Most Cited Cell Regeneration Articles
Volume 3, Issue 1, 四月 2014, Page
Guangming Wu | Hans R. Schöler | Hans R. Schöler
© 2014 Wu and Schöler; licensee BioMed Central Ltd.Oct4 is a key component of the pluripotency regulatory network, and its reciprocal interaction with Cdx2 has been shown to be a determinant of either the self-renewal of embryonic stem cells (ESCs) or their differentiation into trophoblast. Oct4 of maternal origin is postulated to play critical role in defining totipotency and inducing pluripotency during embryonic development. However, the genetic elimination of maternal Oct4 using a Cre-lox approach in mouse revealed that the establishment of totipotency in maternal Oct4-depleted embryos was not affected, and that these embryos could complete full-term development without any obvious defect. These results indicate that Oct4 is not essential for the initiation of pluripotency, in contrast to its critical role in maintaining pluripotency. This conclusion is further supported by the formation of Oct4-GFP- and Nanog- expressing inner cell masses (ICMs) in embryos with complete inactivation of both maternal and zygotic Oct4 expression and the reprogramming of fibroblasts into fully pluripotent cells by Oct4-deficient oocytes.
Volume 3, Issue 1, 八月 2014, Page
Quanmei Yan | Quanjun Zhang | Huaqiang Yang | Qingjian Zou | Chengcheng Tang | Nana Fan | Liangxue Lai | Liangxue Lai
© 2014 Yan et al.; licensee BioMed Central Ltd.The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) is a simple, robust and efficient technique for gene targeting in model organisms such as zebrafish, mice and rats. In this report, we applied CRISPR technology to rabbits by microinjection of Cas9 mRNA and guided RNA (gRNA) into the cytoplasm of pronuclear-stage embryos. We achieved biallelic gene knockout (KO) rabbits by injection of 1 gene (IL2rg) or 2 gene (IL2rg and RAG1) Cas9 mRNA and gRNA with an efficiency of 100%. We also tested the efficiency of multiple gene KOs in early rabbit embryos and found that the efficiency of simultaneous gene mutation on target sites is as high as 100% for 3 genes (IL2rg, RAG1 and RAG2) and 33.3% for 5 genes (IL2rg, RAG1, RAG2, TIKI1 and ALB). Our results demonstrate that the Cas9/gRNA system is a highly efficient and fast tool not only for single-gene editing but also for multi-gene editing in rabbits.
Volume 3, Issue 1, 一月 2014, Page
Eva Harreither | Hanna A. Rydberg | Helene L. Åmand | Vaibhav Jadhav | Lukas Fliedl | Christina Benda | Miguel A. Esteban | Duanqing Pei | Nicole Borth | Nicole Borth | Regina Grillari-Voglauer | Regina Grillari-Voglauer | Regina Grillari-Voglauer | Oliver Hommerding | Frank Edenhofer | Frank Edenhofer | Bengt Nordén | Johannes Grillari | Johannes Grillari | Johannes Grillari
© 2014 Harreither et al.; licensee BioMed Central Ltd.Background: Oct4 is a transcription factor that plays a major role for the preservation of the pluripotent state in embryonic stem cells as well as for efficient reprogramming of somatic cells to induced pluripotent stem cells (iPSC) or other progenitors. Protein-based reprogramming methods mainly rely on the addition of a fused cell penetrating peptide. This study describes that Oct4 inherently carries a protein transduction domain, which can translocate into human and mouse cells.Results: A 16 amino acid peptide representing the third helix of the human Oct4 homeodomain, referred to as Oct4 protein transduction domain (Oct4-PTD), can internalize in mammalian cells upon conjugation to a fluorescence moiety thereby acting as a cell penetrating peptide (CPP). The cellular distribution of Oct4-PTD shows diffuse cytosolic and nuclear staining, whereas penetratin is strictly localized to a punctuate pattern in the cytoplasm. By using a Cre/loxP-based reporter system, we show that this peptide also drives translocation of a functionally active Oct4-PTD-Cre-fusion protein. We further provide evidence for translocation of full length Oct4 into human and mouse cell lines without the addition of any kind of cationic fusion tag. Finally, physico-chemical properties of the novel CPP are characterized, showing that in contrast to penetratin a helical structure of Oct4-PTD is only observed if the FITC label is present on the N-terminus of the peptide.Conclusions: Oct4 is a key transcription factor in stem cell research and cellular reprogramming. Since it has been shown that recombinant Oct4 fused to a cationic fusion tag can drive generation of iPSCs, our finding might contribute to further development of protein-based methods to generate iPSCs.Moreover, our data support the idea that transcription factors might be part of an alternative paracrine signalling pathway, where the proteins are transferred to neighbouring cells thereby actively changing the behaviour of the recipient cell.
Volume 4, Issue 1, 七月 2015, Page
Gunes Ozhan | Gunes Ozhan | Gilbert Weidinger
© 2015 Ozhan and Weidinger.The ability to repair damaged or lost tissues varies significantly among vertebrates. The regenerative ability of the heart is clinically very relevant, because adult teleost fish and amphibians can regenerate heart tissue, but we mammals cannot. Interestingly, heart regeneration is possible in neonatal mice, but this ability is lost within 7 days after birth. In zebrafish and neonatal mice, lost cardiomyocytes are regenerated via proliferation of spared, differentiated cardiomyocytes. While some cardiomyocyte turnover occurs in adult mammals, the cardiomyocyte production rate is too low in response to injury to regenerate the heart. Instead, mammalian hearts respond to injury by remodeling of spared tissue, which includes cardiomyocyte hypertrophy. Wnt/β-catenin signaling plays important roles during vertebrate heart development, and it is re-activated in response to cardiac injury. In this review, we discuss the known functions of this signaling pathway in injured hearts, its involvement in cardiac fibrosis and hypertrophy, and potential therapeutic approaches that might promote cardiac repair after injury by modifying Wnt/β-catenin signaling. Regulation of cardiac remodeling by this signaling pathway appears to vary depending on the injury model and the exact stages that have been studied. Thus, conflicting data have been published regarding a potential role of Wnt/β-catenin pathway in promotion of fibrosis and cardiomyocyte hypertrophy. In addition, the Wnt inhibitory secreted Frizzled-related proteins (sFrps) appear to have Wnt-dependent and Wnt-independent roles in the injured heart. Thus, while the exact functions of Wnt/β-catenin pathway activity in response to injury still need to be elucidated in the non-regenerating mammalian heart, but also in regenerating lower vertebrates, manipulation of the pathway is essential for creation of therapeutically useful cardiomyocytes from stem cells in culture. Hopefully, a detailed understanding of the in vivo role of Wnt/β-catenin signaling in injured mammalian and non-mammalian hearts will also contribute to the success of current efforts towards developing regenerative therapies.
Aberrant DNA methylation reprogramming during induced pluripotent stem cell generation is dependent on the choice of reprogramming factors
Volume 3, Issue 1, 二月 2014, Page
Aline C. Planello | Aline C. Planello | Junfeng Ji | Junfeng Ji | Vivek Sharma | Vivek Sharma | Rajat Singhania | Faridah Mbabaali | Fabian Müller | Javier A. Alfaro | Javier A. Alfaro | Christoph Bock | Christoph Bock | Christoph Bock | Daniel D. De Carvalho | Daniel D. De Carvalho | Nizar N. Batada | Nizar N. Batada
© 2014 Planello et al.; licensee BioMed Central Ltd.The conversion of somatic cells into pluripotent stem cells via overexpression of reprogramming factors involves epigenetic remodeling. DNA methylation at a significant proportion of CpG sites in induced pluripotent stem cells (iPSCs) differs from that of embryonic stem cells (ESCs). Whether different sets of reprogramming factors influence the type and extent of aberrant DNA methylation in iPSCs differently remains unknown. In order to help resolve this critical question, we generated human iPSCs from a common fibroblast cell source using either the Yamanaka factors (OCT4, SOX2, KLF4 and cMYC) or the Thomson factors (OCT4, SOX2, NANOG and LIN28), and determined their genome-wide DNA methylation profiles. In addition to shared DNA methylation aberrations present in all our iPSCs, we identified Yamanaka-iPSC (Y-iPSC)-specific and Thomson-iPSC (T-iPSC)-specific recurrent aberrations. Strikingly, not only were the genomic locations of the aberrations different but also their types: reprogramming with Yamanaka factors mainly resulted in failure to demethylate CpGs, whereas reprogramming with Thomson factors mainly resulted in failure to methylate CpGs. Differences in the level of transcripts encoding DNMT3b and TET3 between Y-iPSCs and T-iPSCs may contribute partially to the distinct types of aberrations. Finally, de novo aberrantly methylated genes in Y-iPSCs were enriched for NANOG targets that are also aberrantly methylated in some cancers. Our study thus reveals that the choice of reprogramming factors influences the amount, location, and class of DNA methylation aberrations in iPSCs. These findings may provide clues into how to produce human iPSCs with fewer DNA methylation abnormalities.
Pharmacological manipulation of transcription factor protein-protein interactions: Opportunities and obstacles
Volume 4, Issue 1, 三月 2015, Page
Frank Fontaine | Jeroen Overman | Mathias François
© 2015 Fontaine et al.; licensee BioMed Central.Much research on transcription factor biology and their genetic pathways has been undertaken over the last 30 years, especially in the field of developmental biology and cancer. Yet, very little is known about the molecular modalities of highly dynamic interactions between transcription factors, genomic DNA, and protein partners. Methodological breakthroughs such as RNA-seq (RNA-sequencing), ChIP-seq (chromatin immunoprecipitation sequencing), RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins), and single-molecule imaging will dramatically accelerate the discovery rate of their molecular mode of action in the next few years. From a pharmacological viewpoint, conventional methods used to target transcription factor activity with molecules mimicking endogenous ligands fail to achieve high specificity and are limited by a lack of identification of new molecular targets. Protein-protein interactions are likely to represent one of the next major classes of therapeutic targets. Transcription factors, known to act mostly via protein-protein interaction, may well be at the forefront of this type of drug development. One hurdle in this field remains the difficulty to collate structural data into meaningful information for rational drug design. Another hurdle is the lack of chemical libraries meeting the structural requirements of protein-protein interaction disruption. As more attempts at modulating transcription factor activity are undertaken, valuable knowledge will be accumulated on the modality of action required to modulate transcription and how these findings can be applied to developing transcription factor drugs. Key discoveries will spawn into new therapeutic approaches not only as anticancer targets but also for other indications, such as those with an inflammatory component including neurodegenerative disorders, diabetes, and chronic liver and kidney diseases.
Vascularization mediated by mesenchymal stem cells from bone marrow and adipose tissue: A comparison
Volume 4, Issue 1, 十月 2015, Page
Karoline Pill | Karoline Pill | Sandra Hofmann | Sandra Hofmann | Sandra Hofmann | Heinz Redl | Heinz Redl | Wolfgang Holnthoner | Wolfgang Holnthoner
© 2015 Pill et al.Tissue-engineered constructs are promising to overcome shortage of organ donors and to reconstruct at least parts of injured or diseased tissues or organs. However, oxygen and nutrient supply are limiting factors in many tissues, especially after implantation into the host. Therefore, the development of a vascular system prior to implantation appears crucial. To develop a functional vascular system, different cell types that interact with each other need to be co-cultured to simulate a physiological environment in vitro. This review provides an overview and a comparison of the current knowledge of co-cultures of human endothelial cells (ECs) with human adipose tissue-derived stem/stromal cells (ASCs) or bone marrow-mesenchymal stem cells (BMSCs) in three dimensional (3D) hydrogel matrices. Mesenchymal stem cells (MSCs), BMSCs or ASCs, have been shown to enhance vascular tube formation of ECs and to provide a stabilizing function in addition to growth factor delivery and permeability control for ECs. Although phenotypically similar, MSCs from different tissues promote tubulogenesis through distinct mechanisms. In this report, we describe differences and similarities regarding molecular interactions in order to investigate which of these two cell types displays more favorable characteristics to be used in clinical applications. Our comparative study shows that ASCs as well as BMSCs are both promising cell types to induce vascularization with ECs in vitro and consequently are promising candidates to support in vivo vascularization.
Generation of tooth-like structures from integration-free human urine induced pluripotent stem cells
Volume 2, Issue 1, 七月 2013, Page
Jinglei Cai | Yanmei Zhang | Pengfei Liu | Pengfei Liu | Shubin Chen | Xuan Wu | Xuan Wu | Yuhua Sun | Ang Li | Ke Huang | Rongping Luo | Lihui Wang | Ying Liu | Ying Liu | Ting Zhou | Shicheng Wei | Shicheng Wei | Guangjin Pan | Duanqing Pei
© 2013 Cai et al.; licensee BioMed Central Ltd.Background: Tooth is vital not only for a good smile, but also good health. Yet, we lose tooth regularly due to accidents or diseases. An ideal solution to this problem is to regenerate tooth with patients' own cells. Here we describe the generation of tooth-like structures from integration-free human urine induced pluripotent stem cells (ifhU-iPSCs). Results: We first differentiated ifhU-iPSCs to epithelial sheets, which were then recombined with E14.5 mouse dental mesenchymes. Tooth-like structures were recovered from these recombinants in 3 weeks with success rate up to 30% for 8 different iPSC lines, comparable to H1 hESC. We further detected that ifhU-iPSC derived epithelial sheets differentiated into enamel-secreting ameloblasts in the tooth-like structures, possessing physical properties such as elastic modulus and hardness found in the regular human tooth. Conclusion: Our results demonstrate that ifhU-iPSCs can be used to regenerate patient specific dental tissues or even tooth for further drug screening or regenerative therapies.
Volume 2, Issue 1, 十一月 2013, Page
Jack W. Lambshead | Jack W. Lambshead | Laurence Meagher | Carmel O'Brien | Carmel O'Brien | Andrew L. Laslett | Andrew L. Laslett | Andrew L. Laslett
© 2013 Lambshead et al.; licensee BioMed Central Ltd.Human pluripotent stem cells (hPSCs) are able to self-renew indefinitely and to differentiate into all adult cell types. hPSCs therefore show potential for application to drug screening, disease modelling and cellular therapies. In order to meet this potential, culture conditions must be developed that are consistent, defined, scalable, free of animal products and that facilitate stable self-renewal of hPSCs. Several culture surfaces have recently been reported to meet many of these criteria although none of them have been widely implemented by the stem cell community due to issues with validation, reliability and expense. Most hPSC culture surfaces have been derived from extracellular matrix proteins (ECMPs) and their cell adhesion molecule (CAM) binding motifs. Elucidating the CAM-mediated cell-surface interactions that are essential for the in vitro maintenance of pluripotency will facilitate the optimisation of hPSC culture surfaces. Reports indicate that hPSC cultures can be supported by cell-surface interactions through certain CAM subtypes but not by others. This review summarises the recent reports of defined surfaces for hPSC culture and focuses on the CAMs and ECMPs involved.
Volume 2, Issue 1, 六月 2013, Page
Renli Ru | Yongchao Yao | Songlin Yu | Benpeng Yin | Wanwan Xu | Siting Zhao | Li Qin | Xiaoping Chen | Xiaoping Chen
© 2013 Ru et al.; licensee BioMed Central Ltd.Background: Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research. However, the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases. A new delivery method that can improve the utility of these nucleases is needed. Results: In this study, we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN, respectively. However, TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture. Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine (C-C motif) receptor 5 (CCR5, a co-receptor for HIV-1 entry into cells). Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency. A 5% modification rate was observed in human induced pluripotent stem cells (hiPSCs) treated with TAT-TALEN as measured by the Surveyor assay. Conclusions: TAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells. This new technique may advance the clinical application of TALEN technology.
Reprogramming barriers and enhancers: Strategies to enhance the efficiency and kinetics of induced pluripotency
Volume 4, Issue 1, 十一月 2015, Page
© 2015 Ebrahimi.Induced pluripotent stem cells are powerful tools for disease modeling, drug screening, and cell transplantation therapies. These cells can be generated directly from somatic cells by ectopic expression of defined factors through a reprogramming process. However, pluripotent reprogramming is an inefficient process because of various defined and unidentified barriers. Recent studies dissecting the molecular mechanisms of reprogramming have methodically improved the quality, ease, and efficiency of reprogramming. Different strategies have been applied for enhancing reprogramming efficiency, including depletion/inhibition of barriers (p53, p21, p57, p16Ink4a/p19Arf, Mbd3, etc.), overexpression of enhancing genes (e.g., FOXH1, C/EBP alpha, UTF1, and GLIS1), and administration of certain cytokines and small molecules. The current review provides an in-depth overview of the cutting-edge findings regarding distinct barriers of reprogramming to pluripotency and strategies to enhance reprogramming efficiency. By incorporating the mechanistic insights from these recent findings, a combined method of inhibition of roadblocks and application of enhancing factors may yield the most reliable and effective approach in pluripotent reprogramming.
Volume 5, Issue 1, 三月 2016, Page
Young Jin Lee | Suresh Ramakrishna | Suresh Ramakrishna | Himanshu Chauhan | Won Sun Park | Seok Ho Hong | Seok Ho Hong | Kye Seong Kim | Kye Seong Kim
© 2016 Lee et al.Increasing evidence indicates that microRNAs (miRNAs), endogenous short non-coding RNAs 19-24 nucleotides in length, play key regulatory roles in various biological events at the post-transcriptional level. Embryonic stem cells (ESCs) represent a valuable tool for disease modeling, drug discovery, developmental studies, and potential cell-based therapies in regenerative medicine due to their unlimited self-renewal and pluripotency. Therefore, remarkable progress has been made in recent decades toward understanding the expression and functions of specific miRNAs in the establishment and maintenance of pluripotency. Here, we summarize the recent knowledge regarding the regulatory roles of miRNAs in self-renewal of pluripotent ESCs and during cellular reprogramming, as well as the potential role of miRNAs in two distinct pluripotent states (naïve and primed).
Volume 3, Issue 1, 六月 2014, Page
Anne Limbourg | Anne Limbourg | Anne Limbourg | Anne Limbourg | Sabine Schnabel | Sabine Schnabel | Vladimir J. Lozanovski | Vladimir J. Lozanovski | Vladimir J. Lozanovski | L. C. Napp | L. C. Napp | Teng Cheong Ha | Teng Cheong Ha | Tobias Maetzig | Tobias Maetzig | Johann Bauersachs | Johann Bauersachs | Hassan Y. Naim | Axel Schambach | Axel Schambach | Axel Schambach | Axel Schambach | Florian P. Limbourg | Florian P. Limbourg | Florian P. Limbourg | Florian P. Limbourg
© 2014 Limbourg et al.; licensee BioMed Central Ltd.The transcription factor Oct4 (Pou5f1) is a critical regulator of pluripotency in embryonic and induced pluripotent stem cells. Therefore, Oct4 expression might identify somatic stem cell populations with inherent multipotent potential or a propensity for facilitated reprogramming. However, analysis of Oct4 expression is confounded by Oct4 pseudogenes or non-pluripotency-related isoforms. Systematic analysis of a transgenic Oct4-EGFP reporter mouse identified testis and skin as two principle sources of Oct4+ cells in postnatal mice. While the prevalence of GFP+ cells in testis rapidly declined with age, the skin-resident GFP+ population expanded in a cyclical fashion. These cells were identified as epidermal stem cells dwelling in the stem cell niche of the hair follicle, which endogenously expressed all principle reprogramming factors at low levels. Interestingly, skin wounding or non-traumatic hair removal robustly expanded the GFP+ epidermal cell pool not only locally, but also in uninjured skin areas, demonstrating the existence of a systemic response. Thus, the epithelial stem cell niche of the hair follicle harbors an expandable pool of Oct4+ stem cells, which might be useful for therapeutic cell transfer or facilitated reprogramming.
Volume 3, Issue 1, 五月 2014, Page
Quanmei Yan | Huaqiang Yang | Dongshan Yang | Bentian Zhao | Zhen Ouyang | Zhaoming Liu | Nana Fan | Hongsheng Ouyang | Weiwang Gu | Liangxue Lai | Liangxue Lai
© 2014 Yan et al.; licensee BioMed Central Ltd.The myxovirus resistance gene (Mx1) has a broad spectrum of antiviral activities. It is therefore an interesting candidate gene to improve disease resistance in farm animals. In this study, we report the use of somatic cell nuclear transfer (SCNT) to produce transgenic pigs over-expressing the Mx1 gene. These transgenic pigs express approximately 15-25 times more Mx1 mRNA than non-transgenic pigs, and the protein level of Mx1 was also markedly enhanced. We challenged fibroblast cells isolated from the ear skin of transgenic and control pigs with influenza A virus and classical swine fever virus (CFSV). Indirect immunofluorescence assay (IFA) revealed a profound decrease of influenza A proliferation in Mx1 transgenic cells. Growth kinetics showed an approximately 10-fold reduction of viral copies in the transgenic cells compared to non-transgenic controls. Additionally, we found that the Mx1 transgenic cells were more resistant to CSFV infection in comparison to non-transgenic cells. These results demonstrate that the Mx1 transgene can protect against viral infection in cells of transgenic pigs and indicate that the Mx1 transgene can be harnessed to develop disease-resistant pigs.
Volume 2, Issue 1, 四月 2013, Page
Thomas Hill | Olga Krougly | Enayat Nikoopour | Stacey Bellemore | Edwin Lee-Chan | Lynette A. Fouser | David J. Hill | David J. Hill | Bhagirath Singh | Bhagirath Singh | Bhagirath Singh
© 2013 Hill et al.; licensee BioMed Central Ltd.Background: In Type 1 diabetes, the insulin-producing β-cells within the pancreatic islets of Langerhans are destroyed. We showed previously that immunotherapy with Bacillus Calmette-Guerin (BCG) or complete Freund's adjuvant (CFA) of non-obese diabetic (NOD) mice can prevent disease process and pancreatic β-cell loss. This was associated with increased islet Regenerating (Reg) genes expression, and elevated IL-22-producing Th17 T-cells in the pancreas. Results: We hypothesized that IL-22 was responsible for the increased Reg gene expression in the pancreas. We therefore quantified the Reg1, Reg2, and Reg 3δ (INGAP) mRNA expression in isolated pre-diabetic NOD islets treated with IL-22. We measured IL-22, and IL-22 receptor(R)- α mRNA expression in the pancreas and spleen of pre-diabetic and diabetic NOD mice. Our results showed: 1) Reg1 and Reg2 mRNA abundance to be significantly increased in IL-22-treated islets in vitro; 2) IL-22 mRNA expression in the pre-diabetic mouse pancreas increased with time following CFA treatment; 3) a reduced expression of IL-22R α following CFA treatment; 4) a down-regulation in Reg1 and Reg 2 mRNA expression in the pancreas of pre-diabetic mice injected with an IL-22 neutralizing antibody; and 5) an increased islet β-cell DNA synthesis in vitro in the presence of IL-22. Conclusions: We conclude that IL-22 may contribute to the regeneration of β-cells by up-regulating Regenerating Reg1 and Reg2 genes in the islets.
Reprogramming somatic cells to cells with neuronal characteristics by defined medium both in vitro and in vivo
Volume 4, Issue 1, 十二月 2015, Page
Songwei He | Yiping Guo | Yixin Zhang | Yuan Li | Chengqian Feng | Xiang Li | Lilong Lin | Lin Guo | Haitao Wang | Chunhua Liu | Yi Zheng | Chuanming Luo | Qiang Liu | Fuhui Wang | Hao Sun | Lining Liang | Lingyu Li | Lingyu Li | Huanxing Su | Jiekai Chen | Duanqing Pei | Hui Zheng
© 2015 He et al.Background: Currently, direct conversion from somatic cells to neurons requires virus-mediated delivery of at least one transcriptional factor or a combination of several small-molecule compounds. Delivery of transcriptional factors may affect genome stability, while small-molecule compounds may require more evaluations when applied in vivo. Thus, a defined medium with only conventional growth factors or additives for cell culture is desirable for inducing neuronal trans-differentiation. Results: Here, we report that a defined medium (5C) consisting of basic fibroblast growth factor (bFGF), N2 supplement, leukemia inhibitory factor, vitamin C (Vc), and β-mercaptoethanol (βMe) induces the direct conversion of somatic cells to cells with neuronal characteristics. Application of 5C medium converted mouse embryonic fibroblasts (MEFs) into TuJ+ neuronal-like cells, which were capable of survival after being transplanted into the mouse brain. The same 5C medium could convert primary rat astrocytes into neuronal-like cells with mature electrophysiology characteristics in vitro and facilitated the recovery of brain injury, possibly by inducing similar conversions, when infused into the mouse brain in vivo. Crucially, 5C medium could also induce neuronal characteristics in several human cell types. Conclusions: In summary, this 5C medium not only provides a means to derive cells with neuronal characteristics without viral transfection in vitro but might also be useful to produce neurons in vivo for neurodegenerative disease treatment.
Volume 3, Issue 1, 四月 2014, Page
Yanding Zhang | Yi Ping Chen | Yi Ping Chen
© 2014 Zhang and Chen; licensee BioMed Central Ltd.A major challenge in stem cell-based bioengineering of an implantable human tooth is to identify appropriate sources of postnatal stem cells that are odontogenic competent as the epithelial component due to the lack of enamel epithelial cells in adult teeth. In a recent issue (2013, 2:6) of Cell Regeneration, Cai and colleagues reported that epithelial sheets derived from human induced pluripotent stem cells (iPSCs) can functionally substitute for tooth germ epithelium to regenerate tooth-like structures, providing an appealing stem cell source for future human tooth regeneration.
Volume 3, Issue 1, 五月 2014, Page
Yu Wang | Nana Fan | Jun Song | Juan Zhong | Xiaogang Guo | Weihua Tian | Quanjun Zhang | Fenggong Cui | Li Li | Philip N. Newsome | Philip N. Newsome | Jon Frampton | Miguel A. Esteban | Liangxue Lai
© 2014 Wang et al.; licensee BioMed Central Ltd.Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.
Glbase: A framework for combining, analyzing and displaying heterogeneous genomic and high-throughput sequencing data
Volume 3, Issue 1, 一月 2014, Page
Andrew P. Hutchins | Ralf Jauch | Ralf Jauch | Mateusz Dyla | Mateusz Dyla | Diego Miranda-Saavedra
© 2014 Hutchins et al.; licensee BioMed Central Ltd.Genomic datasets and the tools to analyze them have proliferated at an astonishing rate. However, such tools are often poorly integrated with each other: each program typically produces its own custom output in a variety of non-standard file formats. Here we present glbase, a framework that uses a flexible set of descriptors that can quickly parse non-binary data files. glbase includes many functions to intersect two lists of data, including operations on genomic interval data and support for the efficient random access to huge genomic data files. Many glbase functions can produce graphical outputs, including scatter plots, heatmaps, boxplots and other common analytical displays of high-throughput data such as RNA-seq, ChIP-seq and microarray expression data. glbase is designed to rapidly bring biological data into a Python-based analytical environment to facilitate analysis and data processing. In summary, glbase is a flexible and multifunctional toolkit that allows the combination and analysis of high-throughput data (especially next-generation sequencing and genome-wide data), and which has been instrumental in the analysis of complex data sets. glbase is freely available at http://bitbucket.org/oaxiom/glbase/.
Volume 1, Issue 1, 六月 2012, Page
Wolfgang H. Cerwinka | Wolfgang H. Cerwinka | Starlette M. Sharp | Barbara D. Boyan | Haiyen E. Zhau | Leland W K Chung | Clayton Yates | Clayton Yates
© 2012 Cerwinka et al. licensee Biomed Central Ltd.To develop and characterize a novel cell culture method for the generation of undifferentiated and differentiated human mesenchymal stem cell 3D structures, we utilized the RWV system with a gelatin-based scaffold. 3 × 106 cells generated homogeneous spheroids and maximum spheroid loading was accomplished after 3days of culture. Spheroids cultured in undifferentiated spheroids of 3 and 10 days retained expression of CD44, without expression of differentiation markers. Spheroids cultured in adipogenic and osteogenic differentiation media exhibited oil red O staining and von Kossa staining, respectively. Further characterization of osteogenic lineage, showed that 10 day spheroids exhibited stronger calcification than any other experimental group corresponding with significant expression of vitamin D receptor, alkaline phosphatase, and ERp 60. In conclusion this study describes a novel RWV culture method that allowed efficacious engineering of undifferentiated human mesenchymal stem cell spheroids and rapid osteogenic differentiation. The use of gelatin scaffolds holds promise to design implantable stem cell tissue of various sizes and shapes for future regenerative treatment.
Volume 1, Issue , 七月 2012, Page
Zhen Ning Zhang | Yang Xu
With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells of individual patients with defined factors, have unlimited potential in cell therapy and in modeling complex human diseases. Significant progress has been achieved to improve the safety of iPSCs and the reprogramming efficiency. To avoid the cancer risk and spontaneous reactivation of the reprogramming factors associated with the random integration of viral vectors into the genome, several approaches have been established to deliver the reprogramming factors into the somatic cells without inducing genetic modification. In addition, a panel of small molecule compounds, many of which targeting the epigenetic machinery, have been identified to increase the reprogramming efficiency. Despite these progresses, recent studies have identified genetic and epigenetic abnormalities of iPSCs as well as the immunogenicity of some cells derived from iPSCs. In addition, due to the oncogenic potential of the reprogramming factors and the reprogramming-induced DNA damage, the critical tumor suppressor pathways such as p53 and ARF are activated to act as the checkpoints that suppress induced pluripotency. The inactivation of these tumor suppression pathways even transiently during reprogramming processes could have significant adverse impact on the genome integrity. These safety concerns must be resolved to improve the feasibility of the clinic development of iPSCs into human cell therapy. © 2012 Zhang and Xu; licensee BioMed Central Ltd.
ETV2 expression increases the efficiency of primitive endothelial cell derivation from human embryonic stem cells
Volume 4, Issue 1, 一月 2015, Page
Anne G. Lindgren | Matthew B. Veldman | Shuo Lin
© 2015 Lindgren et al.; licensee BioMed Central.Background: Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body. Ets variant 2 (ETV2) is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification. Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells. Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal.Findings: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs. We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression.Conclusions: Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification. This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.
SNX17 regulates Notch pathway and pancreas development through the retromer-dependent recycling of Jag1
Volume 1, Issue 1, 六月 2012, Page
Wenguang Yin | Dapeng Liu | Nian Liu | Liangliang Xu | Song Li | Shuo Lin | Shuo Lin | Xiaodong Shu | Duanqing Pei
© 2012 Yin et al.Background: Notch is one of the most important signaling pathways involved in cell fate determination. Activation of the Notch pathway requires the binding of a membrane-bound ligand to the Notch receptor in the adjacent cell which induces proteolytic cleavages and the activation of the receptor. A unique feature of the Notch signaling is that processes such as modification, endocytosis or recycling of the ligand have been reported to play critical roles during Notch signaling, however, the underlying molecular mechanism appears context-dependent and often controversial. Results: Here we identified SNX17 as a novel regulator of the Notch pathway. SNX17 is a sorting nexin family protein implicated in vesicular trafficking and we find it is specifically required in the ligand-expressing cells for Notch signaling. Mechanistically, SNX17 regulates the protein level of Jag1a on plasma membrane by binding to Jag1a and facilitating the retromer-dependent recycling of the ligand. In zebrafish, inhibition of this SNX17-mediated Notch signaling pathway results in defects in neurogenesis as well as pancreas development. Conclusions: Our results reveal that SNX17, by acting as a cargo-specific adaptor, promotes the retromer dependent recycling of Jag1a and Notch signaling and this pathway is involved in cell fate determination during zebrafish neurogenesis and pancreas development.
Volume 4, Issue 1, 十一月 2015, Page
Zhijuan Mao | Suming Zhang | Hong Chen
© 2015 Mao et al.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the loss of motor neurons. Currently, no effective therapy is available to treat ALS, except for Riluzole, which has only limited clinical benefits. Stem-cell-based therapy has been intensively and extensively studied as a potential novel treatment strategy for ALS and has been shown to be effective, at least to some extent. In this article, we will review the current state of research on the use of stem cell therapy in the treatment of ALS and discuss the most promising stem cells for the treatment of ALS.
Combined influence of basal media and fibroblast growth factor on the expansion and differentiation capabilities of adipose-derived stem cells
Volume 3, Issue 1, 十一月 2014, Page
Mark Ahearne | Mark Ahearne | Joanne Lysaght | Amy P. Lynch | Amy P. Lynch
© 2014 Ahearne et al.; licensee BioMed Central Ltd.Background: Interest in adipose-derived stem cells (ASCs) has increased in recent years due to their multi-linage differentiation capabilities. While much work has been done to optimize the differentiation media, few studies have focused on examining the influence of different expansion media on cell behavior. In this study, three basal media (low glucose Dulbecco's modified Eagle's medium (DMEM), high glucose DMEM and DMEM-F12) supplemented with or without fibroblast growth factor 2 (FGF) were examined to assess their suitability for expanding ASCs. Findings: Flow cytometry, colony-forming unit assays (CFU-Fs) and differentiation assays were utilized to study cell behavior. High glucose media CFU-Fs produced fewest colonies while the addition of FGF increased colony size. By passage 2, the majority of cells were positive for CD44, 45, 73, 90 and 105 and negative for CD14, 31 and 45, indicating a mesenchymal phenotype. A sub-population of CD34 positive cells was present among passage 2 cells; however, by passage 4 the cells were negative for CD34. FGF has a negative effective on passage 4 ASC adipogenesis and high glucose media plus FGF-enhanced osteogenic capacity of passage 4 ASCs. FGF supplemented basal media were most suitable for chondrogenesis. High glucose media plus FGF appeared to be the most beneficial for priming ASCs to induce a keratocyte phenotype. Conclusions: These findings demonstrate the reciprocal effect FGF and basal media have on ASCs. This research has implications for those interested regenerating bone, cartilage, cornea or adipose tissues.