Transplantation Window Created by Bone Marrow Irradiation and Its Closure.
Authors: Katarina Forgacova , Sefc Ludek , Michalova Jana , Savvulidi Filipp , Chang Ko-Tung , Necas Emanuel 
Published: October 2019

Abstract
Abstract 3713

Introduction:
Therapeutic bone marrow transplantation (BMT) is usually performed 0–2 days after a conditioning treatment. Our aim was to determine the „transplantation window“, i.e. for how long after conditioning by irradiation are transplanted hematopoietic stem and progenitor cells (HSPCs) able to become engrafted in the microenvironmental niches of the recipient. Following closure of the window, we determined the presence of HSPCs in regenerated bone marrow (BM) by flow cytometry and transplantation assays.

Method:
C57Bl/6 Ly5.2 and congenic Ly5.1, as well as GFP transgenic mice, were used. Total body irradiation (TBI) was carried out from a 60Co source and the doses were 6 Gy (submyeloablative dose) or 9 Gy (ablative dose). Recipients irradiated by 9 Gy received a small „rescue“ syngenic transplant corresponding to 1/200 of femoral BM. With a delay of up to 180 days, the mice then received a large congenic transplant corresponding to 1/2 of femoral BM. In an experiment aimed at a premature closure of the transplantation window, the recipients exposed to 9 Gy were subsequently transplanted with syngenic BM cells in a dose of about 40×106 (2 femurs) cells for 5 consecutive days. On the 15th day, they were transplanted with congenic BM cells corresponding to one femur. Resulting chimerism of donor/recipient derived blood cells was evaluated from 1 to 6 months after the congenic transplantation in peripheral blood and in BM at the end of examination period. Tests for presence of HSPCs in regenerating BM included a competitive repopulation for STRCs and LTRCs (Short and Long Term Repopulating Cells), CFU-S assay and a flow cytometry based determination of BM subpopulations highly enriched for HSPCs.

Result:
Congenic BM engrafted recipients up to 20 days following the submyeloablative irradiation, and up to 30 days following the ablative irradiation and a small rescue transplant. Afterwards the transplantation window closed abruptly. The massive syngeneic transplant, delivered during first 5 days after the ablative irradiation, closed the transplantation window prematurely. After complete closure of the transplantation window, numbers of HSPCs determined by flow cytometry as Lin-Sca-1+ c-Kit+ (LSK) CD150+CD48- either CD34 positive or negative cells were still decreased, as well as LSK SP (Side Population) cells and CFU-S. The most significant difference from normal BM was, however, in an extremely low recovery of repopulating STRCs and LTRCs.

Conclusion:
The period during which BM damaged by conditioning irradiation accepts BM grafts lasts for weeks and significantly extends over recovery of BM cellularity and resumption of blood cell production. Regenerated BM, when it no longer accepts transplanted cells, is still highly deficient in STRC and LTRC repopulating progenitor and stem cells. Supported by projects LC06044, MSM 0021620806 and the grant SVV-2010-254260507.

Disclosures:
No relevant conflicts of interest to declare.