Megakaryocyte Lineage Commitment in Hematopoietic Stem Cells

Abstract 909

Hematopoietic stem cells (HSCs) are able to self-renew and differentiate into all blood lineages. Lineage commitment can take place at any stage from daughter cells of HSCs to multipotent progenitors. We have previously suggested that lineage commitment takes place at the level of HSCs (Takano et al. 2004). According to our asymmetric model, one HSC gives rise to two daughter cells consisting of one HSC and one lineage-committed progenitor. To verify this model, we performed a series of single-cell transplantation, followed by paired daughter cell experiments. The Ly5 congenic system has been commonly used to distinguish test donor cells from recipient cells and competitor cells. Since the Ly5 antigen is only expressed in leukocytes, platelets and erythrocytes have not been examined for evaluation of multi-lineage reconstitution. To assess lineage contribution more precisely, we have recently generated a transgenic mouse line in which Kusabira Orange (KuO) is expressed in all blood lineages including platelets and erythrocytes. CD150⁺CD41⁻CD34^−/lowc-Kit⁺Sca-1⁺Lin⁻ (CD150⁺CD41⁻CD34⁻KSL) cells, in which HSCs are highly enriched (Kiel et al. 2005, Morita et al. 2010), and CD150⁻CD41⁻CD34⁻KSL cells were individually isolated from KuO transgenic mice (B6-Ly5.1), and were transplanted into lethally irradiated mice (B6-Ly5.2), along with 2×10⁵ bone marrow cells (B6-Ly5.1/Ly5.2). For secondary transplantation, 1×10⁷ bone marrow cells from recipient mice were transplanted to lethally irradiated mice. Peripheral blood cells of primary and secondary recipient mice were periodically analyzed after transplantation. In some mice, full-lineage reconstitution was detected after transplantation with single CD150⁺CD41⁻CD34⁻KSL cells. In others, only common myeloid progenitor-type reconstitution was detected whereas common lymphoid progenitor-type reconstitution was not detected at all. Interestingly, megakaryocyte lineage (Meg)-restricted reconstitution and Meg/erythrocyte lineage-restricted (ME) reconstitution were observed after transplantation with single CD150⁺CD41⁻CD34⁻KSL cells, but not after transplantation with single CD150⁻CD41⁻CD34⁻KSL cells. These results suggest that Meg and ME progenitors are closely related to HSCs in their development. Furthermore, we performed paired daughter cell assays combined with single cell transplantation. Following division of CD150⁺CD41⁻CD34⁻KSL cells in culture, individual two daughter cells were separated by micromanipulation techniques and were transplanted into lethally irradiated mice with 2 × 10⁵ competitor cells. Preliminary data showed in effect that the recipient mice of each single daughter cell derived from one CD150⁺CD41⁻CD34⁻KSL cell exhibited asymmetric differentiation; one with full-lineage reconstitution and the other with Meg-restricted reconstitution. Our findings offer a new perspective of HSC differentiation pathways.