人类多潜能干细胞体外分化红细胞发育过程中表型分子的研究

发布时间：2018-01-07 18:44

本文关键词：人类多潜能干细胞体外分化红细胞发育过程中表型分子的研究　出处：《北京协和医学院》2015年博士论文　论文类型：学位论文

【摘要】：研究目的：人类多潜能干细胞(human pluripotent stem cells, hPSCs)主要包括人胚胎干细胞(human embryonic stem cells, hESCs)和人诱导性多潜能干细胞(human induced pluripotent stem cells, hiPSCs),二者兼具体外自我更新、无限增殖及多分化潜能的特性。它们的成功建株,极大地推动了干细胞基础研究和临床应用研究。研究的一个重要方向是将hPSCs向特定谱系的成熟血液细胞定向诱导分化。因为一直没有合适的研究人类早期造血发生的模型,以往的研究主要都是基于小鼠等动物模型。hESCs体外诱导分化红细胞的过程,模拟了人类胚胎期体内红系发生发育过程,为研究红细胞正常发育的调控机理奠定了实验基础。另一方面,利用患者hiPSCs体系建立体外诱导分化红细胞的方法,将为研究红细胞早期发育异常相关疾病的致病机理和开发个体化治疗手段提供理想的平台。在所有血细胞中,成熟红细胞因为不含细胞核,并携带着最小量的遗传物质,寿命较长等特点,有望作为最早的干细胞来源的细胞治疗产品而应用于临床。但在成功实现hPSC来源的红细胞临床应用前,还存在诸多问题需解决,例如：培养体系的不健全导致的体外扩增效率低、成熟程度低；脱核调控机制不明；没有合适的活体移植模型等。这些困难需要通过对hPSC来源红细胞的发生和成熟过程中的关键调控机理的理解来攻克。为了精密地研究hPSCs体外诱导分化红细胞的发育过程,在方法学上有两个亟待解决的问题。(1)现有实验数据已显示不同诱导体系产生的红细胞成熟程度有差异。这种差异指向一个事实,即成体造血微环境来源的基质细胞的诱导对hPSCs产生成熟红细胞是必需的。所以需要建立一套高效并趋于自然的共培养方法,以得到类似于自然发育过程产生的成熟红细胞。(2) hPSC来源的红细胞分化培养体系中,同时存在原始造血及成体造血过程,并且红细胞在早期发育存在不同的发育阶段。为了辨别不同的细胞,需要建立一种快捷、方便且准确的标识方法。在成体干细胞向红细胞分化发育过程的研究领域,已成功利用表型分子来区分红细胞的不同发育阶段,启示我们也可能利用表型分子来区分hPSC来源红细胞的早期不同发育阶段。研究方法：我们比较了不同的成体造血微环境来源的基质细胞,选择了小鼠主动脉-性腺-中肾(Aorta-Gonad-Mesonephros, AGM)细胞作为共培养体系的基质细胞。AGM区域是最早的支持成体造血的位点。我们建立了将hPSCs与AGM来源的细胞系AGM-S3体外向红细胞分化的培养方法。首先将hPSCs细胞与AGM-S3细胞系先共培养诱导造血分化的发生,再经悬浮培养向红细胞定向分化并扩增。以成体干细胞hCB-CD34~+来源的红细胞为对照,用先进的多色流式分析技术,探索hPSCs与AGM-S3共培养来源红细胞特异的表型分子表达谱系。随后以分别表达特异表型分子的红细胞亚群GPA~+CD36-和GPA~+CD34~+为切入点,利用荧光激活细胞分选(Fluorescence Activated Cell Sorting, FACS)技术,精确地将各目标细胞亚群分选出。然后利用瑞姬氏(May-Grunwald-Giemsa, MGG)染色方法观察细胞形态,通过免疫荧光染色方法考察血红蛋白组分来评价红细胞的成熟程度,并采用qRT-PCR技术检测造血及红细胞发育相关的重要基因的转录水平。研究结果：我们建立了高效的hPSC/AGM-S3共培养造血诱导分化体系,可以产生大量的高纯度和高成熟度红细胞,共培养12天再悬浮培养24天时,红细胞数量约为起始未分化H1细胞数量的300倍,其中85%以上表达成体型血红蛋白p。hPSC/AGM-S3共培养体系来源的红细胞p血红蛋白的表达率远远高于其他实验室报道的数据。我们在这个高效体系上进一步研究了hPSC/AGM-S3共培养体系来源红细胞发育过程的表型分子表达谱系,发现共培养阶段红系特有的表型分子GPA阳性(GPA~+)细胞上的其他共表达表型分子模式与已知的成体型红细胞的发育模式不同。其中GPA和成熟红细胞的特定分子CD36和造血干细胞的特有分子CD34的共表达均存在hPSCs分化发育的特有模式。我们根据这两条线索,进行了进一步细致地研究工作。本工作通过精密细致地研究,首次发现并论证了hPSC来源的早期红细胞可根据GPA和CD36抗原的共表达的表达变化指示不同的发育阶段。在共培养阶段相当长一段时间(6-18天)红细胞表型为GPA~+CD36-,悬浮培养后逐渐变为GPA~+CD36low/~+,10~+5天时达到一半左右,然后又再逐渐变为GPA~+CD36-。我们在不同时间点将CD36表达不同的红细胞亚群利用流式分选技术分离出来,通过对特定红细胞亚群Hb组分、表型分子及基因转录相对水平的比较,进一步揭示了这一系列的表型变化过程同时伴随着中胚层及内皮细胞特性的丢失,原始造血向成体造血特性的转变以及红细胞成熟度逐渐升高等变化过程。综合我们的实验数据,提示红细胞的初期发育是源于中胚层向内皮造血的途径,远在成体造血干／祖细胞被确定诞生之前。进一步的研究还发现hPSC/AGM-S3共培养阶段红细胞最初存在于表型为GPA~+CD34~+的细胞亚群中,细胞亚群比例先增加后减少。流式分选出共培养10天中的GPA~+CD34~+,对其细胞形态,人血红蛋白表达以及分化潜能进行了研究。现有数据显示GPA~+CD34~+是带有很强内皮细胞特性的细胞,约60%表达人Hb。我们拟进一步深入研究GPA~+CD34~+细胞与内皮细胞的关系,找出GPA~+CD34~+细胞的前体细胞,有望揭示hPSCs细胞来源最早红细胞的起源和发育过程。
[Abstract]:Objective: To study human pluripotent stem cells (human pluripotent stem cells, hPSCs) mainly includes human embryonic stem cells (human embryonic stem cells, hESCs) and human induced pluripotent stem cells (human induced pluripotent stem cells, hiPSCs), two with self-renewal, unlimited proliferation characteristics and multi differentiation potential. Their success were greatly promoted the stem cell research and clinical application research. An important research direction is to induce hPSCs differentiate to specific lineages of mature blood cells. Because there has been no research orientation of early human hematopoiesis model is appropriate, the previous studies are mainly the process of differentiation of red blood cells animal model of.HESCs in vitro based on Simulation of the human embryo in vivo erythroid development process, lay the regulation mechanism for the normal development of red blood cells Based. On the other hand, establishment method of differentiation of red blood cells in vitro by patients with hiPSCs system, pathogenic mechanism and development of individualized treatment means abnormal development of related diseases for early studies of red blood cells provide an ideal platform. In all blood cells, mature red blood cells because of not containing the nucleus, and carrying the genetic material minimum the long life and other characteristics, is expected as a cell therapy product of the earliest source of stem cells for clinical application. But in clinical application of red blood cells in the successful implementation of hPSC source, there are still many problems need to be solved, such as: the training system is not perfect in the in vitro amplification of low efficiency, low degree of maturity; deribonucleic the molecular mechanism is still unknown; there is no suitable living donor transplantation model. These difficulties through the key regulatory mechanism and process of mature red blood cells in the hPSC source of understanding to overcome. The development process of differentiation of red blood cells on the precision of hPSCs in vitro, the method has two problems to be solved on the school. (1) the existing experimental data have shown that there is a difference in degrees of maturity have different induction system of red blood cells. These differences point to the fact that induce stromal cells in hematopoietic microenvironment of the source body the mature red blood cells on hPSCs is required. So it is necessary to establish a set of effective and tends to natural co culture method to obtain similar mature red blood cells in the natural development process produced. (2) red cell differentiation from hPSC culture system, at the same time are primitive hematopoietic and adult hematopoiesis, and red cells in the early development of different developmental stages. In order to identify the different types of cells, we need to establish a fast, convenient and accurate identification method. In the study of adult stem cells in the red cell differentiation process. Domain, different developmental stages have been successfully used to cell phenotypic molecules into different developmental stages, early enlightenment we may also use phenotypic molecules to differentiate hPSC from red blood cells. Methods: We compared the stromal cell body of hematopoietic microenvironment into different sources, the mouse aorta gonad mesonephros (Aorta-Gonad-Mesonephros, AGM) cells as co cultured stromal cells.AGM system is the first to support hematopoietic sites. We established the culture square hPSCs and AGM derived cell lines AGM-S3 in vitro erythroid differentiation method. Firstly, hPSCs cells and AGM-S3 cells to co culture induction of hematopoietic differentiation occurred, followed by suspension culture of red cell differentiation and amplification. In adult stem cells from the red blood cells of hCB-CD34~+ were type analysis technology with advanced multi-color flow, exploring hPSCs and AGM-S3 Cultured erythroid specific sources of phenotypic molecules. Then the expression of lineage specific expression to phenotypic molecules of red blood cell subsets of GPA~+CD36- and GPA~+CD34~+ as the starting point, using fluorescence activated cell sorting (Fluorescence Activated Cell Sorting FACS) technology, precisely the target cell subsets were selected. Then the other (May-Grunwald-Giemsa, MGG) the cell morphology was observed by staining method, investigation group to evaluate the degree of maturity of hemoglobin in red blood cells by immunofluorescence staining, and the transcription level of qRT-PCR detection of hematopoietic and red blood cell development important genes. Results: we established a highly efficient hPSC/AGM-S3 hematopoietic differentiation of co culture system, can produce a large amount of high purity and the high maturity of red blood cells, co cultured 12 days suspension cultured for 24 days, the number of red blood cells about initiation of undifferentiated H1 cells 300 times the number of more than 85% of them expressed as the expression system of source of co culture of hemoglobin p.hPSC/AGM-S3 red blood cell p type hemoglobin rate is much higher than other reported laboratory data. In this system, we studied the hPSC/AGM-S3 co culture during the development of the source of red cell phenotypic molecules expression system of lineage, found co cultured erythroid specific the molecular phenotype of GPA positive cells (GPA~+) on the other co expression of phenotypic molecules known as development mode and mode shape of red blood cells. Different unique patterns of differentiation and development of hPSCs were co expressed in the specific molecular GPA and mature red blood cells CD36 and hematopoietic stem cell specific molecules CD34. According to the two clues for further detailed research work. In this work, through meticulous precision, first discovered and demonstrated early hPSC sources Red blood cells may indicate different developmental stages according to the changes of the expression of the co expression of GPA and CD36 antigen. In the co culture stage for quite a long period of time (6-18 days) red blood cell phenotype was GPA~+CD36- after suspension culture has gradually changed to GPA~+CD36low/~+, 10~+5 days to about half, and then gradually changed to GPA~+CD36-. we are in different the expression of CD36 in different time points of red blood cell subsets by flow cytometry analysis separated by specific red cell subsets in Hb group, compared the molecular phenotypes and gene transcription relative level, further reveals the phenotypic changes in the process of this series is accompanied by loss of mesoderm and endothelial cells into primitive hematopoietic characteristics. Change characteristics of hematopoietic and red cell maturity gradually rise higher. Comprehensive change process of our experimental data, suggesting that early red blood cell development is derived from mesoderm inward leather Blood way before adult hematopoietic stem / progenitor cells was determined. The birth of further research found that hPSC/AGM-S3 co culture stage of red blood cells initially present in the phenotype of GPA~+CD34~+ cell subsets, the proportion of cell subsets increased firstly and then decreased. Flow cytometry sorting out co cultured for 10 days in GPA~+CD34~+, on the cell morphology, expression of human hemoglobin and differentiation potential were studied. The existing data show that GPA~+CD34~+ is a strong endothelial cells, about 60% Hb. expression we intend to further study of GPA~+CD34~+ cells and endothelial cells, progenitor cells found in GPA~+CD34~+ cells, the process is expected to reveal hPSCs cells derived from the earliest origin of red blood cells and development.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R329.2

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