Tissue Availability of Donor Mesenchymal Stem Cells Determines the Degree of Plasticity Following Transplantation in a Non-Injury Model.
Authors: Chamberlain Jason , Porada Christopher D. , Frias Ana , Yamagami Takashi , Zanjani Esmail D. , Almeida-Porada Graca 
Published: October 2019

Abstract
The ability of bone marrow-derived Hematopoietic and Mesenchymal Stem Cells (MSC) to generate hepatocytes in a large non-injury sheep model of human stem cell transplantation has been demonstrated. Although in our sheep model the liver is still a hematopoietic organ at the time of transplantation, we hypothesized that the human BM-derived MSCs migrated preferentially to the BM, thus leaving few MSCs available to lodge in the liver and give rise to hepatocytes. Thus, in the present studies, we compared the ability and efficiency of clonally-derived BM MSC populations to give rise to liver cells upon either intra-hepatic (IH) or intra-peritoneal (IP) transplantation into fetal sheep. Five MSC clones were established from human adult BM by single cell deposition of Stro-1+ Gly-A-CD45- cells and expanded until sufficient cells were obtained. Phenotypic characterization demonstrated that all clones were positive for CD90, CD13, and CD29, and negative for hematopoietic markers such as CD34, CD133, and CD45. Five different clonal populations were used and each clone was transplanted either IP (n=7) or IH (n=6) into 55-60 days old fetal sheep recipients at 5x105cells/fetus. Evaluation of the recipients at 56-70 days post-transplant showed that clonally-derived MSC were not only able to generate hepatocytes, but they were also able to generate hematopoietic cells detectable both in the BM and in the PB. IP transplanted animals had a significantly higher level of human CD45+ in their BM (CD45=2.7±0.4%) compared to the IH transplanted animals (CD45=0.25±0.05%), demonstrating that IP injection favors the homing of these cells to the BM while upon IH injection, the human blood cells generated remain in circulation (2.6% CD45+). When we evaluated the presence of hematopoietic cells in the livers of these animals, the IP transplanted animals contained almost 2% CD45+ cells within their livers, while IH transplanted animals had levels of only 0.5-0.65%. When we looked at the generation of liver cells in both groups of animals by immunohistochemistry using monoclonal antibodies specific for human hepatocytes and albumin, we observed that IH injection was much more efficient at generating human hepatocytes than IP injection. The levels of human hepatocytes in most of the IH transplanted animals (10-15% positive cells) were roughly 5X higher than their IP counterparts (2.5-3%). Furthermore, human hepatocytes produced following IH injection were widely distributed throughout the liver parenchyma, while IP injection resulted in a periportal pattern of human hepatocytic activity. We also confirmed the functionality of the generated hepatocytes by detecting human albumin in circulation in these animals by ELISA. In conclusion, these studies show that in a non-injury model the number of available donor cells reaching the liver can be controlled by altering the route of administration, and that this availability determines both the levels of donor-derived hepatocytes generated and the pattern of distribution of these hepatocytes throughout the recipient liver.