|
1
|
Ye J, Boileau RM, Parchem RJ, Judson-Torres RL, Blelloch R. The miR-290 and miR-302 clusters are essential for reprogramming of fibroblasts to induced pluripotent stem cells. Stem Cells 2025; 43:sxae080. [PMID: 40037390 PMCID: PMC11879289 DOI: 10.1093/stmcls/sxae080] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2024] [Accepted: 10/24/2024] [Indexed: 03/06/2025]
Abstract
The miR-290 and miR-302 clusters of microRNAs are highly expressed in naïve and primed pluripotent stem cells, respectively. Ectopic expression of the embryonic stem cell (ESC)-specific cell cycle regulating family of microRNAs arising from these two clusters dramatically enhances the reprogramming of both mouse and human somatic cells to induced pluripotency. Here, we used genetic knockouts to dissect the requirement for the miR-290 and miR-302 clusters during the reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPSCs) with retrovirally introduced Oct4, Sox2, and Klf4. Knockout of either cluster alone did not negatively impact the efficiency of reprogramming. Resulting cells appeared identical to their ESC microRNA cluster knockout counterparts. In contrast, the combined loss of both clusters blocked the formation of iPSCs. While rare double knockout clones could be isolated, they showed a dramatically reduced proliferation rate, a persistent inability to fully silence the exogenously introduced pluripotency factors, and a transcriptome distinct from individual miR-290 or miR-302 mutant ESC and iPSCs. Taken together, our data show that miR-290 and miR-302 are essential yet interchangeable in reprogramming to the induced pluripotent state.
Collapse
Affiliation(s)
- Julia Ye
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California at San Francisco, San Francisco, CA 94143, United States
- Center for Reproductive Sciences, University of California at San Francisco, San Francisco, CA 94143, United States
- Department of Urology, University of California at San Francisco, San Francisco, CA 94143, United States
| | - Ryan M Boileau
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California at San Francisco, San Francisco, CA 94143, United States
- Center for Reproductive Sciences, University of California at San Francisco, San Francisco, CA 94143, United States
- Department of Urology, University of California at San Francisco, San Francisco, CA 94143, United States
| | - Ronald J Parchem
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, United States
| | - Robert L Judson-Torres
- Huntsman Cancer Institute, The University of Utah, Salt Lake City, UT 84112, United States
- Department of Dermatology, The University of Utah, Salt Lake City, UT 84112, United States
| | - Robert Blelloch
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California at San Francisco, San Francisco, CA 94143, United States
- Center for Reproductive Sciences, University of California at San Francisco, San Francisco, CA 94143, United States
- Department of Urology, University of California at San Francisco, San Francisco, CA 94143, United States
| |
Collapse
|
|
2
|
Ye J, Boileau RM, Parchem RJ, Judson-Torres RL, Blelloch R. The miR-290 and miR-302 clusters are essential for reprogramming of fibroblasts to induced pluripotent stem cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.09.02.610895. [PMID: 39282363 PMCID: PMC11398367 DOI: 10.1101/2024.09.02.610895] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 10/25/2024]
Abstract
The miR-290 and miR-302 clusters of microRNAs are highly expressed in naïve and primed pluripotent stem cells, respectively. Ectopic expression of the embryonic stem cell-specific cell cycle regulating (ESCC) family of microRNAs arising from these two clusters dramatically enhances the reprogramming of both mouse and human somatic cells to induced pluripotency. Here, we used genetic knockouts to dissect the requirement for the miR-290 and miR-302 clusters during the reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPSCs) with retrovirally introduced Oct4, Sox2, and Klf4. Knockout of either cluster alone did not negatively impact the efficiency of reprogramming. Resulting cells appeared identical to their embryonic stem cell microRNA cluster knockout counterparts. In contrast, the combined loss of both clusters blocked the formation of iPSCs. While rare double knockout clones could be isolated, they showed a dramatically reduced proliferation rate, a persistent inability to fully silence the exogenously introduced pluripotency factors, and a transcriptome distinct from individual miR-290 or miR-302 mutant ESC and iPSCs. Taken together, our data show that miR-290 and miR-302 are essential yet interchangeable in reprogramming to the induced pluripotent state. Impact Statement The process by which somatic cell reprogramming yields induced pluripotent stem cells (iPSCs) is incompletely understood. MicroRNAs from the miR-290 and miR-302 clusters have been shown to greatly increase reprogramming efficiency, but their requirement in the process has not been studied. Here, we examine this requirement by genetically removing the miRNA clusters in somatic cells. We discover that somatic cells lacking either, but not both, of these miRNA clusters can form iPSC cells. This work thus provides new important insight into mechanisms underlying reprogramming to pluripotency.
Collapse
Affiliation(s)
- Julia Ye
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California, 94143, USA
- Center for Reproductive Sciences, University of California, San Francisco, San Francisco, California, 94143, USA
- Department of Urology, University of California, San Francisco, San Francisco, California, 94143, USA
| | - Ryan M. Boileau
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California, 94143, USA
- Center for Reproductive Sciences, University of California, San Francisco, San Francisco, California, 94143, USA
- Department of Urology, University of California, San Francisco, San Francisco, California, 94143, USA
| | - Ronald J. Parchem
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
| | - Robert L. Judson-Torres
- Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA
- Department of Dermatology, University of Utah, Salt Lake City, UT 84112, USA
| | - Robert Blelloch
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California, 94143, USA
- Center for Reproductive Sciences, University of California, San Francisco, San Francisco, California, 94143, USA
- Department of Urology, University of California, San Francisco, San Francisco, California, 94143, USA
| |
Collapse
|
|
3
|
Waldo JJ, Halmai JANM, Fink KD. Epigenetic editing for autosomal dominant neurological disorders. Front Genome Ed 2024; 6:1304110. [PMID: 38510848 PMCID: PMC10950933 DOI: 10.3389/fgeed.2024.1304110] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2023] [Accepted: 02/23/2024] [Indexed: 03/22/2024] Open
Abstract
Epigenetics refers to the molecules and mechanisms that modify gene expression states without changing the nucleotide context. These modifications are what encode the cell state during differentiation or epigenetic memory in mitosis. Epigenetic modifications can alter gene expression by changing the chromatin architecture by altering the affinity for DNA to wrap around histone octamers, forming nucleosomes. The higher affinity the DNA has for the histones, the tighter it will wrap and therefore induce a heterochromatin state, silencing gene expression. Several groups have shown the ability to harness the cell's natural epigenetic modification pathways to engineer proteins that can induce changes in epigenetics and consequently regulate gene expression. Therefore, epigenetic modification can be used to target and treat disorders through the modification of endogenous gene expression. The use of epigenetic modifications may prove an effective path towards regulating gene expression to potentially correct or cure genetic disorders.
Collapse
Affiliation(s)
| | | | - Kyle D. Fink
- Neurology Department, Stem Cell Program and Gene Therapy Center, MIND Institute, UC Davis Health System, Sacramento, CA, United States
| |
Collapse
|
|
4
|
Expression analysis of circulating miR-22, miR-122, miR-217 and miR-367 as promising biomarkers of acute lymphoblastic leukemia. Mol Biol Rep 2023; 50:255-265. [PMID: 36327023 DOI: 10.1007/s11033-022-08016-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2021] [Accepted: 10/07/2022] [Indexed: 11/06/2022]
Abstract
BACKGROUND The role of serum-based biomarkers such as microRNAs in cancer diagnosis has been extensively established. This study aimed to determine the expression levels of bioinformatically selected miRNAs and whether they can be used as biomarkers or a new therapeutic target in patients with acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS The expression levels of serum miR-22, miR-122, miR-217, and miR-367 in 21 ALL patients and 21 healthy controls were measured using quantitative real-time PCR. The receiver operating characteristic (ROC) curve and the associated area under the curve (AUC) was used to assess candidate miRNAs' diagnostic value as a biomarker. RESULTS The results showed that miR-217 was markedly decreased in patients with ALL compared to controls. Moreover, miR-22, miR-122, and miR-367 were found to be upregulated. Furthermore, ROC analysis showed that serum miR-217 and miR-367 could differentiate ALL patients from healthy individuals, while miR-22 has approximate discriminatory power that requires further investigation. CONCLUSION These results provide promising preliminary evidence that circulating miR-217 and miR-367 could be considered potent diagnostic biomarkers and therapeutic goals in this disease.
Collapse
|
|
5
|
Sugawara T, Kawamoto Y, Kawasaki T, Umezawa A, Akutsu H. A single allele of the hsa-miR-302/367 cluster maintains human pluripotent stem cells. Regen Ther 2022; 21:37-45. [PMID: 35702483 PMCID: PMC9162946 DOI: 10.1016/j.reth.2022.05.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2022] [Revised: 04/13/2022] [Accepted: 05/15/2022] [Indexed: 12/03/2022] Open
Abstract
Introduction In a diploid organism, two alleles from a single genetic locus are expressed to generate a normal phenotype. Heterozygous deleterious mutation causes a reduction of functional proteins to a half dose and insufficient amounts of functional proteins can occur to generate an in–normal phenotype, namely haploinsufficiency. Heterozygous deleterious mutation of microRNAs (miRs), non-coding RNAs that regulate the expression level of target transcripts, is still not well understood. The hsa-miR-302/367 cluster is the most abundant and specifically up-regulated miR cluster in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) and plays an important role in the maintenance of pluripotency. Methods We targeted the hsa-miR-302/367 region via a Cas9 nuclease complex with guide RNA and replaced that region with green fluorescent protein (GFP). Using a homologous donor, consisting of left and right arms and GFP, we confirmed deletion of the hsa-miR-302/367 cluster by homologous recombination without cellular destruction by microscopy. We sub-cloned GFP-positive colonies and checked the genotype of each sub-clone by genomic PCR. We then analyzed the pluripotency of heterozygous knockout cells with a hsa-miR-302/367 cluster by assessing cell proliferation ratio, morphology, and undifferentiated marker gene expression. We also used an embryoid body formation assay and transplanted wild-type and heterozygous knockout cells into immune-deficient mice. Furthermore, to analyze the lineage-specific differentiation potential of heterozygous knockout cells, we differentiated both wild-type and heterozygous knockout cells into neural stem cells. Results Here, we show that the half dose of mature miRs from the hsa-miR-302/367 cluster loci was sufficient for the continued self-renewal of hiPSCs. All GFP-positive clones were revealed to be heterozygous knockout cells, suggesting hsa-miR-302/367 cluster homozygous knockout cells were not maintained. The cell proliferation ratio, morphology, and expression of undifferentiated marker genes were comparable between wild-type and heterozygous knockout of undifferentiated human iPSCs. In addition, we found that heterozygous knockout human iPSCs have the capacity to differentiate into three germ layers, including neural stem cells. Conclusions Taken together, a single allele of the hsa-miR-302/367 cluster expresses a sufficient amount of miRs to maintain the pluripotent properties of human stem cells.
hsa-miR-302/367 cluster was deleted with CRISPR/Cas9 in human pluripotent stem cells. Homozygous hsa-miR-302/367 knockout cell was not generated.
Collapse
Affiliation(s)
| | | | | | | | - Hidenori Akutsu
- Corresponding author. Department of Reproductive Medicine, Center for Regenerative Medicine, National Center for Child Health and Development (NCCHD), Okura 2-10-1, Setagaya, Tokyo, 157-8535, Japan. Tel: +81-3-5494-7047, Fax: +81-3-5494-7048.
| |
Collapse
|
|
6
|
Bailly A, Milhavet O, Lemaitre JM. RNA-Based Strategies for Cell Reprogramming toward Pluripotency. Pharmaceutics 2022; 14:317. [PMID: 35214051 PMCID: PMC8876983 DOI: 10.3390/pharmaceutics14020317] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Revised: 01/16/2022] [Accepted: 01/25/2022] [Indexed: 02/04/2023] Open
Abstract
Cell therapy approaches to treat a wide range of pathologies have greatly benefited from cell reprogramming techniques that allow the conversion of a somatic cell into a pluripotent cell. Many technological developments have been made since the initial major discovery of this biological process. Recently reprogramming methods based on the use of RNA have emerged and seem very promising. Thus, in this review we will focus on presenting the interest of such methods for cell reprogramming but also how these RNA-based strategies can be extended to eventually lead to medical applications to improve healthspan and longevity.
Collapse
Affiliation(s)
- Anaëlle Bailly
- IRMB, University Montpellier, INSERM, 34295 Montpellier, France
- INGRAALYS, SA, IRMB, Incubator Cyborg, 34295 Montpellier, France
| | - Ollivier Milhavet
- IRMB, University Montpellier, INSERM, CNRS, 34295 Montpellier, France
- SAFE-iPSC Facility, CHU Montpellier, 34295 Montpellier, France
| | - Jean-Marc Lemaitre
- IRMB, University Montpellier, INSERM, 34295 Montpellier, France
- SAFE-iPSC Facility, CHU Montpellier, 34295 Montpellier, France
| |
Collapse
|
|
7
|
Borgohain MP, Haridhasapavalan KK, Dey C, Adhikari P, Thummer RP. An Insight into DNA-free Reprogramming Approaches to Generate Integration-free Induced Pluripotent Stem Cells for Prospective Biomedical Applications. Stem Cell Rev Rep 2020; 15:286-313. [PMID: 30417242 DOI: 10.1007/s12015-018-9861-6] [Citation(s) in RCA: 48] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
More than a decade ago, a pioneering study reported generation of induced Pluripotent Stem Cells (iPSCs) by ectopic expression of a cocktail of reprogramming factors in fibroblasts. This study has revolutionized stem cell research and has garnered immense interest from the scientific community globally. iPSCs hold tremendous potential for understanding human developmental biology, disease modeling, drug screening and discovery, and personalized cell-based therapeutic applications. The seminal study identified Oct4, Sox2, Klf4 and c-Myc as a potent combination of genes to induce reprogramming. Subsequently, various reprogramming factors were identified by numerous groups. Most of these studies have used integrating viral vectors to overexpress reprogramming factors in somatic cells to derive iPSCs. However, these techniques restrict the clinical applicability of these cells as they may alter the genome due to random viral integration resulting in insertional mutagenesis and tumorigenicity. To circumvent this issue, alternative integration-free reprogramming approaches are continuously developed that eliminate the risk of genomic modifications and improve the prospects of iPSCs from lab to clinic. These methods establish that integration of transgenes into the genome is not essential to induce pluripotency in somatic cells. This review provides a comprehensive overview of the most promising DNA-free reprogramming techniques that have the potential to derive integration-free iPSCs without genomic manipulation, such as sendai virus, recombinant proteins, microRNAs, synthetic messenger RNA and small molecules. The understanding of these approaches shall pave a way for the generation of clinical-grade iPSCs. Subsequently, these iPSCs can be differentiated into desired cell type(s) for various biomedical applications.
Collapse
Affiliation(s)
- Manash P Borgohain
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India
| | - Krishna Kumar Haridhasapavalan
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India
| | - Chandrima Dey
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India
| | - Poulomi Adhikari
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India
| | - Rajkumar P Thummer
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India.
| |
Collapse
|
|
8
|
Sugawara T, Miura T, Kawasaki T, Umezawa A, Akutsu H. The hsa-miR-302 cluster controls ectodermal differentiation of human pluripotent stem cell via repression of DAZAP2. Regen Ther 2020; 15:1-9. [PMID: 32490061 PMCID: PMC7251312 DOI: 10.1016/j.reth.2020.03.011] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2020] [Revised: 03/03/2020] [Accepted: 03/11/2020] [Indexed: 12/27/2022] Open
Abstract
Introduction Recent studies have revealed that microRNAs (miRNAs, miRs) are important for self-renewal, differentiation, and cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSC); however, their functional roles and target genes that are regulated by human PSC-specific miRs including hsa-miR-302 clusters remain largely unknown. Analysis of their target gene will give us the opportunity to understand the functional roles of such miRs. Methods We analyzed the expression profiles of miRs in 4 somatic cell lines, 8 human iPSC lines derived from 4 different cell types, 3 human ESC lines, and embryoid bodies differentiated from the human ESCs to identify human PSC-specific miRs. We also analyzed the simultaneous expression profiles of miRs and mRNAs to identify candidate targets of human PSC-specific miRs. Then, we constructed a vector for overexpressing one of the target gene to dissect the functions of human PSC-specific miR in maintenance of self-renew and differentiation. Results We focused on hsa-miR-302 cluster as a human PSC-specific miR and identified 22 candidate targets of hsa-miR-302 cluster that were moderately expressed in undifferentiated human PSCs and up-regulated in differentiated cells. Deleted in azoospermia-associated protein 2 (DAZAP2), one such target, was directly repressed by hsa-miR-302a, -302b, -302c and -302d, but not by hsa-miR-367. Overexpression of DAZAP2 caused a decrease in cell proliferation of undifferentiated human iPSCs, although morphology and undifferentiated marker gene expression was not affected. In addition, neural differentiation was suppressed in DAZAP2-overexpressing human iPSCs. Conclusion Our study revealed that hsa-miR-302 cluster controls the cell proliferation of human PSCs and the neural differentiation of human PSCs by repression of DAZAP2, thereby highlighting an additional function of human PSC-specific miRs in maintaining pluripotency.
Collapse
Affiliation(s)
| | | | | | | | - Hidenori Akutsu
- Corresponding author. Department of Reproductive Medicine, National Center for Child Health and Developmen, Okura 2-10-1, Setagaya-ku, Tokyo, 157-8535, Japan. Fax: +81-3-5494-7048.
| |
Collapse
|
|
9
|
Jin L, Zhou Y, Han L, Piao J. MicroRNA302-367-PI3K-PTEN-AKT-mTORC1 pathway promotes the development of cardiac hypertrophy through controlling autophagy. In Vitro Cell Dev Biol Anim 2019; 56:112-119. [PMID: 31845077 DOI: 10.1007/s11626-019-00417-5] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2019] [Accepted: 10/21/2019] [Indexed: 12/13/2022]
Abstract
Cardiac hypertrophy at a decompensated state eventually leads to heart failure that mostly contributes to deaths globally. Dysregulated cardiac autophagy is a hallmark of a diseased heart, and a close contact between cardiac autophagy and cardiac hypertrophy is emerging. MicroRNAs (miRNAs) have been recently reported to be prominently implicated in cardiac hypertrophy through regulating cardiac autophagy. However, the role and function of miR302-367 clusters in cardiac autophagy and cardiac hypertrophy remain largely masked. Therefore, to investigate the performance of miR302-367 in cardiac hypertrophy, the specific in vitro hypertrophic model was established in H9c2 cells upon Ang II treatment. Consequently, we discovered a distinct inhibition on autophagy and a remarkable upregulation of miR302-367 expression in hypertrophic H9c2 cells. Besides, loss- and gain-of-function assays demonstrated miR302-367 inhibited autophagy and then aggravated cardiac hypertrophy. Mechanically, PTEN was predicted and confirmed as the shared target of miR302-367. Further, we recognized the apparent inactivation of PI3K/AKT/mTORC1 signaling in the face of miR302-367 suppression in Ang II-induced hypertrophic H9c2 cells. Moreover, co-treatment of PTEN inhibitor re-activated the PI3K/AKT/mTORC1 pathway, therefore counteracting the pro-autophagic and anti-hypertrophic effects of miR302-367 depletion on cardiomyocytes. These findings unveiled the pivotal role of the miR302-367 cluster in regulating cardiac autophagy and therefore modulating cardiac hypertrophy through PTEN/PI3K/AKT/mTORC1 signaling, indicating a promising therapeutic strategy for cardiac hypertrophy and even heart failure. Graphical abstract .
Collapse
Affiliation(s)
- Lianhua Jin
- Cardiology Department of Pediatric of the First Hospital of Jilin University, No.71 Xinmin Street, Changchun City, 130021, Jilin Province, China
| | - Yan Zhou
- Cardiology Department of Pediatric of the First Hospital of Jilin University, No.71 Xinmin Street, Changchun City, 130021, Jilin Province, China
| | - Lizhi Han
- Cardiology Department of Pediatric of the First Hospital of Jilin University, No.71 Xinmin Street, Changchun City, 130021, Jilin Province, China
| | - Jinhua Piao
- Cardiology Department of Pediatric of the First Hospital of Jilin University, No.71 Xinmin Street, Changchun City, 130021, Jilin Province, China.
| |
Collapse
|
|
10
|
Zhang J, Gao X, Yang J, Fan X, Wang W, Liang Y, Fan L, Han H, Xu X, Tang F, Bao S, Liu P, Li X. Xist Intron 1 Repression by Transcriptional-Activator-Like Effectors Designer Transcriptional Factor Improves Somatic Cell Reprogramming in Mice. Stem Cells 2019; 37:599-608. [PMID: 30353613 DOI: 10.1002/stem.2928] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2018] [Revised: 07/06/2018] [Accepted: 08/18/2018] [Indexed: 11/11/2022]
Abstract
Xist is the master regulator of X chromosome inactivation. In order to further understand the Xist locus in the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) and in somatic cell nuclear transfer (SCNT), we tested transcription-activator-like effectors-based designer transcriptional factors (dTFs), which were specific to numerous regions at the Xist locus. We report that the selected dTF repressor 6 (R6) binding the intron 1 of Xist, which caused higher H3K9me3 followed by X chromosome opening and repression of X-linked genes in mouse embryonic fibroblasts, rather than affecting Xist expression, substantially improved the iPSC generation and the SCNT preimplantation embryo development. Conversely, the dTF activator targeting the same genomic region of R6 decreased iPSC formation and blocked SCNT-embryo development. These results thus uncover the critical requirement for the Xist locus in epigenetic resetting, which is not directly related to Xist transcription. This may provide a unique route to improving the reprogramming. Stem Cells 2019;37:599-608.
Collapse
Affiliation(s)
- Jindun Zhang
- Research Center for Animal Genetic Resources of Mongolian Plateau, Inner Mongolia University, Hohhot, People's Republic of China.,Wellcome Trust Sanger Institute, Cambridge, United Kingdom.,Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot, People's Republic of China
| | - Xuefei Gao
- Wellcome Trust Sanger Institute, Cambridge, United Kingdom
| | - Jian Yang
- Wellcome Trust Sanger Institute, Cambridge, United Kingdom
| | - Xiaoying Fan
- Biodynamic Optical Imaging Center (BIOPIC), Peking University, Beijing, People's Republic of China.,College of Life Sciences, Peking University, Beijing, People's Republic of China
| | - Wei Wang
- Wellcome Trust Sanger Institute, Cambridge, United Kingdom
| | - Yanfeng Liang
- Research Center for Animal Genetic Resources of Mongolian Plateau, Inner Mongolia University, Hohhot, People's Republic of China
| | - Lihong Fan
- Research Center for Animal Genetic Resources of Mongolian Plateau, Inner Mongolia University, Hohhot, People's Republic of China
| | - Hongmei Han
- Research Center for Animal Genetic Resources of Mongolian Plateau, Inner Mongolia University, Hohhot, People's Republic of China
| | - Xiaorong Xu
- Research Center for Animal Genetic Resources of Mongolian Plateau, Inner Mongolia University, Hohhot, People's Republic of China
| | - Fuchou Tang
- Biodynamic Optical Imaging Center (BIOPIC), Peking University, Beijing, People's Republic of China.,College of Life Sciences, Peking University, Beijing, People's Republic of China
| | - Siqin Bao
- Research Center for Animal Genetic Resources of Mongolian Plateau, Inner Mongolia University, Hohhot, People's Republic of China
| | - Pentao Liu
- Wellcome Trust Sanger Institute, Cambridge, United Kingdom
| | - Xihe Li
- Research Center for Animal Genetic Resources of Mongolian Plateau, Inner Mongolia University, Hohhot, People's Republic of China.,Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot, People's Republic of China
| |
Collapse
|
|
11
|
Deng P, Carter S, Fink K. Design, Construction, and Application of Transcription Activation-Like Effectors. Methods Mol Biol 2019; 1937:47-58. [PMID: 30706389 DOI: 10.1007/978-1-4939-9065-8_3] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Transcription activator-like effectors (TALEs) are modular proteins derived from the plant Xanthomonas sp. pathogen that can be designed to target unique DNA sequences following a simple cipher. Customized TALE proteins can be used in a variety of molecular applications that include gene editing and transcriptional modulation. Presently, we provide a brief primer on the design and construction of TALEs. TALE proteins can be fused to a variety of different effector domains that alter the function of the TALE upon binding. This flexibility of TALE design and downstream effect may offer therapeutic applications that are discussed in this section. Finally, we provide a future perspective on TALE technology and what challenges remain for successful translation of gene-editing strategies to the clinic.
Collapse
Affiliation(s)
- Peter Deng
- Stem Cell Program and Institute for Regenerative Cures, University of California, Davis, Sacramento, CA, USA.,Genome Center, MIND Institute, and Biochemistry and Molecular Medicine, University of California, Davis, Davis, CA, USA.,Department of Neurology, University of California Davis , Sacramento, CA, USA
| | - Sakereh Carter
- Stem Cell Program and Institute for Regenerative Cures, University of California, Davis, Sacramento, CA, USA.,Department of Neurology, University of California Davis , Sacramento, CA, USA
| | - Kyle Fink
- Stem Cell Program and Institute for Regenerative Cures, University of California, Davis, Sacramento, CA, USA. .,Department of Neurology, University of California Davis , Sacramento, CA, USA.
| |
Collapse
|
|
12
|
Salvatori DCF, Dorssers LCJ, Gillis AJM, Perretta G, van Agthoven T, Gomes Fernandes M, Stoop H, Prins JB, Oosterhuis JW, Mummery C, Looijenga LHJ. The MicroRNA-371 Family as Plasma Biomarkers for Monitoring Undifferentiated and Potentially Malignant Human Pluripotent Stem Cells in Teratoma Assays. Stem Cell Reports 2018; 11:1493-1505. [PMID: 30503260 PMCID: PMC6294243 DOI: 10.1016/j.stemcr.2018.11.002] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2018] [Revised: 11/01/2018] [Accepted: 11/01/2018] [Indexed: 01/09/2023] Open
Abstract
Predicting developmental potency and risk of posttransplantation tumor formation by human pluripotent stem cells (hPSCs) and their derivatives largely rely on classical histological analysis of teratomas. Here, we investigated whether an assay based on microRNAs (miRNA) in blood plasma is able to detect potentially malignant elements. Several hPSCs and human malignant germ cell tumor (hGCT) lines were investigated in vitro and in vivo after mouse xenografting. The multiple conventional hPSC lines generated mature teratomas, while xenografts from induced hPSCs (hiPSCs) with reactivated reprogramming transgenes and hGCT lines contained undifferentiated and potentially malignant components. The presence of these elements was reflected in the mRNA and miRNA profiles of the xenografts with OCT3/4 mRNA and the miR-371 and miR-302 families readily detectable. miR-371 family members were also identified in mouse plasma faithfully reporting undifferentiated elements in the xenografts. This study demonstrated that undifferentiated and potentially malignant cells could be detected in vivo.
Collapse
Affiliation(s)
- Daniela C F Salvatori
- Central Laboratory Animal Facility, Leiden University Medical Center, Einthovenweg 20, Leiden 2333 ZC, the Netherlands.
| | - Lambert C J Dorssers
- Department of Pathology, Laboratory for Experimental Patho-Oncology, Erasmus MC Cancer Institute, Be-432A, PO Box 2040, 3000 CA Rotterdam, the Netherlands
| | - Ad J M Gillis
- Department of Pathology, Laboratory for Experimental Patho-Oncology, Erasmus MC Cancer Institute, Be-432A, PO Box 2040, 3000 CA Rotterdam, the Netherlands
| | - Gemma Perretta
- Fondazione Guido Bernardini, Via Manfredo Camperio, 10, 20123 Milano, Italy
| | - Ton van Agthoven
- Department of Pathology, Laboratory for Experimental Patho-Oncology, Erasmus MC Cancer Institute, Be-432A, PO Box 2040, 3000 CA Rotterdam, the Netherlands
| | - Maria Gomes Fernandes
- Central Laboratory Animal Facility, Leiden University Medical Center, Einthovenweg 20, Leiden 2333 ZC, the Netherlands
| | - Hans Stoop
- Department of Pathology, Laboratory for Experimental Patho-Oncology, Erasmus MC Cancer Institute, Be-432A, PO Box 2040, 3000 CA Rotterdam, the Netherlands
| | - Jan-Bas Prins
- Central Laboratory Animal Facility, Leiden University Medical Center, Einthovenweg 20, Leiden 2333 ZC, the Netherlands
| | - J Wolter Oosterhuis
- Department of Pathology, Laboratory for Experimental Patho-Oncology, Erasmus MC Cancer Institute, Be-432A, PO Box 2040, 3000 CA Rotterdam, the Netherlands
| | - Christine Mummery
- Department of Anatomy & Embryology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
| | - Leendert H J Looijenga
- Department of Pathology, Laboratory for Experimental Patho-Oncology, Erasmus MC Cancer Institute, Be-432A, PO Box 2040, 3000 CA Rotterdam, the Netherlands.
| |
Collapse
|
|
13
|
Zhu H, Zheng L, Wang L, Tang F, Hua J. MiR-302 enhances the viability and stemness of male germline stem cells. Reprod Domest Anim 2018; 53:1580-1588. [PMID: 30070400 DOI: 10.1111/rda.13266] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2018] [Accepted: 06/06/2018] [Indexed: 12/27/2022]
Abstract
MicroRNAs were reported to be able to regulate mGSCs' self-renewal through post-transcriptional inhibition of gene expression. miR-302 worked as one important microRNA family existed mainly in human ESCs, and its role in mGSCs has not been reported yet. To elucidate the role of miR-302 in dairy goat mGSCs, the expression profile of miR-302 was explored through qPCR and FISH. Furthermore, to detect the function of miR-302, the expression vector containing miR-302 was transfected into mGSCs, and then, the cell cycle, the cell apoptosis and the genes associated with mGSCs' self-renewal and differentiation were examined. The results showed that miR-302 expressed in testis moderately and located on the basement of seminiferous tubes which shared the same location as mGSCs. Transfection of the vector containing miR-302 fragment into the immortalized mGSCs obviously enhanced the cell proliferation ability and the attachment ability, also, promoted the expression level of CD49f and OCT4. Also, miR-302 reduced the cell apoptosis and downregulated the expression of P21. miR-302 sustained mGSCs' proliferation in vitro.
Collapse
Affiliation(s)
- Haijing Zhu
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling, China.,Shaanxi Province Engineering and Technology Research Center of Cashmere Goat, Research Center of Life Science in Yulin University, Yulin, China
| | - Liming Zheng
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling, China
| | - Long Wang
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling, China
| | - Furong Tang
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling, China
| | - Jinlian Hua
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling, China
| |
Collapse
|
|
14
|
Zi C, Zeng D, Zhou J, Dai J, Jiang L, Xue F, Jiang Y, Li B. Selected microRNA-192 mutant indicates association with several function genes in bovine cells. Genes Genomics 2018; 40:361-371. [PMID: 29892841 DOI: 10.1007/s13258-017-0635-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2017] [Accepted: 11/13/2017] [Indexed: 10/18/2022]
Abstract
MicroRNAs are implicated in many cellular processes such as cell differentiation and development, tumorigenesis, and immune regulation. In this study, miR192 was detected using quantitative real-time polymerase chain reaction (qRT-PCR) when MDBK cells were exposed to Escherichia coli. Cells with malfunction of bta-miR-192 were established using transcription activator-like effector nuclease (TALEN) technology. Finally, bta-miR-192 mutant cells were screened for differentially expressed genes using RNA-sequencing (RNA-seq). The results showed that miR192 significantly decreased in cells exposed to E. coli F18ac and E. coli K88ac. The RNA-seq results showed that 1673 differentially expressed transcripts were identified; 890 genes were upregulated and 775 genes were downregulated. With the gene ontology enrichment analysis, 431 differentially expressed genes (DEGs) were classified into 937 gene ontology terms. The pathway enrichment analysis showed that 535 genes were involved in 254 pathway terms. Interestingly, most of these DEGs were associated with the pathways in cancers or infectious diseases. When the selected DEGs (n = 162) in these pathways were intersected with 120 differential transcripts, 11 DEGs were identified. Subsequently, several genes associated with regulation, cancers, or viral infections, such as LEF1, AXIN2, MX1, and FCGR2B, were identified among the DEGs using functional analysis. Furthermore, associations between bta-miR-192 and DEGs were detected by intersecting the bta-miR-192's target genes with the DEGs, indicating that three genes including CBL, DICER1 and TRERF1 were involved in this relationship. These findings provided useful guidance for investigating the role played by bta-miR-192 in cellular functionality in bovine cells.
Collapse
Affiliation(s)
- Chen Zi
- MOE Joint International Research Laboratory of Animal Health and Food safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China
- APFIC, Jiangsu Entry-Exit Inspection and Quarantine Bureau, Nanjing, 210001, China
| | - Dexin Zeng
- MOE Joint International Research Laboratory of Animal Health and Food safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China
| | - Jiyong Zhou
- MOE Joint International Research Laboratory of Animal Health and Food safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China
| | - Jianjun Dai
- MOE Joint International Research Laboratory of Animal Health and Food safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China
| | - Luyan Jiang
- APFIC, Jiangsu Entry-Exit Inspection and Quarantine Bureau, Nanjing, 210001, China
| | - Feng Xue
- MOE Joint International Research Laboratory of Animal Health and Food safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.
| | - Yuan Jiang
- Shanghai Entry-Exit Inspection and Quarantine Bureau, Shanghai, 200135, China.
| | - Baoguang Li
- Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Laurel, MD, USA
| |
Collapse
|
|
15
|
Insight into the molecular mechanism of miR-192 regulating Escherichia coli resistance in piglets. Biosci Rep 2018; 38:BSR20171160. [PMID: 29363554 PMCID: PMC5821941 DOI: 10.1042/bsr20171160] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2017] [Revised: 12/31/2017] [Accepted: 01/23/2018] [Indexed: 11/21/2022] Open
Abstract
MicroRNAs (miRNAs) have important roles in many cellular processes, including cell proliferation, growth and development, and disease control. Previous study demonstrated that the expression of two highly homologous miRNAs (miR-192 and miR-215) was up-regulated in weaned piglets with Escherichia coli F18 infection. However, the potential molecular mechanism of miR-192 in regulating E. coli infection remains unclear in pigs. In the present study, we analyzed the relationship between level of miR-192 and degree of E. coli resistance using transcription activator-like effector nuclease (TALEN), in vitro bacterial adhesion assays, and target genes research. A TALEN expression vector that specifically recognizes the pig miR-192 was constructed and then monoclonal epithelial cells defective in miR-192 were established. We found that miR-192 knockout led to enhance the adhesion ability of the E. coli strains F18ab, F18ac and K88ac, meanwhile increase the expression of target genes (DLG5 and ALCAM) by qPCR and Western blotting analysis. The results suggested that miR-192 and its key target genes (DLG5 and ALCAM) could have a key role in E. coli infection. Based on our findings, we propose that further investigation of miR-192 function is likely to lead to insights into the molecular mechanisms of E. coli infection.
Collapse
|
|
16
|
|
|
17
|
Ross K. Towards topical microRNA-directed therapy for epidermal disorders. J Control Release 2017; 269:136-147. [PMID: 29133119 DOI: 10.1016/j.jconrel.2017.11.013] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2017] [Revised: 11/08/2017] [Accepted: 11/09/2017] [Indexed: 01/09/2023]
Abstract
There remains an unmet dermatological need for innovative topical agents that achieve better longterm outcomes with fewer side effects. Modulation of the expression and activity of microRNA (miRNAs) represents an emerging translational framework for the development of such innovative therapies because changes in the expression of one miRNA can have wide-ranging effects on diverse cellular processes associated with disease. In this short review, the roles of miRNA in epidermal development, psoriasis, cutaneous squamous cell carcinoma and re-epithelisation are highlighted. Consideration is given to the delivery of oligonucleotides that mimic or inhibit miRNA function using vehicles such as cell penetrating peptides, spherical nucleic acids, deformable liposomes and liquid crystalline nanodispersions. Formulation of miRNA-directed oligonucleotides with such skin-penetrating epidermal agents will drive the development of RNA-based cutaneous therapeutics for deployment as primary or adjuvant therapies for epidermal disorders.
Collapse
Affiliation(s)
- Kehinde Ross
- School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool L3 3AF, United Kingdom.
| |
Collapse
|
|
18
|
Abstract
The dysregulation of autophagy is implicated in many pathological disorders including infections, aging, neurodegenerative diseases, and cancer. Autophagy can be precisely controlled both transcriptionally and translationally. Accumulating evidences show that the autophagy response is regulated by microRNAs, which therefore becomes subject area of interest in recent years. Herein, we give a brief introduction of the recent advancement in the regulation of microRNA on autophagy, and then we focus on the microRNA regulation of the mitophagy receptor, NIX. Finally, we present the methodology on how to study it in detail.
Collapse
|
|
19
|
Liu Z, Skamagki M, Kim K, Zhao R. Canonical MicroRNA Activity Facilitates but May Be Dispensable for Transcription Factor-Mediated Reprogramming. Stem Cell Reports 2016; 5:1119-1127. [PMID: 26651605 PMCID: PMC4682342 DOI: 10.1016/j.stemcr.2015.11.002] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2014] [Revised: 11/03/2015] [Accepted: 11/12/2015] [Indexed: 12/13/2022] Open
Abstract
MicroRNAs (miRNAs) are important regulators of reprogramming of somatic cells into induced pluripotent stem cells (iPSCs); however, it is unclear whether miRNAs are required for reprogramming and whether miRNA activity as a whole facilitates reprogramming. Here we report on successful reprogramming of mouse fibroblasts and neural stem cells (NSCs) lacking Dgcr8, a factor required for the biogenesis of canonical miRNAs, by Yamanaka factors, albeit at decreased efficiencies. Though iPSCs derived from Dgcr8-deficient mouse fibroblasts or NSCs were able to self-renew and expressed pluripotency-associated markers, they exhibited poor differentiation potential into mature somatic tissues, similar to Dgcr8−/− embryonic stem cells. The differentiation defects could be rescued with expression of DGCR8 cDNA. Our data demonstrate that while miRNA activity as a whole facilitates reprogramming, canonical miRNA may be dispensable in the derivation of iPSCs.
Reprogramming may be initiated and maintained solely by transcription factors miRNA activity as a whole facilitates reprogramming Canonical miRNAs may be dispensable for reprogramming
Collapse
Affiliation(s)
- Zhong Liu
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Maria Skamagki
- Cancer Biology and Genetics Program, Center for Cell Engineering, Center for Stem Cell Biology, Sloan-Kettering Institute, Cell and Developmental Biology Program, Weill Medical College of Cornell University, New York, NY 10065, USA
| | - Kitai Kim
- Cancer Biology and Genetics Program, Center for Cell Engineering, Center for Stem Cell Biology, Sloan-Kettering Institute, Cell and Developmental Biology Program, Weill Medical College of Cornell University, New York, NY 10065, USA.
| | - Rui Zhao
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
| |
Collapse
|
|
20
|
Parr CJC, Katayama S, Miki K, Kuang Y, Yoshida Y, Morizane A, Takahashi J, Yamanaka S, Saito H. MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells. Sci Rep 2016; 6:32532. [PMID: 27608814 PMCID: PMC5016789 DOI: 10.1038/srep32532] [Citation(s) in RCA: 66] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2016] [Accepted: 08/05/2016] [Indexed: 01/28/2023] Open
Abstract
The efficiency of pluripotent stem cell differentiation is highly variable, often resulting in heterogeneous populations that contain undifferentiated cells. Here we developed a sensitive, target-specific, and general method for removing undesired cells before transplantation. MicroRNA-302a-5p (miR-302a) is highly and specifically expressed in human pluripotent stem cells and gradually decreases to basal levels during differentiation. We synthesized a new RNA tool, miR-switch, as a live-cell reporter mRNA for miR-302a activity that can specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 0.05% of hiPSCs in a heterogeneous population and can prevent teratoma formation in an in vivo tumorigenicity assay. Automated and selective hiPSC-elimination was achieved by controlling puromycin resistance using the miR-302a switch. Our system uniquely provides sensitive detection of pluripotent stem cells and partially differentiated cells. In addition to its ability to eliminate undifferentiated cells, miR-302a switch also holds great potential in investigating the dynamics of differentiation and/or reprograming of live-cells based on intracellular information.
Collapse
Affiliation(s)
- Callum J C Parr
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Shota Katayama
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Kenji Miki
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Yi Kuang
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Yoshinori Yoshida
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Asuka Morizane
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Jun Takahashi
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Shinya Yamanaka
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.,Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA
| | - Hirohide Saito
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| |
Collapse
|
|
21
|
Xiao X, Li N, Zhang D, Yang B, Guo H, Li Y. Generation of Induced Pluripotent Stem Cells with Substitutes for Yamanaka's Four Transcription Factors. Cell Reprogram 2016; 18:281-297. [PMID: 27696909 DOI: 10.1089/cell.2016.0020] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Induced pluripotent stem cells (iPSCs) share many characteristics with embryonic stem cells, but lack ethical controversy. They provide vast opportunities for disease modeling, pathogenesis understanding, therapeutic drug development, toxicology, organ synthesis, and treatment of degenerative disease. However, this procedure also has many potential challenges, including a slow generation time, low efficiency, partially reprogrammed colonies, as well as somatic coding mutations in the genome. Pioneered by Shinya Yamanaka's team in 2006, iPSCs were first generated by introducing four transcription factors: Oct 4, Sox 2, Klf 4, and c-Myc (OSKM). Of those factors, Klf 4 and c-Myc are oncogenes, which are potentially a tumor risk. Therefore, to avoid problems such as tumorigenesis and low throughput, one of the key strategies has been to use other methods, including members of the same subgroup of transcription factors, activators or inhibitors of signaling pathways, microRNAs, epigenetic modifiers, or even differentiation-associated factors, to functionally replace the reprogramming transcription factors. In this study, we will mainly focus on the advances in the generation of iPSCs with substitutes for OSKM. The identification and combination of novel proteins or chemicals, particularly small molecules, to induce pluripotency will provide useful tools to discover the molecular mechanisms governing reprogramming and ultimately lead to the development of new iPSC-based therapeutics for future clinical applications.
Collapse
Affiliation(s)
- Xiong Xiao
- 1 College of Animal Science and Technology, Southwest University , Chongqing, China .,2 Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California , Los Angeles, California
| | - Nan Li
- 1 College of Animal Science and Technology, Southwest University , Chongqing, China
| | - Dapeng Zhang
- 1 College of Animal Science and Technology, Southwest University , Chongqing, China
| | - Bo Yang
- 1 College of Animal Science and Technology, Southwest University , Chongqing, China
| | - Hongmei Guo
- 1 College of Animal Science and Technology, Southwest University , Chongqing, China
| | - Yuemin Li
- 1 College of Animal Science and Technology, Southwest University , Chongqing, China
| |
Collapse
|
|
22
|
Liu H, Zeng F, Zhang M, Huang F, Wang J, Guo J, Liu C, Wang H. Emerging landscape of cell penetrating peptide in reprogramming and gene editing. J Control Release 2016; 226:124-137. [PMID: 26849918 DOI: 10.1016/j.jconrel.2016.02.002] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2015] [Revised: 01/31/2016] [Accepted: 02/01/2016] [Indexed: 12/11/2022]
Abstract
The plasma membrane remains a major barrier for intracellular drug delivery, to overcome this issue, a variety of approaches have been developed and used to deliver therapeutic cargos. Among these approaches, cell penetrating peptide (CPP) is promising and affords widely used vector for efficient intracellular delivery of cargos. Moreover, the latter findings including iPS reprogramming and direct transdifferentiation as well as gene editing have gradually become hot research topic; because their application in tissue engineering and disease modeling have great potential to advance innovation in precision medicine. Since the beginning, research on these approaches is mainly based on virus transduction system, while, under the consideration for obviating the risk of mutagenic insertion and enables more accurate controlling, CPP-based efficient virus-free delivery strategy has been used recently. In this review, we summarize the existing CPP-based delivery system, emerging landscape of CPP application in stem cell manipulation and reprogramming, along with CPP contributions to gene editing techniques.
Collapse
Affiliation(s)
- Huiting Liu
- Medical School, China Three Gorges University, Yichang 443002, China; Department of Nuclear Medicine, Chongqing Three Gorges Central Hospital, Wanzhou 404000, China
| | - Fanhui Zeng
- The Central Hospital of Enshi Tujia and Miao Autonomous Prefecture, Enshi 445000, China
| | - Ming Zhang
- Medical School, China Three Gorges University, Yichang 443002, China
| | - Fajun Huang
- School of Medical Science, Hubei University for Nationalities, Enshi 445000, China
| | - Jiajun Wang
- Medical School, China Three Gorges University, Yichang 443002, China; School of Medical Science, Hubei University for Nationalities, Enshi 445000, China.
| | - Jingjing Guo
- Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States
| | - Changbai Liu
- Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang 443002, China.
| | - Hu Wang
- Medical School, China Three Gorges University, Yichang 443002, China; Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States.
| |
Collapse
|
|
23
|
Lee YJ, Ramakrishna S, Chauhan H, Park WS, Hong SH, Kim KS. Dissecting microRNA-mediated regulation of stemness, reprogramming, and pluripotency. ACTA ACUST UNITED AC 2016; 5:2. [PMID: 27006752 PMCID: PMC4802578 DOI: 10.1186/s13619-016-0028-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2015] [Accepted: 02/20/2016] [Indexed: 02/06/2023]
Abstract
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).
Collapse
Affiliation(s)
- Young Jin Lee
- iDream Research Center, MizMedi Women's Hospital, Seoul, 07639 South Korea
| | - Suresh Ramakrishna
- Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763 South Korea.,College of Medicine, Hanyang University, Seoul, South Korea
| | | | - Won Sun Park
- Department of Physiology, School of Medicine, Kangwon National University, Chuncheon, 24341 South Korea
| | - Seok-Ho Hong
- Department of Internal Medicine, School of Medicine, Kangwon National University, 1 Kangwondaehak-gil, Chuncheon-si, Gangwon-do 24341 South Korea.,Stem Cell Institute, Kangwon National University, Chuncheon, 24341 South Korea
| | - Kye-Seong Kim
- Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763 South Korea.,College of Medicine, Hanyang University, Seoul, South Korea
| |
Collapse
|
|
24
|
Chang H, Yi B, Ma R, Zhang X, Zhao H, Xi Y. CRISPR/cas9, a novel genomic tool to knock down microRNA in vitro and in vivo. Sci Rep 2016; 6:22312. [PMID: 26924382 PMCID: PMC4770416 DOI: 10.1038/srep22312] [Citation(s) in RCA: 158] [Impact Index Per Article: 17.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2015] [Accepted: 02/12/2016] [Indexed: 02/06/2023] Open
Abstract
MicroRNAs are small and non-coding RNA molecules with the master role in regulation of gene expression at post-transcriptional/translational levels. Many methods have been developed for microRNA loss-of-function study, such as antisense inhibitors and sponges; however, the robustness, specificity, and stability of these traditional strategies are not highly satisfied. CRISPR/cas9 system is emerging as a novel genome editing tool in biology/medicine research, but its indication in microRNA research has not been studied exclusively. In this study, we clone CRISPR/cas9 constructs with single-guide RNAs specifically targeting biogenesis processing sites of selected microRNAs; and we find that CRISPR/cas9 can robustly and specifically reduce the expression of these microRNAs up to 96%. CRISPR/cas9 also shows an exclusive benefit in control of crossing off-target effect on microRNAs in the same family or with highly conserved sequences. More significantly, for the first time, we demonstrate the long term stability of microRNA knockdown phenotype by CRISPR/cas9 in both in vitro and in vivo models.
Collapse
Affiliation(s)
- Hong Chang
- Mitchell Cancer Institute, University of South Alabama, USA
| | - Bin Yi
- Mitchell Cancer Institute, University of South Alabama, USA
| | - Ruixia Ma
- Mitchell Cancer Institute, University of South Alabama, USA
| | - Xiaoguo Zhang
- Mitchell Cancer Institute, University of South Alabama, USA
| | - Hongyou Zhao
- Mitchell Cancer Institute, University of South Alabama, USA
| | - Yaguang Xi
- Mitchell Cancer Institute, University of South Alabama, USA
| |
Collapse
|
|
25
|
Abstract
During development, cells transition from a pluripotent to a differentiated state, generating all the different types of cells in the body. Development is generally considered an irreversible process, meaning that a differentiated cell is thought to be unable to return to the pluripotent state. However, it is now possible to reprogram mature cells to pluripotency. It is generally thought that reprogramming is accomplished by reversing the natural developmental differentiation process, suggesting that the two mechanisms are closely related. Therefore, a detailed study of cell reprogramming has the potential to shed light on unexplained developmental mechanisms and, conversely, a better understanding of developmental differentiation can help improve cell reprogramming. However, fundamental differences between reprogramming processes and multi-lineage specification during early embryonic development have also been uncovered. In addition, there are multiple routes by which differentiated cells can re-enter the pluripotent state. In this Review, we discuss the connections and disparities between differentiation and reprogramming, and assess the degree to which reprogramming can be considered as a simple reversal of development.
Collapse
Affiliation(s)
- Kazutoshi Takahashi
- Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA
| | - Shinya Yamanaka
- Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA
| |
Collapse
|
|
26
|
Tompkins JD, Jung M, Chen CY, Lin Z, Ye J, Godatha S, Lizhar E, Wu X, Hsu D, Couture LA, Riggs AD. Mapping Human Pluripotent-to-Cardiomyocyte Differentiation: Methylomes, Transcriptomes, and Exon DNA Methylation "Memories". EBioMedicine 2016; 4:74-85. [PMID: 26981572 PMCID: PMC4776252 DOI: 10.1016/j.ebiom.2016.01.021] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2015] [Revised: 01/05/2016] [Accepted: 01/15/2016] [Indexed: 11/17/2022] Open
Abstract
The directed differentiation of human cardiomyocytes (CMs) from pluripotent cells provides an invaluable model for understanding mechanisms of cell fate determination and offers considerable promise in cardiac regenerative medicine. Here, we utilize a human embryonic stem cell suspension bank, produced according to a good manufacturing practice, to generate CMs using a fully defined and small molecule-based differentiation strategy. Primitive and cardiac mesoderm purification was used to remove non-committing and multi-lineage populations and this significantly aided the identification of key transcription factors, lncRNAs, and essential signaling pathways that define cardiomyogenesis. Global methylation profiles reflect CM development and we report on CM exon DNA methylation "memories" persisting beyond transcription repression and marking the expression history of numerous developmentally regulated genes, especially transcription factors.
Collapse
Key Words
- Cardiomyocytes
- Cardiomyogenesis
- DNA methylation
- Differentiation
- Epigenetic
- Good manufacturing practice, GMP, epigenetic memory, WNT, hedgehog, transforming growth factor, ROR2, PDGFRα, demethylation, TET, TDG, HOX, TBOX
- Human embryonic stem cells
- Long non-coding RNA
- Mesoderm
- Methylome
- Pluripotent
- Transcriptome
- lncRNA
Collapse
Affiliation(s)
- Joshua D. Tompkins
- Department of Diabetes Complications and Metabolism, Duarte, CA 91010, USA
- Beckman Research Institute at City of Hope National Medical Center, Duarte, CA 91010, USA
| | - Marc Jung
- Department of Diabetes Complications and Metabolism, Duarte, CA 91010, USA
- Beckman Research Institute at City of Hope National Medical Center, Duarte, CA 91010, USA
| | - Chang-yi Chen
- Center for Biomedicine and Genetics, Duarte, CA 91010, USA
- Sylvia R. and Isador A. Deutch Center for Applied Technology Development, Duarte, CA 91010, USA
- Beckman Research Institute at City of Hope National Medical Center, Duarte, CA 91010, USA
| | - Ziguang Lin
- Center for Biomedicine and Genetics, Duarte, CA 91010, USA
- Sylvia R. and Isador A. Deutch Center for Applied Technology Development, Duarte, CA 91010, USA
- Beckman Research Institute at City of Hope National Medical Center, Duarte, CA 91010, USA
| | - Jingjing Ye
- Center for Biomedicine and Genetics, Duarte, CA 91010, USA
- Sylvia R. and Isador A. Deutch Center for Applied Technology Development, Duarte, CA 91010, USA
- Beckman Research Institute at City of Hope National Medical Center, Duarte, CA 91010, USA
| | - Swetha Godatha
- Department of Diabetes Complications and Metabolism, Duarte, CA 91010, USA
- Beckman Research Institute at City of Hope National Medical Center, Duarte, CA 91010, USA
| | - Elizabeth Lizhar
- Department of Diabetes Complications and Metabolism, Duarte, CA 91010, USA
- Beckman Research Institute at City of Hope National Medical Center, Duarte, CA 91010, USA
| | - Xiwei Wu
- Biomedical Informatics Core, Duarte, CA 91010, USA
- Beckman Research Institute at City of Hope National Medical Center, Duarte, CA 91010, USA
| | - David Hsu
- Center for Biomedicine and Genetics, Duarte, CA 91010, USA
- Sylvia R. and Isador A. Deutch Center for Applied Technology Development, Duarte, CA 91010, USA
- Beckman Research Institute at City of Hope National Medical Center, Duarte, CA 91010, USA
| | - Larry A. Couture
- Center for Biomedicine and Genetics, Duarte, CA 91010, USA
- Sylvia R. and Isador A. Deutch Center for Applied Technology Development, Duarte, CA 91010, USA
- Beckman Research Institute at City of Hope National Medical Center, Duarte, CA 91010, USA
| | - Arthur D. Riggs
- Department of Diabetes Complications and Metabolism, Duarte, CA 91010, USA
- Beckman Research Institute at City of Hope National Medical Center, Duarte, CA 91010, USA
| |
Collapse
|
|
27
|
Jusiak B, Cleto S, Perez-Piñera P, Lu TK. Engineering Synthetic Gene Circuits in Living Cells with CRISPR Technology. Trends Biotechnol 2016; 34:535-547. [PMID: 26809780 DOI: 10.1016/j.tibtech.2015.12.014] [Citation(s) in RCA: 83] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2015] [Revised: 12/15/2015] [Accepted: 12/16/2015] [Indexed: 12/26/2022]
Abstract
One of the goals of synthetic biology is to build regulatory circuits that control cell behavior, for both basic research purposes and biomedical applications. The ability to build transcriptional regulatory devices depends on the availability of programmable, sequence-specific, and effective synthetic transcription factors (TFs). The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR) system, recently harnessed for transcriptional regulation in various heterologous host cells, offers unprecedented ease in designing synthetic TFs. We review how CRISPR can be used to build synthetic gene circuits and discuss recent advances in CRISPR-mediated gene regulation that offer the potential to build increasingly complex, programmable, and efficient gene circuits in the future.
Collapse
Affiliation(s)
- Barbara Jusiak
- Research Laboratory of Electronics, Synthetic Biology Center, Department of Biological Engineering and Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Sara Cleto
- Research Laboratory of Electronics, Synthetic Biology Center, Department of Biological Engineering and Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Pablo Perez-Piñera
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Timothy K Lu
- Research Laboratory of Electronics, Synthetic Biology Center, Department of Biological Engineering and Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA.
| |
Collapse
|
|
28
|
Cellular Engineering and Disease Modeling with Gene-Editing Nucleases. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2016. [DOI: 10.1007/978-1-4939-3509-3_12] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
|
|
29
|
Abstract
The development of a facile genome engineering technology based on transcription activator-like effector nucleases (TALENs) has led to significant advances in diverse areas of science and medicine. In this review, we provide a broad overview of the development of TALENs and the use of this technology in basic science, biotechnology, and biomedical applications. This includes the discovery of DNA recognition by TALEs, engineering new TALE proteins to diverse targets, general advances in nuclease-based editing strategies, and challenges that are specific to various applications of the TALEN technology. We review examples of applying TALENs for studying gene function and regulation, generating disease models, and developing gene therapies. The current status of genome editing and future directions for other uses of these technologies are also discussed.
Collapse
Affiliation(s)
- David G Ousterout
- Department of Biomedical Engineering, Duke University, Durham, NC, 27708, USA
| | - Charles A Gersbach
- Department of Biomedical Engineering, Duke University, Room 136 Hudson Hall, Box 90281, Durham, NC, 27708-0281, USA. .,Center for Genomic and Computational Biology, Duke University, Durham, NC, 27708, USA. .,Department of Orthopaedic Surgery, Duke University Medical Center, Durham, NC, 27710, USA.
| |
Collapse
|
|
30
|
Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research. Int J Mol Sci 2015; 16:23143-64. [PMID: 26404236 PMCID: PMC4632690 DOI: 10.3390/ijms161023143] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2015] [Revised: 08/31/2015] [Accepted: 09/14/2015] [Indexed: 11/16/2022] Open
Abstract
Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing.
Collapse
|
|
31
|
Deng W, Cao X, Chen J, Zhang Z, Yu Q, Wang Y, Shao G, Zhou J, Gao X, Yu J, Xu X. MicroRNA Replacing Oncogenic Klf4 and c-Myc for Generating iPS Cells via Cationized Pleurotus eryngii Polysaccharide-based Nanotransfection. ACS APPLIED MATERIALS & INTERFACES 2015; 7:18957-18966. [PMID: 26269400 DOI: 10.1021/acsami.5b06768] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/04/2023]
Abstract
Induced pluripotent stem cells (iPSCs), resulting from the forced expression of cocktails out of transcription factors, such as Oct4, Sox2, Klf4, and c-Myc (OSKM), has shown tremendous potential in regenerative medicine. Although rapid progress has been made recently in the generation of iPSCs, the safety and efficiency remain key issues for further application. In this work, microRNA 302-367 was employed to substitute the oncogenic Klf4 and c-Myc in the OSKM combination as a safer strategy for successful iPSCs generation. The negatively charged plasmid mixture (encoding Oct4, Sox2, miR302-367) and the positively charged cationized Pleurotus eryngii polysaccharide (CPEPS) self-assembled into nanosized particles, named as CPEPS-OS-miR nanoparticles, which were applied to human umbilical cord mesenchymal stem cells for iPSCs generation after characterization of the physicochemical properties. The CPEPS-OS-miR nanoparticles possessed spherical shape, ultrasmall particle size, and positive surface charge. Importantly, the combination of plasmids Oct4, Sox2, and miR302-367 could not only minimize genetic modification but also show a more than 50 times higher reprogramming efficiency (0.044%) than any other single or possible double combinations of these factors (Oct4, Sox2, miR302-367). Altogether, the current study offers a simple, safe, and effective self-assembly approach for generating clinically applicable iPSCs.
Collapse
Affiliation(s)
- Wenwen Deng
- Department of Pharmaceutics, School of Pharmacy, and Center for Drug/Gene Delivery and Tissue Engineering, Jiangsu University , Zhenjiang 212001, People's Republic of China
| | - Xia Cao
- Department of Pharmaceutics, School of Pharmacy, and Center for Drug/Gene Delivery and Tissue Engineering, Jiangsu University , Zhenjiang 212001, People's Republic of China
| | - Jingjing Chen
- Department of Pharmaceutics, School of Pharmacy, and Center for Drug/Gene Delivery and Tissue Engineering, Jiangsu University , Zhenjiang 212001, People's Republic of China
| | - Zhijian Zhang
- Center for Drug/Gene Delivery and Tissue Engineering, and School of Medical Science and Laboratory Medicine, Jiangsu University , Zhenjiang 212001, People's Republic of China
| | - Qingtong Yu
- School of Life Science & Technology, China Pharmaceutical University , Nanjing 210009, People's Republic of China
| | - Yan Wang
- Department of Pharmaceutics, School of Pharmacy, and Center for Drug/Gene Delivery and Tissue Engineering, Jiangsu University , Zhenjiang 212001, People's Republic of China
| | - Genbao Shao
- Center for Drug/Gene Delivery and Tissue Engineering, and School of Medical Science and Laboratory Medicine, Jiangsu University , Zhenjiang 212001, People's Republic of China
| | - Jie Zhou
- Department of Pharmaceutics, School of Pharmacy, and Center for Drug/Gene Delivery and Tissue Engineering, Jiangsu University , Zhenjiang 212001, People's Republic of China
| | - Xiangdong Gao
- School of Life Science & Technology, China Pharmaceutical University , Nanjing 210009, People's Republic of China
| | - Jiangnan Yu
- Department of Pharmaceutics, School of Pharmacy, and Center for Drug/Gene Delivery and Tissue Engineering, Jiangsu University , Zhenjiang 212001, People's Republic of China
| | - Ximing Xu
- Department of Pharmaceutics, School of Pharmacy, and Center for Drug/Gene Delivery and Tissue Engineering, Jiangsu University , Zhenjiang 212001, People's Republic of China
| |
Collapse
|
|
32
|
Chen L, Heikkinen L, Emily Knott K, Liang Y, Wong G. Evolutionary conservation and function of the human embryonic stem cell specific miR-302/367 cluster. COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY D-GENOMICS & PROTEOMICS 2015; 16:83-98. [PMID: 26363379 DOI: 10.1016/j.cbd.2015.08.002] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/15/2015] [Revised: 08/14/2015] [Accepted: 08/19/2015] [Indexed: 01/06/2023]
Abstract
miRNA clusters define a group of related miRNAs closely localized in the genome with an evolution that remains poorly understood. The miR-302/367 cluster represents a single polycistronic transcript that produces five precursor miRNAs. The cluster is highly expressed and essential for maintenance of human embryonic stem cells. We found the cluster to be highly conserved and present in most mammals. In primates, seed sequence and miRNA structure are conserved, but inter-precursor sequences are evolving. Insertions of new miRNAs, deletions of individual miRNAs, and a cluster duplication observed in different species suggest an actively evolving cluster. Core transcriptional machinery consisting of NANOG and OCT-4 transcription factors that define stem cells are present upstream of the miR-302/367 cluster. Interestingly, we found the miR-302/367 cluster flanking region to be enriched as a target site of other miRNAs suggesting a mechanism for feedback control. Analysis of miR-302 and miR-367 targets demonstrated concordance of gene set enrichment groups at high gene ontology levels. This cluster also expresses isomiRs providing another means of establishing sequence diversity. Finally, using three different kidney tumor datasets, we observed consistent expression of miR-302 family members in normal tissue while adjacent tumor tissue showed a significant lack of expression. Clustering expression levels of miR-302 validated target genes showed a significant correlation between miR-302/367 cluster miRNAs and a subset of validated gene targets in healthy and adjacent tumor tissues. Taken together, our data show a highly conserved and still evolving miRNA cluster that may have additional unrecognized functions.
Collapse
Affiliation(s)
- Liang Chen
- Key Laboratory of Symbolic Computation and Knowledge Engineering of the Ministry of Education, College of Computer Science and Technology, Jilin University, Changchun 130012, China; A.I. Virtanen Institute, Faculty of Health Sciences, University of Eastern Finland, PL 1627, Kuopio 70211, Finland
| | - Liisa Heikkinen
- University of Jyväskylä, Department of Biological & Environmental Science, P.O. Box 35, FI-40014, University of Jyväskylä, Finland
| | - K Emily Knott
- University of Jyväskylä, Department of Biological & Environmental Science, P.O. Box 35, FI-40014, University of Jyväskylä, Finland
| | - Yanchun Liang
- Key Laboratory of Symbolic Computation and Knowledge Engineering of the Ministry of Education, College of Computer Science and Technology, Jilin University, Changchun 130012, China
| | - Garry Wong
- A.I. Virtanen Institute, Faculty of Health Sciences, University of Eastern Finland, PL 1627, Kuopio 70211, Finland; Faculty of Health Sciences, University of Macau, Taipa, Macau S.A.R., China.
| |
Collapse
|
|
33
|
Drozd AM, Walczak MP, Piaskowski S, Stoczynska-Fidelus E, Rieske P, Grzela DP. Generation of human iPSCs from cells of fibroblastic and epithelial origin by means of the oriP/EBNA-1 episomal reprogramming system. Stem Cell Res Ther 2015; 6:122. [PMID: 26088261 PMCID: PMC4515927 DOI: 10.1186/s13287-015-0112-3] [Citation(s) in RCA: 54] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2015] [Revised: 05/25/2015] [Accepted: 06/10/2015] [Indexed: 11/10/2022] Open
Abstract
INTRODUCTION The prospect of therapeutic applications of the induced pluripotent stem cells (iPSCs) is based on their ability to generate virtually any cell type present in human body. Generation of iPSCs from somatic cells has opened up new possibilities to investigate stem cell biology, to better understand pathophysiology of human diseases, and to design new therapy approaches in the field of regenerative medicine. In this study, we focus on the ability of the episomal system, a non-viral and integration-free reprogramming method to derive iPSCs from somatic cells of various origin. METHODS Cells originating from neonatal and adult tissue, renal epithelium, and amniotic fluid were reprogrammed by using origin of replication/Epstein-Barr virus nuclear antigen-1 (oriP/EBNA-1)-based episomal vectors carrying defined factors. The iPSC colony formation was evaluated by using immunocytochemistry and alkaline phosphatase assay and by investigating gene expression profiles. The trilineage formation potential of generated pluripotent cells was assessed by embryoid body-mediated differentiation. The impact of additionally introduced factors on episome-based reprogramming was also investigated. RESULTS Reprogramming efficiencies were significantly higher for the epithelial cells compared with fibroblasts. The presence of additional factor miR 302/367 in episomal system enhanced reprogramming efficiencies in fibroblasts and epithelial cells, whereas the downregulation of Mbd3 expression increased iPSC colony-forming efficiency in fibroblasts solely. CONCLUSIONS In this study, we performed a side-by-side comparison of iPSC colony-forming efficiencies in fibroblasts and epithelial cells transiently transfected with episomal plasmids and demonstrated that iPSC generation efficiency was highest when donor samples were derived from epithelial cells. We determined that reprogramming efficiency of episomal system could be further improved. Considering results obtained in the course of this study, we believe that episomal reprogramming provides a simple, reproducible, and efficient tool for generating clinically relevant pluripotent cells.
Collapse
Affiliation(s)
- Anna M Drozd
- Department of Research and Development, Celther Polska Ltd., Milionowa 23, 93-193, Łódź, Poland.
| | - Maciej P Walczak
- Department of Research and Development, Celther Polska Ltd., Milionowa 23, 93-193, Łódź, Poland.
| | - Sylwester Piaskowski
- Department of Research and Development, Celther Polska Ltd., Milionowa 23, 93-193, Łódź, Poland. .,Department of Tumor Biology, Medical University of Łódź, Żeligowskiego 7/9, 90-752, Łódź, Poland.
| | - Ewelina Stoczynska-Fidelus
- Department of Research and Development, Celther Polska Ltd., Milionowa 23, 93-193, Łódź, Poland. .,Department of Tumor Biology, Medical University of Łódź, Żeligowskiego 7/9, 90-752, Łódź, Poland.
| | - Piotr Rieske
- Department of Research and Development, Celther Polska Ltd., Milionowa 23, 93-193, Łódź, Poland. .,Department of Tumor Biology, Medical University of Łódź, Żeligowskiego 7/9, 90-752, Łódź, Poland.
| | - Dawid P Grzela
- Department of Research and Development, Celther Polska Ltd., Milionowa 23, 93-193, Łódź, Poland.
| |
Collapse
|
|
34
|
Kumar D, Talluri TR, Anand T, Kues WA. Induced pluripotent stem cells: Mechanisms, achievements and perspectives in farm animals. World J Stem Cells 2015; 7:315-328. [PMID: 25815117 PMCID: PMC4369489 DOI: 10.4252/wjsc.v7.i2.315] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/15/2014] [Revised: 08/19/2014] [Accepted: 12/17/2014] [Indexed: 02/06/2023] Open
Abstract
Pluripotent stem cells are unspecialized cells with unlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem (iPS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived iPS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming mRNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of iPS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of iPS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals.
Collapse
|
|
35
|
Zhu P, Liu Q, Liu S, Su X, Feng W, Lei X, Liu J, Cui K, Huang B, Shi D. Generation of Foxo3-targeted Mice by Injection of mRNAs Encoding Transcription Activator-like Effector Nucleases (TALENs) into Zygotes. Reprod Domest Anim 2015; 50:474-83. [PMID: 25800339 DOI: 10.1111/rda.12515] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2014] [Accepted: 02/23/2015] [Indexed: 12/28/2022]
Abstract
In this study, for exploring the mechanism of forkhead box O3(Foxo3) participating in regulation of the activation of primordial oocytes, Foxo3-targeted mice were generated by injection of mRNAs encoding transcription activator-like effector nucleases (TALENs) into mouse zygotes. The TALEN sites were designed with high conservative homologous region among pig, bovine, buffalo and mouse by commercial bio-companies. The TALENs mutagenic non-homologous end-joining (NHEJ) repair activity were determined to be 31.3% in human embryonic kidney 293T (HEK-293T) cells by dual luciferase reporter assay system. Then, we firstly injected TALEN-mRNAs into the cytoplasm of mouse zygotes by micromanipulation, and four of 48 mouse blastocysts were identified as mutation by sequencing. Subsequently, by the method of TALEN-mRNAs injected into the zygotes with pronucleus micromanipulation technique, we obtained seven Foxo3 mutants of 20 FVB/NJ backgrounds mice which were Foxo3-independent alleles with frameshift and deletion mutations. It was very interesting that all seven were heterozygous mutants (Foxo3(-/+) ), and the gene mutagenesis rates of the mice reached 35%. The five Foxo3 mutant females were all infertile in the following 6 months after birth. The histological examination results showed that there were rare primordial follicles and primary follicles in the ovary of Foxo3 mutant compared to that of wide-type female mice. Moreover, one of two mutant males was subfertile and another was fertile normally. Those results suggested that the mutant of Foxo3 severely affected the fertile ability of female and perhaps male in some degree; furthermore, an even more efficient TALENs-based gene mutation method has been established to be poised to revolutionize the study of mouse and other species genetics.
Collapse
Affiliation(s)
- P Zhu
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, Guangxi, China
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
36
|
MicroRNA-302/367 cluster governs hESC self-renewal by dually regulating cell cycle and apoptosis pathways. Stem Cell Reports 2015; 4:645-57. [PMID: 25801506 PMCID: PMC4400607 DOI: 10.1016/j.stemcr.2015.02.009] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2014] [Revised: 02/11/2015] [Accepted: 02/12/2015] [Indexed: 01/31/2023] Open
Abstract
miR-302/367 is the most abundant miRNA cluster in human embryonic stem cells (hESCs) and can promote somatic cell reprogramming. However, its role in hESCs remains poorly understood. Here, we studied functional roles of the endogenous miR-302/367 cluster in hESCs by employing specific TALE-based transcriptional repressors. We revealed that miR-302/367 cluster dually regulates hESC cell cycle and apoptosis in dose-dependent manner. Gene profiling and functional studies identified key targets of the miR-302/367 cluster in regulating hESC self-renewal and apoptosis. We demonstrate that in addition to its role in cell cycle regulation, miR-302/367 cluster conquers apoptosis by downregulating BNIP3L/Nix (a BH3-only proapoptotic factor) and upregulating BCL-xL expression. Furthermore, we show that butyrate, a natural compound, upregulates miR-302/367 cluster expression and alleviates hESCs from apoptosis induced by knockdown of miR-302/367 cluster. In summary, our findings provide new insights in molecular mechanisms of how miR-302/367 cluster regulates hESCs.
Knockdown of the endogenous miR-302/367 cluster attenuates hESC self-renewal Endogenous miR-302/367 cluster dually regulates cell cycle and apoptosis in hESCs miR-302/367 cluster regulates hESC self-renewal by inhibiting apoptosis pathway Butyrate suppresses BNIP3L/Nix expression via miR-302/367 cluster
Collapse
|
|
37
|
Watanabe-Tanaka Y, Asahara H. The role of microRNAs in the pathogenesis of rheumatoid arthritis. Inflamm Regen 2015. [DOI: 10.2492/inflammregen.35.148] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
Affiliation(s)
- Yoko Watanabe-Tanaka
- CREST, Japan Science and Technology Agency, Tokyo, Japan
- Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| | - Hiroshi Asahara
- Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, U S A
- Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo, Japan
- CREST, Japan Science and Technology Agency, Tokyo, Japan
- Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| |
Collapse
|
|
38
|
Deng XY, Wang H, Wang T, Fang XT, Zou LL, Li ZY, Liu CB. Non-viral methods for generating integration-free, induced pluripotent stem cells. Curr Stem Cell Res Ther 2015; 10:153-158. [PMID: 25248676 PMCID: PMC4460285 DOI: 10.2174/1574888x09666140923101914] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2014] [Revised: 09/05/2014] [Accepted: 09/16/2014] [Indexed: 12/11/2022]
Abstract
Induced pluripotent stem (iPS) cells were created from mouse fibroblasts by induced expression of Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. This technique has quickly resulted in an exponential increase in the amount of pluripotency studies, and has provided a valuable tool in regenerative medicine. At the same time, many methodologies to generate iPS cells have been reported, and are comprised mainly of viral methods and non-viral methods. Although viral methods may not be applicable for clinical applications, various nonviral methods have been reported in recent years, including DNA vector-based approaches, transfection of mRNA, transduction of reprogramming proteins, and use of small molecule compounds. This review summarizes and evaluates these non-viral methods.
Collapse
Affiliation(s)
- Xiao-Yue Deng
- Institute of Molecular Biology, China Three Gorges University, Yichang, China
- Medical School, China Three Gorges University, Yichang, China
- Second Clinical Medical School, China Three Gorges University, Yichang, China
| | - Hu Wang
- Medical School, China Three Gorges University, Yichang, China
| | - Tao Wang
- Third Clinical Medical School, China Three Gorges University, Yichang, China
| | - Xian-Tao Fang
- Institute of Molecular Biology, China Three Gorges University, Yichang, China
- Medical School, China Three Gorges University, Yichang, China
- Second Clinical Medical School, China Three Gorges University, Yichang, China
| | - Li-Li Zou
- Medical School, China Three Gorges University, Yichang, China
| | - Zhi-Ying Li
- Second Clinical Medical School, China Three Gorges University, Yichang, China
| | - Chang-Bai Liu
- Institute of Molecular Biology, China Three Gorges University, Yichang, China
- Medical School, China Three Gorges University, Yichang, China
| |
Collapse
|
|
39
|
A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets. Sci Rep 2014; 4:7338. [PMID: 25475013 PMCID: PMC4256643 DOI: 10.1038/srep07338] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2014] [Accepted: 11/14/2014] [Indexed: 02/06/2023] Open
Abstract
Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways.
Collapse
|
|
40
|
Controlling gene networks and cell fate with precision-targeted DNA-binding proteins and small-molecule-based genome readers. Biochem J 2014; 462:397-413. [PMID: 25145439 DOI: 10.1042/bj20140400] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Transcription factors control the fate of a cell by regulating the expression of genes and regulatory networks. Recent successes in inducing pluripotency in terminally differentiated cells as well as directing differentiation with natural transcription factors has lent credence to the efforts that aim to direct cell fate with rationally designed transcription factors. Because DNA-binding factors are modular in design, they can be engineered to target specific genomic sequences and perform pre-programmed regulatory functions upon binding. Such precision-tailored factors can serve as molecular tools to reprogramme or differentiate cells in a targeted manner. Using different types of engineered DNA binders, both regulatory transcriptional controls of gene networks, as well as permanent alteration of genomic content, can be implemented to study cell fate decisions. In the present review, we describe the current state of the art in artificial transcription factor design and the exciting prospect of employing artificial DNA-binding factors to manipulate the transcriptional networks as well as epigenetic landscapes that govern cell fate.
Collapse
|
|
41
|
Cellular reprogramming by transcription factor engineering. Curr Opin Genet Dev 2014; 28:1-9. [DOI: 10.1016/j.gde.2014.07.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2014] [Accepted: 07/03/2014] [Indexed: 12/20/2022]
|
|
42
|
Gao X, Tsang JCH, Gaba F, Wu D, Lu L, Liu P. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res 2014; 42:e155. [PMID: 25223790 PMCID: PMC4227760 DOI: 10.1093/nar/gku836] [Citation(s) in RCA: 146] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
The transcription activator–like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (Cas9) utlilize distinct molecular mechanisms in targeting site recognition. The two proteins can be modified to carry additional functional domains to regulate expression of genomic loci in mammalian cells. In this study, we have compared the two systems in activation and suppression of the Oct4 and Nanog loci by targeting their enhancers. Although both are able to efficiently activate the luciferase reporters, the CRISPR/dCas9 system is much less potent in activating the endogenous loci and in the application of reprogramming somatic cells to iPS cells. Nevertheless, repression by CRISPR/dCas9 is comparable to or even better than TALE repressors. We demonstrated that dCas9 protein binding results in significant physical interference to binding of native transcription factors at enhancer, less efficient active histone markers induction or recruitment of activating complexes in gene activation. This study thus highlighted the merits and drawbacks of transcription regulation by each system. A combined approach of TALEs and CRISPR/dCas9 should provide an optimized solution to regulate genomic loci and to study genetic elements such as enhancers in biological processes including somatic cell reprogramming and guided differentiation.
Collapse
Affiliation(s)
- Xuefei Gao
- Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1HH, UK
| | - Jason C H Tsang
- Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1HH, UK
| | - Fortis Gaba
- Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1HH, UK
| | - Donghai Wu
- Key laboratory of Regenerative Biology, GIBH, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Liming Lu
- Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1HH, UK Shanghai Institute of Immunology, Shanghai Jiaotong University School of Medicine Shanghai 200025. China
| | - Pentao Liu
- Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1HH, UK Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Universityof Cambridge, Tennis Court Rd, Cambridge CB2 1QR, UK
| |
Collapse
|
|
43
|
Targeted repression of AXIN2 and MYC gene expression using designer TALEs. Biochem Biophys Res Commun 2014; 446:1120-5. [PMID: 24667606 DOI: 10.1016/j.bbrc.2014.03.077] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2014] [Accepted: 03/17/2014] [Indexed: 11/23/2022]
Abstract
Designer TALEs (dTALEs) are chimeric transcription factors that can be engineered to regulate gene expression in mammalian cells. Whether dTALEs can block gene transcription downstream of signal transduction cascades, however, has yet to be fully explored. Here we tested whether dTALEs can be used to target genes whose expression is controlled by Wnt/β-catenin signaling. TALE DNA binding domains were engineered to recognize sequences adjacent to Wnt responsive enhancer elements (WREs) that control expression of axis inhibition protein 2 (AXIN2) and c-MYC (MYC). These custom DNA binding domains were linked to the mSin3A interaction domain (SID) to generate TALE-SID chimeric repressors. The TALE-SIDs repressed luciferase reporter activity, bound their genomic target sites, and repressed AXIN2 and MYC expression in HEK293 cells. We generated a novel HEK293 cell line to determine whether the TALE-SIDs could function downstream of oncogenic Wnt/β-catenin signaling. Treating these cells with doxycycline and tamoxifen stimulates nuclear accumulation of a stabilized form of β-catenin found in a subset of colorectal cancers. The TALE-SIDs repressed AXIN2 and MYC expression in these cells, which suggests that dTALEs could offer an effective therapeutic strategy for the treatment of colorectal cancer.
Collapse
|
|
44
|
Zhao W, Ning B, Qian C. Regulatory factors of induced pluripotency: current status. Stem Cell Investig 2014; 1:15. [PMID: 27358861 DOI: 10.3978/j.issn.2306-9759.2014.07.01] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2014] [Accepted: 06/08/2014] [Indexed: 11/14/2022]
Abstract
Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) through enforced expression of four transcription factors [Oct4, Sox2, Klf4, and c-Myc (OSKM)]; however, the reprogramming efficiency is extremely low. This finding raises fundamental questions about the regulators that influence the change in epigenetic stability and endowment of dedifferentiation potential during reprogramming. Identification of such regulators is critical to removing the roadblocks impeding the efficient generation of safe iPSCs and their successful translation into clinical therapies. In this review, we summarize the current progress that has been made in understanding cellular reprogramming, with an emphasis on the molecular mechanisms of epigenetic regulators in induced pluripotency.
Collapse
Affiliation(s)
- Wei Zhao
- Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX 77030, USA
| | - Bo Ning
- Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX 77030, USA
| | - Chen Qian
- Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX 77030, USA
| |
Collapse
|