RasGRP3 Role in Umbilical Cord Blood CD34+ Cell Homing and Engraftment
Year: 2024
Type: article
Abstract: We previously identified RasGRP3 as a regulator of erythropoietin-mediated reduced umbilical cord blood (UCB) CD34+ cell transmigration.1 To examine the effects of manipulating RasGRP3 expression on hematopoietic stem and progenitor cell (HSPC) homing, engraftment, long term reconstitution, and self-renewal, we utilized a mouse model for UCB transplantation. UCB derived hematopoietic cells were lentiviral transduced with shRNA constructs targeting RasGRP3, leading to reduced expression, or with overexpression constructs leading to stabilized expression of RASGRP3. On the day of transplantation and 2 days following lentiviral transduction, UCB units were sorted for CD34+, fluorophore+ cells. These selected cells were then transplanted to immune deficient mice (NSG) which had been sublethally irradiated 1 day prior. Mice were bled every 4 weeks to examine human cell donor chimerism in the PB. For secondary transplantations, we used BM pooled from all primary recipients. This pooled BM was transplanted to immune deficient mice (NSG) or transgenic immune deficient mice that express a trio of human cytokines (NSGS) which had been sublethally irradiated 1 day prior. To examine BM homing, mouse BM was harvested 3 hours after transplantation. At weeks 12, 16, and 20 post transplantation, there were consistent but statistically insignificant trends demonstrating reduced human cell chimerism in the PB when RasGRP3 was knocked down. At weeks 16 and 20, RasGRP3 overexpression trended towards leading to increased human cell chimerism in the PB. Interestingly, both knockdown and overexpression of RasGRP3 trended toward decreased human chimerism in recipient mouse BM 20 weeks post transplantation. There were decreased percentages of primitive HSPC in the BM of mice receiving RasGRP3 shRNA compared to control. At week 16 post-secondary transplantation, mice that received RasGRP3 knockdown cells showed statistically significant decreases in human cell chimerism in the PB of NSG mice, and a similar trend in NSGS mice. Similarly, RasGRP3 recipients receiving RasGRP3 overexpressed cells showed trends toward increased PB chimerism at week 16 in secondary mice. Interestingly, there were no significant differences in human cell chimerism in the BM of either NSG or NSGS receiving cells with RasGRP3R expression manipulation. Mice receiving RasGRP3 knockdown UCB trended toward having lower numbers of total human cell in the BM 3-hours post transplantation, suggesting RasGRP3 expression levels affect in vivo homing. RasGRP3 expression levels play a role in UCB homing and engraftment, but the main phenotypic effects appear to be on PB hematopoietic and hematopoietic derived cells.
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $3,650
OpenAlex ID: W4391599733