Transplantation Conditioning Regimens Induce Different Hematopoietic Stem Cell Differentiation in Mice At the Clonal Level

Abstract 140

Hematopoietic stem cell (HSC) transplantation is a common clinical treatment for patients with cancers or hematopoietic disorders. Yet it remains a risky procedure with many complications that are not well understood. To better understand blood reconstitution after transplantation, we used a mouse model to track individual donor HSCs in vivo. We compared lethal irradiation mediated transplantation, ckit antibody clearance based transplantation and unconditioned transplantation.

To track single HSCs in vivo, we have developed a novel experimental system combining viral cellular labeling, genetic barcoding and high-throughput sequencing. This experimental system allows us to simultaneously track thousands of cells with single cell resolution and also to directly measure the clonality of small cell populations such as HSC. We used a lentiviral vector to insert unique 33-mer DNA codes into HSCs' genomes to serve as genetic barcodes. Barcoded HSCs were transplanted into mice preconditioned with lethal irradiation, with ckit antibody (ACK2) or without any conditioning regimen. Five months post-transplantation, barcodes from HSCs as well as from blood cells and intermediate progenitors were extracted and analyzed using high-throughput sequencing technology.

Our data suggest that all donor-derived mouse HSCs uniformly contributed to granulocytes and B cells in unconditioned mice. However, in irradiated mice, a small subset of HSC clones expanded dramatically to supply the majority of granulocytes and B cells. This clonal expansion was also observed after antibody clearance mediated transplantation, but not as severely. In addition, after unconditioned transplantation only a small subset of HSC clones contributed to T cells. CD4+ T cells and CD8+ T cells were supplied by different HSC clones. In contrast, after irradiation or antibody treatment the HSC clones that contributed to T cells produced CD4+ T cells and CD8+ T cells equally.

We also found that HSCs contribute to blood lineages differently (lineage bias) depending on the transplantation conditioning regimens. In irradiated mice and ckit antibody treated mice, donor HSC clones contributed differently to granulocytes and B cells. However, we did not observe any lineage bias after unconditioned transplantation. This striking difference suggests that irradiation regimens induce unbalanced differentiation of HSCs at the clonal level. To identify the source of lineage bias after irradiation, we examined the intermediate progenitors of the myeloid differentiation and the lymphoid differentiation. We found no lineage bias at the clonal level between granulocyte/monocytic progenitors (GMP), megakaryotic/erythroid progenitors (MEP) and common lymphocyte progenitors (CLP) in unconditioned mice nor in mice treated with any form of pre-conditioning including irradiation. This indicates that lineage bias is induced downstream of these oligopotent progenitors.

In summary, our data suggest that different transplantation conditioning regimens induce strikingly different HSC differentiation at the clonal level. After severe conditioning regimens such as irradiation, a small subset of HSC clones dramatically expands to supply the majority of the blood cells. These HSC clones do not contribute equally to the myeloid and lymphoid lineages. In contrast, after unconditioned transplantation, all HSC clones equally and uniformly contribute to the myeloid and lymphoid lineages. These clonal level differences of blood reconstitution after various conditioning regimens may be related to complications associated with HSC transplantation.