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

造血干细胞（HSC）已经在干细胞移植中使用了几十年，用于挽救白血病的生命，并且代表了应用再生医学的主要例子。然而，尽管对HSC身份和生物学有详细的了解，但HSC自我更新和分化控制的分子复杂性需要进一步了解。这可能有助于制定合理的方案，以实现高级再生医学体外HSC扩增的长期设想目标。在以前的研究中，我们发现了一种新的途径，在开关从自我更新到分化程序的HSC。GADD 45家族成员通过特异性激活p38 MAPK途径介导HSC的生理性分化和DNA损伤诱导分化。一旦GADD 45 α或γ的表达被触发，磷酸化的p38将自我更新程序转换为分化程序并加速血细胞成熟。这些有趣的发现保证了在p38活性阻断下HSC行为的定量测量，用于改进的离体HSC扩增方案的合理设计。在本项目申请中，我们计划a）通过p38 MAPK抑制和优化HSC扩增培养物中的细胞因子条件定量评估鼠和人HSC的自我更新; B）揭示GADD 45-MAP 3 K4-p38激活下游的基因网络，该激活导致HSC的分化诱导，以及c）回答分化是否需要DNA复制（S期）的长期问题。我们建立的技术，将使我们能够调查分子的变化与HSC的命运决定控制在单细胞分辨率。诱导型慢病毒GADD 45表达系统能够定时诱导分化，用于分子和功能事件的详细动力学研究。通过在受体小鼠中连续移植的金标准定量评估在离体培养后高度FACS纯化的鼠和人HSC的适应性和数量。独特的基于视频显微镜的连续细胞跟踪实时阐明了单个HSC及其后代在整个分化过程中的命运。诱导HSC分化的分子网络将使用（单细胞）RNA测序、基于定量质谱的蛋白质组学和单倍体白血病细胞系中的功能性遗传筛选以高时间分辨率破译，利用我们的遗传工具诱导HSC适时分化。最后，我们希望了解细胞周期进入和进展与HSC分化趋势的联系，因此持续追踪HSC是绝对必要的。这些重要问题的答案将为合理调控HSC自我更新，增加HSC的适应性和数量铺平道路。

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.