Early‐acting cytokine‐driven ex vivo expansion of mobilized peripheral blood CD34+ cells generates post‐mitotic offspring with preserved engraftment ability in non‐obese diabetic/severe combined immunodeficient mice

The ability of ex‐vivo expanded peripheral blood stem cells (PBSC) to engraft non‐obese diabetic/severe combined immunodeficient (NOD/SCID) mice has not been evaluated to date. We investigated the maintenance of primitive SCID‐repopulating cells (SRC) and long‐term culture‐initiating cells (LTCIC) in PBSC expanded with early‐acting cytokines, thrombopoietin (TPO), stem cell factor (SCF) and FlT3‐ligand (FL) with or without interleukin 3 (IL‐3) and IL‐6 in short‐term (6 d) stroma‐free serum‐free cultures. TPO + SCF + FL and TPO + SCF + FL + IL‐3 + IL‐6 produced 5·9 ± 1·97 and 18·25 ± 4·49 (mean ± SEM)‐fold increase of CD34 + cells respectively. We tracked cellular division with PKH26 and sorted post‐mitotic CD34 + PKH26 low cells to assess their primitive functional properties. After culture with TPO + SCF + FL, LTCICs among post‐mitotic cells increased 12·08 ± 3·4 times, and 4·3 ± 1·6 times when IL‐3 + IL‐6 were added. CD34 + PKH26 low cells cultured with TPO + SCF + FL provided human multilineage (CD34, CD33 and CD19) engraftment in NOD/SCID mice, whereas no human cells were detected in mice injected with cells cultured with TPO + SCF + FL + IL‐3 + IL‐6. Percentages of CD34 + /CD38, CD34 + /CD33, CD34 + /DR and cells in G 0 /G 1 phase were similar among cells cultured with both cytokine combinations, indicating that the deleterious impact of IL‐3 + IL‐6 on the ability to engraft is not translated into phenotypic or cycling features. In conclusion, TPO + SCF + FL‐expanded PBSC maintain multilineage engraftment ability in NOD/SCID mice, which is abrogated by the addition of IL‐3 + IL‐6.