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接近的障碍是安全有效地将细胞递送到需要/需要它们的地方。尽管将成体干细胞直接注射到疾病/损伤部位对于具有限定解剖边界的某些器官是可行的（例如，心脏），直接插入对于全身性疾病如全身性骨疾病（例如，成骨不全）。血液传播细胞的组织特异性迁移由靶组织内皮处的粘附相互作用引发，所述粘附相互作用由循环细胞上的分子介导，所述分子专门抵抗血流的剪切力，即“归巢受体”。细胞从血管区室向骨的募集发生在骨髓内，由携带唾液酸岩藻糖基化乳糖胺聚糖的归巢受体介导，所述唾液酸岩藻糖基化乳糖胺聚糖与E-选择素结合，E-选择素是一种钙依赖性凝集素，在骨髓窦状血管上组成性表达。在之前的资助期间，我们分析了归巢受体在人造血干/祖细胞（HSPC）亚群和人骨髓间充质干细胞（BMSC）上的表达。我们发现，人类HSPC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BMSC不表达E-选择素配体，仅表达两种特征性归巢受体，VLA-4和CD 44。然而，重要的是，我们观察到BMSC上的CD 44具有（2，3）-唾液酸乳糖胺碳水化合物修饰，其可以作为外源性岩藻糖基化的受体。使用一种新的α-（1，3）岩藻糖基转移酶和附带的缓冲液条件，我们专门制定了治疗BMSC，而不影响细胞活力或多能性，我们将天然CD 44糖型转化为HCELL，一种高效的E-选择素配体。在基于流动的测定中，HCELL* BMSC在E-选择素上显示出深刻的粘附相互作用，并且如在免疫受损小鼠宿主中通过实时活体显微镜观察到的，静脉内输注的HCELL* BMSC稳健地归巢到骨，在输注数小时内浸润明显。因此，单一膜糖蛋白的立体选择性聚糖工程可以引导BMSC导航到预定的解剖隔室。在这项更新申请中，我们将扩展这些令人兴奋的发现，以进一步优化人BMSCs在表达E-选择素的内皮上的粘附相互作用和迁移。我们还将研究改善骨髓间充质干细胞向骨髓运输的生物学效应，并将分析由强制岩藻糖基化产生的离散聚糖结构。我们预计，这些研究的结果将揭示一个易于翻译的路线图编程的人BMSC的向骨性，从而促进这些细胞的临床应用，用于治疗全身性骨骼疾病和增强造血干细胞移植后的植入。

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.