Investigation into the mechanisms of mesendoderm specification during ES cell differentiation

人体内有200多种不同的细胞类型。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，编码所有信息，使所有不同类型的细胞。然而，每种类型的细胞具有不同的特性和不同的功能，以构成个体组织。这是由基因的不同用途，不同的基因表达造成的。通常，终末分化的细胞不分裂，因此大部分组织被供应细胞以通过谱系定向干细胞和/或祖细胞维持和更新。谱系定向干细胞通过保持相似的基因表达谱而持续供应特定类型的细胞，但不是不同谱系中的细胞，因为造血干细胞不产生肌肉。在胚胎发生过程中，存在多能细胞，其可以在体内产生任何类型的细胞，直到围植入期。然而，在发育过程中，这些细胞逐渐失去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定型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，即。G. DNA甲基化之后是许多其他沉默分子的积累，以及组蛋白的修饰，组蛋白充当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性或沉默标记，其功能是限制可塑性。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，并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，并在细胞分裂时由子细胞继承。因此，表观遗传修饰对于维持细胞类型特异性基因表达和限制细胞命运是重要的。胚胎干细胞（Embryonic stem cells，ES细胞）来源于囊胚内细胞团（inner cell mass，ICM），具有多能性，在一定条件下具有无限的自我更新能力。由于它们能够在体内产生任何类型的细胞，ES细胞的使用被认为是再生医学最广泛适用的策略。为了从ES细胞获得某些类型的细胞，通常使用具有特定细胞因子的特定培养条件，其允许ES细胞从自我更新中走出来并促进谱系定型。最近，内胚层、中胚层和神经外胚层的产生效率已经提高，将来可以从中产生胰腺和损伤、肌肉和盒、用于移植的神经元（1，2）。然而，ES细胞除了这个优点之外还有一个缺点，即多能性，用于移植。未分化的ES细胞是致瘤的。移植组织中少量未分化的ES细胞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的机制和控制未分化-分化状态是必不可少的。 Mbd 3是核小体重构和组蛋白脱乙酰酶（NuRD）复合物的组成部分之一，具有改变染色质结构和去除组蛋白上活性表观遗传标记的活性，导致靶基因沉默。最近，我们发现通过基因靶向破坏Mbd 3基因的ES细胞在其分化能力方面存在严重缺陷（4）。野生型ES细胞失去未分化细胞特异性基因的表达，并在不存在LIF的情况下产生谱系定向分化细胞，LIF是一种促进自我更新并阻止ES细胞分化的细胞因子，因此在培养基中提供以维持未分化的ES细胞。然而，Mbd 3-null 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