Detection of donor cells in recipient tissues after stem cell transplantation using FISH and immunophenotyping

Abstract

Haematopoietic stem cell transplantation (HSCT) is curative therapy for many malignant and non-malignant diseases. Effective conditioning regimen is an important component of HSCT in order to achieve a successful engraftment and to pursue a curative treatment. Conditioning regimens consist of chemotherapy with or without total body irradiation. Despite improved techniques and invention of new drugs, HSCT is still associated with transplantation related complications that negatively affect the outcome. Mechanisms behind the engraftment and the onset of the haematopoietic stem cells (HSC) proliferation and differentiation are not fully understood. Some studies have shown an evidence for trans-differentiation of HSC to non-haematopoietic tissues, but other studies claim that it is either cell fusion or transplantation of other progenitor cells.

The aims of this thesis were to investigate plasticity of donor HSC and the occurrence of tissue specific cells of donor origin in recipient using the fluorescens in situ hybridization (FISH) technique and immunophenotyping. We also studied the effect of the different conditioning regimens on the engraftment, chimerism and the outcome of HSCT in mouse model.

In study I, we investigated the effect of the administration sequence of busulphan (Bu) and cyclophosphamide (Cy) on the myeloablative and immunosuppressive effects, engraftment and toxicity in mouse model. Female BALB/c mice were transplanted with syngenic male donors after Bu-Cy or Cy-Bu conditioning and followed up to 90 days after HSCT. Sex mismatch enabled assessment of chimerism by detection of Y-chromosome carrying cells using FISH. Chimerism within specific cell populations, CD11c+ dendritic and CD4+CD25+ regulatory T cells, was assessed using FISH in combination with fluorescence immuno-phenotyping. In both Bu-Cy and Cy-Bu groups, comparable levels of chimerism were detected in the bone marrow with the peak at day 60 after HSCT, and this level remained stable. In spleen, both treatments produced similar levels of chimerism, but at the end of the study the chimerism level in Bu-Cy treated animals was significantly higher compared to the Cy-Bu group. The portion of CD11c+ and CD4+CD25+ of donor origin in the spleens was significantly lower in Cy-Bu treated animals compared to the Bu-Cy group until day +40. However, until day +90 dendritic and regulatory T cells of donor origin in the Cy-Bu group slightly exceeded those in Bu-Cy treated animals. Both dendritic and regulatory T cells play an important role in graft-versus-host disease and graft-versus leukemia effect in HSCT. Immunosuppressive effect and immune reconstitution expressed as spleen cellularity were similar in both groups. Toxicity profile expressed as decrease in body weight and levels of liver enzymes was advantageous for Cy-Bu regimen.

In summary, the patterns of long-term reconstitution of the bone marrow and spleen in Bu-Cy and Cy-Bu treated animals were comparable, with less liver toxicity in Cy-Bu group. Thus, altering the administration order from Bu-Cy to Cy-Bu may be beneficial in clinical use and may have positive impact on the outcome of HSCT.

In study II and III, we approached the plasticity of the stem cells by combining FISH with immunophenotyping. We studied the occurrence of non-haematopotietic cells of donor origin in female recipients transplanted with male donors. We found that 2-6% of pneumocytes type II (cytokeratin +/surfactant protein A+) were carrying Y-chromosome, and thus originating from the donor. In endometrial blood vessels about 10% of endometrial endothelial cells (CD34+/VEGFR2+) were of donor origin. In conclusion, bone marrow derived donor progenitor cells may trans-differentiate to other than haematopoeitic cells.