SPRY1 Is a Negative Regulator of Long-Term In Vivo Engraftment and Ex Vivo Expansion of Primitive Human Umbilical Cord Blood Cells.

Abstract

Hematopoietic stem cells (HSCs) are functionally defined by their capacity to home to the bone marrow microenvironment, proliferate and differentiate to restore normal hematopoiesis in a myeloablated recipient; however, the molecular determinants of these processes are not well understood. By comparing the gene expression profiles of highly purified human HSC-enriched and HSC-depleted cell populations, and subsequently validating the hematopoietic function of a subset of these differentially expressed genes using zebrafish (Danio rerio), we previously identified human Sprouty 1 (SPRY1), an evolutionarily conserved antagonist of RTK signaling, as a potential regulator of mammalian HSC development and/or cell-fate decisions. To directly assess the role of SPRY1 in mammalian HSC and hematopoietic progenitor cell (HPC) function, we constructed a dual-promoter lentiviral vector to co-express SPRY1 and green fluorescent protein (GFP) (SPRY1-LV) and a control lentiviral vector to express GFP alone (GFP-LV) in the CD34+ fraction of human umbilical cord blood (UCB). While the enforced expression of SPRY1 in CD34+ UCB cells had no effect on the frequency or morphology of colonies generated in short-term in vitro colony-forming cell (CFC) assays (SPRY1-LV = 27.6 ± 12.3% and GFP-LV = 28.4 ± 20.0%; n = 3), it profoundly inhibited the capacity of UCB CD34+ cells to engraft in the bone marrow NOD-SCID mice in vivo. The estimated frequency of week 11 SCID-repopulating cells (SRC) (± 1 standard error) for SPRY1-LV (n = 13 mice) and GFP-LV (n = 15 mice) cells was 1 in 12,678 cells (6,167 – 26,064) and 1 in 3,412 cells (2,272 – 5,124), respectively, as determined using limiting dilution conditions and Poisson statistics. Furthermore, in 14 day cultures designed for the ex vivo expansion and/or maintenance of primitive hematopoietic cells, ectopic expression of SPRY1 in CD34+ UCB cells dramatically inhibited the expansion of total nucleated cells (SPRY1-LV = 37.9 ± 10.1 fold; GFP-LV = 71.1 ± 5.8 fold; n = 3; p<0.05) and CFCs (SPRY1-LV = 5.7 ± 1.2 fold; GFP-LV = 20.8 ± 14.9 fold; n = 3), although it had no effect on expansion of CD34+CD38− cells. We are currently investigating potential mechanisms for the observed affects of SPRY1 on primitive hematopoietic cells, paying particular attention to the possible effects of SPRY1 expression on “early-acting” hematopoietic cytokines and growth factors that activate RTKs - including FGF1, FGF2, VEGF, SCF, FLT3L, and ANGPT1. In conclusion, enforced expression of SPRY1 negatively regulates primitive hematopoietic cell engraftment in vivo and expansion in vitro, thereby presenting the first example of a role for a Sprouty family member, SPRY1, in primitive human hematopoietic cell function. Moreover, this data further validates the use of model organisms, such as zebrafish, for evaluating the functional roles of transcripts identified in large-scale gene expression profiling experiments in mice and humans.