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During the process of normal hematopoiesis, proliferation is tightly linked to maturation. The molecular mechanisms that lead to production of mature effector cells with a variety of phenotypes and functions from a single multipotent progenitor are only beginning to be elucidated. It is important to determine how these maturation events are regulated at the molecular level, because this will provide significant insights into the process of normal hematopoiesis as well as leukemogenesis. Transcription factors containing the highly conserved homeobox motif show considerable promise as potential regulators of hematopoietic maturation events. In this study, we focused on identification and characterization of homeobox genes of the HOX family that are important in regulating normal human myeloid differentiation induced by the hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF). We have identified three homeobox genes, HOX A5, HOX B6, and HOX B7, which are expressed during early myelopoiesis. Treating bone marrow cells with antisense oligodeoxynucleotides to HOX A5 resulted in inhibition of granulocytic/monocytic hematopoiesis and increased the generation of erythroid progenitors. Also, overexpression of HOX A5 inhibited erythroid differentiation of the K562 cell line. Based on these observations, we propose that HOX A5 functions as an important regulator of hematopoietic lineage determination and maturation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that stimulates the proliferation, maturation, and functional activity of myeloid cells in peripheral blood and bone marrow. Expression of GM-CSF is tightly regulated and is limited to cells stimulated directly (T cells, macrophages) or indirectly (fibroblasts, endothelial cells) by immune challenge. Several studies of the transcriptional control of GM-CSF expression have elucidated a region of the GM-CSF promoter that mediates positive regulatory activity in a number of cell types. This region contains a direct repeat of the sequence CATTA/T that extends from nucleotides -37 to -48 upstream of the start of mRNA synthesis. Although specific DNA:protein interactions have been shown within this region, neither the nature nor the number of nuclear factors responsible for these interactions have been characterized. In this study, we use DNase I footprinting analysis to demonstrate that point mutations, which inactivate the GM-CSF promoter, disrupt DNA:protein interactions within this region. By combined electrophoretic mobility shift and ultraviolet cross-linking analysis, we have detected several protein species that bind specifically to the positive regulatory sequence.

We and others have previously demonstrated that unrelated cyropreserved banked CB is an effective alternative source of allogeneic stem cells but is limited by delays in myeloid and platelet engraftment and immune reconstitution (Kurtzberg et al, NEJM 1996; Cairo et al, Blood 1997). CB nucleated (nuc) cell count, nucleated RBC and total CFU have been previously demonstrated to be associated with time to hematopoietic reconstitution (Rubinstein et al, NEJM 1998; Stevens et al, Blood 2002). More recently CB CD34+ content has been demonstrated to be significantly correlated to speed of engraftment and survival (Wagner et al, Blood 2002; Styczynski et al, BMT 2004). CB CD34+ subsets (CD38−, CD61+, CD90+) may also be critically important in predicting time to CB hematopoietic reconstitution, ex-vivo CB expansion and/or CB gene therapy. We investigated the relationships of CB CD34+ and CD34+ subsets with CB total CFU, CFU subsets, nuc cell, nuc RBC, ethnicity, gender, birth weight (BWT), gestational age (GA) and type of delivery in over 8,000 CB units. Briefly, CB was collected and processed (Fraser et al, J Hematother 1998) and aliquots were obtained for flow cytometry and in-vitro colony assays. Statistical analysis was based on multiple regression analysis and analysis or variance of logged counts. Approximately 35,000 maternal donors were screened, 20,700 consented, 16,700 CB collected and 8,730 CB units entered long-term cryopreserved storage. Ethnic distribution was 42% Caucasian, 21% Hispanic, 14% African-American, 11% Asian and 12% mixed. CB was collected by vaginal delivery (74%), at 39–40 weeks GA (61%) and 3–4000 Gm BWT (73%). Mean ± SD CD34+ and CD34+/38−, 61+, 90+ were 34±42, 3.4±4.8, 7.3±9.5 and 0.3±1.9 x 105, respectively. Mean SD of total CFU, BFU-E, CFU-GM and CFU-GEMM was 23.2±19.8, 13.3±3.1, 9.3±8.6 and 0.5±1.1, respectively. There was a significant correlation of log CD34+ with log nuc cell count and log nuc RBC count (R= 0.64, p

Several cell surface and cytoplasmic markers specific for the megakaryocyte-platelet lineage have been described. However, as yet, none of these has been shown to be expressed on cells earlier than the committed megakaryocyte progenitor, CFU-Meg. The present study was aimed at determining whether platelet lineage antigens could be detected on human pluripotential stem cells. Rabbit antiserum against human platelets (APS) was extensively absorbed with erythrocytes and either platelets, neutrophils, monocytes, or cells of the monocytic cell line U937. The anti-stem cell antibodies in each absorbed antiserum were determined using a complement-dependent cytotoxic assay for the pluripotential stem cell CFU-mix. Platelets alone removed anti- stem cell antibodies from APS. Absorption of APS with platelets from a patient with Glanzmann's thrombasthenia failed to remove the anti-stem cell activity, providing evidence for involvement of the platelet glycoprotein IIb/IIIa complex. Antiserum against purified glycoprotein IIb and against glycoprotein IIIa also recognized stem cells, resulting in reduced formation of mixed colonies. Absorption of these antisera with normal platelets removed the anti-stem cell activity, indicating that both IIb and IIIa are represented on stem cells. Hence, cell surface antigens specific for the stem cell-megakaryocyte-platelet pathway are expressed on the platelet glycoprotein IIb/IIIa complex.

Erythroid differentiation is mediated by several interacting factors which include the glycoprotein hormone erythropoietin (Epo), interleukin-3 (IL-3) in the mouse, and erythroid-potentiating activity (EPA) in humans. Each of these factors binds to specific cell surface receptors on responsive target cells, but the way in which these factors interact to modulate erythropoiesis is unknown. In the present study, we used the human erythroleukemic cell line K562 to examine expression and regulation of the receptor for Epo using 125I-labeled, bioactive recombinant human Epo. K562 cells expressed low numbers of a single class of high-affinity Epo receptors corresponding to 4 to 6 receptors per K562 cell (KD = 270 to 290 pmol/L). Treatment of K562 cell cultures with medium conditioned by the EPA-secreting cell line U937 (U937CM) increased receptor expression 2.6 to 3.5-fold to 13 to 17 receptors/cell (KD = 260 to 300 pmol/L). That all of the Epo receptor- potentiating activity in U937CM was accounted for by EPA was shown by a similar increase in Epo receptor expression on K562 cells with recombinant EPA. The effect of U937CM on Epo receptors was reversed by culturing cells in inducer-free medium for 3 days. Medium conditioned by the 5637 cell line had no effect on Epo receptors on K562 cells. In methylcellulose culture, U937CM and Epo acted synergistically to increase erythroid differentiation of K562. Similarly, U937CM stimulated human cord blood CFU-E growth under conditions in which Epo was limiting or in excess. Increases in Epo receptor expression on K562 cells and on CFU-E in response to EPA may mediate the effects of Epo on these cells.