Direct Induction of Chondrogenic Cells from Human Dermal Fibroblast Culture by Defined Factors

The repair of large cartilage defects with hyaline cartilage continues to be a challenging clinical issue. We recently reported that the forced expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) can induce chondrogenic cells from mouse dermal fibroblast culture without going through a pluripotent state. We here generated induced chondrogenic (iChon) cells from human dermal fibroblast (HDF) culture with the same factors. We developed a chondrocyte-specific COL11A2 promoter/enhancer lentiviral reporter vector to select iChon cells. The human iChon cells expressed marker genes for chondrocytes but not fibroblasts, and were derived from non-chondrogenic COL11A2-negative cells. The human iChon cells formed cartilage but not tumors in nude mice. This approach could lead to the preparation of cartilage directly from skin in human, without going through pluripotent stem cells.     

Heterogeneity of pluripotent marker gene expression in colonies generated in human iPS cell induction culture

Induction of pluripotent stem cells from human fibroblasts has been achieved by the ectopic expression of two different sets of four genes. However, the mechanism of the pluripotent stem cell induction has not been elucidated. Here we identified a marked heterogeneity in colonies generated by the four-gene (Oct3/4, Sox2, c-Myc, and Klf4) transduction method in human neonatal skin-derived cells. The four-gene transduction gave a higher probability of induction for archetypal pluripotent stem cell marker genes (Nanog, TDGF, and Dnmt3b) than for marker genes that are less specific for pluripotent stem cells (CYP26A1 and TERT) in primary induction culture. This tendency may reflect the molecular mechanism underlying the induction of human skin-derived cells into pluripotent stem cells. Among the colonies induced by the four-gene transduction, small cells with a high nucleus-to–cytoplasm ratio could be established by repeated cloning. Subsequently established cell lines were similar to human embryonic stem cells as well as human induced pluripotent stem (iPS) cells derived from adult tissue in morphology, gene expression, long-term self-renewal ability, and teratoma formation. Genome-wide single-nucleotide polymorphism array analysis of the human iPS cell line indicates that the induction process did not induce DNA mutation.     

Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors

Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.     

两种细胞来源的21-三体综合征诱导多能干细胞系的建立及比较

21-三体综合征是染色体异常导致的疾病,通过重编程21-三体综合征患儿两种组织来源的细胞成为多能干细胞,比较两种组织来源的细胞建立21-三体综合征诱导多能干细胞（T21-iPSCs）系的效率,为进一步研究21-三体综合征发病机制提供细胞模型,并为选择高效制备T21-iPSCs的组织类型提供理论依据。该实验利用慢病毒介导4种转录因子（Oct4、Sox2、Klf4、c-Myc）分别诱导人21-三体综合征的羊水细胞和胎儿皮肤成纤维细胞,建立诱导多能干细胞系（Trisomy 21 human amniotic fl uid induced pluripotent stem cells,T21 hAF-iPSCs;Trisomy 21 human dermal fi broblast induced pluripotent stem cells,T21 hDF-iPSCs）,T21 hAF-iPSCs及T21 hDF-iPSCs在蛋白和mRNA水平上均表达人胚胎干细胞的多能性分子标记,如Oct4、Nanog等,具有在体外及体内分化三个胚层的能力,其在培养过程中能维持异常核型并能维持自我更新状态。结果发现,利用羊水细胞建立T21-iPSCs效率高于皮肤成纤维细胞,羊水细胞可能是制备T21-iPSCs的理想细胞类型。     

体细胞重编程为诱导多能干细胞

诱导多功能性干细胞（induced pluripotent stem cells,iPS细胞）是通过导入特定的转录因子（如Oct3/4、Sox2、c-Myc和Klf4等）将体细胞诱导重编程为多能性干细胞,其功能与胚胎干细胞相似.iPS细胞的建立,在生命科学领域引起了新的轰动.目前,iPS细胞的研究领域在转录因子的优化、iPS细胞的筛选、载体的运用、体细胞种类的选择和iPS细胞的应用等方面取得突破进展,但仍然存在致癌性、效率低等一系列急需解决的问题.     

Rapid induction of pluripotency genes after exposure of human somatic cells to mouse ES cell extracts

The expression of 4 pluripotency genes (Oct4, Sox2, c-Myc and Klf4) in mouse embryonic fibroblasts can reprogramme them to a pluripotent state. We have investigated the expression of these pluripotency genes when human somatic 293T cells are permeabilized and incubated in extracts of mouse embryonic stem (ES) cells. Expression of all 4 genes was induced over 1–8 h. Gene expression was associated with loss of repressive histone H3 modifications and increased recruitment of RNA polymerase II at the promoters. Lamin A/C, which is typically found only in differentiated cells, was also removed from the nuclei. When 293T cells were returned to culture after exposure to ES cell extract, the expression of the pluripotency genes continued to rise over the following 48 h of culture, suggesting that long-term reprogramming of gene expression had been induced. This provides a methodology for studying the de-differentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells to a pluripotent state without genetically altering them.     

Genomic instability in induced stem cells

The ability to reprogram adult cells into stem cells has raised hopes for novel therapies for many human diseases. Typical stem cell reprogramming protocols involve expression of a small number of genes in differentiated somatic cells with the c-Myc and Klf4 proto-oncogenes typically included in this mix. We have previously shown that expression of oncogenes leads to DNA replication stress and genomic instability, explaining the high frequency of p53 mutations in human cancers. Consequently, we wondered whether stem cell reprogramming also leads to genomic instability. To test this hypothesis, we examined stem cells induced by a variety of protocols. The first protocol, developed specifically for this study, reprogrammed primary mouse mammary cells into mammary stem cells by expressing c-Myc. Two other previously established protocols reprogrammed mouse embryo fibroblasts into induced pluripotent stem cells by expressing either three genes, Oct4, Sox2 and Klf4, or four genes, OSK plus c-Myc. Comparative genomic hybridization analysis of stem cells derived by these protocols revealed the presence of genomic deletions and amplifications, whose signature was suggestive of oncogene-induced DNA replication stress. The genomic aberrations were to a significant degree dependent on c-Myc expression and their presence could explain why p53 inactivation facilitates stem cell reprogramming.     

Characterization of bovine induced pluripotent stem cells by lentiviral transduction of reprogramming factor fusion proteins

Pluripotent stem cells from domesticated animals have potential applications in transgenic breeding. Here, we describe induced pluripotent stem (iPS) cells derived from bovine fetal fibroblasts by lentiviral transduction of Oct4, Sox2, Klf4 and c-Myc defined-factor fusion proteins. Bovine iPS cells showed typical colony morphology, normal karyotypes, stained positively for alkaline phosphatase (AP) and expressed Oct4, Nanog and SSEA1. The CpG in the promoter regions of Oct4 and Nanog were highly unmethylated in bovine iPS cells compared to the fibroblasts. The cells were able to differentiate into cell types of all three germ layers in vitro and in vivo. In addition, these cells were induced into female germ cells under defined culture conditions and expressed early and late female germ cell-specific genes Vasa, Dazl, Gdf9, Nobox, Zp2, and Zp3. Our data suggest that bovine iPS cells were generated from bovine fetal fibroblasts with defined-factor fusion proteins mediated by lentivirus and have potential applications in bovine transgenic breeding and gene-modified animals.     

胎儿肺组织来源间充质干细胞的分离、培养、鉴定及重编程初探

该文主要研究将进行胎儿肺部组织来源的间充质干细胞（mesenchymal stem cells derived from fetal lung,FL-MSCs）转变成为诱导多能干细胞（induced pluripotent stem cells,iPS细胞）。首先使用酶消化法对胎儿肺部组织进行分离,然后采用常规方法进行培养并成功获得成纤维细胞样细胞。使用共聚焦技术检测获得的细胞,发现角蛋白表达呈阴性;共聚焦技术检测c-Myc、Oct4、Nanog以及Nestin四个干性相关因子,发现它们呈阳性;检测成纤维细胞样细胞的免疫表型,符合间充质干细胞的表型判断标准;然后进行诱导分化实验,发现这些细胞可以向成脂、成骨细胞分化,经过以上实验鉴定获得的成纤维细胞为FL-MSCs。使用Yamanaka四因子体系对FL-MSCs进行诱导,可以形成类似人胚胎干细胞（human embryonic stem cells,hES细胞）的克隆,采用核型分析、STR检测分析以及畸胎瘤形成实验初步验证获得的克隆为iPS。