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- MATERIALS AND METHODS
In-vitro expansion of human cord blood (CB) cells could enhance peripheral blood recovery and ensure long-term engraftment of larger recipients in the clinical transplant setting. Enrichment of CD34+ cells using the MiniMACS column has been evaluated for the preparation of CB CD34+ cells before and after expansion culture. Repurification of CD34+ cells after culture would assist accurate phenotypic and functional analysis. When fresh CB mononuclear cells (MNC) were separated, the MACS positive (CD34+) fraction (90.1% pure) contained a mean (± SD, n = 5) of 93.0 ± 8.0% of the eluted CD34+ cells, 99.6 ± 0.7% of the CFU-GM and all of the eluted long-term culture-initiating cells (LTC-IC). Cord blood CD34+ cells were then cultured for 14 d with IL-3, IL-6, SCF, G-CSF and GM-CSF, each at 10 ng/ml. The total cell expansion was 2490 ± 200-fold and the CD34+ cell expansion was 49 ± 17-fold. The percentage of CD34+ cells present after expansion culture was 1.2 ± 0.85%. When these cells were repurified on the MiniMACS column, the MACS positive fraction only contained 40.3 ± 13.4% of the eluted CD34+ cells which was enriched for the mature CD34+ CD38+ subset, 24.4 ± 8.8% of the eluted CFU-GM and 79.5 ± 11.0% of the LTC-IC. The remaining cells were eluted in the MACS negative fraction. In conclusion, repurification of cultured CD34+ cells does not yield a representative population and many progenitors are lost in the MACS negative fraction. This can give misleading phenotypic and functional data. Cell losses may be important in the clinical setting if cultured cells were repurified for purging.
Cord blood has been used as a source of haemopoietic stem cells for transplantation since 1989 (Gluckman et al, 1989) to treat a wide variety of malignant and non-malignant haemopoietic disorders (Wagner et al, 1995; Kurtzberg et al, 1996). Despite limited progenitor numbers, sufficient marrow repopulating cells are present, because sustained engraftment in children has been documented (Gluckman et al, 1989). However, the number of nucleated cells infused per kilogram is a major factor in the recovery of neutrophil and platelet counts (Gluckman et al, 1997). We hypothesized that expansion of post-progenitor cells, progenitor cells and primitive cells such as LTC-IC may be required for adequate engraftment in larger recipients. For these reasons we have investigated the in vitro expansion of CB cells.
Enriched CD34+ cells are routinely used as starting material for expansion cultures. We have evaluated the MiniMACS immunomagnetic separation column for the preparation of CD34+ cells prior to and after expansion culture. Enrichment of CD34+ cells from cultures, if successful, would enable accurate phenotypic analysis using a population of cells of similar purity to the starting material.
In this study we enriched CB CD34+ cells using the MiniMACS column. Its performance was assessed by monitoring the positive (CD34+) and negative fractions for cell recovery and the presence of colony forming cells (CFC) and LTC-IC. Enriched CD34+ cells from the MACS positive fraction were then cultured for 14 d in the presence of IL-3, IL-6, G-CSF, GM-CSF and SCF. Expansion of total cells, CFU-GM and LTC-IC was calculated, together with flow cytometric analysis, to estimate the expression of CD38 and HLA-DR in cultured cells. Cells from expansion cultures were also reapplied to the miniMACS column and the performance of the cells on the column investigated as before.
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- MATERIALS AND METHODS
Enriched CD34+ CB cells harvested in the MACS positive fraction had the same clonogenic potential and LTC-IC frequency as the MNC CD34+ population, indicating that they were representive of the CD34+ population found in whole CB. The column processing was also very efficient in that very few CD34+ cells or progenitors were lost either on the column or to the negative fraction. This is in agreement with other studies which conclude that the miniMACS system is superior to CEPRATE avidin-biotin column separation or Dynabeads in terms of purity and yield (De Wynter et al, 1995; Laver et al, 1995).
When CB CD34+ cells were cultured in suspension in the presence of growth factors a substantial expansion of CB progenitors and a small but consistent expansion of 5-week LTC-IC/CAFC was observed. During the culture mature cells were produced at a faster rate than progenitors or LTC-IC so that the resulting percentage of CD34+ cells present and the proportion of cells which were CFU-GM was very similar to the CB MNC fraction. However, when these cells were applied to the MiniMACS column substantial numbers of progenitors and LTC-IC were eluted in the MACS negative fraction. Cells eluted in the positive fraction were highly enriched for the mature CD38+/DR+ subpopulation and were not representative of the cell population applied to the column. If cultured CD34+ cells were enriched by the MiniMACS column for analysis there would be an under estimation of the expansion of CFU-GM and LTC-IC together with misleading phenotypic and functional data.
The CD34 molecule expressed on haemopoietic progenitor cells contains a large number of epitopes.These epitopes can be categorized according to their sensitivity to degredation by neuraminidase, chymopapain and a glycoprotease from Pasteurella haemolytica (Sutherland et al, 1992). Class I epitope is sensitive to all three enzymes, class II, neuraminidase resistant, glycoprotease/chymopapain sensitive and class III is resistant to all three enzymes. Cleavage of the 110 kD CD34 structure by the glycoprotease genereates a cell-bound protein of 75 kD, identified by class III antibodies such as HPCA-2 used for flow cytometric analysis of all our samples. However, the glycoprotease removes the epitope recognized by QBEnd 10, the antibody used in the enrichment of CD34+ cells on the immunomagnetic column. Therefore it is possible for differential epitope expression on haemopoietic cells, which may be related to their maturation or function (Steen et al, 1996), such that some may bind QBEnd 10 and HPCA-2 but some may only bind HPCA-2.
On the immunomagnetic column separation of cells into the MACS positive or negative fraction depended on the binding of QBEnd 10 and detection of CD34+ cells in either fraction used the class III antibody HPCA-2 (which should detect all CD34+ cells). Our experience with fresh CB CD34+ cells suggests that a very high proportion express both class II and class III epitopes. The negative fraction contained at the most 19.5% of the eluted CD34+ cells, but accounted for <2% of eluted CFU-GM, suggesting that these were very mature progenitors. Data of Steen et al (1996) would support this theory, as CD34+ cells detected by HPCA-2 and not QBEnd 10 have high forward scatter consistent with slightly granular cytoplasm.
Analysis of MACS fractions from cultured CD34+ cells showed not only that a much higher proportion of cells did not bind HPCA-2 and were eluted in the MACS negative fraction, but in contrast to fresh CB CD34+ cells, lack of class II epitope was not associated with lack of clonogenicity or LTC-IC activity. Indeed, the MACS negative fraction was enriched for CFU-GM. The CD34+ cells which seemed to stain most strongly with HPCA-2 were CD38+/DR+, and although strict comparison with cultured cells before MiniMACS separation is difficult, it seems that this subpopulation was enriched in the positive fraction (Fig 4). Freshly isolated CB CD34+ cells are quiescent with 70–97% in G0/G1 (Traycoff et al, 1994; Hao et al, 1995; Engelhardt et al, 1997). During exposure to cytokines cell division increases so that after 72 h 40–50% are in cell cycle (Engelhardt et al, 1997) and after 7 d >95% of cells have divided (Traycoff et al, 1995). It is possible that rapidly dividing cells experience an early down-regulation of CD34 class I and II epitope expression out of sychrony with their functional development. This view is supported by the loss of more mature CFC to the MACS negative fraction whereas most of the LTC-IC were retained in the MACS positive fraction. There is also evidence that the CD38 antigen may be lost from CD38 positive cells during expansion culture, leading to falsely high estimates of CD38− cell expansion (Dorrell et al, 1998).
In addition, impaired engraftment of cultured cells has been reported (Brandt et al, 1998; Habibian et al, 1998; Gothot et al, 1998), which may also be due to changes in cell surface antigens induced by in vitro culture. These changes may be a reversible plastic feature correlated with cell cycle progression (Habibian et al, 1998), including decreased expression of adhesion proteins during late S/G2 (Becker et al, 1997) or an increase in peptides which allow cells to undergo endothelial transmigration so that infusion of cells in cycle would lead to the homing of cells in non-haemopoietic organs (Yong et al, 1998).
In conclusion, immunomagnetic columns are ideal for the production of enriched fresh CD34+ cells, but repurification of cultured CD34+ cells by this method does not yield a representative population, with many progenitors being lost in the negative fraction. This can give misleading phenotypic and functional data. These problems may be overcome in the research setting by the use of flowcytometric cell sorting or by using negative selection. In a clinical setting, repurification of CD34+ cells by positive selection on an immunomagnetic column of expansion cultures would lead to large cell losses. It is important that there is careful selection of CD34-specific antibodies for both cell purification and enumeration. Possible abnormal expression of cell surface antigens during processing and culture procedures should also be taken into account as this can alter their behaviour in vitro and in vivo.