Showing papers by "Jamie Case published in 2001"

Characterization of cytokine interactions by flow cytometry and factorial analysis.

Abstract: Background Multiple cytokines are required for the growth and development of hematopoietic cells. The effect of many cytokines depends on the activity of other signaling pathways. These interactions are quantified using factorial experimental design and analysis. Methods Human umbilical cord blood (HUCB) CD34+ cells were cultured in fully defined media containing various combinations of recombinant cytokines as defined by resolution IV factorial (2) or full factorial (24) design experiments. The cytokines studied were stem cell factor (SCF), interleukin (IL)-3, megakaryocyte growth and development factor (MGDF), granulocyte-colony stimulating factor (G-CSF), Flt-3 ligand, IL-6, IL-11, and erythropoietin (EPO). In vitro cell divisions were tracked by staining CD34+ cells with 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, followed by flow cytometric analysis at 4 days of culture. In separate experiments, lineage commitment and differentiation were determined at 7 days by immunophenotype. Results In addition to the main effects of single cytokines, cytokine interactions were identified. There was a negative interaction between IL-3 and MGDF that resulted in a less than additive effect of these factors on erythroid and megakaryocytic development. The effect of Flt-3 ligand and SCF factor on CD34+ cell production was also less than additive, although the response to both cytokines was greater than single cytokines. The only positive interaction that was identified was between EPO and SCF, which resulted in the synergistic production of erythroid cells. Conclusions Factorial analysis provides a powerful methodology to study the integration of multiple signals at the cellular and molecular level. Cytometry 43:69–81, 2001. © 2001 Wiley-Liss, Inc.

Prior Cryopreservation of Ex Vivo-Expanded Cord Blood Cells Is Not Detrimental to Engraftment as Measured in the NOD-SCID Mouse Model

Abstract: Cytokine-mediated expansion has been proposed and successfully used to facilitate engraftment post transplantation. This study examined whether cryopreservation following expansion has a detrimental effect on the ability of cells to engraft, using the NOD-SCID mouse model. Cord blood (CB) CD34+ cells were incubated for 7 days with stem cell factor (SCF), flt-3 ligand (FL), and megakaryocyte growth and development factor (MGDF). Expanded CD34+ cells were transplanted into NOD-SCID mice either fresh or following cryopreservation and thawing. After thawing, recovery of nucleated cells was 94%, of CD34 cells was 63%, and of day-14 progenitors was 17%. The loss of day-14 progenitor cells among the thawed expanded cells did not influence the kinetics of human engraftment in the mouse. Bone marrow (BM) of mice transplanted with thawed expanded CD34+ cells (14 ± 3.9%) showed significantly higher levels of human engraftment than mice transplanted with fresh expanded CD34+ cells (1.5 ± 0.5%, p = 0.0064). Thawed exp...

Prolonged ex vivo culture of cord blood CD34+ cells facilitates myeloid and megakaryocytic engraftment in the non‐obese diabetic severe combined immunodeficient mouse model

Abstract: A clinical goal for ex vivo expansion of cord blood (CB) CD34(+) cells is to shorten the period of neutropenia and thrombocytopenia following myeloablative therapy and transplantation. Prolongation of cytokine expansion leads to the production of greater numbers of cells, and should have an impact on neutrophil and platelet recovery. Furthermore, expansion of CD34(+) cells should support the continued production of neutrophils and platelets in the 6-week period following transplantation. We tested these hypotheses by characterization of the kinetics (human CD45(+) cells in the blood) and phenotype (CD45, CD34, CD61, CD33, CD19 and CD3) of human engraftment in the non-obese diabetic severe combined immunodeficient mouse (NOD-SCID) following 7 or 14 d of ex vivo expansion of CB CD34(+) cells. Mice transplanted with 14 d cells showed greater percentages of human CD45(+) cells in the blood, bone marrow and spleen than mice transplanted with unexpanded cells or 7 d cells. Prolonging cytokine exposure of CD34(+) cells and transplantation with increasing numbers of input cells facilitated the production of absolute numbers of CD34(+), CD33(+), CD61(+) and CD19(+) cells in vivo. Furthermore, analysis of SCID engrafting potential showed that prolongation of culture duration facilitates in vivo expansion of CD45(+), CD34(+) and CD19(+) cells after transplantation. It is anticipated that prolonged (2 weeks) ex vivo culture of CB will have a beneficial clinical effect.

Research Report Prior Cryopreservation of Ex Vivo-Expanded Cord Blood Cells Is Not Detrimental to Engraftment as Measured in the NOD-SCID Mouse Model

Abstract: Cytokine-mediated expansion has been proposed and successfully used to facilitate engraftment post transplantation. This study examined whether cryopreservation following expansion has a detrimental effect on the ability of cells to engraft, using the NOD-SCID mouse model. Cord blood (CB) CD34 1 cells were incubated for 7 days with stem cell factor (SCF), flt-3 ligand (FL), and megakaryocyte growth and development factor (MGDF). Expanded CD34 1 cells were transplanted into NODSCID mice either fresh or following cryopreservation and thawing. After thawing, recovery of nucleated cells was 94%, of CD34 cells was 63%, and of day-14 progenitors was 17%. The loss of day-14 progenitor cells among the thawed expanded cells did not influence the kinetics of human engraftment in the mouse. Bone marrow (BM) of mice transplanted with thawed expanded CD34 1 cells (14 6 3.9%) showed significantly higher levels of human engraftment than mice transplanted with fresh expanded CD34 1 cells (1.5 6 0.5%, p 5 0.0064). Thawed expanded CD34 1 cells had significantly higher SCID Engrafting Potential (SEP) than freshly expanded CD34 1 cells (p , 0.001). Results suggest that prior cryopreservation does not prevent expanded cells engrafting in NODSCID mice.