Lin−CD117+ Hematopoietic Cells Preferentially Home to Spleen and Their Migration Is Affected by Selectins.
Authors: Calado Rodrigo T. , Young Neal S. , Chen Jichun 
Published: April 2020

Abstract
In hematopoietic stem and progenitor cell transplantation, infused donor cells home to recipient bone marrow (BM) where they become residents and sustain normal hematopoiesis. Understanding the molecular mechanisms that regulate hematopoietic cell homing might help to increase homing efficiency and to optimize cell engraftment in patients. When BM cells from enhanced green fluorescence protein (GFP) transgenic mice were transplanted into normal C57BL/6 recipients, GFP+Lin− cell recovery in BM peaked to 17 ± 2 %, 21 ± 8 %, and 22 ± 14 % at 18, 48 and 72 hours after cell infusion; likewise, GFP+Lin− cell recovery in the spleen peaked to 7 ± 3 %, 10 ± 0 %, and 14 ± 3 % at these time points. Conversely, GFP+Lin− cells were not detected in recipient thymus at any time. Of the recovered GFP+Lin− cells in the BM and spleen, GFP+Lin−CD117+ cells preferentially homed to the spleen (7.0 ± 0.5 % recovery in spleen vs. 0.3 ± 0.5 % recovery in BM; P&lt;0.0001) since only 0.5 ± 1.1 % of recovered GFP+Lin− cells in the BM were CD117+, compared to a 25 ± 1.1% CD117+ recovery rate in the spleen (P&lt;0.0001). Total body irradiation did not increase but instead reduced GFP+Lin− cell homing in both BM (P&lt;0.002) and spleen (P&lt;0.005). Also, the proportion of CD117+ cells among recovered GFP+Lin− cells was much lower (P&lt;0.05) in irradiated than in unirradiated recipients. When sorted GFP+Lin−CD117+ and GFP+Lin−CD117− cells were transplanted into sublethally-irradiated recipients at 20–180 ×103 cells per recipient, no GFP+ cells reached detectable level in either the spleen or BM. When the same number of GFP+Lin−CD117+ and GFP+Lin−CD117− cells were co-transplanted with 20 ×106 normal B6 BM cells, we detected 0.15% to 0.86% cell recovery in recipient BM and spleen, indicating that total number of injected cells affects cell homing and recovery. All recovered cells carried the same GFP+Lin−CD117+ or GFP+Lin−CD117− phenotype as they were injected, indicating that there was no phenotype switch during the first 18 hours of homing. In a short-term engraftment assay, 81 ×103 GFP+Lin−CD117+ cells transplanted along with 20 ×106 normal BM cells resulted in 1.7–3.8%, 3.2–5.2% and 11–17% engraftment in recipient spleen, BM and peripheral blood respectively at 10 days after transplantation, while 96 ×103 GFP+Lin−CD117− cells transplanted along with 20 ×106 normal BM cells produced in no detectable engraftment. Similarly, in a long-term engraftment assay, donor engraftment was detected from GFP+Lin−CD117+ but not from GFP+Lin−CD117− cells when recipients were monitored at 15, 30, 60 and 90 days after transplantation. To test the role of selectin proteins in hematopoietic cell homing, we transplanted 20 ×106 GFP+ BM cells to B6 recipients that carry targeted deletions for E-, L- and P-selectins. At 18 hours, GFP+Lin- cell recovery was at 5.0 ± 0.4 % and 9.8 ± 1.8 % in the spleen and 19 ± 1.4 % and 11 ± 1.9 % in the BM for normal and selectin-deficient recipients. In conclusion, GFP+Lin− cells homed to BM and spleen but not to thymus, indicating that an appropriate microenvironment is necessary for adequate homing. Irradiation did not attract Lin− cells to the thymus but reduced Lin− cell homing to BM and spleen. While only a very small fraction of GFP+Lin−CD117+ cells homed to BM, they are likely the cells responsible for short- and long-term hematopoietic engraftment in recipients. Deletion of selectin genes may alter the microenvironment and shift Lin- cell homing to spleen, thus reducing hematopoietic cell homing to BM.