The relationships between transfused cell number of CD34+ cell subpopulations divided by HLA-DR and CD33 antibodies and hematopoietic recovery patterns after peripheral blood progenitor cell transplantation (PBPCT) subsequent to myeloablative chemoradiotherapy were investigated in 14 children with cancer. Both logarithm of transfused CD34+ cell number/106/kg and logarithm of transfused cell number/106/kg of the CD34+HLA-DR+CD33+ sub-population, which is supposed to be myeloid-committed cells, were correlated with myeloid recovery after PBSCT, though they were not correlated with erythroid or platelets recovery. On the other hand, logarithm of transfused cell number/106/kg of CD34+HLA-DR-CD33- subpopulation, which is supposed to be immature progenitor cells, was not correlated with myeloid recovery but correlated with erythroid recovery and platelet recovery. These results suggested that rapid myelopoiesis after PBPCT occurs following transfusion of sufficient numbers of myeloid-committed cells and complete hematopoietic reconstitution occurs after transfusion of sufficient numbers of immature hematopoietic progenitor cells.
To investigate immaturity of hematopoietic progenitor cells in umbilical cord blood mononuclear cells (CB-MNC), the formation of macroscopic colonies and mixedcell colonies was assayed by methylcellulose culture with various combinations of cytokines (stem cell factor [SCF], interleukin [IL]-3, IL-6, granulocytecolony stimulating factor [G-CSF], erythropoietin [EPO]) and compared with bone marrow (BM)-MNC. Moreover, distribution of the subpopulations divided by CD34, CD38, HLA-DR and CD33 was compared by flow-cytometry.Colonies derived from CB-MNC were so large that they could be observed with the naked eye and consisted of a variety of types of hematopoietic cells. Mixedcell colonies were formed to a much greater extent in CB-MNC than in BM-MNC. Addition of EPO, IL-3, and SCF had rapid effects on the growth of mixedcell colonies. The subpopulations of immature hematopoietic progenitor cells (CD34+, CD38-, HLA-DR-), which are supposed to be able to differentiate into hematopoietic precursors and stromal cells, were significantly higher in CB-MNC (8.7 f 6.6%) than in BM-MNC (0.0 f 0.1%; P
Ex vivo expansion of hematopoietic progenitor cells in the umbilical cord blood mononuclear cells (CB‐MNC) was investigated in liquid culture system with various combinations of cytokines (stem cell factor [SCF], interleukin [IL]‐3, IL‐6, granulocyte‐colony stimulating factor [G‐CSF], erythropoietin [EPO], and interferon [INF]‐γ). Non‐lineage‐committed hematopoietic progenitor cells and lineage committed hematopoietic progenitor cells were represented as CD34+CD38− and CD34+CD38+ subpopulations, respectively. Although absolute CD34+CD38− cell numbers decreased even in the presence of multicytokines, the combinations of SCF plus IL‐6 and SCF plus IL‐3 plus IL‐6 plus INF‐γ were significantly effective in maintaining CD34+CD38− cells than the other combinations (P < 0.05). After 4 weeks of culture, CD34+CD38− cells disappeared in all combinations of cytokines. Absolute CD34+CD38+ cell numbers increased in the presence of cytokines. Maximal expansion of CD34+CD38+ cells were observed in the combinations of SCF plus IL‐3 plus IL‐6 plus EPO (19.8 ± 3.3 ‐fold) and SCF plus IL‐3 plus IL‐6 plus G‐CSF (18.3 ± 2.6). The combination of SCF plus IL‐3 plus IL‐6 was also effective to expand CD34+CD38+ cells (15.8 ± 3.9). However, the expansion was transient and they decreased to zero within 3 weeks. In the combinations of SCF plus IL‐6 and SCF plus IL‐3 plus IL‐6 plus INF‐γ, maximal expansion was inferior to the others but CD34+CD38+ cells were maintained more than 4 weeks. These results suggested that the indication of CBT can be expanded into older children by ex vivo augmentation of CB hematopoietic progenitor cells using multi‐cytokines.
We compared the effects of various combinations of cytokines (stem cell factor [SCF], interleukin [IL] −3, IL‐6, granulocyte‐colony stimulating factor [G‐CSF], erythropoietin [EPO]) among the growth of human hematopoietic progenitor cells from cord blood (CB), bone marrow (BM), and peripheral blood mononuclear cells (MNC) mobilized by chemotherapy and G‐CSF (PB) in a semi‐solid medium. Macroscopic colonies, that were visible to the naked eye, were formed from PB‐MNC within 1 week even without cytokines. They consisted of blasts containing macrophage‐like cells with immature nuclei on Wright stain, and were strongly accelerated by IL‐3. Macroscopic colonies were also formed from CB‐MNC. However, they appeared after 1–3 weeks and synergistic effects of SCF with other cytokines, especially EPO, were prominent. Macroscopic colonies were not formed from BM‐MNC. Granulocyte‐colony stimulating factor was effective in increasing colony forming units of granulocyte macrophage from BM‐MNC and they appeared between 1 and 2 weeks. These results suggested that the quality of hematopoietic progenitor cells was different among blood sources. This might lead to different bone marrow recovery patterns after transplantation of each blood source. The appropriate cytokines should be added to evaluate their exact potential.
This retrospective study attempts to clarify the optimal timing for peripheral blood stem cell (PBSC) collection after conventional chemotherapy followed by granulocytecolony stimulating factor (G-CSF) administration. Leukapheresis was performed 32 times in nine children with various cancers during bone marrow recovery phase following transient pancytopenia after chemotherapy. (On two occasions, leukapheresis was excluded because many leukemic blasts were included.) When the number of white blood cells (WBC) exceeded 1.8 x 10lo/L after administration of G-CSF (200 pg/m2, continuous infusion), many more CD34 + cells were contained in the collected peripheral mononuclear cells (P > 0.02) and a sufficient number of PBSC for transplantation (t 10 x 108 CD34 + cellskg) was obtained after one run in 15 of 17 leukapheresis sessions. In contrast, sufficient PBSC were obtained only in one of 13 r~n S of leukapheresis when the number of WBC was < 1.8 x 10IO/L. The number of WBC on the day when PBSC were collected correlated with collected nuclear cell number (r = 0.60), but not with the CD34 + cell ratio. The ratio was higher only when both platelets and reticulocytes increased in parallel with WBc. We conclude that sufficient PBSC collection is possible after conventional chemotherapy using G-CSF, when hematopoietic recovery is parallel, without the use of high-dose chemotherapy.
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