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Autologous peripheral blood progenitor cell (PBPC) transplantation is now commonly used in children. The ontogenic differences in haematopoiesis published in recent years suggest differences in the categories of mobilized PBPCs between children and adults. We investigated the frequency and distribution of mature progenitor cells (colony-forming cells, CFCs) and primitive progenitor cells [CD34+ CD38− and CD34+ Thy-1+ cells, long-term culture-initiating cells (LTC-ICs)] in children and adults mobilized using granulocyte colony-stimulating factor alone. We found similar proportions of granulocyte colony-forming units (CFU-G) and/or macrophage CFUs (CFU-M), mixed lineage CFUs (CFU-Mix) and megakarocyte CFUs (CFU-Mk), CD34+ CD38− and CD34+ Thy-1+ cells, and LTC-ICs (16·5 ± 3·5 vs. 10·65 ± 5 per 104 CD34+ cells), which produced the same number of CFCs (5 ± 1 vs. 6 ± 1 CFCs/LTC-ICs) in PB CD34+ cells from children and adults. However, we noted a higher proportion of erythroid blast-forming units (BFU-E) in PB CD34+ cells from adults (× 1·5, P = 0·003). Using cord blood as a third ageing point, we observed an inverse age-related propensity for commitment to the monocyte/macrophage lineage that was still found after normalizing the data per body weight and processed blood mass. This ontogeny-related programming was detected from the LTC-IC level, which produced 1·7 times more CFU-M in children than in adults (P = 0·048). These subtle differences in commitment between children and adults, shown here for the first time, are of interest for the in vitro manipulation of PBPCs and, in particular, for application in adoptive immunotherapy in children.
Accelerated haematopoietic recovery after peripheral blood progenitor cell (PBPC) transplant has prompted the increasing use of autologous PBPCs in children (Deméocq et al, 1994; Takaue et al, 1995; Diaz et al, 1996). Allowing for the differences in conditioning regimens and chemotherapeutic protocols, the relatively satisfactory outcome in children compared with adults (Beelen et al, 1997; Woolfrey et al, 1998), in particular for immunological reconstitution (Small, 1996), may be partly explained by variations in the physiological properties of haematopoietic stem cells. Several observations support such an explanation. First, one ontogeny-related cell feature is that the proliferative and self-renewal potential of progenitor/stem cells decreases from embryonic to adult age (Lansdorp et al, 1993; Lu et al, 1993; Rebel et al, 1996). A very recent study has reported a continuous decline in the function of circulating mature progenitor cells from birth (Marley et al, 1999). Hence, the efficiency of the mobilization regimen may differ between children and adults. Second, the telomeres, the clipping of which during successive cell divisions is taken as an indicator of cell ageing, shows age-associated shortening in human mature blood cells and progenitor/stem cells (Vaziri et al, 1994; Lansdorp, 1995; Rufer et al, 1999). Third, lower success rates after using transplantation/recipient from older donors have been observed. Only a few comparative studies of steady-state (Geissler et al, 1986) or mobilized PBPCs (Kawano et al, 1998) from children and adults have been published.
Here, we measured the frequency of mature and primitive progenitor cells, that may play a role in both early and late haematopoietic grafting, detected as clonogenic progenitor cells, and as CD34+ CD38− cells (Terstappen et al, 1991; Gonzalez-Requejo et al, 1998; Henon et al, 1998), CD34+ Thy-1+ cells (Craig et al, 1993; Murray et al, 1995; Humeau et al, 1996) and long-term culture-initiating cells (LTC-ICs), in granulocyte colony-stimulating factor (G-CSF)-mobilized PBPCs from both children and adults. As higher G0/G1 cell cycle status (Ponchio et al, 1995; Roberts & Metcalf, 1995; Leitner et al, 1996; Lemoli et al, 1997; Uchida et al, 1997; Croockewit et al, 1998) and downmodulation of adhesion molecules (Asosingh et al, 1998) reported in PBPCs compared with bone marrow (BM) cells argues for substantial functional differences between circulating and non-circulating cells, we considered that cord blood progenitor cells (CBPCs) represented a logical reference point for studying PBPCs in ageing, although CBPCs were not mobilized by high doses of G-CSF.
This first comparative study of G-CSF-mobilized PBPCs taken from children and adults revealed subtle age-related differences in the intrinsic regulation of commitment that are of interest for in vitro manipulation of PBPCs.
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To compare the biological properties of PBPCs mobilized by G-CSF alone in adults and in children, we first evaluated the proportion of mature progenitors, i.e. CFU-G, CFU-M, CFU-GM, BFU-E, CFU-Mix and CFU-Mk, in the CD34+ cell population initially present in the MNC suspension collected from each leukapheresis or separated from cord blood. The proportions of CD34+ cells present in MNCs able to generate granulocyte and/or macrophage (CFU-G + CFU-M + CFU-GM), mixed and Mk colonies were similar in adult and child collections. This observation is consistent with the results of Kawano et al (1999) concerning the CD34+ cells and CFU-GM. However, we observed a significantly higher clonogenic erythroid progenitor proportion in adults, representing the majority of all the clonogenic progenitors in mobilized CD34+ cells. We already made this observation for data expressed per 2 × 105 MNCs (not shown), but it became more evident when we expressed the data per 1 × 103 CD34+ cells. We consider that the normalization of the numbers of CFCs according to the initial number of plated CD34+ cells avoids an apparent higher clonogenicity of MNCs owing to the higher percentage of CD34+ cells, as was the case, e.g. for the CB MNC collections, for which we evaluated the biological properties as a third ontogenic point of reference of the circulating immature cells. We observed a significantly higher proportion of CD34+cells in CB MNCs, but the clonogenicity of the circulating unselected CD34+ cells towards the categories of non-lymphoid lineages was similar to that of the mobilized PB unselected CD34+ cells in both adults and children, even though the CB colonies were bigger. The numbers of BFU-E and CFU-Mk were higher in CB after calculating per 2 × 105 MNCs, but no different after normalizing per 1 × 103 input CD34+ cells. These results conflict with some reports (Ho et al, 1996) in which the efficiency of clonogenicity was calculated per plated MNCs and not per exact seeded CD34+ cell number. The high occurrence of BFU-E in adults and children seems at variance with the studies reporting that G-CSF treatment increases, in particular, the number of G and/or M progenitors in peripheral blood (Prosper et al, 1996). We also observed a marked effect of G-CSF on CFU-GM mobilization, the BFU-E/CFU-GM ratio falling from 4·60 in the steady-state to 1·8 post G-CSF in adults, and from 1·70 to 1·23 in children (data not shown). This already published (Geissler et al, 1986; Ho et al, 1996; Suzuki et al, 1998) preferential commitment to an erythroid lineage of PB progenitor cells, particularly in the adults in this study, remains unexplained. Although the series were as homogeneous as possible (identical mobilization regimens and techniques of progenitor determination), we cannot absolutely exclude a secondary effect of the different prior chemotherapy and conditioning regimens in adults and children. However, this finding might be explained by the physiology of erythropoiesis in adults and children. Children show a relative haemoglobin deficit (Dallman, 1977; Oski, 1982; Hinchliffe, 1992) until 12 years of age, associated with a relative negative iron balance (Saarinien & Siimes et al 1977; Dallman et al, 1993; Oski et al, 1993), and it is possible that this context does not favour the mobilization of BFU-E.
Next, we measured the proportions of primitive progenitors, i.e. CD38− and Thy-1+ subsets, and LTC-ICs. We observed a similar proportion of CD34+ Thy-1+ and CD34+ CD38− subsets between adult and paediatric PBPCs. The LTC-ICs were mobilized at the same levels in peripheral CD34+ cells from both adults and children and produced the same number of CFCs. The proportions of CD34+ CD38− cells and LTC-ICs were higher (× 1·5 and × 7 respectively) in CB CD34+ cells. CB LTC-ICs produced about three times more CFCs after 5 weeks long-term culture. This observation agrees with already published data (Van Epps et al, 1994). In this study, we confirmed that the high proliferation potential of LTC-ICs observed at birth is lost early in life, which argues for the already reported early decline of some haemopoietic progenitor cell functions from birth (Geissler et al, 1986; Migliaccio et al, 1996; Li et al, 1999). In contrast to other reports, we observed a lower CD34+ Thy-1+ subset proportion in CB CD34+ cells, possibly owing to a relative overexpression of Thy-1 antigen on G-CSF-primed CD34+ cells from adults and children (Murray et al, 1995, 1996). There are variable reports in the literature concerning the expression of Thy-1. The results of this study might be explained by a higher heterogenicity of CD34+ Thy-1+ from G-CSF-mobilized cells in both children and adults (Humeau et al, 1996), revealing a possible overexpression of Thy-1 by the more mature progenitor cells.
Finally, we calculated the total number of each category of progenitors in one leukapheresis collection and divided it by body weight to evaluate the graft progenitor content in the same way. We did not observe any significant difference between the absolute numbers of mature and primitive progenitors collected from adults and children. We can relate these data to similar published results of the comparative quantification of CD34+ and CFU-GM in children and adults (Kawano et al, 1998, 1999). The published decrease in the steady-state circulating CFCs with age (Egusa et al, 1998; Marley et al, 1999) has no evident impact on the harvesting of the progenitor cells determined in our study.
Alternatively, when we analysed the production of myeloid CFCs according to whether they belonged to G and/or M lineage, as identified in short-term culture, we observed that the PB CD34+ from children produced a higher number of CFU-M accompanied by a correspondingly lower number of CFU-G, suggesting a possible intrinsic feature at the CFU-GM level. Interestingly, the values lay between adult and cord blood values. Although the overall number of G and/or M progenitors from CB was similar to that observed from the child and adult collections, the distribution of the CFU-G, CFU-M and CFU-GM revealed the highest number of CFU-M and CFU-GM and the lowest number of CFU-G in the CB group among the three series. We observed a similar profile of the distribution of myeloid CFCs derived from LTC-ICs, with an age-related decrease in the proportion of CFU-M-derived LTC-ICs. The propensity to produce monocyte/macrophage lineage cells in culture has already been underlined in cord blood (Lu et al, 1993) and in fetal liver (Nicolini et al, 1999). This propensity does not seem be owing only to accessory cells (Muller et al, 1996; Fietz et al, 1999), but also to intrinsic programming because of its ontogenically related intensity detected from the LTC-IC stage, and shown for the first time in this study. The physiological significance of this finding is unclear. The involvement of the monocyte/macrophage lineage, considered as occurring in the microenvironment, in physiological processes such as haematopoiesis and, in particular, erythropoiesis, indicates a role for it in the development of haematopoiesis during the first stages of life. Furthermore, we now know that this lineage, including in fetal tissues and cord blood, can produce dendritic cells (DCs) (Triozzi & Aldrich, 1997; Banyer & Hapel, 1999), so opening perspectives for the in vitro production of DCs in children. The specialization of the monocyte/macrophage filiation in some tissues suggests that they could be more critical at a fetal age and even in childhood, even perhaps in organs during development. By calculating the number of progenitors/kg body weight, we lost the significant difference in monocyte/macrophage commitment. However, after normalization of the data by dividing by the number of treated blood masses, although we obtained about the same harvested number for each category of progenitor cells, we observed a higher number of CFU-M/kg collected from one child blood mass processed during one leukapheresis, which means that the higher blood volume processed during the leukapheresis procedure in adults compensated for the lower number of harvested CFU-M. We could not identify any dramatically different distribution of G-CSF-mobilized progenitors between adults and children, which could explain the apparently better outcome of haematopoietic transplantation in younger patients (Beelen et al, 1997; Rubinstein et al, 1998). However, we show here for the first time that subtle differences in G/M commitment exist between these two patient series. The monocyte/macrophage cells may play a role in the reconstitution of the bone marrow microenvironment and/or the reconstitution of immunological defences. The ontogenically related commitment towards monocyte/macrophage lineage may be an advantage for the in vitro production of dendritic cells generated from monocytes in a strategy of adoptive immunotherapy in children.