Label-free interrogation of heterogeneities in HSC fate decision signatures

 描述（由申请人提供）：我们正在开发用于造血干细胞（HSC）离体培养的人工骨髓。该平台具有重要的科学价值， 检验关于负责指导骨髓内HSC命运决定的外部信号级联的假设。人工骨髓对于HSC的治疗性扩增或对于造血病理的病因学和治疗的研究也将具有显著的临床价值。然而，复杂的这一努力是有限的信息所发挥的调节作用的连续体的亚生态位，存在于整个骨髓中负责维持造血稳态的紧密空间秩序。我们最近描述了微加工方法，以产生一个工程骨髓（EBM）含有骨髓启发的细胞，生物物理和生物分子线索的重叠模式，开始检查这些信号对HSC静止与激活的协调影响。然而，小规模，使EBM的优势介绍了干细胞的反应，这些线索的未知异质性的关注。虽然HSC可能对小生境信号表现出一系列反应并不奇怪，但在多线索环境中可能会放大效果。因此，我们提出了一种无标记的方法来暂时跟踪和量化单一HSC命运决定的异质性，以响应多路EBM小生境信号。为此，我们将结合联合收割机光子晶体增强显微镜（PCEM）和飞行时间二次离子质谱（TOF-SIMS），以创建一个集成的检测仪器，能够跟踪单个HSC的命运决定。我们假设，在连续的生物材料子生态位中单独分析这些决定将使我们能够更好地预测HSC命运的生态位调节，而不是报告整体平均值的更传统的指标。目的1将整合西姆斯和PCEM，以确定HSC对已证明对HSC群体有影响的生物物理线索的反应的异质性。目的2将采用PCEM/SIMS为基础的生物传感器，以检查在多细胞集落内含有HSC和支持性小生境细胞的HSC的命运决定。将这种新的生物传感器与传统的功能检测相结合，将使我们能够第一次确定HSC对工程化小生境信号反应的异质性。它还提供了一个框架，使用最少数量的细胞快速评估多个信号的影响，以确定这些线索之间的层次结构和/或协同作用。这些数据将为我们理解HSC如何整合小生境信号以及识别工程骨髓的关键设计元素带来新的丰富性。通过量化这些命运决定的异质性水平，我们还将建立沿着单一HSC和集合平均值之间的连续体，未来的调查必须集中在哪里。

 DESCRIPTION (provided by applicant): We are developing an artificial bone marrow for ex vivo culture of hematopoietic stem cells (HSCs). This platform has significant scientific value for testing hypotheses regarding the cascade of external signals responsible for directing HSC fate decisions within the bone marrow. An artificial marrow would also have significant clinical value for therapeutic expansion of HSCs or for study of the etiology and treatment of hematopoietic pathologies. However complicating this effort is limited information regarding the regulatory role played by the continuum of sub-niches that exist in close spatial order across the marrow responsible for maintaining hematopoietic homeostasis. We have recently described microfabrication approaches to generate an engineered bone marrow (EBM) containing overlapping patterns of marrow-inspired cellular, biophysical, and biomolecular cues to begin to examine the coordinated impact of these signals on HSC quiescence vs. activation. However, the small scale that makes the EBM advantageous introduces concerns regarding the unknown heterogeneity of a stem cell's response to these cues. While not surprising HSCs may exhibit a range of responses to a niche signal, effects may be magnified in multi-cue environments. We therefore propose to demonstrate a label-free approach to temporally track and quantify the heterogeneity of single HSC fate decisions in response to multiplexed EBM niche signals. To do this, we will combine photonic crystal enhanced microscopy (PCEM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) to create an integrated detection instrument able to trace single HSC fate decisions. We hypothesize that individually profiling these decisions across a continuum of biomaterial sub-niches will allow us to better predict niche regulation of HSC fate than more traditional metrics that report ensemble averages. Aim 1 will integrate SIMS and PCEM to determine the heterogeneity of HSC response to biophysical cues demonstrated to have an effect on populations of HSCs. Aim 2 will employ the PCEM/SIMS-based biosensor to examine HSC fate decisions within multi-cell colonies containing HSCs and supportive niche cells. Combining this novel biosensor with traditional functional assays will allow us, for the firt time, to determine the heterogeneity of HSC response to engineered niche signals. It also offers a framework to rapidly assess the impact of multiple signals using a minimal number of cells in order to identify hierarchies and/or synergies between these cues. Such data will bring new richness to our understanding of how HSCs integrate niche signals as well as identify critical design elements of an engineered bone marrow. By quantifying the level of heterogeneity in these fate decisions, we will also establish where along the continuum between single HSC and ensemble averages future investigations must focus.