Autologous Transplantation Of Thrombopoietin Derived-Hepatic Hematopoietic Progenitor Cells In Xenopus Laevis

Thrombopoietin (TPO) is required for megakaryopoiesis generating platelets via acting through its receptor c-Mpl, and supports hematopoietic stem cell maintenance. It is unknown whether there are evolutionary conserved signalling pathways and molecular functions governed by TPO/c-Mpl in non-vertebrates. We therefore attempt to develop a non-mammalian animal model in order to investigate thrombocyte development in adult African clawed frog (Xenopus laevis: X. laevis). We previously reported that Xenopus hepatic cells were differentiated to polyploid megakaryocyte-like cells and thrombocyte under X. laevis TPO (xlTPO) stimulation. The aim of this study is to determine the cellular characteristics of TPO-induced immature cells by establishing in vitro and in vivo Xenopus models.

Hepatic cells were cultured in a 35mm diameter dish at 23°C under 5% CO₂ with minimum essential medium containing 0.87% methylcellulose and 20% FCS in the presence of xlTPO (10 ng/ml). After 24 days of culture, the number of colonies reached 9.0±3.0 per dish. The size of colonies was heterogeneous, and a typical large colony was composed of more than 512 cells. Cells recovered from the large colonies at 24-day culture were then serially replated to a 96-well plate at a density of 1x10³ cells/well with semi-solid medium in the presence of xlTPO (10 ng/ml) or X. laevis erythropoietin (xlEPO) (10 ng/ml). At day 8, a significant number of colonies (125±10/100μl culture well) were emerged in the xlTPO culture; but a few colonies in the xlEPO culture (3±1/100μl culture). The colony formation was inhibited by the addition of recombinant xlc-Mpl-FC fusion protein in the culture, indicating that the colony formation was directly stimulated by xlTPO. It was notable that xlTPO alone maintained and expanded the culture for over four months. The component cells in xlTPO-induced colonies resembled megakaryocytes or thrombocytic cells with spindle shapes, suggesting that xlTPO exerted the ability to support long-term maintenance and proliferation of thrombocytic progenitors. We then collected the cells after 24 days of liver cell culture in the presence of xlTPO. The RT-PCR revealed that the cells did not express hepatic markers of albumin and tpo; while they expressed not only mRNAs of thrombocytic markers such as c-mpl, fli-1, integrinαIIb, but also mRNAs of mpo, g-csfr, γ—globin , gata-1, esam and c-kit. The result proved that xlTPO expanded multilineage hematopoietic progenitors; not restricted to cells in the thrombocyte lineage. Subsequently we cultured cells collected from the day-24 colonies in conditioned medium of liver and spleen. After 8 days of culture with xlTPO, approximately 5% of the cells developed morphologies resembling mature eosinophils in the spleen-conditioned medium. As well, about 18% of the cells appeared to be macrophage-like cells, and they phagocytosed latex beads. Taken together, we concluded that xlTPO supported long-term maintenance of multipotent heamatopoietic cells in the Xenopus liver via TPO-c-Mpl signalling. Because inbred strains of X. laevis are not abundantly available, transplantation studies in X. laevis are limited. We therefore attempted to develop autologous cell transplantation of xlTPO-induced cells in X. laevis. Liver cells obtained by partial hepatic resection were cultured in the semi-solid culture system in the presence of xlTPO. After 24 days, the cells labeled with PKH26 were autologously transplanted by intracardiac injection. PKH26-positive cells were detected in the liver and the spleen of the auto-transplanted frogs after 12 days, though the cells were diminished from the body within 4 to 6 days in the allo-transplanted Xenopus. Flow cytometric analysis showed that PKH26-high cells contained much RNA and displayed lower forward scatter (FSC); while PKH 26-low cells were detected in the intermediate FSC. The findings suggested that a subpopulation of long-term maintained liver cells by xlTPO could barely proliferate and differentiate in the liver and the spleen, and the characteristics of these cells agrees with the features of hematopoietic stem cells or progenitors, as in mammals and zebrafish. This unique culture system developed in this study has clues to resolve the functional evolution of TPO-cMpl system shared by thrombocytic development and earlier hematopoietic progenitors.