DOT1L methyltransferase regulates the calcium influx in erythroid progenitor cells in response to erythropoietin

ABSTRACTErythropoietin (EPO) signaling plays a vital role in erythropoiesis by regulating proliferation and lineage-specific differentiation of hematopoietic progenitor cells. An important downstream response of EPO signaling is calcium influx, which is regulated by transient receptor potential channel (TRPC) proteins, particularly TRPC2 and TRPC6. While EPO induces Ca2+influx through TRPC2, TRPC6 inhibits the function of TRPC2. Thus, interactions between TRPC2 and TRPC6 regulate the rate of Ca2+influx in EPO-induced erythropoiesis. In this study, we observed that the expression of TRPC6 in c-KIT positive erythroid progenitor cells is regulated by DOT1L. DOT1L is a methyltransferase that plays an important role in many biological processes during embryonic development, including early erythropoiesis. We previously reported that Dot1L knockout (Dot1L-KO) hematopoietic progenitors in the yolk sac failed to develop properly, which resulted in lethal anemia. In this study, we have detected a marked downregulation of Trpc6 gene expression in Dot1L-KO progenitor cells in the yolk sac compared to wildtype. However, the expression of Trpc2, the positive regulator of Ca2+influx, remained unchanged. The promoter and the proximal region of the Trpc6 gene loci exhibited an enrichment of H3K79 methylation, which is mediated solely by DOT1L. As the loss of DOT1L affects the expression of TRPC6, which inhibits Ca2+influx by TRPC2, Dot1L-KO progenitor cells in the yolk sac exhibit accelerated and sustained high levels of Ca2+influx. Such heightened Ca2+ levels might have detrimental effects on the development of hematopoietic progenitor cells in response to erythropoietin.

Download Full-text

A role for calmodulin in the growth of human hematopoietic progenitor cells

Blood ◽

10.1182/blood.v75.7.1446.1446 ◽

1990 ◽

Vol 75 (7) ◽

pp. 1446-1454 ◽

Cited By ~ 11

Author(s):

N Katayama ◽

M Nishikawa ◽

F Komada ◽

N Minami ◽

S Shirakawa

Keyword(s):

Progenitor Cells ◽

Colony Formation ◽

Hematopoietic Progenitor Cells ◽

Dependent Manner ◽

Hematopoietic Progenitor ◽

Erythroid Progenitor ◽

Gm Csf ◽

Erythroid Progenitor Cells ◽

Dependent Inhibition ◽

Dose Dependent

Abstract A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N- (6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N- (4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.

Download Full-text

A role for calmodulin in the growth of human hematopoietic progenitor cells

Blood ◽

10.1182/blood.v75.7.1446.bloodjournal7571446 ◽

1990 ◽

Vol 75 (7) ◽

pp. 1446-1454

Author(s):

N Katayama ◽

M Nishikawa ◽

F Komada ◽

N Minami ◽

S Shirakawa

Keyword(s):

Progenitor Cells ◽

Colony Formation ◽

Hematopoietic Progenitor Cells ◽

Dependent Manner ◽

Hematopoietic Progenitor ◽

Erythroid Progenitor ◽

Gm Csf ◽

Erythroid Progenitor Cells ◽

Dependent Inhibition ◽

Dose Dependent

A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N- (6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N- (4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.

Download Full-text

Murine yolk sac endoderm- and mesoderm-derived cell lines support in vitro growth and differentiation of hematopoietic cells

Blood ◽

10.1182/blood.v83.9.2436.2436 ◽

1994 ◽

Vol 83 (9) ◽

pp. 2436-2443 ◽

Cited By ~ 49

Author(s):

MC Yoder ◽

VE Papaioannou ◽

PP Breitfeld ◽

DA Williams

Keyword(s):

Progenitor Cells ◽

Cell Lines ◽

Stromal Cell ◽

Hematopoietic Cells ◽

Yolk Sac ◽

Hematopoietic Progenitor Cells ◽

Visceral Endoderm ◽

Hematopoietic Progenitor

Abstract The mechanisms involved in the induction of yolk sac mesoderm into blood islands and the role of visceral endoderm and mesoderm cells in regulating the restricted differentiation and proliferation of hematopoietic cells in the yolk sac remain largely unexplored. To better define the role of murine yolk sac microenvironment cells in supporting hematopoiesis, we established cell lines from day-9.5 gestation murine yolk sac visceral endoderm and mesoderm layers using a recombinant retrovirus vector containing Simian virus 40 large T- antigen cDNA. Obtained immortalized cell lines expressed morphologic and biosynthetic features characteristic of endoderm and mesoderm cells from freshly isolated yolk sacs. Similar to the differentiation of blood island hematopoietic cells in situ, differentiation of hematopoietic progenitor cells in vitro into neutrophils was restricted and macrophage production increased when bone marrow (BM) progenitor cells were cultured in direct contact with immortalized yolk sac cell lines as compared with culture on adult BM stromal cell lines. Yolk sac- derived cell lines also significantly stimulated the proliferation of hematopoietic progenitor cells compared with the adult BM stromal cell lines. Thus, yolk sac endoderm- and mesoderm-derived cells, expressing many features of normal yolk sac cells, alter the growth and differentiation of hematopoietic progenitor cells. These cells will prove useful in examining the cellular interactions between yolk sac endoderm and mesoderm involved in early hematopoietic stem cell proliferation and differentiation.

Download Full-text

Human erythroid progenitor cells express Rhesus antigens

Blood ◽

10.1182/blood.v66.3.660.660 ◽

1985 ◽

Vol 66 (3) ◽

pp. 660-663 ◽

Cited By ~ 12

Author(s):

JH Falkenburg ◽

WE Fibbe ◽

N van der Vaart-Duinkerken ◽

ME Nichols ◽

P Rubinstein ◽

...

Keyword(s):

Progenitor Cells ◽

Cell Sorting ◽

Blood Cells ◽

Hematopoietic Progenitor ◽

Erythroid Progenitor ◽

Positive Fraction ◽

Erythroid Progenitor Cells ◽

Fold Enrichment ◽

Hla Dr ◽

Complement Dependent Cytotoxicity

Abstract The expression of Rhesus antigens on hematopoietic progenitor cells was studied using monoclonal antibodies. Because these antibodies are not capable of lysing mature red blood cells in a complement-dependent cytotoxicity assay, fluorescence-activated cell sorting was performed. Using the monoclonal anti-Rh 29 antibody B10, 68% +/- 6% of the mature erythroid progenitor cells (CFU-E) were sorted into the positive fraction, while only 2% +/- 1% of the relatively immature erythroid progenitor cells (BFU-E), and 3% +/- 1% of the granulocyte-macrophage progenitor cells (CFU-GM) were cultured from this same fraction. Thus up to a 15-fold enrichment of CFU-E could be obtained. In two experiments more than 4% of the cells in the positive fraction consisted of CFU-E; in one experiment even more than 7% did. Using fractionated cell sorting, the Rhesus antigens appeared to have a lower density on CFU-E than HLA-DR determinants. Antibodies against the Rhesus antigens can be applied to enrich erythroid-committed stem cells and to separate mature from immature erythroid progenitor cells.

Download Full-text

Recapitulation of Transient Abnormal Myelopoiesis Using Patient Derived iPSCs

Blood ◽

10.1182/blood.v126.23.4115.4115 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4115-4115

Author(s):

Yoko Nishinaka-Arai ◽

Akira Niwa ◽

Mitsujiro Osawa ◽

Tatsutoshi Nakahata ◽

Megumu K Saito

Keyword(s):

Progenitor Cells ◽

Trisomy 21 ◽

Hematopoietic Cells ◽

Chromosome 21 ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Erythroid Progenitor ◽

Transient Abnormal Myelopoiesis ◽

Gata1 Mutation

Abstract Down syndrome (DS) is characterized by the trisomy of chromosome 21 and complicated with multi-organ dysfunctions including the hematopoietic system. Among them, myeloproliferative disorder is known as a particular feature of the abnormality in hematopoiesis. At birth, about 10% of DS newborns show an extreme increase in blast cell number of peripheral blood and bone marrow, which is called transient abnormal myelopoiesis (TAM), because the blasts spontaneously disappear within 3 months. Morphologically, blast cells in TAM are similar to those typically found in acute megakaryoblastic leukemia (AMKL). Genetic analysis of blasts in TAM usually shows mutation in GATA1 gene. After spontaneous remission of TAM, 20 to 30% of TAM patients develop AMKL within several years. This type of AMKL is especially called DS related-AMKL (DS-AMKL). This leukemogenic transition from TAM to AMKL is considered to be a typical model of multi-step tumorigenesis. In this model, we deal with the initial part of TAM development in relationship with trisomy 21 and GATA1 mutation, especially focusing on how GATA1 mutation promotes TAM development and why hematopoietic progenitors with GATA1 mutation prevail during embryonic hematopoiesis only in the cells with trisomy 21. In order to address these unsolved issues, we have established a strictly controlled human induced pluripotent stem cell (iPSC) lines derived from DS patients with or without TAM. In this study, to recapitulate the phenotype of TAM and to specify the differentiation stage affected in hematopoietic cells in TAM patients, we differentiated established isogenic iPSC lines into megakaryocytes and erythrocytes in a step-wise manner. For this purpose, we employed two-dimensional differentiation system and compared the frequency of hematopoietic progenitor cells at various stages. For megakaryocytic lineage, we traced their differentiation as follows; hematopoietic progenitor cells committed to megakaryocytic lineage (day 9, CD34+CD41a+CD42b-CD235a-), megakaryoblasts (day 16, CD34-CD41a+CD42b-CD235a-) and promegakaryocytes (day 16, CD34-CD41a+CD42b+CD235a-). For erythrocytic differentiation, CD71+CD235a+ cells were defined as erythroid-committed hematopoietic cells. On nine days after the initiation of hematopoietic differentiation (day 9), the frequency of CD41a+CD235a- cells showed no significant differences irrespective of the status of chromosome 21 and GATA1 genotype. However, on the day 16, while the frequency of promegakaryocytes significantly decreased in GATA1-mutated iPSCs, megakaryoblasts, an earlier stage cells than promegakaryocytes, were increased in GATA1-mutated iPSCs. These data suggest that megakaryocytic maturation is arrested in GATA1-mutated iPSCs at the stage of megakaryoblasts. In GATA1-non-mutated clones, iPSCs with trisomy 21 yielded erythroid-committed CD71+CD235a+ cells more frequently than those with disomy 21. However, in GATA1-mutated clones, either trisomy 21 or disomy 21 iPSC clones never yielded the erythroid-committed cells. Taken these results together, we suspected that these in vitro phenotypes observed in both erythroid and megakaryocytic lineages were caused by the impairment of fate decision in their progenitor cells. In conclusion, we successfully recapitulated the phenotypes of TAM in vitro in regard to the abnormal differentiation into megakaryocytic and erythroid lineages. We noticed that the in vitro phenotype were associated with the GATA1 genotype and the ploidy of chromosome 21. Considering these results, analyses of the megakaryocytic and erythroid progenitor cells, such as CMP, MEP and Mk-p, are important to determine which stage of progenitors is responsible for the impairment of hematopoietic cell maturation and subsequent TAM development. Moreover, we believe that the recapitulated TAM model using iPSCs is helpful for the comprehensive understanding of pathogensis in TAM. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from human induced pluripotent stem cells

10.1101/2021.04.25.441298 ◽

2021 ◽

Author(s):

Naritaka Tamaoki ◽

Stefan Siebert ◽

Takuya Maeda ◽

Ngoc-Han Ha ◽

Meghan L. Good ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Progenitor Cells ◽

Pluripotent Stem Cells ◽

Circulatory System ◽

Yolk Sac ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Induced Pluripotent

The human definitive yolk sac is an important organ supporting the early developing embryo through nutrient supply and by facilitating the establishment of the embryonic circulatory system. However, the molecular and cellular biology of the human yolk sac remains largely obscure due to the lack of suitable in vitro models. Here, we show that human induced pluripotent stem cells (hiPSCs) co-cultured with various types of stromal cells as spheroids self-organize into yolk sac-like organoids without the addition of exogenous factors. Yolk sac-like organoids recapitulated a yolk sac specific cellular complement and structures as well as the functional ability to generate definitive hematopoietic progenitor cells (HPCs). Furthermore, sequential hemato-vascular ontogenesis could be observed during organoid formation. Notably, our organoid system can be performed in a scalable, autologous, and xeno-free condition, thereby providing an important model of human definitive yolk sac development and allows for efficient bulk generation of hiPSC-derived HPCs.

Download Full-text

Expression and function of c-Kit in fetal hemopoietic progenitor cells: transition from the early c-Kit-independent to the late c-Kit-dependent wave of hemopoiesis in the murine embryo

Development ◽

10.1242/dev.117.3.1089 ◽

1993 ◽

Vol 117 (3) ◽

pp. 1089-1098 ◽

Cited By ~ 6

Author(s):

M. Ogawa ◽

S. Nishikawa ◽

K. Yoshinaga ◽

S. Hayashi ◽

T. Kunisada ◽

...

Keyword(s):

Monoclonal Antibody ◽

Progenitor Cells ◽

Fetal Liver ◽

Flow Cytometric Analysis ◽

Yolk Sac ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

And Function ◽

Hemopoietic Progenitor ◽

Hemopoietic Progenitor Cells

The protooncogene c-kit encodes a receptor type tyrosine kinase and is allelic with the W locus of mice. SLF, the c-Kit ligand which is encoded by the Sl locus, has growth promoting activity for hemopoietic stem cells. Previous studies demonstrated that c-Kit is functionally required for the proliferation of hemopoietic progenitor cells at various differentiation stages in adult bone marrow. However, the absence of functional SLF and c-Kit in fetuses with mutant alleles of Sl and W loci produces only minor effects on the myeloid and early erythroid progenitor cells in the fetal liver, although the level of the late erythroid progenitor cells is significantly affected. We used an anti-c-Kit monoclonal antibody to investigate the expression and function of c-Kit in murine fetal hemopoietic progenitor cells. Flow-cytometric analysis showed that hemopoiesis in the yolk sac and fetal liver started from cells that express c-Kit. The c-Kit expression decreased upon maturation into erythrocytes in each organ. By fluorescence activated cell sorting, the c-Kit+ cell population was enriched with the hemopoietic progenitor cells clonable in vitro (CFU-E, BFU-E and GM-CFC). To elucidate whether c-Kit functions in these progenitor cells in vivo, we took advantage of the antagonistic anti-c-Kit monoclonal antibody, ACK2, which can block the function of c-Kit. Administration of ACK2 after 12.5 days of gestation rapidly eliminated BFU-E and GM-CFC as well as CFU-E from the fetal liver. However, the number of these progenitor cells in the yolk sac and fetal liver was less affected when the fetuses were given ACK2 before 12.5 days of gestation. Our results provide evidence that there are two waves of hemopoiesis in murine embryos relative to c-Kit dependency. The c-Kit has an essential role on the growth of hemopoietic progenitor cells in the fetal liver after 12.5 days of gestation, whereas the progenitor cells in the liver and yolk sac of the earlier embryo do not depend on c-Kit and its ligand SLF.

Download Full-text

Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells

Development ◽

10.1242/dev.129.8.2003 ◽

2002 ◽

Vol 129 (8) ◽

pp. 2003-2013 ◽

Cited By ~ 4

Author(s):

Maria Teresa Mitjavila-Garcia ◽

Michel Cailleret ◽

Isabelle Godin ◽

Maria Manuela Nogueira ◽

Karine Cohen-Solal ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Fetal Liver ◽

Hematopoietic Cells ◽

Embryonic Stem ◽

Yolk Sac ◽

Embryoid Bodies ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitors ◽

Hematopoietic Progenitor

In this study, we have characterized the early steps of hematopoiesis during embryonic stem cell differentiation. The immunophenotype of hematopoietic progenitor cells derived from murine embryonic stem cells was determined using a panel of monoclonal antibodies specific for hematopoietic differentiation antigens. Surprisingly, the CD41 antigen (αIIb integrin, platelet GPIIb), essentially considered to be restricted to megakaryocytes, was found on a large proportion of cells within embryoid bodies although very few megakaryocytes were detected. In clonogenic assays, more than 80% of all progenitors (megakaryocytic, granulo-macrophagic, erythroid and pluripotent) derived from embryoid bodies expressed the CD41 antigen. CD41 was the most reliable marker of early steps of hematopoiesis. However, CD41 remained a differentiation marker because some CD41– cells from embryoid bodies converted to CD41+ hematopoietic progenitors, whereas the inverse switch was not observed. Immunoprecipitation and western blot analysis confirmed that CD41 was present in cells from embryoid bodies associated with CD61 (β3 integrin, platelet GPIIIa) in a complex. Analysis of CD41 expression during ontogeny revealed that most yolk sac and aorta-gonad-mesonephros hematopoietic progenitor cells were also CD41+, whereas only a minority of bone marrow and fetal liver hematopoietic progenitors expressed this antigen. Differences in CD34 expression were also observed: hematopoietic progenitor cells from embryoid bodies, yolk sac and aorta-gonad-mesonephros displayed variable levels of CD34, whereas more than 90% of fetal liver and bone marrow progenitor cells were CD34+. Thus, these results demonstrate that expression of CD41 is associated with early stages of hematopoiesis and is highly regulated during hematopoietic development. Further studies concerning the adhesive properties of hematopoietic cells are required to assess the biological significance of these developmental changes.

Download Full-text

Deletion of Mtg16, a Target of t(16;21), Alters Hematopoietic Progenitor Cell Proliferation and Lineage Allocation

Molecular and Cellular Biology ◽

10.1128/mcb.00404-08 ◽

2008 ◽

Vol 28 (20) ◽

pp. 6234-6247 ◽

Cited By ~ 49

Author(s):

Brenda J. Chyla ◽

Isabel Moreno-Miralles ◽

Melissa A. Steapleton ◽

Mary Ann Thompson ◽

Srividya Bhaskara ◽

...

Keyword(s):

Cell Proliferation ◽

Progenitor Cells ◽

Cell Fate ◽

Progenitor Cell ◽

Hematopoietic Progenitor Cell ◽

Hematopoietic Progenitor ◽

Erythroid Progenitor ◽

Cell Fate Decisions ◽

Erythroid Progenitor Cells ◽

Progenitor Cell Proliferation

ABSTRACT While a number of DNA binding transcription factors have been identified that control hematopoietic cell fate decisions, only a limited number of transcriptional corepressors (e.g., the retinoblastoma protein [pRB] and the nuclear hormone corepressor [N-CoR]) have been linked to these functions. Here, we show that the transcriptional corepressor Mtg16 (myeloid translocation gene on chromosome 16), which is targeted by t(16;21) in acute myeloid leukemia, is required for hematopoietic progenitor cell fate decisions and for early progenitor cell proliferation. Inactivation of Mtg16 skewed early myeloid progenitor cells toward the granulocytic/macrophage lineage while reducing the numbers of megakaryocyte-erythroid progenitor cells. In addition, inactivation of Mtg16 impaired the rapid expansion of short-term stem cells, multipotent progenitor cells, and megakaryocyte-erythroid progenitor cells that is required under hematopoietic stress/emergency. This impairment appears to be a failure to proliferate rather than an induction of cell death, as expression of c-Myc, but not Bcl2, complemented the Mtg16 − / − defect.

Download Full-text

Human erythroid progenitor cells express Rhesus antigens

Blood ◽

10.1182/blood.v66.3.660.bloodjournal663660 ◽

1985 ◽

Vol 66 (3) ◽

pp. 660-663

Author(s):

JH Falkenburg ◽

WE Fibbe ◽

N van der Vaart-Duinkerken ◽

ME Nichols ◽

P Rubinstein ◽

...

Keyword(s):

Progenitor Cells ◽

Cell Sorting ◽

Blood Cells ◽

Hematopoietic Progenitor ◽

Erythroid Progenitor ◽

Positive Fraction ◽

Erythroid Progenitor Cells ◽

Fold Enrichment ◽

Hla Dr ◽

Complement Dependent Cytotoxicity

The expression of Rhesus antigens on hematopoietic progenitor cells was studied using monoclonal antibodies. Because these antibodies are not capable of lysing mature red blood cells in a complement-dependent cytotoxicity assay, fluorescence-activated cell sorting was performed. Using the monoclonal anti-Rh 29 antibody B10, 68% +/- 6% of the mature erythroid progenitor cells (CFU-E) were sorted into the positive fraction, while only 2% +/- 1% of the relatively immature erythroid progenitor cells (BFU-E), and 3% +/- 1% of the granulocyte-macrophage progenitor cells (CFU-GM) were cultured from this same fraction. Thus up to a 15-fold enrichment of CFU-E could be obtained. In two experiments more than 4% of the cells in the positive fraction consisted of CFU-E; in one experiment even more than 7% did. Using fractionated cell sorting, the Rhesus antigens appeared to have a lower density on CFU-E than HLA-DR determinants. Antibodies against the Rhesus antigens can be applied to enrich erythroid-committed stem cells and to separate mature from immature erythroid progenitor cells.

Download Full-text