We have previously established a stroma-free culture with Flt-3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO) that allows the maintenance and the expansion for several weeks of a cord blood (CB) CD34+ cell population capable of multilineage and long-lasting hematopoietic repopulation in non-obese diabetic/ severe combined immunodeficient (NOD/SCID) mice. In this work the kinetics of megakarocyte (Mk)-engraftment that is often poor and delayed in CB transplantation, and human platelet (HuPlt) generation in NOD/SCID mice of baseline CD34+ cells (b34+), and of CD34+ cells reisolated after a 4-week expansion with FL+SCF+TPO (4w34+) were compared. With b34+ cells Mk-engraftment was first seen at week 3 (CD41+: 0.4%); 4w34+ cells allowed a more rapid Mk-engraftment (at weeks 2 and 3 the CD41+ cells were 0.3% and 0.8%). Circulating HuPlts were first seen at weeks 2 and 1, respectively. Mk-engraftment levels of b34+ and 4w34+ cells 6-8 weeks after transplantation were similar (12 +/- 3.5 versus 15 +/- 5% CD45+; 1.3 +/- 0.5 versus 1.8 +/- 0.5% CD41+ cells). Also serial transplant experiments were performed with expanded and reselected CB cells. In secondary and tertiary recipients the Mk population was detected with bone marrow fluorescence-activated cell sorter analysis; these experiments indicate the effective long-term repopulation of expanded cells. Selected CD34+ cells after a 4-week expansion with FL+SCF+TPO are more efficient in Mk engraftment than the same number of unmanipulated cells.
Fast but durable megakaryocyte repopulation and platelet production in NOD/SCID mice transplanted with ex-vivo expanded human cord blood CD34+ cells
BRUNO, Stefania;GUNETTI, Monica;SANAVIO, Fiorella;FAGIOLI F;AGLIETTA, Massimo;PIACIBELLO, Vanda
2004-01-01
Abstract
We have previously established a stroma-free culture with Flt-3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO) that allows the maintenance and the expansion for several weeks of a cord blood (CB) CD34+ cell population capable of multilineage and long-lasting hematopoietic repopulation in non-obese diabetic/ severe combined immunodeficient (NOD/SCID) mice. In this work the kinetics of megakarocyte (Mk)-engraftment that is often poor and delayed in CB transplantation, and human platelet (HuPlt) generation in NOD/SCID mice of baseline CD34+ cells (b34+), and of CD34+ cells reisolated after a 4-week expansion with FL+SCF+TPO (4w34+) were compared. With b34+ cells Mk-engraftment was first seen at week 3 (CD41+: 0.4%); 4w34+ cells allowed a more rapid Mk-engraftment (at weeks 2 and 3 the CD41+ cells were 0.3% and 0.8%). Circulating HuPlts were first seen at weeks 2 and 1, respectively. Mk-engraftment levels of b34+ and 4w34+ cells 6-8 weeks after transplantation were similar (12 +/- 3.5 versus 15 +/- 5% CD45+; 1.3 +/- 0.5 versus 1.8 +/- 0.5% CD41+ cells). Also serial transplant experiments were performed with expanded and reselected CB cells. In secondary and tertiary recipients the Mk population was detected with bone marrow fluorescence-activated cell sorter analysis; these experiments indicate the effective long-term repopulation of expanded cells. Selected CD34+ cells after a 4-week expansion with FL+SCF+TPO are more efficient in Mk engraftment than the same number of unmanipulated cells.File | Dimensione | Formato | |
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