Defining the Axolotl Hematopoietic Stem Cell

Abstract 1295

Hematopoietic stem cells (HSCs) are the most therapeutically usable stem cells to date. HSC derived cells are involved in wound healing responses throughout the body. The goal of our study is to discovery methods to promote improved regenerative responses from mammalian HSC. The champion of vertebrate regeneration is the axolotl. It can regrow entire limbs, major portions of most internal organs – including the brain and spinal cord. Recent advances in the production of transgenic axolotl, complete mapping of the axolotl transcriptome and production of gene expression arrays finally allow molecular mapping of regeneration pathways. In order to define the role of HSC in regeneration it was first necessary to define and characterize the axolotl HSC. In the current study we develop classic assays such as hematopoietic cell transplantations (HCT) and colony forming cell (CFC) assays to map axolotl hematopoiesis. In addition we take advantage of embryo manipulations possible with the axolotl to generate hematopoietic chimeras (GFP+ vs. white) as non-injury models for regeneration.

Axolotl have a naturally occurring white variant that are nearly as transparent as zebrafish. We used a normal and CMV:GFP+ transgenic white strains to map sites of hematopoiesis and to develop HSC transplant methodology. We also establish protocols for Colony Forming Unit (CFU) assays for hematopoietic progenitors. Liver, spleen, kidney, marrow, and thymus were tested as potential HSC sources. Both the liver and spleen contained transplantable HSC capable of radioprotection and multilineage reconstitution of lethally irradiated (1000 rads) adult axolotls or non-conditioned embryos. As in zebrafish, use of the white axolotl mutant allows direct visualization of engraftment, homing, and hematopoiesis in real time. Fluorescent donor cells were seen homing to the liver and spleen regardless of tissue origin, albeit with better overall engraftment from spleen cells. Hematopoiesis occurred for more than 6 months in embryo transplantations. FACS analysis of these two organs showed that the liver contains relatively even numbers of myeloid and lymphoid populations whereas the spleen has a bias toward lymphoid and erythroid populations. Additionally, early stage white and GFP embryos were transected and the posterior and anterior halves of the two different animals were joined. GFP+ blood was present in animals that contained the liver or spleen within the GFP portion of the body further indicating that hematopoiesis occurs in these organs. These embryo chimeras also provide stable long-term GFP+ blood chimeras without irradiation injury. CFU assays demonstrated that the liver contains predominantly myeloid progenitors. In contrast, the spleen has mostly erythroid and megakaryocytic progenitors but also some myeloid progenitors.

Here we have shown that the axolotl HSC resides in the liver and spleen and begun the basic mapping of hematopoiesis. Moreover, we have established the essential hematopoietic assays for the axolotl. The ability to generate both irradiation and non-injury GFP+ blood chimeras provide powerful tools to determine the role of HSC/progeny in axolotl regeneration.