Influence of in vitro manipulation on the stability of methylation patterns in the Snurf/Snrpn-imprinting region in mouse embryonic stem cells

Recent work on embryonic stem (ES) cells showed that stem cell-derived tissues and embryos, cloned from ES cell nuclei, often fail to maintain epigenetic states of imprinted genes. This deregulation is frequently associated with in vitro manipulations and culture conditions which might affect the cells potential to develop into normal fetuses. Usually, epigenetic instability is reported in differentially methylated regions of mostly growth-related imprinted genes. However, little is known about the epigenetic stability of genes that function late in organogenesis. Hence, we set out to investigate the epigenetic stability of neuronal genes and analyzed DNA methylation patterns in the Snurf/Snrpn imprinted cluster in several cultured mouse ES cell lines. We also determined the effects of in vitro stress factors such as consecutive passaging, trypsination, mechanical handling, single cell cloning, centrifugation, staurosporine-induced neurogenesis and the insertion of viral (foreign) DNA into the host genome. Intriguingly, none of these in vitro manipulations interfered with the stability of the methylation patterns in the analyzed neuronal genes. These data imply that, in contrast to growth-related genes like Igf2, H19, Igf2r or Grb10, the methylation imprints of the analyzed neuronal genes in the Snurf/Snrpn cluster may be particularly stable in manipulated ES cells.     

Analysis of neurogenesis in a mammalian neuroepithelium: proliferation and differentiation of an olfactory neuron precursor in vitro

Development of a culture system for mammalian olfactory epithelium has permitted the process of neurogenesis to be examined in vitro. Antibody markers allowing the unambiguous identification of putative neuroepithelial stem cells (keratin+ basal cells) and differentiated neurons (N-CAM+ olfactory receptor neurons) are described. In combination with [3H]thymidine uptake analysis, these antibodies have been used to characterize the existence, proliferation, and differentiation of the immediate neuronal precursor in this system. This cell is distinct from basal cells and rapidly sorts out from them, dividing as it migrates. Data are presented which suggest that the precursor follows a simple lineage program, dividing to give rise to two N-CAM+ daughter neurons. Although this precursor efficiently generates neurons in defined medium, neurogenesis subsequently ceases because new precursors are not produced, suggesting that epigenetic factors may regulate continual neurogenesis in this system.     

Coordinated regulation of the ribosome and proteasome by PRMT1 in the maintenance of neural stemness in cancer cells and neural stem cells

Previous studies suggested that cancer cells resemble neural stem/progenitor cells in regulatory network, tumorigenicity, and differentiation potential, and that neural stemness might represent the ground or basal state of differentiation and tumorigenicity. The neural ground state is reflected in the upregulation and enrichment of basic cell machineries and developmental programs, such as cell cycle, ribosomes, proteasomes, and epigenetic factors, in cancers and in embryonic neural or neural stem cells. However, how these machineries are concertedly regulated is unclear. Here, we show that loss of neural stemness in cancer or neural stem cells via muscle-like differentiation or neuronal differentiation, respectively, caused downregulation of ribosome and proteasome components and major epigenetic factors, including PRMT1, EZH2, and LSD1. Furthermore, inhibition of PRMT1, an oncoprotein that is enriched in neural cells during embryogenesis, caused neuronal-like differentiation, downregulation of a similar set of proteins downregulated by differentiation, and alteration of subcellular distribution of ribosome and proteasome components. By contrast, PRMT1 overexpression led to an upregulation of these proteins. PRMT1 interacted with these components and protected them from degradation via recruitment of the deubiquitinase USP7, also known to promote cancer and enriched in embryonic neural cells, thereby maintaining a high level of epigenetic factors that maintain neural stemness, such as EZH2 and LSD1. Taken together, our data indicate that PRMT1 inhibition resulted in repression of cell tumorigenicity. We conclude that PRMT1 coordinates ribosome and proteasome activity to match the needs for high production and homeostasis of proteins that maintain stemness in cancer and neural stem cells.     

Neurogenic differentiation of human adipose-derived stem cells: Relevance of different signaling molecules,transcription factors,and key marker genes

Since numerous diseases affect the central nervous system and it has limited self-repair capability, a great interest in using stem cells as an alternative cell source is generated. Previous reports have shown the differentiation of adipose-derived stem cells in neuron-like cells and it has also been proved that the expression pattern of patterning, proneural, and neural factors, such as Pax6, Mash1, Ngn2, NeuroD1, Tbr2 and Tbr1, regulates and defines adult neurogenesis. Regarding this, we hypothesize that a functional parallelism between adult neurogenesis and neuronal differentiation of human adipose-derived stem cells exists. In this study we differentiate human adipose-derived stem cells into neuron-like cells and analyze the expression pattern of different patterning, proneural, neural and neurotransmitter genes, before and after neuronal differentiation. The neuron-like cells expressed neuronal markers, patterning and proneural factors characteristics of intermediate stages of neuronal differentiation. Thus we demonstrated that it is possible to differentiate adipose-derived stem cells in vitro into immature neuron-like cells and that this process is regulated in a similar way to adult neurogenesis. This may contribute to elucidate molecular mechanisms involved in neuronal differentiation of adult human non-neural cells, in aid of the development of potential therapeutic tools for diseases of the nervous system.     

Cell line differences in replication timing of human glutamate receptor genes and other large genes associated with neural disease

There is considerable current interest in the function of epigenetic mechanisms in neuroplasticity with regard to learning and memory formation and to a range of neural diseases. Previously, we described replication timing on human chromosome 21q in the THP-1 human cell line (2n = 46, XY) and showed that several genes associated with neural diseases, such as the neuronal glutamate receptor subunit GluR-5 (GRIK1) and amyloid precursor protein (APP), were located in regions where replication timing transitioned from early to late S phase. Here, we compared replication timing of all known human glutamate receptor genes (26 genes in total) and APP in 6 different human cell lines including human neuron-related cell lines. Replication timings were obtained by integrating our previously reported data with new data generated here and information from the online database ReplicationDomain. We found that many of the glutamate receptor genes were clearly located in replication timing transition zones in neural precursor cells, but this relationship was less clear in embryonic stem cells before neural differentiation; in the latter, the genes were often located in later replication timing zones that displayed DNA hypermethylation. Analysis of selected large glutamate receptor genes (>200 kb), and of APP, showed that their precise replication timing patterns differed among the cell lines. We propose that the transition zones of DNA replication timing are altered by epigenetic mechanisms, and that these changes may affect the neuroplasticity that is important to memory and learning, and may also have a role in the development of neural diseases.