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一个主要挑战。尽管在这一领域进行了广泛的调查，但许多基本问题仍未得到解决。在我们的实验室中，我们将研究重点放在具有某些独特特征的生物体上，这些特征使其成为研究异基因环境中嵌合体和干细胞功能的吸引人的模型。这种原脊索动物（Botryllus schlosseri）展示了体细胞和生殖干细胞寄生的遗传层次。在这种生物体中，具有广泛多态性的组织相容性位点（Fu/HC）允许在亲属之间共享血管系统和创建嵌合体。在嵌合体中，一个配偶体的循环多能细胞可以寄生并取代另一个配偶体的生殖系和/或索马（称为生殖系或体干细胞寄生）。这种替换宿主组织的能力遵循“赢家株”替换“败者株”组织的遗传等级。我们最近的研究已经将捕食性细胞谱系定义为可分离的干细胞，并表明Fu/HC基因座和种系竞争遗传在注射的纯化干细胞水平上起作用。我们克隆了Fu/HC基因，发现它与人类和小鼠有4个同源物：Tie 1、Tek、IgSF 4（小鼠造血干细胞或血管形成干细胞的标志物）和脊髓灰质炎病毒受体CD 155（人类自然杀伤细胞识别的靶标，可能涉及NK细胞作为耐受诱导与组织排斥的介导物）。此外，通过直接可视化的细胞，表现出哺乳动物干细胞生物学的基本和重要方面，我们已经确定了一个主要的成体干细胞在这个有机体的生态位。我们计划研究干细胞竞争潜力的遗传传递，并确定决定这种表型的基因。我们将进行系统的育种实验，并建立遗传定义的干细胞竞争潜力（SCP;赢家与输家纯合株）性状的纯合系。纯合菌株将进一步用于构建SCP区域的遗传图谱。将对与SCP区域共分离的AFLP标记（批量分离分析）进行克隆、测序，并进一步与葡萄球菌基因组序列和EST比对（项目正在进行中）。基于这些数据，将选择候选SCP基因，并通过高通量mRNA测序确定它们在“赢家品系”干细胞及其小生境中相对于“失败者品系”干细胞及其小生境中的表达谱。将使用功能测定直接测试在同种异体干细胞竞争性调节中的一些选定基因的参与。这项对葡萄球菌的全面分子研究将为导致成功的同种异体干细胞竞争的途径提供新的见解，这可能适用于一般的脊椎动物。

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.