Ping Law

Mobilization of blood‐derived stem and progenitor cells in normal subjects by granulocyte‐macrophage‐ and granulocyte‐colony‐stimulating factors

BACKGROUND: It was previously reported that the combination of granulocyte‐macrophage‐colony‐stimulating factor (GM–CSF) and granulocyte‐CSF (G–CSF) for 4 days mobilized more primitive CD34+ subsets than did either G–CSF or GM–CSF alone.

Correlation between IL-3 receptor expression and growth potential of human CD34+ hematopoietic cells from different tissues.

CD123 (α‐subunit of IL‐3 receptor) expression on primitive and committed human hematopoietic cells was studied by multicolor sorting and single‐cell culture. The sources of cells included fetal liver (FLV), fetal bone marrow, umbilical cord blood, adult bone marrow and mobilized peripheral blood. Three subsets of CD34+ cells were defined by the levels of surface CD123: CD123negative, CD123low, and CD123bright. Coexpression of lineage markers showed that a majority of CD34+CD123bright cells were myeloid and B‐lymphoid progenitors, while erythroid progenitors were mainly in the CD34+CD123negative subset. The CD34+CD123low subset contained a heterogeneous distribution of early and committed progenitor cells. Single CD34+ cells from the CD123 subsets were cultured in a cytokine cocktail of stem cell factor, interleukin 3 (IL‐3), IL‐6, GM‐CSF, erythropoietin, insulin‐like growth factor‐1, and basic fibroblast growth factor. After 14 days of incubation, a higher cloning efficiency (CE) was observed in the CD34+CD123negative and CD34+CD123low fractions (37 ± 23% and 44 ± 23%, respectively) than in the CD34+CD123bright fraction (15 ± 21%). Using previously published criteria that colonies containing dispersed, translucent cells (dispersed growth pattern, DGP) were derived from primitive cells and that colonies composed solely of clusters were from committed cells, early precursors were distributed evenly in the CD34+CD123negative and CD34+CD123low subsets. When CD38 and CD90 (Thy‐1) were used for further characterization of CD34+ cells from FLV, CE increased from 37 ± 23% in CD123negative to 70 ± 19% in CD123negativeCD38− and from 44 ± 23% in CD123low to 66 ± 19% in CD123lowCD38−. No significant increase in CE or DGP progenitors was observed when CD34+ cells were sorted by CD90 and CD123. We concluded that: A) high levels of CD123 were expressed on B‐lymphoid and myeloid progenitors; B) early erythroid progenitors had little or no surface CD123, and C) primitive hematopoietic cells are characterized by CD123negative/low expression.

Retention of cellular properties of PBPCs following liquid storage and cryopreservation

BACKGROUND: G–CSF‐mobilized PBPCs are routinely cryopreserved within 24 hours of collection. The ability to hold PBPCs for extended time would offer increased flexibility for patients and hospitals. Retention of PBPC properties following overnight shipping, extended liquid storage at 1 to 6°C, and cryopreservation was evaluated.

Small peptide analogue of SDF-1α supports survival of cord blood CD34+ cells in synergy with other cytokines and enhances their ex vivo expansion and engraftment into nonobese diabetic/severe combined immunodeficient mice

The SDF‐1/CXCR4 axis has been implicated in the chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. We studied the effects of a SDF‐1 peptide analogue CTCE‐0214 on the survival of cord blood CD34+ cells in culture, expansion, and engraftment of expanded cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. Our results demonstrated that CTCE‐0214 synergized with thrombopoietin (TPO), stem cell factor (SCF), or flt‐3 ligand (FL) on the survival of stem and progenitor cells in culture. Adding CTCE‐0214 at a low concentration (0.01 ng/ml) for 4 days together with TPO, SCF, and FL significantly enhanced ex vivo expansion of CD34+ cells to subsets of primitive (CD34+CD38− cells, colony‐forming unit‐mixed [CFU‐GEMMs]), erythroid (CFU‐Es), myeloid (CFU‐GMs), and megakaryocytic (CD61+CD41+ cells, CFU‐MKs) progenitors, as well as their multilineage engraftment in NOD/SCID mice. Interestingly, the short exposure of expanded cells to CTCE‐0214 (100 and 500 ng/ml) for 4 hours did not increase the quantity of progenitor cells but enhanced their engraftment capacity. The proportion of CD34+ cells expressing surface CXCR4 was decreased, but the overall number of this population increased upon expansion. The small peptide analogue of SDF‐1 could be developed for ex vivo expansion and improving engraftment of cord blood transplantation.

Simultaneous administration of G-CSF and GM-CSF for re-mobilization in patients with inadequate initial progenitor cell collections for autologous transplantation

BACKGROUNDnA proportion of candidates for high-dose chemotherapy with autologous PBPC support (HDC-PBPCS) will not provide an adequate PBPC yield from their first mobilization. The value of re-mobilization and the best regimen for re-mobilization in these patients is unclear.nnnMETHODSnIn 23 patients who failed to provide > or = 3 x 10(6) CD34+ cells/kg after their first mobilization, PBPC were re-mobilized using a regimen of simultaneous administration of G-CSF and GM-CSF (10 microg/kg/day each) with leukaphereses (LP) starting Day 4 or 5 of CSF administration. Yields of WBC/kg, MNC/kg and CD34+ cells/kg/L of processed blood were compared between the first and second mobilization in each patient. The ability of the combined yield from the two mobilizations to achieve the desired threshold PBPC yield and the tolerability of the re-mobilization were determined.nnnRESULTSnThe re-mobilization regimen was well-tolerated and no patient discontinued the regimen because of toxicity. Median collected WBC/kg/L (1.37 x 10(7) versus 2.62 x 10(7), p = 0.0065), MNC/kg/L (0.77 x 10(7) versus 1.97 x 10(7), p = 0.0003), CD34+ cells/kg/L (1.64 x 10(7) versus 4.18 x 10(7), p = 0.001) were significantly higher after the second mobilization (G-CSF/GM-CSF combination). Percentage of CD34+ cells in the leukapheresis was also significantly higher after the second mobilization (median 0.104% versus 0.195%, p = 0.036). Twelve of 22 patients achieved the target PBPC dose (> 3 x 10(6)/CD34+ cells/kg) after two mobilizations (six patients achieved the target from the second mobilization alone). A further eight underwent HDC-PBPCS without achieving the target PBPC dose. These patients experienced a significant delay in neutrophil and platelet engraftment when compared with those patients achieving the target dose.nnnDISCUSSIONnThis study demonstrates that the combination of G-CSF and GM-CSF is an effective and tolerable method for re-mobilization of PBPC in patients who fail to provide an adequate yield from their first mobilization.

Peripheral blood progenitor cell mobilization with intermediate-dose cyclophosphamide, sequential granulocyte-macrophage–colony-stimulating factor and granulocyte–colony-stimulating factor, and scheduled commencement of leukapheresis in 225 patients undergoing autologous transplantation

BACKGROUND: Interpatient variability in the kinetics of peripheral blood progenitor cell (PBPC) mobilization is commonly seen with conventional chemotherapy‐based mobilization regimens. This necessitates the availability of leukapheresis (LP) facilities 7u2003days a week.

Mobilization of peripheral blood progenitor cells for human immunodeficiency virus–infected individuals

Gene therapy is becoming one of the most promising modalities for the treatment of acquired immunodeficiency syndrome. The purpose of this study was to investigate the mobilization and collection of peripheral blood progenitor cells from human immunodeficiency virus (HIV)-infected individuals using granulocyte colony-stimulating factor (G-CSF). A total of 10 patients (9 male, 1 female; median age 36.5 years) with varying circulating CD4+ cell counts (13.9-1467/microL) were administered 10 microg/kg G-CSF daily for 6 days. Peripheral white blood cells (WBCs), CD34+ cell counts, lymphocyte subsets, and plasma viremia were monitored before each G-CSF injection. An average sixfold increase in WBCs was observed, which stabilized on day 4 or thereafter. The level of CD34+ cells was increased by 20-fold, and did not differ between days 5 and 6. Smaller increases in CD4+, CD8+, and CD4+CD8+ cells were observed. HIV viral load, as measured by RNA copy number in plasma, was not significantly altered by G-CSF administration. The leukapheresis product (LP), collected on day 7, contained an average of 6.25+/-4.52 (mean +/- standard deviation) x 10(10) WBCs and 3.08+/-2.98 x 10(6) CD34+ cells/kg. The levels of different CD34+ cell subsets were similar to those in the LPs of G-CSF-mobilized healthy individuals from an earlier study. Primitive hematopoietic cells (CD38- and CD38-HLA-DR+ cells) were detected in LPs (1.19+/-0.46% and 0.87+/-0.23%, respectively, of CD34+ cells). All parameters (WBC counts, lymphocyte populations, CD34+ cells, and HIV-1 RNA copies) measured 3 weeks after leukapheresis returned to baseline values. The administration of G-CSF was well tolerated by the HIV patients; side effects included bone pain, headache, flulike symptoms, and fatigue. There were no correlations between baseline CD4+ cell count and the WBCs, mononuclear cells, or CD34+ cells collected in the LP. Similarly, no correlation existed between baseline CD4+ and CD34+ cells, peak CD34+ cells, or days to achieve peak CD34+ cell counts after G-CSF mobilization. Our results showed that: (1) maximal mobilization can be achieved after 4 days of G-CSF administration; (2) therapeutic quantities of hematopoietic cells can be collected and used for gene therapy; and (3) G-CSF administration is well tolerated and does not cause a clinically significant increase in viremia.

Mobilization of Allogeneic Peripheral Blood Progenitor Cells

It is possible to reliably obtain sufficient PBSC from most normal donors to perform allogeneic transplantation. The mobilization regimen, usually administration of a single daily dose of G-CSF at 7.5 to 10 micrograms/kg subcutaneously for 4 to 6 days, is tolerable with acceptable side effects. However, there is wide variability among individuals with respect to the extent of mobilization achieved by the regimen and the optimal timing of apheresis. Studies suggest that the likelihood of obtaining an adequate harvest of CD34+ cells, as defined locally may be enhanced by employing higher doses or different schedules of G-CSF, monitoring the mobilization and/or collection of PBPC, and using apheresis procedures processing 2 or more times blood volume. However, an optimal regimen for mobilization and harvesting for all donors has not yet been identified and a small percentage of donors may not mobilize adequately with G-CSF. Alternative regimens employing combinations of G-CSF and GM-CSF are available that may prove useful in such cases and novel cytokines that are even more effective than G-CSF in mobilizing stem cells are eagerly awaited. Based on currently available experience with normal donors, the short-term safety of G-CSF appears to be acceptable, however there exist several scenarios in which marrow harvesting may be preferable to G-CSF mobilization and apheresis collection of PBPC.

Cell analysis for hematopoietic stem/progenitor cell transplantation

Cytometric analysis has become an important aspect in the quality control of cells in all phases of hematopoietic cell transplantation. In the stage of donor conditioning the counting of stem and progenitor cells is important and several reliable single platform tests for CD34+ cells have become available recently. It has been shown, that the count of certain subsets of CD34 may predict best time for harvesting stem cells better than just CD34. In many cases manipulation of the cell sample after collection from the donor is necessary before the cells are adequate for transplantation. Characterization of the resulting cell preparations requires reliable quantitative analysis of a variety of cell types like the enumeration of T-cells at the level of one in ten thousand for some allogeneic transplantations. It is discussed how these clinical requirements will need a refinement of cytometric procedures to achieve adequate clinical decisions.