Hematopoietic stem cell (HSC) transplantation stands as the pioneering long-term cell therapy used for life threatening blood disorders, such as leukemia and sickle cell disease. Yet, the persistent shortage of compatible grafts, compounded by rising clinical demand and an aging donor population, demands innovation to secure, safe, scalable and renewable HSC sources.

To address this challenge human-induced pluripotent stem cells (hiPSCs) is a transformative approach for HSCs de novo generation. While hiPSCs are able to generate mature blood cells, including red blood cells, platelets, B lymphocytes, and neutrophils, achieving complete and durable hematopoietic reconstitution in vivo remains challenging.

Based on an original Design of Experiment strategy combining defined morphogens and cytokines, we developed a robust, transgene-free differentiation protocol that generates bona fide human HSCs from hiPSCs in 17 days. These HSCs showed durable long-term, multilineage engraftment in immunodeficient NSG mice. Single-cell RNA sequencing revealed that our approach faithfully reproduces the cellular and molecular dynamics that occur during early human embryonic hematopoiesis.

Through comprehensive bioinformatic analysis, we pinpointed the molecular of engrafting HSCs. This profile is characterized by the prominent expression of the transcription factors LMO2 and MEIS1, secretion of APELIN, and surface expression of CD133, FLT3, and MPL. This signature will now help us as a blueprint for developing clinically viable grafts.

To bridge the gap toward clinical translation, we scaled up HSC production under Good Manufacturing Practice (GMP)-like conditions in partnership with Atlantic Bio GMP, an EFS advanced therapy medicinal production platform. The 17-day protocol was validated using flow cytometry and in vivo engraftment assays. Concurrently, we established the Aachen mini-pig as a translational large animal model, optimizing conditioning regimens - including irradiation, immunosuppression, and serial blood sampling – to assess graft feasibility and hematopoietic recovery.

This work represents a landmark achievement in producing hiPSC-derived hematopoietic grafts under clinically compliant conditions. By integrating a transgene-free protocol, functional validation in mice, and a robust large animal model, we lay the groundwork for global, accessible, and scalable autologous hiPSC-derived HSC therapies. This paves the way for accessible, scalable, curative cell therapies worldwide.