Transplantation of Hematopoietic and Endothelial Stem Cell Progenitors in Transgenic Zebrafish.

The zebrafish has emerged as an excellent vertebrate model for analysis of hematopoietic and leukemogenic development. Blood production in adult zebrafish occurs in the kidney marrow (KM), within the peritubular kidney interstitium, while kidney tubules support renal functions. To study the molecular mechanisms of stem cell specification and fate decision, we developed transgenic zebrafish expressing EGFP either at distinct hematopoietic levels using the zebrafish SCL/TAL1, RAG2, and FLI1 promoters, or in all tissues using the zebrafish β-actin promoter. KM progenitor cells from the SCL-EGFP, FLI1-EGFP, RAG2-EGFP, and ZBA-EGFP transgenic zebrafish were separated by FACS based on previously described method. The kinetics of KM progenitor cell transplantation from transgenic zebrafish into adult and embryonic recipients was analyzed. Visualization of donor cells in embryonic and adult recipients allowed us to detect trafficking and multilineage reconstitution in transplanted recipients in real-time. Transplantation of whole KM to unconditioned adult recipients resulted in the disappearance of donor-derived cells within 3 weeks. Thus, efficient engraftment of donor cells required host conditioning using β radiation or busulfan. Dose response experiments indicated that the minimum lethal dose of radiation averaged between 38 and 54 Gy, and was strain-dependent since hematolymphoid ablation of the Ek strain required higher radiation doses compared to the AB* strain. Sublethal irradiation doses of 25 Gy predominantly ablated lymphocytes and permitted transplantation of lethal TEL-AML1-induced pre B-cell leukemia. All irradiated animals not receiving transplants showed 10–50 fold decrease in all hematopoietic lineages at one week, and then died by 3 weeks post-radiation. In contrast, 60% of animals that received a transplant survived for at least 3 months. FACS analysis indicated that the frequency of EGFP-positive KM progenitors from the ZBA-EGFP (Common progenitors), the FLI1-EGFP (Hematopoietic-endothelial progenitors), and the RAG2-EGFP (Lymphoid progenitors) averaged 7%, 0.6%, and 4%, respectively. FACS-sorted EGFP⁺ donor cells were transplanted into adult wild-type irradiated recipients or blastula stage embryos. Transplantation of donor ZBA-EGFP KM cells resulted in engraftment and detection of donor-derived hematopoietic cells of all lineages within 2–3 weeks. No overt signs of graft-versus-host disease or graft rejection were detected. Transplantation of KM endothelial progenitors or angioblasts from the FLI1-EGFP transgenic fish resulted in endothelial homing and appearance of donor-derived intersegmental vessels in recipient fish 3–4 weeks post-transplant. These data demonstrate that marrow-derived angioblasts reconstitute endothelium of adult zebrafish, and represent, to our knowledge, the first evidence for a marrow derived hematopoietic-endothelial precursor homing to the vasculature of a transplanted vertebrate. Embryonic transplantation of 2×10³ to 3×10³ cells to blastula stage embryos resulted in dose-dependent lineage-faithful contribution of the transplanted cells to the developed embryos. We are investigating whether single transplanted progenitor cells sorted from SCL-EGFP KM engraft wild-type and SCL-depleted recipients, and provide long-term survival when transplanted to GATA1⁻/⁻ and RUNX1⁻/⁻ mutants. Transplantation of KM progenitors in zebrafish is sufficient for radio- and chemoprotection, and provides an excellent system to analyze oncogenic transformation, stem cell engraftment and immune cell function.