thus we studied hematopoiesis of 7 to 10 days-old neonate mice, before phenotypic modification and inflammatory cytokine release. Similar results were observed with a decrease in the number of progenitors and in the proliferation of TGF-β1−/− BM cells along with an increased differentiation but without an augmentation in apoptosis. Moreoever, a loss of long term reconstitutive capacity of BM Lineage negative (Lin−) TGF-β1−/− cells along with a diminution of homing of TGF-β1−/− progenitors was found. These results demonstrate that TGF-β1 may play a major role on the HSC/Progenitor compartment in vivo and that this defect does not seem to be linked to the immune disease. To completely overpass the risk of the inflammatory syndrome, we analyzed hematopoiesis of fetal liver (FL) of TGF-β1−/− mice and still found a decrease in progenitors, a profound defect in the proliferative capacities, in long term reconstitutive activity and homing potential of primitive FL hematopoietic cells. Our results demonstrate that TGF-β1 plays an important role during hematopoietic embryonic development. Altogether these findings suggest that TGF-β1 is a potent positive regulator for the in vivo homeostasis of the HSC compartment.

Role of the WT1 tumor suppressor in murine hematopoiesis

Blood ◽  
2003 ◽  
Vol 101 (7) ◽  
pp. 2570-2574 ◽  
Author(s):  
Julia A. Alberta ◽  
Gregory M. Springett ◽  
Helen Rayburn ◽  
Thomas A. Natoli ◽  
Janet Loring ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tumor Suppressor ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Colony Forming Unit ◽  
Organ Systems

The WT1 tumor-suppressor gene is expressed by many forms of acute myeloid leukemia. Inhibition of this expression can lead to the differentiation and reduced growth of leukemia cells and cell lines, suggesting that WT1 participates in regulating the proliferation of leukemic cells. However, the role of WT1 in normal hematopoiesis is not well understood. To investigate this question, we have used murine cells in which the WT1 gene has been inactivated by homologous recombination. We have found that cells lacking WT1 show deficits in hematopoietic stem cell function. Embryonic stem cells lacking WT1, although contributing efficiently to other organ systems, make only a minimal contribution to the hematopoietic system in chimeras, indicating that hematopoietic stem cells lacking WT1 compete poorly with healthy stem cells. In addition, fetal liver cells lacking WT1 have an approximately 75% reduction in erythroid blast-forming unit (BFU-E), erythroid colony-forming unit (CFU-E), and colony-forming unit–granulocyte macrophage–erythroid–megakaryocyte (CFU-GEMM). However, transplantation of fetal liver hematopoietic cells lackingWT1 will repopulate the hematopoietic system of an irradiated adult recipient in the absence of competition. We conclude that the absence of WT1 in hematopoietic cells leads to functional defects in growth potential that may be of consequence to leukemic cells that have alterations in the expression of WT1.

Podocalyxin is a CD34-related marker of murine hematopoietic stem cells and embryonic erythroid cells

Blood ◽  
2005 ◽  
Vol 105 (11) ◽  
pp. 4170-4178 ◽  
Author(s):  
Regis Doyonnas ◽  
Julie S. Nielsen ◽  
Shierley Chelliah ◽  
Erin Drew ◽  
Takahiko Hara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Erythroid Cells ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Nucleated Red Blood Cells ◽  
Erythroid Precursors ◽  
Valuable Marker

Abstract Podocalyxin/podocalyxin-like protein 1 [PCLP1]/thrombomucin/MEP21 is a CD34-related sialomucin. We have performed a detailed analysis of its expression during murine development and assessed its utility as a marker of hematopoietic stem cells (HSCs) and their more differentiated progeny. We find that podocalyxin is highly expressed by the first primitive hematopoietic progenitors and nucleated red blood cells to form in the embryonic yolk sac. Likewise, podocalyxin is expressed by definitive multilineage hematopoietic progenitors and erythroid precursors in fetal liver. The level of podocalyxin expression gradually declines with further embryo maturation and reaches near-background levels at birth. This is followed by a postnatal burst of expression that correlates with the seeding of new hematopoietic progenitors to the spleen and bone marrow. Shortly thereafter, podocalyxin expression gradually declines, and by 4 weeks postpartum it is restricted to a rare population of Sca-1+, c-kit+, lineage marker- (Lin-) cells in the bone marrow. These rare podocalyxin-expressing cells are capable of serially reconstituting myeloid and lymphoid lineages in lethally irradiated recipients, suggesting they have HSC activity. In summary, we find that podocalyxin is a marker of embryonic HSCs and erythroid cells and of adult HSCs and that it may be a valuable marker for the purification of these cells for transplantation.

Human hematopoiesis in murine embryos after injecting human cord blood–derived hematopoietic stem cells into murine blastocysts

Blood ◽  
2002 ◽  
Vol 99 (2) ◽  
pp. 719-721 ◽  
Author(s):  
Friedrich Harder ◽  
Reinhard Henschler ◽  
Ilse Junghahn ◽  
Marinus C. Lamers ◽  
Albrecht M. Müller
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Developmental Stages ◽  
Specific Gene ◽  
Human Cord Blood ◽  
Human Donor ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Murine Embryos

Abstract At different developmental stages, candidate human hematopoietic stem cells (HSCs) are present within the CD34+ CD38− population. By means of xenotransplantation, such CD34+CD38− cells were recently shown to engraft the hematopoietic system of fetal sheep and nonobese diabetic severe combined immunodeficient adult mice. Here it is demonstrated that, after their injection into murine blastocysts, human cord blood (CB)–derived CD34+and CD34+ CD38− cells repopulate the hematopoietic tissues of nonimmunocompromised murine embryos and that human donor contribution can persist to adulthood. It is further observed that human hematopoietic progenitor cells are present in murine hematopoietic tissues of midgestational chimeric embryos and that progeny of the injected human HSCs activate erythroid-specific gene expression. Thus, the early murine embryo provides a suitable environment for the survival and differentiation of human CB CD34+ CD38− cells.

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