File Name: nuclear reprogramming and stem cells file.zip
- Paying the Toll in Nuclear Reprogramming
- Reprogramming somatic cells into stem cells
- Nuclear reprogramming: A key to stem cell function in regenerative medicine
Evolving challenges in promoting stem cell based cardiovascular repair and regeneration View all 6 Articles.
Paying the Toll in Nuclear Reprogramming
Metrics details. Nuclear reprogramming reinstates totipotency or pluripotency in somatic cells by changing their gene transcription profile.
This technology is widely used in medicine, animal husbandry and other industries. However, certain deficiencies severely restrict the applications of this technology. Using single-embryo RNA-seq, our study provides complete transcriptome blueprints of embryos generated by cumulus cell CC donor nuclear transfer NT , embryos generated by mouse embryonic fibroblast MEF donor NT and in vivo embryos at each stage zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst.
According to the results from further analyses, NT embryos exhibit RNA processing and translation initiation defects during the zygotic genome activation ZGA period, and protein kinase activity and protein phosphorylation are defective during blastocyst formation.
Among these constant genes, genes are continuously mis-transcribed throughout all developmental stages. These differential genes may be reprogramming barrier genes RBGs and more studies are needed to identify. These embryonic transcriptome blueprints provide new data for further mechanistic studies of somatic nuclear reprogramming. These findings may improve the efficiency of somatic cell nuclear transfer. Utilizing somatic cell nuclear transfer SCNT technology, somatic cells have been reprogrammed not only to totipotency but also to form live cloned offspring [ 1 ].
Furthermore, nuclear transfer NT blastocysts have been used to produce NT embryonic stem cells [ 5 ], regardless of whether the NT blastocysts were obtained from a healthy control [ 6 ] or a patient [ 7 ].
Therefore, the SCNT technique also has wide prospective applications in human therapeutic cloning [ 8 , 9 ]. Although the NT technique displays great potential in various applications, the developmental ability of NT embryos remains very poor [ 3 ]. The poor developmental ability is attributed to transcriptional defects and reprogramming barriers. A normal fertilized embryo undergoes a series of transcriptional regulatory steps after fertilization.
The well-known transcriptional period ZGA is mainly regulated by maternal factors [ 10 , 11 ] and epigenetic patterns [ 12 , 13 ]. NT embryos appear to undergo abnormal gene regulation during this period.
Certain somatic genes are constitutively expressed in 2-cell NT embryos [ 14 ]. Maternal factors are not degraded properly in NT embryos [ 15 ]. NT embryos display an abnormally high DNA methylation level in more than 20 genes [ 16 ]. Even the Oct4 regulatory region appears to exhibit an abnormally high methylation level [ 17 , 18 ]. The level of methylation on H3K9 [ 13 , 19 ] and H3K27 [ 20 ] is noticeably higher.
This increased methylation also leads to the abnormal positioning of the PRC2 complex [ 20 ] and the abnormal expression of the polycomb-associated gene in NT embryos [ 21 ]. These epigenetic abnormalities are often regarded as reprogramming barriers.
The abnormal epigenetic patterns further cause abnormal gene transcription in NT embryos [ 22 , 23 ]. In conclusion, the abnormal transcriptional regulation in NT embryos may be responsible for their poor developmental ability. Transcriptomic analyses are appropriate methods for studying transcriptional regulation.
However, transcriptomic analysis of NT embryos is challenging to perform using the traditional transcriptomic analysis method, as a sufficient number of NT embryos are not typically accumulated for use in this analysis. Since the invention of single-embryo transcriptome analysis technology RNA-seq [ 24 ], many transcriptomic analyses of embryos have been successfully performed [ 25 , 26 , 27 , 28 ]. Several RNA-seq studies have focused on specific stages of NT embryos [ 13 , 29 ], but no transcriptome blueprint describing the entire developmental period of NT embryos has been published.
Then, the differences in gene transcripts among these embryos and donor cells were extensively explored. This study provides a foundation for studies of the mechanism of nuclear reprogramming and promotes the application of NT technology in livestock production, therapeutic cloning and regenerative medicine.
All chemicals used in this study were purchased from Sigma St. COCs were digested in the M2 drop containing 0. CCs were removed from the oocytes by gentle pipetting. MEFs were obtained from MEFs were used at passages 2—5 in this experiment. Synchronized single-embryo MEFs were obtained by treating the cells with 0. SCNT was performed using the micro-manipulator system. As shown in Fig. Female C57 mice were mated with male DBA mice, and These MEFs with the BDF1 genetic background were sub-cultured, and cells at the 3rd-5th passages were used as donor cells.
Six stages of reconstituted NTM embryos were collected after different culture periods. The derivation of embryos and cells with the same genetic background. Twenty different samples shown in the two dashed boxes were used for RNA-seq analyses. All samples were on BDF1 genetic background. The study included 18 groups to represent the six stages of development in each of the three types of embryos.
Three biological replicates from each group were prepared. Then, we used 54 embryos for the single-embryo RNA-seq. These 54 embryos were chosen from embryos based on stringent morphological criteria.
Reverse transcription was performed directly on the cytoplasmic lysate. Beijing, China. A Perl script was used to filter the original data raw data and guarantee the quality of the data for the analysis. The filtering step using the Perl script included the removal of adapters, redundant reads and low-quality reads. After the filtering process, the clean data were assessed for data quality, including Q30 statistics, data quantity statistics, and base content statistics.
The reference gene and genome annotation files were downloaded from UCSC and used to build the reference genome library using Bowtie2 v2. Then, the clean data were mapped to the reference genome using TopHat v2. In addition, Bowtie2 software was used to map and compare the data obtained from TopHat to enhance the accuracy of the mapping results.
IGV Integrative Genomics Viewer was used to view the mapped results in a heatmap, histogram, scatter plot and other formats. The significance of the differentially expressed genes was calculated using DESeq software by performing pair-wise comparisons among the different types of embryos. The P -value adjusted for multiple tests and absolute value of log2 fold change were obtained to determine whether the genes were significantly differentially expressed.
DAVID bioinformatics resource version 6. The significance of the differential GO terms was calculated using above mentioned software by performing pair-wise comparisons, and three independent examples were used in each groups. Venn diagrams were generated using Venny2. Then, the batch-corrected data were subjected to a PCA clustering analysis.
Embryos and cells were collected using the methods shown in Fig. Therefore, the genetic background of all three types of embryos was BDF1. Sixty samples were analyzed. Therefore, the mouse preimplantation embryo expresses more than half of the known genes in the mouse.
A principal component analysis PCA was performed to examine the obtained data Fig. The two types of donor cells were clustered together and segregated from the other samples, conforming to the biological laws of cell differentiation. The mouse ZGA occurred at the 2-cell stage.
The transcription patterns were obviously different between the 1—2 cell stage and subsequent stages. Our PCA findings are consistent with these data. The three types of 1-cell embryos and 2-cell embryos clustered together, but the three types of 4-cell, 8-cell, morula and blastula embryos were clustered in another region.
Single-cell transcriptional profiles of in vivo embryos, NT embryos and donor cells. Smaller symbols represent each sample. Bigger symbols represent the average of samples from each group. Blue indicates lower expression and red indicates higher expression. The hierarchical clustering analysis of the samples is shown on top of the heatmap. The hierarchical clustering analysis of the genes is shown on the left.
The gene transcription patterns were significantly changed during embryonic development Fig. The major ZGA stage occurs in the late 2-cell embryos in mouse, which corresponds to our 2—4 cell stage embryos. The largest transcriptional difference appeared in this stage, as the transcription of approximately genes exhibited changes. Significantly fewer up-regulated genes were observed in both NT embryos than in the in vivo embryos, indicating that many genes failed to be activated in both NT groups during the major ZGA stage.
The lack of these transcripts may be a reason for the poor development of NT embryos. During the morula-blastula period, more than 3, transcripts were altered in the in vivo group, but only approximately differences were observed in both NT groups. The first cell differentiation step occurred during this period. These erroneous transcription steps may affect the differentiation of trophoblastic cells, which may further cause the placental abnormality after NT embryo implantation.
Many genes are differentially expressed between NT embryos and in vivo embryos Fig. The greatest difference was observed between the blastula stage and 4-cell stage. In the blastula stage, more than 3, genes were differentially expressed between NT groups and the in vivo group. In the 4-cell stage, approximately genes were differentially expressed between the NT groups and the in vivo group. This stage is the initial step in cellular reprogramming. The functions of genes were analyzed using the GO method.
Thus, the transcriptional differences between 1-cell NTC and NTM embryos were more likely due to the use of different donor cells. Based on these findings, the cellular reprogramming was not complete and substantial differences were observed between two NT groups. Invivo were shown in Additional file 4 : Figure S2B.
Reprogramming somatic cells into stem cells
Oncotarget a primarily oncology-focused, peer-reviewed, open access, biweekly journal aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. Its scope is unique. The term "oncotarget" encompasses all molecules, pathways, cellular functions, cell types, and even tissues that can be viewed as targets relevant to cancer as well as other diseases. The term was introduced in the inaugural Editorial , Introducing OncoTarget. Sponsored Conferences. Impact Journals is a member of the Society for Scholarly Publishing. Keywords: somatic cell nuclear transfer SCNT , gene co-expression analysis, enrichment of GO category, pathway aberrant activation, reprogramming barriers.
Metrics details. Nuclear reprogramming reinstates totipotency or pluripotency in somatic cells by changing their gene transcription profile. This technology is widely used in medicine, animal husbandry and other industries. However, certain deficiencies severely restrict the applications of this technology. Using single-embryo RNA-seq, our study provides complete transcriptome blueprints of embryos generated by cumulus cell CC donor nuclear transfer NT , embryos generated by mouse embryonic fibroblast MEF donor NT and in vivo embryos at each stage zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst. According to the results from further analyses, NT embryos exhibit RNA processing and translation initiation defects during the zygotic genome activation ZGA period, and protein kinase activity and protein phosphorylation are defective during blastocyst formation.
Nuclear reprogramming: A key to stem cell function in regenerative medicine
During embryonic development pluripotency is progressively lost irreversibly by cell division, differentiation, migration and organ formation. Terminally differentiated cells do not generate other kinds of cells. Pluripotent stem cells are a great source of varying cell types that are used for tissue regeneration or repair of damaged tissue. The pluripotent stem cells can be derived from inner cell mass of blastocyte but its application is limited due to ethical concerns.
Research into the field of stem cell biology has developed exponentially over recent years, and is beginning to offer significant promise for unravelling the molecular basis of a multitude of disease states. Importantly, in addition to offering the opportunity to delve deeply into the mechanisms that drive disease aetiology the research is realistically opening the doors for development of targeted and personalized therapeutic applications that many considered, until recently, to be nothing more that a far fetched dream. This volume provides a timely glimpse into the methods that have been developed to instigate, and the mechanisms that have been identified to drive, the process of nuclear reprogramming, chronicling how the field has developed over the last years.
Recent scientific achievements in cell and developmental biology have provided unprecedented opportunities for advances in biomedical research. The demonstration that fully differentiated cells can reverse their gene expression profile to that of a pluripotent cell, and the successful derivation and culture of human embryonic stem cells ESCs have fuelled hopes for applications in regenerative medicine. These advances have been put to public scrutiny raising legal, moral and ethical issues which have resulted in different levels of acceptance. In this article, we will review the present state of these reprogramming technologies and discuss their relative success. We also overview reprogramming events after somatic cell nuclear transfer SCNT , as they may further instruct ex ovo strategies for cellular manipulation.
With advances in cancer therapies, survival rates in prepubescent patients have steadily increased.
Table of contents
Стратмор знал, что его следующий шаг имеет решающее значение. От него зависела жизнь Сьюзан, а также будущее Цифровой крепости. Стратмор также понимал, что первым делом нужно разрядить ситуацию. Выдержав паузу, он как бы нехотя вздохнул: - Хорошо, Грег. Ты выиграл.
Их количество удваивалось каждую минуту. Еще немного, и любой обладатель компьютера - иностранные шпионы, радикалы, террористы - получит доступ в хранилище секретной информации американского правительства. Пока техники тщетно старались отключить электропитание, собравшиеся на подиуме пытались понять расшифрованный текст. Дэвид Беккер и два оперативных агента тоже пробовали сделать это, сидя в мини-автобусе в Севилье. ГЛАВНАЯ РАЗНИЦА МЕЖДУ ЭЛЕМЕНТАМИ, ОТВЕТСТВЕННЫМИ ЗА ХИРОСИМУ И НАГАСАКИ Соши размышляла вслух: - Элементы, ответственные за Хиросиму и Нагасаки… Пёрл-Харбор. Отказ Хирохито… - Нам нужно число, - повторял Джабба, - а не политические теории. Мы говорим о математике, а не об истории.
Выдержав долгую паузу, Мидж шумно вздохнула. - Возможны ли другие варианты. - Конечно.