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Efficient and Directive Generation of Two Distinct Endoderm Lineages from Human ESCs and iPSCs by Differentiation Stage-Specific SOX17 Transduction  [PDF]
Kazuo Takayama,Mitsuru Inamura,Kenji Kawabata,Katsuhisa Tashiro,Kazufumi Katayama,Fuminori Sakurai,Takao Hayakawa,Miho Kusuda Furue,Hiroyuki Mizuguchi
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0021780
Abstract: The establishment of methods for directive differentiation from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is important for regenerative medicine. Although Sry-related HMG box 17 (SOX17) overexpression in ESCs leads to differentiation of either extraembryonic or definitive endoderm cells, respectively, the mechanism of these distinct results remains unknown. Therefore, we utilized a transient adenovirus vector-mediated overexpression system to mimic the SOX17 expression pattern of embryogenesis. The number of alpha-fetoprotein-positive extraembryonic endoderm (ExEn) cells was increased by transient SOX17 transduction in human ESC- and iPSC-derived primitive endoderm cells. In contrast, the number of hematopoietically expressed homeobox (HEX)-positive definitive endoderm (DE) cells, which correspond to the anterior DE in vivo, was increased by transient adenovirus vector-mediated SOX17 expression in human ESC- and iPSC-derived mesendoderm cells. Moreover, hepatocyte-like cells were efficiently generated by sequential transduction of SOX17 and HEX. Our findings show that a stage-specific transduction of SOX17 in the primitive endoderm or mesendoderm promotes directive ExEn or DE differentiation by SOX17 transduction, respectively.
Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods  [PDF]
Toshikazu Araoka, Shin-ichi Mae, Yuko Kurose, Motonari Uesugi, Akira Ohta, Shinya Yamanaka, Kenji Osafune
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0084881
Abstract: The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM), an embryonic germ layer that gives rise to kidneys. In order to achieve this goal, establishing an efficient, stable and low-cost method for differentiating IM cells using small molecules is required. In this study, we identified two retinoids, AM580 and TTNPB, as potent IM inducers by high-throughput chemical screening, and established rapid (five days) and efficient (80% induction rate) IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator, CHIR99021, combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys, and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step, directly inducing IM cells by activating Wnt, retinoic acid (RA), and bone morphogenetic protein (BMP) pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy.
The gene expression profiles of induced pluripotent stem cells (iPSCs) generated by a non-integrating method are more similar to embryonic stem cells than those of iPSCs generated by an integrating method
Liu, Yajun;Cheng, De;Li, Zhenzhen;Gao, Xing;Wang, Huayan;
Genetics and Molecular Biology , 2012, DOI: 10.1590/S1415-47572012005000050
Abstract: induced pluripotent stem cells (ipscs) obtained by the ectopic expression of defined transcription factors have tremendous promise and therapeutic potential for regenerative medicine. many studies have highlighted important differences between ipscs and embryonic stem cells (escs). in this work, we used meta-analysis to compare the global transcriptional profiles of human ipscs from various cellular origins and induced by different methods. the induction strategy affected the quality of ipscs in terms of transcriptional signatures. the ipscs generated by non-integrating methods were closer to escs in terms of transcriptional distance than ipscs generated by integrating methods. several pathways that could be potentially useful for studying the molecular mechanisms underlying transcription factor-mediated reprogramming leading to pluripotency were also identified. these pathways were mostly associated with the maintenance of esc pluripotency and cancer regulation. numerous genes that are up-regulated during the induction of reprogramming also have an important role in the success of human preimplantation embryonic development. our results indicate that hipscs maintain their pluripotency through mechanisms similar to those of hescs.
大家畜诱导多潜能干细胞(ipscs)研究进展  [PDF]
王峰,刘灿,潘传英
农业生物技术学报 , 2014,
Abstract: ?向分化的体细胞内导入特定的诱导因子,可将其重编程为诱导多潜能干细胞(inducedpluripotentstemcells,ipscs),ipscs同胚胎干细胞(embryonicstemcells,escs)一样具有自我更新并维持未分化状态的能力。ipscs的出现,有效地解决了escs研究领域存在的伦理道德限制和免疫排斥问题。自2006年国际上首次成功获得小鼠(musmusculus)诱导多潜能干细胞以来,ipscs研究发展迅猛。从利用病毒载体到普通质粒载体,从导入dna、rna和蛋白质,再到利用小分子化合物组合进行体细胞重编程,ipscs诱导技术正在向多元化发展;同时,研究者们对细胞重编程机理的认识也在加深。与此同时,大家畜ipscs研究领域也相继获得了猪(susscrofa)、牛(bostaurus)和绵羊(ovisaries)等动物的ipscs,并得到了ipscs嵌合猪和ipscs嵌合羊。由于猪等大家畜不仅在解剖和生理结构等方面与人相似,还是人类(homosapiens)最主要的肉类和奶类等食物来源,关系着人类的健康,因此大家畜ipscs在临床应用和生产实践上具有重大价值。鉴于此,本文对ipscs诱导方法、效率、机制和大家畜ipscs研究现状做一综述。
A Modified EpiSC Culture Condition Containing a GSK3 Inhibitor Can Support Germline-Competent Pluripotency in Mice  [PDF]
Tomoyuki Tsukiyama, Yasuhide Ohinata
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0095329
Abstract: Embryonic stem cells (ESCs) can contribute to the tissues of chimeric animals, including the germline. By contrast, epiblast stem cells (EpiSCs) barely contribute to chimeras. These two types of cells are established and maintained under different culture conditions. Here, we show that a modified EpiSC culture condition containing the GSK3 inhibitor CHIR99021 can support a germline-competent pluripotent state that is intermediate between ESCs and EpiSCs. When ESCs were cultured under a modified condition containing bFGF, Activin A, and CHIR99021, they converted to intermediate pluripotent stem cells (INTPSCs). These INTPSCs were able to form teratomas in vivo and contribute to chimeras by blastocyst injection. We also induced formation of INTPSCs (iINTPSCs) from mouse embryonic fibroblasts by exogenous expression of four reprogramming factors, Oct3/4, Sox2, Klf4, and c-Myc, under the INTPSC culture condition. These iINTPSCs contributed efficiently to chimeras, including the germline, by blastocyst injection. The INTPSCs exhibited several characteristic properties of both ESCs and EpiSCs. Our results suggest that the modified EpiSC culture condition can support growth of cells that meet the most stringent criteria for pluripotency, and that germline-competent pluripotency may depend on the activation state of Wnt signaling.
Myc and Max Genome-Wide Binding Sites Analysis Links the Myc Regulatory Network with the Polycomb and the Core Pluripotency Networks in Mouse Embryonic Stem Cells  [PDF]
Anna Krepelova, Francesco Neri, Mara Maldotti, Stefania Rapelli, Salvatore Oliviero
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0088933
Abstract: Myc is a master transcription factor that has been demonstrated to be required for embryonic stem cell (ESC) pluripotency, self-renewal, and inhibition of differentiation. Although recent works have identified several Myc-targets in ESCs, the list of Myc binding sites is largely incomplete due to the low sensitivity and specificity of the antibodies available. To systematically identify Myc binding sites in mouse ESCs, we used a stringent streptavidin-based genome-wide chromatin immunoprecipitation (ChIP-Seq) approach with biotin-tagged Myc (Bio-Myc) as well as a ChIP-Seq of the Myc binding partner Max. This analysis identified 4325 Myc binding sites, of which 2885 were newly identified. The identified sites overlap with more than 85% of the Max binding sites and are enriched for H3K4me3-positive promoters and active enhancers. Remarkably, this analysis unveils that Myc/Max regulates chromatin modifiers and transcriptional regulators involved in stem cell self-renewal linking the Myc-centered network with the Polycomb and the Core networks. These results provide insights into the contribution of Myc and Max in maintaining stem cell self-renewal and keeping these cells in an undifferentiated state.
The State of Play with iPSCs and Spinal Cord Injury Models  [PDF]
Stuart I. Hodgetts,Michael Edel,Alan R. Harvey
Journal of Clinical Medicine , 2015, DOI: 10.3390/jcm4010193
Abstract: The application of induced pluripotent stem cell (iPSC) technologies in cell based strategies, for the repair of the central nervous system (with particular focus on the spinal cord), is moving towards the potential use of clinical grade donor cells. The ability of iPSCs to generate donor neuronal, glial and astrocytic phenotypes for transplantation is highlighted here, and we review recent research using iPSCs in attempts to treat spinal cord injury in various animal models. Also discussed are issues relating to the production of clinical grade iPSCs, recent advances in transdifferentiation protocols for iPSC-derived donor cell populations, concerns about tumourogenicity, and whether iPSC technologies offer any advantages over previous donor cell candidates or tissues already in use as therapeutic tools in experimental spinal cord injury studies.
Cell-Type Independent MYC Target Genes Reveal a Primordial Signature Involved in Biomass Accumulation  [PDF]
Hongkai Ji, George Wu, Xiangcan Zhan, Alexandra Nolan, Cheryl Koh, Angelo De Marzo, Hoang Mai Doan, Jinshui Fan, Christopher Cheadle, Mohammad Fallahi, John L. Cleveland, Chi V. Dang, Karen I. Zeller
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0026057
Abstract: The functions of key oncogenic transcription factors independent of context have not been fully delineated despite our richer understanding of the genetic alterations in human cancers. The MYC oncogene, which produces the Myc transcription factor, is frequently altered in human cancer and is a major regulatory hub for many cancers. In this regard, we sought to unravel the primordial signature of Myc function by using high-throughput genomic approaches to identify the cell-type independent core Myc target gene signature. Using a model of human B lymphoma cells bearing inducible MYC, we identified a stringent set of direct Myc target genes via chromatin immunoprecipitation (ChIP), global nuclear run-on assay, and changes in mRNA levels. We also identified direct Myc targets in human embryonic stem cells (ESCs). We further document that a Myc core signature (MCS) set of target genes is shared in mouse and human ESCs as well as in four other human cancer cell types. Remarkably, the expression of the MCS correlates with MYC expression in a cell-type independent manner across 8,129 microarray samples, which include 312 cell and tissue types. Furthermore, the expression of the MCS is elevated in vivo in Eμ-Myc transgenic murine lymphoma cells as compared with premalignant or normal B lymphocytes. Expression of the MCS in human B cell lymphomas, acute leukemia, lung cancers or Ewing sarcomas has the highest correlation with MYC expression. Annotation of this gene signature reveals Myc's primordial function in RNA processing, ribosome biogenesis and biomass accumulation as its key roles in cancer and stem cells.
Extended passaging increases the efficiency of neural differentiation from induced pluripotent stem cells
Karl R Koehler, Philippe Tropel, Jonathan W Theile, Takako Kondo, Theodore R Cummins, Stéphane Viville, Eri Hashino
BMC Neuroscience , 2011, DOI: 10.1186/1471-2202-12-82
Abstract: Our gene expression and morphological analyses revealed that neural conversion was temporally delayed in iPSC lines and some iPSC lines did not properly form embryoid bodies during the first stage of differentiation. Notably, these deficits were corrected by continual passaging in an iPSC clone. iPSCs with greater than 20 passages (late-passage iPSCs) expressed higher expression levels of pluripotency markers and formed larger embryoid bodies than iPSCs with fewer than 10 passages (early-passage iPSCs). Moreover, late-passage iPSCs started to express neural marker genes sooner than early-passage iPSCs after the initiation of neural induction. Furthermore, late-passage iPSC-derived neurons exhibited notably greater excitability and larger voltage-gated currents than early-passage iPSC-derived neurons, although these cells were morphologically indistinguishable.These findings strongly suggest that the efficiency neuronal conversion depends on the complete reprogramming of iPSCs via extensive passaging.Induced pluripotent stem cells (iPSCs) are somatic cells that have been epigenetically reprogrammed to a pluripotent state using the ectopic expression of defined factors (Oct3/4, Sox2, Klf4, c-myc, Nanog or Lin28) or small molecule treatments [1-5]. Like embryonic stem cells (ESCs), iPSCs have the ability to differentiate into all three germ layers and thus, represent a viable option for autologous cell replacement therapies. A number of groups have investigated the potential of iPSCs for generating in vitro models of neurodegenerative maladies, such as, Parkinson's disease, retinal degeneration, amyotrophic lateral sclerosis and Rett Syndrome [6-14]. Although these studies are encouraging, little is currently known about the molecular underpinnings of reprogramming and the faithfulness with which iPSCs can recapitulate neuronal differentiation.Although iPSCs of both mouse and human origins appear morphologically indistinguishable from ESCs, several reports have emerged
Induced Pluripotent Stem Cells Generated from Human Adipose-Derived Stem Cells Using a Non-Viral Polycistronic Plasmid in Feeder-Free Conditions  [PDF]
Xinjian Qu, Tianqing Liu, Kedong Song, Xiangqin Li, Dan Ge
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0048161
Abstract: Induced pluripotent stem cells (iPSCs) can be generated from somatic cells by ectopic expression of defined transcription factors (TFs). However, the optimal cell type and the easy reprogramming approaches that minimize genetic aberrations of parent cells must be considered before generating the iPSCs. This paper reports a method to generate iPSCs from adult human adipose-derived stem cells (hADSCs) without the use of a feeder layer, by ectopic expression of the defined transcription factors OCT4, SOX2, KLF4 and C-MYC using a polycistronic plasmid. The results, based on the expression of pluripotent marker, demonstrated that the iPSCs have the characteristics similar to those of embryonic stem cells (ESCs). The iPSCs differentiated into three embryonic germ layers both in vitro by embryoid body generation and in vivo by teratoma formation after being injected into immunodeficient mice. More importantly, the plasmid DNA does not integrate into the genome of human iPSCs as revealed by Southern blotting experiments. Karyotypic analysis also demonstrated that the reprogramming of hADSCs by the defined factors did not induce chromosomal abnormalities. Therefore, this technology provides a platform for studying the biology of iPSCs without viral vectors, and can hopefully overcome immune rejection and ethical concerns, which are the two important barriers of ESC applications.
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