Utilizing the GADD45-MAP3K4-P38 MAPK axis to control hematopoietic stem cell self-renewal and differentiation for therapeutic stem cell expansion

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Hematopoietic stem cells (HSCs) have been utilized for decades in stem cell transplantations for the life-saving treatment of leukemia, and represent a prime example for applied regenerative medicine. However, despite the detailed knowledge on HSC identity and biology, the molecular complexity of HSC self-renewal and differentiation control requires further understanding. This might facilitate the development of rational protocols for the long-term envisioned goal of HSC expansion ex vivo for advanced regenerative medicine. In previous studies we identified a novel pathway in the switch from the self-renewal into the differentiation program in HSCs. Members of the Growth arrest and DNA-damage inducible 45 (GADD45) family mediate the physiological cytokine-induced and the DNA-damage-induced differentiation in HSCs by specifically activating the p38 MAPK pathway. Once the expression of GADD45 Alpha or Gamma is triggered, phosphorylated p38 switches the self-renewal program into a differentiation program and accelerates blood cell maturation. These intriguing findings warrant a quantitative measurement of HSC behavior under the block of p38 activity for the rational design of improved ex vivo HSC expansion protocols. In this project application we plan a) to quantitatively assess the self-renewal of murine and human HSCs by p38 MAPK inhibition and optimized cytokine conditions in HSC expansion cultures; b) to enlighten the gene network downstream of the GADD45-MAP3K4-p38 activation that leads to differentiation induction in HSCs, and c) to answer the long-standing question whether differentiation requires DNA replication (S-Phase). We established technologies that will allow us to investigate molecular changes in conjunction with HSC fate decision control at single cell resolution. Inducible lentiviral GADD45 expression systems enable the timed induction of differentiation for detailed kinetic studies on molecular and functional events. The fitness and number of highly FACS-purified murine and human HSCs after ex vivo culture are quantitatively assessed by the gold standard of serial transplantation in recipient mice. Unique video-microscopy-based continuous cell tracking elucidates the fate of individual HSCs and their progeny during the whole differentiation process in realtime. The molecular network that induces differentiation in HSCs will be deciphered in high temporal resolution using (single cell) RNA sequencing, a quantitative mass spectrometry-based proteomics, and a functional genetic screen in a haploid leukemic cell line, utilizing our genetic tools to induce a well-timed differentiation in HSCs. Last, we want to understand the connection of cell cycle entry and progression and the tendency of HSCs to differentiate, whereby continuous HSC tracking is absolutely essential. The answers to these important questions will pave the way for rationally manipulate HSC self-renewal to increase the fitness and number of HSCs ex vivo.