Transplantation of Ex Vivo Expanded Cord Blood Progenitor Cells: First Experience in Two Children Affected by Hemoglobinopathies.
Authors: Lazzari Lorenza , Giordano Rosaria , Giorgiani Giovanna , Montemurro Tiziana , Giorgetti Alessandra , Lisini Daniela , Maccario Rita , Rebulla Paolo , Locatelli Franco 
Published: October 2019

Abstract
The main limitations of CB transplantation (CBT) are represented, in comparison to bone marrow transplantation, by a lower probability of engraftment and a slower hematopoietic recovery which can favor the occurrence of fatal complications. Several studies have clearly documented that the higher the number of cells infused, the faster the rate of engraftment and the lower the risk of transplant-related mortality. In order to increase the number of CB cells infused, approaches based on ex vivo expansion of CB progenitor cells are, in principle, particularly promising. In this regard, we developed a clinical protocol to expand the number of CB hematopoietic progenitor cells (HPCs), based on the use of a cytokine cocktail including interleukin-6 (10 ng/ml), stem cell factor (50 ng/ml), Flt-3 ligand (50 ng/ml) and thrombopoietin (10 ng/ml), in the presence of a serum-free medium and 10% AB allogeneic plasma. This combination induced significant and sustained ex vivo expansion of primitive CB HPCs. After clinical grade immunoselection (CliniMACS, Miltenyi Biotech), CD34+ cells were cultured at a concentration of 5x103 cells/ml for 2 weeks. Cytokines were added at the onset of culture and replaced twice a week. The purity of CD34+ cells (median and range) after immunoselection of thawed CB samples was 79.5% (47–96). After culture, the median fold expansion of total nucleated cells (NC) and CFU-GM was around 2 logs, while that of CD34+ cells was around 1 log. The expanded cells had levels of endotoxin below the required limit of 0.05IU/mL (E.Ph.) and were negative for mycoplasma, bacterial and fungal contamination.
Two children, one with thalassemia and one with sickle cell disease (SCD), were given ex vivo expanded CD34+ cells from an HLA-identical sibling. In both cases the conditioning regimen included busulfan, thiotepa and fludarabine, while GVHD prophylaxis consisted of cyclosporine-A. The first patient, a 6-year-old boy with thalassemia, was given 2 CB units derived from 2 HLA-identical fraternal (i.e. non syngeneic) twins (1 unmanipulated CB unit and 1 CB unit infused after ex vivo expansion). The fold expansions of NC and CD34+ cells were 623 and 56.6 respectively. The patient had neutrophil recovery (i.e. above 500/cmm) on day +16, while the platelet recovery (i.e. above 30,000/cmm) occurred on day +31. The second patient, a 7-year-old girl with SCD, was given 2/3 of the CB unit unmanipulated and 1/3 after ex vivo expansion. The fold expansions of NC and CD34+ cells were 189.3 and 30.5 respectively. Neutrophil and platelet recovery occurred on day +18 and +33 respectively. Neither acute nor chronic GVHD occurred in both patients, who are alive and disease free at 12 and 5 months after CBT respectively. In the first patient, chimerism evaluation showed the presence of erythroid and myeloid colonies, as well as of T cells, derived from the ex vivo expanded cells up to 7 months after CBT. Mesenchymal stem cells remained of patient origin in both cases. These preliminary results indicate that our expansion procedure is safe and able to ex vivo expand CB HPCs; the expanded cells seem to accelerate hematopoietic recovery and, for the first time, we demonstrate that these cells contribute to sustain hematopoietic recovery after CBT. Altogether these results could widen the applicability of CBT in adults.