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: 7929581
• 负责人: WILLIAM M MILLER
• 金额: $ 34.3万
• 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 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7929581
• 关键词: Acetylation; Address; Alloimmunization; Antigens; Apoptosis; Autologous; Biological Assay; Biomedical Engineering; Blood; Blood Component Removal; Blood Platelets; Blood donor; Blood-Borne Pathogens; Bone Marrow; CD34 gene; CSF3 gene; Cell Cycle; Cell Cycle Regulation; Cell Proliferation; Cell physiology; Cells; Collection; Development; Dose; Equipment; Europe; Flow Cytometry; Gene Expression Regulation; Generations; Goals; Growth Factor; Harvest; Hematopoietic; Hematopoietic stem cells; Histones; Human; Immune response; Individual; Lead; Lentivirus Vector; Leukocytes; Mediating; Megakaryocytopoieses; Messenger RNA; Microscopic; Niacinamide; Patients; Platelet Transfusion; Ploidies; Population; Process; Production; Proteins; RNA Interference; Reporting; Research Personnel; Risk; Role; Stem cells; Testing; Transfusion; Transplantation; Umbilical Cord Blood; Viral; Vitamins; base; cytokine; in vivo; knock-down; large scale production; peripheral blood; progenitor; response; 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.

血小板输注用于广泛的血栓形成缺陷，但 问题血小板采集通常需要汇集来自多个供体的收获物。 血小板输注的风险来自细菌污染、血源性病原体和 同种免疫是复杂的，因为患者从许多供体接受血小板。 通过巨核细胞（Mks）衍生的自体或相容性血小板的生产 来自培养的造血干细胞和祖细胞（HSPC或CD 34+细胞）的细胞将 大大降低了这些风险。然而，为一个人产生5000亿个血小板， 使用产生相对纯（e 75%）Mk群体的培养条件进行输血 需要2.5亿个CD 34+细胞这相当于50多条脐带 从接受治疗的供体的外周血中采集血液或采集1-2次HSPC（或 动员）与生长因子。为了使培养物衍生的血小板生产能够 经济上可行的是，每个CD 34+产生更多的Mk祖细胞是必要的。 细胞，获得更大数量的Mk/Mk祖细胞，并增加Mk倍性（血小板- 生产潜力）。我们的目标是增加培养衍生的Mk的倍性， 与人类骨髓中的水平相似。我们已经证明维生素 烟酰胺（NIC）大大增加培养物中的Mk倍性。由于Mks在体内产生 几千个血小板，我们预计在NIC培养中产生的Mks可以 生成1000个血小板。理解NIC介导的 Mk倍性的增加将促进使用NIC生产 血小板移植，并可能导致发现更有效的 Mk多倍体化的条件。我们建议使用RNA干扰介导的 击倒测试的假设，根据我们的初步结果，NIC增加 通过抑制SIRT 1和SIRT 2 III类组蛋白/蛋白脱乙酰酶的MK倍性。 然后，我们将研究SIRT靶蛋白乙酰化的变化， 调节细胞周期和/或凋亡。最后，我们将研究 最有希望的SIRT靶点的巨核细胞生成。