Molecular Characterization of Predatory Stem Cells in a Chimeric Protochordate

了解宿主中异基因干细胞活性的调节是移植医学的主要挑战。尽管在这一领域进行了广泛的调查，但许多根本问题仍然没有得到解决。在我们的实验室里，我们的研究集中在一个具有某些独特功能的有机体上，这使得它成为研究同种异体环境中嵌合体和干细胞功能的一个有吸引力的模型。这种原索动物(Botryllus Schlosseri)展示了体细胞和生殖系干细胞寄生的遗传等级。在这种生物中，一个具有广泛多态性的组织相容性基因座(FU/HC)允许共享血管系统并在亲缘关系中创建嵌合体。在嵌合体中，一个伴侣的循环多能细胞可以寄生和取代另一个伴侣的生殖系和/或胞体(称为生殖系或体细胞寄生)。这种替换寄主组织的能力遵循着“赢家菌株”取代“输家菌株”组织的遗传等级。我们最近的研究将捕食性细胞谱系定义为预期可分离的干细胞，并表明FU/HC基因座和生殖系竞争遗传在注射的、纯化的干细胞水平上起作用。我们克隆了FU/HC基因，发现它与人和小鼠有4个同源物：Tie 1、Tek、IgSF4(小鼠造血干细胞或血管形成干细胞的标记)和脊髓灰质炎病毒受体CD155(人类自然杀伤细胞识别的靶点，潜在地涉及NK细胞作为耐受诱导和组织排斥的介体)。此外，通过对显示哺乳动物干细胞生物学基本和重要方面的细胞的直接可视化，我们已经确定了这种生物中一个主要的成体干细胞生态位。我们计划研究干细胞竞争潜力的传递遗传学，并确定决定这一表型的基因。我们将进行系统的育种实验，并建立干细胞竞争潜力(SCP；获胜者与失败者纯合子品系)特性纯合子的遗传定义系。纯合菌株将进一步用于构建SCP区域的遗传图谱。AFLP与SCP区共分离的标记(批量分离分析)将被克隆、测序，并与Botryllus基因组序列和EST(正在进行的项目)进一步比对。基于这些数据，我们将选择候选的SCP基因，并通过高通量的信使核糖核酸测序来确定它们在“优胜株”干细胞及其利基细胞中的表达谱，以及在“失败株”干细胞及其利基细胞中的表达谱。一些选定的基因参与同种异体干细胞的竞争调节，将直接使用功能分析进行测试。这项对Botryllus的全面分子研究将为导致成功的同种异基因干细胞竞争的途径提供新的见解，这可能适用于一般脊椎动物。

Understanding the regulation of allogeneic stem cell activity in a host is a major challenge of transplantation medicine. Despite extensive investigation in this area, many fundamental issues remain unresolved. In our lab, we have focused our studies on an organism with certain unique features that make it an appealing model for studying chimerism and stem cell functionality in allogeneic environment. This protochordate (Botryllus schlosseri) demonstrates' genetic hierarchies for somatic and germline stem cell parasitism. In this organism, a histocompatibility locus (Fu/HC) with extensive polymorphism allows sharing of vascular systems and creation of chimera among kin. In the chimeras, the circulating pluripotent cells of one partner can parasitize and replace the germline and/or the soma of the other partner (termed germline or somatic stem cell parasitism). This ability to replace host tissues follows genetic hierarchies of "winner strain" that replace "loser strain" tissues. Our recent studies have defined the predatory cell lineages as prospectively isolatable stem cells and showed that the Fu/HC locus and germline competition inheritance operates at the level of injected, purified stem cells. We cloned the Fu/HC gene and found that it has 4 homologues to the human and mouse: Tie 1,Tek, IgSF4, (markers for either mouse blood forming stem cells or blood vessel forming stem cells) and the poliovirus receptor, CD155 (a target for human natural killer cell recognition, potentially implicating NK cells as mediators of tolerance induction versus tissue rejection). In addition by direct visualization of cells that exhibit fundamental and important aspects of mammalian stem cell biology, we have identified a major adult somatic stem cell niche in this organism. We plan to investigate the transmission genetics of stem cell competitive potential, and to identify the genes which determine this phenotype. We will perform systematic breeding experiments and establish genetically defined lines that are homozygous for the trait of stem cell competitive potential (SCP; winner versus loser homozygous strains). The homozygous strains will be further used to construct a genetic map of the SCP region. AFLP's markers which co-segregate with the SCP region (bulk segregation analysis) will be cloned, sequenced and further aligned with the Botryllus genome sequence and EST's (project in process). Based on these data, candidate SCP genes will be selected and their expression profiles in "winner strain" stem cells and their niches, versus "loser strain" stem cells and their niches will be determined via high throughput mRNA sequencing. The involvement of a few selected genes in allogeneic stem cell competitive regulation will be tested directly using functional assays. This comprehensive molecular study in Botryllus will provide novel insights into the pathways that lead to successful allogeneic stem cell competition that will likely be applicable to vertebrates in general.