Alexandra Rolletschek

Embryonic stem cell-derived neurogenesis. Retinoic acid induction and lineage selection of neuronal cells.

Abstract. Embryonic stem (ES) cells are able to differentiate in vitro into endodermal, mesodermal, and ectodermal cell types. However, the spontaneous development of neuronal cells from ES cells is rather limited. Therefore, specific protocols to increase the differentiation of neuronal cells have been established, such as retinoic acid (RA) induction and lineage selection of neuronal cells. High concentrations of RA resulted in efficient neuronal differentiation paralleled by the expression of tissue-specific genes, proteins, ion channels, and receptors in a developmentally controlled manner. Because the developmental pattern and survival capacity of RA-induced neuronal cells were limited, specific differentiation protocols by lineage selection of neuronal cells have been established using growth and extracellular matrix factors. After formation of cells of the three primary germ layers, mesodermal differentiation was inhibited by serum depletion, and neural precursor cells were generated by addition of basic fibroblast growth factor, followed by differentiation induction by neuronal differentiation factors. Further application of survival-promoting factors such as neurotrophic factors and cytokines at terminal stages resulted in a significant increase, survival, and maintenance of dopaminergic neurons. In the future, these cellular systems will be applicable: (1) for studying commitment and neuronal specification in vitro, (2) as pharmacological assays for drug screening, and (3) for the selective isolation of differentiated neuronal cells which may be used as a source for cell and tissue grafts.

Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors.

Here, we describe the generation of viable and dopamine-producing neurons derived from pluripotent mouse embryonic stem cells. Neurotrophic factors in combination with survival-promoting factors, such as interleukin-1beta, glial cell line-derived neurotrophic factor, neurturin, transforming growth factor-beta(3) and dibutyryl-cyclic AMP, significantly enhanced Nurr1 and tyrosine hydroxylase (TH) mRNA levels, whereas En-1, mash-1 and dopamine-2-receptor mRNA levels were not upregulated. In parallel, mRNA levels of the anti-apoptotic gene bcl-2 were found to be upregulated at terminal stages. Double immunofluorescence analysis revealed increased numbers of TH- and dopamine transporter-, but not gamma-aminobutyric acid- and serotonin-positive neurons in relation to synaptophysin-labeled cells by survival-promoting factors. Moreover, high-performance liquid chromatography analysis showed detectable levels of intracellular dopamine. We conclude that survival-promoting factors enhance differentiation, survival and maintenance of dopaminergic neurons derived from embryonic stem cells.

Electromagnetic fields affect transcript levels of apoptosis-related genes in embryonic stem cell-derived neural progenitor cells

Mouse embryonic stem (ES) cells were used as an experimental model to study the effects of electromagnetic fields (EMF). ES‐derived nestin‐positive neural progenitor cells were exposed to extremely low frequency EMF simulating power line magnetic fields at 50 Hz (ELF‐EMF) and to radiofrequency EMF simulating the Global System for Mobile Communication (GSM) signals at 1.71 GHz (RF‐EMF). Following EMF exposure, cells were analyzed for transcript levels of cell cycle regulatory, apoptosis‐related, and neural‐specific genes and proteins; changes in proliferation; apoptosis; and cytogenetic effects. Quantitative RT‐PCR analysis revealed that ELF‐EMF exposure to ES‐derived neural cells significantly affected transcript levels of the apoptosis‐related bcl‐2, bax, and cell cycle regulatory “growth arrest DNA damage inducible” GADD45 genes, whereas mRNA levels of neural‐specific genes were not affected. RF‐EMF exposure of neural progenitor cells resulted in down‐regulation of neural‐specific Nurr1 and in up‐regulation of bax and GADD45 mRNA levels. Short‐term RF‐EMF exposure for 6 h, but not for 48 h, resulted in a low and transient increase of DNA double‐strand breaks. No effects of ELF‐ and RF‐EMF on mitochondrial function, nuclear apoptosis, cell proliferation, and chromosomal alterations were observed. We may conclude that EMF exposure of ES‐derived neural progenitor cells transiently affects the transcript level of genes related to apoptosis and cell cycle control. However, these responses are not associated with detectable changes of cell physiology, suggesting compensatory mechanisms at the translational and posttranslational level.

Potential of embryonic and adult stem cells in vitro

Abstract Recent developments in the field of stem cell research indicate their enormous potential as a source of tissue for regenerative therapies. The success of such applications will depend on the precise properties and potentials of stem cells isolated either from embryonic, fetal or adult tissues. Embryonic stem cells established from the inner cell mass of early mouse embryos are characterized by nearly unlimited proliferation, and the capacity to differentiate into derivatives of essentially all lineages. The recent isolation and culture of human embryonic stem cell lines presents new opportunities for reconstructive medicine. However, important problems remain; first, the derivation of human embryonic stem cells from in vitro fertilized blastocysts creates ethical problems, and second, the current techniques for the directed differentiation into somatic cell populations yield impure products with tumorigenic potential. Recent studies have also suggested an unexpectedly wide developmental potential of adult tissuespecific stem cells. Here too, many questions remain concerning the nature and status of adult stem cells both in vivo and in vitro and their proliferation and differentiation/ transdifferentiation capacity. This review focuses on those issues of embryonic and adult stem cell biology most relevant to their in vitro propagation and differentiation. Questions and problems related to the use of human embryonic and adult stem cells in tissue regeneration and transplantation are discussed.

Pluripotency Associated Genes Are Reactivated by Chromatin‐Modifying Agents in Neurosphere Cells

Chromatin architecture in stem cells determines the pattern of gene expression and thereby cell identity and fate. The chromatin‐modifying agents trichostatin A (TSA) and 5‐Aza‐2′‐deoxycytidine (AzaC) affect histone acetylation and DNA methylation, respectively, and thereby influence chromatin structure and gene expression. In our previous work, we demonstrated that TSA/AzaC treatment of neurosphere cells induces hematopoietic activity in vivo that is long‐term, multilineage, and transplantable. Here, we have analyzed the TSA/AzaC‐induced changes in gene expression by global gene expression profiling. TSA/AzaC caused both up‐ and downregulation of genes, without increasing the total number of expressed genes. Chromosome analysis showed no hot spot of TSA/AzaC impact on a particular chromosome or chromosomal region. Hierarchical cluster analysis revealed common gene expression patterns among neurosphere cells treated with TSA/AzaC, embryonic stem (ES) cells, and hematopoietic stem cells. Furthermore, our analysis identified several stem cell genes and pluripotency‐associated genes that are induced by TSA/AzaC in neurosphere cells, including Cd34, Cd133, Oct4, Nanog, Klf4, Bex1, and the Dppa family members Dppa2, 3, 4, and 5. Sox2 and c‐Myc are constitutively expressed in neurosphere cells. We propose a model in which TSA/AzaC, by removal of epigenetic inhibition, induces the reactivation of several stem cell and pluripotency‐associated genes, and their coordinate expression enlarges the differentiation potential of somatic precursor cells.

The FunGenES database: a genomics resource for mouse embryonic stem cell differentiation.

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the “Functional Genomics in Embryonic Stem Cells” consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in “Expression Waves” and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells.

Induced human pluripotent stem cells: promises and open questions

Abstract Adult cells have been reprogrammed into induced pluripotent stem (iPS) cells by introducing pluripotency-associated transcription factors. Here, we discuss recent advances and challenges of in vitro reprogramming and future prospects of iPS cells for their use in diagnosis and cell therapy. The generation of patient-specific iPS cells for clinical application requires alternative strategies, because genome-integrating viral vectors may cause insertional mutagenesis. Moreover, when suitable iPS cell lines will be available, efficient and selective differentiation protocols are needed to generate transplantable grafts. Finally, we point to the requirement of a regulatory framework necessary for the commercial use of iPS cells.

Generation of pancreatic insulin-producing cells from embryonic stem cells — ‘Proof of principle’, but questions still unanswered

The generation of insulin-producing cells from differentiating mouse embryonic stem (ES) cells was described some years ago [1], but subsequent studies could not replicate the results. Instead, using the same differentiation protocol, it was found that insulin immunoreactivity occurred as a consequence of insulin uptake from the medium [2], neuronal cells were formed [2–4], or insulin was released as an artefact from differentiated ES cells [2, 3]. Functional pancreatic cells, however, were successfully generated using lineage selection strategies based on pancreas-specific promoters [5, 6], by modified protocols in combination with transgene expression [7–9], or by addition of a phosphoinositol-3 kinase inhibitor [10]. The differentiated cells showed properties of (neonatal) beta cells, such as insulin transcripts and C-peptide/insulin co-expression, insulin-secretory granules, ion channel activity of embryonal beta cells, and normalisation of blood glucose level after transplantation into diabetic mice [5–10]. Most of the differentiation protocols required a long cultivation period, including 4–5 days of embryoid body (EB) formation, followed by 3–4 weeks of differentiation, and some protocols required genetic manipulation. Recently, a relatively short procedure of pancreatic differentiation by a three-step experimental approach was published [11]. The strategy is based on the combined treatment by activin A, all-trans-retinoic acid, and other factors such as basic fibroblast growth factor (bFGF), which induced murine ES cells to differentiate into insulin-producing cells within 2 weeks. The authors presented results on insulin transcripts, C-peptide/insulin co-expression, glucose-induced insulin release, and the normalisation of glycaemia following transplantation into diabetic mice.

Signals from embryonic fibroblasts induce adult intestinal epithelial cells to form nestin-positive cells with proliferation and multilineage differentiation capacity in vitro

The intestinal epithelium has one of the greatest regenerative capacities in the body; however, neither stem nor progenitor cells have been successfully cultivated from the intestine. In this study, we applied an “artificial niche” of mouse embryonic fibroblasts to derive multipotent cells from the intestinal epithelium. Cocultivation of adult mouse and human intestinal epithelium with fibroblast feeder cells led to the generation of a novel type of nestin‐positive cells (intestinal epithelium‐derived nestin‐positive cells [INPs]). Transcriptome analyses demonstrated that mouse embryonic fibroblasts expressed relatively high levels of Wnt/bone morphogenetic protein (BMP) transcripts, and the formation of INPs was specifically associated with an increase in Lef1, Wnt4, Wnt5a, and Wnt/BMP‐responsive factors, but a decrease of BMP4 transcript abundance. In vitro, INPs showed a high but finite proliferative capacity and readily differentiated into cells expressing neural, pancreatic, and hepatic transcripts and proteins; however, these derivatives did not show functional properties. In vivo, INPs failed to form chimeras following injection into mouse blastocysts but integrated into hippocampal brain slice cultures in situ. We conclude that the use of embryonic fibroblasts seems to reprogram adult intestinal epithelial cells by modulation of Wnt/BMP signaling to a cell type with a more primitive embryonic‐like stage of development that has a high degree of flexibility and plasticity.

Characterization of mouse embryonic stem cell differentiation into the pancreatic lineage in vitro by transcriptional profiling, quantitative RT-PCR and immunocytochemistry

We have previously shown that mouse embryonic stem (ES) cells differentiate into insulin-positive cells via multi-lineage progenitors. Here, we used Affymetrix chips and quantitative RT-PCR analysis to determine transcriptional profiles of undifferentiated wildtype (wt) and Pax4 expressing (Pax4+) ES cells and differentiated cells of committed progenitor and advanced stages. From undifferentiated to the committed stage, 237 (wt) and 263 (Pax4+) transcripts were 5- or more-fold up-regulated, whereas from the committed to the advanced stage, 28 (wt) and 5 (Pax4+) transcripts, respectively, were two- or more-fold up-regulated. Transcripts were classified into main subclasses including transcriptional regulation, signalling/growth factors, adhesion/extracellular matrix, membrane/transport, metabolism and organogenesis. Remarkably, endoderm-specific Sox17 and early pancreas-specific Isl1 transcripts were up-regulated at an earlier stage of multi-lineage progenitors, whereas highly up-regulated probe sets and transcripts of genes involved in endoderm, pancreatic, hepatic, angiogenic and neural differentiation were detected at the committed progenitor stage. Pax4+ cells showed specific differences in transcript up-regulation and a lower amount of up-regulated neural-specific transcripts in comparison to wt cells, but no enhanced gene expression complexity. Immunocytochemical analysis of selected proteins involved in endoderm and pancreatic differentiation, such as chromogranin B, transthyretin, Foxa1 and neuronatin revealed co-expression with insulin- or C-peptide-positive cells. The comparison of transcript profiles of ES cells differentiating in vitro with those of the embryonic and adult pancreas in vivo suggested that in vitro differentiated cells resemble an embryonal stage of development, supporting the view that ES-derived pancreatic cells are unable to complete pancreatic differentiation in vitro.