Animal Serum-Free Culture of Purified Human CD34+ Cells: Amplification of Progenitors from G-CSF and GM-CSF-Mobilized Peripheral Blood

Abstract

The isolation and culture of human CD34+ cells could have broad clinical application for hematologic support following high-dose chemotherapy or bone marrow transplantation. The need for reproducible, animal product-free conditions for the culture of progenitors is crucial to the widespread clinical implementation of ex vivo cell therapies. In these studies, we explored the use of animal serum-free (ASF) medium for the culture of isolated human bone marrow and peripheral blood CD34+ cells. In this ASF system, isolated CD34+ cells were cultured using a variety of different growth factor combinations. Such ASF culture conditions yielded equivalent to superior cell and progenitor growth when directly compared with culture containing 10% fetal calf serum (FCS). In cultures containing IL-1, IL-3, and stem cell factor, total cell numbers increased, on average, 33-fold over the first 2 weeks. On phenotypic analysis, the ASF cultures demonstrated sustained proliferation of CD33+ myeloid cells throughout the culture period. CD34+ cell numbers increased during the first 7-10 days of culture, with a mean 3.4-fold expansion. Concomitant with the CD34+ cell expansion was an average 8.2-fold expansion of colony-forming unit-granulocyte-macrophage (CFU-GM) and a 102.0-fold increase in burst-forming units-erythrocytes (BFU-E). Likewise, a mean 4929-fold expansion of CD41a+ megakaryocyte progenitors was observed in these CD34+ cultures. Different combinations of growth factors affected the fold increase in cell and progenitor number. When CD34+ cell cultures from normal healthy volunteers mobilized with either G-CSF or GM-CSF were compared, similar expansions of total cell and progenitor cells resulted. However, CD41+ cells expansions were greater in those samples from G-CSF-mobilized volunteers in every case tested. These studies established the feasibility of this ASF CD34+ cell culture system to generate a population of maturing progenitors for potential use in transfusion support during cytopenic periods following high-dose chemotherapy or bone marrow transplantation.

Tolerance induction by megadose hematopoietic progenitor cells: expansion of veto cells by short-term culture of purified human CD34+ cells

“…Considering that a large number of early myeloid progenitors can be expanded ex vivo by short-term cultures using different cytokine combinations, [25][26][27][28][29][43][44][45][46][47] we hypothesized that if the veto activity could be retained along the myeloid differentiation pathway, then a small fraction of the CD34 ϩ cells used for transplantation could provide a substantial source of veto cells. Our current results confirmed this hypothesis, demonstrating that it is possible to harvest about 28-to 80-fold more veto cells on culturing of purified CD34 ϩ cells for 7 to 12 days with an early-acting cytokine mixture including FL, SCF, and TPO.…”