Controversy persists about mixed chimerism (mCh) occurring in the hearts of patients after orthotopic cardiac transplantation in comparison with allogeneic bone marrow (BM) and peripheral blood stem-cell (PBSC) transplants. Cadaver hearts were examined after sex-mismatched transplantation by immunophenotyping combined with dual color fluorescence in situ hybridization (X and Y chromosome-specific probes). A striking disparity in the extent of mCh depending on the different transplantation procedures was recognizable. After allografting with PBSCs, 1.7% chimeric cardiomyocytes were detectable contrasting 5.4% of donor cells after full BM transplantation. In cardiac transplants, host-type endothelial cells (16.2%) and myocytes (14.3%) of the vessel walls were more often encountered than after BM and PBSC allografting. A sprouting of vascular structures into the donor heart after orthotopic cardiac transplantation has to be assumed, as does a pivotal role of the mesenchymal stem cells of the BM in the development of mCh.

Experimental findings support the hypothesis that within the functional network of the bone marrow (BM) microenvironment the endothelial cells (ECs) exert a pivotal role as gatekeepers by controlling the trafficking and homing of progenitor cells. However, little information is available concerning the origin of ECs after allogeneic bone marrow transplantation (BMT) in CML. To determine the extent of mixed chimerism (MCh) a simultaneous immunohistochemical and fluorescence in-situ hybridization (FISH) study was performed on BM biopsies derived from patients following sex-mismatched BMT with full unmanipulated BM. ECs were identified by their staining with CD34 and the myofibroblasts (MFs) of large vessels were labeled by an antibody against alpha-smooth muscle actin. For sex-typing and demonstration of the bcr/abl fusion product appropriate commercially available probes and detection systems were applied. Contrasting a total congruence of labeling in control samples five patients showed donor type ECs in the early posttransplant period in about 20%. In the remaining four patients the amount of donor type ECs increased slightly after the third month up to 30%. A total of 26 MFs could be identified lining large capillaries and arterioles that exclusively revealed a host origin. Following successful engraftment only very few of the persistent host-derived ECs also displayed the bcr/abl gene. In five patients, a conversion of MCh from donor to host type ECs was recognizable during the evolution of leukemic relapse. This finding was accompanied by a bcr/abl rearrangement of about 10% of these cells. In conclusion, following myelo-ablative therapy, a survival of a considerable number of ECs and MFs of the vessel walls has been found implying persistence of host-derived vascular structures of the BM stroma. However, in only a small proportion bcr/abl+ ECs and thus minimal residual disease was detectable. Evolution of leukemic relapse was characterized by conversion of MCh with almost total loss of donor type ECs and increase in number of bcr/abl+ ECs.

The lineage-restricted MCh of progenitors after BMT is in keeping with the assumption that leukemic (bcr/abl ) precursors represent only a fraction of the total host-derived (chimeric) CD34 cells. These residual clonally transformed progenitors survive myeloablative treatment and thus may be the source for a later relapse.

Until now, studies on mixed chimerism (MCh) after allogeneic bone marrow transplantation (BMT) have predominantly focused on the B- and T-lymphocyte population, but not on distinct myeloid cell lineages like nucleated erythroid precursors and megakaryocytes. To evaluate the lineage-restricted MCh more explicitly in 10 patients with chronic myelogenous leukemia (CML), a quantitative analysis was performed on bone marrow biopsies following a sex-mismatched host/donor constellation. Techniques included immunophenotyping (antiglycophorin C, CD61) for the identification of erythro- and megakaryopoiesis and a simultaneously conducted genotyping with x- and y-chromosome-specific DNA probes. Normal bone marrow and specimens taken before BMT served as controls. Contrasting a total gender-dependent sex-typing in the latter samples in the early and late posttransplant period (up to 586 days), 3–9% erythroid precursors and about 16% megakaryocytes revealed a host-type origin. This significantly higher number of host megakaryocytes is explained by their polyploidy generating an increased probability to detect positive signals at a certain section level of the corresponding biopsies. A striking conversion of MCh to a recipient cell type was found in leukemic relapse with a more than 90% host-derived erythroid and megakaryocytic cell population in 4 patients approximately 643 days after BMT.

The CD68+ resident bone marrow macrophage population including PGCs are involved in the lineage-specific chimerism and minimal residual disease after BMT in CML.