Enhanced generation of endothelial cells from CD34+ cells of the bone marrow in rheumatoid arthritis: Possible role in synovial neovascularization

Abstract Human brain endothelial cells (HUBEC), a U.S. Navy proprietary cell line, was reported previously by Chute et al as a promising co-culture ex vivo expansion system for both adult bone marrow (ABM) and cord blood (CB) hematopoietic stem cells (HSC).a,b,c We report here our results of using HUBEC in ex vivo expansion and in vivo engraftment assay using NOD-SCID mice. CD34+ enriched fresh ABM was obtained using the method as described previously.a,b However, we used frozen CB and the same cytokines for both ABM and CB expansion whereas Chute et al used fresh CB and different cytokines. Ex vivo expansion studies for both ABM and CB were performed for 7 days in the HUBEC coated plates with previously reported cell density and cytokine cocktail containing GM-CSF, IL-3, IL-6, SCF, and flt-3 (GM36SF) in IMDM 10% FBS media.a HSC injections and BM harvesting of NOD-SCID mice as well as flow cytometric analysis were performed using the methods of Chute et al.a NOD-SCID mice were transplanted with limiting doses of either fresh ABM CD34+ cells or freshly thawed CB CD34+. The progeny of the identical doses of ABM CD34+ or the progeny of the identical doses of CB CD34+ cells was then transplanted. Culture with GM36SF alone resulted in a 15.5-fold and 70-fold increase in total cells, a 3.4-fold and 32-fold increase in CD34+ cells, and a 4.8-fold and 4.1-fold increase in CD34+/CD38- cells for ABM and CB, respectively. In contrast, HUBEC co-culture with GM36SF yielded a 25-fold and 48-fold increase in total cells, a 8.9-fold and 13-fold increase in CD34+ cells, and 114-fold and 106-fold increase in CD34+/CD38- cells for ABM and CB, respectively. HUBEC co-culture without GM36SF supported a 1.0-fold and 1.0-fold increase in total cells, a 0.06-fold and 0.1-fold increase in CD34+ cells, and 0.25-fold and 0.2-fold increase in CD34+/CD38- cells for ABM and CB. HUBEC co-culture with GM36SF and transwell (non-contact culture) resulted in a 20-fold and 48-fold increase in total cells, a 6-fold and 8-fold increase in CD34+ cells, and a 32-fold and 38-fold increase in CD34+/CD38- cells for ABM and CB. Overall, the transwell expansion of CD34+/CD38- population in both ABM and CB was reduced to 30% of that achieved in the contact culture. ABM CD34+ cells (5 x 105) engrafted 60% and the progeny of 5 x 105 cultured in the HUBEC monolayer with GM36SF engrafted in 90% of transplanted mice. CB CD34+ cells (1 x 104) engrafted 27% and the progeny 1 x 104 CB CD34+ cells cultured in the HUBEC monolayer with GM36SF engrafted 64% of NOD-SCID mice. SRC frequencies calculated as a 3.12-fold and 2.7-fold increase in CD34+ enriched ABM and CB, respectively, which was less than reported previously.a,b In summary, HUBEC supports and expands SRC mainly through cell-to-cell contact between HSC and endothelial cells, with HUBEC-secreted factors playing a minor role.

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Abnormalities of Bone Marrow Immune Micro-Environment in Patients with Immune Thrombocytopenia

Blood ◽

10.1182/blood.v126.23.3464.3464 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3464-3464

Author(s):

Yang Song ◽

Yu-tong Wang ◽

Xiao-jun Huang ◽

Yuan Kong

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Endothelial Cells ◽

Th17 Cells ◽

Immune Thrombocytopenia ◽

Perivascular Cells ◽

Platelet Production ◽

Cd34 Cells ◽

Significant Difference

Abstract Background: Immune thrombocytopenia (ITP) is an immune-mediated disease that is characterized by excessive platelet destruction and decreased platelet production. Although antiplatelet antibodies are considered as the primary immunologic defect in ITP, dysfunctional cellular immunity is also important in the pathophysiology of ITP. The current publications have observed excessive activation and proliferation of platelet auto-antigen-reactive CTLs, production abnormal Th cells, abnormal numbers and function of Tregs in peripheral blood of ITP, but no one focus on the bone marrow (BM) micro-environment in ITP patients. Many cell types including osteoblastic, perivascular, endothelial cells, and various mature immune cells contribute to the BM micro-environment. We have recently reported that the impaired BM vascular micro-environment may affect the thrombopoiesis of CD34+ cells by disrupting the interaction between megakaryocytes and BM endothelial cells (BMECs), resulting in the delayed platelet engraftment in allotransplant patients with prolonged isolated thrombocytopenia (Kong Y, et al. Biol Blood Marrow Transplant. 2014; 20:1190-1197). In mice model, the cross-talk between megakaryocytes and BMECs in BM vascular micro-environment regulates the megakaryocyte maturation and thrombopoiesis. Therefore, we hypothesized that the abnormal BM vascular micro-environment and immune micro-environment may operate in the occurrence of ITP. Aims: To investigate whether abnormal BM vascular and immune micro-environment are involved in ITP patients. Methods: The compartments of BM immune micro-environment were analyzed by flow cytometry in 26 untreated ITP patients and 26 healthy donors (HD). The fractions of T cells, including Th1, Tc1,Th2, Tc2 ,Th17 and Treg were identified as CD3+ CD8- IFN-gama+, CD3+ CD8- IFN-gama+, CD3+ CD8+ IL4+, CD3+ CD8+ IL-4+, CD3+ CD8- IL17A+ and CD3+ CD4+ CD25+ Foxp3+, respectively. The BMECs and perivascular cells, acting as key elements of vascular micro-environment, were identified as CD45- CD34+ VEGFR2+ and CD45- CD34- CD146+, respectively. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) using rabbit anti-human CD34 and CD146 primary antibodies were performed on each BM trephine biopsies (BMB) derived from the patients and controls. Results: The proportion of Th1 cells and Tc1 cells among the bone marrow mononuclear cells (BMMNCs) was significantly increased in ITP patients compared to HD (27.7% ± 11.6% vs. 16.3% ± 7.7%, P<0.001; 39.8%±17.7% vs. 24.1%±11.8%, P<0.005), whereas there was no significant difference in the percentages of Th2 and Tc2 cells. In addition, the proportion of Th17 cells in ITP patients was remarkable higher than HD (3.2%±0.51%1.5%vs 1.7%±1.0%, P<0.0001). We also found the significantly decreased percentage of Treg in ITP patients compared to HD (2.5%±2.0% vs 3.7%±2.6%, P<0.001). However, the frequency of CD34+ cells as well as BMECs and perivascular cells were similar in BM between the ITP patients and HD. Consistent with our flow cytometry data, histological analysis of the recipient BMBs in situ showed no significant differences in CD34-positive BMECs and CD146-positive perivascular cells between ITP patients and HD. Summary/Conclusion: The BM CD34+ cells and vascular micro-environment were normal in ITP patients. However, the abnormal BM immune micro-environment, including the excessive polarization of Th1, Tc1 and Th17 cells and a remarkable decrease of Treg cells were observed in ITP patients. Our data indicated that the desregulated T cells responses in BM may abrogate the thrombopoiesis through the impaired megakaryocytes maturation and decreased platelet production, and eventually contributing to the occurrence of ITP. Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), and the Beijing Municipal Science and Technology Program (grant nos. Z141100000214011& Z151100004015164& Z151100001615020). Disclosures No relevant conflicts of interest to declare.

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Enhanced expression of mRNA for FLT3 in bone marrow CD34+ cells in rheumatoid arthritis

Inflammation and Regeneration ◽

10.2492/inflammregen.35.255 ◽

2015 ◽

Vol 35 (5) ◽

pp. 255-260

Author(s):

Shunsei Hirohata ◽

Tamiko Yanagida ◽

Tetsuya Tomita ◽

Hideki Yoshikawa

Keyword(s):

Rheumatoid Arthritis ◽

Bone Marrow ◽

Cd34 Cells ◽

Enhanced Expression

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Human Embryonic Stem Cell-Derived CD34+ Cells Preferentially Develop into Endothelial Cells When Transplanted into Neonatal Immunodeficient Mice

Blood ◽

10.1182/blood.v112.11.69.69 ◽

2008 ◽

Vol 112 (11) ◽

pp. 69-69

Author(s):

Xinghui Tian ◽

Melinda K. Hexum ◽

Vesselin R. Penchev ◽

Russell J. Taylor ◽

Leonard Shultz ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endothelial Cells ◽

Cord Blood ◽

Embryonic Stem ◽

Post Transplantation ◽

Time Points ◽

Bioluminescent Signal ◽

Cd34 Cells

Abstract Selection for CD34+ cells from human bone marrow, umbilical cord blood and mobilized peripheral blood cells has been used to enrich for putative hematopoietic stem cells that are typically characterized by the ability to provide long-term multi-lineage engraftment after transplantation into NOD/SCID mice. In vitro studies have shown that CD34+ cells derived from human embryonic stem cells (hESC) have the similar capacity to develop into diverse mature blood cell lineages under defined conditions, though they may represent an even more primitive progenitor population with bi-potent ability to developed into both hematopoietic and endothelial cells. Previously, we and others have demonstrated in vivo hematopoietic engraftment of an unsorted heterogeneous population of differentiated hESC-derived cells injected into NOD/SCID mice. Here, to determine whether sorted CD34+ cells isolated from differentiated hESC have the equivalent SCID-repopulating potential, we directly delivered the CD34+ cells into the liver of neonatal NOD/SCID/gamma c (null) mice. Stable expression of firefly luciferase (luc) in the hESC derived-CD34+ cells allowed us to non-invasively track the engraftment patterns by in vivo bioluminescent imaging. Luc+ CD34+ cells were produced by co-culture of hESC with M2-10B4 mouse bone marrow-derived stromal cells. Each mouse (n=18) was injected with 2.5 – 9.4 × 10^5 CD34+ cells isolated at an early stage of differentiation (day 6–9 of co-culture). Successful injections were routinely confirmed by the bioluminescent signal detected in the liver at 1 week post-transplantation. There was typically a decrease of luc signal observed between 2–5 weeks post-transplantation, followed by the stabilizing and increasing of luc-expressing cells at later time points. Dramatic increase of bioluminescent signal in the livers was routinely demonstrated 8–12 weeks post-transplantation. The bioluminescence foci were initially maintained in the liver. Migration of luc+ cells from liver to bone marrow, spleen, kidney and mediastinum/thymus was also detected at later time points (10+ weeks post-transplantation). Flow cytometric analysis demonstrated a significant population of human HLA-class I+ cells (0.1–5.35%) in the engrafted livers (11 out of 15 mice), with the majority of the human CD34+CD31+ endothelial cells (0.03–4.78%). In most of cases, these endothelial cells also co-expressed CD73. No human CD45+ cells were found in any of the tissues evaluated. This is in contrast to CD34+ cells obtained from human cord blood utilized as a positive control cell populations that routinely demonstrated high levels of CD45+ cells (86.92±10.5%) of diverse hematopoietic lineages in the liver when transplanted in this model. Immunohistochemical staining showed that the hESC-derived CD34+CD31+ cells localized in vessels of the engrafted liver. Remarkably, the hESC-derived luc+CD34+CD31+ cells could be isolated several months post-transplant and re-cultured to clearly demonstrate typical endothelial cell characteristics including expression of vonWillebrand factor and VE-Cadherin, taking up of ac-LDL, and formation of capillary-like tubes when cultured in Matrigel. Additionally, ex vivo imaging of the exercised spleens and their surrounding connective tissues demonstrated luc+ cells located in the surrounding connective/mesenchymal tissues. Similar luc+ mesenchymal tissues were also found around the kidneys. Importantly, no visible teratomas were found in any of the transplanted mice. Together, these results demonstrate hESC-derived CD34+ cells preferentially develop into endothelial cells after transplantation into the fetal liver environment, with some additional development of mesenchymal tissues, but minimal hematopoietic cells. These studies demonstrate hESC-derived CD34+ cells are suitable for in vivo models to correct of vascular disease and further studies are underway to define signals required for improved hematopoietic engraftment of purified hESC-derived CD34+ cells.

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CD45-CD34+ Endothelial Progenitor Cells (EPCs) from Human Adipose Tissue Promote Tumor Growth and Metastases

Blood ◽

10.1182/blood.v118.21.2208.2208 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2208-2208

Author(s):

Patrizia Mancuso ◽

Ines Martin Padura ◽

Giuliana Gregato ◽

Paola Marighetti ◽

Angelica Calleri ◽

...

Keyword(s):

Breast Cancer ◽

Bone Marrow ◽

Endothelial Cells ◽

Adipose Tissue ◽

Breast Reconstruction ◽

Progenitor Cells ◽

Human Breast Cancer ◽

Human Adipose Tissue ◽

Human Breast ◽

Cd34 Cells

Abstract Abstract 2208 A catalytic role has been proposed in neoplastic angiogenesis and cancer progression for bone marrow-derived endothelial progenitor cells (EPCs). However, in preclinical and clinical studies the quantitative role of marrow-derived EPCs in cancer vascularization was found to be extremely variable. Adipose tissue represents an attractive source of autologous adult stem cells due to its abundance and surgical accessibility. Lipotransfer aspirates (LAs) from patients undergoing breast reconstruction after breast cancer surgery were analyzed by six colors flow cytometry and tissue culture. After collagenase digestion, cells were stained with the nuclear binding antigen Syto16 and 7-AAD and with CD34, CD45, CD133, CD31, CD140b, CD105, CD90, CD44, CD13, CD144, CD10, CD29, CD109, CD117, CD146,CD16, CD11c, CD14, CD38, CXCR4, VEGFR-1, VEGFR-2, VEGFR-3, Tie-2. The absolute count of CD45-CD34+ cells was obtained using reference beads in Trucount tubes (BD, Mountain View, CA). LAs were found to contain a large amount of CD45-CD34+ cells fulfilling the most recent criteria for EPC identification. These CD45-CD34+ cells included two subpopulations: CD45-CD34++ CD13+ CD140b+ CD44+ CD90++ cells and CD45-CD34+ CD31+CD105+ cells. We found in the adipose tissue about 263 fold more CD45-CD34+ EPCs/mL when compared to the bone marrow. In particular, the median of CD45-CD34+CD31- cells/mL was 181,046 (range 35,970–465,357), and the median of CD45-CD34+CD31+ cells/mL was 76,946 (range13,982-191,287). When compared to marrow-derived CD34+ cells, purified CD45-CD34+ adipose cells expressed similar levels of stemness-related genes such as NANOG, SOX2, Lin28 and significantly increased levels of angiogenesis-related genes such as CD144, VEGFR2, ALK-1. In vitro, CD45-CD34+ cells generated mature endothelial cells and capillary tubes as well as mature mesenchymal cells. When coinjected with triple negative human breast cancer MDA-MB-436 and HCC1937 cells in the mammary fat of a murine model of human breast cancer, purified CD45-CD34+ cells significantly increased tumor growth, and immunohistochemistry studies demonstrated the presence of human CD31+, CD34+, CD105+ endothelial cells lining the vessels of orthotopic breast cancers growing in mice co-injected with human adipose tissue-derived CD45-CD34+ cells. Moreover, in a mouse model of breast cancer metastatization we found an increased number of lung and axillary lymph node metastases when purified CD34+ WAT cells were injected into the third mammary fat pad after the primary tumor resection. In conclusion our data demonstrate that the phenotype of adipose derived EPCs is consistent with that reported for both bone marrow and circulating EPCs, but their frequency in adipose tissue is more than 250 fold higher. Further studies are ongoing to clarify what cell populations residing in the adipose tissue can be used safely for breast reconstruction and what are at risk for supporting the growth of otherwise quiescent cancer cells still resident after surgery. Disclosures: No relevant conflicts of interest to declare.

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Evidence for Circulating Bone Marrow-Derived Endothelial Cells

Blood ◽

10.1182/blood.v92.2.362.414k38_362_367 ◽

1998 ◽

Vol 92 (2) ◽

pp. 362-367 ◽

Cited By ~ 26

Author(s):

Qun Shi ◽

Shahin Rafii ◽

Moses Hong-De Wu ◽

Errol S. Wijelath ◽

Cong Yu ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Growth Factor ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Peripheral Circulation ◽

Dacron Graft ◽

Cd34 Cells

It has been proposed that hematopoietic and endothelial cells are derived from a common cell, the hemangioblast. In this study, we demonstrate that a subset of CD34+ cells have the capacity to differentiate into endothelial cells in vitro in the presence of basic fibroblast growth factor, insulin-like growth factor-1, and vascular endothelial growth factor. These differentiated endothelial cells are CD34+, stain for von Willebrand factor (vWF), and incorporate acetylated low-density lipoprotein (LDL). This suggests the possible existence of a bone marrow-derived precursor endothelial cell. To demonstrate this phenomenon in vivo, we used a canine bone marrow transplantation model, in which the marrow cells from the donor and recipient are genetically distinct. Between 6 to 8 months after transplantation, a Dacron graft, made impervious to prevent capillary ingrowth from the surrounding perigraft tissue, was implanted in the descending thoracic aorta. After 12 weeks, the graft was retrieved, and cells with endothelial morphology were identified by silver nitrate staining. Using the di(CA)n and tetranucleotide (GAAA)n repeat polymorphisms to distinguish between the donor and recipient DNA, we observed that only donor alleles were detected in DNA from positively stained cells on the impervious Dacron graft. These results strongly suggest that a subset of CD34+ cells localized in the bone marrow can be mobilized to the peripheral circulation and can colonize endothelial flow surfaces of vascular prostheses.

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Bone marrow CD34+ cells expanded on human brain endothelial cells reconstitute lethally irradiated baboons in a variable manner

Leukemia & Lymphoma ◽

10.3109/10428191003786774 ◽

2010 ◽

Vol 51 (6) ◽

pp. 1121-1127 ◽

Cited By ~ 3

Author(s):

Hiroto Araki ◽

John P. Chute ◽

Benjamin Petro ◽

Lisa Halliday ◽

Ronald Hoffman ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Human Brain ◽

Brain Endothelial Cells ◽

Cd34 Cells

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Prognostic Impact of Percentage CD34 Expression in Bone Marrow Trephines of Patients with Chronic Myeloid Leukaemia in Chronic Phase Treated with Imatinib.

Blood ◽

10.1182/blood.v104.11.1019.1019 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1019-1019

Author(s):

David Marin ◽

Donna Horncastle ◽

Catherina Andreasson ◽

Victoria Elliot ◽

Jonhn M. Goldman ◽

...

Keyword(s):

Bone Marrow ◽

Overall Survival ◽

Endothelial Cells ◽

Risk Group ◽

Chronic Phase ◽

Bone Marrow Examination ◽

Imatinib Therapy ◽

Cd34 Cells ◽

Haematological Remission ◽

Cd34 Expression

Abstract CD34 is a surface glycophosphoprotein expressed on developmentally early lymphohematopoietic stem and progenitor cells, small-vessel endothelial cells, and embryonic fibroblasts. We studied the prognostic value of presence of CD34 expressing cells in the marrow trephine biopsies of 58 patients with chronic myeloid leukemia in chronic phase treated with imatinib (21 newly diagnosed and 37 IFN failures). Briefly, patients received 400 mg daily and the dose was then adjusted according to tolerance and response. Bone marrow examinations were performed before starting therapy with imatinib and then every 3–6 months. All patients achieved complete haematological remission and 48% achieved complete cytogenetic remission. Chronic, accelerated and blastic phases were defined as described by Kantarjian 1988. Paraffin embedded sections were stained with the monoclonal antibody QBEND10. A minimum of 500 nucleated cells were counted per trephine section. Results were expressed as percentage of CD34+ vs all nucleated marrow cells. The expression of CD34 by the endothelial cells was used as an internal control. The median CD34 percentage before the onset of the imatinib therapy was 0.8% (25 and 75 percentiles 0.8 and 2%). We performed univariate and multivariate analysis for PFS with variables defined at the time of starting imatinib. Sokal risk group (defined at diagnosis), presence or absence of additional cytogenetic abnormalities, percentage of blasts in the bone marrow aspirate and the percentage of CD34+ cells in the trephine (considered as a continuos variable) were significant in the univariate analysis. Only CD34 expression and Sokal risk group were significant in the multivariate model. Sokal risk group lost significance when the percentage of CD34 cells was categorized as <=2% and >2% (RR for PFS=11.7, 95 CI=1.9–70.4, p=0.007; RR for survival = 10.5, 95CI= 1.01–80.3, p=0.05). The 3 years PFS for patients with a CD34 percentage <= 2% before the onset of the imatinib therapy (n= 44) was 77% while for patients with a CD34 >2% (n=14) it was 36% (p=0.006), similarly the three year overall-survival was 80 and 45% respectively (p=0.009). The median value of CD34 in the first bone marrow examination performed after the start of imatinib (typically performed at 3–6 months) was 0.8 (25 and 75 percentiles 0.8 and 1.5%). The degree of reduction from the baseline level achieved in the first bone marrow examination had favourable prognostic significance (as a continuos variable) with a RR for PFS of 0.88, 95CI 0.53–0.93 (p= 0.006) and RR for overall survival of 0.93, 95CI 0.19–0.998 (p=0.05). During follow up, an increment of 2 percentage points over the pre therapy CD34 value in patients in otherwise complete haematological remission had an adverse prognostic implication with a RR for PFS of 10.2 95 CI 2.1–17, (p=0.006) and a RR for survival of 6.8 95 CI 1.01–101-(p=0.05). We conclude that the use of CD34 staining in bone marrow trephines is a valuable tool for both diagnosis and monitoring in patients with CML treated with imatinib.

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