Efficient ex vivo expansion of NOD/SCID-repopulating cells with lympho-myeloid potential in hematopoietic grafts of children with solid tumors.
BACKGROUND
The ex vivo expansion of hematopoietic grafts could be an important therapeutic tool for accelerating hematopoietic recovery after administration of high-dose chemotherapy regimens. The fate of the long-term repopulating cells during the ex vivo manipulation of grafts is a critical issue and will ultimately define the clinical applicability of this technology to hematopoietic transplantation.


METHODS
To study the effects of a clinically applicable ex vivo expansion protocol in the proliferative potential of the most primitive human hematopoietic cells, both LTC-IC and NOD/SCID-RC assays were used to determine LTC-IC and NOD/SCID-RC contents of hematopoietic grafts, both before and after expansion (SCF, IL-3, PEG-MGDF Flt3-L and 5% AB serum), in four children with non-hematological malignancies.


RESULTS
The mean percentage of CD34+ cells after expansion was 16%. The numbers of nucleated cells increased 20-fold with a mean three-fold increase in the numbers of CD34+ cells during the expansion period. The CFC content of the samples showed a mean 11-fold increase (range: 5-17) after ex vivo expansion. The primitive hematopoietic stem cell content of the expanded cell fraction evaluated by LTC-IC assays was found to be increased in two patients out of three, with maintenance of the LTC-IC frequency in the third patient. The NOD/SCID-RC potential, evaluated in five experiments from four patients using 109 mice injected 5-6 weeks earlier with human hematopoietic cells, increased from a mean percentage of 36% (range: 7-75%) before expansion, to a mean percentage of 70% (range: 37-100%) after expansion (P < 0.00001). The frequency of NOD/SCID-RC calculated with pooled data from all patients was 1/80,000 at day 0 and 1/40,000 after seven days of culture. The full phenotypic analysis of human hematopoietic cells obtained in NOD/SCID mice injected with expanded cells showed the presence of significant numbers of CD34+, CD19+ and CD15+ cells, suggesting the persistent lympho-myeloid potential of the expanded hematopoietic cells.


CONCLUSIONS
Our results suggest that efficient expansion of NOD/SCID-RC with lympho-myeloid potential can be achieved not only in cord blood or normal marrow as previously reported, but also in hematopoietic grafts obtained from children exposed to high-dose chemotherapy.
Tourino
C
C
Departamento Basico de Medicina, Hospital de Clinicas, Montevideo, Uruguay.
Pflumio
F
F
Novault
S
S
Massé
A
A
Guiller
M
M
Bonnet
M L
ML
Valteau-Couanet
D
D
Hartmann
O
O
Vainchenker
W
W
Beaujean
F
F
Coulombel
L
L
Turhan
A G
AG
D000818:Animals; D002675:Child, Preschool; D005260:Female; D005312:Fetal Blood / Q000166:cytology; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007223:Infant; D038041:Lymphopoiesis; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D038042:Myelopoiesis; D009369:Neoplasms / Q000188:drug therapy / Q000503:physiopathology*