Precise Surface Patterning to Investigate Cell-Cell Interactions that Regulate Ne

描述（由申请人提供）：干细胞在广泛的发育阶段和谱系中存在于成体和胚胎组织中，因此它们天然暴露于不同的微环境或小生境，这些微环境或小生境调节它们自我更新和分化成一种或多种成熟谱系的标志性行为。在每个这样的微环境中，干细胞感知和处理多种生化和生物物理线索，这些线索可以施加冗余的、竞争的或正交的影响，以共同调节细胞命运和功能。这些无数的调节信号的正确呈现是组织发育和体内平衡所必需的，并且它们的不适当的出现可能会导致疾病。在该领域的相当大的进展已经导致了普遍认识到，可溶性蛋白质线索是重要的调控信号;然而，干细胞驻留在“固相”微环境中，该微环境还呈现来自细胞外基质和来自邻近细胞的关键调控信息。例如，成人脑中的神经干细胞（NSC）被许多其他细胞类型包围，包括其他NSC、分化过程中的细胞、成熟神经元、星形胶质细胞、少突胶质细胞和内皮细胞，其中许多细胞参与向NSC传递调节信号。然而，细胞-细胞相互作用的生物学对于使用传统的细胞培养系统来控制和剖析可能是具有挑战性的，因为目前难以在细胞集合内建立和维持精确的几何排列足够的时间以使丰富的干细胞行为能够出现。 我们建议开发新的培养系统，以在基底上以高空间精度模式化一种或多种细胞类型-神经干细胞和其生态位的其他组成细胞。首先，我们最近已经表明，一种新的陷阱和围栏技术，包括微威尔斯与PDMS网包含细胞内的威尔斯延长的时间段。我们将利用这个系统来研究同型， NSC-NSC相互作用对细胞命运的决定。第二，我们将利用最近开发的技术，使用DNA寡核苷酸碱基配对，在我们发现NSC可以维持一段时间的表面上建立细胞模式，从而能够分析细胞分化。我们将使用这种技术来改变与NSC接触的细胞类型和数量，从而分析细胞-细胞接触的数量和性质如何动态调节NSC的命运决定。因此，这种新型生物表面图案化技术的混合物将使当前和未来的研究能够进入干细胞龛中的细胞-细胞接触调节NSC行为的基本机制，对干细胞和发育生物学以及干细胞疗法开发的生物医学应用都具有基本意义。

DESCRIPTION (provided by applicant): Stem cells reside in adult and embryonic tissues in a broad spectrum of developmental stages and lineages, and they are thus naturally exposed to diverse microenvironments or niches that modulate their hallmark behaviors of self-renewal and differentiation into one or more mature lineages. Within each such microenvironment, stem cells sense and process multiple biochemical and biophysical cues, which can exert redundant, competing, or orthogonal influences to collectively regulate cell fate and function. The proper presentation of these myriad regulatory signals is required for tissue development and homeostasis, and their improper appearance can potentially lead to disease. Considerable advances in the field have led to the general appreciation that soluble protein cues are important regulatory signals; however, stem cells reside in a "solid phase" microenvironment that also presents key regulatory information from the extracellular matrix and from neighboring cells. For example, neural stem cells (NSCs) in the adult brain are surrounded by many other cell types - including other NSCs, cells in the process of differentiating, mature neurons, astrocytes, oligodendrocytes, and endothelial cells - many of which have been implicated in conveying regulatory cues to the NSCs. However, the biology of cell-cell interactions can be challenging to control and dissect using traditional cell culture systems, as it is currently diffiult to establish and maintain a precise geometrical arrangement within an ensemble of cells for sufficient times to enable rich stem cell behaviors to emerge. We propose to develop novel culture systems to pattern one or more cell types - NSCs and other constituent cells of their niche - with high spatial precision on a substrate. First, we have recently shown that a novel trap-and-corral technology composed of microwells with a PDMS mesh to contain cells within the wells for extended time periods. We will utilize this system to study the effects of homotypic, NSC-NSC interactions on cell fate decisions. Second, we will utilize a recently developed technology that uses DNA oligonucleotide base pairing to establish a cell pattern on a surface that we have found NSCs can maintain for time periods that enable analysis of cell differentiation. We will use this technology to vary the numbers and cell types that contact a NSC and thereby analyze how the number and nature of cell-cell contacts dynamically regulate NSC fate decisions. This blend of novel biosurface patterning technologies will therefore enable current and future investigations into basic mechanisms by which cell-cell contacts in the stem cell niche regulate NSC behavior, work with both basic implications for stem cell and developmental biology and biomedical applications for the development of stem cell therapies.