Hematopoietic Progenitor Cell Marker Research Articles

Blood Flow Restriction Using a Pneumatic Tourniquet Is Not Associated With a Cellular Systemic Response

PurposeThe purpose of this study was to determine the effects of blood flow restriction (BFR) using a pneumatic tourniquet on CD34+ cells, platelets, white blood cells, neutrophils, lymphocytes, lactate, and glucose compared with standard exercise.MethodsFifteen healthy volunteers (8 males and 7 females, 28.6 ± 3.6 years old) who were able to perform the exercise sessions on a VersaClimber participated. Participants were randomized to undergo an experimental (EXP) occluded testing session using the pneumatic tourniquets on all 4 extremities and a control (CON) session. The exercise protocol concluded after 9 minutes or when participants reached a rating of perceived exertion of 20. Blood draws were performed before testing and immediately after the exercise session. Blood analysis consisted of complete blood counts as well as flow cytometry to measure peripheral CD34+ counts as a marker for hematopoietic progenitor cells (HPCs).ResultsA significant increase from before to after exercise values was observed in both the EXP and CON groups with CD34+, WBC counts, platelets, and lymphocytes; however, no differences existed between EXP and CON groups for any variable. CD34+ increased in the EXP (3.1 ± 1.6 vs. 4.3 ± 1.8 cells · L–1; P < .001) and CON (3.3 ± 1.9 vs. 4.4 ± 1.4 cells · L–1; P < .001) sessions. White blood cells also significantly increased in both the EXP (7.8 ± 1.4 vs. 11.8 ± 2.5 K · L–1 K · L–1; P < .001) and CON (7.5 ± 1.8 vs. 11.3 ± 3.0 K · L–1; P < .001) sessions. Platelets also increased in both the EXP (258.6 ± 52.5 vs. 309.9 ± 52.7 K · L–1; P < .001) and CON (263.1 ± 44.7 vs. 316.1 ± 43.9 K · L–1; P < .001) sessions, and conversely, a significant decrease in the average neutrophil counts in the EXP (mean difference = –13.7%; P < .001) and CON (mean difference = –13.2%; P < .001) sessions was observed. Lymphocyte counts in the EXP (mean difference = 22.8%; P < .001) and CON (mean difference = 19.3%; P < .001) sessions increased significantly.ConclusionsThere were no significant differences in systemic cellular responses when undergoing aerobic-based exercise with and without a pneumatic tourniquet system.Level of Evidence2, prospective comparative study.

Pou5f3.3 is involved in establishment and maintenance of hematopoietic cells during Xenopus development

Three POU family class V gene homologues are expressed in the development of Xenopus. In contrast to the expression of Pou5f3.1 and Pou5f3.2 in organogenesis, Pou5f3.3 is expressed during oogenesis in ovary. We investigated the expression and function of Pou5f3.3 in organogenesis of Xenopus laevis. RT-PCR and immunohistochemical analysis indicated that Pou5f3.3 was expressed in a small number of adult liver cells and blood cells. Immunocytochemical investigation proved that Bmi1, a marker for hematopoietic progenitor cells, was co-expressed in Pou5f3.3-expressing small spherical cells in the peripheral blood. In anemic induction by intraperitoneal injection of phenyl hydrazine, the number of Pou5f3.3-expressing cells significantly increased within 3 days after phenyl hydrazine injection. In CRISPR/Cas mutagenesis of Pou5f3.3, Bmi1-positive hematopoietic progenitor cell count decreased in the hematopoietic dorsal-lateral plate (DLP) region, resulting in a considerable reduction in peripheral blood cells. CRISPR/Cas-induced hematopoietic deficiency was completely rescued by Pou5f3.3 supplementation, but not by Pou5f3.1 or Pou5f3.2. Transplantation experiments using the H2B-GFP transgenic line demonstrated that DLP-derived Pou5f3.3-positive and Bmi1-positive cells were translocated into the liver and bone through the bloodstream. These results suggest that Pou5f3.3 plays an essential role in the establishment and maintenance of hematopoietic progenitor cells during Xenopus development.

A Comparative Immunofluorescence Study Demonstrating that Peri‐arteriolar Glioma Stem Cell Niches in Human Glioblastoma are a Mimic of Peri‐arteriolar Hematopoietic Stem Cell Niches in Human Bone Marrow

Glioblastoma is the most aggressive type of brain tumor with poor patient survival. A fraction of glioblastoma cells within glioblastoma tumors consists of slowly dividing and therefore therapy‐resistant glioma stem cells (GSCs) residing in protective peri‐arteriolar niches and are held responsible for tumor recurrence. The hypothesis of this study is that GSC niches are a mimic of hematopoietic stem cell (HSC) niches in human bone marrow as in both types of niches, similar niche factors and molecular interactions are involved in retention and release of stem cells. Immunofluorescence was performed on paraffin sections of 10 glioblastoma patient samples and 2 human bone marrow samples to localize GSC niches and HSC niches and to determine whether the two types of niches are similar or not. Smooth muscle actin (SMA) was used to detect smooth muscle cells in the arteriolar wall. The markers CD133 and CD150 were used as HSC markers and CD244 as hematopoietic progenitor cell marker. CD133 and SOX2 were used as markers to detect GSCs and CD105 and CD73 were used as markers to detect mesenchymal stem cells (MSCs). In addition, expression of chemoattractants stromal‐derived factor‐1α (SDF‐1α) and osteopontin (OPN), and their receptors, C‐X‐C receptor type 4 (CXCR4) and CD44, respectively, was localized. Expression of hypoxia‐inducible factor(HIF)‐1α, HIF‐2α and vascular endothelial growth factor (VEGF) was localized to determine hypoxic conditions. We found CD133‐CD150‐CXCR4‐CD44‐positive HSCs localized adjacent to bone around SMA‐positive arterioles in SDF‐1α‐ and OPN‐rich niches. CD244‐positive hematopoietic progenitor cells were localized at a distance from bone near sinusoids. CD105‐ and CD73‐positive MSCs were localized in peri‐arteriolar niches as well. Expression of HIF‐1α, HIF‐2α and VEGF confirmed hypoxia. Similarly, we found CD133‐SOX2‐CXCR4‐CD44‐positive GSCs around SMA‐positive arterioles in SDF‐1α‐ and OPN‐rich niches. CD105‐positive MSCS were localized around the arterioles. Hypoxic conditions were confirmed by HIF‐1α, HIF‐2α and VEGF expression in GSC niches. In both niche types, SDF‐1α and OPN retain HSCs and GSCs in hypoxic peri‐arteriolar niches by interacting with their receptors CXCR4 and CD44 on the HSCs and GSCs, respectively. MSCs are major SDF‐1α producers, attracting and maintaining HSCs and GSCs in niches. We conclude that GSC niches in glioblastoma are a mimic of HSC niches in bone marrow. We aim to disrupt interactions between GSCs and their protective niches to induce GSC differentiation and proliferation to render GSCs more sensitive to anti‐glioblastoma therapies.Support or Funding InformationKWF; UVA 2014‐6839This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelial Colony-Forming Cells (ECFC) As an Autologous Model for Studying Endothelial Pathophysiology in Sickle Cell Anemia and Myeloproliferative Neoplasms

Background: Endothelial colony-forming cells (ECFC) are an important source of autologous endothelial cells to study its implication in the pathophysiology of diseases with risk of vaso-occlusive events. Currently, our research group began to elucidate the ECFC mechanisms that contribute to the complex clinical vascular manifestations in two diseases, sickle cell anemia (SCA) and myeloproliferative neoplasms (MPN).Aims: In this study, we analyzed functional in vitro assays of endothelial cells. The adhesion to red blood cell (RBC), migration and angiogenesis process of ECFC isolated from patients with SCA and MPN, as well as from healthy individuals (CTR) were evaluated in seeking an expanded understanding of the biology of the endothelial cell and its role in vascular events.Methods: ECFC were obtained through the isolation and culture of human peripheral blood mononuclear cells. ECFC were isolated from 8 patients with SCA under regular transfusion, 6 patients with MPN and 10 CTR. Human umbilical vein endothelial cells (HUVEC) were used as additional controls. Flow cytometry of ECFC indicated that all cells were highly positive for endothelial cell markers CD31, CD144, CD146 and KDR with no indication of CD45 (leukocyte antigens), CD133 (endothelial progenitor cell marker) and CD34 (hematopoietic progenitor cell marker). RBCs from healthy individuals were obtained after centrifugation of whole blood. Cellular adhesion was evaluated after incubation of ECFCs with RBCs in the presence or absence of inflammatory stimulus (TNF-α). Endothelial adhesion molecules were analyzed by flow cytometry (ICAM-1, VCAM-1, E and P-selectin). ECFC migration was assessed using a scratch-wound healing assay and wound regression was analyzed by time-lapse videos. Angiogenesis capacity was evaluated through three-dimensional ECFC cultures in Matrigel. Network parameters (segments, junctions and meshes) were characterized during 24h after seeding. All experiments were performed in triplicate.Results: In total, 48 ECFC colonies were established, 10 from SCA, 25 MPN and 13 CTR. We observed a higher percentage of adhered RBCs to ECFCs isolated from patients with SCA (14.0%) and MPN (23.4%) without TNF-ɑ stimulus, when compared to ECFC from CTR, (8.4%, p< 0.04 and p< 0.001, respectively), and HUVEC (9.4%, p< 0.05 and p< 0.02, respectively). No differences were detected in the expression of adhesion molecules in ECFC.Mean wound regression rates at 14h were 79.9% for SCA, 84.4% for MPN, and 88.8% for CTR. The high variability among colonies in each group could explain why this difference was not statistically significant. Finally, HUVECs had a shorter time for wound closure, with complete wound regression at 10h.The angiogenesis analysis at 15h ECFC from SCA and MPN had, respectively, 20% and 50% less network parameters than CTR. Then beyond 15h post-seeding, the network parameters regressed until reaching a plateau. At 24h the segments began to disappear progressively, leading to a marked reduction in 40h. Unlike ECFC, HUVEC presented a high network formation.Conclusions: Our findings reveal distinct functional properties and behavior between the ECFCs from two diseases with vascular manifestations, SCA and MPN. ECFC do provide access to patient vascular endothelium and enable us to validate the use of these cells as investigative models. In contrast, HUVECs showed a unique behavior, which differed from both diseased and controls ECFCs. This highlights the differences between autologous in vitro and established cell lines as experimental models for vascular diseases. This raises the question of which is the most representative model of human endothelial pathophysiology in vascular diseases. DisclosuresOzelo:BioMarin: Honoraria, Speakers Bureau; Grifols: Honoraria; Novo Nordisk: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Shire: Honoraria, Research Funding, Speakers Bureau; Bioverativ: Honoraria, Research Funding.

216 - High-throughput immunophenotypic characterization of bone marrow- and cord blood-derived mesenchymal stromal cells (MSC): Common and differentially expressed markers. relevance of hematopoietic progenitor cell marker (CD143)

216 - High-throughput immunophenotypic characterization of bone marrow- and cord blood-derived mesenchymal stromal cells (MSC): Common and differentially expressed markers. relevance of hematopoietic progenitor cell marker (CD143)

Interleukin-3 greatly expands non-adherent endothelial forming cells with pro-angiogenic properties

Circulating endothelial progenitor cells (EPCs) provide revascularisation for cardiovascular disease and the expansion of these cells opens up the possibility of their use as a cell therapy. Herein we show that interleukin-3 (IL3) strongly expands a population of human non-adherent endothelial forming cells (EXnaEFCs) with low immunogenicity as well as pro-angiogenic capabilities in vivo, making their therapeutic utilisation a realistic option. Non-adherent CD133+ EFCs isolated from human umbilical cord blood and cultured under different conditions were maximally expanded by day 12 in the presence of IL3 at which time a 350-fold increase in cell number was obtained. Cell surface marker phenotyping confirmed expression of the hematopoietic progenitor cell markers CD133, CD117 and CD34, vascular cell markers VEGFR2 and CD31, dim expression of CD45 and absence of myeloid markers CD14 and CD11b. Functional experiments revealed that EXnaEFCs exhibited classical properties of endothelial cells (ECs), namely binding of Ulex europaeus lectin, up-take of acetylated-low density lipoprotein and contribution to EC tube formation in vitro. These EXnaEFCs demonstrated a pro-angiogenic phenotype within two independent in vivo rodent models. Firstly, a Matrigel plug assay showed increased vascularisation in mice. Secondly, a rat model of acute myocardial infarction demonstrated reduced heart damage as determined by lower levels of serum creatinine and a modest increase in heart functionality. Taken together, these studies show IL3 as a potent growth factor for human CD133+ cell expansion with clear pro-angiogenic properties (in vitro and in vivo) and thus may provide clinical utility for humans in the future.

Basaloid follicular hamartoma on the upper eyelid.

Basaloid follicular hamartoma on the upper eyelid.

Regulation of cell shape and adhesion by CD34

We previously reported that expression of CD43/leukosialin induces cell rounding and microvillus formation via inhibition of cell adhesion. Here, we found that CD34, a cell surface sialomucin and marker for hematopoietic progenitor cells, also inhibited cell adhesion and induced cell rounding and microvillus formation. Forced expression of CD34-induced cell rounding, microvillus formation, and phosphorylation of ezrin/radixin/moesin (ERM) proteins in HEK293T cells, while inhibiting integrin-mediated cell re-attachment. Furthermore, CD34+ blood cells and KG-1 cells, which express endogenous CD34 on their surface, were spherical in shape, surrounded by microvilli, and non-adherent to substrata. In addition, cleavage of O-sialomucin augmented integrin-mediated cell adhesion of KG-1 cells. These results suggest the involvement of CD34 in the inhibition of integrin-mediated cell adhesion and formation of the cell surface structure. The inhibitory function of CD34 in cell adhesion may affect cell shape organization via phosphorylation of ERM proteins. Cellular structures such as the spherical shape and microvilli of CD34+ cells may also contribute to regulation of cell adhesion.

Characterization of a Distinct Population of Circulating Human Non-Adherent Endothelial Forming Cells and Their Recruitment via Intercellular Adhesion Molecule-3

Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133+ population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.

Abstract 9122: Heparin Mobilizes Multipotent Human Mesoangioblasts

The identification of clinically applicable drugs which mobilize endogenous mesenchymal progenitor cells may offer novel therapeutic application in cardiovascular diseases. We recently showed that hepatocyte growth factor (HGF) induces the mobilization of multipotent circulating mesenchymal cells that co-express endothelial markers (so-called circulating mesoangioblasts, cMABs). Since HGF only has a short half life, we investigated whether heparin might be useful to endogenously increase HGF levels thereby inducing the mobilization of cMABs. Heparin was infused in patients with coronary artery disease at a concentration of 200 U/kg (n=11 patients) and 300 U/kg (n=11 patients) and serum HGF levels were determined by ELISA. Consistent with published studies, heparin dose-dependently increased HGF levels to 34.4+1.9 in patients receiving 200U/kg heparin and 42.8+0.9 ng/ml in the patients treated with 300 U/kg. Moreover, heparin dose-dependently increased the number of circulating mesenchymal cell colonies. Patients receiving 200U/kg showed 0.30+0.12 colonies / mononuclear cells (x10 6 ). Further increasing the heparin dosage to 300U/kg increased the number of colonies to 0.63+0.17 colonies / mononuclear cells (x10 6 ). In contrast, we were never able to obtain colonies in patients receiving heparin concentrations below 100 U/kg. The mesenchymal origin of the colonies was characterised by the lack of hematopoietic progenitor cell markers CD45 and CD34. Consistent with the original characterisation of the mesoangioblasts, the colonies expressed the mesenchymal marker CD73, the HGF-receptor c-Met as well as the endothelial marker KDR. In conclusion, we demonstrate that heparin can dose-dependently mobilize mesenchymal progenitor cells in humans. These findings might open up an avenue to non-invasively obtain multipotent mesenchymal cells for therapeutic application in patients with cardiovascular disease.

A Role for Activation of the Kallikrein-Kinin System in the Homing Property of Endothelial Progenitor Cells.

The plasma kallikrein–kinin system (KKS) plays an important role in inflammation, thrombosis and vascular pathology. Activation of the KKS results in bradykinin release, which increases vascular permeability and modulates inflammatory responses. Circulating endothelial progenitor cells (EPCs) home to ischemic and inflamed tissues and participate in the vasculogenesis. However, the homing mechanism remains poorly understood. We now report that activation of the KKS regulates the homing property of EPCs using in vivo and in vitro approaches. We have previously shown that the Lewis rat, which displays a mutant kininogen (Ser511Ala), is susceptible to peptidoglycan-polysaccharide (PG-PS)- induced systemic inflammation including arthritis and displays activation of the KKS and increased bradykinin levels. CD34-positive EPCs were isolated from bone marrow of disease-free Lewis rats by Ficoll gradient centrifugation. After culture in EGM2 on collagen surfaces for 7 days, a single colony with cobblestone-like morphology appeared and was subjected to large-scale expansion. Compared with differentiated rat lung microvessel endothelial cells (rLMECs), rat EPCs expressed similar levels of endothelial cell lineage marker mRNA such as VE-cadherin, CD31, vWF and uPAR. EPCs exclusively expressed hematopoietic progenitor cell markers, Sca-1 and CD34. Both of EPCs and rLMECs expressed bradykinin type 2 receptor (B2R) but not B1R. To observe the in vivo mobilization of EPCs, EPCs were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) and injected into Lewis rats in which inflammation/arthritis was induced by PG-PS. Two weeks after PG-PS injection, pannus formed in the synovial tissues, representing the entry into the chronic phase of arthritis. The fluorescently-labeled EPCs were selectively detected in the inflamed synovium of PG-PS-treated rats, but not that of disease-free rats. As detected by confocal microscopy, implanted EPCs formed new blood vessels in the inflamed synovial tissues. To further investigate the mechanism by which the KKS regulates EPC mobilization, we observed that bradykinin (a B2R agonist), but not des-Arg9-bradykinin (a B1R agonist), stimulated EPC transmigration through confluent rLMEC layer at 100 and 1000 nM (P<0.01 and P<0.001, respectively). Bradykinin did not affect EPC adhesion to rLMECs. Priming by tumor necrosis factor a (TNFa), a major inflammatory cytokine, induced transendothelial migration of EPCs by 100 nM des-Arg9- bradykinin (168.56 ± 9.56 vs 96.02 ± 10.23, P<0.001). The priming effect resulted from TNFa induction of B1R mRNA. As determined by RT-PCR, rat EPCs expressed mRNA of multiple homing receptors including chemokine (C-X-C motif) receptor 4, CD49d and CD62-E, but not CD11a. Thus, activation of the KKS regulates the homing property of EPCs, which constitutes an additional mechanism for involvement of the KKS in the vasculogenesis. B2R predominantly mediates transendothelial migration of normal EPCs, whereas B1R seems responsible for that of inflamed EPCs. Kinins and cytokines such as TNFa may synergize to regulate the mobilization of EPCs. These observations provide a new insight into understanding the role of the KKS in vascular pathology and inflammation.

Comparison of hepatic properties and transplantation of Thy-1+ and Thy-1− cells isolated from embryonic day 14 rat fetal liver

Thy-1, a marker of hematopoietic progenitor cells, is also expressed in activated oval cells of rat liver. Thy-1(+) cells are also in rat fetal liver and exhibit properties of bipotent hepatic epithelial progenitor cells in culture. However, no information is available concerning liver repopulation by Thy-1(+) fetal liver cells. Therefore, we isolated Thy-1(+) and Thy-1(-) cells from embryonic day (ED) 14 fetal liver and compared their gene expression characteristics in vitro and proliferative and differentiation potential after transplantation into adult rat liver. Fetal liver cells selected for Thy-1 expression using immunomagnetic microbeads were enriched from 5.2%-87.2% Thy-1(+). The vast majority of alpha fetoprotein(+), albumin(+), cytokine-19(+), and E-cadherin(+) cells were found in cultured Thy-1(-) cells, whereas nearly all CD45(+) cells were in the Thy-1(+) fraction. In normal rat liver, transplanted Thy-1(+) cells produced only rare, small DPPIV(+) cell clusters, very few of which exhibited a hepatocytic phenotype. In retrorsine-treated liver, transplanted Thy-1(+) fetal liver cells achieved a 4.6%-23.5% repopulation. In contrast, Thy-1(-) fetal liver cells substantially repopulated normal adult liver and totally repopulated retrorsine-treated liver. Regarding the stromal cell-derived factor (SDF)-1/chemokine (C-X-C motif) receptor 4 (CXCR4) axis for stem cell homing, Thy-1(+) and Thy-1(-) fetal hepatic epithelial cells equally expressed CXCR4. However, SDF-1alpha expression was augmented in bile ducts and oval cells in retrorsine/partial hepatectomy-treated liver, and this correlated with liver repopulation by Thy-1(+) cells. Highly enriched Thy-1(+) ED14 fetal liver cells proliferate and repopulate the liver only after extensive liver injury and represent a fetal hepatic progenitor cell population distinct from Thy-1(-) stem/progenitor cells, which repopulate the normal adult liver.

Hepatic properties and transplantation of Thy-1+ cells isolated from ED14 rat fetal liver

Thy–1, a phenotypic marker of hematopoietic progenitor cells, is also expressed in activated oval cells of the adult rat liver. Thy1+ cells have also been observed in the rat fetal liver and these cells exhibit properties of bipotent hepatic epithelial progenitor cells in vitro. However, the stem-like properties of Thy–1+ fetal liver cells have not yet been fully established and there is no information available concerning the liver repopulation capacity of Thy1+ fetal liver cells. We therefore isolated Thy–1+ cells from ED14 fetal liver and examined their hepatic gene expression profile and characteristics in vitro and proliferative and differentiation potential after transplantation into the adult rat liver in comparison to Thy–1- fetal liver cells.

The p67 laminin receptor identifies human erythroid progenitor and precursor cells and is functionally important for their bone marrow lodgment

AbstractThe laminins are a group of extracellular matrix proteins with constitutive expression in all tissues, including bone marrow stroma. A functional role for the nonintegrin laminin receptor p67 has been described for cancer metastasis and lymphocyte trafficking. Expression of p67 was also reported for other subsets of mature leukocytes and for malignant hematopoietic or nonhematopoietic cells. We explored p67 expression on normal hematopoietic progenitor cells (HPCs) and its putative role in bone marrow retention of transplanted HPCs. We found p67 expression on a subset of primary human CD34+ cells coexpressing erythroid markers. Of importance, p67 recognizes early erythroid progenitors, since sorted p67+ cells were significantly enriched for burst-forming units–erythroid (BFU-Es) and depleted of colony-forming units–granulocyte/macrophage (CFU-GMs). Blockade of p67 binding of donor cells, using antifunctional antibody, reduced bone marrow homing of BFU-Es. These studies identify p67 as a novel phenotypic marker for erythroid HPCs of functional importance for lineage-specific homing/retention among adult transplanted HPCs.

CD41+ and Vascular Endothelial (VE)-Cadherin+ Cells: Two Developmental Origins of Hematopoietic Cell Lineages That Show Different Hematogenic Potentials.

It has been proposed that the definitive hematopoietic cell lineages are derived from hemogenic endothelial cells. Recently, CD41 was identified as an earliest cell surface marker of hematopoietic progenitor cells during mouse embryogenesis. We examined relationship between VE-cadherin+ hemogenic endothelial cells and CD41+ progenitors as developmental origins of hematopoietic cells by using in vitro differentiation system of ES cells as well as mouse embryos.FACS analyses on ES cells differentiating on OP9 stromal cells identified two cell populations, CD41+CD45− and CD41lowVE-cadherin+CD45−. The CD41+ cell population was derived from Flk1+ cells that represent lateral plate mesoderm but not from PDGFRa+ cells that represent paraxial mesoderm. CD41 expression on CD41+CD45− cells was weak at Day4 of ES cell culture. CD41+CD45− cells rapidly increased in number and CD41 expression became higher after Day5. CD45+ cells became detectable as a subpopulation of CD41+ cells two days after the appearance of the CD41+CD45− cell population. A significant proportion of the purified CD41lowVE-cadherin+CD45− cells differentiated to cells with CD41+CD45− phenotype in short-term culture, while CD41+CD45− cells did not differentiate into VE-cadherin+ cells. Unsurprisingly only CD41lowVE-cadherin+CD45− population had potential to produce endothelial cell colonies on OP9 cell layer. Liquid cultures and methylcellulose colony assay with a proper combination of cytokines showed that primitive erythroid colony forming cells were highly enriched in the CD41+CD45− cell population. CD41+CD45− cells also differentiated to Ter119+ definitive erythrocytes and Gr-1+ and Mac-1+ myeloid cells. In contrast, CD41lowVE-cadherin+CD45− cells produced only few hematopoietic cells in the same condition. However, CD41lowVE-cadherin+CD45− cells were capable of differentiating into multi-lineage hematopoietic cells including B lymphocytes when cultured with OP9 stromal cells. CD41+CD45− cells did not show any B lymphogenic potentials even when cultured with OP9 cells.We examined hemogenic potentials of phenotypically equivalent cells purified from mouse embryos. FACS analyses on cells dissociated from yolk sac and lower trunk of embryos proper revealed two distinct populations, CD41+CD45− cells and CD41−/lowVE-cadherin+CD45− cells. Both populations were already detectable in 8.5 dpc embryos. CD41−/lowVE-cadherin+CD45− cells but not CD41+CD45− cells produced endothelial cell colonies in vitro. The CD41−/lowVE-cadherin+CD45− cell population isolated from yolk sac was able to differentiate into multi-lineage hematopoietic cells when cultured with OP9 cells. However, the same population that was isolated from embryos proper had very poor potential to generate erythroid and myeloid cells although it still initiated robust production of B lymphocytes. Nevertheless, hemogenic activities of this population declined to undetectable level on 11.5 dpc. In contrast, CD41+CD45− cells isolated from yolk sac and embryos proper gave rise to multilineage hematopoietic cells and those potentials were stronger than that of yolk sac-derived CD41−/lowVE-cadherin+CD45− cells. Our results suggest that two distinct precursors, hemogenic endothelial cells and CD41+ progenitor cells, may contribute to the initiation of definitive hematopoiesis in mouse ontogeny, although activity of hemogenic endothelial cells in embryo proper might be unexpectedly limited.

Co-Expression of BAALC with ERG, TCF4, and CD133 Indicates a Shared Pathway in Acute Myeloid and Lymphoblastic Leukemia.

The human gene BAALC (Brain And Acute Leukemia, Cytoplasmic) is a molecular marker of hematopoietic progenitor cells and is aberrantly expressed in subsets of acute myeloid (AML) and lymphoblastic (ALL) leukemias. High mRNA expression levels of BAALC have been shown to adversely impact outcome in newly diagnosed AML patients (pts) with normal cytogenetics. To gain insight into the functional role of BAALC and its significance to normal hematopoiesis and leukemogenesis we compared gene expression profiles of normal CD34+ progenitors with those of AML and ALL blasts (using oligonucleotide microarrays; HG-U133 plus 2.0, Affymetrix, Santa Clara, CA). First we explored the regulation of BAALC expression during lineage specific maturation of in vitro differentiated human CD34+ bone marrow cells selected from healthy individuals. Microarray analyses were carried out using CD34+ cells stimulated in vitro with EPO, TPO, or G/GM-CSF to induce lineage-specific differentiation. At day 0 of culture and at three different time points during differentiation (days 4, 7, 11) cells were harvested, and if necessary purified by immunomagnetic beads and used for microarray studies. Experiments of all lineages and time points were done in triplicates. A total of 276 genes were identified showing similar changes in expression (with downregulation during differentiation) as BAALC at the three time points in all lineages with a correlation coefficient of R>0.95. This set of 276 BAALC co-expressed genes was investigated in an AML expression dataset generated from 51 adult pts with newly diagnosed de novo AML and normal cytogenetics (Cancer and Leukemia Group B). After exclusion of probesets expressed in fewer than 20% of pt samples, 21 probesets representing 14 named genes 6 of which are known to be involved in AML (BAALC, CD34, CD133, SOX4, ERG, SEPT6) and 4 implicated in lymphoid development (TCF4, SH2D1A, ITM2A, ITM2C) were found to be overexpressed (a significance level of P=0.01 was used) in pts of the highest third compared to pts of the lowest third of BAALC expression values as measured by real-time RT-PCR. We next applied these same 21 BAALC co-expressed probesets to an ALL expression dataset generated from 66 adult pts with newly diagnosed standard risk B-lineage precursor ALL (from the German ALL GMALL study group). A BAALC specific cluster uncovered 7 probesets representing 4 different co-expressed genes: BAALC, CD133, and the transcription factors ERG and TCF4. Thus, applying a BAALC specific expression signature to AML and ALL gene expression profiles revealed 3 genes (CD133, ERG, TCF4), which are highly associated with BAALC in myeloid and lymphoid blasts. Interestingly in non-malignant lymphoid and myeloid cells the oncogeneic ETS transcription factor ERG has shown specificity to immature cells, while its mechanistical role in leukemogenesis remains unknown. ERG and TCF4 may directly regulate BAALC and indicate a specific pathway implicated in leukemogenesis, while co-expression of CD133 and BAALC suggests shared stem cell characteristics. Functional studies are in progress to further explore these findings.

Distinguishing between proliferating nodal lymphoid blasts in chronic myelogenous leukemia and non‐Hodgkin lymphoma: Report of three cases and detection of a bcr/abl fusion signal by single‐cell analysis

Lymph node biopsies were analyzed from three patients with chronic myelogenous leukemia (CML) showing nodal blast proliferation. Immunohistochemically, the blasts from all three patients had an immature marker profile with a T-blast population (cCD3+, CD4-, CD7+, CD8-, CD99+, terminal deoxynucleotidyl transferase +) and a hematopoietic progenitor cell marker (CD34). In two patients, the blasts also expressed myeloid lineage specificity (naphthol AS-D chloroacetate esterase activity and myeloperoxidase positivity). However, it was difficult to distinguish between blast proliferation in CML and non-Hodgkin lymphoma from these immunohistopathological findings alone. Subsequently, bcr gene rearrangement and bcr/abl mRNA expression were detected by Southern blot and reverse transcription-polymerase chain reaction analysis of the lymph nodes. Fluorescence in situ hybridization (FISH) analysis of lymph node touch smears also disclosed bcr/abl gene fusion signals in the blasts of all patients, confirming that the blasts were derived from Philadelphia chromosome-positive CML. Accurate discrimination between the proliferating nodal blasts of CML and non-Hodgkin lymphoma is essential for determining subsequent therapy. FISH analysis of bcr/abl in single-cell blast preparations is an efficient tool that allows rapid, accurate cytopathological diagnosis of extramedullary blast-phase CML and its discrimination from non-Hodgkin lymphoma.

Characterization of CD34+ cells isolated from human fetal lung.

The large capillary mass of the newborn lung demands the presence of endothelial cell precursors in lung tissue before development of the pulmonary capillary bed. The objective of this investigation was to isolate and characterize putative endothelial cell precursors from developing human lung. CD34, a cell surface marker for hematopoietic progenitor cells, endothelial precursor cells, and small vessel endothelial cells, was employed as an immunological "handle" for the selection of the desired cells. When CD34+ cells were isolated from midtrimester human fetal lung tissue, then maintained in culture, the isolated cells expressed immunoreactivity for the endothelial cell marker von Willebrand factor and the vascular endothelial growth factor receptors KDR and Flt-1. However, only 5% or fewer of the cells expressed PECAM, an important factor in cell-cell interactions and a marker for endothelial cells associated with vessels. The CD34+ cells endocytosed acetylated low-density lipoprotein and formed capillary-like structures when incubated in a cushion of Matrigel. RT-PCR analysis of mRNA for endothelial cell-related proteins Flt-1, Tie-2, and endothelial nitric oxide synthase demonstrated expression of these mRNAs by the isolated cells for at least 16 cell passages. These observations demonstrate that capillary endothelial cell precursors can be isolated from developing human lung and maintained in cell culture. These cells represent a potentially important tool for investigating the regulation of mechanisms governing development of the air-blood barrier in the human lung.

Immunoblot Analysis of CD34 Expression in Histologically Diverse Neoplasms

CD34 is a heavily glycosylated transmembrane protein of approximately 110 kd whose function is essentially uncharacterized. First identified in a myeloid leukemia cell line, immunohistological reactivity with anti-CD34 antibodies is also encountered in a histologically diverse subset of nonhematolymphoid neoplasms including angiosarcoma, solitary fibrous tumors, epithelioid sarcomas, spindle cell lipomas, dermatofibrosarcoma protuberans, and myofibroblastomas. Immunohistological reactivity for CD34 in hematopoietic stem cells and endothelial cells has been shown to correspond to the expression of the CD34 protein. With the exception of gastrointestinal stromal tumors, CD34 protein expression has not been investigated in other CD34 immunohistologically reactive nonhematolymphoid neoplasms. We undertook this study to examine whether the observed reactivity for anti-CD34 antibodies in apparently unrelated tumors is due to the expression of the same protein or whether shared epitopes elaborated by other proteins could account for this reactivity. Immunoblot analyses with anti-CD34 antibodies of six different CD34 immunohistologically reactive lesions show the same approximately 110-kd molecular weight protein. In addition, two cases of dermatofibrosarcoma protuberans show double bands at approximately 110 kd. Laser-capture microdissection of CD34 immunohistologically reactive epithelioid sarcoma and nonreactive epidermal cells illustrates that this reactivity is specific to tumor cells. These results show that the observed immunohistological reactivity with anti-CD34 antibodies is due to the expression of the CD34 protein and not to shared epitopes on unrelated proteins.

KDR receptor: a key marker defining hematopoietic stem cells.

Studies on pluripotent hematopoietic stem cells (HSCs) have been hindered by lack of a positive marker, comparable to the CD34 marker of hematopoietic progenitor cells (HPCs). In human postnatal hematopoietic tissues, 0.1 to 0.5% of CD34(+) cells expressed vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR). Pluripotent HSCs were restricted to the CD34+KDR+ cell fraction. Conversely, lineage-committed HPCs were in the CD34+KDR- subset. On the basis of limiting dilution analysis, the HSC frequency in the CD34+KDR+ fraction was 20 percent in bone marrow (BM) by mouse xenograft assay and 25 to 42 percent in BM, peripheral blood, and cord blood by 12-week long-term culture (LTC) assay. The latter values rose to 53 to 63 percent in LTC supplemented with VEGF and to greater than 95 percent for the cell subfraction resistant to growth factor starvation. Thus, KDR is a positive functional marker defining stem cells and distinguishing them from progenitors.