Reconstitution of human haematopoiesis in non-obese diabetic/severe combined immunodeficient mice by clonal cells expanded from single CD34+CD38- cells expressing Flk2/Flt3.
In the present study, we examined the expression of Flk2/Flt3, a tyrosine kinase receptor, on human cord blood CD34+ haematopoietic progenitor/stem cells. In flow cytometric analysis, Flk2/Flt3 was expressed on 80% of CD34+ cells and their immature subpopulations, CD34+CD33- and CD34+CD38- cells. Methycellulose clonal culture of sorted CD34+Flk2/Flt3+ and CD34+Flk2/Flt3- cells showed that most of myelocytic progenitors expressed Flk2/Flt3, but erythroid and haematopoietic multipotential progenitors were shared by both fractions. When 1 x 10(4) lineage marker-negative (Lin-)CD34+Flk2/Flt3- cells were transplanted into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, none of the recipients possessed human CD45+ cells in bone marrow 11-12 weeks after the transplantation. In contrast, all recipients transplanted with 1 x 10(4) Lin-CD34+Flk2/Flt3+ cells showed successful engraftment. Furthermore, clonal cells expanded from single Lin-CD34+CD38-Flk2/Flt3+ cells in the culture with Flk2/Flt3 ligand, stem cell factor, thrombopoietin, and a complex of interleukin 6/soluble interleukin 6 receptor were individually transplanted into NOD/SCID mice. At 20 to 21 weeks after the transplantation, three out of 10 clones harvested at d 7 of culture, and three out of six clones at d 14 could reconstitute human haematopoiesis in recipient marrow. These results demonstrated that Flk2/Flt3 was expressed on a wide variety of human haematopoietic cells including long-term-repopulating haematopoietic stem cells.
Ebihara
Yasuhiro
Y
Division of Cellular Therapy, Advanced Clinical Research Centre, The Institute of Medical Science, The University of Tokyo, Japan.
Wada
Mika
M
Ueda
Takahiro
T
Xu
Ming-Jiang
MJ
Manabe
Atsushi
A
Tanaka
Ryuhei
R
Ito
Mamoru
M
Mugishima
Hideo
H
Asano
Shigetaka
S
Nakahata
Tatsutoshi
T
Tsuji
Kohichiro
K
D000818:Animals; D018952:Antigens, CD34; D002478:Cells, Cultured; D002999:Clone Cells; D006410:Hematopoiesis; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000276:immunology*; D006801:Humans; D008565:Membrane Proteins; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D014183:Transplantation, Heterologous
D018952:Antigens, CD34; D008565:Membrane Proteins; C084881:flt3 ligand protein