Pluripotency Genes Overexpressed in Primate Embryonic Stem Cells Are Localized on Homologues of Human 16, 17, 19 and X. Macaca mulatta

While human embryonic stem cells (hESCs) are predisposed towards chromosomal aneploidities on 12, 17, 20 and X, rendering them susceptible to transformation, the specific genes expressed are not yet known. Here, by identifying the genes over expressed in pluripotent rhesus ESCs (nhpESCs) and comparing them to both their genetically-identical differentiated progeny (teratoma fibroblasts) as well as genetically-related differentiated parental cells (parental skin fibroblasts from whom gametes were used for ESC derivation), we find that some of those over expressed genes in nhpESCs cluster preferentially on rhesus chromosomes 16, 19, 20 and X, homologues of human chromosomes 17, 19, 16 and X respectively. Differentiated parental skin fibroblasts display gene expression profiles closer to nhpESC profiles than to teratoma cells, which are genetically identical to the pluripotent nhpESCs. Twenty over and under expressed pluripotency modulators, some implicated in neurogenesis, have been identified. The over expression of some of these genes discovered using pedigreed nhpESCs derived from prime embryos generated by fertile primates, which is impossible to perform with the anonymously donated clinically-discarded embryos from which hESCs are derived, independently confirms the importance of chromosome 17 and X regions in pluripotency and suggests specific candidates for targeting differentiation and transformation decisions. Overall design: To overcome the genetic background heterogeneity which might mask some aspects of the actual stemness signature, we utilized a newly established bank of pedigreed non-human primate embryonic stem cells (nhpESCs) to generate a unique non-human primate “stemness gene” list generated by the comparison of nhpESC gene expression to two different sources of genetically-identical or genetically-related differentiated fibroblasts. The first were grown out of skin explants taken from the nonhuman primate parents from which gametes were used to generate the stem cell lines. The second were teratoma explants. RNA was isolated from all three types of cells: 1. Skin fibroblasts; these samples are indicated by the letter M (monkey) before the monkey identification number and sample label (a-f). 2. nhpESCs denoted by C (cell line) followed by the cell line and sample. 3. Fibroblast explants from teratomas generated by the injection of nhpESC into immune compromised mice. Since there were a number of teratomas for every stem cell line, we designated the nomenclature as follows: T (teratoma) - number of teratoma from the line - line name - sample. Therefore, TA31b denotes that this is the second sample of the first teratoma from line 3106. A total of 39 samples were used for this comparison: Pluripotent pedigreed rhesus ESCs (female ESC line C3806; male ESC line C3106) were compared to genetically identical differentiated progeny grown from their resultant teratomas (five male teratomas and three female teratomas: ‘T’, i.e. TA31, TB31, TC31, TD31, TE31 and TA38, TB38, TC38 respectively) as well as to differentiated cells from their macaque parents (‘M’). therefore, 2 cell lines (3106 and 3806) 3 types of skin fibroblasts and 8 teratoma fibroblasts (5 generated from line 3106 and 3 from 3806). All samples were run in biological triplicates for a total of 39 samples. A total of 39 samples were used for this comparison: Pluripotent pedigreed rhesus ESCs (female ESC line C3806; male ESC line C3106) were compared to genetically identical differentiated progeny grown from their resultant teratomas (five male teratomas and three female teratomas: ‘T’, i.e. TA31, TB31, TC31, TD31, TE31 and TA38, TB38, TC38 respectively) as well as to differentiated cells from their macaque parents (‘M’). therefore, 2 cell lines (3106 and 3806) 3 types of skin fibroblasts and 8 teratoma fibroblasts (5 generated from line 3106 and 3 from 3806). All samples were run in biological triplicates for a total of 39 samples.

人类胚胎干细胞（human embryonic stem cells, hESCs）易在12、17、20号染色体及X染色体发生染色体非整倍体变异，使其易于发生恶性转化，但目前其特异性表达基因仍未明确。本研究通过鉴定多能性恒河猴胚胎干细胞（pluripotent rhesus ESCs, nhpESCs）中高表达的基因，并将其与两类分化细胞进行比对：一类为遗传同源的分化子代（畸胎瘤（teratoma）成纤维细胞），另一类为遗传相关的分化亲本细胞（即用于生成ESC系的配子供体的亲本皮肤成纤维细胞），发现nhpESCs中的部分高表达基因优先富集于恒河猴16、19、20号染色体及X染色体，分别对应人类17、19、16号染色体及X染色体的同源区域。
分化的亲本皮肤成纤维细胞的基因表达谱与nhpESCs的表达谱更为接近，而非与遗传同源的多能nhpESCs来源的畸胎瘤细胞。本研究已鉴定出20个与多能性调控相关的差异表达基因（包括高表达与低表达基因），其中部分基因与神经发生过程相关。本研究使用由可育灵长类动物繁育的优质胚胎构建的谱系明确的nhpESCs，这一实验体系无法通过匿名捐赠的临床废弃胚胎（即传统hESCs的来源）实现；通过该体系验证的部分基因高表达结果，独立证实了17号染色体及X染色体区域在多能性维持中的重要性，并提出了可用于调控细胞分化与转化决策的潜在候选靶点。
总体实验设计：为克服可能掩盖真实干细胞干性特征的遗传背景异质性，本研究利用新构建的谱系明确的非人灵长类胚胎干细胞（nhpESCs）库，通过将nhpESC的基因表达谱与两种不同的遗传同源或遗传相关的分化成纤维细胞进行比对，得到了独特的非人灵长类"stemness gene"列表。第一种成纤维细胞源自用于生成干细胞系的配子供体的皮肤外植体；第二种则源自畸胎瘤外植体。
我们从三类细胞中分离总RNA：1. 皮肤成纤维细胞：此类样本以字母M（monkey，猴）作为前缀，标注猴编号及样本标签（a-f）；2. nhpESCs：以字母C（cell line，细胞系）作为前缀，标注细胞系及样本信息；3. 畸胎瘤来源成纤维细胞：由nhpESC注射至免疫缺陷小鼠体内生成的畸胎瘤分离得到的成纤维细胞。由于每个干细胞系可生成多个畸胎瘤，我们采用如下命名规则：T（teratoma，畸胎瘤）- 该细胞系来源的畸胎瘤编号 - 细胞系名称 - 样本编号。例如TA31b表示来自3106细胞系的第一个畸胎瘤的第二个样本。
共计39份样本用于本项比对研究：将多能性谱系明确的恒河猴ESC（雌性细胞系C3806；雄性细胞系C3106）与其畸胎瘤来源的遗传同源分化子代（5份雄性畸胎瘤样本与3份雌性畸胎瘤样本，分别为TA31、TB31、TC31、TD31、TE31以及TA38、TB38、TC38），以及其亲本猕猴来源的分化细胞（M组）进行比对。因此本实验包含2株细胞系（3106与3806）、3组皮肤成纤维细胞样本以及8组畸胎瘤成纤维细胞样本（其中5株源自3106细胞系，3株源自3806细胞系）。所有样本均进行三次生物学重复，共计39份样本。
共计39份样本用于本项比对研究：将多能性谱系明确的恒河猴ESC（雌性细胞系C3806；雄性细胞系C3106）与其畸胎瘤来源的遗传同源分化子代（5份雄性畸胎瘤样本与3份雌性畸胎瘤样本，分别为TA31、TB31、TC31、TD31、TE31以及TA38、TB38、TC38），以及其亲本猕猴来源的分化细胞（M组）进行比对。因此本实验包含2株细胞系（3106与3806）、3组皮肤成纤维细胞样本以及8组畸胎瘤成纤维细胞样本（其中5株源自3106细胞系，3株源自3806细胞系）。所有样本均进行三次生物学重复，共计39份样本。