Bioengineering challenges for platelet production from hematopoietic stem cells

造血干细胞生产血小板的生物工程挑战

• 批准号: 7731886
• 负责人: WILLIAM M MILLER
• 金额: $ 34.5万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-15 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7731886
• 关键词: Acetylation; Address; Alloimmunization; Antigens; Apoptosis; Autologous; Biomedical Engineering; Biotechnology; Blood; Blood Cells; Blood Component Removal; Blood Platelets; Blood donor; Blood-Borne Pathogens; Bone Marrow; Budgets; CD34 gene; CSF3 gene; Calendar; Canada; Cell Count; Cell Cycle; Cell Cycle Regulation; Cells; Chemical Engineering; Collection; Commit; Computers; Data Analyses; Delaware; Development; Direct Costs; Doctor of Philosophy; Dose; Equipment; Europe; Experimental Designs; Facilities and Administrative Costs; Fees; First Name; Flow Cytometry; Fred Hutchinson Cancer Research Center; Freezing; Fringe Benefit; Funding; Generations; Growth Factor; Harvest; Health Insurance; Hematopoietic; Hematopoietic stem cells; Histones; Human; Human Resources; Immune response; Institutes; Institution; Last Name; Lead; Lentivirus Vector; Leukocytes; Manuscripts; Mediating; Megakaryocytopoieses; Messenger RNA; Mexico; Modification; Monoclonal Antibodies; Names; Nature; Niacinamide; Participant; Patients; Persons; Plastics; Platelet Transfusion; Play; Ploidies; Poly(ADP-ribose) Polymerases; Population; Postdoctoral Fellow; Preparation; Principal Investigator; Production; Proteins; Publications; RNA Interference; Reagent; Reporting; Research; Research Personnel; Risk; Role; Serum-Free Culture Media; Services; Sirtuins; Stem cells; Students; Sum; Telephone; Testing; Transfusion; Transplantation; Travel; Umbilical Cord Blood; Universities; Vial device; Viral; Vitamins; Wages; Western Blotting; Work; base; cost; cytokine; expiration; graduate student; in vivo; inhibitor/antagonist; knock-down; large scale production; meetings; peripheral blood; professor; progenitor; programs; research study; small hairpin RNA; stem

Platelets are transfused for a wide range of thrombotic deficiencies, but there are problems. Platelet collection typically requires pooling harvests from multiple donors. Platelet transfusion risks from bacterial contamination, blood-borne pathogens, and alloimmunization are compounded because patients receive platelets from many donors. Production of autologous or compatible platelets by megakaryocytic cells (Mks) derived from cultured hematopoietic stem and progenitor cells (HSPCs or CD34+ cells) would greatly decrease these risks. However, generating 500 billion platelets for a single transfusion using culture conditions that yield relatively pure (e 75%) Mk populations would require 250 million CD34+ cells. This is equivalent to more than 50 umbilical cord blood harvests or 1-2 harvests of HSPCs from the peripheral blood of donors treated (or mobilized) with growth factors. In order for culture-derived platelet production to be economically feasible, it will be necessary to produce more Mk progenitors per CD34+ cell, obtain a greater number of Mks per Mk progenitor, and increase Mk ploidy (platelet- producing potential). Our objective is to increase the ploidy of culture-derived Mks to levels similar to those found in human bone marrow. We have shown that the vitamin nicotinamide (NIC) greatly increases Mk ploidy in culture. Since Mks in vivo produce several thousand platelets, we anticipate that Mks produced in culture with NIC could generate 1000 platelets. Understanding the mechanisms responsible for NIC-mediated increases in Mk ploidy will facilitate regulatory approval for using NIC to produce platelets for transplantation and is likely to lead to the discovery of even more effective conditions for Mk polyploidization. We propose to use RNA-interference-mediated knockdown to test the hypothesis, based on our preliminary results, that NIC increases Mk ploidy via inhibition of the SIRT1 and SIRT2 Class III histone/protein deacetylases. We will then examine changes in the acetylation of SIRT target proteins involved in the regulation of the cell cycle and/or apoptosis. Finally, we will investigate the roles in megakaryopoiesis of the most promising SIRT targets.

血小板可用于治疗广泛的血栓缺乏症，但有 有问题。采集血小板通常需要汇集来自多个捐赠者的采集。 血小板输注有细菌污染、血液传播病原体和 由于患者接受来自许多捐赠者的血小板，所以异基因免疫是复合的。 来源的巨核细胞产生自体或相容的血小板 从培养的造血干细胞和祖细胞(HSPC或CD34+细胞)中 大大降低了这些风险。然而，一个人产生5000亿个血小板 使用培养条件进行输血，以产生相对纯净(e 75%)的Mk群体 需要2.5亿个CD34+细胞。这相当于50多条脐带 采血或从接受治疗的献血者的外周血中采集1-2个HSPC(或 被动员的)与生长因子。为了使培养衍生的血小板生产 在经济上可行的情况下，每个CD34+产生更多的MK祖细胞是必要的 细胞，每个Mk祖细胞获得更多的MKs，并增加Mk倍体(血小板- 生产潜力)。我们的目标是将培养来源的MKs的倍性增加到 与人类骨髓中发现的水平相似。我们已经证明，维他命 烟酰胺(NIC)可显著提高Mk倍性。由于MKS在体内产生 几千个血小板，我们预计在NIC的培养中产生的MKS可能 产生1000个血小板。了解NIC中介的负责机制 Mk倍体的增加将有助于监管部门批准使用NIC生产 血小板用于移植，很可能会导致发现更有效的 Mk多倍化的条件。我们建议使用RNA干扰介导的 根据我们的初步结果，测试假设：NIC增加 通过抑制SIRT1和SIRT2-III类组蛋白/蛋白去乙酰基酶实现MK倍性。 然后，我们将研究参与SIRT靶蛋白乙酰化的变化 调节细胞周期和/或细胞凋亡。最后，我们将调查在 最有希望的SIRT靶点的巨核生成。