Neuronal differentiation of cryopreserved neural progenitor cells derived from mouse embryonic stem cells

Embryonic stem cells (ES cells) are developmentally pluripotent cells isolated from pre-implantation mammalian embryos. In cell culture ES cells can be easily differentiated to generate cultures of neural progenitors. We present a simple method for the cryopreservation of these ES-derived neural progenitors. Cryopreserved neural progenitor stocks can be thawed, expanded with FGF2, and differentiated into functional neurons. This method will facilitate studies using ES-derived neural progenitor cells as a cell culture model system for neural development and differentiation. It will also aid studies designed to test the ability of these progenitor cells to functionally engraft and repair damaged neural tissue.     

Chondrocytes derived from mouse embryonic stem cells

Our knowledge of cellular differentiation processes during chondro- and osteogenesis, in particular the complex interaction of differentiation factors, is still limited. We used the model system of embryonic stem (ES) cell differentiation in vitro via cellular aggregates, so called embryoid bodies (EBs), to analyze chondrogenic and osteogenic differentiation. ES cells differentiated into chondrocytes and osteocytes throughout a series of developmental stages resembling cellular differentiation events during skeletal development in vivo. A lineage from pluripotent ES cells via mesenchymal, prechondrogenic cells, chondrocytes and hypertrophicchondrocytes up to osteogenic cells was characterized. Furthermore, we found evidence for another osteogenic lineage, bypassing the chondrogenic stage. Together our results suggest that this in vitro system will be helpful to answer so far unacknowledged questions regarding chondrogenic and osteogenic differentiation. For example, we isolated an as yet unknown cDNA fragment from ES cell-derived chondrocytes, which showed a developmentally regulated expression pattern during EB differentiation. Considering ES cell differentiation as an alternative approach for cellular therapy, we used two different methods to obtain pure chondrocyte cultures from the heterogenous EBs. First, members of the transforming growth factor (TGF)-β family were applied and found to modulate chondrogenic differentiation but were not effective enough to produce sufficient amounts of chondrocytes. Second, chondrocytes were isolated from EBs by micro-manipulation. These cells initially showed dedifferentiation into fiboblastoid cells in culture, but later redifferentiated into mature chondrocytes. However, a small amount of chondrocytes isolated from EBs transdifferentiated into other mesenchymal cell types, indicating that chondrocytes derived from ES cells posses a distinct differentiation plasticity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells

Retinoic acid (RA) is one of the most important morphogens, and its embryonic distribution correlates with neural differentiation and positional specification in the developing central nervous system. To investigate the concentration-dependent effects of RA on neural differentiation of mouse embryonic stem cells (ES cells), we investigated the precise expression profiles of neural and regional specific genes by ES cells aggregated into embryoid bodies (EBs) exposed to various concentrations of RA or the BMP antagonist Noggin. RA promoted both neural differentiation and caudalization in a concentration-dependent manner, and the concentration of RA was found to regulate dorso-ventral identity, i.e., higher concentrations of RA induced a dorsal phenotype, and lower concentrations of RA induced a more ventral phenotype. The induction of the more ventral phenotype was due to the higher expression level of the N-terminus of sonic hedgehog protein (Shh-N) when treated with low concentration RA, as it was abrogated by an inhibitor of Shh signaling, cyclopamine. These findings suggest that the concentration of RA strictly and simultaneously regulates the neuralization and positional specification during differentiation of mouse ES cells and that it may be possible to use it to establish a strategy for controlling the identity of ES-cell-derived neural cells.     

Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells

Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest-related disorders.     

Gene expression profiles during early differentiation of mouse embryonic stem cells

Background  

Understanding the mechanisms controlling stem cell differentiation is the key to future advances in tissue and organ regeneration. Embryonic stem (ES) cell differentiation can be triggered by embryoid body (EB) formation, which involves ES cell aggregation in suspension. EB growth in the absence of leukaemia inhibitory factor (LIF) leads EBs to mimic early embryonic development, giving rise to markers representative of endoderm, mesoderm and ectoderm. Here, we have used microarrays to investigate differences in gene expression between 3 undifferentiated ES cell lines, and also between undifferentiated ES cells and Day 1–4 EBs     

Developmental potential of defined neural progenitors derived from mouse embryonic stem cells

The developmental potential of a uniform population of neural progenitors was tested by implanting them into chick embryos. These cells were generated from retinoic acid-treated mouse embryonic stem (ES) cells, and were used to replace a segment of the neural tube. At the time of implantation, the progenitors expressed markers defining them as Pax6-positive radial glial (RG) cells, which have recently been shown to generate most pyramidal neurons in the developing cerebral cortex. Six days after implantation, the progenitors generated large numbers of neurons in the spinal cord, and differentiated into interneurons and motoneurons at appropriate locations. They also colonized the host dorsal root ganglia (DRG) and differentiated into neurons, but, unlike stem cell-derived motoneurons, they failed to elongate axons out of the DRG. In addition, they neither expressed the DRG marker Brn3a nor the Trk neurotrophin receptors. Control experiments with untreated ES cells indicated that when colonizing the DRG, these cells did elongate axons and expressed Brn3a, as well as Trk receptors. Our results thus indicate that ES cell-derived progenitors with RG characteristics generate neurons in the spinal cord and the DRG. They are able to respond appropriately to local cues in the spinal cord, but not in the DRG, indicating that they are restricted in their developmental potential.     

Expression profiles of Wnt genes during neural differentiation of mouse embryonic stem cells

The Wnt family of secreted signaling proteins regulates many aspects of animal development and the behavior of several types of stem cells, including embryonic stem (ES) cells. Activation of canonical Wnt signaling has been shown to either inhibit or promote the differentiation of ES cells into neurons, depending on the stage of differentiation. Here, we describe the expression of all 19 mouse Wnt genes during this process. Using the well-established retinoic acid induction protocol we found that all Wnt genes except Wnt8b are expressed as ES cells differentiate into neurons, many of them in dynamic patterns. The expression pattern of 12 Wnt genes was analyzed quantitatively at 2-day intervals throughout neural differentiation, showing that multiple Wnt genes are expressed at each stage. A large proportion of these, including both canonical and noncanonical Wnts, are expressed at highest levels during later stages of differentiation. The complexity of the patterns observed indicates that disentangling specific roles for individual Wnt genes in the differentiation process will be a significant challenge.     

Proteomic analysis of neural differentiation of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) can differentiate into different types of cells, and serve as a good model system to study human embryonic stem cells (hESCs). We showed that mESCs differentiated into two types of neurons with different time courses. To determine the global protein expression changes after neural differentiation, we employed a proteomic strategy to analyze the differences between the proteomes of ES cells (E14) and neurons. Using 2-DE plus LC/MS/MS, we have generated proteome reference maps of E14 cells and derived dopaminergic neurons. Around 23 proteins with an increase or decrease in expression or phosphorylation after differentiation have been identified. We confirmed the downregulation of translationally controlled tumor protein (TCTP) and upregulation of alpha-tubulin by Western blotting. We also showed that TCTP was further downregulated in derived motor neurons than in dopaminergic neurons, and its expression level was independent of extracellular Ca(2+) concentration during neural differentiation. Potential roles of TCTP in modulating neural differentiation through binding to Ca(2+), tubulin and Na,K-ATPase, as well as the functional significance of regulation of other proteins such as actin-related protein 3 (Arp3) and Ran GTPase are discussed. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.     

Gene expression profiling revealed specific spermatogonial stem cell genes in mouse

Mammalian spermatogenesis originates from spermatogonial stem cells (SSCs), which undergo mitosis, meiosis and spermiogenesis in order to generate mature spermatozoa. SSCs are adult stem cells that can both self‐renew and differentiate. To maintain pluripotency, SSCs are regulated by both extrinsic factors secreted from surrounding somatic cells and intrinsic factors including specific gene expression programs. Using fluorescent labeled germ line stem cells, mouse gonocytes and SSCs were purified up to 97% by improved FACS method. Through microarray analyses, global gene expression profiles of gonocytes, SSCs, and differentiated cells were compared. A large number of distinctive genes were found to be enriched in respective cell populations, indicating different functional requirements of each cell type. Functional clustering analyses revealed that while gonocytes and SSCs preferentially express genes implicated in gene expression regulation and epigenetic modifications, differentiated cells including somatic cells are enriched with genes encoding proteins involved in various cellular activities. Further in situ hybridization and RT‐PCR experiments confirmed SSC specific expression of several genes of which functions have not been characterized in SSCs. The comparative gene expression profiling provides a useful resource for gene discovery in relation to SSC regulation and opens new avenues for the study of molecular mechanisms underlying SSC self‐renewal and differentiation. genesis 51:83–96, 2013. © 2012 Wiley Periodicals, Inc.     

MicroRNA expression profiling during neural differentiation of mouse embryonic carcinoma P19 cells

MicroRNAs （or miRNAs） are small non-coding RNAs （21-25 nucleotides） that are involved in a wide range of activities related to the development and differentiation of cells. Comparison of the miRNA expression profiles of mouse P19 embryonic carcinoma cells with those of differentiated neural stem cells showed that the expression level of 65 miRNAs changed （2-fold） after differentiation. MiR-124a was dramatically upregulated （more than 20-fold） while miRNAs of the miR-302 family and those in the miR-290-295 cluster were strongly down-regulated. Further analysis revealed that some important factors such as Oct4 and Sox2 appeared to be involved in the regulation of these miRNAs. These results may contribute to a better understanding of miRNA-regulated neural differentiation in early mouse embryos.     

Circular RNA expression profiles during the differentiation of mouse neural stem cells

Background

Circular RNAs (circRNAs) have recently been found to be expressed in human brain tissue, and many lines ofevidence indicate that circRNAs play regulatory roles in neurodevelopment. Proliferation and differentiation of neural stem cells (NSCs) are critical parts during development of central nervous system (CNS).To date, there have been no reports ofcircRNA expression profiles during the differentiation of mouse NSCs. We hypothesizethat circRNAs mayregulate gene expression in the proliferation anddifferentiation of NSCs.

Results

In this study, we obtained NSCs from the wild-type C57BL/6 J mouse fetal cerebral cortex. We extracted total RNA from NSCs in different differentiation stagesand then performed RNA-seq. By analyzing the RNA-Seq data, we found 37circRNAs and 4182 mRNAs differentially expressedduringthe NSC differentiation. Gene Ontology (GO) enrichment analysis of thecognate linear genes of these circRNAsrevealed that some enriched GO terms were related to neural activity. Furthermore, we performed a co-expression network analysis of these differentially expressed circRNAs and mRNAs. The result suggested a stronger GO enrichmentin neural features for both the cognate linear genes of circRNAs and differentially expressed mRNAs.

Conclusion

We performed the first circRNA investigation during the differentiation of mouse NSCs. Wefound that12 circRNAs might have regulatory roles duringthe NSC differentiation, indicating that circRNAs might be modulated during NSC differentiation.Our network analysis suggested the possible complex circRNA-mRNA mechanisms during differentiation, and future experimental workis need to validate these possible mechanisms.

     

Spontaneous differentiation of mouse embryonic stem cells in vitro: characterization by global gene expression profiles

We characterized the temporal gene expression changes during four weeks of spontaneous differentiation of mouse ES cells in a monolayer culture in order to obtain better insight into the differentiation process. The overall gene expression pattern was changed dramatically during the first two weeks of spontaneous differentiation, but stabilized after the second week. Most of the genes regulated within the first two weeks of spontaneous differentiation were genes related to development including morphogenesis, cell differentiation, embryonic development, pattern specification, mesoderm development, post-embryonic development, and blastocyst development. While most of the ectoderm lineage related genes were down-regulated, genes related to the mesoderm or endoderm lineage were up-regulated through the first week and second week, respectively. This study revealed that the development of ectoderm lineage is a recessive process during the spontaneous differentiation of mouse ES cells in monolayer culture. Our time-course characterization might provide a useful time line for directed differentiation of mouse ES cells.