Majority Of CD34 Research Articles

Using digital RNA counting to establish flow cytometry diagnostic criteria for subtypes of CD34+ canine acute leukaemia.

Canine acute leukaemia is a heterogeneous neoplasm with multiple phenotypes. Criteria to subtype acute leukaemia by flow cytometry have not been validated. The goal of this study was to develop a panel of antibodies and objective antigen expression criteria for the assignment of lymphoid or myeloid lineage by flow cytometry. We isolated mRNA from the blood of 45 CD34+ acute leukaemia cases and measured expression of 43 genes that represent lymphoid and myeloid lineages using NanoString technology. We determined differentially expressed genes between major groups identified by unsupervised hierarchical clustering. We then evaluated the expression of antigens by flow cytometry to determine if cases could be assigned to a lineage. Two groups were identified by gene expression. Group 1/LYMPH overexpressed lymphoid‐associated genes (ex. DNTT) and had a higher percentage of CD5 + CD3‐ cells by flow cytometry. Group 2/MYELO overexpressed myeloid‐associated genes (ex. ANPEP/CD13) and had a higher percentage of class II major histocompatibility complex (MHCII)‐ CD14+ and/or CD18 + CD4‐ cells. We proposed that >12.5% CD5 + CD3‐ cells in the blood was indicative of lymphoid lineage, and > 3.0% CD14 + MHCII‐ cells or > 18% CD18 + MHCII‐CD4‐ cells was indicative of myeloid lineage. 15/15 cases that met the proposed criteria for acute lymphocytic leukaemia were in LYMPH group and 12/15 cases that met the proposed criteria for acute myeloid leukaemia were in MYELO group. The majority of CD34+ cases that did not meet either immunophenotyping lineage criterion (12/13) clustered within the LYMPH group. In conclusion, currently available antibodies can be useful for determining canine acute leukaemia subtypes.

C-MPL Is a Candidate Surface Marker and Confers Self-Renewal, Quiescence, Chemotherapy Resistance, and Leukemia Initiation Potential in Leukemia Stem Cells

Acute myeloid leukemia (AML) is initiated and maintained by a unique, small subset of leukemia cells known as leukemia stem cells (LSCs). Self-renewal, quiescence, and chemotherapy resistance are key stemness properties of LSCs that are essential for poor clinical responses to conventional therapies. Identifying LSC surface markers and targeting LSCs are important for the development of potential therapies. In this study, application of chemotherapy treatment in AML-ETO9a (AE9a) leukemia mice led to the enrichment of a chemotherapy-resistant cell population identified as Lin- c-Kit+ c-MPL+ . In addition, this c-MPL-positive cell population within Lin- c-Kit+ leukemia cells included a high percentage of cells in a quiescent state, enhanced colony formation ability, and increased homing efficiency. Serial transplantation demonstrated that Lin- c-Kit+ c-MPL+ cells displayed a significantly high potential for leukemia initiation. Furthermore, it was demonstrated that in AML patients, c-MPL was expressed on the majority of CD34+ leukemia cells and that the proportion of c-MPL+ cells in CD34+ leukemia cells is associated with poor prognosis. Finally, AMM2, an inhibitor of c-MPL, was shown to significantly enhance the survival of AE9a leukemia mice when combined with chemotherapeutic agent. These results indicate that c-MPL is a candidate LSC surface marker that may serve as a therapeutic target for the elimination of LSCs. Stem Cells 2018;36:1685-1696.

Abstract 4635: The LSD1 Specific Inhibitor INCB059872 enhances the activity of immune checkpoint blockade by reshaping the myeloid compartment in the syngeneic 4T1 mouse mammary tumor model

Abstract Immune checkpoint blockade has shown considerable therapeutic promise in the clinic. However, single agent activity is compromised by the presence of suppressive myeloid cells, including myeloid derived suppressor cells (MDSC), tumor associated macrophages (TAM) and polymorphonuclear (PMN) cells, in the tumor microenvironment. Epigenetic alterations can significantly contribute to the development of the immunosuppressive tumor microenvironment and recent data have suggested that combining epigenetic-based therapies with immunotherapeutic agents can lead to improved efficacy in preclinical models. Since Lysine Specific Demethylase 1 (LSD1) has been shown to play a critical role in hematopoiesis, we hypothesized that inhibition of LSD1 could have a direct effect on myeloid cell differentiation and potentially restore normal myelopoiesis in cancer patients. To test this hypothesis, we evaluated INCB059872, a potent, selective and orally available FAD-directed covalent inhibitor of LSD1 in several experimental models. In an in vitro differentiation assay, the majority of CD34+ progenitor cells were driven to a monocytic phenotype in the presence of INCB059872, while control treated cells differentiated toward granulocytic PMN cells. Similar results were observed in vivo. Using the orthotopic 4T1 mammary cancer model, the myeloid compartment was characterized in tumor tissues following treatment with INCB059872. Notably, the population of PMN-MDSC was significantly decreased in tumor tissues following oral administration of INCB059872, whereas the macrophage population was increased. These data suggest that INCB059872 can redirect myeloid differentiation toward monocyte/macrophages and inhibit the differentiation of PMN-MDSC in this syngeneic tumor microenvironment. Consistently, intratumoral T lymphocyte infiltration was increased following INCB059872 treatment. The combination of INCB059872 and α-PD-L1 antibody enhanced anti-tumor efficacy in the 4T1 orthotopic tumor model. Collectively, these data suggest that inhibition of LSD1 with INCB059872 can directly affect myeloid differentiation to reduce the accumulation of myeloid suppressive cells, restoring the tumor microenvironment to be more responsive to PD-1/PD-L1 axis blockade. This study supports the therapeutic potential for the combination of an LSD1 inhibitor with immuno-therapeutic agents to improve overall clinical response in cancer patients. Citation Format: Thomas Condamine, Steve Wang, Melody Diamond, Leslie Hall, Huiqing Liu, Antony Chadderton, Jin Lu, Chunhong He, Liangxing Wu, Timothy Burn, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle, Holly Koblish, Sang Hyun Lee. The LSD1 Specific Inhibitor INCB059872 enhances the activity of immune checkpoint blockade by reshaping the myeloid compartment in the syngeneic 4T1 mouse mammary tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4635. doi:10.1158/1538-7445.AM2017-4635

Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells

Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1× 108 to 1× 109 CD34+ cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34+ cells uses LV inefficiently, because the majority of CD34+ cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34+CD38- cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34+CD38- cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34+ purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34+CD38- cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38+ cells. Importantly, LV modification and transplantation of IB-purified CD34+CD38- cells/non-modified CD38+ cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34+ cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases.

ESAM Is a Novel Human Hematopoietic Stem Cell Marker Associated with a Subset of Human Leukemias

Murine hematopoietic stem cells (HSCs) can be isolated with high efficiency as Lineage- Sca-1+ c-kitHigh (LSK) CD34-/Low CD150+ CD48- cells. In humans, however, the same method is not useful because of critical differences between murine and human HSC phenotypes. Such discrepancy has hampered the translation of findings in mice into a human preclinical or clinical context. Therefore, the identification of common HSC antigens between the two species would be a significant advance with respect to translational studies of HSC biology. We previously identified endothelial cell-selective adhesion molecule (ESAM) as a novel maker for HSCs in mice (Blood, 2009). We also found that ESAM is functionally important for murine HSCs to reconstitute hematopoiesis after 5-FU treatment (J Immunol, 2012). In the present study, we have extended our research of ESAM to human HSCs and leukemia.We first examined whether ESAM expression showed potential as a marker of human HSCs. In addition to adult BM, the majority of CD34+ CD38- cells in cord blood (CB) and G-CSF mobilized peripheral blood expressed ESAM. The addition of anti-CD90 and CD45RA antibodies divides the adult BM CD34+ CD38- fraction into three subpopulations, namely HSCs, multipotent progenitors (MPPs), and multi-lymphoid progenitors (MLPs). We found that HSCs expressed high levels of ESAM whereas MPPs expressed lower levels and many MLPs lost ESAM expression. Functional assessment for ESAM-/Low and ESAMHigh cells in the CD34+ CD38- fractionconfirmed that high ESAM expression distinguishes progenitors that are more primitive and multipotent.We also identified a subset of CD34+ CD38- cells in adult BM and CB that expressed extremely high levels of ESAM, namely ESAMBright cells. Gene expression profiles of the CD34+ CD38- ESAMHigh and CD34+ CD38- ESAMBright populations showed that the former cells expressed HSC-related genes whereas the latter showed more endothelial-related profiles. Indeed, the CD34+ CD38- ESAMBright cells produced CD31+ endothelial cells, but not CD45+ hematopoietic cells, in co-culture with MS5 stromal cells. These results suggest that the CD34+ CD38- fraction, which is conventionally considered the human HSC fraction, also contains a substantial number of non-hematopoietic progenitors. Thus, the inclusion of ESAM provides a more accurate estimation of HSC numbers.Since some of HSC-related antigens are useful for determining leukemia lineage and have utility as prognostic indicators, we determined whether ESAM might also be a valuable addition to this antigen panel. First, we examined human leukemia cell lines. Tested myeloid leukemia lines including KG-1a, HL60, THP1, U937 and Kasumi were uniformly negative for ESAM expression. Jurkat and MOLT4, lymphoid lineage lines were also negative. On the other hand, HEL, an erythroid leukemia cell line, and CMK, a megakaryocytic leukemia cell line, exhibited high expression of ESAM. Additionally, K562 cells, which originated from CML that subsequently transformed into acute erythro-leukemia, also express ESAM. We then evaluated ESAM expression on primary acute leukemia cells, which were isolated from patients upon diagnosis. Interestingly, while all of ALL cases were virtually negative for ESAM, more than half of AML cases were ESAM-positive. Notably, the ESAM expression pattern on AML cases substantially differs even in the same FAB classification.We inferred that AML cells might change their ESAM expression levels according to cell intrinsic features and/or the surrounding environment in vivo. Therefore, we inoculated ESAM- KG-1a cells into NOD/SCID mice and harvested reconstituted KG-1a (rKG-1a) cells after the inoculation. They were then cultured in vitro and inoculated again into NOD/SCID mice. FACS analyses revealed that, although parental KG-1a cells were ESAM-negative, rKG-1a cells expressed a substantial amount of ESAM. Notably, rKG-1a cells were more aggressive and killed the recipient mice in a shorter period. This observation indicates that leukemia cells change their surface phenotype according to the environment, and that ESAM expression may be related to the acquisition of a more aggressive phenotype.In conclusion, we demonstrate that ESAM is a reliable marker of HSCs in humans as well as in mice. Additionally, ESAM is expressed on some of human acute leukemia cells and might be useful for lineage determination and as prognostic indicator. DisclosuresYokota:SHIONOGI & CO., LTD.: Research Funding. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Novel and rapid enumeration method of peripheral blood stem cells using automated hematology analyzer

The number of infused CD34(+) cells is crucial to the success of peripheral blood stem cell transplantation (PBSCT). Here, we present, for the first time, a new method of enumerating hematopoietic progenitor cells (HPCs) for PBSCT. This novel method is based on hemolysis and chemical staining, followed by flow cytometry-based optical detection, conducted using an automated hematology analyzer (XN series, Sysmex). CD34(+) cells and HPCs were compared in 76 granulocyte colony-stimulating factor (G-CSF)-mobilized blood or apheresis samples taken from healthy donors (n=18) or patients undergoing autologous PBSCT (n=6). There was a strong correlation between the numbers of HPCs and CD34(+) cells (R(2) =0.958). The expected total number of HPCs in the final products, which was estimated from HPCs in pre-apheresis PB or mid-apheresis products, also correlated well with the total number of CD34(+) cells in the final products. The change in HPCs in PB closely resembled that of CD34(+) cells during mobilization. Experiments using immunomagnetic beads suggested that the majority of CD34(+) cells existed in HPCs, and vice versa. Hematopoietic progenitor cells may serve as surrogates for CD34(+) cells in PBSCT. However, further investigations are required to verify this.

Single Cell Analysis Of JAK2V617F Positive MPN Stem/Progenitor Cells In Chronic Phase and Leukemic Transformation

IntroductionEssential thrombocytosis (ET), polycythemia vera (PV), and myelofibrosis (MF) share the JAK2V617F mutation, but differ with regard to clinical phenotype, rate of disease progression, and risk of leukemic transformation. Variation in the JAK2V617F neutrophil allele burden does not account for these observed differences in clinical behavior. We therefore investigated JAK2V617Fallele burden and genotype in the stem/progenitor populations of MPN patients in chronic and leukemic phases. MethodsWe studied 39 JAK2V617Fpositive MPN patients evaluated between 2005 and 2013, including 8 patients at leukemic transformation. CD34+ cells isolated from peripheral blood were flow sorted based on CD34, CD38, and the stem cell marker aldehyde dehydrogenase (ALDH). JAK2V617F allele percentages were calculated using an allele specific real time PCR assay. Cells were sorted into 96 well plates and single cell JAK2V617Fgenotypes were obtained using a nested PCR assay. Additional genomic lesions and chromosomal copy number variation were investigated in the sorted fractions when applicable. ResultsIn all MPN cases, the JAK2V617F mutation was detected in the CD34+CD38-ALDHhigh fraction – the same population in which the normal hematopoietic stem cell resides. Quantitative JAK2V617F allele burdens in this fraction were highest in MF > PV > ET. Single cell JAK2V617F genotyping revealed a higher proportion of JAK2V617F-/- cells in ET and PV than in MF, but JAK2V617F-/- cells were detectable in the CD34+CD38-ALDHhigh fraction of all cases. In most cases of PV and MF, this fraction contained a mixture of JAK2V617F-/-, JAK2V617F-/+, and JAK2V617F+/+ cells. Additional chronic phase lesions (including mutations of ASXL1 &TET2) were also found in the CD34+CD38-ALDHhighfraction.Two patterns of leukemic transformation were observed. The first pattern (in 7/8 patients) was identical to that of de novo AML (Gerber JM, Blood 2012), with emergence of a unique CD34+CD38-ALDHint fraction, which was clonal by JAK2V617F genotype and contained leukemia-specific lesions (e.g., 5q deletion). In contrast, the residual CD34+CD38-ALDHhigh population lacked the leukemic abnormalities and was oligoclonal with respect to JAK2V617F. In 3 of these AML cases, the CD34+CD38-ALDHint fraction was JAK2V617F-/-, while the JAK2V617F mutation remained detectable in the CD34+CD38-ALDHhigh fraction. Single cell genotyping performed during the leukemic phase of a PV patient revealed only JAK2V617F-/- CD34+CD38-ALDHint cells but identified JAK2V617F-/-, JAK2V617F-/+, and JAK2V617F+/+ CD34+CD38-ALDHhigh cells; JAK2V617F levels were barely detectable in the progenitors and neutrophils during this leukemic phase. Upon achievement of complete remission from AML, high JAK2V617F allele burdens were then found in the progenitors and neutrophils, as well as in the CD34+CD38-ALDHhigh fraction. A second pattern of leukemic transformation was seen in one patient, in whom no CD34+CD38-ALDHint population was present. The CD34+CD38-ALDHhigh population was expanded in this case and harbored JAK2V617F+/+ positive cells with the leukemia-specific lesion. ConclusionsUnlike CML, in which the BCR/ABL oncogene is typically present in the majority of CD34+CD38-ALDHhigh cells at diagnosis (Gerber JM, Am J Hematol 2011), the JAK2V617F mutation was present in only a minority of CD34+CD38-ALDHhigh cells in JAK2V617F positive ET and PV. Moreover JAK2V617F-/- cells were detected even in longstanding, advanced phase PV and MF. Lower JAK2V617F clonal burdens in the primitive CD34+CD38-ALDHhigh compartment as compared to neutrophils in most cases of MPN suggest that the JAK2V617F mutation does not confer a significant advantage at the stem cell level and that other genetic lesions may drive expansion of this population. JAK2V617F negative leukemias occur in about 35% of PV patients, apparently arising from the residual JAK2V617F negative CD34+CD38-ALDHhigh reservoir. We conclude that primitive stem/progenitor cells are mosaic with regard to JAK2V617F mutation status in the majority of MPN patients. Furthermore, acquisition of JAK2V617F, development of JAK2V617F homozygosity, and accrual of other acquired lesions in chronic phase MPN all occur in the primitive CD34+CD38-ALDHhigh compartment. Lesions specific to post MPN AML segregate to a distinct CD34+CD38-ALDHint population. Disclosures:Jones:Cytomedix: Patent holder for Aldefluor reagent, which is licensed by Cytomedix. This relationship is managed by the Johns Hopkins Office of Policy Coordination., Patent holder for Aldefluor reagent, which is licensed by Cytomedix. This relationship is managed by the Johns Hopkins Office of Policy Coordination. Patents & Royalties.

Successful Engraftment in HLA-Mismatched Bone Marrow Transplantation Despite the Persistence of High-Level Donor-Specific Anti-HLA-DR Antibody

Successful Engraftment in HLA-Mismatched Bone Marrow Transplantation Despite the Persistence of High-Level Donor-Specific Anti-HLA-DR Antibody

Predicting the quality of transplantable cord blood collections through prefreeze and postthaw Apgar scoring

Predicting the quality of transplantable cord blood collections through prefreeze and postthaw Apgar scoring

Increased CD271+ CXCL12 Chemokine Overproducing Mesenchymal Stromal Cells Maintain Distinctive Association with CD34+ Hematopoietic Progenitor/Stem Cells in Myelodysplastic Syndrome

Abstract 2789 Background:The bone marrow microenvironment provides a crucial hematopoietic niche for survival and differentiation of hematopoietic progenitor/stem cells (HPSCs). The dysfunctional microenvironment in myelodysplastic syndrome (MDS) may provide a new source of prognostic and diagnostic information and even provide insight into the pathophysiology of the disease. We used tissue microarray (TMA) techniques combined with semi-automated image acquisition using double immunofluorescence and confocal microscopy to methodically and quantitatively analyze the interrelationships among key components of the bone marrow microenvironment and their relationships to CD34+ hematopoietic progenitor/stem cells (HPSCs) in intact human bone marrow derived from archival diagnostic paraffin-embedded core biopsy specimens representing benign (NL), MDS, and acute myeloid leukemia (AML) diagnoses. Methods:Whole bone marrow core biopsies (13 NL, 11 MDS, 7 AML) and 1 mmTMA cores (5 NL, 6 MDS, 4 AML) were evaluated. The area occupied by stromal cell populations was assessed using freely available ImageJ software. Adjacency of CD34+ HPSCs to specific cell populations was manually quantitated on double immunofluorescent-stained whole TMA core images. Results:CD271 (low-affinity nerve growth factor receptor/pNGFR) highlights an extensive arborizing MSC population, whereas nestin and CD146 (melanoma cell adhesion molecule, MCAM) localize to specific vascular compartments. In MDS, CD271+ MSCs are markedly increased as compared to benign marrow (p=0.012), whereas there is no significant change in CD146, nestin, or CD163+ macrophage populations (Table 1). We document an intimate and conserved relationship of the CD271+ MSC population to CD34+ HPSCs and quantitate the preservation of this relationship in MDS (Fig 1, Table 2). Remarkably, the CD271+ MSC population is in intimate contact with the majority of CD34+ HPSCs (86% on average) across NL, MDS and AML marrows, whereas no significant contact is seen with nestin+ or CD146+ vascular elements. In benign bone marrow expression of the pro-survival chemokine CXCL12 is largely restricted to the vasculature, whereas in MDS and AML CD271+ MSCs abnormally express CXCL12. [Display omitted] Conclusions:We propose that CD271+ MSCs are candidates for a pathophysiologic role in MDS, and are candidate prognostic/diagnostic markers. We hypothesize that the relationship of MDS and AML CD34+ blasts with abnormal CD271+ MSC likely exposes neoplastic CD34+ HPSCs to aberrant contact-dependent signaling via the chemokine CXCL12 or other signals. We propose that the increased, dysfunctional, and spatially disarranged CD271+ arborizing MSC infrastructure may contribute to the ineffective hematopoiesis that is a defining feature of MDS by promoting inappropriate proliferation/survival of CD34+ HPSCs and by failing to support trafficking of maturing hematopoietic cells to microenvironmental compartments where they receive specific contact-dependent signals.Table 1:Quantification of % bone marrow area encompassed by CD271, CD146, nestin, and CD163+ cellsCD271CD146nestinCD163Nmean+/−std devnmean+/−std devnmean+/−std devnmean+/−std devNL143.1 +/− 3.2%160.6 +/− 0.9%140.0 +/− 0.0%612.6 +/− 4.9%MDS139.1 +/− 8.7%100.6 +/− 0.6%100.1 +/− 0.1%523.5 +/− 17.8%AML107.1 +/− 5.3%100.6 +/− 1.0%90.7 +/− 0.2%411.5 +/− 2.2%Total376.3 +/− 6.6%360.6 +/− 0.8%330.1 +/− 0.4%1515.9 +/− 11.4%Mann-Whitney U test p valuesNormal vs MDS0.0120.280.290.40Normal vs AML0.0640.790.160.40CD34+ HPSCs are in intimate and conserved contact with CD271+ MSCs at baseline and in myeloid neoplasia. CD34+ HPSCs (red) are in intimate contact with NGFR+ arborizing stromal cells (green). Representative photomicrographs of benign (NL), low-grade myelodysplastic syndrome/5Q minus (RA5Q), and high grade myelodysplastic syndrome/refractory anemia with excess blasts 2 (RAEB2).Table 2:Quantification of % CD34+ HPSCs in contact with CD271, CD146, nestin, and CD163+ cell typesCD271CD146nestinCD163nmean+/−std devnmean+/−std devnmean+/−std devnmean+/−std devNL682 +/− 10%50.0 +/− 0.0%41.0 +/− 2.1%627 +/− 23%MDS588 +/− 9%50.2 +/− 0.5%41.3 +/− 1.6%516 +/− 16%AML488 +/− 5%40.2 +/− 0.3%30.5 +/− 0.8%415 +/− 6%Total1586 +/− 7%140.1 +/− 0.3%121.0 +/− 0.2%1520 +/− 17%Kruskall Wallis test p values, adjusted for multiple comparisonsversus CD271—<0.001<0.0010.003 Disclosures:Greenberg:amgen: Consultancy, Research Funding; onconova: Research Funding; glaxosmithkline: Research Funding; novartis: Consultancy, Research Funding.

Mobilization of Plasma Cells in Healthy Individuals Treated with G-CSF for Hematopoietic Stem Collection.

Abstract 3679Poster Board III-615In patients with advanced hematologic malignancies, allogenic transplantation with hematopoietic stem cell (HSC) can improve survival. Granulocyte Colony-Stimulating Factor (G-CSF) makes it possible the mobilization of bone marrow HSCs mobilized cells into peripheral blood (PB) and the harvesting of large HSC numbers. G-CSF stimulates BM neutrophils to produce enzymes that cleave adhesion molecules and chemokines inducing HSC exit from the BM environment. Tokoyoda et al. have shown that plasma cells (PC) use similar BM niches as HSC, which involve CXCR4 ligand - CXCL12 - producing cells. Evidences have been obtained that G-CSF mobilization process may impart CXCR12/CXCR4 signaling. In the present study, we have monitored the PC mobilization after G-CSF treatment of healthy donors.Every day hematopoietic niches release a few HSCs in the peripheral circulation (2 HSC/μL) - in the PB of healthy individuals, 2 CD38++ PC circulate per μL. Twenty four healthy donors treated with G-CSF in order to mobilize and harvest HSCs were analyzed by multiparametric flow cytometry. After 5 day mobilization by subcutaneous administration of 5-10 μg/kg/day of G-CSF, a median value of 110 CD34+ HSC/μL and 18 CD38++ PC/μL were detected in PB. A median value of 1510 CD34+ HSC/μL and 140 CD38++ PC/μL were collected in one leukapheresis and patients were allografted with 5.9 106 HSC/kg and 0.5 106 CD38++PC/kg. These mobilized PCs have a slightly different phenotype than that from steady state blood-borne PCs. Among these mobilized PCs, two sub-populations CD138− and CD138+ were identified, respectively 63.3% and 36.7%. Compared to CD138− PCs, CD138+ PCs present a more mature phenotype: lower CD45 expression, strong cytoplasmic Igs, as well as CD38 and CD27 expression. As opposed to B cells, both PC populations are cell cycling (KI-67+), and express CD43. Compared to steady state circulating CD38++ PCs, mobilized PCs lose CD62L expression, and are less cycling. Besides, it has previously been shown that a large part of CXCR4 protein is sequestered intracellularly in CD34+ HSC. In leukapheresis, the majority of CD34+ HSC and CD38++ PC do not expressed CXCR4 on the cell surface while it is sequestered intracellularly.In this study, we demonstrated that as HSCs, PCs are mobilized by G-CSF (less efficiently, respectively 55 fold and 9 fold increase), and CXCR4 intracellular sequestration is observed in mobilized cells. The CXCR4 sequestration mechanism may be an important control mechanism for HSC and PC mobilization. Steady state circulating PCs do not express CXCR4 on the cell surface but CXCR4 is also sequestered intracellularly. This data seems to indicate that this HSC and PC mobilization mechanism is likely a physiological mechanism. How G-CSF mobilization affect CXCR4 expression on cell surface remains undefined.This study raises several open questions. When donor PCs are injected to a patient, do they also confer on the patient the donor's humoral immunity? Moreover, where do the donor mobilized PCs migrate after transplantation in the recipient? Disclosures:No relevant conflicts of interest to declare.

Successful Stem Cell Remobilization Using Plerixafor (Mozobil) Plus Granulocyte Colony-Stimulating Factor in Patients with Non-Hodgkin Lymphoma: Results from the Plerixafor NHL Phase 3 Study Rescue Protocol

In a phase 3 multicenter, randomized, double-blinded, placebo-controlled study of 298 patients with non-Hodgkin lymphoma (NHL), granulocyte colony-stimulating factor (G-CSF) plus plerixafor increased the proportion of patients who mobilized >or=5 x 10(6) CD34(+) hematopoietic stem cells (HSCs)/kg compared with placebo plus G-CSF (P < .001). Patients in either study arm who failed mobilization (< 0.8 x 10(6) CD34(+) cells/kg in 2 collections or <2 x 10(6) CD34(+) cells/kg in 4 collections) were eligible to enter the opened-label rescue protocol. Following a 7-day minimum rest period, these patients received G-CSF (10 microg/kg/day) for 4 days, followed by daily plerixafor (0.24 mg/kg) plus G-CSF and apheresis for up to 4 days. Of the 68 patients failing initial mobilization (plerixafor, n = 11; placebo, n = 57), 62 patients (91%) entered the rescue procedure (plerixafor, n = 10; placebo, n = 52). Four of 10 patients (40%) from the plerixafor group and 33 of 52 (63%) from the placebo group mobilized sufficient CD34(+) cells (>or= 2 x 10(6) cells/kg) for transplantation from the rescue mobilization alone (P = .11). Engraftment of neutrophils (11 days) and platelets (20 days) was similar to that in patients who did not fail initial mobilization, and all patients had durable grafts at the 12-month follow-up. Common plerixafor-related adverse events (AEs) included mild gastrointestinal (GI) effects and injection site reactions. There were no drug-related serious AEs. These data support that plerixafor plus G-CSF can safely and effectively remobilize patients with NHL who have failed previous mobilization.

Flow Cytometry Analysis of Peripheral Blood CD34+ Cells in Patients with Primary Myelofibrosis

Primary myelofibrosis (PMF) is a clonal chronic myeloproliferative disorder characterized by the accumulation of megakaryocytes in the bone marrow (BM), variable degrees of BM fibrosis, tear-drop erythrocytes, increased numbers of CD34+ hematopoietic progenitors in the peripheral blood (PB), and extramedullary hematopoiesis. Since the antigenic properties of the circulating CD34 cells may yield clues to disease pathogenesis and have not been extensively studied, we used five-color flow cytometry to examine these cells from 20 well characterized patients with PMF and 10 normal controls. Bone marrow biopsies, molecular and cytogenetic studies were also reviewed. As expected, the percentages of peripheral-blood CD34 cells were significantly higher in the PMF patients (mean 1.4%, range, range 0.065–7.15) compared to the controls (mean 0.05%, range 0.01–0.57). The mean fluorescence intensity (MFI) values related to HLA-DR expression were increased (more than 3 fold) on the CD34+ cells in 12/20 (60%) PMF patients relative to normal control levels, while increased levels of CD13 were seen in 5/20 (25%) of PMF patients. CD33 and CD117 expression were similar on the CD34+ cells in both groups. Aberrant expression of lymphoid antigens was observed in 6/20 (30%) with CD7, 6/20 (30%) with CD4, and 3/20 (15%) with CD56 on CD34 positive cells in PMF. In the18 cases also studied with antibodies against CD45RA and CD123, the majority of CD34+ CD38 + cells phenotypically resembled megakaryocyte-erythroid precursors (CD45RA−, CD123−) in 5 cases, common myeloid progenitors (CD45RA−, CD123+) in 12 cases, and granulocyte-macrophage progenitors (CD45RA+, CD123 +) in 1 case. JAK2-V617F mutations were detected in 9 of 20 cases, but were present in only 1 of 5 cases showing predominately megakaryocyte-erythroid precursors. The percentage of CD34+ cells also expressing CXCR4 (CD184) appears to be increased in some patients relative to normal controls in contrast to other reported studies. In conclusion, the peripheral blood CD34+, progenitor cells in PMF patients are heterogeneous phenotypically resembling megakaryocyte-erythroid precursors in approximately 30% of cases, and common myeloid progenitors in approximately 70% of cases. In addition, these cells often show phenotypic abnormalities (increased intensity of HLA-DR and CD13 expression) that can be detected with flow cytometry relative to normal peripheral blood CD34+ cells. Patterns of antigen expression in PMF also appear to differ from those reported for CD34 positive cells in other myeloproliferative disorders which may help in early diagnosis and/or monitoring treatment responses.

Imatinib Induced Molecular Response in CD34+ Hematopoietic Stem and Progenitor Cells of Patients with Chronic Myeloid Leukemia Following the First Week of Therapy

Imatinib is a selective bcr-abl tyrosine-kinase inhibitor, successfully used as standard therapy for patients with chronic myelogenous leukemia (CML) in chronic phase. We examined the effect of Imatinib on the gene expression transcriptome pattern of CD34 positive hematopoietic stem and progenitor cells of 6 patients with newly diagnosed CML following the first week of therapy. Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyze and compare the gene expression profiles of CD34+ cells from peripheral blood of 6 patients with newly diagnosed CML in chronic phase before and 7 days after the initiation of Imatinib first line therapy. One patient was female and 5 male. Their median age was 61 years (range 32–79 years). In addition, quantitative molecular monitoring of bcr-abl was performed using Light Cycler technology. Using a supervised hierarchical cluster analysis of the gene expression data we found that 303 genes were differentially expressed with a lower bound of at least 1.2 fold (−2.58 to −1.2 and 1.2 to 2.09). Of these, 183 genes were down-regulated and 120 genes were up-regulated comparing day 1 with day 7. A considerable number of the down-regulated genes are involved in the regulation of cell cycle (CDC2, CDC20, CCNB1, CCNB2) and DNA replication (TOP2A, POLE2, MCM-complex). In contrast, genes coding for proteins governing adhesion and cell migration were up-regulated (CD44, L-Selectin). The observations of the gene expression data of the aforementioned genes could be confirmed by quantitative real-time PCR. After 10 months of Imatinib therapy (range 6–13 months) all 6 patients reached in median a 3 log reduction (range 1.5–3 log reduction) of their bcr-abl mRNA. Bcr-abl FISH analysis confirmed that the majority of CD34+ cells examined were Ph-Chromosome positive. These data indicate that in the early phase of treatment Imatinib mainly acts as an inhibitor of cell cycle and replication which results in a decreased proliferation. The silencing of the Ph-Chromosome positive cells is associated with an up-regulation of genes coding for adhesion during the first week of therapy.

A Population of Multipotent CD34-Positive Adipose Stromal Cells Share Pericyte and Mesenchymal Surface Markers, Reside in a Periendothelial Location, and Stabilize Endothelial Networks

It has been shown that stromal-vascular fraction isolated from adipose tissues contains an abundance of CD34+ cells. Histological analysis of adipose tissue revealed that CD34+ cells are widely distributed among adipocytes and are predominantly associated with vascular structures. The majority of CD34+ cells from freshly isolated stromal-vascular fraction were CD31-/CD144- and could be separated from a distinct population of CD34+/CD31+/CD144+ (endothelial) cells by differential attachment on uncoated plastic. The localization of CD34+ cells within adipose tissue suggested that the nonendothelial population of these cells occupied a pericytic position. Analysis of surface and intracellular markers of the freshly isolated CD34+/CD31-/CD144- adipose-derived stromal cells (ASCs) showed that >90% coexpress mesenchymal (CD10, CD13, and CD90), pericytic (chondroitin sulfate proteoglycan, CD140a, and CD140b), and smooth muscle (alpha-actin, caldesmon, and calponin) markers. ASCs demonstrated polygonal self-assembly on Matrigel, as did human microvascular endothelial cells. Coculture of ASCs with human microvascular endothelial cells on Matrigel led to cooperative network assembly, with enhanced stability of endothelial networks and preferential localization of ASCs on the abluminal side of cords. Bidirectional paracrine interaction between these cells was supported by identification of angiogenic factors (vascular endothelial growth factor, hepatocyte growth factor, basic fibroblast growth factor), inflammatory factors (interleukin-6 and -8 and monocyte chemoattractant protein-1 and -2), and mobilization factors (macrophage colony-stimulating factor and granulocyte/macrophage colony-stimulating factor) in media conditioned by CD34+ ASCs, as well a robust mitogenic response of ASCs to basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor-BB, factors produced by endothelial cells. These results demonstrate for the first time that the majority of adipose-derived adherent CD34+ cells are resident pericytes that play a role in vascular stabilization by mutual structural and functional interaction with endothelial cells.

Different levels of CD52 antigen expression evaluated by quantitative fluorescence cytometry are detected on B‐lymphocytes, CD 34+ cells and tumor cells of patients with chronic B‐cell lymphoproliferative diseases

The success of treatment using monoclonal antibodies in oncology is influenced by, among other factors, the level of target antigen expression on tumor cells. The authors analyzed the intensity of the CD52 antigen expression in patients with chronic lymphoproliferative diseases and compared them with B-lymphocytes of a healthy population and CD34(+) cells in peripheral blood stem cells (PBSC) grafts. Recently diagnosed and previously untreated patients with B-cell chronic lymphocytic leukemia (B-CLL), mantle-cell lymphoma (MCL), or small lymphocytic lymphoma (SLL) were evaluated and compared with control group and CD34(+) cells. The intensity of CD52 was expressed in molecules of equivalent soluble fluorochrome units (MESF) and antibody-binding capacity (ABC). In the group of patients with B-CLL, the CD52 level on tumor cells (245 x 10(3) MESF; 107 x 10(3) ABC) was significantly lower than on B-lymphocytes of the control group (446 x 10(3) MESF; 194 x 10(3) ABC; P < 0.001) and SLL tumor cells (526 x 10(3) MESF; 229 x 10(3) ABC; P < 0.001). The CD52 antigen was expressed on a majority of CD34(+) cells, but its expression intensity was low (101 x 10(3) MESF; 44 x 10(3) ABC). Our data demonstrate differences in the intensity of the CD52 antigen expression between B-lymphocytes and tumor lymphocytes of B-CLL patients, and between B-CLL and SLL tumor cells. CD52 antigen is expressed at low level on CD34(+) cells.

CD133-enriched CD34 − (CD33/CD38/CD71) − cord blood cells acquire CD34 prior to cell division and hematopoietic activity is exclusively associated with CD34 expression

We compared the cell division behavior of CD34(-) and CD34(+) (CD33/CD38/CD71)-negative (Lin(-)) CD133(+) cord blood cells stimulated with the cytokines Flt3-ligand, stem cell factor, and thrombopoietin. Within a 4-day time frame, Lin(-)CD34(-) CD133(+) (CD34(-)) cells underwent more cell divisions in serum-free culture than their Lin(-)CD34(+) CD133(+) (CD34(+)) counterparts. The majority of CD34(-) cells acquired expression of CD34 in vitro, including most undivided cells. Moreover, hematopoietic activity from both CD34(-) and CD34(+) cells was exclusively retained within the cell fraction expressing CD34 after 4 days in culture. Most strikingly, in cultures from Lin(-)CD34(-) cells hematopoietic activity was associated with the fraction of divided cells, whereas in cultures of CD34(+) cells, hematopoietic activity associated with the undivided cell fraction. Therefore, clonogenic CD34(+) cells either do not divide or lose their clonogenic capacity upon cell division in vitro, while CD34(-) cells divide and retain this capacity under the same specific conditions. In conclusion, we demonstrate that CD133-enriched Lin(-)CD34(-) cord blood cells acquire CD34 prior to cell division and that long-term hematopoietic activity is associated exclusively with expression of CD34.

CD96 is a leukemic stem cell-specific marker in human acute myeloid leukemia.

Permanent cure of acute myeloid leukemia (AML) by chemotherapy alone remains elusive for most patients because of the inability to effectively eradicate leukemic stem cells (LSCs), the self-renewing component of the leukemia. To develop therapies that effectively target LSC, one potential strategy is to identify cell surface markers that can distinguish LSC from normal hematopoietic stem cells (HSCs). In this study, we employ a signal sequence trap strategy to isolate cell surface molecules expressed on human AML-LSC and find that CD96, which is a member of the Ig gene superfamily, is a promising candidate as an LSC-specific antigen. FACS analysis demonstrates that CD96 is expressed on the majority of CD34(+)CD38(-) AML cells in many cases (74.0 +/- 25.3% in 19 of 29 cases), whereas only a few (4.9 +/- 1.6%) cells in the normal HSC-enriched population (Lin(-)CD34(+)CD38(-)CD90(+)) expressed CD96 weakly. To examine whether CD96(+) AML cells are enriched for LSC activity, we separated AML cells into CD96(+) and CD96(-) fractions and transplanted them into irradiated newborn Rag2(-/-) gamma(c)(-/-) mice. In four of five samples, only CD96(+) cells showed significant levels of engraftment in bone marrow of the recipient mice. These results demonstrate that CD96 is a cell surface marker present on many AML-LSC and may serve as an LSC-specific therapeutic target.

Simultaneous Isolation of Human Bone Marrow Hematopoietic, Endothelial, and Mesenchymal Progenitor Cells by Flow Sorting Based on Aldehyde Dehydrogenase Activity.

ALDH bright [ALDHbr] cells exhibit low side scatter and high activity of the intracellular enzyme aldehyde dehydrogenase [ALDH] in flow cytometric analysis. ALDH regulates gene expression in many developing systems by producing retinoic acids. ALDHbr populations from human umbilical cord [UCB] and mobilized peripheral blood preparations [PBSC] are highly enriched for cells that express CD34 and CD133 and for hematopoeitic progenitor cells. Because human bone marrow [BM] contains a highly heterogeneous population of stem and progenitor cells, we used multiparameter flow analysis and cell sorting to measure expression of cell surface markers and lineage specific developmental potential of BM ALDHbr populations. The ALDHbr population comprised 1.2 ± 0.8% (mean ± SD, n=39) of the nucleated BM cells and was significantly (p<0.05) enriched in cells expressing CD34, CD117, CD105, CD127, CD133, and CD166. ALDHbr populations were also significantly enriched in cells expressing primitive progenitor phenotypes including CD34+CD38− and CD34+CD133+ cells. The cell surface antigen expression of BM ALDHbr populations differed significantly from UCB and PBSC populations. In particular, the BM ALDHbr populations had more CD34− cells and CD45− cells and fewer CD133+ cells than the UCB and PBSC ALDHbr populations. Next, we compared the ability of sorted ALDHbr cells and ALDHdim cells derived from each bone marrow to develop into erythroid and myeloid cells, megakaryocytes, endothelial cells, and mesenchymal cells in culture. The ALDHdim population was the residual cell population produced when we sorted ALDHbr cells out of BM. ALDHbr populations were 144-fold more active in primary hematopoietic colony forming activity than ALDHdim cells, and all detectable BM cells that could form megakaryocyte colonies were ALDHbr. Even though hematopoietic progenitor activity was concentrated in the ALDHbr population, the majority of CD34+ and CD133+ cells in BM were ALDHdim. Cultures seeded with 50,000 ALDHbr cells gave rise to confluent growth of endothelial cells that expressed surface Ulex lectin receptors and von Willebrand factor, formed tubules on Matrigel, and took up acetylated LDL in 34 of 39 BM tested. In contrast, only 18 of 33 ALDHdim BM populations tested gave rise to endothelial cultures, and 500,000 ALDHdim cells were required to generate confluent cultures in 21 days. Cells that gave rise to early endothelial growth colonies at day 7 were 435-fold more frequent in the ALDHbr than the ALDHdim population. Confluent cultures of cells expressing cell surface antigens and functional attributes of multipotential mesenchymal cells were also routinely established with 50,000 ALDHbr cells. Again, ten-fold more ALDHdim cells than ALDHbr cells were needed to establish mesenchymal cultures. The mesenchymal cultures could be driven to differentiate into adipocytes, chondrocytes, osteoblasts, and neural lineage cells in standard culture assays. These results show that most of the hematopoietic, endothelial, and mesenchymal progenitor cell activities in human BM can be simultaneously isolated by cell sorting based on ALDH activity. Such ALDHbr populations may be useful in a variety of cell therapy indications.

Activation of C/EBPα in Hematopoietic Stem Cells Leads to Growth Suppression and Impairment of Megakaryocyte Differentiation.

C/EBPα is a critical regulator of early myeloid differentiation. Resent study showed that the loss of C/EBPα led to an augmentation of competitive repopulating activity of hematopoietic stem cells (HSCs). Our previous study using the transgenic (Tg) mice expressing an inducible form of C/EBPα (C/EBPα-ER) also showed that in vivo activation of C/EBPα by 4-hydroxy tamoxifen (4-HT) led to remarkable decrease of HSC population (Abstract #671, Blood 104, 193a, 2004). In order to investigate further the role of C/EBPα in HSC homeostasis, we purified CD34− c-Kithi Sca-1hi lineage− cells (CD34−KSLs) from the C/EBPα-ER Tg mice and examined the effect of C/EBPα activation on their growth, self-renewal, and differentiation potential. Clonal proliferation assay showed that the growth of about 95 % of CD34−KSLs was suppressed in the presence of 4-HT. Single cell colony assay showed that the percentage of CFU-GEMM and CFU-MK remarkably decreased from 80 % to 2.1 % and from 2.1 % to 0 %, respectively by 4-HT treatment. In contrast, percentage of CFU-GEM increased from 0 % to 74 %, indicating that differentiation potential to megakaryocytes (MKs) was selectively lost by C/EBPα activation. To gain more insights into the cell fate determination at clonal level, we performed paired daughter cell colony assay. Sorted CD34−KSLs were clonally deposited into 96-well plates and allowed to divide once to generate daughter cell pairs, which were then separated by micromanipulation, and transplanted into control or 4-HT containing methylcellulose. 26 daughter cell pairs out of assessable 41 colony pairs lost MK differentiation capacity in the presence of 4-HT. In contrast, remaining 15 pairs took the same fate of differentiation in control and 4-HT. Surprisingly, while most of the CD34−KSLs stopped their growth upon C/EBPα activation, approximately 5 % of the cells proliferated even in the presence of 4-HT. These cells grew from a single cell to generate more than 45 cells and sustained multipotentiality to generate GEMM-colonies even after 8-days of culture. To further examine the effect of C/EBPα activation on self-renewal potential of HSCs, clonally sorted CD34−KSLs were allowed to divide once in 4-HT- or control-media, and the fate of each daughter cell was traced in methylcellulose. The result showed that GEMM-colony pairs were generated at similar frequency in control and 4-HT culture, indicating that the self-renewal capacity of CD34−KSLs was not affected by C/EBPα. These results indicated that activation of C/EBPα caused growth suppression and impaired differentiation to MKs in the majority of CD34−KSLs. However, small fraction does not respond to C/EBPα activation, indicating CD34−KSLs could be classified into two subpopulations by their sensitivity to C/EBPα.