Dataset Information

Comparison of in vitro Neuronal Differentiation Capacity Between Mouse Epiblast Stem Cells Derived From Nuclear Transfer and Naturally Fertilized Embryos.

ABSTRACT: Somatic cell nuclear transfer (SCNT) can give rise to fertile adults, but the successful perinatal and postnatal developmental rates are inefficient, including delayed developmental behaviors, and respiratory failure. However, the molecular and cellular mechanisms remain elusive. Mouse epiblast stem cells (mEpiSCs) from E5.5-6.5 epiblasts share defining features with human embryonic stem cells (hESCs), providing a new opportunity to study early mammalian development in vitro. In this study, mEpiSCs were established from naturally fertilized mouse embryos (F-mEpiSCs) and SCNT mouse embryos (NT-mEpiSCs). Also, the in vitro neuronal differentiation capacity of F-mEpiSCs and NT-mEpiSCs was compared. Morphology analysis showed less and smaller neurospheres formation and lower percentage of early neurons generation in NT-mEpiSCs. The immunocytochemical analysis and altered mRNA expression levels of the neuronal markers in differentiated cells further confirmed that neurogenesis was slower in NT-mEpiSCs than in F-mEpiSCs. Moreover, neuronal differentiation capacity was correlated with the basal expression levels of Atox1 and Vinculin but not Brachyury and Otx2, emphasizing that developmental aberrations in neurogenesis were associated with the NT technique but not random variations between clones. This study provided an important in vitro platform using mEpiSCs to study early epigenetic and developmental processes associated with neurogenesis.

SUBMITTER: Li T

PROVIDER: S-EPMC6218595 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Comparison of <i>in vitro</i> Neuronal Differentiation Capacity Between Mouse Epiblast Stem Cells Derived From Nuclear Transfer and Naturally Fertilized Embryos.

Li Tong T Zheng Yi Y Li Yan Y Ye Danna D

Frontiers in molecular neuroscience 20181030

Somatic cell nuclear transfer (SCNT) can give rise to fertile adults, but the successful perinatal and postnatal developmental rates are inefficient, including delayed developmental behaviors, and respiratory failure. However, the molecular and cellular mechanisms remain elusive. Mouse epiblast stem cells (mEpiSCs) from E5.5-6.5 epiblasts share defining features with human embryonic stem cells (hESCs), providing a new opportunity to study early mammalian development <i>in vitro.</i> In this stud ...[more]

PMID: 30425619

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Project description:Trophoblast stem cells (TSCs) are critical to mammalian embryogenesis by providing the cell source of the placenta. TSCs can be derived from trophoblast cells. However, the efficiency of TSC derivation from somatic cell nuclear transfer (NT) blastocysts is low. The regulatory mechanisms underlying transcription dynamics and epigenetic landscape remodeling during TSC derivation remain elusive. Here, we derived TSCs from the blastocysts by natural fertilization (NF), NT, and a histone deacetylase inhibitor Scriptaid-treated NT (SNT). Profiling of the transcriptomes across the stages of TSC derivation revealed that fibroblast growth factor 4 (FGF4) treatment resulted in many differentially expressed genes (DEGs) at outgrowth and initiated transcription program for TSC formation. We identified 75 transcription factors (TFs) that are continuously upregulated during NF TSC derivation, whose transcription profiles can infer the time course of NF not NT TSC derivation. Most DEGs in NT outgrowth are rescued in SNT outgrowth. The correct time course of SNT TSC derivation is inferred accordingly. Moreover, these TFs comprise an interaction network important to TSC stemness. Profiling of DNA methylation dynamics showed an extremely low level before FGF4 treatment and gradual increases afterward. FGF4 treatment results in a distinct DNA methylation remodeling process committed to TSC formation. We further identified 1,293 CpG islands (CGIs) whose DNA methylation difference is more than 0.25 during NF TSC derivation. The majority of these CGIs become highly methylated upon FGF4 treatment and remain in high levels. This may create a barrier for lineage commitment to restrict embryonic development, and ensure TSC formation. There exist hundreds of aberrantly methylated CGIs during NT TSC derivation, most of which are corrected during SNT TSC derivation. More than half of the aberrantly methylated CGIs before NT TSC formation are inherited from the donor genome. In contrast, the aberrantly methylated CGIs upon TSC formation are mainly from the highly methylated CGIs induced by FGF4 treatment. Functional annotation indicates that the aberrantly highly methylated CGIs play a role in repressing placenta development genes, etc., related to post-implantation development and maintaining TSC pluripotency. Collectively, our findings provide novel insights into the transcription dynamics, DNA methylation remodeling, and the role of FGF4 during TSC derivation.

Project description:Somatic cell nuclear transfer and transcription factor overexpression can induce reprogramming of somatic cells, whereby one cell fate is changed into another cell fate of choice. Yet the efficiency of this process for generating functional cells is low, limiting their therapeutic applications. The persistence of transcriptional memory from the cell's prior identity is thought to be a major hindrance to effective reprogramming and differentiation to functional cell types. To explore the effects of transcriptional memory on cell fate specification of reprogrammed cells, we analyzed epidermal tissue development in nuclear transfer embryos derived from endoderm nuclei. Our findings reveal variations in the success rate of differentiation to functional cells across cell types in cloned embryos. While some cell types, such as goblet cells, differentiated normally, a specific subset of cells resisted cell fate reprogramming, adopting a new endoderm-like state and disrupting normal body patterning. Furthermore, inefficient transcriptional reprogramming correlated with reduced basal stem cell populations, faulty differentiation of basal stem cell-derived fates, and increased cell death in the epidermis of cloned embryos. Crucially, we identified that the memory of active transcriptional states linked to key endoderm transcription factors plays a significant role in these issues. Mimicking active state transcriptional memory of these genes through the forced expression of Sox17b and Foxa4 in the epidermis of fertilized embryos produced the same defects. On the contrary, reducing transcriptional memory by interfering with the expression of Sox17b led to the rescue of observed epidermal defects. In summary, our study suggests that transcriptional memory tends to persist predominantly in specific reprogrammed cell types, hindering their differentiation into functional cells and embryonic structures. This underscores the critical need to assess and reduce transcriptional memory from the previous somatic identity during reprogramming, to efficiently generate functional cell types for therapeutic applications.

Dataset Information

Comparison of in vitro Neuronal Differentiation Capacity Between Mouse Epiblast Stem Cells Derived From Nuclear Transfer and Naturally Fertilized Embryos.

Publications

Comparison of <i>in vitro</i> Neuronal Differentiation Capacity Between Mouse Epiblast Stem Cells Derived From Nuclear Transfer and Naturally Fertilized Embryos.

Similar Datasets

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