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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

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Abstract:

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+ PKH26low 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+ PKH26low 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 G0/G1 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.

Keywords: NOD; PBSC; SCID mice; cytokine-mediated expansion; early-acting growth factors; serum-free culture

Document Type: Research Article

Affiliations: Department of Haematology, Reina Sofia University Hospital, Cordoba, Spain

Publication date: September 1, 2001

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