Background In 1877, Paul Ehrlich devised a system that identified hematopoietic stem cells (HSCs). A century later, a system for the in vitro culture of HSCs was devised. Over the past four decades, great progress in HSC biology has resulted from murine models. Unfortunately, human HSC differentiation models have been difficult to produce. Specifically, the in vitro culture of polymorphonuclear leukocytes (PMNs) from human HSCs has been difficult, and to date, specific methods for this culture have not been defined. Here we describe a precise model for reliable differentiation of PMNs from human umbilical cord blood stem cells.
Methods HSCs were isolated from human umbilical cord blood from full-term neonates. The cord blood underwent magnetic bead-mediated positive selection for CD34 HSCs. HSCs were cultured in X-Vivo 20 with 10% PHS for 16 days. The media was augmented with SCF 100 ng/mL and IL-3 10 ng/mL for the initial 8 days, and G-CSF 20 ng/mL for the entire culture. The cell population was purified by magnetically selecting CD16 cells. Cell morphology was verified with Wright-Giemsa staining and immunocytochemistry (ICC). Cell surface markers were verified with flow cytometry. Functional assay of respiratory burst was performed using dihydrorhodamine (DHR) and phorbol myristate acetate (PMA) to determine stimulation index. Finally, adhesion characteristics were verified with a P-selectin-mediated cell adhesion assay.
Results Morphologic evaluation demonstrated cultures with a high percentage of leukocytes with multilobed nuclei, consistent with mature PMNs. ICC probing for CD18 confirmed the presence of this PMN-specific adhesion molecule. Flow cytometric analysis indicated that the cultured cells express CD16 and CD18, indicating the presence of mature neutrophils. Respiratory burst activity following DHR/PMA assay indicated intact free radical production. Adhesion of cells to P-selectin-coated plates demonstrated integrity of P-selectin/PSGL-1 adhesion.
Conclusions These results demonstrate that mature and functional PMNs can be efficiently cultured from human umbilical cord blood stem cells. Although HSC culture of mature human PMNs has been difficult for a generation of cell biologists, this investigation demonstrates detailed methodology for consistent culturing of PMNs from human HSCs. The utility of this model to delineate ontogenic events in PMN differentiation will afford greater understanding of variable phenotypes encountered in human disease.
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