Dual gold and photoredox catalysed C-H activation of arenes for aryl-aryl cross couplings.

A mild and fully catalytic aryl-aryl cross coupling via gold-catalysed C-H activation has been achieved by merging gold and photoredox catalysis. The procedure is free of stoichiometric oxidants and additives, which were previously required in gold-catalysed C-H activation reactions. Exploiting dual gold and photoredox catalysis confers regioselectivity via the crucial gold-catalysed C-H activation step, which is not present in the unselective photocatalysis-only counterpart.

Biosynthesis of O-glycans in leukocytes from normal donors and from patients with leukemia: increase in O-glycan core 2 UDP-GlcNAc:Gal beta 3 GalNAc alpha-R (GlcNAc to GalNAc) beta(1-6)-N-acetylglucosaminyltransferase in leukemic cells.

We have studied the biosynthesis of altered O-glycan structures on leukocytes from patients with chronic myelogenous leukemia and with acute myeloblastic leukemia (AML). It has been shown previously that the activity of CMP-NeuAc:Gal beta 1-3GalNAc alpha-R (sialic acid to galactose) alpha(2-3)-sialytransferase (EC 2.4.99.4) is increased in leukocytes from patients with chronic myelogenous leukemia (M. A. Baker, A. Kanani, I. Brockhausen, H. Schachter, A. Hindenburg, and R. N. Taub, Cancer Res., 47: 2763-2766, 1987) and with AML (A. Kanani, D. R. Sutherland, E. Fibach, K. L. Matta, A. Hindenburg, I. Brockhausen, W. Kuhns, R. N. Taub, D. van den Eijnden and M. A. Baker, Cancer Res., 50: 5003-5007, 1990). This increased activity may in part be responsible for the hypersialylation observed in leukemic leukocytes; however, hypersialylation may also be due to changes in underlying O-glycan structures. To test this hypothesis, we have assayed in normal human granulocytes and leukemic leukocytes several glycosyltransferases involved in the synthesis and elongation of the four common O-glycan cores. UDP-GlcNAc:Gal beta 1-3GalNAc-R (GlcNAc to GalNAc) beta(1-6)-GlcNAc transferase (EC 2.4.1.102), which synthesizes O-glycan core 2 (GlcNAc beta 1-6[Gal beta 1-3]GalNAc alpha), is significantly elevated in chronic myelogenous leukemia (4-fold) and AML (18-fold) leukocytes relative to normal human granulocytes. Neither normal nor leukemic cells show detectable activities of GlcNAc transferases which synthesize O-glycan core 3 (GlcNAc beta 1-3GalNAc-R) and core 4 (GlcNAc beta 1-6[GlcNAc beta 1-3] GalNAc-R) or the blood group I structure. The beta 3-GlcNAc transferase which elongates core 1 and core 2 was found at low levels in normal granulocytes but was not detectable in leukemic cells. The beta 3-GlcNAc transferase and beta 4-Gal transferase involved in poly-N-acetyllactosamine synthesis, as well as the beta 3-Gal transferase synthesizing core 1 (Gal beta 3 GalNAc), were present in all samples but were significantly increased in patients with AML. The observed changes are consistent with hypersialylation in leukemia.

Human leukemic myeloblasts and myeloblastoid cells contain the enzyme cytidine 5'-monophosphate-N-acetylneuraminic acid:Gal beta 1-3GalNAc alpha (2-3)-sialyltransferase.

We have examined the role of CMP-NeuAc:Gal beta 1-3GalNAc-R alpha(2-3)-sialyltransferase in fresh leukemia cells and leukemia-derived cell lines. Enzyme activity in normal granulocytes using Gal beta 1-3GalNAc alpha-o-nitrophenyl as substrate was 1.5 +/- 0.7 nmol/mg/h whereas activity in morphologically mature granulocytes from 6 patients with chronic myelogenous leukemia (CML) was 4.2 +/- 1.6 nmol/mg/h (P less than 0.05). Myeloblasts from 5 patients with CML in blast crisis showed enzyme activity levels of 6.5 +/- 2.5 nmol/mg/h. From 2 patients with CML, both blasts and granulocytes were obtained, with higher enzyme activity in the patients' blasts (7.1 nmol/mg/h) than in their granulocytes (4.9 nmol/mg/h) in both cases, suggesting that the increase in enzyme activity is related to the differentiation or proliferation status of the CML cells. However, similarly high enzyme levels were also seen in myeloblasts from acute myeloblastic leukemia patients (5.6 +/- 1.4 nmol/mg/h) and in some acute myeloblastic leukemia-derived cell lines (KG1a and HL60), suggesting that increased levels of this enzyme are not directly correlated with the presence of the Ph1 chromosome. This alpha(2-3)-sialyltransferase activity can also be detected in normal peripheral blood lymphocytes and exhibits increased activity in chronic lymphocytic leukemia cells and acute lymphoblastic leukemia. These data suggest that the level of enzyme activity may vary with growth rate and maturation status in myeloid and lymphoid hemopoietic cells. Finally, we have identified a glycoprotein in acute myeloblastic leukemia cells that serves as a substrate for the alpha(2-3)-sialyltransferase. The desialylated form of the glycoprotein was resialylated in vitro by the purified placental form of this alpha(2-3)-sialyltransferase and exhibits a molecular weight of about 150,000.

Biochemical characterisation of leukaemia-associated antigen p24 defined by the monoclonal antibody BA-2.

The leukaemia-associated cell surface antigen p24/BA-2 is a single polypeptide chain with a molecular weight of 24,000. Treatment with glycosidases or exposure of cells to tunicamycin failed to show any change in the molecular weight of the antigen when examined by SDS-polyacrylamide gel electrophoresis. In addition, it failed to bind to lectin affinity columns of concanavalin A, lentil lectin or ricinus communis lectin. This is consistent with the absence of N-asparagine linked oligosaccharide chains on the antigen. Pulse-chase labelling of protein p24 shows a post-translational modification resulting in a molecular weight increase of approx. 500-1000. Alkaline treatment resulted in a decrease in molecular weight of approximately the same amount, suggesting that p24 contain some O-glycosidically linked oligosaccharide. Protein p24 has a basic pI of 7.3 which is unchanged after neuraminidase treatment. Protein P24/Ba-2 cannot be labelled by either the lipophilic photoactivatable nitrene reagent, hexanoyldiiodo-N-(4-azido-2-nitrophenyl)tyramine, or with [32P]phosphate. This suggests that the molecule is non-integral in nature and that it does not form an intimate association with the lipid matrix. Identical molecular weights, when reduced and non-reduced antigens were compared, suggest that it contains no internal disulphide linkages and failure to detect any other band on gradient gel SDS-polyacrylamide gel electrophoresis from 5-15% suggests that is is not strongly associated with any other structure.

Retention of progenitor cell function in CD34+ cells purified using a novel O-sialoglycoprotease.

We previously showed that the sialoglycoprotein, CD34, which is expressed on primitive human hematopoietic progenitor cells, is cleaved by a unique glycoprotease from Pasteurella haemolytica (P.h. glycoprotease). This proteolytic enzyme specifically cleaves glycoproteins rich in O-sialoglycans. Glycoproteins containing only N-linked glycans are not cleaved. Cleavage of the CD34 antigen results in the loss of epitopes detected by five of seven CD34-designated antibodies. In this study, we investigated the role of the P.h. glycoprotease in isolating CD34+ cells from unfractionated normal human bone marrow mononuclear cells (MNCs), and determined the effect of the glycoprotease on the proliferative capacity of the progenitor-enriched fraction. CD34+ cells were isolated from MNCs using immunomagnetic beads attached via a CD34 antibody whose epitope is susceptible to removal by the cleavage with the glycoprotease. Subsequent cleavage with P.h. glycoprotease for 30 min at 37 degrees C released the CD34+ cells from the beads with a recovery of up to 78%. Using a CD34 antibody whose epitope was not removed by the glycoprotease, up to 95% of the recovered cells expressed CD34. Compared to unseparated MNCs, the CD34+ cells showed the following enrichment of committed hematopoietic progenitors, as assayed in semi-solid media: CFU-GM, 45-fold; CFU-M, 13-fold; BFU-E, 26-fold and CFU-GEMM, 81-fold. Hematopoiesis was also studied in two-stage long-term bone marrow cultures in which the CD34+ cells were co-cultured over irradiated, allogeneic adherent layers. Output of CFU-GM over a seven week period from these cultures was similar to that from control cultures with autologous adherent-cell-depleted marrow MNCs. These data suggest that the loss of O-sialo-glycosylated peptide moieties from P.h. glycoprotease-released CD34+ cells neither affects the functional capacity of committed progenitors, nor impairs the proliferation of long-term culture-generating cells. The P.h. glycoprotease can be used to facilitate the isolation and recovery of functionally competent CD34+ cells at high yield and purity, without prior removal of other adherent cells. The ability to rapidly purify CD34+ cells using this non-cytotoxic enzyme has important implications for bone marrow transplantation as well as for gene transfer studies in vitro.

Chronic myeloid leukemia arising in a progenitor common to T cells and myeloid cells.

Until recently, T cells were believed not to be involved in chronic myeloid leukemia. We describe an example of CML in T lymphoblastic crisis with massive generalized lymphadenopathy in which the blasts were CD2(+), CD5(+), and CD7(+), variably CD1(+) and CD3(+), and both responded to and could be induced to produce the T cell growth factor, interleukin-2. Additionally, the blasts were shown to contain the CML-related tyrosine kinase P210bcr-abl rather than the smaller kinase associated with Ph1(+) ALL. Finally, the participation of the T lymphoid lineage in the CML clone was proven by the presence of the same BCR rearrangement in blasts as in granulocytes, suggesting the existence of a bone marrow progenitor common to the T cell and myeloid lineages.

Structural and partial amino acid sequence analysis of the human hemopoietic progenitor cell antigen CD34.

Monoclonal antibodies of the CD34 class all recognize a monomeric cell surface antigen of approximately Mr 110,000 which is selectively expressed on human hemopoietic progenitor cells. This structure can be readily surface-labeled with [125I]actoperoxidase and by periodate-[3H]borohydride, but it labels only weakly with [35S]methionine, [35Sl]cysteine, 3H-amino acids, or 3H-mannose, even after prolonged labeling periods. However, the antigen is more efficiently labeled by [3H]glucosamine. Lectin binding studies, sensitivity to certain glycosidases, and gel filtration analysis of glycans released by alkaline hydrolysis indicate that this glycoprotein contains several complex-type N-linked glycans as well as several highly sialylated O-linked glycans. Western blotting experiments show that various CD34 antibodies fail to efficiently detect desialylated and/or de-N-glycosylated forms of the antigen. Experiments involving the use of tunicamycin, together with metabolic labeling studies, strongly suggest that this structure "turns over" very slowly in vivo. The CD34 antigen is not detectably labeled by 32P-phosphate in vivo, nor are immune complexes containing it associated with phosphokinase activity in vitro. Sequential immunoprecipitation and Western blotting studies indicate that this antigen is not a member of the leukosialin/sialophorin family despite the fact that these molecules share several structural similarities. Partial amino acid analysis of highly purified CD34 antigen revealed no significant sequence similarity with any previously described structures.

Distribution and epitope analysis of the cell membrane glycoprotein (HPCA-1) associated with human hemopoietic progenitor cells.

Monoclonal antibodies My10, BI.3C5, 12.8, and ICH3 identify a monomeric cell surface glycoprotein (HPCA-1) of 100-120 kD, which is selectively expressed on human hemopoietic progenitor cells. Other tissues are nonreactive with the exception of capillary endothelia and basement membrane in some sites. In addition, the antigen can be detected on cell lines that exhibit characteristics associated with early T cell precursors. HPCA-1 is therefore associated with myeloid, B, and T lineage precursors. Sequential immunoprecipitation and Western blotting studies demonstrate that BI.3C5, ICH3, My10, and an antibody directed against endothelial cells, 188.27, all react with the same glycoprotein species, although the epitopes involved may be distinct. The epitope recognized by BI.3C5 is sialic acid dependent, whereas that recognized by ICH3 is not. The My10 epitope has partial sensitivity to neuraminidase. Competitive/additive binding experiments suggest that these epitopes, although probably distinct, may be closely associated.

Sensitive detection and enumeration of CD34+ cells in peripheral and cord blood by flow cytometry.

Peripheral blood stem cell autografts are increasingly used to reconstitute hematopoiesis after intensive, potentially marrow-ablative therapy. Assessment of autograft adequacy by enumeration of hematopoietic progenitors in colony-forming assays is handicapped by lack of reproducibility and prolonged assay time. Alternative approaches of graft assessment by flow-cytometric enumeration of stem/progenitor cells bearing the CD34 antigen can be hampered by low specificity and sensitivity. Here, we report a rapid and reliable multiparameter flow-cytometric approach to accurately enumerate CD34+ cells in peripheral blood (PB) mononuclear cells (MNCs). Total nucleated white blood cells (WBCs) are quantified by staining with fluorescein isothiocyanate (FITC)-conjugated CD45 antibody. Simultaneous staining by phycoerythrin (PE)-conjugated CD34 antibody defines an approximate number for the CD34+ progenitor/stem cell subfraction. When starting CD34+ cell numbers are low (0.01-0.5%), other nonspecifically stained leukocytes make accurate enumeration impossible. However, when the CD34+ fraction is analyzed for CD45 expression vs. side scatter (granularity), true CD34+ blast cells form a discrete cluster exhibiting low-density CD45 expression and low side-scatter characteristics. Cells within this "blast region" can be readily distinguished from lymphocytes, monocytes, granulocytes, and other events that can contaminate the CD34+ population. Here, we used this sensitive procedure to enumerate CD34+ cells in steady-state PB samples (0.03-0.09%), normal bone marrow (BM) aspirates, and umbilical cord blood collections (0.33-1.98%). This approach thus provides a means to analyze CD34+ cells in specimens from patients who have been extensively treated with chemotherapy and those undergoing PB stem cell mobilization with cytokines. Additionally, it is useful for assessment of CD34+ cells in a variety of clinical samples exhibiting perturbations of the hematopoietic progenitor/stem cell compartments.

Differential sensitivity of CD34 epitopes to cleavage by Pasteurella haemolytica glycoprotease: implications for purification of CD34-positive progenitor cells.

Our previous studies have shown that a unique glycoprotease from Pasteurella haemolytica specifically cleaves only proteins containing sialylated O-linked glycans. The hematopoietic progenitor cell antigen, CD34, which is heavily glycosylated with both N- and O-linked glycans, is readily cleaved by this protease. In this study, we demonstrate that the epitopes detected by five of the seven CD34 monoclonal antibodies are removed by the glycoprotease. The differential sensitivity of the CD34 epitopes to cleavage with either neuraminidase and/or glycoprotease establishes three classes of epitopes: 1) (class I) those identified by MY10, B1.3C5, 12.8, and ICH3 that are differentially affected by neuraminidase and removed by the glycoprotease; 2) (class II) the epitope detected by QBEND 10 that is removed only by the glycoprotease; and 3) (class III) those identified by TUK3 and 115.2 that are not removed by either enzyme. Cleavage of the 110-kd CD34 structure by the glycoprotease generates a major cell-bound fragment of about 75 kd, identified by the class III antibodies. We have also used the enzyme to improve the rapid recovery of CD34+ cells selected by immunomagnetic affinity techniques. In a preclinical model, we separated CD34+ KG1 cells with high yield (90%-95%) and high purity (94%-98%) from sham mixtures containing 50% CD34- cells. We also separated CD34+ blast cells from a patient in megakaryoblastic crisis of chronic myelogenous leukemia. In this case, the purity and yield were 93% and 94%, respectively. Enzyme treatment had no detrimental effect on cell viability, and the treated cells showed a normal quantitative expression and distribution of CD34 antigen as assessed with class III antibodies. We conclude that the P. haemolytica glycoprotease has potential to improve the isolation, from human bone marrow, of primitive hematopoietic cells that carry the CD34 antigen.

Optimizing the CD34+ and CD34+Thy-1+ stem cell content of peripheral blood collections.

We have previously described a sensitive and specific CD34 enumeration assay and report here a prospective analysis of 25 myeloma patients undergoing PBSC mobilization using this assay to determine the optimal days for collection of CD34+ and CD34+Thy-1+ cells after chemotherapy and growth factor mobilization. Correlations between frequency of peripheral blood CD34+ cells, circulating white blood cell (WBC) count, apheresis CD34+ cell count, nucleated cell count (NCC), and numbers of apheresis colony-forming units granulocyte/macrophage (CFU-GM) were determined. To assess levels of the more primitive subsets of CD34+ cells in the PBSC collections, coexpression of the Thy-1 antigen (CDw90) on CD34+ cells was also assessed. Marked heterogeneity was noted between patients with apheresis samples containing a median NCC of 4.2 x 10(8)/kg (range 1.3-8.1), median CFU-GM 17 x 10(4)/kg (range 0.15-32 x 10(4)/kg), and median CD34+ cell count of 1.39 x 10(6)/kg (range 0.02-6.6). The frequency of CD34+ cells in PBSC collections coexpressing Thy-1 was also heterogenous (6.2-50% of CD34+ cells), median 21.6%, mean 24.7 +/- 2%. The apheresis CD34+ cell count correlated with the peripheral blood CD34+ cell percentage (r = 0.71, p < 0.0001) but only weakly with the peripheral WBC. Apheresis CD34+Thy-1+ cell numbers correlated strongly with the circulating CD34+ cell numbers (r = 0.80), but no correlation was noted between these candidate stem cells and the peripheral WBC. In contrast, apheresis CFU-GM levels correlated most strongly with the peripheral WBC count (r = 0.61, p < 0.0001). The apheresis CD34+ cell count correlated with apheresis CFU-GM (r = 0.75, p < 0.0001) but not with the apheresis NCC. Apheresis CD34+Thy-1+ counts significantly correlated only with the apheresis CD34+ cell count and not with the apheresis CFU-GM or NCC. A higher percentage of circulating and apheresis CD34+ cells expressing Thy-1 were found on day 1 of collection, and the percentage of CD34+ cells expressing Thy-1 decreased on each subsequent day of measurement: median of 22% day 1 vs. 16.6% day 4, p = 0.04. This study therefore confirms that accurate quantitation of circulating CD34+ cells best predicts the optimal day for apheresis collection of CD34+ and CD34+Thy-1+ cells and is superior to the WBC count in this regard. Furthermore, the candidate stem cell (CD34+Thy-1+) subset is most prevalent during the earliest phases of CD34+ cell mobilization.

Differences in cell cycle kinetics of candidate engrafting cells in human bone marrow and mobilized peripheral blood.

Patients undergoing hematopoietic stem cell transplantation (HSCT) with mobilized peripheral blood (MPB) engraft quicker than those receiving bone marrow (BM). Our objective was to determine whether candidate engrafting cells--primitive hematopoietic progenitors (PHPs)--from MPB and BM exhibit different responses to cytokines that could explain this observation. We compared the cell cycle kinetics and ex vivo expansion of PHP-enriched cells obtained from MPB (n = 12) and BM (n = 10) by fluorescence-activated sorting of CD90+, AC133+ or CD38(dull) subsets of pre-selected CD34(+) cells. Cell cycle status, before and after 40 hours of serum-free culture with a cytokine cocktail, was assessed by multiparameter flow cytometry following incubation with Hoechst 33342 and pyronin Y. We found that 0.2% +/- 0.3% of MPB CD34(+)CD90(+) cells were in S/G(2)/M phases at hour 0, compared with 5% +/- 2.5% of those from BM (p = 0.0001), and 86.3% +/- 9.7% were in G(0), compared with 65.3% +/- 10% of those in BM (p = 0.0001). After 40 hours of culture, CD34(+)CD90(+) cells from MPB were more mitotically active than those from BM, with 29% +/- 4.9% in S/G(2)/M and 20% +/- 11.4% in G(0), compared to 19% +/- 6.5% (p = 0.001) and 39.2% +/- 22% (p = 0.027) of cells from BM. There was greater expansion of both total CD34(+) cells and the CD90(+) subset from MPB samples (p = 0.001 and 0.0001, respectively). Results from PHPs defined on the basis of AC133 expression correlated well with results obtained in CD90(+) subsets (r(2) = 0.81; p = 0.014).MPB PHPs appear to be primed for a greater acceleration in mitotic activity upon cytokine exposure. This qualitative difference may contribute to the earlier engraftment seen after HSCT using MPB grafts.

Efficient adenovirus-mediated gene expression in malignant human plasma cells: relative lymphoid cell resistance.

Although adenoviruses offer several potential advantages as gene transfer vectors, some hematopoietic cells, particularly lymphoid cells, are considered relatively resistant to adenovirus-mediated gene transfer. To examine the role of adenovirus-mediated gene transfer in the lymphoid malignancy multiple myeloma (MM), we used E1- and E3-deleted adenoviral vectors to infect myeloma and lymphoma cell lines and subsequently primary bone marrow plasma cells and lymphocytes from patients with MM. Adenoviral vectors expressing LacZ or luciferase (AdCA18) reporter genes were used initially. Subsequently, we studied adenoviral vectors expressing genes of potential value in therapeutic immunomodulation, i.e., CD80 (AdB7-1) and interleukin-2 (AdIL-2). A human plasma cell line (OCI-My5) infected with LacZ or AdB7-1 vectors expressed the corresponding gene product in 95% and 85% of exposed cells, respectively. Time course experiments indicated that maximum expression of adenoviral transgenes in plasma cells was reached 3 days after infection. IL-2 was detected in the supernatant of AdIL-2-infected plasma cells, was functional, and could be detected for at least 30 days after infection. In contrast, three lymphoma cell lines (OCI-Ly2, OCI-Ly13.2, and OCI-Ly17) were significantly less sensitive to adenovirus infection, with relatively low efficiencies of gene transfer even using high adenoviral titers: Surface CD80 expression (13-25% of infected cells) and positive LacZ staining (0-5% of infected cells). Indeed, expression of luciferase was 96-168 times higher in AdCA18-infected OCI-My5 cells than in the OCI-Ly2 lymphoma cell line. Similar patterns were observed in primary plasma cells and lymphocytes from 19 MM patient bone marrow samples. After infection with AdB7-1, increased levels of CD80 expression on CD38 bright bone marrow plasma cells were observed in 84% of patients, with a 33% average increase in the number of plasma cells expressing CD80. In contrast, although increased CD80 expression was also detected on AdB7-1-infected CD19+ B lymphocytes from 63% of the MM patients, an average of only 14% of the infected lymphocytes demonstrated increased expression of CD80. Circulating T lymphocytes could not be transduced with AdB7-1. The relative resistance of B and T lymphocytes to adenovirus-mediated gene transfer warrants further investigation. Adenoviral vectors can efficiently infect malignant plasma cells and may be useful vehicles for therapeutic gene transfer.

Identification of CD34+ subsets after glycoprotease selection: engraftment of CD34+Thy-1+Lin- stem cells in fetal sheep.

Epitopes on the CD34 molecule detected by some CD34 antibodies can be cleaved by a unique glycoprotease from Pasteurella haemolytica, which cleaves only glycoproteins rich in O-linked glycans. A method to isolate CD34+ cells from adult bone marrow was developed subsequently, in which CD34+ cells were isolated in high purity and yield following immunomagnetic bead selection and detachment with the glycoprotease. Using a variety of other cell-surface markers shown here to be insensitive to glycoprotease, committed progenitors of T lymphoid, B lymphoid, monomyeloid, megakaryoblastic, or erythroid lineages could be identified. Significantly, candidate hematopoietic stem cells (HSC) that are contained within a CD34+Lin- (CD2-, CD14-, CD15-, CD16-, CD19-) (or CD34+CD38-) subset expressing the Thy-1 antigen (CDw90), c-kit receptor (CD117), and CDw109 but lacking expression of CD71 and HLA-DR antigens also were detected. Functionally distinct subsets of glycoprotease-selected CD34+ cells were identified and subfractionated using flow cytometry and fluorescence-activated cell sorting (FACS). These subsets included candidate HSCs expressing the CD34+Thy-1+Lin- phenotype, which were sorted from a CD34+ fraction of a mobilized peripheral blood (MPB) sample. In a fetal sheep model, when CD34+Thy-1+Lin- cells were injected intraperitoneally, they were capable of homing to the marrow, where they generated long-term multilineage hematopoiesis and maintained human CD34+ cells, indicating that candidate HSC subsets of CD34+ cells selected with this highly specific enzyme were capable of engraftment in vivo. The ability to identify and purify virtually any phenotypically defined subset of glycoprotease-selected CD34+ stem/progenitor cells should facilitate the study of hematopoiesis in vitro and in animal models in vivo as well as the development of novel genetic techniques for the correction of specific blood cell disorders in humans.

Fetal bone marrow CD34+CD41+ cells are enriched for multipotent hematopoietic progenitors, but not for pluripotent stem cells.

We have investigated the expression of CD41a (gpIIbIIIa) on a subpopulation of human fetal bone marrow (FBM) CD34+ progenitor cells. Human FBM CD34+Lin- cells were subfractionated into CD41a+ and CD41a- subpopulations by flow cytometry. All the megakaryocyte colony-forming cells (CFU-MK) and almost all the burst-forming units-megakaryocyte (BFU-MK) were found within the CD41a+ subpopulation. In addition, a 14-fold greater number of granulocyte-macrophage colony-forming units (CFU-GM) and a five-fold greater number of mixed lineage progenitor cells (CFU-mix) were observed within the CD34+Lin-CD41a+ subpopulation compared to the CD34+Lin-CD41a- subpopulation. The high proliferative potential of CD34+Lin-CD41a+ cells was demonstrated by their capacity to expand in in vitro culture containing human plasma and recombinant Mpl ligand (thrombopoietin [Tpo]) with production of over 80% CD41b+ (gpIIb+) MKs. However, in long-term bone marrow cultures, the CD34+Lin-CD41a- population contained a significantly higher frequency of cobblestone area-forming cells (CAFC) than the CD34+Lin-CD41a+ population, indicating the presence of a primitive hematopoietic stem cell (HSC) population within the CD34+Lin-CD41a- subset. These data suggest that fetal CD34+Lin-CD41a+ cells are enriched for MK progenitor cells (CFU-MK and BFU-MK), myeloid progenitors, and CFU-mix but do not contain the more primitive CAFC.

CD109 is expressed on a subpopulation of CD34+ cells enriched in hematopoietic stem and progenitor cells.

CD109 is a monomeric cell surface glycoprotein of 170 kD that is expressed on endothelial cells, activated but not resting T-lymphocytes, activated but not resting platelets, leukemic megakaryoblasts, and a subpopulation of bone marrow CD34+ cells. Observing an apparent association between CD109 expression and the megakaryocyte lineage (MK), we sought to determine whether CD109 was expressed on MK progenitors. In fetal bone marrow (FBM), a rich source of MK progenitors, CD109 is expressed on a mean of 11% of CD34- cells. Fluorescence activated cell sorting (FACS) of FBM CD34+ cells into CD109+ and CD109- fractions revealed that the CD34+CD109+ subset contained virtually all assayable MK progenitors, including the colony-forming unit-MK (CFU-MK) and the more primitive burst-forming unit-MK (BFU-MK). The CD34+CD109+ subset also contained all the assayable burst-forming units-erythroid (BFU-E), 90% of the colony-forming units-granulocyte/macrophage (CFU-GM), and all of the more primitive mixed lineage colony-forming units (CFU-mix). In contrast, phenotypic analysis of the CD34+CD109- cells in FBM, adult bone marrow (ABM) and cytokine-mobilized peripheral blood (MPB) demonstrated that this subset comprises lymphoid-committed progenitors, predominantly of the B-cell lineage. CD109 was expressed on the brightest CD34 cells identifiable not only in FBM, but also in ABM and MPB indicating that the most primitive, candidate hematopoietic stem cells (HSC) might also be contained in the CD109+ subset. In long-term marrow cultures of FBM CD34+ cells, all assayable cobblestone area forming cell (CAFC) activity was contained within the CD109+ cell subset. Further phenotypic analysis of the CD34+CD109+ fraction in ABM indicated that this subset included candidate HSCs that stain poorly with CD38, but express Thy-1 (CD90) and AC133 antigens, and efflux the mitochondrial dye Rhodamine 123 (Rho123). When selected CD34+ cells were sorted for CD109 expression and Rho123 staining, virtually all CAFC activity was found in the CD109+ fraction that stained most poorly with Rho123. CD34+ cells were also sorted into Thy-1 CD109+ and Thy-1 CD109+ fractions and virtually all the CAFC activity was found in the Thy-1+CD109+ subset. In contrast, the Thy-1-CD109+ fraction contained most of the short-term colony-forming cell (CFC) activity. CD109, therefore, is an antigen expressed on a subset of CD34+ cells that includes pluripotent HSCs as well as all classes of MK and myelo-erythroid progenitors. In combination with Thy-1, CD109 can be used to identify and separate myelo-erythroid and all classes of MK progenitors from candidate HSCs.

Tools to cleave glycoproteins.

There are a variety of enzymes available that are able to cleave glycoproteins, including enzymes that are specific for carbohydrate-carbohydrate linkages, carbohydrate-protein bonds and the peptide backbone. Such enzymes are useful for determining the sites of glycosylation within proteins, and for releasing glycan structures for subsequent carbohydrate analysis. One protease has been identified as being specific for O-sialoglycoproteins and can be used to identify such molecules and their epitope regions. The lack of cytotoxicity and the narrow specificity of this enzyme provides an improved method for the immunomagnetic selection of human bone-marrow stem-cells.

Engraftment of gene-marked hematopoietic progenitors in myeloma patients after transplant of autologous long-term marrow cultures.

We conducted a phase I hematopoietic stem cell (HSC) gene-marking trial in patients undergoing autologous blood or marrow stem cell transplant for the treatment of multiple myeloma. Between 500 and 1000 ml of bone marrow was harvested from each of 14 myeloma patients and 1 syngeneic donor. A mean of 3.3x10(9) cells per patient were plated in 20 to 50 long-term marrow culture (LTMC) flasks and maintained for 3 weeks. LTMCs were exposed on days 8 and 15 to clinical-grade neo(r)-containing retrovirus supernatant (G1Na). A mean of 8.23x10(8) day-21 LTMC cells containing 5.2x10(4) gene-marked granulocyte-macrophage progenitor cells (CFU-GM) were infused along with an unmanipulated peripheral blood stem cell graft into each patient after myeloablative therapy. Proviral DNA was detected in 71% of 68 tested blood and bone marrow samples and 150 of 2936 (5.1%) CFU-GM derived from patient bone marrow samples after transplant. The proportion of proviral DNA-positive CFU-GM declined from a mean of 9.8% at 3 months to a mean of 2.3% at 24 months postinfusion. Southern blots of 26 marrow and blood samples were negative. Semiquantitative PCR analysis indicated that gene transfer was achieved in 0.01-1% of total bone marrow and blood mononuclear cells (MNCs). Proviral DNA was also observed in EBV-transformed B lymphocytes, in CD34+ -enriched bone marrow cells, and in CFUs derived from the latter progenitors. Gene-modified cells were detected by PCR in peripheral blood and bone marrow for 24 months after infusion of LTMC cells. Sensitivity and specificity of the PCR assays were independently validated in four laboratories. Our data confirm that HSCs may be successfully transduced in stromal based culture systems. The major obstacle to therapeutic application of this approach remains the overall low level of genetically modified cells among the total hematopoietic cell pool in vivo.

Flow cytometric enumeration and immunophenotyping of hematopoietic stem and progenitor cells.

Flow cytometric enumeration of CD34(+) hematopoietic stem and progenitor cells (HPC) is widely used to evaluate the adequacy of peripheral blood stem cell grafts and is also useful for planning the apheresis sessions needed to obtain these grafts. A state-of-the-art method to enumerate CD34(+) cells has been developed that makes use of a multiparameter definition of HPC, based on their light scatter characteristics and dim expression of CD45, utilizing fluorescent counting beads. This approach allows the absolute CD34(+) cell count to be determined directly from a flow cytometer. The method can be extended with a viability stain and additional markers for further immunologic characterization of CD34(+) cells, and has been successfully implemented in multicenter trials. Using such a standardized assay, it should be possible to define more accurately the lower threshold for a safe HPC graft in terms of short- and long-term hematopoietic reconstitution. Semin Hematol 38:139-147.

In vitro biosynthesis of the human cell surface receptor for transferrin.

The human cell surface receptor for transferrin is a transmembrane phosphoglycoprotein composed of two disulphide linked and apparently identical subunits of Mr 90 000. Using an affinity purified, polyclonal rabbit antibody, we have studied the in vitro biosynthesis of this receptor. The primary translation product, synthesised in a rabbit reticulocyte lysate programmed with human placental RNA, appears to have the same Mr (78 000) as the unglycosylated molecule immunoprecipitated from tunicamycin-treated cells. In the presence of a dog pancreatic microsomal system the cell free system accurately reproduces the glycosylation and the asymmetric transmembrane integration.