Circulation of CD34+ hematopoietic stem cells in the peripheral blood of high-dose cyclophosphamide-treated patients: Enhancement by intravenous recombinant human granulocyte-macrophage colony-stimulating factor

We report that hematopoietic pregenitor cells expressing the CD34 antigen (CD34⁺ cells) transiently circulate in the peripheral blood (PB) of cancer patients treated with 7 g/m² cyclophosphamide (HD-CTX) with or without recombinant human granulocyte macrophage-colony stimulating factor (rHuGM-CSF). In adult humans, CD34⁺ cells represent a minor fraction (1% to 4%) of bone marrow (BM) cells, comprising virtually all hematopoietic colony-forming progenitors in vitro and probably also stem cells capable of restoring hematopoiesis of lethally irradiated hosts. We show that CD34⁺ cell circulation is fivefold enhanced by rHuGM-CSF 5.5 protein μg/kg/day by continuous intravenous infusion for 14 days after HD-CTX. During the third week after HD-CTX (ie, when CD34⁺ cells peak in the circulation), large-scale collection of PB leukocytes by three to four continuous-flow leukaphereses allows the yield of 2.19 to 2.73 x 10⁹ or 0.45 to 0.56 x 10⁹ CD34⁺ cells depending on whether or not patients receive rHuGM-CSF. The number of CD34⁺ cells retrieved from the circulation by leukaphereses exceeds the number that can be harvested by multiple BM aspirations under general anesthesia. Thus, after therapy with HD-CTX and rHuGM-CSF, PB represents a rich source of hematopoietic progenitors possibly usable for restoring hematopoiesis after myeloablative chemoradiotherapy. To determine whether CD34⁺ cells found in the PB are equivalent to their marrow counterpart, we evaluated their in vitro growth characteristics and immunological phenotype by colony assays and dual-color immunofluorescence, respectively. We show that PB CD34⁺ cells possess qualitatively normal hematopoietic colony growth and high cloning efficiency comparable to that observed with BM CD34⁺ cells. In addition, PB CD34⁺ cells display heterogeneous surface membrane differentiation antigens analogous to BM CD34⁺ cells. The availability of large quantities of CD34⁺ cells by leukapheresis is relevant to the field of stem cell transplantation and possibly to genetic manipulations of the hematopoietic system in humans.