scholarly journals FACS and immunomagnetic isolation of early erythroid progenitor cells from mouse fetal liver

2022 ◽  
Vol 3 (1) ◽  
pp. 101070
Author(s):  
Tatum W. Braun ◽  
Michael K. Kuoch ◽  
Eugene Khandros ◽  
Hojun Li
Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3381-3387 ◽  
Author(s):  
Chris P. Miller ◽  
Zi Y. Liu ◽  
Constance T. Noguchi ◽  
Don M. Wojchowski

Signals provided by the erythropoietin (Epo) receptor are essential for the development of red blood cells, and at least 15 distinct signaling factors are now known to assemble within activated Epo receptor complexes. Despite this intriguing complexity, recent investigations in cell lines and retrovirally transduced murine fetal liver cells suggest that most of these factors and signals may be functionally nonessential. To test this hypothesis in erythroid progenitor cells derived from adult tissues, a truncated Epo receptor chimera (EE372) was expressed in transgenic mice using a GATA-1 gene-derived vector, and its capacity to support colony-forming unit-erythroid proliferation and development was analyzed. Expression at physiological levels was confirmed in erythroid progenitor cells expanded ex vivo, and this EE372 chimera was observed to support mitogenesis and red blood cell development at wild-type efficiencies both independently and in synergy with c-Kit. In addition, the activity of this minimal chimera in supporting megakaryocyte development was tested and, remarkably, was observed to approximate that of the endogenous receptor for thrombopoietin. Thus, the box 1 and 2 cytoplasmic subdomains of the Epo receptor, together with a tyrosine 343 site (each retained within EE372), appear to provide all of the signals necessary for the development of committed progenitor cells within both the erythroid and megakaryocytic lineages.


Endocrinology ◽  
2014 ◽  
Vol 155 (11) ◽  
pp. 4521-4530 ◽  
Author(s):  
Wei Ying ◽  
Haiqing Wang ◽  
Fuller W. Bazer ◽  
Beiyan Zhou

Abstract Uteroferrin (UF) is a progesterone-induced acid phosphatase produced by uterine glandular epithelia in mammals during pregnancy and targeted to sites of hematopoiesis throughout pregnancy. The expression pattern of UF is coordinated with early fetal hematopoietic development in the yolk sac and then liver, spleen, and bone to prevent anemia in fetuses. Our previous studies suggested that UF exerts stimulatory impacts on hematopoietic progenitor cells. However, the precise role and thereby the mechanism of action of UF on hematopoiesis have not been investigated previously. Here, we report that UF is a potent regulator that can greatly enhance fetal erythropoiesis. Using primary fetal liver hematopoietic cells, we observed a synergistic stimulatory effect of UF with erythropoietin and other growth factors on both burst-forming unit-erythroid and colony-forming unit-erythroid formation. Further, we demonstrated that UF enhanced erythropoiesis at terminal stages using an in vitro culture system. Surveying genes that are crucial for erythrocyte formation at various stages revealed that UF, along with erythropoietin, up-regulated transcription factors required for terminal erythrocyte differentiation and genes required for synthesis of hemoglobin. Collectively, our results demonstrate that UF is a cytokine secreted by uterine glands in response to progesterone that promotes fetal erythropoiesis at various stages of pregnancy, including burst-forming unit-erythroid and colony-forming unit-erythroid progenitor cells and terminal stages of differentiation of hematopoietic cells in the erythroid lineage.


Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5372-5372
Author(s):  
Alvaro A Elorza ◽  
Brigham B Hyde ◽  
Hanna Mikkola ◽  
Sheila Collins ◽  
Orian S Shirihai

Abstract UCP2, an inner membrane mitochondrial protein, has been implicated in bioenergetics and Reactive Oxygen Species (ROS) modulation. UCP2 has been previously hypothesized to function as a facilitator of heme synthesis and iron metabolism by reducing ROS production. While UCP2 has been found to be induced by GATA1 during erythroid differentiation its role in erythropoiesis in vivo or in vitro has not been reported thus far. Here we report on the study of UCP2 role in erythropoiesis and the hematologic phenotype of UCP2 deficient mouse. In vivo we found that UCP2 protein peaks at early stages of erythroid maturation when cells are not fully committed in heme synthesis and then becomes undetectable at the reticulocyte stage. Iron incorporation into heme was unaltered in erythroid cells from UCP2 deficient mice. While heme synthesis was not influenced by UCP2 deficiency, mice lacking UCP2 had a delayed recovery from chemically induced hemolytic anemia. Analysis of the erythroid lineage from bone marrow and fetal liver revealed that in the UCP2 deficient mice the R3 (CD71high/Ter119high) population was reduced by 24%. The count of BFU-E and CFU-E colonies, scored in an erythroid colony assay, was unaffected, indicating an equivalent number of early erythroid progenitor cells in both UCP2 deficient and control cells. Ex-vivo differentiation assay revealed that UCP2 deficient c-kit+ progenitor cells expansion was overall reduced by 14% with population analysis determining that the main effect is at the R3 stage. No increased rate of apoptosis was found indicating that expansion rather than cell death is being compromised. Reduced expansion of c-kit+ cells was accompanied by 30% reduction in the phosphorylated form of ERK, a ROS dependent cytosolic regulator of cell proliferation. Analysis of ROS in UCP2 null erythroid progenitors revealed altered distribution of ROS resulting in 14% decrease in cytosolic and 32% increase in mitochondrial ROS. Restoration of the cytosolic oxidative state of erythroid progenitor cells by the pro-oxidant Paraquat reversed the effect of UCP2 deficiency on cell proliferation in in vitro differentiation assays. Together, these results indicate that UCP2 is a regulator of erythropoiesis and suggests that inhibition of UCP2 function may contribute to the development of anemia.


Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 458-458
Author(s):  
Tatiana Kouznetsova ◽  
Kestis Vaitkus ◽  
Vinzon Ibanez ◽  
Joseph DeSimone ◽  
Donald Lavelle

Abstract Abstract 458 Increased fetal hemoglobin (HbF) levels associated with acute erythropoietic stress in man and experimental baboons have been proposed to result from increased commitment of early progenitors that preferentially express γ-globin to the terminal erythroid differentiation pathway. The increased propensity of early progenitors to preferentially express γ-globin has been hypothesized to be due to the presence of trans-acting factors favoring γ-globin expression. Because increased HbF in response to acute erythropoietic stress does not occur in transgenic human β-globin gene locus mouse models, investigation of the mechanism responsible for this phenomenon requires the use of a primate model system. We investigated the role of DNA methylation and the trans-acting factor BCL11A in the mechanism responsible for increased HbF in a primary cell culture system designed to mimic conditions associated with acute erythropoietic stress. Erythroid progenitor cells (EPC) derived from CD34+ baboon bone marrow (BM) cells cultured in Iscove's medium containing 30% fetal bovine serum supplemented with 2 U/ml Epo, 200ng/ml SCF, and 1uM dexamethasone express high levels of γ-globin (0.47+ 0.09 γ/γ+β; n=6). Bisulfite sequence analysis performed to determine whether changes in DNA methylation of 5 CpG residues within the 5' γ-globin promoter regions were associated with increased γ-globin expression showed that DNA methylation levels were similar in BM erythroid cells from normal baboons expressing very low levels of HbF (<1%), bled baboons expressing moderately elevated levels of HbF (5-10%), and cultured erythroid progenitor cells expressing highly elevated levels of HbF (30-50%). Changes in γ-globin promoter DNA methylation were thus not associated with increased γ-globin expression in EPC cultures. Further experiments were therefore performed to investigate whether differences in BCL11A expression were associated with increased γ-globin in EPC cultures. Western blot assays performed using three different anti-BCL11A monoclonal antibodies recognizing epitopes present in the N terminus, core, and C terminus detected different BCL11A isoforms in cultured EPC and normal BM erythroid cells. The size of the predominant protein band detected in cultured EPC was 125kDa, corresponding to the reported size of the in vitro transcription/translation product encoded by the BCL11A-XL transcript (Liu et al, Mol Cancer 16:18, 2006). In contrast, the size of the predominant band observed in BM erythroid cells was 220kDa. The 220kDa isoform was not observed in cultured EPC. Higher molecular weight forms of BCL11A have been observed following co-transfection of vectors encoding BCL11A and SUMO-1 (Kuwata and Nakamura, Genes Cells 13:931, 2008). Therefore we investigated whether the post-translational modification SUMOylation was responsible for the difference in the size of the 125 and 220kDa isoforms. Immunoprecipitation experiments performed using either SUMO-1 or SUMO 2/3 antibodies followed by Western blot with anti-BCL11A antibody showed that the 220 kDa isoform, but not the 125kDa isoform, was immunoprecipitated by either anti-SUMO-1 or anti-SUMO-2/3 antibody, confirming that the 220 kDA isoform, but not the 125 kDa isoform, was SUMOylated. Western blot assays performed to investigate the relative levels of these isoforms in BM erythroid cells of normal baboons, phlebotomized baboons, and early gestational age (53d) baboon fetal liver showed that expression of the 125kDa isoform was increased in bled compared to normal unbled baboons, suggesting that the deSUMOylated BCL11A isoform was increased by erythropoietic stress. The relative levels of the 125 and 220 kDa isoforms were similar in bled BM and fetal liver, indicating that SUMOylation of BCL11A was not developmentally regulated. The absolute level of BCL11A was reduced in fetal liver erythroid cells compared to BM erythroid cells consistent with observations showing that the level of BCL11A expression is developmentally regulated in man (Sankaran et al, Nature epub 2009). We conclude that BCL11A is post-translationally modified by SUMOylation in primary BM erythroid cells, but not in cultured EPC expressing high levels of HbF and suggest that modulation of the level of BCL11A SUMOylation is important in the mechanism responsible for increased HbF levels during recovery from acute erythropoietic stress. Disclosures: No relevant conflicts of interest to declare.


Development ◽  
1993 ◽  
Vol 117 (3) ◽  
pp. 1089-1098 ◽  
Author(s):  
M. Ogawa ◽  
S. Nishikawa ◽  
K. Yoshinaga ◽  
S. Hayashi ◽  
T. Kunisada ◽  
...  

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.


Development ◽  
1990 ◽  
Vol 110 (2) ◽  
pp. 379-384
Author(s):  
O. Ohneda ◽  
N. Yanai ◽  
M. Obinata

Mouse stromal cell lines (FLS lines), established from the livers of 13-day gestation mouse fetus, supported the proliferation and differentiation of the erythroid progenitor cells from mouse fetal livers and bone marrow in a semisolid medium in the presence of erythropoietin. A large erythroid colony of over 1000 benzidine-positive erythroid cells was developed from a single erythroid progenitor cell on the FLS cell layer after 4 days of culture. When in close contact with the layer, the erythroid progenitor cells divided rapidly with an average generation time of 9.6 h and mature erythroid cells, including enucleated erythrocytes, were produced. The present studies demonstrate that the microenvironment created by the stromal cells can support the rapid expansion of erythropoietic cell population in the fetal liver of mice.


Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 376-386 ◽  
Author(s):  
NA Nicola ◽  
D Metcalf ◽  
H von Melchner ◽  
AW Burgess

Abstract Hemopoietic progenitor cells (colony- and cluster-forming cells in semisolid agar) were purified from light density CBA murine fetal liver cells using fluorescein-conjugated pokeweed mitogen (PWM) and a rhodamine-conjugated antineutrophil serum sandwich (alpha N) and three- parameter fluorescence-activated cell sorting. All clonable progenitor cells were highly enriched (36–50-fold) in PWM-positive (greater than channel 15), alpha N-negative (less than channel 30) fractions with relatively high intensity (greater than 100) low angle light scatter. No separation was achieved between different types of progenitor cells (granulocyte-macrophage and erythroid colony-forming cells). The enriched fraction was a pure population of large, basophilic, undifferentiated blast cells, and in agar cultures stimulated with colony-stimulating factors, up to 90% of the enriched cells were hemopoietic progenitor cells capable of varying levels of clonal proliferation. Further fractionation based on increasing fluorescence with PWM separated into discrete populations, nonproliferative morphologically recognizable erythroid cells, late erythroid progenitor cells (day 2 CFU-E), and cells forming pure or mixed erythroid burst colonies. In addition, the majority of pluripotential hemopoietic stem cells (CFU-SS) were clearly separated from progenitor cells forming colonies in vitro. The present techniques provide suitable numbers of enriched progenitor cells for a variety of biological and biochemical studies.


Blood ◽  
2001 ◽  
Vol 97 (4) ◽  
pp. 901-910 ◽  
Author(s):  
Justin N. Geiger ◽  
Geoffry T. Knudsen ◽  
Leigh Panek ◽  
Ajay K. Pandit ◽  
Michael D. Yoder ◽  
...  

Abstract DYRKs are a new subfamily of dual-specificity kinases that was originally discovered on the basis of homology to Yak1, an inhibitor of cell cycle progression in yeast. At present, mDYRK-3 and mDYRK-2 have been cloned, and mDYRK-3 has been characterized with respect to kinase activity, expression among tissues and hematopoietic cells, and possible function during erythropoiesis. In sequence, mDYRK-3 diverges markedly in noncatalytic domains from mDYRK-2 and mDYRK-1a, but is 91.3% identical overall to hDYRK-3. Catalytically, mDYRK-3 readily phosphorylated myelin basic protein (but not histone 2B) and also appeared to autophosphorylate in vitro. Expression of mDYRK-1a, mDYRK-2, and mDYRK-3 was high in testes, but unlike mDYRK1a and mDYRK 2, mDYRK-3 was not expressed at appreciable levels in other tissues examined. Among hematopoietic cells, however, mDYRK-3 expression was selectively elevated in erythroid cell lines and primary pro-erythroid cells. In developmentally synchronized erythroid progenitor cells, expression peaked sharply following exposure to erythropoietin plus stem cell factor (SCF) (but not SCF alone), and in situ hybridizations of sectioned embryos revealed selective expression of mDYRK-3 in fetal liver. Interestingly, antisense oligonucleotides to mDYRK-3 were shown to significantly and specifically enhance colony-forming unit–erythroid colony formation. Thus, it is proposed that mDYRK-3 kinase functions as a lineage-restricted, stage-specific suppressor of red cell development.


Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 376-386
Author(s):  
NA Nicola ◽  
D Metcalf ◽  
H von Melchner ◽  
AW Burgess

Hemopoietic progenitor cells (colony- and cluster-forming cells in semisolid agar) were purified from light density CBA murine fetal liver cells using fluorescein-conjugated pokeweed mitogen (PWM) and a rhodamine-conjugated antineutrophil serum sandwich (alpha N) and three- parameter fluorescence-activated cell sorting. All clonable progenitor cells were highly enriched (36–50-fold) in PWM-positive (greater than channel 15), alpha N-negative (less than channel 30) fractions with relatively high intensity (greater than 100) low angle light scatter. No separation was achieved between different types of progenitor cells (granulocyte-macrophage and erythroid colony-forming cells). The enriched fraction was a pure population of large, basophilic, undifferentiated blast cells, and in agar cultures stimulated with colony-stimulating factors, up to 90% of the enriched cells were hemopoietic progenitor cells capable of varying levels of clonal proliferation. Further fractionation based on increasing fluorescence with PWM separated into discrete populations, nonproliferative morphologically recognizable erythroid cells, late erythroid progenitor cells (day 2 CFU-E), and cells forming pure or mixed erythroid burst colonies. In addition, the majority of pluripotential hemopoietic stem cells (CFU-SS) were clearly separated from progenitor cells forming colonies in vitro. The present techniques provide suitable numbers of enriched progenitor cells for a variety of biological and biochemical studies.


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