Individual stem cell quality in leukapheresis products is related to the number of mobilized stem cells

Abstract

Peripheral blood stem cells (PBSC) are used for stem cell support in patients after intensive chemotherapy and generally permit faster hematopoietic recovery than bone marrow. The development of different protocols for chemotherapy conditioning, mobilization, and ex vivo manipulation of PBSC may potentially lead to loss of primitive hematopoietic stem cells or reduction of their quality. To characterize the frequency of different stem cell subsets and their quality per mobilized PBSC, we have studied 47 leukapheresis products (LPs) of 21 cancer patients using stroma-dependent long-term culture (LTC) and limiting dilution-type cobblestone area forming cell (CAFC) assays. A large variation in CAFC week-type frequencies between the LPs was observed. The frequencies of CAFC week 2 as a tentative indicator of progenitor cells and transiently repopulating hematopoietic stem cells ranged from 0.89 to 205 per 10(5) mobilized nucleated cells and the frequencies of more primitive CAFC week 6 varied between 0.37 and 48. The average total colony-forming cell (CFC) production per CAFC at week 6 varied between 1.2 and 730, as determined in parallel LTC. In contrast to LPs, bone marrow samples generated 4.2 to 48 CFC per CAFC at week 6. Notably, a poor stem cell quality was consistently found in LPs that contained less than 5,000 CAFC week 6 per kilogram of body weight. Frequency analyses of CFCs, CAFC subtypes, and immunophenotypic subsets showed a good level of mutual correlation, suggesting identical mobilization kinetics of different stem cell subsets. The premobilization chemotherapy intensity was directly correlated with both decreasing frequency and quality of the CAFC week 6 in LPs. The frequency of CFCs, immunophenotypic subsets, and CAFC subsets transplanted and the transplant quality as determined in LTC assays was related to the neutrophil and platelet recovery time after PBSC transplantation. Although the number of progenitor cells transplanted and the in vitro transplant quality showed the best correlation with early hematopoietic recovery, the data did not permit determination of which stem cell subsets are predominantly responsible for early posttransplantation recovery. As a result, frequency and quality analysis of stem cell subsets may be a useful tool to monitor and calibrate the efficacy of novel mobilization regimens and ex vivo manipulation of PBSC.

Delineation of Cell Cycle State and Correlation to Adhesion Molecule Expression of Human CD34+Cells from Steady‐State Bone Marrow and Peripheral Blood Mobilized Following G‐CSF‐Supported Chemotherapy

“…The proliferative activity of the BM-and PB-derived CD34 + cells was assessed in CFC assays and in a group of 6 of the 14 patients in stroma-dependent long-term cultures plated after two and six weeks of cultivation as well as in a limiting dilution set-up with the direct visual endpoint cobblestone area formation two and six weeks after overlay. The CAFC frequencies determined here were in the previously reported ranges for BM and LP samples [22,30] (data not shown). The mean concentration of lineage-committed and primitive progenitor cell subsets contained in the LP CD34 + cells tended to be greater than that contained in the BM CD34 + cells as has been found by other investigators that compared normal BM and LP of tumor patients [30] (Table 2).…”

“…The CAFC frequencies determined here were in the previously reported ranges for BM and LP samples [22,30] (data not shown). The mean concentration of lineage-committed and primitive progenitor cell subsets contained in the LP CD34 + cells tended to be greater than that contained in the BM CD34 + cells as has been found by other investigators that compared normal BM and LP of tumor patients [30] (Table 2). Significance was reached in the week-2 replate (p < 0.05).…”

Separation of G‐CSF‐mobilized PBSC transplants by counterflow centrifugal elutriation: modest enrichment of CD34+ cells but no loss of primitive haemopoietic progenitors

“…However, in contrast to what has been found for human bone marrow cells (Wagner et al, 1995), these small CD34 þ cells were not enriched with CD34 þ /CD38 ¹ cells. Since nearly all CD34 þ cells were CD38 þ we also analysed the CD38 lo population, which also contains many cells capable of long-term haemopoietic activity in vitro (Breems et al, 1996;Reems & Torok Storb, 1995), although these may be of a less immature state of differentiation (Hao et al, 1995;Prosper et al, 1996). Moreover, expression of Thy-1, which also has been shown to mark a subpopulation of CD34 þ cells enriched for primitive stem cells in bone marrow (Craig et al, 1993), cord blood (Mayani & Lansdorp, 1994) and in PBSC (Murray et al, 1995;Humeau et al, 1996), was similar in both elutriation fractions.…”

“…The frequencies of CAFC clones of normal appearance observed in cultures inoculated with cells from lymphocyteenriched CCE fractions decreased during the course of the cultures. Week type 6 CAFC, which, as in the well-validated murine CAFC-assay, may be considered as indicators of stem cells capable of long-term haemopoietic reconstitution (Breems et al, 1994(Breems et al, , 1996Murray et al, 1995;Ploemacher et al, 1993;Van der Loo et al, 1994;Ploemacher, 1994), were nearly exclusively recovered in the elutriation fractions containing the large cells. These results indicate that primitive G-CSF mobilized stem cells were not separated from colony-forming units in the CCE procedure used in this study.…”

“…CAFC frequencies were determined in a limiting dilution type culture assay by inoculating varying numbers of cells (12 dilutions, 2-fold apart, with 15 wells per dilution) in microwell plates on adherent layers of the murine preadipose cell line FBMD-1 as described by Breems et al (1994Breems et al ( , 1996. The percentages of wells with at least one cobblestone area beneath the stromal layer were determined at weeks 2, 4 and 6 and CAFC frequencies calculated using Poisson statistics.…”