The major obstacle against wider use of cord blood (CB) hematopoietic stem cells (HSC) is low level of HSC counts causing delay in both hematopoietic and immunological recovery. In recent years, HSC expansion strategies with small molecules ie Valproic acid (VPA) or nicotinamide (NAM) have yielded the most promising results. In Ankara University Cord Blood Bank, we have previously demonstrated increase in CD34 content and CFU-GM in the presence of VPA and/or NAM.
To ascertain the functionality of expanded HSCs, we have initiated a mice stem cell transplant model using non-obese diabetic (NOD)Cg-Prkdcscid IL2rgtmWjl stocks of Rag2null mice (NSG mice).
Following consent CD34+ cells were isolated by positive selection using MACS CD34 MicroBead Kit-UltraPure system (Miltenyi Biotec) from fresh CBUs collected at Ankara University or Hacettepe University, Departments of Obstetrics and Gynecology. Subsequently, 3.500 CD34+ cells per ml were plated in StemSpan medium in the presence of cytokine cocktail containing SCF, Flt3L, IL-3 and IL-6 (StemCell Technologies) w/wo VPA (1 mM) and/or NAM (5 mM). VPA, NAM and VPA + NAM added cultures were incubated at 37 °C and 5% CO2 for 8 days, 21 days and either 8 or 21 days, respectively. Following incubation CD34+ cells were washed and evaluated against their colony forming capacities using MethoCult Enriched wo EPO (StemCell Technologies) medium and cell viabilities were detected with 7-AAD and HSC phenotyping were performed by flow cytometry (Beckman Coulter) using labeled CD34 and CD45 monoclonal antibodies. Expanded cells were transplanted to 8- to 12 weeks old NOD.Cg-Prkdcscid IL2rgtmWjl/Sz (NSG) mice, purchased from The Jackson Laboratory and maintained under specific pathogen- free conditions at Laboratory of Animal Research Center in Ankara University. 24 hours prior to transplantation, mice were injected with busulfan through the tail vein (25 mg/kg, 50–100 g per dose). The next day, stem cell harvests (2,5 × 104 - 5 × 105 total nucleated cells (TNC) suspended in 200 μl PBS) were transplanted by intravenous injection. Following transplantation complete blood count was performed on peripheral blood drawn from the tail vein in every 3-7 days. As control unexpanded CD34 HSCs and cytokine only expanded HSCs were used.
Two CBUs were successfully expanded with or without VPA and/or NAM in the presence of cytokine cocktail. All expanded HSCs were found to be competent on CFU-GM assays. Either freshly isolated or expanded HSCs were transplanted into 12 mice in total. One mouse was transplanted with non-expanded HSCs; 2 mice with cytokine-only-expanded HSCs; 2 mice with VPA-expanded HSCs and 2 mice with VPA + NAM expanded HSCs on the 8th day of the expansion. 2 mice with NAM-expanded HSCs and 1 mouse with VPA + NAM expanded HSCs on the 21st day of expansion. WBC counts of mice in different time points are shown in Figure 1.
Our preliminary findings show that expanded CB HSCs are capable of increasing the peripheral WBC counts. VPA and/or NAM mediated expanded HSC led to an increase in WBC counts (>1 × 109/L) earlier than the non-transplanted or unexpanded HSC transplanted mice. Since VPA mediated expansion can be achieved within 8 days, VPA-expanded HSCs seem to be the most rapid way to achieve early engraftment in vivo.