CD34 ⁺ cell expansion and expression of lineage markers during liquid culture of human progenitor cells

Abstract

A 96-well-based suspension culture system for human hematopoietic progenitor cells has been developed to monitor the commitment and differentiation of CD34+ cells to specific lineages and the maintenance and expansion of CD34+ cells in vitro. The expression of maturation and lineage markers on the cells in culture was measured by enzyme-linked immunosorbent assay (ELISA). CD34+ cells were isolated from umbilical cord blood and fetal liver (90% purity) and were grown in liquid culture in 96-well plates for 10 days. The cells were then fixed with a glutaraldehyde-paraformaldehyde mixture, attaching the cells firmly to the plastic. An ELISA was performed, using appropriate primary antibodies directed against cell surface markers. The expression of four different lineage markers was measured: CD14 (monocyte), CD15 (neutrophil), platelet glycoprotein (GP) IIb/IIIa (CD41a, megakaryocyte) and glycophorin A (erythroid). The two-growth factor combination of interleukin 3 (IL-3) and stem cell factor (SCF) stimulated expression of CD14, CD15 and GP IIb/IIIa. Lineage-restricted growth factors such as erythropoietin (EPO), in combination with SCF, stimulated expression of glycophorin A. The three-factor combination of IL-3, SCF and EPO stimulated expression of all four lineage markers. Other multiple growth factor combinations all stimulated myeloid and megakaryocyte growth, as measured by ELISA and flow cytometry, but erythroid growth was present only when EPO was included in the growth factor mixture. In serum-free medium or plasma-containing medium, CD14 expression was markedly reduced, whereas glycophorin A expression was greatly elevated in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Tolerance induction by megadose hematopoietic progenitor cells: expansion of veto cells by short-term culture of purified human CD34+ cells

“…Considering that a large number of early myeloid progenitors can be expanded ex vivo by short-term cultures using different cytokine combinations, [25][26][27][28][29][43][44][45][46][47] we hypothesized that if the veto activity could be retained along the myeloid differentiation pathway, then a small fraction of the CD34 ϩ cells used for transplantation could provide a substantial source of veto cells. Our current results confirmed this hypothesis, demonstrating that it is possible to harvest about 28-to 80-fold more veto cells on culturing of purified CD34 ϩ cells for 7 to 12 days with an early-acting cytokine mixture including FL, SCF, and TPO.…”

“…[25][26][27][28][29] To test whether CD34 ϩ cells can be expanded ex vivo without loss of veto activity, CD34 ϩ cells were cultured for a short period (7-12 days) in the presence of an early-acting cytokine mixture including FL, SCF, and TPO 30 (concentrations of 50, 50, and 1 ng/mL, respectively). Phenotyping Responder cells (1 ϫ 10 6 /mL) were simultaneously cultured with irradiated stimulators (0.7 ϫ 10 6 /mL) from the donor of the CD34 ϩ cells (E) and a third party (‚) for 5 days in the absence (E,‚) or presence (F,OE) of CD34 ϩ cells added at a veto-to-responder ratio of 0.4:1.…”

A novel I-branching β-1,6-N-acetylglucosaminyltransferase involved in human blood group I antigen expression

“…Lineage-specific differentiation of hematopoietic cells was conducted from adult bone marrow CD34 ϩ cells, and cDNA from the cells was prepared according to the previous reports 17,18 -that is, purified CD34 ϩ cells were differentiated into the myeloid, erythroid, or megakaryocytic lineage using an optimal combination of hematopoietic growth factors for each lineage in serum-free liquid medium. Cells were cultured for 6 days in Iscove modified Dulbecco medium supplemented with 1% bovine serum albumin, 10 g/mL bovine pancreatic insulin, 200 g/mL human transferrin (BIT 9500; StemCell Technologies, Vancouver, BC, Canada), 40 g/mL human low-density lipoproteins (Chemicon International, Temecula, CA), and 10 Ϫ4 M 2-mercaptoethanol.…”

Post-chemotherapy and cytokine pretreated marrow stromal cell layers suppress hematopoiesis from normal donor CD34+ cells

“…Umbilical cord blood was collected and processed as previously described. 36 Enrichment of CD34 + bone marrow cells CD34 + cells were selected by positive immunomagnetic selection using a high-gradient magnetic separation column and MiniMacs CD34 progenitor cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). The purity of the recovered cells was determined by staining with CD34 (anti-HPCA-2) phycoerythrin (PE; Becton Dickinson, San Jose, CA, USA), and flow cytometry analyses demonstrated that purity was у90%.…”