Faculty Opinions recommendation of Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta.

2002 ◽  
Author(s):  
Roger Patient
Keyword(s):  
Stem Cells ◽  
Endothelial Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Layer ◽  
Hematopoietic Stem ◽  
Endothelial Cell Layer ◽  
Mouse Aorta

scholarly journals Hematopoietic Stem Cells Localize to the Endothelial Cell Layer in the Midgestation Mouse Aorta

Immunity ◽  
2002 ◽  
Vol 16 (5) ◽  
pp. 673-683 ◽  
Author(s):  
Marella F.T.R de Bruijn ◽  
Xiaoqian Ma ◽  
Catherine Robin ◽  
Katrin Ottersbach ◽  
Maria-Jose Sanchez ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Layer ◽  
Hematopoietic Stem ◽  
Endothelial Cell Layer ◽  
Mouse Aorta

Endothelial Cell-Selective Adhesion Molecule Marks Human Hematopoietic Stem Cells Regardless Of The HSC Sources

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2429-2429
Author(s):  
Tomohiko Ishibashi ◽  
Takafumi Yokota ◽  
Michiko Ichii ◽  
Yusuke Satoh ◽  
Takao Sudo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Endothelial Cell ◽  
Hematopoietic Stem Cells ◽  
Adhesion Molecule ◽  
The Other ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Human Hscs ◽  
Nog Mice

Abstract Identification of novel markers associated with hematopoietic stem cells (HSCs) is important to progress basic and clinical research regarding the HSC biology. We previously reported that endothelial cell-selective adhesion molecule (ESAM) marks HSCs throughout life in mice (Yokota et al. Blood, 2009). We also demonstrated that ESAM can be a useful indicator of activated HSCs after bone marrow (BM) injury and that ESAM is functionally important for recovering hematopoiesis by using ESAM knockout mice (Sudo et al. J Immunol, 2012). However, the discrepancy between species has been a long-standing obstacle to apply findings in mice to human. For example, established murine HSC markers such as Sca-1 or CD150 are not expressed on human HSCs. Thus, it is important to know if ESAM marks HSCs beyond species and serves as a functional molecule for the HSC property, but information regarding ESAM expression in human HSCs has been quite limited. In this study, we have examined the ESAM expression pattern on human HSCs derived from diverse sources. In addition, we have performed functional assessment of the ESAM-expressing cells. Cord blood (CB), aspirated BM, and granulocyte-colony stimulating factor-mobilized peripheral blood (GMPB) were obtained from healthy donors. BM was also obtained from head of femora of patients who received the hip replacement surgery. All of the protocols were approved by the Institutional Review Board of Osaka University School of Medicine, and we obtained the written agreement form with informed consent from all participants. Mononuclear cells were separated using Ficoll centrifugation from CB, aspirated BM and GMPB. For preparation of BM cells adjacent to bone tissues, trabecular tissues of femora were treated with 2 mg/ml collagenase IV and DNase and gently agitated for 1 hour at 37 °C. Collected cells were analyzed using flow cytometry for cell surface expression of ESAM and other markers. Further, the CD34+ CD38−cells were fractionated according to the intensity of ESAM expression and evaluated in vivo and in vitro functional assays. Flow cytometry analyses revealed that the majority of CB CD34+ CD38− cells expressed ESAM. According to the expression level, CB CD34+ CD38− cells could be subdivided into three populations, namely ESAM−/Low, ESAMHigh, and ESAMBright. While all CB contained a robust ESAMHigh population in CD34+ CD38− cells, the percentage of ESAMBright cells varied widely among CB samples. The ESAMHigh CD34+ CD38− cells also expressed CD90 and CD133, which are known as HSC markers. Methylcellulose colony-forming assays and limiting dilution assays revealed that ESAMHigh fraction enriches primitive hematopoietic progenitors. Further, ESAMHigh cells also reconstituted the long-term human hematopoiesis in NOD/Shi-scid, IL-2Rγnull (NOG) mice. Therefore, as in mice, ESAMHighmarks authentic HSCs in human. On the other hand, ESAMBright CD34+ CD38− cells showed low colony-forming activities and no reconstitution of human hematopoiesis in NOG mice. These ESAMBright CD34+ CD38− cells expressed CD118/leukemia inhibitor factor receptor and endothelial markers such as VE-Cadherin, Flk-1, and CD146, but not CD45. These results suggested that ESAMBright cells in the CB CD34+ CD38− fraction are non-hematopoietic cells. With respect to the other HSC sources such as aspirated BM and GMPB, almost all CD34+ CD38− cells were ESAMHigh and ESAMBright cells were not found in this fraction. Interestingly, however, ESAMBright cells were found in the CD34+ CD38− fraction isolated from collagenase-treated femora. These BM-derived ESAMBright CD34+ CD38− cells expressed endothelial markers as did the CB-derived cells. They could generate CD31+endothelial cells, but not hematopoietic cells in coculture with MS5 stromal cells with vascular endothelial growth factor, stromal-cell-derived factor, and interleukin 16. In conclusion, ESAM expression serves as a marker to enrich HSCs in human regardless of the HSC sources. In addition, the very high intensity of this marker might be useful to isolate non-hematopoietic progenitors from CD34+ CD38− cells, which has been conventionally used as human HSCs. The common feature of ESAM expression of murine and human HSCs suggests a possibility that functional significance of ESAM expression obtained from mouse studies could be applicable to human. Disclosures: No relevant conflicts of interest to declare.

Immunolocalization of microfibril and microfibril-associated proteins in the subendothelial matrix of the developing mouse aorta

1994 ◽  
Vol 107 (3) ◽  
pp. 727-736 ◽  
Author(s):  
E.C. Davis
Keyword(s):  
Endothelial Cell ◽  
Structural Integrity ◽  
Cell Layer ◽  
Endothelial Cell Layer ◽  
Associated Proteins ◽  
Subendothelial Matrix ◽  
Oriented Parallel ◽  
Mouse Aorta ◽  
Structural Entity ◽  
Elastic Lamina

In the developing aorta, endothelial cell connecting filaments extend from the abluminal surface of the endothelial cell to the subjacent elastic lamina. The connecting filaments are in alignment with intracellular stress fibers and are oriented parallel to the direction of blood flow. In the present study, the composition of the endothelial cell connecting filaments was investigated by indirect immunogold labeling with antibodies to the microfibril proteins, MP340 (fibrillin) and MAGP, and to fibronectin and heparan sulfate proteoglycan (HSPG). In the subendothelial matrix of both 15-day gestational and 5-day post-natal mouse aortae, the connecting filaments showed moderate immunoreactivity with anti-MP340; however, no significant immunoreaction was seen with anti-MAGP. Anti-fibronectin strongly labeled the connecting filaments and a weak immunoreaction was seen with anti-HSPG. In contrast, the adjacent ‘elastin-associated microfibrils’ showed a very strong immunoreaction with anti-MP340 and a moderate reaction with anti-MAGP. Little or no reaction was seen with anti-fibronectin or anti-HSPG. The filaments that connect endothelial cells to the subjacent elastic lamina during aortic development are thus microfibrillar in nature and related to elastin-associated microfibrils as evidenced by their positive immunoreaction with anti-MP340. The absence of labeling with anti-MAGP, however, suggests that either these fibrillin-containing filaments do not contain MAGP or that the immunoreactive epitopes are blocked by the proteins that coat the connecting filaments such as fibronectin. These results suggest that microfibrils not in association with elastin may play a role in cell anchorage and, more specifically, in the aorta may be involved in maintaining the structural integrity of the endothelial cell layer during early development of the vessel wall. Furthermore, the absence of immunoreactivity with anti-MAGP on the fibrillin-containing endothelial cell connecting filaments raises the possibility that microfibrils may consist of a family of related filaments rather than a single structural entity.

The Endothelial Antigen ESAM Marks Hematopoietic Stem Cells throughout Life

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 727-727 ◽  
Author(s):  
Takafumi Yokota ◽  
Kenji Oritani ◽  
Stefan Butz ◽  
Koichi Kokame ◽  
Paul W Kincade ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cell ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Side Population ◽  
Expression Profiles ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSC) are an important cell type with the capacity for self-renewal as well as differentiation into multi-lineage blood cells, maintaining the immune system throughout life. Many studies have attempted to identify unique markers associated with these extremely rare cells. In bone marrow of adult mice, the Lin-c-kitHi Sca1+ CD34−/Lo Thy1.1Lo subset is known to include HSC with long-term repopulating capacity. However, several of these parameters differ between strains of mice, change dramatically during developmental age and/or are expressed on many non-HSC during inflammation. Efficient HSC-based therapies and the emerging field of regenerative medicine will benefit from learning more about what defines stem cells. We previously determined that the most primitive cells with lymphopoietic potential first develop in the paraaortic splanchnopleura/aorta-gonad-mesonephros (AGM) region of embryos using Rag1/GFP knock-in mice. We also reported that Rag1/GFP-c-kitHi Sca1+ cells derived from E14.5 fetal liver (FL) reconstituted lympho-hematopoiesis in lethally irradiated adults, while Rag1/GFPLo c-kitHi Sca1+ cells transiently contributed to T and B lymphopoiesis. To extend those findings, microarray analyses were conducted to search for genes that characterize the initial transition of fetal HSC to primitive lymphopoietic cells. The comparisons involved mRNA from Rag1Lo ckitHi Sca1+, early lymphoid progenitors (ELP) and the HSC-enriched Rag1-ckitHi Sca1+ fraction isolated from E14.5 FL. While genes potentially related to early lymphopoiesis were discovered, our screen also identified genes whose expression seemed to correlate with HSC. Among those, endothelial cell-selective adhesion molecule (ESAM) attracted attention because of its conspicuous expression in the HSC fraction and sharp down-regulation on differentiation to ELP. ESAM was originally identified as an endothelial cell-specific protein, but expression on megakaryocytes and platelets was also reported (J. Biol. Chem., 2001, 2002). Flow cytometry analyses with anti-ESAM antibodies showed that the HSC-enriched Rag1-c-kitHi Sca1+ fraction could be subdivided into two on the basis of ESAM levels. The subpopulation with the high density of ESAM was enriched for c-kitHi Sca1Hi cells, while ones with negative or low levels of ESAM were found in the c-kitHi Sca1Lo subset. Among endothelial-related antigens on HSC, CD34 and CD31/PECAM1 were uniformly present on Rag1-c-kitHi Sca1+ cells in E14.5 FL and neither resolved into ESAMHi and ESAM−/Lo fractions. Expression profiles of Endoglin and Tie2 partially correlate with ESAM. The primitive ESAMHi fraction uniformly expressed high levels of Endoglin and Tie2, but many of the more differentiated ESAM−/Lo cells still retained the two markers. ESAM expression correlated well with HSC activity. Cells in the ESAMHi Rag1-ckitHi Sca1+ fraction formed more and larger colonies than those in the ESAM-/Lo Rag1-ckitHi Sca1+ fraction. Particularly, most CFU-Mix, primitive progenitors with both myeloid and erythroid potential, were found in the ESAMHi fraction. In limiting dilution stromal cell co-cultures, we found that 1 in 2.1 ESAMHi Rag1-ckitHi Sca1+ cells and 1 in 3.5 ESAM−/Lo Rag1-ckitHi Sca1+ cells gave rise to blood cells. However, while only 1 in 125 ESAM−/Lo Rag1-ckitHi Sca1+ cells were lymphopoietic under these conditions, 1 in 8 ESAMHi Rag1-ckitHi Sca1+ cells produced CD19+ B lineage cells. In long-term reconstituting assays, ESAMHi Rag1-ckitHi Sca1+ cells contributed highly to the multi-lineage recovery of lympho-hematopoiesis in recipients, but no chimerism was detected in mice transplanted with ESAM−/Lo Rag1-ckitHi Sca1+ cells. These results suggested that HSC in E14.5 FL are exclusively present in the ESAMHi fraction. Tie2+ c-kit+ lympho-hematopoietic cells of E10.5 AGM also expressed high levels of ESAM. Furthermore, ESAM expression in adult bone marrow was detected on primitive progenitors and cells in the side population within the Lin-ckitHi Sca1+ fraction. Interestingly, the expression was up-regulated in aged mice. Based on these observations, we conclude that ESAM marks HSC throughout life in mice. We also observed that many of human cord blood CD34+ CD38− cells express ESAM, suggesting potential application for the purification of human HSC.

scholarly journals Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Christina M. Termini ◽  
Amara Pang ◽  
Tiancheng Fang ◽  
Martina Roos ◽  
Vivian Y. Chang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Vascular Regeneration ◽  
Vascular Niche ◽  
Hematopoietic Reconstitution ◽  
Neuropilin 1

AbstractIonizing radiation and chemotherapy deplete hematopoietic stem cells and damage the vascular niche wherein hematopoietic stem cells reside. Hematopoietic stem cell regeneration requires signaling from an intact bone marrow (BM) vascular niche, but the mechanisms that control BM vascular niche regeneration are poorly understood. We report that BM vascular endothelial cells secrete semaphorin 3 A (SEMA3A) in response to myeloablation and SEMA3A induces p53 – mediated apoptosis in BM endothelial cells via signaling through its receptor, Neuropilin 1 (NRP1), and activation of cyclin dependent kinase 5. Endothelial cell – specific deletion of Nrp1 or Sema3a or administration of anti-NRP1 antibody suppresses BM endothelial cell apoptosis, accelerates BM vascular regeneration and concordantly drives hematopoietic reconstitution in irradiated mice. In response to NRP1 inhibition, BM endothelial cells increase expression and secretion of the Wnt signal amplifying protein, R spondin 2. Systemic administration of anti - R spondin 2 blocks HSC regeneration and hematopoietic reconstitution which otherwise occurrs in response to NRP1 inhibition. SEMA3A – NRP1 signaling promotes BM vascular regression following myelosuppression and therapeutic blockade of SEMA3A – NRP1 signaling in BM endothelial cells accelerates vascular and hematopoietic regeneration in vivo.

Transplanted adult hematopoietic stems cells differentiate into functional endothelial cells

Blood ◽  
2004 ◽  
Vol 103 (1) ◽  
pp. 13-19 ◽  
Author(s):  
Alexis S. Bailey ◽  
Shuguang Jiang ◽  
Michael Afentoulis ◽  
Christina I. Baumann ◽  
David A. Schroeder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Hematopoietic Stem Cells ◽  
Blood Vessels ◽  
Cell Potential ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Adult Bone

Abstract During early embryogenesis, blood vessels and hematopoietic cells arise from a common precursor cell, the hemangioblast. Recent studies have identified endothelial progenitor cells in the peripheral blood, and there is accumulating evidence that a subset of these cells is derived from precursors in the bone marrow. Here we show that adult bone marrow–derived, phenotypically defined hematopoietic stem cells (c-kit+, Sca-1+, lineage–) give rise to functional endothelial cells. With the exception of the brain, donor-derived cells are rapidly integrated into blood vessels. Durably engrafted endothelial cells express CD31, produce von Willebrand factor, and take up low-density lipoprotein. Analysis of DNA content indicates that donor-derived endothelial cells are not the products of cell fusion. Self-renewal of stem cells with hematopoietic and endothelial cell potential was revealed by serial transplantation studies. The clonal origin of both hematopoietic and endothelial cell outcomes was established by the transfer of a single cell. These results suggest that adult bone marrow–derived hematopoietic stem cells may serve as a reservoir for endothelial cell progenitors.

scholarly journals Generation of AGM-derived Akt-EC (AGM-EC)

2020 ◽  
Author(s):  
Tessa Dignum ◽  
Barbara Varnum-Finney ◽  
Stacey Dozono ◽  
Brandon Hadland
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Cell Layer ◽  
Hematopoietic Stem ◽  
Embryonic Tissues ◽  
Endothelial Cell Layer ◽  
Integral Role ◽  
Ec Cells ◽  
Signaling Interactions

Abstract During murine embryonic development, the first hematopoietic stem cells (HSCs) emerge within the major arterial vasculature, including the aorta-gonad-mesonephros (AGM) region. Throughout their emergence and subsequent maturation, HSCs retain a close physical association with the surrounding endothelial cell layer, suggesting that signaling interactions between HSC and the surrounding vascular niche may play an integral role in HSC development. Indeed, we have previously shown that co-culture with AGM-derived endothelial cells (AGM EC) engineered to constitutively express Akt (AGM Akt-EC) is sufficient to mature non-engrafting HSC precursors from hemogenic endothelium to fully functional HSCs1-3. Here, we describe how to generate these AGM Akt-EC cells for use in co-culture experiments, providing detailed instructions from the isolation of AGM EC from embryonic tissues, to their infection with the PGK.myr-AKT lentivirus and subsequent characterization by flow cytometry.

Cell staining for sorting of hematopoietic stem cells (HSC) and myeloid progenitors and isolating RNA from sorted cells

2006 ◽  
Author(s):  
Hideyo Hirai ◽  
Pu Zhang ◽  
Tajhal Dayaram ◽  
Christopher Hetherington ◽  
Shin-ichi Mizuno ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Cell Staining ◽  
Myeloid Progenitors
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