Characterization of the Impact of Culture on the Thrombopoietic Potential of Cord Blood Progenitors.

Abstract 3710

Co-transplantation of ex vivo expanded progenitors with unmanipulated hematopoietic stem cells improves the recovery of neutrophils while that of platelets remains problematic especially for cord blood (CB) transplantation. The lack of efficacy on platelet recovery could be the result of poor expansion of megakaryocyte progenitors (Mk-P) and Mks induced by the cytokine cocktails currently used, or to the lack of optimization of this procedure. Therefore, we recently developed two cytokine cocktails by statistical design of experiment, OMPC and BS1 for the expansion of CB Mk-P and for the differentiation of CD34+ cells into Mk, respectively. Our objectives were now to compare the short term thrombopoietic potential of CB cells expanded with these cocktails to that of unexpanded cells, and to investigate the impact of two culture parameters, the temperature of incubation and the length of expansion, on platelets recovery.

CB CD34+ cells were expanded in serum-free medium with the cocktail BS1 (SCF, TPO, IL-6, IL-9) or OMPC (SCF, FL, TPO). The total progeny of 130,000 expanded CD34+ cells were transplanted into irradiated NOD/SCID mice (9 mice/group). Unexpanded CD34+ cells were transplanted as control to determine whether this procedure procures any advantages to platelet recovery. Human platelets (hPLT) were monitored in the peripheral blood by cytometry, as well as human bone marrow (BM) engraftment.

Since we previously showed that Mk expansion and Mk purity can be improved by incubating CB cultures at 39°C, we first assessed the impact of mild hyperthermia (MH) on the thrombopoietic potential of OMPC-expanded cells. hPLTs levels 4 days post-transplant (PT) achieved with cells expanded at 37°C were low and insignificant vs the PBS-control group (p>0.05). In contrast, hPLT levels were 20-fold greater in mice transplanted with cells cultured at 39°C (median of 0.7 vs 13.7 hPLT/uL, respectively p<0.002). No differences were then observed up to 36 days-PT, at which time hPLT levels became greater (8.5-fold) in mice injected with cells expanded at 37°C (p<0.04). Next, the short-term hPLT potentials of CB cells expanded for 6 days were compared to that of unmanipulated CD34+ cells. Only low and insignificant hPLT levels (p>0.05 vs PBS-control group) were detected 4- and 7 days-PT with the unexpanded cells (median < 1.4 hPLT/uL). In contrast, hPLT were readily detected in 94±8% of mice injected with OMPC- and BS1-expanded cells (9- and 5-fold greater at day 4, and 26- and 32-fold greater at day 7, respectively; p<0.05 vs unexpanded and PBS controls). Conversely, hPLT levels in mice injected with unexpanded cells became and remained greater by 11 days PT (p<0.05). Finally, we investigated the impact of the length of expansion (6, 10 and 14-days) on the thrombopoietic potential of expanded CB cells. Mice transplanted with day-6, day-10 and day-14 BS1-expanded cells received an average dose of 1.3, 11.3 and 22.7 millions CD41+ Mks. Despite the increasing Mk cell doses, an inverse correlation between the length of expansion and hPLT levels were observed 14- (p<0.001) and 28-days PT (p<0.005). A similar correlation was observed for the number of huCD41+ cells in the BM 14-days PT (p<0.01). An inverse correlation between the length of culture and the total number of CD34+ and GPA+ human BM cells was also evident. As previously observed, hPLTs levels 14-days PT were the highest in mice transplanted with the unexpanded cells (p<0.002). High hPLT levels were detected in all cohorts up to 16 weeks PT, with the highest level observed in the unmanipulated and day-10 expanded groups.

In summary, hPLT levels derived from unmanipulated CB cells were insignificant prior to 11 days PT, while hPLTs were readily detected with OMPC- and BS1- expanded CB cells. However, hPLTs production from unmanipulated cells became and remained dominant by 11–14 days PT. These results also confirm that cytokine cocktails promoting Mk-P expansion can improve short term platelet recovery and that expansion under MH is of added benefit for short term hPLT recovery.

In conclusion, this work reveals that the hPLT reconstitution kinetics differ considerably between expanded and unmanipulated CB cells. Importantly, this difference support the concept that co-transplantation of both cell source could be of significant benefit for the short term platelet recovery in the context of CB transplantation. We are now in the process of evaluating this potential synergy.