Abstract
Background: Hematopoietic stem cells are able to regenerate hematopoiesis in all of its lineages. They are clinically used in transplantation of bone marrow or peripheral blood stem cells (PBSC) in patients with diagnosis of leukemia or lymphoma. While amount of hematopoietic stem cells is critical for the long-term engraftment, the amount of progenitor and precursor cells can influence time to engraftment, and it is critical for duration of neutropenia after transplantation. The methods of expansion of hematopoietic stem cells could reduce time to engraftment and decrease the risk of early post-transplant complications.
Methods: Authors analyzed expansion of enriched hematopoietic stem cells (HSC), selected by immunomagnetic separation of Lin− cells from PBSC, in the culture of serum-free medium in vitro with all combinations of 5 cytokines (SCF, Flt-3-L, IL-3, IL-6, TPO) with and without G-CSF. Cell counts, morphology, immunophenotyping, and CFU-GM and CFU-Meg cultures were performed. Clinical transplantation protocol based on these results was developed. 10 patients with diagnosis of non-Hodgkin’s or Hodgkin’s lymphoma indicated for autologous transplantation, who signed the informed consent, were enrolled into the protocol. Except of standard PBSC graft, additional cells were collected, Lin− cells were selected by immunomagnetic separation, frozen and stored at Tissue bank. At day -14, Lin− cells were thawed and expanded in culture of serum-free medium with cytokines SCF, Flt-3-L, IL-3, IL-6 and G-CSF. Patients received high-dose chemotherapy regimen BEAM from day −7 to day −2. Progenitor cells expanded ex vivo in culture from Lin− cells were infused at day 0, following transplantation of PBSC. Patients were monitored, blood counts were performed, side effects were observed, and times to engraftment in granulocytes and platelets were calculated.
Results: In experiments, the highest number of CFU-GM colonies was observed at day +14 with cytokine combination SCF+IL-3+Flt-3-L+ IL-6. The highest number of CFU-Meg colonies was observed in cytokine combination SCF+IL-3+TPO. G-CSF increased count and maturation of cells. Number of total cells grew 200 to 350 times at day +14. In the clinical protocol, 2 patients were excluded for technical reasons and 1 patient was excluded because of disease progression. 7 patients completed the protocol. The clinical procedure was free of serious adverse effects in all patients. Patients received doses from 5•107 to 3•109 cells. The group of patients was compared with historical controls - 142 patients treated at our department with autologous PBSC transplantation after BEAM regimen for diagnosis of lymphoma. Control group received higher average dose of CD34+ cells than experimental group: 7.7•106 / kg versus 6.6•106 / kg. Engraftment in granulocytes > 1000 / μl was shortened from 11 to 7.3 days in experimental group. Engraftment in platelets was not changed significantly (12 versus 11 days). Duration of neutropenia was shortened from 8 to 5.6 days. In patients, who received higher doses of infused cells > 2•109, average engraftment in granulocytes occurred at 6.3 days and duration of neutropenia was 5 days.
Conclusions: HSC can be enriched from PBSC grafts, cultured and expanded ex vivo, and safely used in the cellular therapy protocols. The procedure resulted in significant shortening of critical period of neutropenia.
Angiopoietin-like 5 and IGFBP2 stimulate ex vivo expansion of human cord blood hematopoietic stem cells as assayed by NOD/SCID transplantation