scholarly journals Endomucin, a CD34-like sialomucin, marks hematopoietic stem cells throughout development

2005 ◽  
Vol 202 (11) ◽  
pp. 1483-1492 ◽  
Author(s):  
Azusa Matsubara ◽  
Atsushi Iwama ◽  
Satoshi Yamazaki ◽  
Chie Furuta ◽  
Ryutaro Hirasawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Signal Sequence ◽  
Time Frame ◽  
Mouse Bone Marrow ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
High Level

To detect as yet unidentified cell-surface molecules specific to hematopoietic stem cells (HSCs), a modified signal sequence trap was successfully applied to mouse bone marrow (BM) CD34−c-Kit+Sca-1+Lin− (CD34−KSL) HSCs. One of the identified molecules, Endomucin, is an endothelial sialomucin closely related to CD34. High-level expression of Endomucin was confined to the BM KSL HSCs and progenitor cells, and, importantly, long-term repopulating (LTR)–HSCs were exclusively present in the Endomucin+CD34−KSL population. Notably, in the yolk sac, Endomucin expression separated multipotential hematopoietic cells from committed erythroid progenitors in the cell fraction positive for CD41, an early embryonic hematopoietic marker. Furthermore, developing HSCs in the intraembryonic aorta-gonad-mesonephros (AGM) region were highly enriched in the CD45−CD41+Endomucin+ fraction at day 10.5 of gestation (E10.5) and in the CD45+CD41+Endomucin+ fraction at E11.5. Detailed analyses of these fractions uncovered drastic changes in their BM repopulating capacities as well as in vitro cytokine responsiveness within this narrow time frame. Our findings establish Endomucin as a novel cell-surface marker for LTR-HSCs throughout development and provide a powerful tool in understanding HSC ontogeny.

Expression of Endomucin, a CD34-Like Sialomucin, Marks Definitive Hematopoietic Stem Cells throughout Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 563-563
Author(s):  
Azusa Maeda ◽  
Atsushi Iwama ◽  
Koji Eto ◽  
Hideo Ema ◽  
Toshio Kitamura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Signal Sequence ◽  
High Yield ◽  
Specific Gene ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract In order to identify cell surface molecules specific to hematopoietic stem cells (HSCs), a modified signal sequence trap was applied to mouse bone marrow (BM) CD34− c-Kit+ sca-1+ lineage− (CD34−KSL) cells which is highly enriched for HSCs. Among the identified genes, mRNA expression of Endomucin, an endothelium-specific gene encoding a CD34-like sialomucin, appeared highly specific to CD34-KSL HSCs. To further investigate the expression of Endomucin, we generated two rat anti-mouse Endomucin monoclonal antibodies that recognize different epitopes (AE2D4, AE7F2). Taking advantage of these and another monoclonal antibody, V7c7 (1999, Blood, 93; 1; 165), detailed expression analysis was performed. Endomucin expression was largely confined to lineage markers-negative (Lin−) cells. Approximately 7 % of Lin− cells were Endomucin-positive. Cells strongly expressing Endomucin represented 30% of c-kit+ sca-1+ cells. Gating out CD34+ cells from Lin− Endomucin+ population resulted in high yield of KSL cells. High correlation between Lin− Endomucin+CD34− cells and KSL cells was confirmed by in vivo bone marrow transplantation. When Lin− cells were fractionated by their expression of CD34 and Endomucin, only Lin− Endomucin+CD34− cells contributed to long-term repopulation (LTR), and as few as 100 cells were enough to obtain engraftment. Furthermore, the majority of CD34−KSL cells were Endomucin+, and again, only CD34−KSL-Endomucin+ cells had LTR activity. These data indicate two facts: 1) A single positive marker, Endomucin can substitute for c-kit+ sca-1+, 2) All LTR -HSCs express Endomucin. We then analyzed the expression of Endomucin during embryonic development of the hematopoietic system. Definitive HSCs arise from the hemogenic endothelium lining the wall of the dorsal aorta in embryonic aorta-gonads-mesonephros (AGM) region, then seed to the fetal liver. E10.5 AGM CD45− cells were segregated into subpopulations by their expression of Endomucin and CD41, an early marker of embryonic hematopoiesis. In vitro coculture system with a stromal cell line, OP9, was applied to detect the ability of hematopoietic potential. Hematopoietic activity was exclusively found in the CD41+Endomucin+ population, that represents 24% of CD41+ cells. Taken together, these data indicate that Endomucin marks both embryonic and adult HSCs, providing a novel useful cell surface marker for definitive HSCs throughout development. Figure Figure

Implication of the Distinct Differentiation Pathway of Megakaryocytes From Hematopoietic Stem Cells in the Mouse Bone Marrow

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1204-1204
Author(s):  
Hidekazu Nishikii ◽  
Kenji Matsushita ◽  
Yosuke Kanazawa ◽  
Yasuhisa Yokoyama ◽  
Takayasu Kato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Dominant Negative Mutant ◽  
Mouse Bone Marrow ◽  
Alpha Cells ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Abstract 1204 Background. Hematopoietic progenitor cells are the progeny of hematopoietic stem cells (HSC) that coordinate the production of precise number of mature blood cells of diverse functional lineages. Megakaryocytes (Meg) are mapped at the downstream of bilineage progenitors for erythroid and megakaryocyte (MEP) in the most widely accepted scenarios, although different notions have also been suggested. Thrombopoietin (TPO) is thought to be the master cytokine for megakaryopoiesis. In mice lacking cMpl, the receptor for TPO, production of platelets and Meg is severely impaired. However, Meg are known to be still present in the bone marrow of these mice. These findings suggested that TPO independent signaling for Meg differentiation would exist. Purpose. To clarify the differentiation pathway of the Meg lineage, we focused on GPIb (CD42)-V-IX complex, expression of which has not been characterized in any progenitor cells whereas it is well known to be expressed on mature Meg and platelets. We also investigated how TPO-cMpl signaling would affect at MEP or pure megakaryocyte progenitor (MKP) stage using the cMpl deficient mice. Results and Discussion. GPIb alpha (CD42b) was expressed on 3–6 % of a mouse bone marrow population characterized as common myeloid progenitors (CMP), i.e., Lin-c-Kit+Sca1-CD34+CD16/32low cells. The GPIb alpha+ CMP (thereafter designated 34-alpha) population also expresses CD9, SLAM1, and CD41. These 34-alpha cells showed a restricted differentiation capacity to the mature Meg in in vitro culture. By intravenously infusing 34-alpha cells derived from CAG promoter-driven GFP-expressing mice into sublethally irradiated syngenic mice, GFP-expressing platelets were generated in vivo. Thus, we designate the 34-alpha cells as 34-alpha MKP. Gene expression analysis also supported that 34-alpha MKP has a restricted capacity of megakaryopoiesis. In vitro colony-forming assay and short-term liquid culture assay suggested that they are not derived from MEP but from the SLAM1+Flt3-c-Kit+Sca1+Lin- population, which highly contain HSC. When experimental thrombocytopenia was induced by injecting 5-fluorouracil into mice, the frequency of 34-alpha MKP was rapidly increased compared to that of MEP. These data imply a distinct pathway of Meg differentiation, which originates at the proximity of HSC. We next investigated whether generation of 34-alpha MKP and MEP is differently impaired in cMpl-deficient mice. The frequency of MEP was only mildly reduced. In contrast, 34-alpha MKP were much severely reduced. Notably, in vitro Meg differentiation was markedly impaired from both MEP and 34-alpha MKP derived from cMpl-deficient mice. These data suggested that discordance between Meg and platelet production is caused by the different dependence on TPO-cMpl signaling between the pathways generating MEP and 34-alpha MKP from HSC. We also found that Hes1, a transcription factor that is the best characterized effector functioning downstream of the Notch signaling pathway, is highly expressed in 34-alpha MKP. Conversely, Meg differentiation was abrogated by retroviral transduction of a dominant-negative mutant of Hes1. Taken together, our data imply the presence of two distinct Meg differentiation pathways from HSC and further suggest that the dependency of TPO-cMpl signaling is different in these pathways and Notch-Hes signaling plays an additional role in them. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 4998-5005 ◽  
Author(s):  
E Sitnicka ◽  
N Lin ◽  
GV Priestley ◽  
N Fox ◽  
VC Broudy ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Synergistic Effects ◽  
Early Time ◽  
Proliferative Potential ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation ◽  
Vitro Effect

In this study, we explored whether thrombopoietin (Tpo) has a direct in vitro effect on the proliferation and differentiation of long-term repopulating hematopoietic stem cells (LTR-HSC). We previously reported a cell separation method that uses the fluorescence-activated cell sorter selection of low Hoescht 33342/low Rhodamine 123 (low Ho/low Rh) fluorescence cell fractions that are highly enriched for LTR-HSC and can reconstitute lethally irradiated recipients with fewer than 20 cells. Low Ho/low Rh cells clone with high proliferative potential in vitro in the presence of stem cell factor (SCF) + interleukin-3 (IL-3) + IL-6 (90% to 100% HPP-CFC). Tpo alone did not induce proliferation of these low Ho/low Rh cells. However, in combination with SCF or IL-3, Tpo had several synergistic effects on cell proliferation. When Tpo was added to single growth factors (either SCF or IL-3 or the combination of both), the time required for the first cell division of low Ho/low Rh cells was significantly shortened and their cloning efficiency increased substantially. Moreover, the subsequent clonal expansion at the early time points of culture was significantly augmented by Tpo. Low Ho/low Rh cells, when assayed in agar directly after sorting, did not form megakaryocyte colonies in any growth condition tested. Several days of culture in the presence of multiple cytokines were required to obtain colony-forming units-megakaryocyte (CFU-Mk). In contrast, more differentiated, low Ho/high Rh cells, previously shown to contain short- term repopulating hematopoietic stem cells (STR-HSC), were able to form megakaryocyte colonies in agar when cultured in Tpo alone directly after sorting. These data establish that Tpo acts directly on primitive hematopoietic stem cells selected using the Ho/Rh method, but this effect is dependent on the presence of pluripotent cytokines. These cells subsequently differentiate into CFU-Mk, which are capable of responding to Tpo alone. Together with the results of previous reports of its effects on erythroid progenitors, these results suggest that the effects of Tpo on hematopoiesis are greater than initially anticipated.

scholarly journals Diabetes impairs the interactions between long-term hematopoietic stem cells and osteopontin-positive cells in the endosteal niche of mouse bone marrow

2013 ◽  
Vol 305 (7) ◽  
pp. C693-C703 ◽  
Author(s):  
Hironori Chiba ◽  
Koji Ataka ◽  
Kousuke Iba ◽  
Kanna Nagaishi ◽  
Toshihiko Yamashita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Mouse Bone Marrow ◽  
Diabetic Mice ◽  
Hematopoietic Stem ◽  
Coculture System ◽  
Endosteal Niche

Hematopoietic stem cells (HSCs) are maintained, and their division/proliferation and quiescence are regulated in the microenvironments, niches, in the bone marrow. Although diabetes is known to induce abnormalities in HSC mobilization and proliferation through chemokine and chemokine receptors, little is known about the interaction between long-term HSCs (LT-HSCs) and osteopontin-positive (OPN) cells in endosteal niche. To examine this interaction, LT-HSCs and OPN cells were isolated from streptozotocin-induced diabetic and nondiabetic mice. In diabetic mice, we observed a reduction in the number of LT-HSCs and OPN cells and impaired expression of Tie2, β-catenin, and N-cadherin on LT-HSCs and β1-integrin, β-catenin, angiopoietin-1, and CXCL12 on OPN cells. In an in vitro coculture system, LT-HSCs isolated from nondiabetic mice exposed to diabetic OPN cells showed abnormal mRNA expression levels of Tie2 and N-cadherin. Conversely, in LT-HSCs derived from diabetic mice exposed to nondiabetic OPN cells, the decreased mRNA expressions of Tie2, β-catenin, and N-cadherin were restored to normal levels. The effects of diabetic or nondiabetic OPN cells on LT-HSCs shown in this coculture system were confirmed by the coinjection of LT-HSCs and OPN cells into bone marrow of irradiated nondiabetic mice. Our results provide new insight into the treatment of diabetes-induced LT-HSC abnormalities and suggest that the replacement of OPN cells may represent a novel treatment strategy.

scholarly journals Asymmetric Division and Lineage Commitment at the Level of Hematopoietic Stem Cells

2004 ◽  
Vol 199 (3) ◽  
pp. 295-302 ◽  
Author(s):  
Hina Takano ◽  
Hideo Ema ◽  
Kazuhiro Sudo ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Single Cell ◽  
Asymmetric Division ◽  
Lineage Commitment ◽  
Mouse Bone Marrow ◽  
Differentiation Potential ◽  
Hematopoietic Stem

How hematopoietic stem cells (HSCs) commit to a particular lineage is unclear. A high degree of HSC purification enabled us to address this issue at the clonal level. Single-cell transplantation studies revealed that 40% of the CD34−/low, c-Kit+, Sca-1+, and lineage marker− (CD34−KSL) cells in adult mouse bone marrow were able, as individual cells, to reconstitute myeloid and B- and T-lymphoid lineages over the long-term. Single-cell culture showed that >40% of CD34−KSL cells could form neutrophil (n)/macrophage (m)/erythroblast (E)/megakaryocyte (M) (nmEM) colonies. Assuming that a substantial portion of long-term repopulating cells can be detected as nmEM cells within this population, we compared differentiation potentials between individual pairs of daughter and granddaughter cells derived in vitro from single nmEM cells. One of the two daughter or granddaughter cells remained an nmEM cell. The other showed a variety of combinations of differentiation potential. In particular, an nmEM cell directly gave rise, after one cell division, to progenitor cells committed to nm, EM, or M lineages. The probability of asymmetric division of nmEM cells depended on the cytokines used. These data strongly suggest that lineage commitment takes place asymmetrically at the level of HSCs under the influence of external factors.

scholarly journals Highly potent human hematopoietic stem cells first emerge in the intraembryonic aorta-gonad-mesonephros region

2011 ◽  
Vol 208 (12) ◽  
pp. 2417-2427 ◽  
Author(s):  
Andrejs Ivanovs ◽  
Stanislav Rybtsov ◽  
Lindsey Welch ◽  
Richard A. Anderson ◽  
Marc L. Turner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Adult Organism ◽  
Human Hscs ◽  
High Level ◽  
Very High

Hematopoietic stem cells (HSCs) emerge during embryogenesis and maintain hematopoiesis in the adult organism. Little is known about the embryonic development of human HSCs. We demonstrate that human HSCs emerge first in the aorta-gonad-mesonephros (AGM) region, specifically in the dorsal aorta, and only later appear in the yolk sac, liver, and placenta. AGM region cells transplanted into immunodeficient mice provide long-term high level multilineage hematopoietic repopulation. Human AGM region HSCs, although present in low numbers, exhibit a very high self-renewal potential. A single HSC derived from the AGM region generates at least 300 daughter HSCs in primary recipients, which disseminate throughout the entire recipient bone marrow and are retransplantable. These findings highlight the vast regenerative potential of the earliest human HSCs and set a new standard for in vitro generation of HSCs from pluripotent stem cells for the purpose of regenerative medicine.

scholarly journals Ontogeny stage-independent and high-level clonal expansion in vitro of mouse hematopoietic stem cells stimulated by an engineered NUP98-HOX fusion transcription factor

Blood ◽  
2011 ◽  
Vol 118 (16) ◽  
pp. 4366-4376 ◽  
Author(s):  
Sanja Sekulovic ◽  
Maura Gasparetto ◽  
Véronique Lecault ◽  
Corinne A. Hoesli ◽  
David G. Kent ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Single Cells ◽  
Hematopoietic Stem ◽  
Image Tracking ◽  
Large Numbers ◽  
High Level

Abstract Achieving high-level expansion of hematopoietic stem cells (HSCs) in vitro will have an important clinical impact in addition to enabling elucidation of their regulation. Here, we couple the ability of engineered NUP98-HOXA10hd expression to stimulate > 1000-fold net expansions of murine HSCs in 10-day cultures initiated with bulk lin−Sca-1+c-kit+ cells, with strategies to purify fetal and adult HSCs and analyze their expansion clonally. We find that NUP98-HOXA10hd stimulates comparable expansions of HSCs from both sources at ∼ 60% to 90% unit efficiency in cultures initiated with single cells. Clonally expanded HSCs consistently show balanced long-term contributions to the lymphoid and myeloid lineages without evidence of leukemogenic activity. Although effects on fetal and adult HSCs were indistinguishable, NUP98-HOXA10hd–transduced adult HSCs did not thereby gain a competitive advantage in vivo over freshly isolated fetal HSCs. Live-cell image tracking of single transduced HSCs cultured in a microfluidic device indicates that NUP98-HOXA10hd does not affect their proliferation kinetics, and flow cytometry confirmed the phenotype of normal proliferating HSCs and allowed reisolation of large numbers of expanded HSCs at a purity of 25%. These findings point to the effects of NUP98-HOXA10hd on HSCs in vitro being mediated by promoting self-renewal and set the stage for further dissection of this process.

Unexpected and Sustained Symmetry in the Repopulation Programs Displayed by Clonally Amplified Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1651-1651
Author(s):  
Brad Dykstra ◽  
David Kent ◽  
Melisa Hamilton ◽  
Merete Kristiansen ◽  
Kristin Lyons ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Adult Life ◽  
Mouse Bone Marrow ◽  
Recipient Mouse ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Over Time

Abstract Heterogeneity in progeny output by individual pluripotent hematopoietic cells is a well documented but poorly understood paradigm. Importantly, the extent to which this functional heterogeneity is pre-determined by intrinsic mechanisms that specify distinct programs, as opposed to conditions that result in a series of stochastic events, is still debated. The prospective isolation of phenotypically defined subpopulations with more restricted behaviors has lent recent support to the concept of predetermined hierarchies with preset, but alternative pathways of lineage restriction and differentiated cell output. Here we have used highly purified starting populations to compare the long-term cell output dynamics of individual multipotent repopulating cells in sublethally irradiated W41/W41 mice transplanted with single Ly-5 congenic CD45midlin−Rho−SP adult mouse bone marrow cells (158 mice) or their clonal progeny generated after 4 days in vitro in 300 ng/ml SF, 20 ng/ml IL-11 and 1 ng/ml Flt3-L (194 mice). WBC samples collected 4, 8, 12, 16, and 24 weeks post-transplant were analyzed for donor contributions to the myeloid (GM) and lymphoid (B and T) lineages. In 49 of the 158 mice (31%) and 44 of the 194 mice (23%), the cells produced in vivo contributed ≥1% of all the WBCs present at ≥16 weeks. The overall and lineage-specific contributions to the WBCs in each recipient mouse varied widely both over time post-transplant and between mice. However, examination of the ratio of the donor contributions to the myeloid and lymphoid lineages (GM:B+T) in each mouse at 16 weeks post-transplant allowed 4 patterns to be readily identified: α and β with GM:B+T ratios of ≥2 and 0.25–2, respectively; γ, with a GM:B+T ratio of <0.25 including a ≥1% contribution to both lymphoid and myeloid lineages at 16 weeks; and δ, also with a GM:B+T ratio of <0.25, but with contribution only to the lymphoid lineages at this time. Secondary transplants performed after 24 weeks showed long-term repopulation (≥16 weeks) of most recipients of type α and β progeny (10/11 and 11/12, respectively) but none of the recipients of type γ and δ progeny were repopulated (0/6 and 0/17, respectively). Interestingly, the variation over time in both the overall and lineage-specific contributions was remarkably similar in pairs of secondary recipients injected with cells from the same primary donor. In addition, the lineage contribution ratios seen in the secondary recipients tended to recapitulate that of the primary donors (i.e., α or β), and these trends remained obvious when tertiary transplants were performed. Preservation of stem cell programming was also evident from sequential analyses of multiple mice injected with aliquots of the same clones generated in vitro after 10 days from single CD45midlin−Rho−SP cells. Very similar patterns of total and lineage-specific contributions were again observed amongst the different recipients of cells from the same clones. Collectively, these findings indicate that by early adult life hematopoietic stem cells have acquired intrinsically fixed patterns of lineage specification that can be stably transmitted through many self-renewal generations.

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