Optimizing Osteotropism of Human Mesenchymal Stem Cells

描述（由申请人提供）：实现基于成体干细胞的再生治疗的巨大潜在益处的最近障碍是安全有效地将细胞输送到需要的地方。虽然将成体干细胞直接注射到疾病/损伤部位对于某些具有明确解剖边界的器官（如心脏）是可行的，但对于全身性骨病（如成骨不全症）等全身性疾病，直接插入是不切实际的。血源性细胞的组织特异性迁移是由靶组织内皮的粘附相互作用启动的，这种相互作用是由循环细胞上的分子介导的，这些分子专门抵抗血流的剪切力，即“归巢受体”。细胞从血管腔室向骨的募集发生在骨髓内，由携带sialo聚焦的乳糖胺聚糖的归巢受体介导，这些乳糖胺聚糖与e -选择素（一种在骨髓窦状血管上组成表达的Cadependent凝集素）结合。在之前的资助期内，我们分析了归巢受体在人造血干细胞/祖细胞（HSPCs）亚群和人骨髓间充质干细胞（BMSCs）上的表达。我们发现，人造血干细胞表达多种归巢受体，包括几种作为e -选择素配体的糖蛋白，而骨髓间充质干细胞不表达e -选择素配体，只表达两种具有特征的归巢受体，即VLA-4和CD44。然而，重要的是，我们观察到骨髓间充质干细胞上的CD44具有(2,3)-唾液基乳胺碳水化合物修饰，可以作为外源性聚焦化的受体。我们使用一种新的α -(1,3)聚焦转移酶和相应的缓冲条件，在不影响细胞活力或多能性的情况下处理骨髓间充质干细胞，将天然CD44糖型转化为HCELL，一种高效的e -选择素配体。在基于流动的实验中，HCELL*骨髓间充质干细胞与e -选择素表现出深刻的粘附相互作用，并且，在免疫功能低下的小鼠宿主中通过实时活体显微镜观察到，静脉注射的HCELL*骨髓间充质干细胞牢固地附着在骨上，在注射后数小时内浸润明显。因此，单一膜糖蛋白的立体选择性聚糖工程可以指导骨髓间充质干细胞导航到预定的解剖腔室。在这项更新应用中，我们将扩展这些令人兴奋的发现，进一步优化人骨髓间充质干细胞在表达e -选择素的内皮细胞上的粘附相互作用和迁移。我们还将研究骨髓间质干细胞向骨髓转运的生物学效应，并分析强制聚焦所产生的离散聚糖结构。我们预期这些研究的结果将揭示一个易于翻译的人类骨髓间充质干细胞的成骨性编程路线图，从而促进这些细胞在治疗广泛性骨骼疾病和加强造血干细胞移植后的植入的临床应用。

DESCRIPTION (provided by applicant): The proximate obstacle to realizing the enormous potential benefits of adult stem cell-based regenerative therapeutics is to safely and efficiently deliver the cells where they are needed/required. Though direct injection of adult stem cells into site(s) of disease/injury is feasible for certain organs with defined anatomic boundaries (e.g., heart), direct insertion is impractical for systemic conditions such as generalized bone diseases (e.g., Osteogenesis Imperfecta). Tissue-specific migration of blood-borne cells is initiated by adhesive interactions at target tissue endothelium mediated by molecules on circulating cells that are specialized to resist the shear forces of blood flow, the "homing receptors". Recruitment of cells from the vascular compartment to bone occurs within marrow, mediated by homing receptors bearing sialofucosylated lactosaminoglycans that engage E-selectin, a Cadependent lectin that is constitutively expressed on marrow sinusoidal vessels. In the prior funding period, we analyzed the expression of homing receptors on subsets of human hematopoietic stem/progenitor cells (HSPCs) and on human bone marrow-derived mesenchymal stem cells (BMSCs). We found that whereas human HSPCs express multiple homing receptors, including several glycoproteins that function as E-selectin ligands, BMSCs express no E-selectin ligands and only two characterized homing receptors, VLA-4 and CD44. Importantly, however, we observed that CD44 on BMSCs possesses a(2,3)-sialyllactosamine carbohydrate modifications that can serve as an acceptor for exogenous fucosylation. Using a novel alpha-(1,3) fucosyltransferase enzyme and attendant buffer conditions specifically formulated by us for treating BMSCs without affecting cell viability or multipotency, we converted the native CD44 glycoform into HCELL, a highly potent E-selectin ligand. In flow-based assays, HCELL* BMSCs displayed profound adhesive interactions on E-selectin, and, as observed by real-time intravital microscopy in immunocompromised mouse hosts, intravenously infused HCELL* BMSCs homed robustly to bone, with infiltrates evident within hours of infusion. Thus, stereoselective glycan engineering of a single membrane glycoprotein can direct navigation of BMSCs to a predetermined anatomic compartment. In this renewal application, we will extend these exciting findings to further optimize adhesive interactions and transmigration of human BMSCs on endothelium expressing E-selectin. We will also examine the biological effects of improved trafficking of BMSCs to marrow, and will analyze the discrete glycan structures created by enforced fucosylation. We anticipate that results of these studies will unveil a readily translatable roadmap for programming osteotropism of human BMSCs, thereby facilitating clinical use of these cells for treatment of generalized skeletal diseases and for enhancing engraftment following hematopoietic stem cell transplantation.