Enrichment of human hematopoietic stem cell activity in the CD34+Thy-1+Lin- subpopulation from mobilized peripheral blood.
The number of CD34+ cells in the peripheral blood of cancer patients is known to be increased following the administration of high dose chemotherapy and hematopoietic growth factors. These so-called peripheral blood stem cell grafts are now frequently used for autologous transplantation of patients with malignancies. In this report, we address the question of whether true long-term repopulating pluripotent hematopoietic stem cells (PHSC) are mobilized into peripheral blood following chemotherapy plus granulocyte/macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) mobilization. We have examined the presence of stem cells in mobilized peripheral blood (MPB) by using an antibody to the human Thy-1 molecule to stain the CD34+Lineage- (Lin-) population. The kinetics of mobilization of CD34+Thy-1+ Lin- cells into peripheral blood were studied, and the percentage of cells with this phenotype was found to vary widely depending on the day of leukapheresis. A CD34+Thy-1+Lin- cell population, potentially containing PHSCs, was isolated by fluorescence activated cell sorting (FACS) and analyzed for activity. The multilineage differentiative capacity of this candidate stem cell-containing population in MPB was determined using an in vitro long-term culture system, in which cobblestone area formation was used as a means of detecting PHSCs. We also measured repopulating capacity by using two in vivo models in which severe combined immunodeficiency (SCID)-hu mice were implanted with human fetal bone or thymus grafts. Using these assays, we show that the highest frequency of cobblestone area-forming cells (CAFC) after 7 weeks of culture was observed in a subpopulation of CD34+Lin- cells, which expressed low levels of Thy-1. This cell population was capable of producing both B and myeloid cells, and maintaining CD34+Lin- cells in these long term cultures. Moreover, the CD34+Thy-1+Lin- cell subset possessed a higher ability to engraft and to demonstrate multilineage differentiative potential at 8 weeks in the SCID-hu bone assay. However, in the SCID-hu thymus model, both Thy-1+ and Thy-1- subpopulations were capable of donor T-cell engraftment at 6 weeks, suggesting the presence of cells capable of initiating T lymphopoiesis in both populations.(ABSTRACT TRUNCATED AT 400 WORDS)
Murray
L
L
SyStemix Inc., Palo Alto, CA 94304.
Chen
B
B
Galy
A
A
Chen
S
S
Tushinski
R
R
Uchida
N
N
Negrin
R
R
Tricot
G
G
Jagannath
S
S
Vesole
D
D
D000818:Animals; D015703:Antigens, CD; D018952:Antigens, CD34; D001772:Blood Cell Count; D001853:Bone Marrow / Q000473:pathology; D002678:Chimera; D003520:Cyclophosphamide / Q000494:pharmacology*; D005047:Etoposide / Q000494:pharmacology*; D005434:Flow Cytometry; D006085:Graft Survival; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D006410:Hematopoiesis / Q000187:drug effects*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D009369:Neoplasms / Q000097:blood / Q000628:therapy; D018800:Thy-1 Antigens; D013997:Time Factors; D014183:Transplantation, Heterologous
D015703:Antigens, CD; D018952:Antigens, CD34; D018800:Thy-1 Antigens; D016179:Granulocyte Colony-Stimulating Factor; D005047:Etoposide; D003520:Cyclophosphamide