3D NICHE ENGINEERING FOR REGULATION OF STEM CELL FATE DECISIONS

骨髓容纳造血干细胞（HSC），负责在人的一生中不断再生血液组织。骨髓移植，也被称为造血干细胞移植（HSCT），是一种拯救生命的手术，用于治疗白血病患者，最近也用于治疗多发性硬化症等自身免疫性疾病，可以导致持久的无疾病缓解。然而，造血干细胞移植受到供应有限、组织错配并发症和高死亡率（高达10%的患者不能在移植后存活）的困扰。如果每个供体样本中的HSC数量可以增加，更多的患者可以从更安全的HSCT中受益。为了实现这一进展，需要更详细地了解HSC与其原生3D骨髓环境或生态位之间的相互作用。当从其原生骨髓龛中取出并置于培养中时，HSC和祖细胞（HSPC）很快失去其干细胞功能（24-72小时）。越来越多的证据表明，骨髓细胞外基质（ECM）是一个关键的，但未被充分认识的，可能对干细胞调控有指导意义的生态位组成部分。为了使HSPCs在体外扩增，需要开发能够再现原生ECM环境的培养条件。该项目旨在开发发现体外干细胞生态位条件的技术，并应用这些知识来阐明干细胞- ecm相互作用。组合化学方法系统地组装和测试个体和ECM成分的组合，将用于阐明每个成分对HSPC功能的贡献。这些发现将在一种新的3D筛选试验中进一步实施，该试验使用“非专利”药物，能够识别调节干细胞功能的分子靶点。最后，将开发3D打印技术，以创建具有ECM的血管系统，并支持HSPC在厚3D结构中存活。该项目将为个性化造血干细胞移植医学开发组织匹配的工程骨髓组织。加拿大白血病和自身免疫性疾病患者受益最多的是更短的等待时间和更安全的程序。通过加速患者的治疗和康复，从而降低管理患病和病情恶化患者的医疗成本，还可以实现加拿大医疗保健方面的节省。此外，这项研究可以应用于在批准研究新药之前测试无意中引起骨髓抑制毒性的新药，这是一种潜在的致命副作用。这种额外的监管批准测试可以减少动物试验，促进更安全的新药物。在项目的前5年，将培养3名博士、3名硕士和10名本科生研究助理，从事干细胞和组织工程方面的培训，并将他们安置在再生医学和生物技术等高影响力领域的学术或行业就业机会。

Bone marrow houses hematopoietic stem cells (HSC) that are responsible for continuously regenerating blood tissue throughout one's lifetime. Bone marrow transplants, also called HSC transplants (HSCT), are life-saving procedures used to treat leukemia patients and more recently, autoimmune diseases like multiple sclerosis, which can lead to durable disease-free remission. However, HSCT are plagued by limited supply, tissue mismatching complications and high mortality rates (up to 10% of patients do not survive the transplant). Many more patients could benefit from safer HSCT if the number of HSC in each donor sample could be augmented. For this advancement to be engineered a more detailed understanding of the interplay between HSC and their native 3D bone marrow environment, or niche, is required. When removed from their native bone marrow niche and placed in culture, HSC and progenitor (HSPC) cells very quickly lose their stem cell function  (24-72h). Mounting evidence suggests that the bone marrow extracellular matrix (ECM) is a critical, yet underappreciated, component of the niche that may be instructive to stem cell regulation. In order for HSPCs to be expanded ex vivo, culture conditions that recapitulate the native ECM environment need to be developed. This program aims to develop technology that discovers ex vivo stem cell niche conditions and apply this knowledge to elucidate stem cell-ECM interactions. Combinatorial chemistry methods that systematically assemble and test individual, and combinations of ECM components will be employed with the intent of elucidating the contribution of each component to HSPC function. These findings will further be implemented in a novel 3D screening assay using "off-patent" drugs able to identify molecular targets that regulate stem cell function. Finally, 3D Printing technology will be developed to create a vascular system with the ECM and support HSPC survival in thick 3D constructs. This program leads to the development of tissue-matched engineered bone marrow tissue for personalized HSCT medicine. Canadian leukemia and autoimmune disease patients stand to benefit the most with shorter waiting times and safer procedures. Canadian healthcare savings can also be realized by accelerating patient treatment and recovery, thereby reducing healthcare costs of managing sick and deteriorating patients. Furthermore, this research can be applied to test new drugs that unintentionally cause myelosuppression toxicity, a potentially lethal side-effect, before approving investigational new drugs. This additional test for regulatory approval can reduce animal testing and promote safer news drugs. Within the program's first 5 years, a total of 3 PhDs, 3 MASc and 10 undergraduate research assistants will be will be trained in stem cells and tissue engineering and position them for academic or industry employment opportunities in high-impact areas such as regenerative medicine and biotechnology.