Investigation into the mechanisms of mesendoderm specification during ES cell differentiation

人体内有两百多种不同类型的细胞。所有细胞都来自一个受精卵，因此所有细胞都有相同的基因组，基因组编码所有信息，形成所有不同类型的细胞。然而，每种类型的细胞都有不同的性质和组成单个组织的不同功能。这是由于不同的基因使用，不同的基因表达造成的。一般来说，终末分化的细胞不会分裂，因此大多数组织都提供细胞，由致力于谱系的干细胞和/或祖细胞维持和更新。血统相关的干细胞通过保持相似的基因表达谱来持续供应特定类型的细胞，而不是不同血统的细胞，因为造血干细胞不会长出肌肉。在胚胎发育期间，有多能细胞，可以在体内产生任何类型的细胞，直到围着床期。然而，在发育过程中，这些细胞逐渐失去了产生不同类型细胞的能力。这种可塑性丧失的一部分是由谱系特异性转录因子解释的，这些转录因子促进谱系承诺或抑制对其他谱系重要的基因表达。此外，还有一种称为表观遗传修饰的机制，例如DNA甲基化，随后积累了许多其他沉默分子，以及组蛋白修饰，它充当DNA缠绕的线轴，如乙酰化、甲基化、磷酸化。表观遗传修饰是染色质上可遗传的活跃或沉默的标记，具有限制可塑性的功能。染色质上的这些标记被其他分子识别和/或改变染色质结构，导致基因激活或沉默。虽然这些修饰不会引起基因组序列的任何变化，但它们在复制过程中以未知的机制复制到新合成的染色质中，并在细胞分裂时被子细胞继承。因此，表观遗传修饰对于维持细胞类型特异性基因的表达和限制细胞命运具有重要意义。胚胎干细胞(ES细胞)来源于胚泡内细胞团(ICM)，在一定条件下具有遗传多能性和无限自我更新的能力。由于胚胎干细胞能够在体内产生任何类型的细胞，因此使用胚胎干细胞有望成为再生医学中最广泛应用的策略。为了从ES细胞中获得特定类型的细胞，通常使用特定的培养条件和特定的细胞因子，使ES细胞从自我更新中走出来，促进谱系承诺。近年来，内胚层、中胚层和神经外胚层的生成效率已经提高，未来可以从内胚层、中胚层和神经外胚层生成胰腺和损伤、肌肉和神经细胞，用于移植(1，2)。然而，除了这种优势外，ES细胞在移植方面还有一个缺点，即多能性。未分化的ES细胞是致瘤细胞。移植组织中未分化的ES细胞的少量污染可能是肿瘤的高风险(3)。为了避免这种风险，了解ES细胞分化的初始步骤和控制未分化-分化状态的机制是必不可少的。MBD3是核小体重塑和组蛋白脱乙酰酶(NuRD)复合体的组成部分之一，具有改变染色质结构和去除组蛋白表面活性表观遗传标记，导致靶基因沉默的活性。最近我们发现，通过基因打靶干扰MBD3基因的ES细胞分化能力存在严重缺陷(4)。野生型ES细胞在没有LIF的情况下失去未分化细胞特异性基因的表达，并产生谱系决定的分化细胞，LIF是一种促进自我更新和阻止ES细胞分化的细胞因子，因此提供在培养液中以维持未分化的ES细胞。然而，MBD3缺失的ES细胞停止

There are over two hundred different cell types in a human body. All cells are derived from one fertilized egg, therefore all cells have the same genome, which encode all information to make all different type of cells. However each type of cells has different character and different function to constitute individual tissues. This is caused by different usage of genes, different gene expression. In general, terminally differentiated cells do not divide, and therefore most of tissues are supplied cells to be maintained and renewed by lineage-committed stem cells and/or progenitor cells. The Lineage-committed stem cells continuously supply specific type of cells, but not cells in different lineage by keeping similar gene expression profiles, as hematopoietic stem cells do not give rise to muscles. During embryogenesis there are pluripotent cells, which can give rise to any type of cells in a body until periimplantation stage. However, during development those cells gradually lose the ability to give rise different type of cells. Some part of this loss of plasticity is explained by lineage specific transcription factors, which promote the lineage commitment or repress gene expression important for other lineages. In addition, there is a mechanism called ‘epigenetic modification‘, e. g. DNA methylation followed by accumulation of many other silencing molecules, and modification of histone, which acts as spools around which DNA winds, such as acetylation, methylation, phosphorylation. Epigenetic modification is inheritable active or silent marks on chromatin and function to restrict plasticity. These marks on chromatin are recognized by other molecules and/or change chromatin structure, and resulting in gene activation or silencing. Although these modifications do not introduce any changing in genomic sequence, they are copied in newly synthesized chromatin during replication with unknown mechanism and inherited by daughter cells when cells are divided. Therefore epigenetic modifications are important to maintain cell type specific gene expression and restrict cell fate. Embryonic stem (ES) cells, which are derived from a pluripotent population, inner cell mass (ICM) of blastocyst, inherit the pluripotency and possess indefinite self-renewal ability in a certain condition. Because of their ability to give rise any type of cells in a body use of ES cells is expected as the most widely applicable strategy for regenerative medicine. To obtain certain type of cells from ES cells, specific culture conditions with specific cytokines, which allow ES cells go out from self-renewal and promote lineage commitment, are often used. Recently efficiency to generate endoderm, mesoderm and neuroectoderm has been improved, from which pancreas and hurt, muscle and cartridge, neuron for transplantation could be generated in future (1, 2). However, ES cells have a disadvantage in addition to this advantage, pluripotency, for the transplantation. Undifferentiated ES cells are tumorgenic. Little amount contamination of undifferentiated ES cells in transplanted tissue can be a high risk of tumor (3). To avoid this risk, understanding the mechanism of an initial step of ES cell differentiation and controlling undifferentiated-differentiated state is indispensable. Mbd3 is a one of components of nucleosome remodeling and histone deacetylase (NuRD) complex, which possess activity to change chromatin structure and remove active epigenetic mark on histone, resulting in silence of target genes. Recently we found that ES cells disrupted Mbd3 gene by gene targeting have severe defect in their differentiation ability (4). Wild-type ES cells lose expression of undifferentiated cell specific genes and generate lineage committed differentiated cells in the absence of LIF, a cytokine which promotes self-renewal and prevents ES cell differentiation, therefore supplied in a culture medium to maintain undifferentiated ES cells. However, Mbd3-null ES cells stop to