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Autologous stem cell transplantation, in the setting of hematologic malignancies such as lymphoma, improves disease-free survival if the graft has undergone tumor purging. Here we show that flowing hematopoietic cells through pulsed electric fields (PEFs) effectively purges myeloma cells without sacrificing functional stem cells. Electric fields can induce irreversible cell membrane pores in direct relation to cell diameter, an effect we exploit in a flowing system appropriate for clinical scale. Multiple myeloma (MM) cell lines admixed with human bone marrow (BM) or peripheral blood (PB) cells were passed through PEFs at 1.35 kV/cm to 1.4 kV/cm, resulting in 3- to 4-log tumor cell depletion by flow cytometry and 4.5- to 6-log depletion by tumor regrowth cultures. Samples from patients with MM gave similar results by cytometry. Stem cell engraftment into nonobese diabetic-severe combined immunodeficient (NOD/SCID)/beta2m-/- mice was unperturbed by PEFs. Flowing cells through PEFs is a promising technology for rapid tumor cell purging of clinical progenitor cell preparations.

Differentiation of pluripotent embryonic stem (ES) cells is associated with expression of fate-specifying gene products. Coordinated development, however, must involve modifying factors that enable differentiation and growth to adjust in response to local microenvironmental determinants. We report here that the ephrin receptor, EphB4, known to be spatially restricted in expression and critical for organized vessel formation, modifies the rate and magnitude of ES cells acquiring genotypic and phenotypic characteristics of mesodermal tissues. Hemangioblast, blood cell, cardiomyocyte, and vascular differentiation was impaired in EphB4-/- ES cells in conjunction with decreased expression of mesoderm-associated, but not neuroectoderm-associated, genes. Therefore, EphB4 modulates the response to mesoderm induction signals. These data add differentiation kinetics to the known effects of ephrin receptors on mammalian cell migration and adhesion. We propose that modifying sensitivity to differentiation cues is a further means for ephrin receptors to contribute to tissue patterning and organization.

T-lymphocyte depletion of bone marrow grafts compromises engraftment, suggesting a facilitating mechanism provided by the T cells that has been shown to associate with CD8(+) but not CD4(+) T cells. Explanations for this phenomenon have focused on immune targeting of residual host cells or cytokine production. We provide evidence for an alternative mechanism based on cooperative effects on cell motility. We observed that engraftment of CD34(+) cells in a beta(2)-microglobulin-deficient nonobese diabetic/severe combined immunodeficiency (beta(2)m(-/-) NOD/SCID) mouse model paralleled clinical observations in humans, with an enhancing effect noted from the addition of CD8(+) cells but not CD4(+) cells. This correlated with CD8(+) augmentation of CD34(+) cell homing to the bone marrow in vivo and CD8(+) cell-associated increases of CD34(+) cell transmigration through a bone marrow endothelial cell line in vitro. The cooperative interaction was not sensitive to brefeldin A inhibition of protein secretion. However, cytochalasin D-induced inhibition of CD8(+) cytoskeletal rearrangements abrogated CD34(+) transendothelial migration and impaired CD34(+) cell homing in vivo. CD8(+) cells did not migrate in tandem with CD34(+) cells or alter endothelial barrier integrity; rather, they affected phosphotyrosine-mediated signaling in CD34(+) cells in response to the chemokine stromal derived factor-1alpha (SDF-1alpha). These data demonstrate cell-cell cooperativity between different cell types in mediating chemotactic events and provide one potential explanation for the clinically observed effect of CD8(+) cells on bone marrow transplantation. This modification of cell migration by neighboring cells provides broad possibilities for combinatorial effects between cells of different types to influence cell localization.

Hematopoietic stem cells sequentially pass through a series of decision points affecting self-renewal or lineage-specific differentiation. Notch1 receptor is a known modulator of lineage-specific events in hematopoiesis that we assessed in the context of in vivo stem cell kinetics. Using RAG-1(-/-) mouse stems cells, we documented increased stem cell numbers due to decreased differentiation and enhanced stem cell self-renewal induced by Notch1. Unexpectedly, preferential lymphoid over myeloid lineage commitment was noted when differentiation occurred. Therefore, Notch1 affects 2 decision points in stem cell regulation, favoring self-renewal over differentiation and lymphoid over myeloid lineage outcome. Notch1 offers an attractive target for stem cell manipulation strategies, particularly in the context of immunodeficiency and acquired immunodeficiency syndrome.