Cluster characterization of mouse embryonic stem cell-derived pluripotent embryoid bodies in four distinct developmental stages

The formation of embryoid bodies (EBs) is the principal step in the differentiation of embryonic stem (ES) cells. In this study, the morphological characteristics and gene expression patterns of EBs related to the sequential stages of embryonic development were well defined in four distinct developmental groups over 112 days of culture: early-stage EBs groups (1–7 days of differentiation), mid-stage EBs groups (9–15 days of differentiation), maturing EBs groups (17–45 days of differentiation) and matured EBs groups (50 days of differentiation). We first determined definite histological location of apoptosis within EBs and the sequential expression of molecular markers representing stem cells (Oct4, SSEA-1, Sox-2 and AKP), germ cells (Fragilis, Dazl, c-kit, StellaR, Mvh and Stra8), ectoderm (Neurod, Nestin and Neurofilament), mesoderm (Gata-1, Flk-1 and Hbb) and endoderm (AFP and Transthyretin). Our results revealed that developing EBs possess either pluripotent stem cell or germ cell states and that three-dimensional aggregates of EBs initiate mES cell differentiation during prolonged culture in vitro. Therefore, we suggest that this EB system to some extent recapitulates the early developmental processes occurring in vivo.     

Search for nave human pluripotent stem cells

Normal mouse pluripotent stem cells were originally derived from the inner cell mass(ICM) of blastocysts and shown to be the in vitro equivalent of those pre-implantation embryonic cells, and thus were called embryonic stem cells(ESCs). More than a decade later, pluripotent cells were isolated from the ICM of human blastocysts. Despite being called human ESCs, these cells differ significantly from mouse ESCs, including different morphology and mechanisms of control of pluripotency, suggesting distinct embryonic origins of ESCs from the two species. Subsequently, mouse pluripotent stem cells were established from the ICMderived epiblast of post-implantation embryos. These mouse epiblast stem cells(Epi SCs) are morphological and epigenetically more similar to human ESCs. This raised the question of whether cells from the human ICM are in a more advanced differentiation stage than their murine counterpart, or whether the available culture conditions were not adequate to maintain those human cells in their in vivo state, leading to a transition into Epi SC-like cells in vitro. More recently, novel culture conditions allowed the conversion of human ESCs into mouse ESC-like cells called nave(or ground state) human ESCs, and the derivation of nave human ESCs from blastocysts. Here we will review the characteristics of each type of pluripotent stem cells, how(and whether) these relate to different stages of embryonic development, and discuss the potential implications of nave human ESCs in research and therapy.     

Link between embryonic stem cell pluripotency and homologous allelic pairing of Oct4 loci

The Oct4 gene is a master regulator of the pluripotent properties of embryonic stem cells (ESCs). Recently, Oct4 loci were shown to frequently localize in close proximity to one another during the early stage of cellular differentiation, implicating this event as an important prerequisite step for ESCs to exert their full differentiation potential. Although the differentiation capacity of embryonal carcinoma cells (ECCs), such as F9 and P19 ECC lines, is severely restricted compared with ESCs, ECCs bear a highly similar expression profile to that of ESCs including expression of Oct4 and other pluripotency marker genes. Therefore, we examined whether allelic pairing of Oct4 loci also occurs during differentiation of F9 and P19 ECCs. Our data clearly demonstrate that this event is only observed within ESCs, but not ECCs, subjected to induction of differentiation, indicating transient allelic pairing of Oct4 loci as a specific feature of pluripotent ESCs. Moreover, our data revealed that this pairing did not occur broadly across chromosome 17, which carries the Oct4 gene, but occurred locally between Oct4 loci, suggesting that Oct4 loci somehow exert a driving force for their allelic pairing.     

Dazl Functions in Maintenance of Pluripotency and Genetic and Epigenetic Programs of Differentiation in Mouse Primordial Germ Cells In Vivo and In Vitro

Background

Mammalian germ cells progress through a unique developmental program that encompasses proliferation and migration of the nascent primordial germ cell (PGC) population, reprogramming of nuclear DNA to reset imprinted gene expression, and differentiation of mature gametes. Little is known of the genes that regulate quantitative and qualitative aspects of early mammalian germ cell development both in vivo, and during differentiation of germ cells from mouse embryonic stem cells (mESCs) in vitro.

Methodology and Principal Findings

We used a transgenic mouse system that enabled isolation of small numbers of Oct4ΔPE:GFP-positive germ cells in vivo, and following differentiation from mESCs in vitro, to uncover quantitate and qualitative phenotypes associated with the disruption of a single translational regulator, Dazl. We demonstrate that disruption of Dazl results in a post-migratory, pre-meiotic reduction in PGC number accompanied by aberrant expression of pluripotency genes and failure to erase and re-establish genomic imprints in isolated male and female PGCs, as well as subsequent defect in progression through meiosis. Moreover, the phenotypes observed in vivo were mirrored by those in vitro, with inability of isolated mutant PGCs to establish pluripotent EG (embryonic germ) cell lines and few residual Oct-4-expressing cells remaining after somatic differentiation of mESCs carrying a Dazl null mutation. Finally, we observed that even within undifferentiated mESCs, a nascent germ cell subpopulation exists that was effectively eliminated with ablation of Dazl.

Conclusions and Significance

This report establishes the translational regulator Dazl as a component of pluripotency, genetic, and epigenetic programs at multiple time points of germ cell development in vivo and in vitro, and validates use of the ESC system to model and explore germ cell biology.     

Simplified three-dimensional culture system for long-term expansion of embryonic stem cells

AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging.METHODS: Mouse embryonic stem cells (ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional (3-D) self-assembling scaffolds and compared with traditional two-dimentional (2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate (PEG-4-Acr) and thiol-functionalized dextran (Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoBlue (PB) assays. Genetic expression of pluripotency markers (Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D culture conditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining (Oct4 and Nanog) and western blot analysis (Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers.RESULTS: Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH (1:1 v/v) to a final concentration of 5% (w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels (P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury (mesoderm), NCAM (ectoderm), and GATA4 (endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively.CONCLUSION: This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.     

Retinoic acid and/or progesterone differentiate mouse induced pluripotent stem cells into male germ cells in vitro

Numerous reagents were employed for differentiating induced pluripotent stem cells (iPSCs) into male germ cells; however, the induction procedure was ineffective. The aim of this study was to improve the in vitro differentiation of mice iPSCs (miPSCs) into male germ cells with retinoic acid (RA) and progesterone (P). miPSCs were differentiated to embryoid bodies (EBs) in suspension with RA with or without progesterone for 0, 4, and 7 days. Then, the expression of certain genes at different stages of male germ cell development including Ddx4 (pre meiosis), Stra8 (meiosis), AKAP3 (post meiosis), and Mvh protein was examined in RNA and/or protein levels by real-time polymerase chain reaction or flow cytometry, respectively. The Stra8 gene expression increased in the RA groups on all days. But, expression of this gene declined in RA + P groups. In addition, an increased expression of Ddx4 gene was observed on day 0 in the P group. Also, a significant upregulation was observed in the expression of AKAP3 gene in the RA + P group on days 0 and 4. However, gene expression decreased in P and RA groups on day 7. The expression of Mvh protein significantly increased in the RA group on day 7. The Mvh expression was also enhanced in the P group on day 4, but it decreased on day 7, while this protein upregulated on day 0 and 7 in the RA + P group. The miPSCs have the capacity for in vitro differentiation into male germ cells by RA and/or progesterone. However, the effects of these inducers depend on the type of combination and an effective time.     

When X-inactivation meets pluripotency: An intimate rendezvous

The integration of X-inactivation with development is a crucial aspect of this classical paradigm of epigenetic regulation. During early female mouse development, X-inactivation reprogramming occurs in pluripotent cells of the inner cell mass of the blastocyst and in pluripotent primordial germ cells. Here we discuss the developmental strategies which ensure the coupling of the regulation of X-inactivation to the acquisition of pluripotency through the regulation of the master of X-inactivation, the non-coding Xist gene, by the key factors which support pluripotency Nanog, Oct4 and Sox2.     

胚胎干细胞向造血干/祖细胞定向诱导分化的研究进展

胚胎干细胞(embryonic stem cell,ES细胞)是指由胚胎内细胞团(inner cell mass,ICM)细胞经体外抑制培养而筛选得到的细胞,具有无限增殖潜能,在体外可以向造血细胞分化,有可能为造血干细胞移植和血细胞输注开辟新的来源．此外,ES细胞向造血干/祖细胞的定向诱导分化也为阐明哺乳动物造血发育的细胞和分子机制提供了良好的体外模型．对ES细胞向造血干/祖细胞定向分化的研究进展进行了综述．     

Epidermal growth factor and three‐dimensional scaffolds provide conducive environment for differentiation of mouse embryonic stem cells into oocyte‐like cells

Three‐dimensional (3D) culture provides a biomimicry of the naive microenvironment that can support cell proliferation, differentiation, and regeneration. Some growth factors, such as epidermal growth factor (EGF), facilitate normal meiosis during oocyte maturation in vivo. In this study, a scaffold‐based 3D coculture system using purified alginate was applied to induce oocyte differentiation from mouse embryonic stem cells (mESCs). mESCs were induced to differentiate into oocyte‐like cells using embryoid body protocol in the two‐dimensional or 3D microenvironment in vitro. To increase the efficiency of the oocyte‐like cell differentiation from mESCs, we employed a coculture system using ovarian granulosa cells in the presence or absence of epidermal growth factor (+EGF or ?EGF) for 14 days and then the cells were assessed for germ cell differentiation, meiotic progression, and oocyte maturation markers. The cultures exposed to EGF in the alginate‐based 3D microenvironment showed the highest level of premeiotic (Oct4 and Mvh), meiotic (Scp1, Scp3, Stra8, and Rec8), and oocyte maturation (Gdf9, Cx37, and Zp2) marker genes (p < .05) in comparison to other groups. According to the gene‐expression patterns, we can conclude that alginate‐based 3D coculture system provided a highly efficient protocol for oocyte‐like cell differentiation from mESCs. The data showed that this culture system along with EGF improved the rate of in vitro oocyte‐like cell differentiation.     

胚胎干细胞及诱导多能干细胞向心肌细胞分化和调控的研究进展

胚胎干细胞（embryonic stem cells,ESCs）具有自我更新、无限增殖和多向分化的特性,包括分化成心脏组织的多种类型细胞。经体细胞重编程产生的诱导多能干细胞（induced pluripotent stem cells,iPS）也被证明有类似胚胎干细胞的特性。但这些多能干细胞向心肌细胞自发分化的效率非常低,因此,如何有效地诱导这些多能干细胞向心肌细胞的定向分化对深入认识心肌发生发育的关键调控机制和实现其在药物发现和再生医学,如心肌梗塞、心力衰竭的细胞治疗以及心肌组织工程中的应用均具有非常重要的意义。该文重点综述了近年来胚胎干细胞及诱导多能干细胞向心肌细胞分化和调控的研究进展,并探讨了这一研究领域亟待解决的关键问题和这些多能干细胞的应用前景。     

DAZ Family Proteins,Key Players for Germ Cell Development

DAZ family proteins are found almost exclusively in germ cells in distant animal species. Deletion or mutations of their encoding genes usually severely impair either oogenesis or spermatogenesis or both. The family includes Boule (or Boll), Dazl (or Dazla) and DAZ genes. Boule and Dazl are situated on autosomes while DAZ, exclusive of higher primates, is located on the Y chromosome. Deletion of DAZ gene is the most common causes of infertility in humans. These genes, encoding for RNA binding proteins, contain a highly conserved RNA recognition motif and at least one DAZ repeat encoding for a 24 amino acids sequence able to bind other mRNA binding proteins. Basically, Daz family proteins function as adaptors for target mRNA transport and activators of their translation. In some invertebrate species, BOULE protein play a pivotal role in germline specification and a conserved regulatory role in meiosis. Depending on the species, DAZL is expressed in primordial germ cells (PGCs) and/or pre-meiotic and meiotic germ cells of both sexes. Daz is found in fetal gonocytes, spermatogonia and spermatocytes of adult testes. Here we discuss DAZ family genes in a phylogenic perspective, focusing on the common and distinct features of these genes, and their pivotal roles during gametogenesis evolved during evolution.     

Functional screen reveals essential roles of miR‐27a/24 in differentiation of embryonic stem cells

MicroRNAs play important roles in controlling the embryonic stem cell (ESC) state. Although much is known about microRNAs maintaining ESC state, microRNAs that are responsible for promoting ESC differentiation are less reported. Here, by screening 40 microRNAs pre-selected by their expression patterns and predicted targets in Dgcr8-null ESCs, we identify 14 novel differentiation-associated microRNAs. Among them, miR-27a and miR-24, restrained by c-Myc in ESC, exert their roles of silencing self-renewal through directly targeting several important pluripotency-associated factors, such as Oct4, Foxo1 and Smads. CRISPR/Cas9-mediated knockout of all miR-27/24 in ESCs leads to serious deficiency in ESC differentiation in vitro and in vivo. Moreover, depleting of them in mouse embryonic fibroblasts can evidently promote somatic cell reprogramming. Altogether, our findings uncover the essential role of miR-27 and miR-24 in ESC differentiation and also demonstrate novel microRNAs responsible for ESC differentiation.