Human Thrombopoietin Knockin Mice Efficiently Support Human Hematopoiesis In Vivo

Abstract 403

Hematopoietic stem cells (HSCs) both self-renew and give rise to all blood cells for the lifetime of an individual. Xenogeneic mouse models are currently broadly used to experimentally study human hematopoietic stem and progenitor cell biology in vivo. However, maintenance, differentiation, and function of human hematopoietic cells are suboptimal in these hosts. More specifically, (i) human cell engraftment is only transient, not lasting for the life of recipient mice, (ii) there is an unphysiological bias towards the lymphoid lineage as well as poor differentiation of myeloid cells, and (iii) there is an important variability in the engraftment levels between different individual animals. Thrombopoietin (TPO) has been demonstrated as a crucial cytokine supporting maintenance and self-renewal of HSCs. Although TPO is mouse to human cross-reactive at supraphysiological levels, we speculated that species differences would lead to insufficient TPO activity on human cells in the xenogeneic environment. We thus generated RAG2^−/−γ_c^−/− mice in which we replaced the gene encoding mouse TPO by its human homologue. This led to the expression of human TPO at human physiological levels in the serum and tissues of TPO knockin mice. Homozygous humanization of TPO (TPO^h/h) led to significantly increased levels of human engraftment in the bone marrow of the hosts (an approximately 2-fold increase). TPO^h/h recipients also displayed a lower engraftment variability, with an at least 80% human chimerism in 75% of the mice, and engraftment levels were maintained for longer periods of time, up to 6–7 months, while they declined after 4 months in control recipient mice. Multilineage differentiation of hematopoietic cells was also improved, with an increased ratio between granulocytes versus and lymphocytes that better reflects the physiological human blood composition. Thus, TPO^h/h recipient mice provide significant improvements compared to previously available models in all three limitations listed above.

Importantly, we performed phenotypical and functional analyses of human hematopoietic stem and progenitor cells in TPO^h/h compared to control recipients. We observed a significant increase in the fraction of human Lin⁻CD34⁺CD38^loCD90⁺CD45RA⁻ cells, a population previously identified as highly enriched in functional long-term HSC. Because serial transplantation is the most stringent protocol to functionally measure the self-renewal capacity of HSCs, we purified human CD34⁺ cells from TPO^h/h and control primary recipients and transplanted them into secondary recipients. Human CD34⁺ cells isolated from control primary recipients had a very low capacity to serially engraft (with human CD45⁺ cells detected in only 2 of 11 secondary recipients). By contrast, CD34⁺ cells isolated from TPO^h/h primary recipients had an increased capacity to efficiently engraft secondary recipients (with human CD45⁺ cells present in the bone marrow of 15 of 19 secondary recipients). This result indicates that the presence of human TPO in the primary recipient favored the maintenance of human cells with enhanced self-renewal capacity.

In conclusion, we demonstrate here that RAG2^−/−γ_c^−/− TPO-humanized mice efficiently support a population of cells immunophenotypically and functionally enriched in hematopoietic stem and progenitor cells. This leads to enhanced engraftment levels, better maintenance of human chimerism and improved multilineage differentiation. Therefore, RAG2^−/−γ_c^−/− TPO-humanized mice represent a novel model to study human hematopoiesis in vivo. We anticipate that this model will be useful to study human hematopoietic stem cells in vivo, with applications in the fields of hematopoiesis, hematology and hematolo-oncology.