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

利用 GADD45-MAP3K4-P38 MAPK 轴控制造血干细胞自我更新和分化以实现治疗性干细胞扩增

• 批准号: 317011487
• 负责人: Professor Dr. Michael Rieger
• 金额: --
• 依托单位: Klinik 2 - Hämatologie/Onkologie, Rheumatologie und Infektiologie/HIV
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/317011487?language=en
• 关键词: Utilizing; GADD45; MAP3K4; P38; MAPK

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.

几十年来，造血干细胞(HSCs)一直被用于干细胞移植，用于挽救白血病的生命，是应用再生医学的一个典型例子。然而，尽管对HSC的身份和生物学有详细的了解，但HSC自我更新和分化调控的分子复杂性还需要进一步了解。这可能有助于为高级再生医学的HSC体外扩增这一长期预期目标开发合理的方案。在以前的研究中，我们发现了一条新的途径，在HSC从自我更新到分化程序的转换中。生长停滞和DNA损伤诱导45(GADD45)家族成员通过特异性激活p38 MAPK通路介导生理性细胞因子诱导和DNA损伤诱导的HSCs分化。一旦GADD45 Alpha或Gamma的表达被触发，磷酸化的p38就会将自我更新程序切换为分化程序，并加速血细胞的成熟。这些耐人寻味的发现为合理设计改进的体外HSC扩增方案提供了定量测量p38活性阻断下HSC行为的依据。在本项目的应用中，我们计划a)通过抑制p38 MAPK和优化细胞因子条件来定量评估小鼠和人HSC在扩增培养中的自我更新；b)揭示GADD45-MAP3K4-p38激活下游的基因网络，从而诱导HSC分化；c)回答长期存在的分化是否需要DNA复制的问题(S期)。我们建立的技术将使我们能够在单细胞分辨率下结合HSC命运决策控制来研究分子变化。可诱导的慢病毒GADD45表达系统能够定时诱导分化，用于详细的分子和功能事件的动力学研究。以受体小鼠连续移植为金标准，定量评价体外培养的高纯度FACS纯化的小鼠和人HSC的适合性和数量。独特的基于视频显微镜的连续细胞跟踪实时阐明了单个HSCs及其后代在整个分化过程中的命运。诱导造血干细胞分化的分子网络将利用(单细胞)RNA测序、基于定量质谱学的蛋白质组学和单倍体白血病细胞系中的功能遗传筛选，以高时间分辨率破译，利用我们的遗传工具诱导造血干细胞适时分化。最后，我们想要了解细胞周期进入和进展的联系以及HSC分化的趋势，因此持续的HSC跟踪是绝对必要的。这些重要问题的解答将为合理操作HSC的自我更新，提高体外HSC的适合性和数量铺平道路。