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1
Ogata, Kiyoyuki, Chikako Satoh, Mikiko Tachibana, Hideya Hyodo, Hideto Tamura, Kazuo Dan, Takafumi Kimura, Yoshiaki Sonoda, and Takashi Tsuji. "Identification and Hematopoietic Potential of CD45-Negative Clonal Cells with Very Immature Phenotype (CD45−CD34−CD38−Lin−) in Patients with Myelodysplastic Syndromes." Blood 104, no. 11 (November 16, 2004): 3426. http://dx.doi.org/10.1182/blood.v104.11.3426.3426.
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Abstract CD45 is a hematopoietic lineage-restricted antigen that is expressed on all hematopoietic cells except for some mature cell types. Cells expressing CD45 and CD34 but lacking CD38 and lineage antigens (CD45+CD34+CD38−Lin− cells) are well-documented hematopoietic stem cells (HSCs), and CD45+CD34−CD38−Lin− cells are probably less mature HSCs. In myelodysplastic syndromes (MDS), the malignant transformation site was reported to be committed myeloid progenitors and, more recently, the CD45+CD34+CD38−Lin− HSCs. In this study, we detected CD45−CD34−CD38−Lin− cells in the peripheral blood and bone marrow of MDS patients. Fluorescence in situ hybridization showed that CD45−CD34−CD38−Lin− cells had the same chromosomal aberration as the myeloblasts. In addition to CD45- and CD34-negativity, they lacked CD117 and CD133 expressions. Generally, MDS cells have extremely reduced hematopoietic potential compared with normal hematopoietic cells, but we documented the following in some cases. Freshly-isolated CD45−CD34−CD38−Lin− cells did not form any hematopoietic colonies but had long-term culture-initiating cell activity. When these cells were co-cultured with stroma cells, CD45−CD34−CD38−Lin− cells showed only weak potential for proliferation/differentiation, yet differentiated to CD34+ cells and then mature myeloid cells. This newly-identified cell population represents the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the malignant clone in MDS.
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Lange, Andrzej, Dorota Dlubek, Barbara Wysoczanska, Daria Drabczak-Skrzypek, and Emilia Jaskula. "An Evidence That Mesenchymal Stem Cells Are Not Replaced by Peripheral Blood Stem Cell Allografts in CML Patients." Blood 106, no. 11 (November 16, 2005): 4875. http://dx.doi.org/10.1182/blood.v106.11.4875.4875.
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Abstract Four CML patients were investigated for post transplant chimerism for total marrow (BM) cells and Mesenchymal Stem Cell (MSC) populations purified and propagated from BM cells. Patients were conditioned with BuFluATG and after allogeneic PBPCT were disease free with proven hematological, cytogenetical and genetical remission. For the study 15–30 ml fresh BM aspirates were phenotypically characterized for the presence of cell populations which may contain MSC. We found (median values): 11.7% CD45-CD34− 1.7% CD45-CD34-CD105+, 0.13% CD45-CD34-CD90+ and 0.03% CD45-CD34-CD73+ cells. These BM populations contained 0.75% CD34+ cells. Genetical work showed that BM cells were BCR-ABL negative and their STR informative allele patterns were consistent with those of donors. BM cells were processed as follows: incubation with Glycophorin A, CD3, CD14, CD19, CD66b, CD38 antibody cocktail which by cross-linking unwanted cells with red blood cells led to rosette formation (RosetteSep MSC Enrichment Cocktail, StemCell Technology), unrosetted cells (MSC enriched) were recovered from the interface after buoyant gradient centrifugation and contained (median values): 68.7% CD45-CD34-, 21.6% CD45-CD34-CD105+, 1.1% CD45-CD34-CD90+ and 0.4% CD45-CD34-CD73+. MSC enriched BM populations were cultured in Medium for Human MSC with Stimulatory Supplements (StemCell Technology). After 10–14 days CFU-F colonies were scored (median value was 77 CFU-F/106 cells) and cells were further cultured until >90% confluence of fibroblast-like cells were reached (usually 3 to 4 weeks after culture initiation). The cells were detached with 0.05% trypsin-EDTA and studied for STR allele patterns, the presence of BCR-ABL transcripts (at that time cells showed STR alleles of the recipient pattern for the first time - mixed chimerism) and the bulk of cells were further passaged. Usually after 3–4 passages (within 7–8 weeks) when fibroblast-like stromal cell populations reached the level of 3x106 cells, cultures were terminated and the cells were studied. These cells were in (median values) 27.0% CD45-CD34-, 23.8% CD45-CD34-CD105+, 26.5% CD45-CD34-CD90+ and 24.3% CD45-CD34-CD73+. The cells had a fibroblast-like morphology but only 26% had phenotype features of MSC on average. Therefore, the population consisted of MSC and more differentiated cells originated from CFU-F (MSC). RNA and DNA were isolated from the cells propagated for 7–8 weeks from the MSC enriched BM populations were BCR-ABL negative. However, their STR informative allele patterns were consistent with those of the recipients in variance to primary BM cell populations which was in all 4 cases of donor origin. Conclusions: with the use of the RosetteSep MSC enrichment purification system BM cells can be enriched in CFU-F which paralleled with an increase in the proportion of CD45-CD34-, CD45-CD34-CD90+, CD45-CD34-CD73+ and CD45-CD34-CD105+ cells, CFU-F BM enriched populations can be cultured with Medium for Human MSC with Stimulatory Supplements for successful propagation of fibroblast-like cells with kinetics documenting ex potential growth after 38 days (median) of culture, Cells originated from CFU-F were in contrast to the BM hematopoietic compartment of the recipients origin and were also BCR-ABL negative, MSC were not replaced by allogeneic PBPC-graft derived MSC.
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Timmermans, Frank, Magda De Smedt, Robrecht Raedt, Jean Plum, and Bart Vandekerckhove. "Endothelial Cells Are Not Derived from Hematopoietic Precursor Cells." Blood 108, no. 11 (November 16, 2006): 1815. http://dx.doi.org/10.1182/blood.v108.11.1815.1815.
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Abstract Endothelial outgrowth cells (EOC) can be generated from mononuclear blood cells. Based on proliferative and functional characteristics, EOC were claimed to derive from an immature endothelial progenitor cell or angioblast. Several investigators have claimed that these cells constitute a subpopulation of CD34+ hematopoietic stem cells(HSC). However, the EOC-precursor is not well defined and its nature remains elusive. Methods and results: Umbilical cord blood CD34+ cells were sorted into a small (< 1 %) CD34+CD45− non-hematopoietic cell fraction (purity > 99.5%) and CD34+CD45+ HSC (purity > 99.2 %) (n=5). The cell fractions were cultured separately in EBM2/EGM2 medium (Cambrex, Verviers, Belgium) onto gelatine coated 24 wells. EOC were exclusively derived from the CD34+CD45− cell fraction and not from the CD45+ HSC. We further analysed the CD34+CD45− cell fraction for expression of endothelial progenitor genes. Analysis showed the presence of VEGFR2, VE-Cadherine and CD146 on the CD34+CD45− precursor population whereas CD45+ HSC were consistantly negative for these markers. CD133, which was claimed to be a marker for endothelial progenitors was negative on the CD34+CD45− cells. No VEGFR2+ CD133+ cells could be detected either by flowcytometry or at the mRNA level. In adult bone marrow, EOC only derived from CD45− CD31+ cells, and not from the CD45+ HSC or CD45− CD31− mesenchymal cells. CD34+CD45+ HSC or CD14+ CD45+ monocytes generated under the same conditions large flat adherent cells positive for CD31, LDL uptake and the lectin UEA-1. On RT-PCR and real time RT-PCR analysis, cells were positive for VEGFRII, CD146 and VE cadherin. However, membrane staining was consistently negative for VE-cadherin on flowcytometric analysis and positive for monocytic markers such as CD14 and CD45. In functional assays, the majority of the cells were shown to be phagocytic and were unable to form vascular tubes in the matrigel angiogenesis assay. These data demonstrate that monocytes may acquire a phenotype in vitro which is difficult to discriminate from endothelial cells. Conclusion : Endothelial cell generated in vitro from cord blood or bone marrow derive from a CD45− nonhematopoietic precursor.
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Dao, Mo A., Jesusa Arevalo, and Jan A. Nolta. "Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution." Blood 101, no. 1 (January 1, 2003): 112–18. http://dx.doi.org/10.1182/blood-2002-01-0025.
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Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.
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Mancuso, Patrizia, Ines Martin Padura, Giuliana Gregato, Paola Marighetti, Angelica Calleri, Chiara Corsini, Giancarlo Pruneri, Visnu Lohsiriwat, Jean Yves Petit, and Francesco Bertolini. "CD45-CD34+ Endothelial Progenitor Cells (EPCs) from Human Adipose Tissue Promote Tumor Growth and Metastases." Blood 118, no. 21 (November 18, 2011): 2208. http://dx.doi.org/10.1182/blood.v118.21.2208.2208.
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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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Fritsch, G., P. Buchinger, D. Printz, FM Fink, G. Mann, C. Peters, T. Wagner, A. Adler, and H. Gadner. "Rapid discrimination of early CD34+ myeloid progenitors using CD45-RA analysis." Blood 81, no. 9 (May 1, 1993): 2301–9. http://dx.doi.org/10.1182/blood.v81.9.2301.2301.
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Abstract Mononuclear cells (MNC) isolated by density centrifugation of cord blood and healthy bone marrow, and of peripheral blood (PB) from patients treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF after chemotherapy, were double-stained with anti CD34 monoclonal antibody (MoAb) (8G12) versus anti CD45, CD45-RB, CD45- RO, and CD45-RA, respectively, and analyzed by flow cytometry. In all specimens, CD34+ MNC co-expressed CD45 at a low level and the expression of CD45-RB was similar or slightly higher. Most CD34+ MNC were negative for CD45-RO, a weak coexpression was only seen in some bone marrow (BM) and blood samples. In contrast, CD45-RA could subdivide the CD34+ population into fractions negative, dim (+), and normal positive (++) for these subgroups, and typical staining patterns were observed for the different sources of hematopoietic cells: in BM, most CD34+ MNC were RA++. In PB, their majority was RA++ after G-CSF but RA+ or RA- after GM-CSF. In cord blood, the hematopoietic progenitors were mainly RA-/RO-. Semisolid culture of sorted CD34+ MNC showed that clusters and dispersed (late) colony-forming unit-GM (CFU- GM) originated from 34+/RA++ cells, while the 34+/RA- MNC formed compact and multicentric, both white and red colonies derived from early progenitors. Addition of 20 ng stem cell factor per milliliter of medium containing 34+/RA- cord blood MNC led to a change of many burst- forming unit-erythrocyte (BFU-E) to CFU-mix which was not, at least to this extent, seen in blood and BM. We conclude that early myeloid CD34+ cells are 45+/RA-. Because this population excludes 34+/19+ B cells and 33+ myeloid cells, both of which are RA++, two-color flow cytometric analysis using CD34 and CD45-RA facilitates the characterization and quantification of early myeloid progenitor cells.
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Fritsch, G., P. Buchinger, D. Printz, FM Fink, G. Mann, C. Peters, T. Wagner, A. Adler, and H. Gadner. "Rapid discrimination of early CD34+ myeloid progenitors using CD45-RA analysis." Blood 81, no. 9 (May 1, 1993): 2301–9. http://dx.doi.org/10.1182/blood.v81.9.2301.bloodjournal8192301.
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Mononuclear cells (MNC) isolated by density centrifugation of cord blood and healthy bone marrow, and of peripheral blood (PB) from patients treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF after chemotherapy, were double-stained with anti CD34 monoclonal antibody (MoAb) (8G12) versus anti CD45, CD45-RB, CD45- RO, and CD45-RA, respectively, and analyzed by flow cytometry. In all specimens, CD34+ MNC co-expressed CD45 at a low level and the expression of CD45-RB was similar or slightly higher. Most CD34+ MNC were negative for CD45-RO, a weak coexpression was only seen in some bone marrow (BM) and blood samples. In contrast, CD45-RA could subdivide the CD34+ population into fractions negative, dim (+), and normal positive (++) for these subgroups, and typical staining patterns were observed for the different sources of hematopoietic cells: in BM, most CD34+ MNC were RA++. In PB, their majority was RA++ after G-CSF but RA+ or RA- after GM-CSF. In cord blood, the hematopoietic progenitors were mainly RA-/RO-. Semisolid culture of sorted CD34+ MNC showed that clusters and dispersed (late) colony-forming unit-GM (CFU- GM) originated from 34+/RA++ cells, while the 34+/RA- MNC formed compact and multicentric, both white and red colonies derived from early progenitors. Addition of 20 ng stem cell factor per milliliter of medium containing 34+/RA- cord blood MNC led to a change of many burst- forming unit-erythrocyte (BFU-E) to CFU-mix which was not, at least to this extent, seen in blood and BM. We conclude that early myeloid CD34+ cells are 45+/RA-. Because this population excludes 34+/19+ B cells and 33+ myeloid cells, both of which are RA++, two-color flow cytometric analysis using CD34 and CD45-RA facilitates the characterization and quantification of early myeloid progenitor cells.
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Vodyanik, Maxim A., James A. Thomson, and Igor I. Slukvin. "Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures." Blood 108, no. 6 (September 15, 2006): 2095–105. http://dx.doi.org/10.1182/blood-2006-02-003327.
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AbstractDuring hematopoietic differentiation of human embryonic stem cells (hESCs), early hematopoietic progenitors arise along with endothelial cells within the CD34+ population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays, their phenotype has not been defined. Here, using hESC differentiation in coculture with OP9 stromal cells, we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45, persisted on differentiating hematopoietic cells, and reliably separated the hematopoietic CD34+ population from CD34+CD43–CD31+KDR+ endothelial and CD34+CD43–CD31–KDR– mesenchymal cells. Furthermore, we demonstrated that the first-appearing CD34+CD43+CD235a+CD41a+/–CD45– cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34+CD43+CD41a–CD235a–CD45– cells. These cells were negative for lineage-specific markers (Lin–), expressed KDR, VE-cadherin, and CD105 endothelial proteins, and expressed GATA-2, GATA-3, RUNX1, C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34+CD43+CD45–Lin– cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34+CD43+CD45+Lin– cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3highGATA3lowRUNX1lowPU1highMPOhighIL7RAhigh gene expression profile.
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Ciraci, Elisa, Silvia Della Bella, Ombretta Salvucci, Cristina Rofani, Marta Segarra, Caterina Bason, Agnese Molinari, Dragan Maric, Giovanna Tosato, and Anna C. Berardi. "Adult human circulating CD34−Lin−CD45−CD133− cells can differentiate into hematopoietic and endothelial cells." Blood 118, no. 8 (August 25, 2011): 2105–15. http://dx.doi.org/10.1182/blood-2010-10-316596.
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Abstract A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast, we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34, lineage markers, CD45 and CD133 (CD34−Lin−CD45−CD133− cells), endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34−Lin−CD45−CD133− cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo, CD34−Lin−CD45−CD133− cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34−Lin−CD45−CD133− cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials, characterized by the expression of c-Kit and CXCR4 as well as EphB4, EphB2, and ephrinB2. Further molecular and functional characterization of CD34−Lin−CD45−CD133− cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life.
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Case, Jamie, Laura E. Mead, Hilary A. White, Mohammad R. Saadatzadeh, Mervin C. Yoder, Laura S. Haneline, and David A. Ingram. "CD34+AC133+VEGFR-2+ Cells Are Primitive Hematopoietic Progenitors, Not Functional Endothelial Progenitor Cells." Blood 108, no. 11 (November 16, 2006): 1796. http://dx.doi.org/10.1182/blood.v108.11.1796.1796.
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Abstract Endothelial progenitor cells (EPCs) are currently used for angiogenic therapies or as biomarkers to assess cardiovascular disease risk and progression. However, there is no uniform definition of an EPC, which complicates interpretation of prior EPC studies. EPCs are primarily defined by expression of cell surface antigens. The most widely cited definition of an EPC is a cell which co-expresses CD34, AC133 and VEGFR-2. Importantly, these antigens are also expressed on the most primitive population of hematopoietic progenitor cells (HPCs), including high proliferative potential- (HPP-) and low proliferative potential-colony forming cells (LPP-CFCs). Remarkably, CD34+AC133+VEGFR-2+ cells have never been isolated and plated in endothelial cell (EC) or hematopoietic cell clonogenic assays to determine what cell progeny can be derived from a CD34+AC133+VEGFR-2+ cell. Utilizing human umbilical cord blood (CB), an enriched source of both EPCs and HPCs, we isolated and purified CD34+AC133+VEGFR-2+ cells by FACS and assayed for the presence of clonogenic endothelial CFCs (ECFCs) plus HPP- and LPP-CFCs. Surprisingly, CD34+AC133+VEGFR-2+ cells do not form ECFCs under any culture conditions previously described for outgrowth of EPCs. However, consistent with a HPC phenotype, CD34+AC133+VEGFR-2+ cells formed both HPP- and LPP-CFCs in multiple independent assays. In addition, all CD34+AC133+VEGFR-2+ cells were shown to co-express the specific hematopoietic cell surface antigen, CD45, which is not present on ECs. Based on this information, we plated CD34+CD45+ or CD34+CD45− cells to determine if EPCs could be separated from HPCs on the basis of CD45 expression. In multiple independent assays, CD34+CD45+ cells consistently formed both HPP- and LPP-CFCs but not EC colonies. In contrast, CD34+CD45− cells form EC colonies but not hematopoietic cell colonies. Taken together, these data demonstrate that circulating CD34+AC133+VEGFR-2+ cells are HPCs and the biologic mechanism for their correlation with cardiovascular disease needs to be re-examined. Furthermore, studies focused on determining the angiogenic potential of CD34+CD45− cells are needed given that this cell population harbors ECFCs.
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Gao, Xingcui, Bin Wei, Yan Deng, Yan Lan Huang, and Weifeng Wu. "Increased Mobilization of CD45+CD34+VLA-4+ Cells in Acute Viral Myocarditis Induced by Coxsackievirus B3." Cardiology 138, no. 4 (2017): 238–48. http://dx.doi.org/10.1159/000477655.
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Objectives: Bone marrow-derived cells (BMCs) have recently been identified to play a vital role in repairing damaged myocardium; however, it is not known whether or not mobilization of BMCs is involved in the pathogenesis of acute viral myocarditis (VMC). Thus, we analyzed the expression of CD45+CD34+VLA-4+ cells and vascular cell adhesion protein-1 (VCAM-1) in a murine model of acute VMC. Methods: Male BALB/c mice were intraperitoneally infected with coxsackievirus B3 to establish acute VMC. The frequency of CD45+CD34+VLA-4+ cells in the heart, peripheral blood, and bone marrow was examined by flow cytometry 3, 7, 14, and 28 days after injection. Cardiac VCAM-1 and pathology scores were determined by immunohistochemistry, and myocardial VCAM-1, IL-1β, and TNF-α were analyzed by RT-PCR and Western blot. Results: In mice with acute VMC, the CD45+CD34+VLA-4+ cell population in the heart was significantly increased by day 7 and then decreased; in contrast, the CD45+CD34+VLA-4+ cell population decreased in the bone marrow and peripheral blood by day 3 and then increased. High expression of VCAM-1 was detected in the heart in parallel with CD45+CD34+VLA-4+ cell expression. Conclusions: In mice with acute VMC, VCAM-1-induced CD45+CD34+VLA-4+ cell mobilization into the injured heart is involved in the repair of injured myocardium.
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Vodyanik, Maxim A., James A. Thomson, and Igor I. Slukvin. "CD43 Defines Early Hematopoietic Progenitors Derived from Human Embryonic Stem Cells (hESC)." Blood 104, no. 11 (November 16, 2004): 3216. http://dx.doi.org/10.1182/blood.v104.11.3216.3216.
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Abstract We have demonstrated that co-culture of hESCs with OP9 bone-marrow stromal cells induced efficient hESC differentiation into hematopoietic colony-forming cells (CFCs). During hESC/OP9 co-culture, the first CFCs were detectable 1–2 days after emergence of CD34+ cells (day 4–5 of co-culture) and 3–4 days before generation of CD45+ cells, indicating that early CFCs arose in the CD45- population. To define phenotype of early hematopoietic progenitors, we examined the expression of CD41a, CD43, CD61 and glycophorin A (CD235a) on the CD34+ cells in the course of hESC/OP9 co-culture. The first detectable CD34+ cells expressed a high level of VEGF-R2 (KDR) and were CD41a, CD43, CD61 and CD235a negative. Appearance of CFCs was accompanied with down-regulation of KDR and almost simultaneous expression of CD41a, CD43 and CD235a on the CD34+ cells. CD43+ cells gradually increased thereafter, whereas the expression of CD41a was retained on the subset of CD43+ cells that co-express CD235a and CD61. CD45+ cells emerged within CD43+ population and the most of CD45+ cells did not express CD235a, CD41a or CD61. The expression of CD34 was decreased progressively on CD43+CD41a+CD235a+CD61+ cells, but retained by CD43+CD45+ cells. To assay hematopoietic potential of defined subsets, CD34+ cells were isolated by magnetic sorting and further separated into CD34+ CD43+ and CD34+CD43− cell fractions. In addition, CD34−CD43+ cells were isolated from CD34− fraction. CD34+ selection markedly enriched all types CFCs except of E-CFCs. All GEMM-CFCs were found within CD34+ population and none within CD34−. However, CD34− cell fraction contained E-CFCs and minimal numbers of M-CFCs and GM-CFCs. CD34+ cells were also heterogenous in morphologic appearance and contained endothelial cells that were capable to endocytose Ac-LDL. Positive selection of CD43+ cells resulted in total recovery of all types of CFCs. E-CFCs, which consistently detected in the CD34- fraction, were also recovered by CD43 selection. Morphologically, CD43+ cells comprised a homogenous blast-like population and were lack of endothelial-like cells. Thus, expression of CD43 molecule (leukosialin/sialophorin) is detected on the earliest clonogenic hematopoietic progenitors before expression of CD45, persists on developing hematopoietic cells after their lineage specification and most likely defines the divergence of hematopoiesis from endothelial cells during early development in humans.
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Sonoda, Yoshiaki, Takafumi Kimura, Rumiko Asada, Takashi Kimura, Miho Morioka, Yutaka Sasaki, and Susumu Ikehara. "Different Proliferative Potential and Redistribution Kinetics of Human Cord Blood-Derived CD34- SCID-Repopulating Cells (SRCs) in Comarison with CD34+CD38+/− SRCs Using Intra-Bone Marrow Injection." Blood 108, no. 11 (November 16, 2006): 1650. http://dx.doi.org/10.1182/blood.v108.11.1650.1650.
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Abstract Using the intra-bone marrow injection (IBMI) method, we have identified human cord blood (CB)-derived CD34-negative (CD34−) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with multi-lineage repopulating ability (Blood101:2924,2003). Functional studies revealed that these CD34− SRCs have different hematopoietic stem cell (HSC) characteristics from CD34+ SRCs. In order to further clarify the HSC characteristics of CD34− SRCs, here we investigate the proliferative potential and redistribution kinetics of human CB-derived CD34− SRCs, and compare them with those of CD34+CD38+/− SRCs using IBMI. First, we performed limiting dilution analyses and revealed that the incidence of CD34+CD38− SRCs in CB-derived Lin−CD34+CD38− cells was 1 out of 41 cells by IBMI. In contrast, the incidence of CD34− SRCs in Lin−CD34− cells was 1 out of 24,100, as we previously reported. Based on these data, we transplanted 200 to 5,000 Lin−CD34+CD38− cells (containing 5 to 120 SRCs), 15,000 to 50,000 Lin−CD34+CD38+ cells (containing 10 to 30 SRCs), or 60,000 to 70,000 Lin−CD34− cells (containing 3 SRCs) into primary recipient NOD/Shi-scid mice. After 5 weeks, all mice that received transplants of Lin−CD34+CD38+/− cells showed the human CD45+ cell repopulation in the other bones as well as the injected left tibiae. However, the human CD45+ cells were only detected in the injected left tibiae in mice that received transplants of Lin−CD34− cells 5 weeks after the transplantation. In the mice that received transplants of 200 Lin−CD34+CD38− cells (containing 5 SRCs), the CD45+CD34+ as well as CD45+CD34− cells were detected in both sites. In contrast, only CD45+CD34− cells were detected in the mice that received transplants of 70,000 Lin−CD34− cells (3 SRCs). These results suggested that CD34− SRCs might remain or slowly proliferate as CD34− cells at the site of injection for at least 5 weeks. Next, we serially investigated the human CD45+ cell repopulation in the injected site and the other bones, separately. Very interestingly, CD34+CD38+/− SRCs began to migrate 2 weeks after the transplantation. The human cell repopulation in these mice was observed in other bones by 3 weeks after transplantation. Moreover, these CD34+ SRCs actively proliferated at both sites and produced CD34+ progenies. In contrast, CD34− SRCs began to migrate 5 weeks after the transplantaion. Furthermore, these CD34− SRCs showed significantly higher proliferative potential 8 weeks after transplantation than CD34+ SRCs and produced more CD34+ progenies not only at the site of injection, but also in the other bones. These results indicated that CD34− SRC as well as CD34+CD38+/− SRCs could actively migrate from the injected site to the other bones. However, the time of initiation of migration was different between CD34+/− SRCs. All these findings indicate that CD34− SRCs show different proliferative potential and redistribution kinetics, and suggest that our identified CD34− SRCs are distinct class of primitive HSCs in comparison with CD34+CD38+/− SRCs. We are now in the progress of clarifying whether the CD34− SRCs migrate to other bones with the CD34− immunophenotype or after their conversion (differentiation) to the CD34+ cells.
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Valverde-Villegas, Jacqueline María, Mélusine Durand, Anne-Sophie Bedin, David Rutagwera, Chipepo Kankasa, Edouard Tuaillon, Nicolas Nagot, Philippe Vande Perre, and Jean-Pierre Molès. "Large Stem/Progenitor-Like Cell Subsets can Also be Identified in the CD45- and CD45+/High Populations in Early Human Milk." Journal of Human Lactation 36, no. 2 (December 9, 2019): 303–9. http://dx.doi.org/10.1177/0890334419885315.
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Background Stem/progenitor cells have been identified in human milk. However, characterization and percentages of cell subsets in human milk using hematopoietic stem and progenitor cell markers according to the differential expression of CD45, i.e., as CD45dim/+ (mainly hematopoietic stem/progenitor cells) and CD45- (mainly non-hematopoietic stem/progenitor cells), have not been assessed to date. Research aim To characterize stem/progenitor-like cell phenotypes in human milk and to report the percentages of these cells at two different lactation stages compared to peripheral blood. Methods Human milk samples paired with peripheral blood samples ( N = 10) were analyzed by flow cytometry using CD45, CD34, CD133, CD38, and lineage-negative markers. The percentage of cell subsets was analyzed in colostrum (Day 3 postpartum) and transitional milk (Day 5/6 postpartum) and compared with the peripheral blood counterpart. Results The percentage of CD45-CD34+ cells was predominant in both colostrum and transitional milk. The percentage of CD45+/highCD133+ cells was high in colostrum while the percentage of CD45-CD133+ cells was high in transitional milk. Furthermore, the median percentages of the CD45-CD34+, CD45-CD133+, and CD45dimCD133+ cell subsets were higher in colostrum than its peripheral blood counterpart (0.11% vs. 0.002%; 0.17% vs. 0.0005%; 0.09% vs. 0.05%, p = .04, respectively); also CD45-CD34-CD133+ and CD45dimCD34-CD133+ cell subsets were higher in colostrum than peripheral blood (1.32% vs. 0.0% and 2.4% vs. 0.06%, p = .04), respectively). Conclusion Early human milk is an abundant reservoir of hematopoietic stem/progenitor-like cells in the CD45+/high population and non-hematopoietic stem/progenitor-like cells in the CD45- population.
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McKinney-Freeman, Shannon L., Olaia Naveiras, Frank Yates, Sabine Loewer, Marsha Philitas, Matthew Curran, Peter J. Park, and George Q. Daley. "Surface antigen phenotypes of hematopoietic stem cells from embryos and murine embryonic stem cells." Blood 114, no. 2 (July 9, 2009): 268–78. http://dx.doi.org/10.1182/blood-2008-12-193888.
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Abstract Surface antigens on hematopoietic stem cells (HSCs) enable prospective isolation and characterization. Here, we compare the cell-surface phenotype of hematopoietic repopulating cells from murine yolk sac, aorta-gonad-mesonephros, placenta, fetal liver, and bone marrow with that of HSCs derived from the in vitro differentiation of murine embryonic stem cells (ESC-HSCs). Whereas c-Kit marks all HSC populations, CD41, CD45, CD34, and CD150 were developmentally regulated: the earliest embryonic HSCs express CD41 and CD34 and lack CD45 and CD150, whereas more mature HSCs lack CD41 and CD34 and express CD45 and CD150. ESC-HSCs express CD41 and CD150, lack CD34, and are heterogeneous for CD45. Finally, although CD48 was absent from all in vivo HSCs examined, ESC-HSCs were heterogeneous for the expression of this molecule. This unique phenotype signifies a developmentally immature population of cells with features of both primitive and mature HSC. The prospective fractionation of ESC-HSCs will facilitate studies of HSC maturation essential for normal functional engraftment in irradiated adults.
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Brunner, Stefan, Hans Diogenes Theiss, Alfons Murr, Thomas Negele, and Wolfgang-Michael Franz. "Primary hyperparathyroidism is associated with increased circulating bone marrow-derived progenitor cells." American Journal of Physiology-Endocrinology and Metabolism 293, no. 6 (December 2007): E1670—E1675. http://dx.doi.org/10.1152/ajpendo.00287.2007.
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Recently, parathyroid hormone (PTH) was shown to support survival of progenitor cells in bone marrow. The release of progenitor cells occurs in physiological and pathological conditions and was shown to contribute to neovascularization in tumors and ischemic tissues. In the present study we sought to investigate prospectively the effect of primary hyperparathyroidism (PHPT) on mobilization of bone marrow-derived progenitor cells. In 22 patients with PHPT and 10 controls, defined subpopulations of circulating bone marrow-derived progenitor cells (BMCs) were analyzed by flow cytometry (CD45+/CD34+/CD31+cells indicating endothelial progenitor cells, CD45+/CD34+/c-kit+cells indicating hematopoietic stem cells, and CD45+/CD34+/CXCR4+cells indicating progenitor cells with the homing receptor CXCR4). Cytokine serum levels (SCF, SDF-1, VEGF, EPO, and G-CSF) were assessed using ELISA. Levels of PTH and thyroid hormone as well as serum electrolytes, renal and liver parameters, and blood count were analyzed. Our data show for the first time a significant increase of circulating BMCs and an upregulation of SDF-1 and VEGF serum levels in patients with PHPT. The number of circulating BMCs returned to control levels measured 16.7 ± 2.3 mo after surgery. There was a positive correlation of PTH levels with the number of CD45+/CD34+/CD31+, CD45+/CD34+/c-kit+, and CD45+/CD34+/CXCR4+cells. However, there was no correlation between cytokine serum concentrations (SDF-1, VEGF) and circulating BMCs. Serum levels of G-CSF, EPO, and SCF known to mobilize BMCs were even decreased or remained unchanged, suggesting a direct effect of PTH on stem cell mobilization. Our data suggest a new function of PTH mobilizing BMCs into peripheral blood.
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Carvalho, Jerusa Martins, Marlon Knabben de Souza, Valéria Buccheri, Cláudia Viviane Rubens, José Kerbauy, and José Salvador Rodrigues de Oliveira. "CD34-positive cells and their subpopulations characterized by flow cytometry analyses on the bone marrow of healthy allogenic donors." Sao Paulo Medical Journal 127, no. 1 (January 2009): 12–18. http://dx.doi.org/10.1590/s1516-31802009000100004.
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CONTEXT AND OBJECTIVE: Counting and separating hematopoietic stem cells from different sources has importance for research and clinical assays. Our aims here were to characterize and quantify hematopoietic cell populations in marrow donors and to evaluate CD34 expression and relate this to engraftment. DESIGN AND SETTING: Cross-sectional study on hematopoietic stem cell assays, using flow cytometry on donor bone marrow samples, for allogenic transplantation patients at two hospitals in São Paulo. METHODS: Immunophenotyping of marrow cells was performed in accordance with positive findings of CD34FITC, CD117PE, CD38PE, CD7FITC, CD33PE, CD10FITC, CD19PE, CD14FITC, CD13PE, CD11cPE, CD15FITIC, CD22PE, CD61FITC and CD56PE monoclonal antibodies in CD45PerCP+ cells, searching for differentiation and maturation regions. CD34+ sorting cells were analyzed for CD38 and CD117. Rh-123 retention was done before and after sorting. Antigen expression and CD34+ cells were correlated with engraftment. RESULTS: In region R1, 0.1% to 2.8% of cells were CD34+/CD45+ and 1.1%, CD34+/CD45-. The main coexpressions of CD45+ cells were CD38, CD22, CD19 and CD56 in R2 and CD33, CD11c, CD14, CD15 and CD61 in R3 and R4. After sorting, 2.2x10(6) CD34+ cells were equivalent to 4.9% of total cells. Coexpression of CD34+/CD38+ and CD34+/CD117+ occurred in 94.9% and 82% of events, respectively. There was a positive relationship between CD34+ cells and engraftment. More than 80% of marrow cells expressed high Rh-123. CD34+ cell sorting showed that cells in regions of more differentiated lineages retained Rh-123 more intensively than in primitive lineage regions. CONCLUSION: We advocate that true stem cells are CD34+/CD45-/CD38-/low-Rh-123 accumulations.
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Nakajima-Takagi, Yaeko, Mitsujiro Osawa, Motohiko Oshima, Haruna Takagi, Satoru Miyagi, Mitsuhiro Endoh, Takaho A. Endo, et al. "Role of SOX17 in hematopoietic development from human embryonic stem cells." Blood 121, no. 3 (January 17, 2013): 447–58. http://dx.doi.org/10.1182/blood-2012-05-431403.
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Abstract To search for genes that promote hematopoietic development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), we overexpressed several known hematopoietic regulator genes in hESC/iPSC-derived CD34+CD43− endothelial cells (ECs) enriched in hemogenic endothelium (HE). Among the genes tested, only Sox17, a gene encoding a transcription factor of the SOX family, promoted cell growth and supported expansion of CD34+CD43+CD45−/low cells expressing the HE marker VE-cadherin. SOX17 was expressed at high levels in CD34+CD43− ECs compared with low levels in CD34+CD43+CD45− pre-hematopoietic progenitor cells (pre-HPCs) and CD34+CD43+CD45+ HPCs. Sox17-overexpressing cells formed semiadherent cell aggregates and generated few hematopoietic progenies. However, they retained hemogenic potential and gave rise to hematopoietic progenies on inactivation of Sox17. Global gene-expression analyses revealed that the CD34+CD43+CD45−/low cells expanded on overexpression of Sox17 are HE-like cells developmentally placed between ECs and pre-HPCs. Sox17 overexpression also reprogrammed both pre-HPCs and HPCs into HE-like cells. Genome-wide mapping of Sox17-binding sites revealed that Sox17 activates the transcription of key regulator genes for vasculogenesis, hematopoiesis, and erythrocyte differentiation directly. Depletion of SOX17 in CD34+CD43− ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a key role in priming hemogenic potential in ECs, thereby regulating hematopoietic development from hESCs/iPSCs.
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Edinger, James, Qian Ye, Ajai Pal, Andy Zeitlin, Wolfgang Hofgartner, Robert Hariri, Derek Ragusa, Ted Burke, and Colleen Delaney. "Phenotype and Function of Placenta Derived Stem Cells (PDSC)." Blood 108, no. 11 (November 16, 2006): 4182. http://dx.doi.org/10.1182/blood.v108.11.4182.4182.
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Abstract We developed a proprietary procedure to recover ethical and non-controversial stem cells from full term, postpartum human placentas (Placenta Derived Stem Cells, PDSC). In this work, we describe the phenotype and function of PDSC as compared to Human Umbilical Cord Blood, (HUCB) derived stem cells. As compared to HUCB, PDSC contains a high percentage of live, post thaw CD34+ cells (2.3% ± 3.5 %, N = 83 versus an average pre-freeze CD34% in HUCB at our bank of 0.3% ± 0.2%; N=220). Notably, 85 to 90% of the CD34+ cells in PDSC but not HUCB were CD45 negative. CD34+ cells derived from PDSC were flow sorted into CD34+CD45+ and CD34+CD45− and colony forming assays performed. Both CD34+CD45− and CD34+CD45+ cells gave rise to hematopoietic colonies including CFU-E, CFU-GM and CFU-GEMM, suggesting that CD34+CD45− cells derived from PDSC were lineage negative, immature stem cells that differentiate into hematopoietic progenitor cells. Using multiparameter flow cytometry, we demonstrated that these cells were CD133+ and CD38−. To determine the hematopoietic repopulating ability of PDSC and to examine if PDSC can augment HUCB engraftment in vivo, sub lethally irradiated NOD/SCID mice were infused with freshly thawed, non-sorted PDSC alone, HUCB alone, or a mixture of PDSC and HUCB. Overall human engraftment was determined by assessment of human CD45+ in the bone marrow of recipient mice at four (short-term engraftment) and 12 weeks. Mice were considered engrafted if the percentage of huCD45 was >0.5%. Human engraftment (>0.5% CD45) was observed in all groups 4 weeks post infusion, including PDSC alone, with 2 out of 6 mice positive for engraftment in the HUCB group (mean CD45% of 0.62%), 2 out of 8 mice in the PDSC group (mean CD45% of 0.52%), and 8 out of 9 mice in the group that received both HUCB and PDSC (mean CD45% of 2.84%). There was a significant increase in human engraftment observed when comparing either the PDSC group alone to the HUCB+PDSC group (p=0.006) and the HUCB group to the HUCB+PDSC group (p=0.02). At 12 weeks post transplant, sustained engraftment in the PDSC group alone was not observed with only 1 out of 8 animals engrafted at >0.5% CD45. In contrast, although there was no statistical difference observed in the overall level of human engraftment between mice that received HUCB alone versus the HUCB+PDSC group (mean CD45% of 15.1% and 13.1%, respectively; p=0.82), only 3 out of 6 mice were engrafted in the HUCB group as compared to 9 out of 9 mice in the HUCB+PDSC group. These data indicate that co-infusion of PDSC and HUCB results in significant enhancement of both short-and longer-term human engraftment as compared to PDSC or HUCB alone. Although it remains unclear whether the observed enhanced engraftment is due to an increased number of repopulating cells or the presence of facilitator cells provided by the PDSC, Studies are ongoing and aimed at investigating the mechanism(s) by which PDSC enhances HUCB engraftment. Delayed engraftment following cord blood transplantation remains a significant clinical problem, even in the case of double unit myeloablative cord blood transplantation, where the median time to neutrophil engraftment is 23 days (Barker et al, Blood.2005; 105:1343–1347). These results also suggest clinical investigation of co-infusion of PDSC with either single or double cord blood units for transplantation as a potential method to facilitate more rapid engraftment.
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Jiang, Yajuan, David Mallinson, Daria Olijnyk, Sarah Paterson, Susan Ridha, Lisa Tang, Vincent O'Brien, Timothy C. Fong, and David W. O'Neill. "Analysis Of The Lin- CD45- CD34/CD133+ Cell Population In G-CSF-Mobilized Peripheral Blood By Polychromatic Flow Cytometry and Micro RNA Profiling." Blood 122, no. 21 (November 15, 2013): 4841. http://dx.doi.org/10.1182/blood.v122.21.4841.4841.
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Abstract Introduction There has been controversy over the existence of rare somatic stem cells in mouse and human bone marrow and in human umbilical cord blood that have been described to be pluripotent. These cells have been observed to lack expression of CD45 and blood cell lineage markers (Lin-), to express markers associated with both hematopoietic (CD34, CD133) and pluripotent (Oct4, Nanog) stem cells, to be smaller than a blood lymphocyte, and have been associated with the ability to differentiate into cells and tissues of all three germ layers. They have been given a variety of names, including Very Small Embryonic/Epiblast-Like (VSEL) cells. Methods and Results To better characterize these cells, we fractionated G-CSF mobilized adult peripheral blood by elutriation, CD34/CD133 immunomagnetic selection (Miltenyi Biotec, Inc., STEMCELL Technologies, Inc.) and fluorescence-activated cell sorting (FACS) using a MoFlo XDP cell sorter (Beckman Coulter, Inc.). Cell fractions were analyzed on a Beckman Coulter Gallios flow cytometer using a 6-color cocktail of antibodies to CD45, CD34, CD133 and blood cell lineage markers, together with membrane-permeable (DRAQ5) and impermeable (7-AAD) nuclear dyes that distinguish live nucleated cells from dead cells, extracellular vesicles and cell debris. We observe that over 95% of Lin- CD45- flow cytometry events are extracellular vesicles rather than nucleated cells, and have isolated a population of Lin- CD45- CD34+ vesicles from the earliest elutriation fractions (<35 ml/min counterflow). Rare Lin- CD45- live nucleated cells are also clearly evident, many of which express CD34 but not CD133. This population of Lin- CD45- CD34+ CD133- cells makes up approximately 0.003% of the mononuclear cell population in G-CSF mobilized peripheral blood (1 in 300,000 mononuclear cells, or approximately 1 for every 1,000 CD34+ CD45+ hematopoietic stem/progenitor cells). Lin- CD45- CD133+ live nucleated cells are also identified but are considerably more rare (approximately 1 in 10 million mononuclear cells). Similar cell and vesicle populations are also found in umbilical cord blood, although with frequencies about 10-fold higher than in mobilized adult blood. To begin to characterize these cell and vesicle fractions, we isolated total RNA from FACS-sorted Lin- CD45- CD34/CD133+ (CD34+ and/or CD133+) cells for miRNA expression profiling (Agilent SurePrint G3 Human v16 microRNA 8x60K microarray, representing 1205 Human miRNAs, 1199 verified as real miRNAs in miRbase 18). Array data were processed using a proprietary normalization algorithm (Sistemic, Ltd.) to generate miRNA expression profiles which were analyzed by microRNA-based fingerprinting (SistemQC™). A robust miRNA profile was generated from the initial Lin- CD45- CD34/CD133+ cell sample, with 107 miRNAs reliably detected (a number consistent with other cellular profiles). The detected miRNAs showed a range of expression levels and were expressed well above the limit of detection for the arrays. Further samples will be analyzed to confirm these preliminary findings. Conclusion The Lin- CD45- population observed by flow cytometry analysis of human mobilized peripheral blood and umbilical cord blood is heterogeneous, and made up of distinct populations of vesicles and live nucleated cells that variably express CD34 and CD133. The ability to determine miRNA profiles from rare sorted populations such as Lin- CD45- CD34/CD133+ cells will enable the possible further understanding of the function of these cells, as well as the role of miRNAs in regulating their cellular processes. It is also hoped that the data will enhance the understanding of the potential clinical utility of such cells isolated from human blood products. Disclosures: Jiang: NeoStem, Inc.: Employment. Mallinson:Sistemic, Ltd.: Employment, Equity Ownership. Olijnyk:Sistemic, Ltd.: Employment. Paterson:Sistemic, Ltd.: Employment. Ridha:Sistemic, Ltd.: Employment. Tang:NeoStem, Inc.: Employment. O'Brien:Sistemic, Ltd.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Fong:NeoStem, Inc.: Employment. O'Neill:NeoStem, Inc.: Employment.
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Michurova, Marina Sergeevna, Victor Yur'evich Kalashnikov, Olga Michailovna Smirnova, Olga Nikolaevna Ivanova, and Sergey Anatol'evich Terekhin. "Mobilization of endothelial progenitor cells after endovascular interventions in patients with type 2 diabetes mellitus." Diabetes mellitus 17, no. 4 (December 8, 2014): 35–42. http://dx.doi.org/10.14341/dm2014435-42.
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Aim. To investigate the mobilisation of endothelial progenitor cells (EPC) in patients with type 2 diabetes mellitus (T2DM) after endovascular interventions for coronary and peripheral arteries. Materials and Methods. The levels of EPC in peripheral blood were determined by flow cytometry in 42 patients prior to endovascular intervention and 2?4 days after surgery. EPC were defined as CD34+ VEGFR2+ CD45- and CD34+ CD133+CD45- cells. Twenty-three patients with T2DM were included in group 1, and 19 patients without metabolic disorders were included in group 2. Results. The levels of EPC in the peripheral blood of patients with T2DM before and after endovascular interventions were not significantly different. In the subgroup of patients without TDM2, the levels of CD34+VEGFR2 +CD45- cells increased after surgery to 55,5% (p
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Harashima, Akira, Motoyuki Suzuki, Ayumi Okochi, Mayuko Yamamoto, Yoshinobu Matsuo, Ryuichi Motoda, Tamotsu Yoshioka, and Kunzo Orita. "CD45 tyrosine phosphatase inhibits erythroid differentiation of umbilical cord blood CD34+ cells associated with selective inactivation of Lyn." Blood 100, no. 13 (December 15, 2002): 4440–45. http://dx.doi.org/10.1182/blood-2002-03-0864.
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CD45 is a membrane-associated tyrosine phosphatase that dephosphorylates Src family kinases and Janus kinases (JAKs). To clarify the role of CD45 in hematopoietic differentiation, we examined the effects of anti-CD45 monoclonal antibody NU-LPAN on the proliferation and differentiation of umbilical cord blood CD34+ cells. NU-LPAN showed a prominent inhibition of the proliferation of CD34+ cells induced by the mouse bone marrow stromal cell line MS-5 or erythropoietin (EPO). However, NU-LPAN did not affect the proliferation induced by interleukin 3. NU-LPAN also inhibited MS-5–induced or EPO-induced erythroid differentiation of CD34+ cells. The cells stimulated with EPO in the presence of NU-LPANmorphologically showed differentiation arrest at the stage of basophilic erythroblasts after 11 days of culture, whereas the cells treated with EPO without NU-LPAN differentiated into mature red blood cells. The Src family kinase Lyn and JAK2 were phosphorylated when erythroblasts obtained after 4 days of culture of CD34+ cells in the presence of EPO were restimulated with EPO. Overnight NU-LPAN treatment before addition of EPO reduced the phosphorylation of Lyn but not that of JAK2. Simultaneously, the enhancement of Lyn kinase activity after restimulation with EPO was reduced by NU-LPAN treatment. These results indicate selective inactivation of Lyn by CD45 activated with NU-LPAN and could partly explain the inhibitory mechanism on erythropoiesis exhibited by EPO. These findings suggest that CD45 may play a pivotal role in erythropoiesis.
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Udi, Josefina, Martina Kleber, Dagmar Wider, Ralph Wäsch, and Monika Engelhardt. "Higher Vascular Endothelial Growth Factor (VEGF) and Endothelial Progenitor Cells (EPCs) in Multiple Myeloma (MM) Patients (pts) as a Reflection of Their Governing Role in Pathological Angiogenesis: Comparison of VEGF and EPC Levels Between Healthy Donors (HD), MGUS and MM Pts and Correlation Analysis with MM Activity." Blood 112, no. 11 (November 16, 2008): 5132. http://dx.doi.org/10.1182/blood.v112.11.5132.5132.
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Abstract Introduction: In MM pathogenesis, angiogenesis and growth factors have a governing role. VEGF, secreted by malignant bone marrow (BM) plasma cells (PCs) and stroma, acts as an important mediator of tumor angiogenesis. VEGF has been suggested as an adverse prognostic factor, being elevated in advanced and plasmablastic MM. Circulating as well as BM-residing endothelial cells (ECs) have been shown to contribute to angiogenesis in MM as well as other tumors. Moreover, endothelial progenitor cells (EPC) have been demonstrated to contribute to vascular repair and to be decreased in number and function with end-stage renal impairment (RI). Whereas VEGF and/or EPCs have been described in small former analysis in MM, larger comparisons with MGUS pts and healthy donors (HD) are lacking, differences in various MM subsets (such as BM; peripheral blood and apheresis [AP] samples) have not been addressed, nor the role of EPCs and hemangioblasts in advanced vs. mild RI in MM pts. Methods: We sought to characterize ECs (namely VEGF+ cells, EPCs as VEGFR2+/CD133+/CD34+, hemangioblasts as VEGF+/CD34+) and other subtypes (CD34+, CD45+, CD38+, CD45+/CD38+, CD45−/CD38+) via multiparametric flow cytometry. This was performed in MM pts (n=70), MGUS pts (n=8) and HD (n=14). In MM, BM (n=70), PB (n=14) and AP (n=21) specimens were compared, as well as changes in EPCs and hemangioblasts with and without mild or severe RI. Renal function was determined via estimated glomerular filtration rate (eGFR), classifying mild RI as eGFR <90ml/min/1.73m2 and severe RI as eGFR <30ml/min/1.73m2. Results: MM pts’ age, BM-infiltration and serum creatinine were 63 years (range: 35–84), 15% (0–96) and 0.91mg/dl (0.5–8.6), respectively. BM specimens from MM and MGUS pts showed 4-fold and 2-fold higher VEGF levels, respectively as compared to HD (Table 1). EPCs were also elevated in MM as compared to MGUS and HD (Table 1). Hemangioblasts, CD45+/CD38+ and CD45−/CD38+ were increased in MM BM specimens as compared to MGUS and HD, whereas CD45+ and CD34+ cell numbers were decreased in myeloma specimens (Table 1). Table 1. Median endothelial cells and other subtypes in MM, MGUS and healthy donors BM MM (n=70) BM MGUS (n=8) BM healthy donors (n=14) VEGF+ (%) 0.38 (0 – 12.9) 0.18 (0 – 0.7) 0.09 (0 – 0.6) EPCs (%) 0.03 (0 – 0.4) 0.02 (0 – 0.07) 0.01 (0 – 0.2) VEGF+/CD34+ (%) 0.21 (0 – 2.2) 0.11 (0 – 0.4) 0.04 (0 – 0.5) CD34+ (%) 0.65 (0 – 6.6) 1.23 (0.02 – 4.0) 1.50 (0.07 – 2.8) CD45+ (%) 39.54 (2.8 – 99.1) 39.34 (13.8 – 69.7) 51.70 (10.3 – 90.9) CD45+/CD38+ (%) 24.61 (0.9 – 89.7) 21.68 (1.4 – 49.4) 17.20 (2.6 – 56.9) CD45−/CD38+ (%) 1.54 (0 – 77.7) 1.15 (0 – 2.4) 0.62 (0.1 – 5.6) The comparison of BM, PB, AP specimens in MM showed similar VEGF levels in BM and PB with 0.38% which were increased in AP specimens with 0.5%. This was similarly observed for EPCs with 0.03% in BM and PB as compared to 0.04% in AP samples. Other markers showed similar values for CD34, CD45, CD38+ cells in BM and PB; similar hemangioblast numbers in all 3 subsets, and higher CD34+ and CD45+ cells, and lower CD45−/CD38+ cells in AP specimens. Correlation of EPCs and hemangioblasts with renal function revealed that EPCs decreased with RI, whereas hemangioblasts remained comparable (Table 2). Table 2. Median EPCs and hemangioblasts (VEGF/CD34) with and without RI EPCs (%) VEGF+/CD34+ (%) eGFR >90 (n=41) 0.050 (0 – 0.41) 0.195 (0 – 0.86) eGFR <90 (n=46) 0.025 (0 – 0.41) 0.230 (0 – 0.8) eGFR >30 (n=81) 0.030 (0 – 0.41) 0.210 (0 – 2.23) eGFR <30 (n=6) 0.025 (0 – 0.41) 0.190 (0 – 2.23) Conclusions: These results demonstrate that all ECs, namely VEGF+ cells, EPCs and hemangioblasts are higher in MM than MGUS and HD. Lower CD34+ and CD45+ cells in MM suggest this as a result of the disease and most likely also due to anti-MM therapy. We observed differences in BM, PB and AP specimens in MM pts. RI influenced EC numbers. These results suggest that elevated ECs in MM may reflect disease activity and may be useful as MM biomarkers. The quantification of ECs in MM may also be informative to monitor the efficacy of anti-angiogenic treatment, such as thalidomide and lenalidomide. Further analyses will evaluate the prognostic significance of EPCs, hemangioblasts and other markers in MM, their role in mild and severe RI is ongoing, as well the correlation with disease outcome.
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Kaufman, Dan S., Petter S. Woll, Colin H. Martin, and Jeffrey S. Miller. "Human Embryonic Stem Cells Differentiate into Functional Natural Killer Cells with the Capacity To Mediate Anti-Tumor Activity." Blood 106, no. 11 (November 16, 2005): 763. http://dx.doi.org/10.1182/blood.v106.11.763.763.
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Abstract Hematopoiesis from human embryonic stem cells (hESCs) follows developmental kinetics similar to what is observed during normal human ontogeny. Myeloid, erythroid and megakaryocytic progenitors can be routinely generated from hESCs. However, little is known about the ability of hESCs to differentiate into the lymphoid lineage. Natural killer (NK) cells are important mediators of donor anti-host alloreactivity seen after allogeneic transplant for myeloid leukemias. Our studies use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. CD34+ and CD34+CD45+ hESC-derived hematopoietic progenitor cells were co-cultured with inactivated AFT024 stromal cells in medium supplemented with IL-7, IL-15, SCF and FL. Generation of NK cells was established by phenotypic and functional analysis. CD34+ umbilical cord blood (UCB) cells were utilized as a positive control. After 14 days of culture of CD34+ hESC-derived cells, more than 90% of the cells express CD45, a pan-hematopoietic cell marker, but few CD56+ cells are observed. At 21 days of culture a distinct CD56+CD45+ cell population develops (14.9%), which increases to 37.5% of cells after 28 days of culture. Similar results are observed for CD34+CD45+ hESC-derived cells, characterizing that both CD34+ and CD34+CD45+ cell populations contain hematopoietic progenitors with NK cell developmental potential. Limiting dilution analysis of hESC-derived progenitor cells demonstrates CD34+ hESC-derived cells have a low NK cell progenitor frequency. However, sorting for CD34+CD45+ hESC-derived cells significantly increased the NK cell cloning frequency (1.92% ± 1.20%) to a level comparable to the frequency observed for CD34+ UCB cells cultured in the same manner (3.57% ± 1.68%). The hESC-derived NK cells also express receptors known to regulate NK cell cytolytic activity, including killer-Ig-like receptors (KIRs), C-type lectin-like receptors (CD94 and NKG2A) and natural cytotoxicity receptors (NKp30, NKp44, and NKp46). Furthermore, hESC-derived NK cells also express CD16, an Fc-receptor typically expressed on more mature NK cells. The expression of KIRs is significantly higher for the hESC-derived NK cells compared to the UCB-derived NK cells. This may lead to future strategies to generate selective alloreactive NK cell populations for therapy. To investigate the functional properties of the hESC-derived NK cells, cytolytic activity was tested against K562 erythroleukemia cells and Raji B-lymphoblastoid cells. hESC-derived NK cells effectively killed K562 cells, with activity similar to that seen with UCB-derived NK cells. As expected, Raji cells were resistant to direct cytotoxicity by both hESC and UCB-derived NK cells. However, treatment of Raji cells with anti-CD20 antibody results in effective antibody-dependent cell-mediated cytoxicity by the hESC-derived NK cells. The hESC-derived NK cells also demonstrate ability to upregulate production of cytokines such as IFN-γ upon stimulation. Furthermore, we also find that hESC-derived progenitors also have T cell and/or B cell potential based on cells that express Ikaros, Rag1, and IL7Rα. These results demonstrate that the CD34+ and CD34+CD45+ hESC-derived cell populations contain lymphoid progenitor cells that can develop into both innate and adaptive immune cells. The ability to generate functional NK cells that can target and lyse human tumor cells via two distinct mechanisms suggests potentially novel anti-cancer therapy applications of hESCs.
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El-Bassiouni, Nora, Noha Amin, S. H. Rizk, M. K. Abo El Azayem, Mona Madkour, Hasan Garem, Raafat Ibrahim, and Ola Abo El Nil. "Role of Circulating Hematopoietic Fibrocytes in Chronic Hepatitis C Patients Induced Liver Fibrosis." Open Access Macedonian Journal of Medical Sciences 10, B (February 6, 2022): 1222–29. http://dx.doi.org/10.3889/oamjms.2022.8123.
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Background: Bone marrow derived fibrocytes may play an important role in pathogenesis and resolution of liver fibrosis. These cells may offer new approaches for better understanding the pathogenesis of liver fibrosis.
Aim of the work: To define the proportion of circulating fibrocytes with hematopoietic progenitor origin as defined by CD45 and CD34 positivity and to assess whether they are increased in patients with chronic C hepatitis in correlation to the degree of liver fibrosis.
Subjects and Methods: Sixty HCV patients were classified according to METAVIR score into 4 stages of liver fibrosis, 15 age and sex-matched controls were included. Flowcytometric analysis for circulating levels of fibrocytes CD34+ve cells, CD45+ve cells, collagen type I+ve cells and CXCR4+ve cells was carried out using monoclonal antibodies (anti-CD34, CD45, collagen type I and CD184). GM-CSF, TGF-β and α-SMA were assessed using ELISA.
Results and Conclusions: A significant increase in the circulating levels of GM-CSF, TGF- β and α-SMA, with a significant increase in the percentage of cells express CXCR4and in the co expression of CD34, CD45 and collagen type I positive cells in different groups of patients compared to control group, denoting the presence of an increased proportion of circulating fibrocytes in peripheral blood of these patients. The percentage of fibrocytes that positively expression CD34, CD45, collagen type I and CXCR4, were increased in step wise fashion in conjunction with worsening severity of liver disease.
Liver fibrosis is associated with increased levels of circulating TGF-β1 and lipopolysaccharide, activation of myofibroblasts, and extensive deposition of extracellular matrix, mostly collagen Type I. TGF-β and LPS play a critical role in fibrogenesis and trigger fibrocyte recruitment to the injured liver promoting their differentiation into collagen type I producing myofibroblast, supporting that fibrocytes may become a novel target for anti fibrotic therapy.
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Wognum, AW, G. Krystal, CJ Eaves, AC Eaves, and PM Lansdorp. "Increased erythropoietin-receptor expression on CD34-positive bone marrow cells from patients with chronic myeloid leukemia." Blood 79, no. 3 (February 1, 1992): 642–49. http://dx.doi.org/10.1182/blood.v79.3.642.642.
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Abstract Erythropoietin-receptor (EpR) expression on bone marrow cells from normal individuals and from patients with chronic myeloid leukemia (CML) was examined by multiparameter flow cytometry after stepwise amplified immunostaining with biotin-labeled Ep, streptavidin- conjugated R-phycoerythrin, and biotinylated monoclonal anti-R- phycoerythrin. This approach allowed the detection of EpR-positive cells in all bone marrow samples studied. Most of the EpR-positive cells in normal bone marrow were found to be CD45-dull, CD34-negative, transferrin-receptor-positive and glycophorin-A-intermediate to - positive. This phenotype is characteristic of relatively mature erythroid precursors, ie, colony-forming units-erythroid and erythroblasts recognizable by classic staining procedures. Approximately 5% of normal EpR-positive cells displayed an intermediate expression of CD45, suggesting that these represented precursors of the CD45-dull EpR-positive cells. Some EpR-positive cells in chronic myeloid leukemia (CML) bone marrow had a phenotype similar to the major EpR-positive phenotype in normal bone marrow, ie, CD34-negative and CD45-dull. However, there was a disproportionate increase in the relative number of EpR-positive/CD45-intermediate cells in CML bone marrow. Even more striking differences between normal individuals and CML patients were observed when EpR-expression on CD34-positive marrow cells was analyzed. Very few EpR-positive cells were found in the CD34- positive fraction of normal bone marrow, whereas a significant fraction of the CD34-positive marrow cells from five of five CML patients expressed readily detectable EpR. These findings suggest that control of EpR expression is perturbed in the neoplastic clone of cells present in patients with CML. This may be related to the inadequate output of mature red blood cells typical of CML patients and may also be part of a more generalized perturbation in expression and/or functional integrity of other growth factor receptors on CML cells.
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Wognum, AW, G. Krystal, CJ Eaves, AC Eaves, and PM Lansdorp. "Increased erythropoietin-receptor expression on CD34-positive bone marrow cells from patients with chronic myeloid leukemia." Blood 79, no. 3 (February 1, 1992): 642–49. http://dx.doi.org/10.1182/blood.v79.3.642.bloodjournal793642.
Full textAbstract:
Erythropoietin-receptor (EpR) expression on bone marrow cells from normal individuals and from patients with chronic myeloid leukemia (CML) was examined by multiparameter flow cytometry after stepwise amplified immunostaining with biotin-labeled Ep, streptavidin- conjugated R-phycoerythrin, and biotinylated monoclonal anti-R- phycoerythrin. This approach allowed the detection of EpR-positive cells in all bone marrow samples studied. Most of the EpR-positive cells in normal bone marrow were found to be CD45-dull, CD34-negative, transferrin-receptor-positive and glycophorin-A-intermediate to - positive. This phenotype is characteristic of relatively mature erythroid precursors, ie, colony-forming units-erythroid and erythroblasts recognizable by classic staining procedures. Approximately 5% of normal EpR-positive cells displayed an intermediate expression of CD45, suggesting that these represented precursors of the CD45-dull EpR-positive cells. Some EpR-positive cells in chronic myeloid leukemia (CML) bone marrow had a phenotype similar to the major EpR-positive phenotype in normal bone marrow, ie, CD34-negative and CD45-dull. However, there was a disproportionate increase in the relative number of EpR-positive/CD45-intermediate cells in CML bone marrow. Even more striking differences between normal individuals and CML patients were observed when EpR-expression on CD34-positive marrow cells was analyzed. Very few EpR-positive cells were found in the CD34- positive fraction of normal bone marrow, whereas a significant fraction of the CD34-positive marrow cells from five of five CML patients expressed readily detectable EpR. These findings suggest that control of EpR expression is perturbed in the neoplastic clone of cells present in patients with CML. This may be related to the inadequate output of mature red blood cells typical of CML patients and may also be part of a more generalized perturbation in expression and/or functional integrity of other growth factor receptors on CML cells.
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Vašíček, Jaromír, Andrej Baláži, Miroslav Bauer, Andrea Svoradová, Mária Tirpáková, Ľubomír Ondruška, Vladimír Parkányi, Alexander V. Makarevich, and Peter Chrenek. "Enrichment of Rabbit Primitive Hematopoietic Cells via MACS Depletion of CD45+ Bone Marrow Cells." Magnetochemistry 7, no. 1 (January 13, 2021): 11. http://dx.doi.org/10.3390/magnetochemistry7010011.
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Hematopoietic stem and progenitor cells (HSC/HPCs) of human or few animal species have been studied for over 30 years. However, there is no information about rabbit HSC/HPCs, although they might be a valuable animal model for studying human hematopoietic disorders or could serve as genetic resource for the preservation of animal biodiversity. CD34 marker is commonly used to isolate HSC/HPCs. Due to unavailability of specific anti-rabbit CD34 antibodies, a novel strategy for the isolation and enrichment of rabbit HSC/HPCs was used in this study. Briefly, rabbit bone marrow mononuclear cells (BMMCs) were sorted immunomagnetically in order to remove all mature (CD45+) cells. The cells were depleted with overall purity about 60–70% and then cultured in a special medium designed for the expansion of CD34+ cells. Quantitative Polymerase Chain Reaction (qPCR) analysis confirmed the enrichment of primitive hematopoietic cells, as the expression of CD34 and CD49f increased (p < 0.05) and CD45 decreased (p < 0.001) at the end of culture in comparison to fresh BMMCs. However, cell culture still exhibited the presence of CD45+ cells, as identified by flow cytometry. After gating on CD45− cells the MHCI+MHCII−CD38+CD49f+CD90−CD117− phenotype was observed. In conclusion, rabbit HSC/HPCs might be isolated and enriched by the presented method. However, further optimization is still required.
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Reyes, Morayma, and Jeffrey S. Chamberlain. "Perivascular CD45−:Sca-1+:CD34− Cells Are Derived from Bone Marrow Cells and Participate in Dystrophic Skeletal Muscle Regeneration." Blood 106, no. 11 (November 16, 2005): 394. http://dx.doi.org/10.1182/blood.v106.11.394.394.
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Abstract Multiple mechanisms may account for bone marrow (BM) cell incorporation into myofibers following muscle damage. Here, we demonstrated that CD45−:Sca-1+:CD34− cells may play a role in the regeneration of mdx4cv skeletal muscles, an animal model for Duchenne muscular dystrophy. To understand the origin of CD45−:Sca-1+:CD34− cells in skeletal muscle, we reconstituted lethally irradiated wild type (wt) or mdx4cv mice with unfractionated BM cells from transgenic mice ubiquitously expressing green fluorescence protein (GFP). 1, 2, and 6 months post-transplantation, we analyzed the skeletal muscle mononuclear cells from the recipients by flow cytometry for GFP, CD45-PerCP, Sca-1-PE, and CD34-APC. To our surprise, we found a small percentage of BM-derived (GFP+) CD45−:Sca-1+:CD34− cells in the skeletal muscles of these GFP+ BM transplant recipients. These BM-derived cells are localized in the perivascular tissue by immunostaining and their frequency increases with time. We were interested in the potential of these cells for clinical application for muscle diseases. Thus, we FACS-sorted CD45−:Sca-1+:CD34− cells from GFP transgenic mouse skeletal muscles and transplanted them in the tibialis anterior (TA) muscle of mdx4cv mice. In ten days we found significant muscle engraftment. In addition, we studied the response of this population upon acute muscle injury. For this, we injected cardiotoxin in the right TA muscle of mdx4cv and wt mice, followed by BrdU administration in drinking water for three days. After 3.5 days, mice were sacrificed and the right and left (control) TA muscles were harvested and muscle CD45−:Sca-1+:CD34− cells were FACS-sorted, fixed and stained for BrdU and Myf-5. In the injured muscle (right TA), more than 70% of these cells were BrdU+ and more than 50% were Myf-5+, compared to baseline levels (close to zero) in the left TA. This indicates that this population can undergo proliferation and myogenic commitment upon muscle injury. To understand how these BM cells migrate to the muscle and once in the muscle how they mobilize, we investigated the in vitro chemotatic response of GFP+ (BM derived) CD45−:Sca-1+ cells isolated from muscles of GFP+ BM transplant recipients. We found that these cells were highly chemoattrated to stroma derived factor, SDF-1, a chemo-attractant for cells expressing CXCR4. We also observed higher frequency of BM-derived CD45−:Sca-1+:CD34− cells in dystrophic muscle than wt muscle, which may be explained by higher expression levels of SDF-1 in dystrophic muscles. In an effort to determine the identity of these cells when ex vivo cultured, we cultured them in several stem cell media, including a low-serum medium containing specific cytokines for the isolation and expansion of multipotent adult progenitor cells (MAPCs). MAPCs can be isolated from skeletal muscle and BM and can differentiate into multiple tissue cells. Strikingly, we found that MAPCs were enriched up to 40 folds by sorting this population from skeletal muscle. The frequency of BM-derived muscle MAPCs also increases with time post-transplantation in dystrophic muscles. These BM-derived muscle MAPCs displayed the typical MAPC immunophenotypes, displayed a normal diploid karyotype and were capable to differentiate into endothelial cells, hepatocytes and neurons. Taken together, our results suggest that dystrophic muscles recruit BM cells that localize in perivascular tissues and can be defined as CD45-:Sca-1+:CD34-. This population when cultured enriches for MAPCs and can participate in muscle regeneration in dystrophic muscles.
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Yoshimoto, Momoko, Chang Hsi, Katsutsugu Umeda, Midori Iida, Toshio Heike, and Tatsutoshi Nakahata. "Bonr Marrow Reconstitution from Mouse Embryonic Stem Cell-Derived Hematopoietic Cells." Blood 104, no. 11 (November 16, 2004): 4145. http://dx.doi.org/10.1182/blood.v104.11.4145.4145.
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Abstract Differentiation of embryonic stem (ES) cells in vitro yield different kinds of hematopoietic progenitors including primitive and definitive hematopoietic cells. It has been reported that HOXB4 induction enable york sac (YS) cells and embryoid body-derived cells to engraft in the irradiated adult mice, however, since the characteristic of ES-derived transplantable cells is not clear, generating hematopoietic stem cells (HSCs) in vitro still remains to be resolved. We previously reported the generation of definitive HSCs from both early YS and intraembryonic paraaortic splanchnopleures (P-Sp) on AGM-S3 stromal cells derived from the aorta-gonad-mesonephros (AGM) region at 10.5 days post coitum (Matsuoka, et al; Blood 2001) Co-cultureing on AGM-S3, these YS cells and PS-Sp cells acquired the reconstituting potential of adult irradiated mice. Here we intended to make HSCs using this stromal cells. We differentiated ES cells labeled with GFP on OP9 stromal cell, which is supportive for Hematopoietic differentiation. After 4 days, we sorted Flk1+ cells, which is considered as a marker of hemangilblasts, and transferred them onto A-9, subline of AGM-S3, or OP9 stromal cell layer with cytokines. After several days incubation, we examined the emergence of CD34+ c-kit+ cells and colony forming ability of CD34+ or CD34− cells. CD34+ cells contained more CFU-Mix than CD34− cells. When compared on A-9 or OP9, cultured cells on OP9 contained more CFU activity than on A-9. We sorted and cultured CD34+ c-kit+ cells on OP9 for 7–10 days, and confirmed Ter119+, Gr-1+, or Mac-1+ cells differentiated from CD34+ c-kit+ cells by FACS analysis. Next, we cultured Flk1+ cells on A-9 or OP9 for 7–15 days and transplanted all the collected cells into 2.4Gy irradiated NOD-SCID mice. After 3 months after transplantation, FACS analysis showed no GFP+ cells in the recipient BM. However, PCR analysis detected donor derived DNAs in BM when Flk1+ cells were cocultured on A-9. We next transplanted 1×104 of CD34+ CD45+ or CD34+ CD45− cells from Flk1+ cells cocultured on A-9 or OP9 into 2.4 Gy irradiated NOD-SCID mice. PCR analysis revealed donor derived DNAs in mice transplanted with CD34+ CD45+ cells on A-9 and with CD34+ CD45− cell on OP9. These data suggested that CD34+ cells differentiated from Flk1+ cells have powerful hematipoietic activity and showed different potential cultured between on A-9 and on OP9.
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Benton, Christopher B., Ahmed Al-Rawi, Taejin L. Min, Rui-Yu Wang, Wendy Schober, Zhiqiang Wang, Zhihong Zeng, et al. "Lineage-negative, CD34-negative, CD45-negative (Lin-CD34-CD45-) leukemia cells from primary adult AML samples have distinct stem cell-like properties." Clinical Lymphoma Myeloma and Leukemia 15 (September 2015): S21—S22. http://dx.doi.org/10.1016/j.clml.2015.07.047.
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Cox, Charlotte Victoria, Paraskevi Diamanti, and Allison Blair. "Assessing CD97 and CD99 As Markers of Leukaemia Initiating Cells in Paediatric ALL." Blood 120, no. 21 (November 16, 2012): 1882. http://dx.doi.org/10.1182/blood.v120.21.1882.1882.
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Abstract Abstract 1882 Overall survival rates in paediatric acute lymphoblastic leukaemia (ALL) have dramatically improved but around 20% do not respond to current therapies and subsequently relapse. Leukaemia initiating cells (LIC) are the topic of much investigation, as these cells can self-renew and may have the potential to cause relapse. It has been shown that multiple subpopulations of ALL cells have the ability to initiate the disease in immune deficient mouse models. Therefore, treatment should be targeted at all cells with this capacity, if the disease is to be eradicated. Minimal residual disease (MRD) detection is an invaluable tracking tool to assess early treatment response and recent studies have highlighted potential markers that may improve the sensitivity of MRD detection by flow cytometry. CD97 and CD99 are two markers which were over expressed in paediatric ALL. Incorporating these markers into investigations of LIC may allow discrimination of leukaemia cells from normal haemopoietic stem cells (HSC). In this study we evaluated the expression of CD34 in combination with CD97 in B cell precursor (BCP) ALL cases and CD99 in T-ALL cases and subsequently assessed the functional capacity of the sorted subpopulations in vitro and in vivo. Ten ALL samples (6 B-ALL & 4 T-ALL) with a median age 7 years (range 2–15 years) were studied. One B-ALL case and 3 T-ALL cases were considered high risk by molecular assessment of MRD at day 28 of treatment. Flow cytometric analyses of the ALL samples and 8 normal haemopoietic cell samples demonstrated that both CD97 and CD99 were over expressed in ALL patients (78.9±14.8% & 76.4±32.8%, respectively) when compared to normal haemopoietic cells (14.1±25.4%; p=0.001, 47.1±10%; p=0.03, respectively). Cells were sorted for expression/lack of expression of these markers and proliferation of the sorted cells was assessed in suspension culture over a 6 week period. In the B-ALL patients the CD34+/CD97+ subpopulation represented the bulk of leukaemia cells (65.2±32.1%), the CD34−/CD97+ the smallest fraction (3.3±2.4%) with the CD34+/CD97− and CD34−/CD97− subpopulations representing 21.1±31.5% and 10.5±5.8% of cells, respectively. When the functional capacity of these subpopulations was assessed in vitro greatest expansion was observed in cells derived from CD34+/CD97− subpopulation (2–173 fold) from 9.4×103 at initiation up to 1.5×106 cells at week 6. Expansion was also observed, to a lesser extent in the CD34−/CD97− subpopulation (3.4–28 fold) from 8×103 up to 1.4×106 cells. No expansion was observed in cultures of CD34+/CD97+ and CD34−/CD97− subpopulations but cells were maintained throughout the culture period. These sorted subpopulations were also inoculated into NOD/LtSz-SCID IL-2Rγc null (NSG) mice to evaluate repopulating capacity. To date, engraftment has been achieved with 3 subpopulations; CD34+/CD97+ (3–28.8% CD45+), CD34+/CD97− (0.5–25.5% CD45+) and CD34−/CD97+ (23.8% CD45+) cells. When the functional capacity of T-ALL cases was assessed the CD34+/CD99+ subpopulation represented the bulk of cells at sorting (51.87±47.2%), the CD34+/CD99- subpopulation was the smallest (0.9±0.8%) and the CD34−/CD99+ and CD34−/CD99− subpopulations represented 32.1±38.9% and 27.2±33.4% of cells, respectively. Greatest expansion was observed in cultures of CD34+/CD99- cells (4.6–1798 fold) from 7.5×103 up to 2.6×106 cells at week 6. The other 3 subpopulations expanded to a lesser extent (1.3–216 fold) from 5×103 up to 1.8×106 cells. When the functional capacity of these cells was assessed in NSG mice, engraftment was achieved in all subpopulations; CD34+/CD99+ (87–90.5% CD45+), CD34+/CD99− (1.5–84.9% CD45+), CD34−/CD99+ (31.3–98.6% CD45+) and CD34−/CD99− (3–92.9% CD45+). In some cases, cells recovered from BM of NSG inoculated with CD99− cells had high expression of CD99, typical of the patient samples at diagnosis, indicating that the inoculated CD99− cells had differentiated in vivo. Studies are ongoing to assess the self-renewal capacity of these subpopulations by serial transplantation. The findings to date indicate that targeting CD97 and CD99, either alone or in combination with CD34 would not eliminate all cells with the capacity to initiate and maintain B-ALL and T-ALL, respectively. Further developments in therapy may require targeting leukaemogenic pathways, rather than only cell surface markers to improve survival outcome in paediatric ALL. Disclosures: No relevant conflicts of interest to declare.
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Sei, Janet J., Blake S. Moses, Abigail Harris-Becker, MinJung Kim, Navjot Kaur, Mohan C. Vemuri, and Curt I. Civin. "Optimized Culture Medium for Enhanced Ex Vivo expansion of Human Hematopoietic Stem Cells." Blood 134, Supplement_1 (November 13, 2019): 1965. http://dx.doi.org/10.1182/blood-2019-126174.
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Increasing the numbers of donor hematopoietic stem cells (HSCs) would accelerate hematopoietic recovery in many HSC transplant recipients, thereby reducing mortality, morbidity and costs. HSC gene therapies would further benefit from high transplant donor HSC numbers, due to inefficiencies in genetic modification of HSCs and losses during transfection protocols. Unfortunately, worldwide attempts to optimize culture parameters, such as hematopoietic growth factor combinations, feeder cells and bioengineered chambers, have failed to result in the substantial HSC self-renewal needed clinically, although it was reported recently that substitution of polyvinyl alcohol for albumin resulted in massive expansion of mouse HSCs (Wilkinson et al. Nature 571:117;2019).Since the same few base hematopoietic culture media have been used for decades, we set out to develop a culture medium specifically for ex vivo expansion of human HSCs. In a Design of Experiments approach, media constituents were systematically varied and each iteration evaluated with the goal to maximize ex vivo expansion of hematopoietic stem-progenitor cell (HSPC) immunophenotypes. After 1 week (wk) culture of human mPB-, CB- or BM-derived CD34+ cells in optimized xeno-free, serum-free StemPro™ HSC Expansion Medium (Prototype) (SPHSC), supplemented with FLT3L, KITL, TPO, IL3, and IL6 (FKT36), there were on average 96-, 178- or 80-fold, respectively, greater numbers (yields) of viable nucleated cells, as compared to day (d)0 (Fig 1). Average yields of the CD34+CD45+Lin- HSPC immunophenotype were increased similarly, by 80-, 104- or 42-fold, respectively. Average yields of the CD34+CD45+CD45RA+CD90+ early HSPC immunophenotype were much more highly increased, by 2300-, 6047- or 1248-fold, respectively. CB-derived CD34+ cells typically generated greater fold increases in these parameters than did CD34+ cells from mPB or BM. In addition, we consistently observed (a) the presence of very early CD34+++CD90+ cells and (b) few Lin+ cells in the ex vivo cultured populations. Furthermore, compared to 2 different FKT36-containing commercial standard media, SPHSC supported statistically greater yields of nucleated cells, CD34+CD45+Lin- HSPCs and CD34+CD45+CD45RA+CD90+ early HSPCs. We focused on the challenge of expanding HSCs from mPB, the most common clinical source. Yields of nucleated cells, CD34+CD45+Lin- HSPCs, and CD34+CD45+CD45RA+CD90+ early HSPCs expanded progressively from d4-d10. Transplant of 1 wk-cultured mPB CD34+ HSPCs generated donor cell dose-dependent increases in %hCD45+mCD45- cells in sublethally-irradiated NRG mouse bone marrows and spleens at 26 wks post-transplant. Human HSPCs and CD33+ myeloid and CD19+ lymphoid progeny were present in hematopoietic organs of the transplanted mice (Fig 2). The long-term (LT)-HSC engraftment capacity of the entire 1 wk-cultured cell population was 10-fold greater than at d0. These results for 1 wk ex vivo expansion of mPB CD34+ cells in SPHSC are similar or superior to reported 12d or 15d expansions of CB CD34+ cells in cytokine-supplemented standard media containing UM171 or SR1 (Fares et al. Science 345:1509;2014; Cohen et al. Biol Blood Marrow Transplant 24:S190;2018; Wagner et al.Cell Stem Cell 18:144;2016). Thus, SPHSC medium may by itself be impactful in clinical transplantation and gene therapy and would also be an optimized platform medium for additional approaches to further enhance ex vivo expansion of human HSCs for transplant and gene therapies. Disclosures Sei: Thermo Fisher Scientific Inc: Employment. Harris-Becker:Thermo Fisher Scientific Inc: Employment. Kaur:Thermo Fisher Scientific Inc: Employment. Vemuri:Thermo Fisher Scientific Inc: Employment.
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Ye, Zhaohui, Xiaobing Yu, and Linzhao Cheng. "Efficient Production of Human Hematopoietic Progenitors from Human Pluripotent Stem Cells Using Chemically Defined Media without Serum or Feeder Cells." Blood 112, no. 11 (November 16, 2008): 2463. http://dx.doi.org/10.1182/blood.v112.11.2463.2463.
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Abstract As established cell lines, human pluripotent stem cells such as embryonic stem (ES) or induced pluripotent stem (iPS) cells can divide indefinitely while retaining their potential to differentiate into many cell types in culture. The induction of mesoderm formation and hematopoietic differentiation was achieved either via embryoid body (EB) formation by culturing ES cell aggregation in suspension or by co-cultures with mouse stromal cell lines. Traditionally serum factors are also added for mesoderm induction and hematopoietic differentiation. Although hematopoietic progenitors are obtained from multiple lines of human ES cells, the low efficiency and high variability have hindered the progress of using human ES cells as a model for studying human hematopoiesis. Normally <15% of cells obtained from a primary culture expressed CD34 (a marker for endothelial cells and hematopoietic progenitors) and even less for CD45 (a pan-leukocyte marker). To generate maximal output of CD34+CD45+ hematopoietic progenitors, we decided to adopt the serum-free and spin-EB formation method (Ng, Blood, 2005) and systematically improved culture conditions. 3,000 human ES cells were added into each well in 96-well plates and formed an aggregate after centrifugation. BMP4 and bFGF were added at day 1, and VEGF and hematopoietic cytokines was added at day 3–9. VEGF was then withdrawn after day 9. Single EB (occasionally 2) grew in each well. By day 8, small blast (or lymphocyte-) like cells were observed on the edge of EBs. By day 12–14, we observed the outgrowth of blast cells (in hundreds to thousands) surrounding each EB (Panel A). By FACS analysis (Panel B), we observed nearly 50% of the total cells express CD45 at day 12, and many co-express CD34. The lymphocytelike cells can be easily separately from EBs by passing through a 40-micron strainer, and nearly all the isolated cells express CD45 (and 50–75% of them co-express CD34). We obtained 6 million CD45+ cells from 0.9 million human ES cells 14 days after EB formation. We also observed that the conditional HES1-ER transgene expression further increased the frequency of CD34+CD45+ cells as we observed under a different culture condition (Yu. Cell Stem Cells, 2008). The isolated CD45+ cells formed efficiently hematopoietic colonies in the methylcellulose medium with a frequency of ~58+/− 4 colonies per 3,000 cells, with or without the HES1-ER transgene. We are currently testing in vivo activities of isolated CD45+ cell populations (+/− HES1-ER) in the NOD/SCID/γC−/− mice. We are also testing if this improved and defined method would also be applicable to hematopoietic differentiation of human iPS cells we recently derived (Mali, Stem Cells, 2008). The significantly improved method using defined media in the absence of serum factors or feeder cells warrants further investigation whether it is better and more reproducible to elucidate mechanisms that regulate early human hematopoiesis, and to generate a large quantity of CD34+CD45+ human hematopoietic progenitor cells for various applications. Figure Figure
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Verstegen, Monique M. A., Paula B. van Hennik, Wim Terpstra, Cor van den Bos, Jenne J. Wielenga, Nico van Rooijen, Rob E. Ploemacher, Gerard Wagemaker, and Albertus W. Wognum. "Transplantation of Human Umbilical Cord Blood Cells in Macrophage-Depleted SCID Mice: Evidence for Accessory Cell Involvement in Expansion of Immature CD34+CD38−Cells." Blood 91, no. 6 (March 15, 1998): 1966–76. http://dx.doi.org/10.1182/blood.v91.6.1966.
Full textAbstract:
Abstract In vivo expansion and multilineage outgrowth of human immature hematopoietic cell subsets from umbilical cord blood (UCB) were studied by transplantation into hereditary immunodeficient (SCID) mice. The mice were preconditioned with Cl2MDP-liposomes to deplete macrophages and 3.5 Gy total body irradiation (TBI). As measured by immunophenotyping, this procedure resulted in high levels of human CD45+ cells in SCID mouse bone marrow (BM) 5 weeks after transplantation, similar to the levels of human cells observed in NOD/SCID mice preconditioned with TBI. Grafts containing approximately 107 unfractionated cells, approximately 105purified CD34+ cells, or 5 × 103 purified CD34+CD38− cells yielded equivalent numbers of human CD45+ cells in the SCID mouse BM, which contained human CD34+ cells, monocytes, granulocytes, erythroid cells, and B lymphocytes at different stages of maturation. Low numbers of human GpA+ erythroid cells and CD41+ platelets were observed in the peripheral blood of engrafted mice. CD34+CD38+ cells (5 × 104/mouse) failed to engraft, whereas CD34− cells (107/mouse) displayed only low levels of chimerism, mainly due to mature T lymphocytes. Transplantation of graded numbers of UCB cells resulted in a proportional increase of the percentages of CD45+ and CD34+ cells produced in SCID mouse BM. In contrast, the number of immature, CD34+CD38− cells produced in vivo showed a second-order relation to CD34+graft size, and mice engrafted with purified CD34+CD38− grafts produced 10-fold fewer CD34+ cells without detectable CD34+CD38− cells than mice transplanted with equivalent numbers of unfractionated or purified CD34+ cells. These results indicate that SCID repopulating CD34+CD38− cells require CD34+CD38+ accessory cell support for survival and expansion of immature cells, but not for production of mature multilineage progeny in SCID mouse BM. These accessory cells are present in the purified, nonrepopulating CD34+CD38+ subset as was directly proven by the ability of this fraction to restore the maintenance and expansion of immature CD34+CD38− cells in vivo when cotransplanted with purified CD34+CD38−grafts. The possibility to distinguish between maintenance and outgrowth of immature repopulating cells in SCID mice will facilitate further studies on the regulatory functions of accessory cells, growth factors, and other stimuli. Such information will be essential to design efficient stem cell expansion procedures for clinical use.
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Verstegen, Monique M. A., Paula B. van Hennik, Wim Terpstra, Cor van den Bos, Jenne J. Wielenga, Nico van Rooijen, Rob E. Ploemacher, Gerard Wagemaker, and Albertus W. Wognum. "Transplantation of Human Umbilical Cord Blood Cells in Macrophage-Depleted SCID Mice: Evidence for Accessory Cell Involvement in Expansion of Immature CD34+CD38−Cells." Blood 91, no. 6 (March 15, 1998): 1966–76. http://dx.doi.org/10.1182/blood.v91.6.1966.1966_1966_1976.
Full textAbstract:
In vivo expansion and multilineage outgrowth of human immature hematopoietic cell subsets from umbilical cord blood (UCB) were studied by transplantation into hereditary immunodeficient (SCID) mice. The mice were preconditioned with Cl2MDP-liposomes to deplete macrophages and 3.5 Gy total body irradiation (TBI). As measured by immunophenotyping, this procedure resulted in high levels of human CD45+ cells in SCID mouse bone marrow (BM) 5 weeks after transplantation, similar to the levels of human cells observed in NOD/SCID mice preconditioned with TBI. Grafts containing approximately 107 unfractionated cells, approximately 105purified CD34+ cells, or 5 × 103 purified CD34+CD38− cells yielded equivalent numbers of human CD45+ cells in the SCID mouse BM, which contained human CD34+ cells, monocytes, granulocytes, erythroid cells, and B lymphocytes at different stages of maturation. Low numbers of human GpA+ erythroid cells and CD41+ platelets were observed in the peripheral blood of engrafted mice. CD34+CD38+ cells (5 × 104/mouse) failed to engraft, whereas CD34− cells (107/mouse) displayed only low levels of chimerism, mainly due to mature T lymphocytes. Transplantation of graded numbers of UCB cells resulted in a proportional increase of the percentages of CD45+ and CD34+ cells produced in SCID mouse BM. In contrast, the number of immature, CD34+CD38− cells produced in vivo showed a second-order relation to CD34+graft size, and mice engrafted with purified CD34+CD38− grafts produced 10-fold fewer CD34+ cells without detectable CD34+CD38− cells than mice transplanted with equivalent numbers of unfractionated or purified CD34+ cells. These results indicate that SCID repopulating CD34+CD38− cells require CD34+CD38+ accessory cell support for survival and expansion of immature cells, but not for production of mature multilineage progeny in SCID mouse BM. These accessory cells are present in the purified, nonrepopulating CD34+CD38+ subset as was directly proven by the ability of this fraction to restore the maintenance and expansion of immature CD34+CD38− cells in vivo when cotransplanted with purified CD34+CD38−grafts. The possibility to distinguish between maintenance and outgrowth of immature repopulating cells in SCID mice will facilitate further studies on the regulatory functions of accessory cells, growth factors, and other stimuli. Such information will be essential to design efficient stem cell expansion procedures for clinical use.
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Carlo-Stella, Carmelo, Cristiana Lavazza, Arianna Giacomini, Daniela Sia, Loredana Cleris, Massimo Di Nicola, Paolo Longoni, et al. "Human CD34+ Cells Expressing Membrane-Bound Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Exert a Potent Anti-Lymphoma Effects by Targeting Tumor Vasculature." Blood 110, no. 11 (November 16, 2007): 527. http://dx.doi.org/10.1182/blood.v110.11.527.527.
Full textAbstract:
Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expected to play a key role in cancer therapy due to its high cancer cell-specificity and potent antitumor activity. We have previously demonstrated that CD34+ cells transduced with an adenovirus encoding the full-length human TRAIL gene (CD34-TRAIL+) exert a potent anti-lymphoma effect in NOD/SCID mice. To investigate the mechanism of action of CD34-TRAIL+ cells, in vivo experiments were perfomed to analyze the tumor homing capacity of CD34-TRAIL+ cells and the effects of transduced cells on tumor vasculature. Tumor homing of CD34-TRAIL+ cells was investigated in NOD/SCID mice bearing subcutaneous lymphoid tumors. Following a single intravenous injection of CD34-TRAIL+ cells (5 x 106), nodules were excised at different time-points and immunostained with an anti-human CD45 antibody. Sections were digitally recorded and the total number of CD45+ cells per tissue section was then counted using the imaging software ImageJ (http://rsb.info.nih.gov/ij/). Tumor homing of CD34-TRAIL+ cells peaked 24 hours after cell injection when a mean of 200 CD34-TRAIL+ cells was recorded per 1 x 105 tumor cells (0.2% CD45+ cells per 1 x 105 tumor cells). Mice pretreatment with the anti-VCAM-1 antibody or the CXCR4 antagonist AMD3100 significantly reduced tumor homing of CD34-TRAIL+ cells, with 0.09% and 0.05% CD45+ cells being recorded per 1 x 105 tumor cells, respectively. To analyze the effects of CD34-TRAIL+ cells on tumor vasculature, mice were injected with sulfo-biotin (0.1 mg, IV, 15 min) and tumor endotelial cells (TEC) were then revealed by staining frozen sections with horseradish peroxidase (HRP)-conjugated streptavidin. As compared with CD34-mock- or soluble (s)TRAIL-treated mice, treatment with CD34-TRAIL+ cells induced within 24 hours a significant (P ≤.001) increase of the thickness of the vessell wall (3.7 ± 1 μm vs 3.4 ± 1 μm vs 6 ± 1 μm, respectively) as well as the endothelial surface (10 ± 4% vs 7 ± 4% vs 21 ± 6% of total tissue section, respectively), suggesting that CD34-TRAIL+ cells induce an early vascular disruption. Confocal microscopic imaging of tumor sections double-stained with anti-CD31 and anti-TRAIL-R2 showed that this receptor was expressed by 8–12% of large tumor vessels. Interestingly, upon treatment with CD34-TRAIL+ cells, but not sTRAIL, TUNEL staining revealed an extensive apoptosis of CD31+/TRAIL-R2+ TEC. Forty-eight hours following injection of CD34-TRAIL+ cells, a 21-fold increase of apoptotic index was detected, which was associated with extensive necrotic areas (20% to 25% of tissue section). These data show that: tumor homing of CD34-TRAIL+ cells induces extensive vascular damage, hemorrhagic necrosis and tumor destruction; the antitumor effect of CD34-TRAIL+ cells is mediated by both indirect vascular-disrupting mechanisms and direct tumor cell killing.
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van Hensbergen, Yvette, Helen de Boer, Manon C. Slot, Laurus F. Schipper, Anneke Brand, and Willem E. Fibbe. "Mesenchymal Stem Cells Promote the Engraftment of Cord Blood CD34+ Cells but Do Not Accelarate Platelet Recovery NOD/SCID Mice." Blood 106, no. 11 (November 16, 2005): 1267. http://dx.doi.org/10.1182/blood.v106.11.1267.1267.
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Abstract Aim: Delayed platelet reconstitution in the peripheral blood (PB) remains a problem in transplantation with umbilical cord blood (CB)-derived stem cells. Previously, we have shown that transplantation with ex-vivo expanded CB CD34+ cells (CD34exp) with thrombopoietin for 10 days, results in an accelerated platelet reconstitution in NOD/SCID mice. It has been shown that mesenchymal stem cells (MSC) are able to enhance the overall engraftment when co-transplanted with CB CD34+ cells. Therefore, we investigated whether co-transplantation of MSC with CD34+ cells or CD34exp cells may have an additive effect in shortening the time to platelet recovery and on the total number of platelets in the PB at 6 weeks after transplantation. Methods: To evaluate the time to platelet recovery and the total number of platelets at 6 weeks after transplantation, we used 4 groups of irradiated NOD/SCID mice, divided according to the transplant received: 1) CD34+ 2) MSC+CD34+ 3) CD34exp 4) MSC+CD34exp. Human platelet recovery was measured twice a week for the first three weeks and once a week thereafter, using an assay that reliably detects 1x106plt/L. The percentage of human CD45+ cells in the bone marrow (BM) was evaluated at 6 weeks after transplantation. Results: In accordance with previous experiments, platelet recovery started earlier in mice transplanted with CD34exp cells compared to CD34+ cells (Table 1). Co-transplantation of MSC with CD34+ cells did not result in an accelerated platelet recovery during the first 2 weeks after transplantation, as was observed for expanded cells. However, co-transplantation of MSC did enhance the number of platelets at 6 weeks after transplantation (454.2±264.5 plt/μ l for MSC+CD34+ vs. 101.9±78.4 plt/μ l for CD34+). MSC had no affect on either the time to platelet recovery nor the total number of human platelets at 6 weeks after transplantation when co-transplanted with CD34exp cells. To assess the overall efficacy of the MSC on the engraftment of human CB cells, we evaluated the percentage of human CD45+ cells in the BM of the NOD/SCID mice at 6 weeks after transplantation. In mice transplanted with MSC+CD34+, the percentage of human CD45+ cells was higher compared to controls transplanted with CD34+ cells only (30.4% for MSC+CD34+ vs. 17.8% for CD34+). No further engraftment enhancing effect of MSC was observed following transplantation of CD34exp cells only (32.1% for CD34exp vs. 35.7% for MSC+CD34exp). Conclusion: Our results show that transplantation with CD34exp cells results in an accelerated platelet recovery in NOD/SCID mice, an effect that can not be achieved by co-transplantation of MSC+CD34+ cells. However, at 6 weeks after transplantation co-transplantation with MSC+CD34+ cells results in a higher number of platelets in the PB. In addition, the level of engraftment of human CD45+ cells in the BM of NOD/SCID mice is increased by co-transplantation of MSC+CD34+ cells. In contrast, MSC did not affect the time to platelet recovery, the number of human platelets at 6 weeks after transplantation, or the engraftment of human CD45+ cells in the BM when co-transplanted with CD34exp. Table 1: % of mice with ≥ 1x106 platelets/L in the PB Days post transplantation 6 9 13 16 CD34+ 0% 20% 67% 100% MSC+CD34+ 20% 0% 80% 100% CD34exp 83% 100% 100% 100% MSC+CD34exp 60% 100% 100% 100%
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Michurova, Marina Sergeevna, Victor Yurievich Kalashnikov, Olga Michailovna Smirnova, Sergey Anatol'evich Terekhin, Olga Nikolaevna Ivanova, Svetlana Michailovna Stepanova, Aleksandr Victorovich Ilin, and Ivan Ivanovich Dedov. "Endothelial progenitor cells and vascular endothelial growth factor after endovascular interventions in patients with type 2 diabetes." Diabetes mellitus 20, no. 1 (May 11, 2017): 59–67. http://dx.doi.org/10.14341/dm8173.
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Aim. To study the quantity of endothelial progenitor cells (EPCs) and levels of vascular endothelial growth factor A (VEGF-A) in patients with type 2 diabetes mellitus (T2DM) after endovascular interventions on coronary and peripheral arteries.
Materials and methods. We observed 68 patients with stable angina pectoris and critical limb ischaemia, admitted for elective percutaneous coronary intervention and endovascular revascularisation of the lower extremity. The number of CD34+VEGFR2+CD45- and CD34+CD133+CD45- cells and levels of VEGF-A were determined before endovascular intervention and 24 days after the surgery.
Results. We found that in patients without diabetes, the levels of EPCs increased significantly after endovascular interventions (CD34+VEGFR2+CD45-cells, p 0.0001; CD34+ CD133+CD45-cells p = 0.041). The levels of EPCs in the peripheral blood of patients with T2DM before and after endovascular interventions did not significantly differ. The analysis of VEGF-A showed a statistically significant increase after intervention in both groups. In addition, in patients with an HbA1c level of 8% and duration of diabetes of 10 years, the levels of EPCs significantly increased (p = 0.001 and 0.005, respectively). In patients with an HbA1c level of 8% and duration of diabetes of 10 years, the levels of EPCs before and after endovascular interventions did not significantly differ.
Conclusions. Patients with diabetes exhibited impaired EPC mobilisation after endovascular interventions. Poor glycaemic control and a long duration of diabetes are among the risk factors of EPC mobilisation.
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Gori, Jennifer L., Jason M. Butler, Devikha Chandrasekaran, Brian C. Beard, Daniel J. Nolan, Michael Ginsberg, Jennifer E. Adair, Shahin Rafii, and Hans-Peter Kiem. "In Vivo Selection and Long-Term Engraftment Of Hematopoietic Stem Cells Generated Via Vascular Niche Induction Of Nonhuman Primate Induced Pluripotent Stem Cells." Blood 122, no. 21 (November 15, 2013): 466. http://dx.doi.org/10.1182/blood.v122.21.466.466.
Full textAbstract:
Clinical use of human pluripotent stem cell (PSC)-hematopoietic stem cells (HSCs) is impeded by low engraftment potential. This block suggests that additional vascular derived angiocrine signals and hematopoietic cues must be provided to produce authentic HSCs. In addition, gene modification of induced (i)PSCs with a chemotherapy resistance transgene would provide a selective mechanism to stabilize or increase engraftment of HSCs. We therefore hypothesized that modifying iPSCs to express the O6-benzylguanine (O6BG)-resistant P140K variant of methylguanine methyltransferase (MGMT), would support in vivo selection of early-engrafted iPSC-HSCs. We further postulated that Akt-activated human endothelial cells afforded by transduction of the E4ORF1 gene (E4ORF1+ECs) through angiocrine upregulation of Notch and IGF ligands would provide the necessary signals under xenobiotic-free conditions to promote definitive hematopoiesis. This vascular induction platform could drive the emergence of true HSCs. We focused on pigtail macaque (Mn)iPSCs, as a scalable, clinically relevant nonhuman primate model. MniPSCs modified to express P140K had 15-fold higher MGMT levels compared to levels in human peripheral blood mononuclear cells. P140K-MniPSCs differentiated into chemoresistant CD34+ hematopoietic progenitors (50% CD34+) with a predominant long-term (LT)-HSC-like phenotype (CD34+CD38-Thy1+CD45RA-CD49f+). Hematopoietic progenitors maintained colony forming potential after O6BG and bis-chloroethylnitrosourea (BCNU) treatment. HSCs expanded on E4ORF1+ECs maintained colony forming potential, in contrast to cells cultured with cytokines alone, with a 22-fold increase in CD34+ cell content and 10-fold increase in LT-HSC-like cells. Importantly, MniPSC-HSCs expanded with the E4ORF1+ECs had long-term engraftment in NSG mice at levels comparable to Mn bone marrow HSC engrafted mice. O6BG/BCNU treatment increased engraftment to 35% CD45+ cells the blood of mice transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs, which was maintained 16 weeks post transplantation. Primate CD45+ cell levels in the blood after selection were significantly higher for this cohort compared to mice transplanted with P140K-MniPSC-HSCs expanded in the “cytokines alone” condition (18% vs. 3% CD45+, P<0.05). On average, 15% CD34+ and 37% CD45+ cells were detected in the bone marrow of mice transplanted with E4ORF1+EC-expanded P140K-MniPSC HSCs, which is significantly higher than levels detected in the other cohorts (Table 1). CD45+ cells in the marrow were predominantly myeloid but lymphoid subsets were also present (10-25% CD3+ cells). Remarkably, the level of gene marking in CFCs and number of gene marked CFCs from mouse bone marrow was substantially higher for mice transplanted with E4ORF1+EC expanded compared to cytokine expanded P140K-MniPSC-HSCs (Table 1). Finally, to confirm engraftment of authentic HSCs, secondary transplants were established. Although engraftment was achieved in all secondary transplanted cohorts, the level of nonhuman primate cells detected was significantly higher in animals transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs. Significantly more lymphocytes (CD45+CD3+ and CD45+CD56+) and monocytes (CD45+CD14+) were detected in the blood of these secondary transplant recipients. These findings confirm generation of bona fide HSCs derived from nonhuman primate iPSCs and demonstrate that O6BG/BCNU chemotherapy supports in vivo selection of P140K-MniPSC-HSCs generated by co-culture with the E4ORF1+EC vascular platform. Our studies mark a significant advance toward clinical translation of PSC-based blood therapeutics and the development of a nonhuman primate preclinical model. Table 1 CD34+ and CD45+ engraftment and gene marking in the bone marrow of mice transplanted with nonhuman primate HPSCs from MniPSCs and bone marrow. HSCs E4ORF1+ECs O6BG/BCNU Mean %CD34+ Mean %CD45+ % gene marking in CFCs (lentivirus+) total lentivirus+ CFCs per 105 cells GFP-MniPSC + - 3 16 9 ± 2 13 ± 2 P140K-MniPSC + - 4 19 12 ± 5 17 ± 7 P140K-MniPSC - + 0.4 24 3 ± 2 2 ± 1 P140K-MniPSC + + 15 37 27 ± 24 111 ± 96 Mn BM CD34+ - - 2 21 0 0 Disclosures: Nolan: Angiocrine Bioscience: Employment. Ginsberg:Angiocrine Bioscience: Employment. Rafii:Angiocrine Bioscience: Founder Other.
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Shah, V. O., C. I. Civin, and M. R. Loken. "Flow cytometric analysis of human bone marrow. IV. Differential quantitative expression of T-200 common leukocyte antigen during normal hemopoiesis." Journal of Immunology 140, no. 6 (March 15, 1988): 1861–67. http://dx.doi.org/10.4049/jimmunol.140.6.1861.
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Abstract We have correlated the intensity of expression of CD45 Ag (T200 common leukocyte Ag) with mAb reactive with various lineages of hemopoietic cells in normal human bone marrow by using two-color immunofluorescence on a flow cytometer. Mature T lymphocytes (CD3+) and NK cells (CD16+ or CD11b+) expressed CD45 at the highest intensity. B lymphoid cells (CD19+) had three distinct levels of CD45 Ag expression. The bright CD45(3+) cells were mature B cells (CD19+, CD20+), whereas the less intense CD45(2+) cells were less mature B lymphoid cells (CD19+, CD10+). The dim CD45+ cells were very early, B lymphoid precursor cells (CD19+, CD10(2+), CD34+). The intensity of CD45 expression increased as cells matured in the monocytic lineage (CD14+, CD11b+). Among marrow granulocytic cells, CD45 intensity did not change on cells during maturation. Within the erythroid lineage, the most immature cells were CD45+ dim, and CD45 expression decreased during erythroid maturation to become undetectable on mature E. Hemopoietic progenitor cells (CD34+) expressed low levels of CD45 Ag. Expression of CD45R on marrow cells also showed intensity differences on different lineages. All NK cells (CD16+) were positive for CD45R, whereas only about one-half of the T lymphocytes (CD3+) were positive for CD45R. Almost all the cells in the erythroid and myelomonocytic lineages were CD45R-. Quantitative differences in expression of CD45R were observed on marrow B lymphoid cells which were correlated with the expression of CD45. The results show that quantitative changes in CD45 Ag expression accompany the differentiation and maturation of cells in the bone marrow. Comparisons with CD45R showed that this Ag was not always correlated with CD45. Since these Ag are the products of the same gene, these data indicate that the regulation of expression of the T200 molecules during normal hemopoietic development must be both quantitative and qualitative.
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Barquinero, Jordi, José Carlos Segovia, Manuel Ramı́rez, Ana Limón, Guillermo Güenechea, Teresa Puig, Javier Briones, Juan Garcı́a, and Juan Antonio Bueren. "Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice." Blood 95, no. 10 (May 15, 2000): 3085–93. http://dx.doi.org/10.1182/blood.v95.10.3085.
Full textAbstract:
Abstract In an attempt to develop efficient procedures of human hematopoietic gene therapy, retrovirally transduced CD34+ cord blood cells were transplanted into NOD/SCID mice to evaluate the repopulating potential of transduced grafts. Samples were prestimulated on Retronectin-coated dishes and infected with gibbon ape leukemia virus (GALV)-pseudotyped FMEV vectors encoding the enhanced green fluorescent protein (EGFP). Periodic analyses of bone marrow (BM) from transplanted recipients revealed a sustained engraftment of human hematopoietic cells expressing the EGFP transgene. On average, 33.6% of human CD45+ cells expressed the transgene 90 to120 days after transplantation. Moreover, 11.9% of total NOD/SCID BM consisted of human CD45+ cells expressing the EGFP transgene at this time. The transplantation of purified EGFP+ cells increased the proportion of CD45+ cells positive for EGFP expression to 57.7% at 90 to 120 days after transplantation. At this time, 18.9% and 4.3% of NOD/SCID BM consisted of CD45+/EGFP+ and CD34+/EGFP+ cells, respectively. Interestingly, the transplantation of EGFP− cells purified at 24 hours after infection also generated a significant engraftment of CD45+/EGFP+ and CD34+/EGFP+ cells, suggesting that a number of transduced repopulating cells did not express the transgene at that time. Molecular analysis of NOD/SCID BM confirmed the high levels of engraftment of human transduced cells deduced from FACS analysis. Finally, the analysis of the provirus insertion sites by conventional Southern blotting indicated that the human hematopoiesis in the NOD/SCID BM was predominantly oligoclonal.
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Barquinero, Jordi, José Carlos Segovia, Manuel Ramı́rez, Ana Limón, Guillermo Güenechea, Teresa Puig, Javier Briones, Juan Garcı́a, and Juan Antonio Bueren. "Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice." Blood 95, no. 10 (May 15, 2000): 3085–93. http://dx.doi.org/10.1182/blood.v95.10.3085.010k01_3085_3093.
Full textAbstract:
In an attempt to develop efficient procedures of human hematopoietic gene therapy, retrovirally transduced CD34+ cord blood cells were transplanted into NOD/SCID mice to evaluate the repopulating potential of transduced grafts. Samples were prestimulated on Retronectin-coated dishes and infected with gibbon ape leukemia virus (GALV)-pseudotyped FMEV vectors encoding the enhanced green fluorescent protein (EGFP). Periodic analyses of bone marrow (BM) from transplanted recipients revealed a sustained engraftment of human hematopoietic cells expressing the EGFP transgene. On average, 33.6% of human CD45+ cells expressed the transgene 90 to120 days after transplantation. Moreover, 11.9% of total NOD/SCID BM consisted of human CD45+ cells expressing the EGFP transgene at this time. The transplantation of purified EGFP+ cells increased the proportion of CD45+ cells positive for EGFP expression to 57.7% at 90 to 120 days after transplantation. At this time, 18.9% and 4.3% of NOD/SCID BM consisted of CD45+/EGFP+ and CD34+/EGFP+ cells, respectively. Interestingly, the transplantation of EGFP− cells purified at 24 hours after infection also generated a significant engraftment of CD45+/EGFP+ and CD34+/EGFP+ cells, suggesting that a number of transduced repopulating cells did not express the transgene at that time. Molecular analysis of NOD/SCID BM confirmed the high levels of engraftment of human transduced cells deduced from FACS analysis. Finally, the analysis of the provirus insertion sites by conventional Southern blotting indicated that the human hematopoiesis in the NOD/SCID BM was predominantly oligoclonal.
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Ojeda-Uribe, Mario, Christophe Desterke, Laura Jung, Pierre Bories, Hanna Sovalat, Laurent Mauvieux, Jean Claude Eisenmann, Bruno Lioure, and Marie-Caroline Le Bousse-Kerdiles. "Blood Traffic of Endothelial, Mesenchymal, Hematopoietic and Lin-/CD45-CD34+AC133+CXCR4+ Progenitor Cell Subsets Show Different Patterns in Pre-Fibrotic and Fibrotic Primary Myelofibrosis." Blood 124, no. 21 (December 6, 2014): 3230. http://dx.doi.org/10.1182/blood.v124.21.3230.3230.
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Abstract Introduction. Primary myelofibrosis (PMF) can be associated to increased bloodstream traffic of some adult progenitor cells. The extramedullary hematopoiesis developed in this setting might remind some features of the fetal hematopoiesis. Material and methods. Some evidence of this hypothesis was searched by flow-cytometry and molecular analysis of blood samples from 12 untreated patients with PMF, 6 pre-fibrotic (PF) and 6 fibrotic (F). The PF or F phase was defined according to the WHO and Thiele histopathology scores. Results. As expected we detected high numbers of CD34+ cells (258x103±402x103/ml; range 1.2-1218x103/ml) as well as small mean numbers of Lin-/CD45-CD34+AC133+CXCR4+ progenitor cells (130±275/ml; range 0-882/ml) that are related to VSELs (very-small embryonic-like stem cells), which have been described as putative pluripotent adult stem cells. When we looked at some embryonic molecular markers by RT-PCR, NANOG and OCT4 expression was detected in the MNC fraction of all the patients tested (hES and CPRE2 cell lines were the positive and negative controls). OCT4 expression was half the level of hES being stronger in F-PMF than in PF-PMF. In all the tested patients NANOG expression was similar to that of hES, whereas SOX2 and LIN28 were not expressed in most patients. Regarding other circulating progenitor cells we observed that PF-PMF had higher mean values of progenitor endothelial cells (PEC) (130±271/ml; range 0-683) than F-PMF (7±16/ml; range 0-37). Mesenchymal progenitor cells (MPC) had also higher mean values in PF-PMF (289±452/ml; range 0-1165) than in F-PMF (111±199/ml; range 0-458). Conversely, Lin-/CD45-CD34+AC133+CXCR4+ cell subset detection was higher in F-PMF (185±381/ml; range 0-882) than in PF-PMF (38±60/ml; range 0-140). Patients V617F-JAK2pos presented lower levels of PEC and MPC (16±19/ml and 43±61/ml respectively) than those V617F-JAK2neg (173±340/ml and 515±470/ml respectively). Similar numbers of Lin-/CD45-CD34+AC133+CXCR4+ cells were observed in both V617F-JAK2pos and V617F-JAK2neg patients (133±331/ml and 123±115/ml respectively). Patients with elevated IPSS (>3) showed higher levels of PEC, MPC and Lin-/CD45-CD34+AC133+CXCR4+ cells (respectively 180±335/ml, 334±560/ml and 256±422/ml) than those with low IPSS (<2) (respectively 12±16/ml, 134±183/ml and 46±92/ml). Based on these findings we were able to define some putative patterns of bloodstream progenitor cell traffic. Thus, PEC appeared to circulate more in PF-PMF, V617F-JAK2neg with a high IPSS. The same pattern was observed regarding MPC. Conversely, Lin-/CD45-CD34+AC133+CXCR4+ cells appeared to circulate more in F-PMF, V617F-JAK2pos with a high IPSS (Table 1). No mutations were detected in the hot-spot of SRSF2 (codons 93-95) which have been associated to a fibrotic progression of PMF. We also performed a bio-informatics analysis of microarray databases comparing the molecular libraries of the gene expression ommibus dataseries GSE3410 and GSE31869 (VSELs (Lin-CD45-CXCR4+CD34-) and CD34+ VSELs (Lin-CD45-CXCR4+CD34+) with the molecular profiling of circulating CD34+ cells from PMF patients and bone marrow CD34+ cells from healthy donors and we observed a tight clustering between PMF and VSELs cells samples (Figure 1). Conclusions. Variations in the blood traffic of some progenitor cell subsets in patients with PMF were observed, including Lin-/CD45-CD34+AC133+CXCR4+ cells. The fact this cell subset was detected in higher number in F-PMF and in patients with a high IPSS, as well as a higher expression of OCT4 in F-PMF supports in part our hypothesis that PMF evolution can be associated to the recruitment and circulation of some primitive progenitors (dormant in the adult life) as it can be observed during the fetal period. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Leonard, Alexis, Aylin Bonifacino, Venina Marcela Dominical, Anna Conrey, Wynona Coles, Mary Link, Matthew Hsieh, et al. "Bone Marrow Characterization in Sickle Cell Disease: Inflammation and Stress Erythropoiesis Lead to Suboptimal CD34 Recovery Compared to Normal Volunteer Bone Marrow." Blood 130, Suppl_1 (December 7, 2017): 966. http://dx.doi.org/10.1182/blood.v130.suppl_1.966.966.
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Abstract Introduction Gene therapy for sickle cell disease (SCD) requires modification of a high number of long term engrafting hematopoietic stem cells (LT-HSCs) sufficient to sustain production of the gene of interest at levels capable of overcoming the pathogenic HbSS phenotype. Unlike β-Thalassemia, the inflammatory bone marrow (BM) environment and stress erythropoiesis associated with SCD may have significant impacts on HSC quality and yield necessary for disease amelioration. Important work to optimize gene therapy through improvement in gene transfer efficiency, editing strategies, or transplant conditioning can only improve gene therapy in SCD if enough autologous HSCs are LT-HSCs, thus characterization of SCD BM and CD34+ HSCs is required. Collection type, storage, and delays in processing may further impact CD34+ recovery and should be investigated as a strategy to maximize LT-HSC recovery. Methods Twenty milliliters of BM from subjects with SCD (HbSS genotype) and normal volunteers was collected in different anticoagulants (Heparin, ACD-A) and processed immediately(day 0) or stored at 40C and processed the following day(day 1). After isolation via Ficoll density gradient centrifugation, the mononuclear (MN) layer was stained with antibodies against inflammatory markers (CD36, CD35, CD11b, CD62L, CD62P), non-MN cells (GPA, CD66b, CD41/61), or processed for CD34+ selection using a magnetic microbead CD34+ selection kit and stained for CD34, CD45, and GPA expression. Data were analyzed by conventional and imaging flow cytometry, the latter confirming post-CD34+ selection flow data and demonstrating antibody intensity as a characterization of HSC heterogeneity and progenitor lineage. Complete blood count and hemoglobin (Hb) electrophoresis were obtained at the time of BM collection. Statistical analyses were performed using unpaired t-tests. Results BM was collected from 18 subjects (16 with SCD; 11M; age 21-41 years). Median Hb (8.6 vs. 13.5 gm/dL, p<0.01) and white blood cell count (8.8 vs. 4.2 K/mcL, p<0.05) differed significantly between SCD and non-SCD subjects. Median percent sickle Hb in SCD subjects was 62%. Inflammatory markers and contamination with red cell and platelet markers in the post-Ficoll MN layer were higher in SCD vs. non-SCD BM regardless of anticoagulant (CD35 24% vs. 13%, p<0.05; CD36 22% vs. 11%, p<0.05; CD62P 16% vs. 3%, p<0.05; GPA 16% vs. 4%, p<0.05; CD41/61 19% vs. 3%, p<0.05), and trended higher on day 1 in SCD BM in both anticoagulants, significantly in Heparin (GPA 23% vs. 33% on day 1, p<0.05). Total CD45 expression was lower in SCD vs. non-SCD BM in both anticoagulants (p<0.05) and on day 0 (p<0.05) and 1 (p<0.01), with Amnis data confirming a higher CD34+CD45- population in SCD BM (4 ± 2% vs. 0.5 ± 0.3%, p<0.05). While there was no significant difference in total CD34+ cell count between SCD and non-SCD BM after selection post-Ficoll, there was a trend for lower CD34+ count in SCD in both anticoagulants (2.6x10^5 vs. 4.7x10^5, p=0.1). SCD CD34+ cells were characterized by higher GPA expression (28 ± 5% vs. 13 ± 3% in non-SCD BM, p<0.01) that worsened in Heparin on day 1 (22 ± 6.3% vs. 35 ± 12.4%, p<0.05). Image cytometry confirmed a majority of GPA expression in SCD BM is from single cell CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs in addition to red cell aggregates, with an increase in CD34+CD45-GPA+ HSCs on day 1 (10 ± 5% vs. 0.6 ± 0.2 % on day 0, p<0.05). Furthermore, the percentage of CD34hi HSCs was lower in SCD vs. non-SCD BM, with >50% SCD HSCs characterized as CD34dim (56% vs. 4% in non-SCD BM, p<0.001). Lastly, the purity of CD34+ selection worsened from day 0 to day 1 in SCD BM in heparin (94% vs. 68 ± 8%, p<0.05) and ACD-A (88% vs. 68 ± 0.7%, p<0.05). Conclusions SCD BM is characterized by increased inflammation and cell contamination in the MN layer regardless of anticoagulant that worsens over time in Heparin more significantly than in ACD-A. Compared to non-SCD BM, CD34+ HSC yield post-Ficoll is lower in SCD subjects, and is characterized by a larger proportion of CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs that rise with delays in processing. This indication of early differentiation along the erythroid lineage, with more than 50% of HSCs losing CD34+ intensity suggesting they are not LT-HSCs, suggests suppression of inflammation and stress erythropoiesis, combined with early cell processing may be critical for maximal HSC recovery necessary for successful gene therapy in SCD. Disclosures Luo: bluebird bio Inc.: Employment. Pierciey: bluebird bio: Employment.
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Bender, JG, KL Unverzagt, DE Walker, W. Lee, DE Van Epps, DH Smith, CC Stewart, and LB To. "Identification and comparison of CD34-positive cells and their subpopulations from normal peripheral blood and bone marrow using multicolor flow cytometry." Blood 77, no. 12 (June 15, 1991): 2591–96. http://dx.doi.org/10.1182/blood.v77.12.2591.2591.
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Abstract Four-color flow cytometry was used with a cocktail of antibodies to identify and isolate CD34+ hematopoietic progenitors from normal human peripheral blood (PB) and bone marrow (BM). Mature cells that did not contain colony forming cells were resolved from immature cells using antibodies for T lymphocytes (CD3), B lymphocytes (CD20), monocytes (CD14), and granulocytes (CD11b). Immature cells were subdivided based on the expression of antigens found on hematopoietic progenitors (CD34, HLA-DR, CD33, CD19, CD45, CD71, CD10, and CD7). CD34+ cells were present in the circulation in about one-tenth the concentration of BM (0.2% v 1.8%) and had a different spectrum of antigen expression. A higher proportion of PB-CD34+ cells expressed the CD33 myeloid antigen (84% v 43%) and expressed higher levels of the pan leukocyte antigen CD45 than BM-CD34+ cells. Only a small fraction of PB-CD34+ cells expressed CD71 (transferrin receptors) (17%) while 94% of BM-CD34+ expressed CD71+. The proportion of PB-CD34+ cells expressing the B-cell antigens CD19 (10%) and CD10 (3%) was not significantly different from BM-CD34+ cells (14% and 17%, respectively). Few CD34+ cells in BM (2.7%) or PB (7%) expressed the T-cell antigen CD7. CD34+ cells were found to be predominantly HLA-DR+, with a wide range of intensity. These studies show that CD34+ cells and their subsets can be identified in normal PB and that the relative frequency of these cells and their subpopulations differs in PB versus BM.
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Bender, JG, KL Unverzagt, DE Walker, W. Lee, DE Van Epps, DH Smith, CC Stewart, and LB To. "Identification and comparison of CD34-positive cells and their subpopulations from normal peripheral blood and bone marrow using multicolor flow cytometry." Blood 77, no. 12 (June 15, 1991): 2591–96. http://dx.doi.org/10.1182/blood.v77.12.2591.bloodjournal77122591.
Full textAbstract:
Four-color flow cytometry was used with a cocktail of antibodies to identify and isolate CD34+ hematopoietic progenitors from normal human peripheral blood (PB) and bone marrow (BM). Mature cells that did not contain colony forming cells were resolved from immature cells using antibodies for T lymphocytes (CD3), B lymphocytes (CD20), monocytes (CD14), and granulocytes (CD11b). Immature cells were subdivided based on the expression of antigens found on hematopoietic progenitors (CD34, HLA-DR, CD33, CD19, CD45, CD71, CD10, and CD7). CD34+ cells were present in the circulation in about one-tenth the concentration of BM (0.2% v 1.8%) and had a different spectrum of antigen expression. A higher proportion of PB-CD34+ cells expressed the CD33 myeloid antigen (84% v 43%) and expressed higher levels of the pan leukocyte antigen CD45 than BM-CD34+ cells. Only a small fraction of PB-CD34+ cells expressed CD71 (transferrin receptors) (17%) while 94% of BM-CD34+ expressed CD71+. The proportion of PB-CD34+ cells expressing the B-cell antigens CD19 (10%) and CD10 (3%) was not significantly different from BM-CD34+ cells (14% and 17%, respectively). Few CD34+ cells in BM (2.7%) or PB (7%) expressed the T-cell antigen CD7. CD34+ cells were found to be predominantly HLA-DR+, with a wide range of intensity. These studies show that CD34+ cells and their subsets can be identified in normal PB and that the relative frequency of these cells and their subpopulations differs in PB versus BM.
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Colletti, Evan, Deena ElShabrawy, Esmail D. Zanjani, Christopher D. Porada, and Graca Almeida-Porada. "Characterization of the Human Hematopoietic Niche During Ontogeny." Blood 114, no. 22 (November 20, 2009): 3626. http://dx.doi.org/10.1182/blood.v114.22.3626.3626.
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Abstract Abstract 3626 Poster Board III-562 Studies thus far have shown that there are at least two anatomically and physiologically distinct hematopoietic niches within the bone marrow (BM); the osteoblastic and the vascular. The former is thought to provide the appropriate support for quiescent hematopoietic stem cells (HSC), while the latter allows/induces expansion of the HSC pool. However, it is not entirely known which subpopulations of HSC interact with each niche, and what physiological role each niche type plays in the regulation of stem cell hierarchy and function. We hypothesized that the developmental process whereby the fetal marrow acquires the ability to support hematopoiesis can be used as a model for understanding both the interactions that occur between HSC and the cells comprising the marrow niches, and the role of these niche elements in the initiation/maintenance of hematopoiesis. To this end, human fetal bones between 10- 22 weeks of gestation (gw) were analyzed by flow cytometry and confocal microscopy to identify and characterize stromal/vascular/osteoblastic and hematopoietic elements. Since the development of the vascular bed has been reported to occur in humans between 9-10.5gw, we started the analysis of the human fetal bones at this time point (n=3). CD44 was the only marker widely expressed at this time point, suggesting that these bone rudiments were in an earlier (cartilaginous) stage of development. At 12gw (n=5), 47±4% of the cells isolated from the long bones were CD34+, but less than 1% of these cells were positive for CD45. Further characterization of the CD45−CD34+ population showed that 74±5.4% of these cells were CD106+; 65±7.2% were CD102+; 10±0.5% were CD31+; and 6.7±2.1 % KDR+. These data suggest that prior to the onset of hematopoiesis, a significant percentage of the cells in the BM are part of the vascular niche, and have an endothelial cell phenotype. Furthermore, the existence of a population of cells with a CD34+CD31+KDR+CD45− phenotype may indicate that, during this stage of development, a hemangioblast-like cell exists in the emergent BM. In addition, the existence of a large mesenchymal/stromal cell population was also found, with 21±2.9% of the cells expressing Stro-1+. CD44 was still widely expressed (55±7.8%) at this time point. Of particular note is the discovery of a cell population in which CD44+ cells co-expressed CD34 in the absence of CD45 (8±1.8%), and another in which CD34+ cells were co-expressing Stro-1 (9±1 %), suggesting the possibility that a common ancestor may exist for these cells. At 16gw (n=6) the overall percentage of CD34+ cells decreased to 15±3.1%, and the majority (72±7%) of the cells harvested from the long bones expressed CD45, showing the change to a predominantly hematopoietic marrow. Furthermore, 12±4.2% of the cells had a CD45+CD34+ phenotype consistent with that of HSC. At this time point, only 2.38±1% of the cells were CD34+CD106+; 3.5±1.5% were CD34+CD102+; and 1.34±0.8% were CD34+CD31+. In addition, the CD44+CD45- population decreased to only 20±1.2%. At 22gw (n=6), a similar flow cytometric profile was found except that the CD45+CD34+ population had decreased to 6±1.3%. Since phenotypic analysis showed a decrease in the percentage of BM stromal cell precursors from 12 to 22gw, we performed CFU-F assays and found that the frequency of CFU-F in 12gw BM was 0.34-0.45%, the highest found at any time point. At 16gw, the frequency of CFU-F was 0.23-0.25%, and at 22gw, it had decreased to 0.1-0.16%. Nevertheless, even this low level at 22gw was still higher than that of adult BM (0.001-0.02%). Thus, the results from CFU-F assays correlate well with the flow cytometry/confocal microscopy data showing that there is a decrease in mesenchymal/stromal cell precursor frequency/potential as the BM niches mature. To investigate the ability of the stromal layers of different gestational ages to interact with/retain HSC, adhesion assays (n=3) were performed with cord blood (CB) and adult BM-derived CD34+ cells. These studies showed that CB CD34+ cells adhered at similar levels to all of the fetal stromal layers, while adult BM-derived CD34+ cells only adhered efficiently to the 22gw stromal layers. Further studies addressing the cellular interactions that occur within each of the niches at the phenotypic, functional, and transcriptional levels are currently underway. Disclosures: No relevant conflicts of interest to declare.
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Metheny, Leland, Marcos de Lima, Stanton Gerson, Saada Eid, and Alex Yee-Chen Huang. "Intra-Osseous Co-Transplantation of CD34-Selected Umbilical Cord Blood and Mesenchymal Stromal Cells." Blood 124, no. 21 (December 6, 2014): 3811. http://dx.doi.org/10.1182/blood.v124.21.3811.3811.
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Abstract Introduction. Intra-Osseous (IO) transplantation has been proposed as a strategy to enhance human UCB (umbilical cord blood; hUCB) engraftment in NOD SCID-gamma (Nude Obesity Diabetic Severe Combined Immunodeficient, NSG) mice. Human Mesenchymal Stromal Cells (hMSC) have been shown to support the growth and differentiation of hematopoietic stem cells. We hypothesize that IO co-transplantation of hMSC and CD34+ selected hUCB may be effective in improving early engraftment in a NOD/SCID-gamma (NSG) mouse model. This concept would provide the basis for postulating that IO co-transplantation of hUCB and hMSC may improve engraftment in patients undergoing umbilical cord transplant. Methods. Umbilical cord grafts were obtained from our cord blood procurement program at University Hospitals Case Medical Center under an IRB approved protocol. CD34+ cells were labeled using the Miltenyi CD34+ Microbead Kit (Miltenyi Biotec, Bergiiisch Gladbach, Germany) and magnetically separated using the Miltenyi MACS system. CD34+ cells recovered from the column were re-suspended to the appropriate concentration per cohort and infused fresh. Human MSC (CD105+ CD73+ CD45- CD14-) were obtained from bone marrow donors using Percoll gradient isolation and culture-expanded to a homogeneous population under approved protocols in the Cellular Therapy Laboratory. MSCs were preserved in DMSO and when needed were thawed and re-suspended at the appropriate concentration per cohort. We used a NSG mouse (Jackson Laboratories) model of transplant to compare the rates of engraftment amongst 6 mouse cohorts by examining bone marrow and peripheral blood cellular composition at 6 weeks following transplant. Following non-lethal irradiation (300rads), 6 groups of NSG mice (5 mice per cohort) were studied: 1) intravenous (IV) 5x105 hUCB CD34+ cells, 2) IV 5x105 hUBC CD34+ cells and 1x106 hMSC, 3) IO 5x105 hUCB CD34+ cells, 4) IO 5x105 hUCB CD34+ cells and IO 1x106 hMSC, 5) IO 5x105 hUCB CD34+ cells and IV 1x106 hMSC, and 6) IV 5x105 hUCB CD34+ and IO 1x106 hMSC. hMSC dose was arbitrarily set at 2:1 with hUCB CD34+ cell dose. IV injections: cells were administered via tail vein injection suspended in a total volume of 200ul. IO injections: cells were administered via bilateral tibia IO injections suspended in a total volume of 40ul. At 6 weeks, the mice were sacrificed and their peripheral blood and bone marrow were analyzed for human hematopoietic cells using antibodies against human CD45, CD3, CD13, CD14 and CD19 (Becton Dickinson Biosciences). Human engraftment was expressed as percentage of CD45+ cells compared to total bone marrow cells. Results. All cohorts showed evidence of human hematopoietic engraftment. Analysis of human CD45, CD3 and CD19 revealed the highest level of engraftment in the IO hUCB /IO hMSC cohort, which was significantly improved over the IV hUCB cohort (Table 1, Percent human CD45, CD3 and CD19 in the right tibial bone marrow at 6 weeks, p values as compared to IV hUCB cohort). The right tibial bone marrow analysis of human CD13 and CD14 revealed no significant difference between cohorts. We observed that IO co-transplantation of hMSC and hUCB led to superior engraftment of CD45, CD3 and CD19 lineage cells in the bone marrow at 6 weeks as compared with the IV hUCB cohort controls (Figure 1, human CD45, CD3 and CD19 analysis of right tibial bone marrow at 6 weeks; * = p <0.05, ** = p<0.001). The frequency of human hematopoietic cells within the peripheral blood was not statistically different among various cohorts at 6 weeks. Conclusions. This study suggests that intra-osseous co-transplantation of hMSC and hUCB may improve early engraftment in NSG mice. The hypothesis that this strategy may improve engraftment of single unit CB transplantation in humans in intriguing and will be investigated in a phase I clinical trial. Disclosures No relevant conflicts of interest to declare.
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Kucharska-Mazur, Jolanta, Marcin Jabłoński, Maciej Tarnowski, Barbara Dołęgowska, Z. Ratajczak Mariusz, and Jerzy Samochowiec. "Searching for new markers of panic disorder – the examination of stem cells mobilization and levels of factors involved in their trafficking." European Psychiatry 33, S1 (March 2016): S73. http://dx.doi.org/10.1016/j.eurpsy.2016.01.009.
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IntroductionRegeneration processes are the new target in looking for biological markers of psychiatric disorders.AimsIn this study, we considered the role of stem cells and factors responsible for their trafficking in panic disorder (PD).MethodsA group of 30 patients with panic disorder was examined and compared with a group of 30 healthy volunteers. In peripheral blood we have analysed: the number of hematopoetic stem cells – HSC (Lin−/CD45+/CD34+) and HSC (Lin−/CD45+/AC133+), the number of very small embryonic – like stem cells – VSEL (Lin−/CD45−/CD34+) and VSEL (Lin−/CD45−/CD133+) and concentration of stromal derived factor-1 (SDF-1), sphingosine-1-phosphate (S1P), and some proteins of the complement cascade.ResultsPeripheral blood concentration of HSCs (Lin−/CD45+/AC133+) was significantly lower in PD group compared to control group, before and after antidepressant treatment. Peripheral blood concentration of VSEL (Lin−/CD45−/CD133+) was significantly lower in PD group before treatment compared to concentration after treatment. In PD group concentrations of factors involved in stem cell trafficking were statistically significant lower in PD group (before and after treatment) compared to control group.ConclusionExamination of regeneration system seems to be useful in PD diagnostics.Disclosure of interestThe authors have not supplied their declaration of competing interest.