Developing Immature Nucleus Pulposus Cells for Regenerative Therapy

描述（由申请人提供）：未成熟髓核（NP）细胞具有生物活性，在椎间盘（IVD）发育和衰老过程中调节基质生物合成发挥重要作用。未成熟的NP含有大量高度空泡化的脊索细胞，这些细胞随着年龄的增长而发生早期形态变化。在动物模型中，未成熟NP的细胞，而不是成熟NP的细胞，已被证明可以增强多种细胞类型的基质合成，并减轻变性。这种独特的生物合成效应可能是由于未成熟NP中存在大量的脊索细胞，这些细胞随后随着成熟NP的衰老而消失。因此，在NP中识别一个独特的脊索样细胞群的存在和独特的特征是很有兴趣的。我们开发了一种荧光激活细胞分选（FACS）方案（基于细胞大小和自身荧光），该方案识别出一种独特的脊索样NP细胞群体，这种细胞具有独特的形态外观和分子表型（即独特的整合素和mRNA表达模式），与动物组织的NP和AF中的其他细胞类型不同。我们建议使用这种方法在人类NP中基于独特分子标记的表达来鉴定脊索样NP细胞群。该建议的中心假设是脊索样NP细胞是代谢和表型上不同的NP细胞群，具有独特的细胞表面受体、基质和分泌蛋白的表达模式，随着年龄的增长而改变。我们建议利用这些独特的细胞表面受体选择或富集脊索样NP细胞，并研究其生物学行为和体外再生NP样基质的潜力。在Aim 1中，我们将基于不同的形态学，利用FACS在大鼠中鉴定一种独特的脊索样NP细胞群，以确定该细胞群中细胞表面受体（即整合素亚基和选择的CD抗原）的独特表达。细胞存活、粘附、形态和分泌蛋白合成的生物学行为也将在体外对这些独特细胞进行2D或3D培养。从未成熟大鼠到老年大鼠的分子表型和生物学行为的差异将被用来测试一种独特的未成熟细胞表型的存在及其随年龄的变化。在Aim 2中，将根据在大鼠脊索样NP细胞中确定的一组独特标记的表达来评估少年和成人IVD中独特的脊索样NP细胞表型的存在。一个独特的表达谱将首先通过免疫染色和流式细胞术在人类中得到证实，随后NP细胞分选产生一个独特的脊索样NP细胞亚群。细胞存活、粘附、形态、分泌蛋白合成和细胞外基质再生潜力的生物学行为也将在体外对这些独特的facs分类NP细胞进行2D或3D培养。该研究结果将揭示NP细胞亚群的存在，并精确表征其分子特征和生物学行为，该细胞亚群具有调节椎间盘再生的假设潜力。基于旨在促进“健康NP”细胞存活和基质合成的新方案和技术的发展，这项工作将为临床转译自体NP细胞和前体细胞治疗椎间盘病理的多种途径奠定基础。

DESCRIPTION (provided by applicant): Cells of the immature nucleus pulposus (NP) are biologically active and play an important role in regulating matrix biosynthesis during intervertebral disc (IVD) development and aging. The immature NP contains a large amount of highly vacuolated cells of notochordal origin that undergo early morphological changes with age. Cells of the immature NP, but not mature NP, have been shown to enhance matrix synthesis in multiple cell types and to attenuate degeneration in animal models. This unique biosynthetic effect may be due to the presence of a large amount of notochordal cells present in the immature NP which subsequently disappear with aging in mature NP. Thus, there is great interest in identifying the existence of, and unique features of a distinct notochordal-like cell population in the NP. We have developed a fluorescence-activated cell sorting (FACS) protocol (based on cell size and auto-fluorescence) that identifies a unique population of notochordal-like NP cells with a distinct morphological appearance and molecular phenotype (i.e. unique integrin and mRNA expression pattern) that is distinct from other cell types in the NP and AF of animal tissues. We propose to use this method to identify a notochordal-like NP cell population within the human NP based on expression of unique molecular markers identified here. The central hypothesis of this proposal is that notochordal-like NP cells are a metabolically and phenotypically distinct NP cell population, with unique expression patterns for cell surface receptors, matrix and secreted proteins that are altered upon aging. We propose to select or enrich notochordal-like NP cells by these unique cell surface receptors and study their biological behaviors and the potential for regeneration of NP-like matrix in vitro. In Aim 1, a unique notochordal-like NP cell population will be identified in the rat using FACS based on the distinct morphology in order to define the unique expression of cell surface receptors (i.e., integrin subunits and select CD antigens) for this population. Biological behaviors of cell survival, adhesion, morphology, and secreted protein synthesis will also be examined for 2D or 3D culture of these unique cells in vitro. Differences in molecular phenotype and biological behaviors from immature to aged rat will be used to test for the presence of a unique immature cell phenotype and its changes with age. In Aim 2, the existence of a unique notochordal-like NP cell phenotype in the juvenile and adult human IVD will be evaluated based on expression of a unique set of markers determined in the notochordal- like NP cells of the rat. A unique expression profile will first be confirmed by immunostaining and flow cytometry in the human, followed NP cell sorting to yield a unique notochordal-like NP cell subpopulation. Biological behaviors of cell survival, adhesion, morphology, secreted protein synthesis, and the extracellular matrix regeneration potential will also be examined for 2D or 3D culture of these unique FACS-sorted NP cells in vitro. Results from the study will reveal the existence of, and precisely characterize the molecular features and biological behaviors of an NP cell subpopulation with hypothesized potential to regulate disc regeneration. This work will build a foundation for multiple paths to clinical translation of autologous NP cell and precursor cell therapies for the treatment of disc pathology, based on the development of new protocols and technologies designed to promote "healthy NP" cell survival, and matrix synthesis. PUBLIC HEALTH RELEVANCE: Intervertebral disc degenerative disorders and resulting pain may be associated with aging- or pathology- related cell loss, and changes in cell phenotype. Cellular therapies for biologically-driven regeneration of intervertebral disc are of great interest, whereby disc cells from human patients or progenitor cells are used to generate new matrix in vitro or following re-implantation in vivo. This project will use a cell sorting technology to identify a population of disc cells which retain characteristics of an immature cell phenotype, and to investigate the key contributors or therapeutic targets for maintaining this immature cell-like phenotype and matrix synthesis during cell regeneration.