Campath-1H-Regulated Ex Vivo Expansion of Cord Blood: Selection of Stem Cells, Depletion of Lymphocytes and Preferential Expansion of Myeloid, Megakaryocytic and Erythroid Precursors.
Authors: Qi Feng , Lim Che K. , Sun Li , Hwang William Y.K. 
Published: October 2019

Abstract
Human umbilical cord blood (CB) represents a unique source of transplantable hematopoietic cells. Unfortunately, when the cell dose is less than optimal, delayed or failed engraftment may result. To overcome this limitation, ex vivo expansion of CB products in an attempt to increase the number of progenitor cells has been evaluated. However, current clinical conditions for ex vivo expansion of CB cells require selection of the CD34+ or CD133+ subset, as unfractionated or mononuclear cells (MNC) do not expand well. Such cell selection processes often result in low CD34+ cell recoveries and suboptimal purities, with a consequent lowering of total overall cell expansion. CD52 is an abundant antigen specifically expressed on lymphocytes, monocytes, eosinophils, thymocytes and macrophages; and there is significant evidence for the lack of CD52 expression on hematopoietic progenitor cells. In our current study, Campath-1H, a humanized anti-CD52 antibody, was added to Ficoll-separated cord blood cells to promote active selection of hematopoietic stem cells and curtailment of excess lymphocyte expansion in ex vivo cultures. Prior to start of the cultures, initial Ficoll separation of cord blood removed most of the granulocytes (CD33+ cells: 2.3%, CD13+ cells: 3.1%) though 90.3% percent of the cells remained CD52 positive. Megakaryocytic (CD41+), erythroid (GlyA+) and CD34+ cell percentages in the starting cultures were 12.8%, 1.8% and 0.4% respectively. Cells were not subjected to CD34 or CD133 column selection prior to ex vivo expansion and Campath-1H was added every 3 days to the expansion culture media (incorporating 50 ng/ml SCF, TPO and Flt-3 ligand in StemSpan SFEM medium). CD52+ cells decreased from 90.3% to 3.1% after treatment with Campath-1H antibody (Ab), maintaining at 64.4% in control cultures. After 2 weeks of culture, CD34 cells expanded 12.56 fold in cultures with Campath-1H added versus 7.10 fold in cultures without Campath. There was a 1.49 fold decrease in total cell number in ex-vivo cultures without Campath-1H added, which was consistent with the current literature on expansion with non-selected cells. With Campath-1H in the culture system, there was a 1.60 fold increase in total cell number, which was 2.39 fold higher than control cultures. Decrease in the lymphoid cell percentage was accompanied by a relative increase in CD33+ (50.2% with Ab vs. 41.9% without Ab) and CD13+ (82% with Ab vs. 61.1% without Ab) myeloid cells, CD41+ megakaryocytes (34.5% with Ab vs 13.0% without Ab) and GlyA+ erythrocytes (9.9% with Ab vs. 4.5% without Ab). The relative expansion of non-lymphocyte populations observed in our cultures system could potentially reduce graft-versus-host disease (GVHD) and promote enhanced engraftment of myeloid, megakaryocytic and erythroid precursors after transplantation. The ability to use a CD34/CD133 column-independent expansion system through Ab-regulated culture with a clinically licensed antibody is also compelling.