The Microarray Signature of Rhesus Macaque CD34+CD123 (IL-3 Receptor)+ Hematopoietic Stem/Progenitor Cells Isolated Following Mobilization with Plerixafor Alone or in Combination with Granulocyte Colony-Stimulating Factor (G-CSF)
Authors: Donahue Robert E. , Jin Ping , Klinman Johanna I. , Bonifacino Aylin C. , Metzger Mark E. , Stroncek David 
Published: October 2019

Abstract
Abstract 1301
CD34+ cells are a heterogeneous population of cells which contain the hematopoietic stem cell (HSC). Previously we determined that the microarray signature of CD34+ cells mobilized by G-CSF differed from those mobilized with either plerixafor (AMD3100) alone or in combination with G-CSF (Donahue RE, et al. Blood 2009; 114:2530–2541). Recently we also determined that a novel subpopulation of CD34+CD123+ cells was mobilized with plerixafor and not G-CSF in rhesus macaques (Uchida N, et al. Exp Hematol 2011; 39:795–805). To explore differences between CD34+CD123+ and CD34+CD123- cells, we evaluated their gene expression signatures. Following mobilization with plerixafor alone or in combination with G-CSF, we collected mononuclear cells by leukapheresis, isolated rhesus CD34+ cells by immunoselection, and further isolated CD34+CD123+ and CD34+CD123- by cell sorting. CD34+CD123+ cells were found not to express CXCR4 on their cell surface. Total RNA was isolated from cells and amplified to cRNA. Control cRNA was labeled with Cy3 dye and experimental cRNA was labeled with Cy5 dye. Control and experimental labeled cRNA were co-hybridized to a custom-made 17.5K cDNA (UniGene cluster) microarray. Gene expression was analyzed by quantifying the fluorescence from the microarray. The raw data set was filtered according to a standard procedure to exclude spots with minimum intensity. The relationship between the cells was analyzed using an unsupervised Eisen's hierarchical clustering method. Statistical analysis was done using Array Class Comparison analysis. Pathway analysis was carried out using Ingenuity Pathway Analysis. Principal Component Analysis demonstrated that CD34+CD123+ and CD34+CD123- cells cluster separately and are not distinguishable on the basis of mobilization regimen. Using Supervised Hierarchical Cluster Analysis for genes which were significantly different (p<0.05) there was further confirmation of clustering of the CD34+CD123+ cells versus the CD34+CD123- cells. Microarray analysis revealed that pathways including retinoic acid receptor activation and TGF-β and MAPK signaling were up-regulated in CD34+CD123+ cells, whereas glucocorticoid receptor and TREM-1 signaling, and genes involved in dendritic cell maturation and production of nitric oxide and reactive oxygen by macrophages were down regulated. The up regulated pathways and the down regulated pathways for the CD34+CD123+ population suggest that CD34+CD123+ cells are lymphoid progenitors. Upon comparing pathways between those previously reported to be unique to CD34+ cells mobilized with plerixafor alone or in combination with G-CSF, the majority are those that have been identified also to be in the CD34+CD123+ subpopulation. It appears as if the CD34+CD123+ population accounts for many of the differences in CD34+ cells mobilized with plerixafor observed in the earlier study. In conclusion, the microarray signature of the CD34+CD123+ cells subpopulation suggests that these cells are lymphoid progenitors which can be effectively mobilized with plerixafor. This may account for the more rapid lymphoid recovery we observed for rhesus macaques transplanted with CD34+ cells mobilized with plerixafor and suggests a therapeutic approach through immunoselection to selectively target CD34+CD123+ progenitor populations.

Disclosures:
No relevant conflicts of interest to declare.