Definitive Erythro-Myeloid Progenitors (EMP) Emerge in the Yolk Sac From Hemogenic Endothelium and Share Transcriptional Regulators with Adult Hematopoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 910-910
Author(s):  
Kathleen E McGrath ◽  
Katherine H Fegan ◽  
Jenna M Frame ◽  
Paul D Kingsley ◽  
James Palis
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Fetal Liver ◽  
Transcriptional Regulators ◽  
Yolk Sac ◽  
Erythroid Cells ◽  
Hemogenic Endothelium ◽  
Cell Potential ◽  
Myeloid Progenitors

Abstract Abstract 910 In the mammalian embryo, hematopoietic stem cells (HSC) emerge from vascular beds and colonize the fetal liver. The first HSC are found in the murine fetal liver by embryonic day 12.5 (E12.5). However blood function is required before HSC have formed, and two earlier waves of hematopoietic potential arise to sustain the embryo. The first wave of hematopoietic progenitors are formed in the yolk sac between E7.25 and E8.5 and are termed “primitive” because, along with megakaryocyte and macrophage potential, they differentiate into primitive erythroid cells that mature in the circulation and express embryonic globins. A second lineage of hematopoietic potential has been characterized in the murine and human yolk sac as well as in zebrafish. These cells have been termed “EMP”, erythro-myeloid progenitors, that generate definitive erythroid and myeloid lineages, including granulocytes. We find that EMP emerging in the mouse embryo express many of the markers associated with HSC emergence from hemogenic endothelium. At early stages of their emergence (E8.5), EMP express not only kit and CD41, but also VE-cadherin, CD31 and CD34. A day later, EMP constitute a robust population of over 1,000 cells in the yolk sac and display diminished expression of VE-cadherin and increased expression of CD45. However, unlike HSC, EMP do not express Sca1, but do express the myeloid progenitor marker CD16/32 (low affinity FCgamma receptor II/III). Like CD45, CD16/32 is expressed on a subset of the CD41+/kit+ cells at E8.5. Colony forming assays confirm that EMP potential is found in both CD16/32 positive and negative CD41+/kit+ cells. By E9.5, over 90% of CD41+/kit+ cells also express CD16/32 and all of the hematopoietic colony-forming potential at E9.5 is found in this triple-positive population. The transition from endothelial-associated to hematopoietic-associated genes suggests that EMP may emerge from hemogenic endothelial intermediates. Between E9.5 and E11.5, cells with the EMP immunophenotype are found in the bloodstream and become concentrated in the liver, where evidence of very robust erythro-myeloid differentiation precedes HSC colonization. In culture, EMP rapidly expand, dividing twice daily, and within 6 days generate predominately erythroid cells as well as smaller numbers of megakaryocyte, macrophage and mast cells, but only rare granulocytes. This is in contrast to lin-/kit+ ScaI- bone marrow progenitors grown in the same culture conditions that generate predominately myeloid cells, particularly granulocytes, and rarely mast cells. In agreement with data in the zebrafish, we also do not see evidence of lymphoid potential or RAG2 expression in EMP cultures or expression of the lymphoid markers Flt3 and IL7 receptor on the cell surface of EMP. In order to better understand the lineage potential of EMP, we examined the expression of known transcriptional regulators of bone marrow hematopoiesis. In the adult, relative levels of GATA1 versus Pu.1 are proposed to determine fate between megakaryocyte/erythroid progenitors (MEP- GATA1 hi) and granulocyte/macrophage progenitors (GMP-Pu.1 hi). Consistent with their erythro-myeloid potential, EMP expressed both GATA1 and Pu.1 at intermediate levels compared to adult marrow-derived MEP and GMP. In the adult, Gfi-1 and C/EBPalpha are both proposed to upregulate granulocyte versus macrophage differentiation. We found lower levels of these regulators in EMP compared to GMP, consistent with EMP cultures generating small numbers of granulocytes versus macrophages. In addition, the GATA1 expression within the Pu.1+ GMP is found to increase mast cell potential and, thus, the high GATA1 and Pu.1 expression in EMP may account for their high mast cell potential. Taken together, these data suggest that, like HSC, EMP emerge from hemogenic endothelium and their erythro-myeloid potential is governed by the action of shared regulatory networks. However, the transcription factors and markers are present in the EMP in unique combinations consistent with their specific role in providing a transient initial wave of definitive hematopoiesis in the embryo. Disclosures: No relevant conflicts of interest to declare.

Definitive Hematopoiesis In the Mammalian Embryo Prior to HSC Formation.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1599-1599
Author(s):  
Kathleen E McGrath ◽  
Jenna M Frame ◽  
Anne Koniski ◽  
Paul D Kingsley ◽  
James Palis
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Differentiation ◽  
Blood Cells ◽  
Fetal Liver ◽  
Yolk Sac ◽  
Es Cell ◽  
Hematopoietic Progenitors ◽  
Adult Bone ◽  
Myeloid Progenitors

Abstract Abstract 1599 The ontogeny of hematopoiesis in mammalian embryos is complicated by the requirement for functional blood cells prior to the emergence of hematopoietic stem cells or the bone marrow microenvironment. In the murine embryo, transplantable HSC are first evident at embryonic day (E) 10.5 and the first few HSC are found in the fetal liver hematopoietic environment by E12.5. However, two overlapping waves of hematopoietic potential arise in the yolk sac before E10.5. The first “primitive” wave produces progenitors from E7.25 to E8.5 with primitive erythroid, megakaryocyte and macrophage potentials. The resulting primitive erythroid cells mature within the circulation and support embryonic growth past E9.5. At E8.5, a second wave of hematopoiesis begins in the yolk sac and generates definitive erythroid and multiple myeloid progenitors that are the proposed source of the hematopoietic progenitors seeding the fetal liver before HSC colonization. We have identified a cell population displaying a unique cell surface immunophenotype in the E9.5 yolk sac that contains the potential to form definitive erythroid cells, megakaryocytes, macrophages and all forms of granulocytes within days of in vitro culture. Furthermore, all definitive hematopoietic colony-forming cells (BFU-E, CFC-myeloid and HPP-CFC) in the E9.5 yolk sac have this immunophenotype. These erythro-myeloid progenitors (EMP) are lineage-negative and co-express ckit, CD41, CD16/32 and Endoglin. Interestingly, this is not an immunophenotype evident in the adult bone marrow. Other markers that have been associated with HSC formation (AA4.1, ScaI) or with lymphoid potential (IL7R, Flt3) are not present on these cells at E9.5. Consistent with the lack of lymphoid markers, we also do not observe short-term development of B-cells (CD19+B220+ expressing Rag2 RNA) in cultures of the E9.5 sorted EMP, while bone marrow Lin-/ckit+/ScaI- cells do form B-cells under the same conditions. Clonal analysis of sorted EMP cells revealed single cells with both erythroid and granulocyte potential, similar to the common myeloid progenitors in adult bone marrow. Though these EMP are enriched at E9.5 in the yolk sac, they are also found at low levels in the fetal blood, embryo proper and placenta, consistent with their entrance into the circulation. By E10.5, EMP were most highly enriched in the newly formed fetal liver. Additionally by E12.5, a time when the first few HSCs are detected in the fetal liver, we find active erythropoiesis and granulopoiesis in the liver and the first definitive red blood cells and neutrophils in the bloodstream. Therefore, we believe the yolk sac definitive progenitors' fate is to populate the fetal liver and thus provide the first definitive erythrocytes and granulocytes for the embryo. The differentiation of embryonic stem cells (ES) and induced pluripotent stem cells (iPS) cells into mature cells types offers the hope of cell-based therapies. Analysis of differentiating murine ES cells reveals overlapping waves of primitive and definitive hematopoietic colony forming potential. We demonstrate the appearance of an EMP-like (ckit+/CD41+/FcGR+) population coincident with the emergence of definitive hematopoietic progenitors during murine ES cell differentiation as embryoid bodies. We have confirmed with colony forming assays that definitive hematopoietic potential is associated with this immunophenotypic group. Our studies support the concept that blood cell emergence during ES cell differentiation closely mimics pre-HSC hematopoiesis in the yolk sac. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ezh2 is essential for the generation of functional yolk sac derived erythro-myeloid progenitors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wen Hao Neo ◽  
Yiran Meng ◽  
Alba Rodriguez-Meira ◽  
Muhammad Z. H. Fadlullah ◽  
Christopher A. G. Booth ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Epigenetic Regulation ◽  
Yolk Sac ◽  
Specific Role ◽  
Primary Reason ◽  
Erythroid Cells ◽  
Hemogenic Endothelium ◽  
Epigenetic Regulator ◽  
Myeloid Progenitors

AbstractYolk sac (YS) hematopoiesis is critical for the survival of the embryo and a major source of tissue-resident macrophages that persist into adulthood. Yet, the transcriptional and epigenetic regulation of YS hematopoiesis remains poorly characterized. Here we report that the epigenetic regulator Ezh2 is essential for YS hematopoiesis but dispensable for subsequent aorta–gonad–mesonephros (AGM) blood development. Loss of EZH2 activity in hemogenic endothelium (HE) leads to the generation of phenotypically intact but functionally deficient erythro-myeloid progenitors (EMPs), while the generation of primitive erythroid cells is not affected. EZH2 activity is critical for the generation of functional EMPs at the onset of the endothelial-to-hematopoietic transition but subsequently dispensable. We identify a lack of Wnt signaling downregulation as the primary reason for the production of non-functional EMPs. Together, our findings demonstrate a critical and stage-specific role of Ezh2 in modulating Wnt signaling during the generation of EMPs from YS HE.

Balance of Transcription Factors Downstream of Notch Signaling Determines the Fate of Myeloid Progenitors toward Differentiation to Mast Cells or Immortalization without Differentiation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 676-676
Author(s):  
Mamiko Sakata-Yanagimoto ◽  
Fumio Nakahara ◽  
Etsuko Yamaguchi-Nakagami ◽  
Keiki Kumano ◽  
Toshiki Saito ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Cell Differentiation ◽  
Mast Cells ◽  
Notch Signaling ◽  
Cell Fate ◽  
Myeloid Cell ◽  
Cell Generation ◽  
Notch Activation ◽  
Myeloid Progenitors

Abstract Notch signaling represents one of the fundamental communication channels in various types of cells. While Notch activation has been shown to inhibit myeloid differentiation in a subset of hematopoietic progenitors, the role of Notch signaling in mast cell differentiation is not clear. When common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) purified from mouse bone marrow cells were stimulated with Delta1-Fc, a soluble form of Notch ligand, in the presence of stem cell factor, IL-3, IL-6, and thrombopoietin, granulocyte and macrophage differentiation, which is observed at day 7 of culture in the absence of Delta1-Fc, was markedly inhibited. Instead, Lin-c-Kit+FcεR+ mast cells dominated in the culture. Delta1-Fc did not increase mast cell generation from either CMPs or GMPs of the bone marrow of pI:pC-treated Mx-Cre x Notch2 flox/flox (N2-MxcKO) mice, in contrast to littermate Notch2 flox/flox mice treated with pI:pC, which suggests that Notch2 is responsible for the Delta1-Fc-augmented mast cell generation from CMPs and GMPs in culture. Retroviral transfer of constitutive active form of Notch2 (aN2) into CMPs and GMPs resulted in the complete loss of granulocyte-macrophage colony-forming cells and the emergence of basophilic granules-containing blast like cells, indicating the cell fate instruction. Real-time PCR analysis revealed that Delta1-Fc stimulation and aN2 introduction up-regulated the expression of Hes1, a transcriptional suppressor that is known to be a direct target of Notch activation in several cell types, within 12 h. Moreover, among GATA genes, Delta1-Fc stimulation and aN2 introduction resulted in increase of GATA3 mRNA, while expression levels of GATA1 and GATA2, which have been suggested to play a role in regulating mast cell differentiation, were unchanged. Next, we retrovirally expressed Hes1 and/or a GATA gene into CMPs and GMPs to see if the same effects were observed. Mast cells were increased only when both genes were expressed. On the other hand, when Hes1 alone was transduced, we observed rapid growth and immortalization of these cells without differentiation. C/EBPa, which is known to be suppressed in mast cell differentiation and upregulated in myeloid cell differentiation, was down-regulated within 48 h from the initiation of Hes1 retroviral transduction, suggesting that C/EBPa is a downstream target of Hes1 in this myeloid cell fate determination. Theses results indicate that, at the downstream of Notch activation, there are a C/EBPa down-regulation pathway that is Hes1-dependent and a GATA3 up-regulation pathway. Balanced regulation of these pathways should play a physiological role in myeloid and mast cell differentiation, while imbalance between these two pathways might provide a new model of myeloid transformation.

Complete Myeloid Potential Arises First in the Mammalian Yolk Sac

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 730-730 ◽  
Author(s):  
Kathleen E. McGrath ◽  
Jenna M. Cacciatori ◽  
Anne D. Koniski ◽  
James Palis
Keyword(s):  
Mast Cells ◽  
Fetal Liver ◽  
Yolk Sac ◽  
Receptor Expression ◽  
Erythroid Cells ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Second Wave ◽  
Definitive Hematopoiesis

Abstract In the mouse embryo, hematopoietic function is required by E10.5 (embryonic day 10.5) before adult-repopulating hematopoietic stem cells (HSC) exist. The earliest erythroid function is provided by a wave of primitive erythroid progenitors that arise at E7.5, in association with megakaryocyte and macrophage progenitors. Intriguingly, a second wave of hematopoietic potential arises between the first primitive hematopoietic wave and functional HSC formation. This second progenitor wave also forms in the yolk sac but is distinguished from the primitive wave by its slightly later onset (E8.25), generation of definitive erythroid cells, and its additional association with granulocyte and mast cell progenitors. The proposed function of these “wave 2” progenitors is to colonize the newly formed fetal liver (beginning at E10) and differentiate into the first mature definitive erythroid cells observed in circulation at E12. However, it is unclear how much definitive hematopoiesis arising in the yolk sac recapitulates the paradigm of later HSC-derived myeloid potential, progenitor hierarchy and immunophenotype. To investigate this question, we examined markers of adult myeloid progenitor maturation in the yolk sac and early fetal liver. As previously described by others, all definitive hematopoietic progenitors in the yolk sac, unlike those in the bone marrow, express CD41, which we found associated with Fc gamma receptor expression (FcγR, CD16/32) beginning at E8.5. By E9.5, definitive hematopoietic progenitors can be identified by their surface co-expression of ckit, CD41, FcγR, as well as endoglin. When cultured in vitro, these cells can differentiate into all myeloid lineages, including neutrophils, eosinophils, basophils and mast cells as identified by morphology, immunophenotype and gene expression. Preliminary clonal analysis confirms that a common erythroid/granulocyte progenitor exists in this population. Consistent with adult myelopoiesis, we found a qualitative association of higher FcγR expression with granulocyte fate and higher endoglin expression associated with erythroid fate. However, the unusual co-expression of these four markers and the prevalence of erythroid fate, even in FcγRhi cells, suggest the definitive hematopoietic progenitors in the yolk sac may be quite plastic and highly predisposed to an erythroid fate. Consistent with the concept that these “wave 2” progenitors colonize the fetal liver, we also found similar ckit+CD41+FcγR+endoglin+ cells in the early liver (E11.5) with the potential to produce a variety of myeloid cells when cultured in vitro. The emergence of enucleated definitive erythrocytes by E12, within 24 hours HSC fetal liver colonization, implies that these first erythrocytes are derived from the yolk sac definitive progenitors found in the liver by E10.5. We therefore asked whether the multiple myeloid potentials associated with “wave 2” progenitors are similarily realized the early fetal liver. Beginning at E11.5 we found a population of Gr1+Mac1+ cells in the liver with morphological and histological characteristics of neutrophils, and increase 100-fold in number between E12.5 and E14.5. In contrast, we did not observe eosinophils, basophils or mast cells by morphology, immunophenotype or by RT-PCR for lineage-specific messages. We conclude that complete definitive myeloid potential first arises in the yolk sac from a unique population of ckit+FcγR+CD41+endoglin+ progenitors. Our data suggest that these progenitors then enter the fetal liver and differentiate into a subset of their potential fates producing the first mature definitive erythromyeloid cells. This second wave of hematopoietic progenitors emerging from the yolk sac thus serves as a novel model of mulitpotential definitive hematopoiesis.

Development of hematopoietic cells lacking transcription factor GATA-1

Development ◽  
1995 ◽  
Vol 121 (1) ◽  
pp. 163-172 ◽  
Author(s):  
L. Pevny ◽  
C.S. Lin ◽  
V. D'Agati ◽  
M.C. Simon ◽  
S.H. Orkin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mast Cell ◽  
Cell Differentiation ◽  
Mast Cells ◽  
Blood Cell ◽  
Embryonic Stem ◽  
Sequence Motif ◽  
Erythroid Cells ◽  
Gata Factor

GATA-1 is a zinc-finger transcription factor believed to play an important role in gene regulation during the development of erythroid cells, megakaryocytes and mast cells. Other members of the GATA family, which can bind to the same DNA sequence motif, are co-expressed in several of these hemopoietic lineages, raising the possibility of overlap in function. To examine the specific roles of GATA-1 in hematopoietic cell differentiation, we have tested the ability of embryonic stem cells, carrying a targeted mutation in the X-linked GATA-1 gene, to contribute to various blood cell types when used to produce chimeric embryos or mice. Previously, we reported that GATA-1- mutant cells failed to contribute to the mature red blood cell population, indicating a requirement for this factor at some point in the erythroid lineage (L. Pevny et al., (1991) Nature 349, 257–260). In this study, we have used in vitro colony assays to identify the stage at which mutant erythroid cells are affected, and to examine the requirement for GATA-1 in other lineages. We found that the development of erythroid progenitors in embryonic yolk sacs was unaffected by the mutation, but that the cells failed to mature beyond the proerythroblast stage, an early point in terminal differentiation. GATA-1- colonies contained phenotypically normal macrophages, neutrophils and megakaryocytes, indicating that GATA-1 is not required for the in vitro differentiation of cells in these lineages. GATA-1- megakaryocytes were abnormally abundant in chimeric fetal livers, suggesting an alteration in the kinetics of their formation or turnover. The lack of a block in terminal megakaryocyte differentiation was shown by the in vivo production of platelets expressing the ES cell-derived GPI-1C isozyme. The role of GATA-1 in mast cell differentiation was examined by the isolation of clonal mast cell cultures from chimeric fetal livers. Mutant and wild-type mast cells displayed similar growth and histochemical staining properties after culture under conditions that promote the differentiation of cells resembling mucosal or serosal mast cells. Thus, the mast and megakaryocyte lineages, in which GATA-1 and GATA-2 are co-expressed, can complete their maturation in the absence of GATA-1, while erythroid cells, in which GATA-1 is the predominant GATA factor, are blocked at a relatively early stage of maturation.

scholarly journals Adenosine A2Areceptor activation reduces infarct size in the isolated, perfused mouse heart by inhibiting resident cardiac mast cell degranulation

2008 ◽  
Vol 295 (5) ◽  
pp. H1825-H1833 ◽  
Author(s):  
Tyler H. Rork ◽  
Kori L. Wallace ◽  
Dylan P. Kennedy ◽  
Melissa A. Marshall ◽  
Amy R. Lankford ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Mast Cell Degranulation ◽  
Chimeric Mice ◽  
Cgs 21680 ◽  
Perfused Hearts

Mast cells are found in the heart and contribute to reperfusion injury following myocardial ischemia. Since the activation of A2Aadenosine receptors (A2AARs) inhibits reperfusion injury, we hypothesized that ATL146e (a selective A2AAR agonist) might protect hearts in part by reducing cardiac mast cell degranulation. Hearts were isolated from five groups of congenic mice: A2AAR+/+mice, A2AAR−/−mice, mast cell-deficient (KitW-sh/W-sh) mice, and chimeric mice prepared by transplanting bone marrow from A2AAR−/−or A2AAR+/+mice to radiation-ablated A2AAR+/+mice. Six weeks after bone marrow transplantation, cardiac mast cells were repopulated with >90% donor cells. In isolated, perfused hearts subjected to ischemia-reperfusion injury, ATL146e or CGS-21680 (100 nmol/l) decreased infarct size (IS; percent area at risk) from 38 ± 2% to 24 ± 2% and 22 ± 2% in ATL146e- and CGS-21680-treated hearts, respectively ( P < 0.05) and significantly reduced mast cell degranulation, measured as tryptase release into reperfusion buffer. These changes were absent in A2AAR−/−hearts and in hearts from chimeric mice with A2AAR−/−bone marrow. Vehicle-treated KitW-sh/W-shmice had lower IS (11 ± 3%) than WT mice, and ATL146e had no significant protective effect (16 ± 3%). These data suggest that in ex vivo, buffer-perfused hearts, mast cell degranulation contributes to ischemia-reperfusion injury. In addition, our data suggest that A2AAR activation is cardioprotective in the isolated heart, at least in part by attenuating resident mast cell degranulation.

scholarly journals Changes in numbers and types of mast cell colony-forming cells in the peritoneal cavity of mice after injection of distilled water: evidence that mast cells suppress differentiation of bone marrow-derived precursors

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 573-580 ◽  
Author(s):  
Y Kanakura ◽  
A Kuriu ◽  
N Waki ◽  
T Nakano ◽  
H Asai ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Peritoneal Cavity ◽  
Bone Marrow Cells ◽  
Water Injection ◽  
Lymphoid Cells ◽  
Distilled Water ◽  
Peritoneal Mast Cells ◽  
Chediak Higashi Syndrome

Abstract Two different types of cells in the peritoneal cavity of mice produce mast cell colonies in methylcellulose. “Large” mast cell colonies are produced by bone marrow-derived precursors resembling lymphoid cells by light microscopy (L-CFU-Mast), whereas “medium” and “small” mast cell colonies are produced by morphologically identifiable mast cells (M-CFU- Mast and S-CFU-Mast, respectively). In the present study we eradicated peritoneal mast cells by intraperitoneal (IP) injection of distilled water. The regeneration process was investigated to clarify the relationship between L-CFU-Mast, M-CFU-Mast, and S-CFU-Mast. After injection of distilled water, M-CFU-Mast and S-CFU-Mast disappeared, but L-CFU-Mast increased, and then M-CFU-Mast and S-CFU-Mast appeared, suggesting the presence of a hierarchic relationship. When purified peritoneal mast cells were injected two days after the water injection, the L-CFU-Mast did not increase. In the peritoneal cavity of WBB6F1-+/+ mice that had been lethally irradiated and rescued by bone marrow cells of C57BL/6-bgJ/bgJ (beige, Chediak-Higashi syndrome) mice, L-CFU-Mast were of bgJ/bgJ type, but M-CFU-Mast and S-CFU-Mast were of +/+ type. The injection of distilled water to the radiation chimeras resulted in the development of bgJ/bgJ-type M-CFU-Mast and then S-CFU-Mast. The presence of mast cells appeared to suppress the recruitment of L-CFU- Mast from the bloodstream and to inhibit the differentiation of L-CFU- Mast to M-CFU-Mast.

The effect of IL-3 and SCF mixture on cell proliferation and rat mast cell protease-???? synthesis of rat bone marrow derived mast cells

2007 ◽  
Vol &NA; ◽  
pp. S187
Author(s):  
Haneul Nari Lee ◽  
Ju Hyeon Lee ◽  
Chul Hwan Kim ◽  
Yoon Gyu Kang ◽  
Kyung-Whan Joo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Mast Cell ◽  
Mast Cells ◽  
Mast Cell Protease ◽  
Rat Bone

scholarly journals Immunomagnetic Isolation of Rat Bone Marrow-derived and Peritoneal Mast Cells

1997 ◽  
Vol 45 (12) ◽  
pp. 1715-1722 ◽  
Author(s):  
Maria Celia Jamur ◽  
Ana Cristina G. Grodzki ◽  
Andrea N. Moreno ◽  
William D. Swaim ◽  
Reuben P. Siraganian ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Cell Types ◽  
Ultrastructural Level ◽  
Immunomagnetic Separation ◽  
Specific Cell ◽  
Morphological Examination ◽  
Immunomagnetic Isolation ◽  
Rat Bone

Mast cells are difficult to purify from heterogeneous cell populations and to preserve, especially for pre-embedding immunostaining at the ultrastructural level. We have developed a technique that permits the isolation of a pure population of mast cells suitable for immunocytochemical studies. A rat mast cell-specific monoclonal antibody (MAb AA4) conjugated to tosylactivated Dynabeads 450 was used to immunomagnetically separate mast cells from rat bone marrow and peritoneal cell suspensions. Approximately 85% of the mast cells were recovered in the positive population that comprised virtually pure mast cells. After microwave fixation, morphological examination showed that the cells were intact and retained their ultrastructural detail. Mast cells in all stages of maturation were immunolabeled with a panel of antibodies after immunomagnetic separation. The combination of immunomagnetic separation followed by immunostaining should prove useful for the study of mast cell maturation and for the characterization of other specific cell types that are present in tissues in only limited numbers.

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