Our studies have focused specifically on the ability of SDF-1 to act as a chemoattractant for primitive progenitor/stem cells circulating in the neonate and obtained in CB, or for their adult equivalent mobilized into the peripheral blood. We chose the CAFC assay to detect the committed progenitors at 2 weeks and candidate stem cells at 5 weeks. In addition, we performed secondary colony assays at 5 weeks to enumerate LTC-ICs, again as a candidate stem cell assay. The formal demonstration that SDF-1 acts as a chemoattractant for both committed progenitors and primitive progenitor/stem cells was obtained by the use of recombinant SDF-1 in the lower chamber of the transwell system, with CB CD34+
cells in the upper chamber. Within 3 hours, 25–30% of the 2-week and 5-week CAFCs had migrated across the bare membrane with virtually no migration in this time in the absence of the chemokine. Substantial inhibition of the migration was obtained by treating the cells with mAb to CXCR4. SDF-1 also promoted transendothelial chemotaxis of both 2-week CAFCs and 5-week CAFCs, again partially inhibitable by antibody to CXCR4. These data suggest that SDF-1 is effective as a chemotactic factor on both committed progenitors and primitive progenitor/stem cells signaling through CXCR4. This is in good agreement with a recent report that shows dependence of the engraftment of human NOD/SCID repopulating cells on CXCR4 (38
Under serum-free conditions, no spontaneous transendothelial migration of 5-week CAFCs occurred in 24 hours, but it was seen when recombinant SDF-1 was added to the lower chamber; this characteristic response was completely abolished by antibody to CXCR4. The same population of CD34+ cells was also evaluated for transendothelial chemotactic response to MS-5 monolayer with either fresh serum-containing medium or with old medium “conditioned” by the MS-5 cells. A comparably high degree of transmigration (~50%) of 5-week CAFCs was seen under both conditions, being partially blocked by antibody to CXCR4. The greater chemotactic response to MS-5 versus SDF-1 may reflect (a) a serum requirement for optimal transendothelial migration; (b) the role of another chemoattractant produced by MS-5 not acting through CXCR4 and therefore not blocked by the antibody; (c) an effect of diffusible cytokines produced by MS-5 influencing the viability, mobility, adhesion molecule expression, or CXCR4 expression of CD34+ cells; and (d) selective loss of transmigrated cells in the lower chamber on transfer to secondary MS-5 cultures in the X-VIVO (Bio-Whittaker, Walkersville, Maryland, USA) SDF-1 study versus the direct enumeration of CAFC on the in situ MS-5 layer.
We established a self-contained assay for measurement of transmembrane and the transendothelial migration of CAFCs and LTC-ICs, using 2 standardized cell lines to permit reproducibility: BMEC-1 for the endothelial monolayer, and MS-5 as both a standard source of SDF-1 and as a reproducible assay for both CAFCs and LTC-ICs. The major advantage of the self-contained system was that there was no cell loss associated with transfer of cells from either the upper or lower chamber to secondary MS-5 stroma. The transmigration of LTC-ICs to an SDF-1 gradient, which shows no further migration after 6 hours, suggests a possibility that both chemotaxis and chemokinesis are present in this MS-5–based closed transmigration system. Three-hour migration of CAFCs and LTC-ICs could be mainly attributed to chemotaxis and the increase at 24 hours could be attributed to chemokinesis provided by other soluble factors released from MS-5 or BMEC-1. Therefore, this experimental system might simulate the in vivo situation much better than SDF-1–based chemotaxis. Evaluation of transmigration across the endothelium showed that at nearly all time points, CB cells migrated to a greater degree than cells of mobilized PB. Enhanced transendothelial chemotaxis of CB primitive progenitor/stem cells relative to adult PB cells may provide an explanation for the more efficient engraftment of NOD-SCID mice by neonatal than by adult CD34+
Ex vivo expansion of primitive human hematopoietic cells has been extensively evaluated over the last 5 years (35
). Autologous transplantation of ex vivo expanded PB CD34+
cells in myelosuppressed individuals showed comparable time and extent of platelet and neutrophil recovery to those achieved with 10-fold more nonexpanded cells (27
). However, recent reports indicate that long-term engraftment with ex vivo expanded PB CD34+
cells in vigorously myeloablated individuals was compromised, despite otherwise optimal numbers of CD34+
cells and LTC-IC infused (26
). We present data suggesting that primitive progenitor/stem cells may exhibit impairment in chemotactic responsiveness when cultured for prolonged periods, particularly in the presence of IL-3. Flow cytometric analysis showed clearly downmodulation of CXCR4 on ex vivo expanded CD34+
cells without notable difference among the different cytokine combinations. The CD34+
cells, however, do not represent primitive hematopoietic cells. Analyzing the cells of primitive phenotype, such as CD34+
, might provide the information on primitive hematopoietic cells, but this option is not available after expansion, especially in the presence of IL-3, because of very low frequency of the cells. Flow cytometric analysis still does not address functional status of CXCR4. Therefore, we need a functional assay system. There could be a concern that the attenuation in the migration potential of expanded cells especially in the presence of IL-3 might be attributed to the high cell densities in the upper chamber. The surface area of 6-well transwell inserts, however, is quite enough for a large number of cells. Furthermore, we could not see differences in the migration of CAFCs or LTC-ICs between 5 × 103
cells and up to 2 × 105
matched mononuclear cells in the upper chamber. In 7-day culture, the cell expansion was minimal or modest even in the presence or IL-3, but marked decrease in transmigration was still observed in cultured cells with IL-3. In addition, the cell expansion with TPO + FL + IL-3 was similar to or minimally higher than that with TPO + FL + KL, but the CAFCs and LTC-ICs from the former showed markedly attenuated transmigration. Taken together, the defective transmigration of ex vivo expanded cells in our system cannot be attributed to high cell density or heterogeneity of the cells.
There are conflicting reports on the effects of brief cytokine exposure on marrow repopulating ability. Tavassoli et al. (43
) reported that 2–3 hour preincubation of mouse bone marrow cells with IL-3 enhanced repopulating ability, possibly due to upregulation of homing receptors. In contrast, van der Loo and Ploemacher (2
) found that a similar preincubation with IL-3 or IL-3 + IL-12 + KL led to a sustained decrease in marrow and spleen seeding of both early and late CAFCs as well as day-12 CFU-S, together with an impairment in long-term repopulation. Although our data involved much longer exposure of cells to IL-3, the decreased chemotactic responsiveness of CAFCs could account for this decreased seeding efficiency. In some studies, in vivo repopulating capacity of expanded CB cells increased at 4 days by 2- to 4-fold but was lost by 9 days (44
), or increased by 2-fold at 5–8 days (45
). In stromal coculture, a 6-fold loss of repopulating capacity was reported with CB cells over 14 days despite LTC-ICs being higher than input (26
). CB cells expanded for 6 days with IL-3, and KL failed to engraft when injected intravenously in NOD/SCID mice but did so when engrafted intraperitoneally (39
). This observation supports our concept that ex vivo expansion of primitive progenitor/stem cells does occur as indicated by LTC-IC data but that defects in marrow homing capacity induced by IL-3 prevent their localization in the marrow, possibly resulting in their clearance and destruction in nonhematopoietic tissue. Ex vivo expansion of adult CD34+
populations with IL-3–containing cytokine cocktails showed a major loss of NOD/SCID repopulating capacity at 6–7 weeks with bone marrow (37
) or mobilized PB (26
). In contrast, the cytokine combination of TPO + KL + FL for 6 days resulted in an increase in CAFC numbers and a capacity for NOD/SCID repopulation similar to fresh bone marrow (37
). This result parallels our observation that a similar cytokine combination expanded CAFCs and LTC-ICs for 7 and 14 days without loss of chemotactic responsiveness and that these features were compromised by addition of IL-3.
Our data point to the importance of CXCR4 signaling in transendothelial chemotaxis of primitive progenitor/stem cells and the significance of this to stem cell transplantation. Modulation of expression of CXCR4 is thus likely to have major effects on the efficiency with which stem cells localize to the marrow.