523. Human Umbilical Vein Endothelial Cell, HUVEC, Co-Culture Promotes Robust Expansion and Maintains Phenotypic Integrity of Rhesus Hematopoietic Stem and Progenitor Cells, HSPC, Prior to Autologous Tansplantation
Authors: Sandhya R. Panch, Idalia Yabe, Diego Espinoza, Chuanfeng Wu, So gun Hong, Stephanie Sellers, Allen Krouse, Aylin Bonifacino, Robert Donahue, Cynthia Dunbar
Journal: Molecular Therapy (2016)

The development of ex-vivo HSPC expansion techniques is particulary relevant for improving cord blood transplantation and gene therapy. Despite successful long-term, multilineage reconstitution of expanded human cord blood HSPC in immunodeficient mice, early phase clinical trials have failed to demonstrate improved outcomes. Thus, it is critical to develop a robust pre-clinical model to study ex-vivo expansion strategies, particularly function of long term HSPC and engrafment of all lineages, difficult in xenograft models. We have previously used retroviral insertion site retrieval and autologous competitive transplantation in rhesus macaques to track hematopoietic engraftment and ontogeny at a single HSPC level, in addition to comparing ex-vivo expanded and unexpanded HSPC (Gomes et al, Mol Ther). More recently we have developed retrieval of 31bp diverse barcodes as a more robust and quantitative in vivo HSPC tracking approach (Wu et al, Cell Stem Cell). Modified human endothelial cells (HUVEC) with the ability to be maintained in serum free media for prolonged periods have shown to support and expand human HSPC in murine Butler et al, Blood). We have developed a barcoded rhesus autologous transplant model to evaluate expansion of HPSC on HUVEC versus unexpanded or cytokine expanded rhesus HSPC. We first tested the feasibility of rhesus CD34+ cell expansion on HUVEC versus cytokines/fibronectin for 8 days. We also evaluated for the diversity of expanded HSPC by retrieving transduced viral barcode DNA tags from individual cells by low cycle PCR followed by Illumina sequencing. On average, rhesus CD34+ cells expanded 76 fold (± 53) on HUVEC versus 13 fold (±7) in cytokines (n=4; p=0.03). By morphologic assessment, the HUVEC expanded cell fraction contained higher numbers of progenitors and differentiating cells, with up to 97% CD34+CD45+ cells in the HUVEC expanded fraction and 65% in the cytokine expanded fraction. The HUVEC expanded rhesus CD34+ cells were capable of multi-lineage colony formation after 8 days of expansion. Within the HUVEC expanded cell fractions, fold expansion was similar whether CD34+ cells were frozen after (C1) or before (C2) transduction-expansion. However, viability and percentages of CD34+CD45+ cells were higher in C2 compared to C1 (55% versus 26%). GFP transduction efficiency was also higher in C2 compared to C1 (43% vs. 28%). We demonstrated a slightly lower percentage but higher absolute number of CD34+CD38-CD45RA-CD90+CD49f+Rho-low putative long-term HSC after expansion on HUVEC compared to in cytokines over 26 days in culture (0.018% vs. 0.016% pre and post expansion, respectively). Retrieved barcode analysis and quantitation revealed that in the HUVEC expanded fraction, the top 20 clones constituted up 15% of the total valid reads by day 8, in contrast to only 3.9% of total clones in the cytokine expanded fraction. Cells have been transplanted into autologous macaques, in a competitive model comparing non-expanded, cytokine-expanded and HUVEC-expanded conditions, and in vivo expansion and barcode analysis will be presented. Our data thus far show that peripheral blood CD34+ cells from rhesus macaques expand more robustly in the presence of HUVEC and cytokines as compared to expansion in cytokines alone, and barcode analysis suggests that the HUVEC fraction selectively expanded a subset of highly proliferative cells, which may represent true HSCs within the CD34+ cell fraction.