br Materials and methods br Results and

Materials and methods
Results and discussion By analyzing surface markers of farnesoid x receptor isolated from PD effluents, we identified a Lin−/CD34+/CD38−/CD90+ (~0.14±0.03%) subpopulation (Fig. 1A), which are known to enrich for human HSCs. PD cells also contain phenotypic hematopoietic progenitors including common myeloid progenitors (CMP), megakaryocyte–erythroid progenitors (MEP), granulocyte/monocyte progenitors (GMP), and common lymphoid progenitors (CLP) (Fig. 1B). The overall peritoneal cells further include CD3+T-lymphocytes, CD19+/CD20+B-lymphocytes, CD15+/CD66b+ myeloid cells, and CD71+ erythroid cells (Fig. 1C). After analyzing all samples, we found that the phenotypic HSCs were consistently present in the PD effluents isolated from different patients at different times (Fig. 1D–F). Statistically, analyses revealed that a patient\'s gender, age, prior hemodialysis (HD) treatment and duration following catheter transplantation had no influence on the numbers of Lin−/CD34+/CD38−/CD90+ cells found in the PD effluents. The existence of hematopoietic progenitors was also confirmed by colony formation assays (Supplemental Fig. 1) (Zheng et al., 2011b, 2012). Additional analyses revealed that, on average, about 5.6×104 Lin−/CD34+/CD38−/CD90+ cells can be recovered from one PD effluent sample. There were on average 2.8×105 phenotypic HSCs acquired from 5 effluents of each patient during the PD training period. The frequency and number of HSCs acquired from PD effluents and patients varies greatly (Supplemental Fig. 2). The reason(s) for such variations remain undetermined. However, statistical analyses have excluded the influence of prior hemodialysis, gender, and ages on PD-derived HSC numbers. In addition, the numbers of phenotypic HSCs recovered from PD effluents did not significantly reduce for up to 100days after surgery. These results suggest that HSCs are either resident cells or continuously replenished in the peritoneal cavity. The functionality of these phenotypic HSCs isolated from PD effluents was evaluated by transplantation into sub-lethally irradiated NOD/SCID/gamma−/− (NSG) mice (Zheng et al., 2012). Fig. 2A shows multi-lineage engraftment of total hematopoietic (CD45/71+), myeloid (CD15/66b+), B-lymphoid (CD34−CD19/20+), and primitive (CD34+) human cells. Collectively, for 107 donor cells, there were 2.87±1.66% total human hematopoietic cells, 0.80±0.70% myeloid cells, 0.19±0.09% lymphoid cells, and 0.92±0.53% primitive cells in recipient bone marrow (Fig. 2B), attesting the existence of functional HSCs in the donor population. To assess self-renewal and long-term functionality of PD-derived HSCs, we collected bone marrow cells from the primary mice and then transplanted them into sub-lethally irradiated secondary recipients. The donor-derived cells showed comparable positive engraftment of B-lymphoid cells (0.97±0.54%) in bone marrow as in the primary recipients, and lower engraftment of myeloid cells (0.070±0.031%) and primitive cells (0.019±0.007%) (Fig. 2C). Together, our results imply that PD-derived HSCs contain repopulating HSCs. Finally, the transplantation routes (intraperitoneal and intravenous injection) have no effect on the final outcome of chimerisms. To investigate whether PD-derived HSCs behave differently from HSCs from other sources, we compared the engraftment of 107 human PD cells and 105 human cord blood (CB) CD34+ cells in different tissues/organs of the recipient mice via intravenous injection. At 2months post-transplantation, the indicated number of CB mononuclear cells had a greater engraftment in the recipient bone marrow, whereas the PD cells repopulated better in the peritoneal cavity and spleen, and produced higher percentages of myeloid cells and B-lymphocytes than CB counterparts (Fig. 3A). We suspect that, after residing in the peritoneal cavities, PD-derived HSCs are “primed” to initiate adaptive and innate immune responses against potential invasion of pathogens through intestinal mucosa. It is also possible that PD HSCs have a certain disadvantage while homing to recipient bone marrow. While we did carry out intrafemoral transplantation, this type of injection can only administer a volume less than 20μl. This small injection volume is insufficient for PD cells to produce detectable reconstitution. To explain the “primed” properties, we believe that donor PD HSCs have distinct intrinsic potential asc compared to bone marrow or cord blood HSCs. We also suspect that the recipient peritoneal and extramedullary sites provide unique microenvironment for PD HSCs to home and differentiate.