Current biomarker-associated procedures of cancer modeling-a reference in the context of IDH1 mutant glioma.

Isocitrate dehydrogenases (IDH1/2) are central molecular markers for glioblastoma. Providing in vitro or in vivo models with mutated IDH1/2 can help prepare facilities to understand the biology of these mutated genes as glioma markers, as well as help, improve therapeutic strategies. In this review, we first summarize the biology principles of IDH and its mutations and outline the core primary findings in the clinical context of neuro-oncology. Given the extensive research interest and exciting developments in current stem cell biology and genome editing, the central part of the manuscript is dedicated to introducing various routes of disease modeling strategies of IDH mutation (IDHMut) glioma and comparing the scientific-technological findings from the field using different engineering methods. Lastly, by giving our perspective on the benefits and limitations of patient-derived and donor-derived disease modeling respectively, we aim to propose leading research questions to be answered in the context of IDH1 and glioma.

Acquisition of chromosome 1q duplication in parental and genome-edited human-induced pluripotent stem cell-derived neural stem cells results in their higher proliferation rate in vitro and in vivo.

OBJECTIVES: Genetic engineering of human-induced pluripotent stem cell-derived neural stem cells (hiPSC-NSC) may increase the risk of genomic aberrations. Therefore, we asked whether genetic modification of hiPSC-NSCs exacerbates chromosomal abnormalities that may occur during passaging and whether they may cause any functional perturbations in NSCs in vitro and in vivo. MATERIALS AND METHODS: The transgenic cassette was inserted into the AAVS1 locus, and the genetic integrity of zinc-finger nuclease (ZFN)-modified hiPSC-NSCs was assessed by the SNP-based karyotyping. The hiPSC-NSC proliferation was assessed in vitro by the EdU incorporation assay and in vivo by staining of brain slices with Ki-67 antibody at 2 and 8 weeks after transplantation of ZFN-NSCs with and without chromosomal aberration into the striatum of immunodeficient rats. RESULTS: During early passages, no chromosomal abnormalities were detected in unmodified or ZFN-modified hiPSC-NSCs. However, at higher passages both cell populations acquired duplication of the entire long arm of chromosome 1, dup(1)q. ZNF-NSCs carrying dup(1)q exhibited higher proliferation rate than karyotypically intact cells, which was partly mediated by increased expression of AKT3 located on Chr1q. Compared to karyotypically normal ZNF-NSCs, cells with dup(1)q also exhibited increased proliferation in vivo 2 weeks, but not 2 months, after transplantation. CONCLUSIONS: These results demonstrate that, independently of ZFN-editing, hiPSC-NSCs have a propensity for acquiring dup(1)q and this aberration results in increased proliferation which might compromise downstream hiPSC-NSC applications.

Ketamine Increases Proliferation of Human iPSC-Derived Neuronal Progenitor Cells via Insulin-Like Growth Factor 2 and Independent of the NMDA Receptor.

The N-methyl-D-aspartate (NMDA) receptor antagonist ketamine offers promising perspectives for the treatment of major depressive disorder. Although ketamine demonstrates rapid and long-lasting effects, even in treatment-resistant patients, to date, the underlying mode of action remains elusive. Thus, the aim of our study was to investigate the molecular mechanism of ketamine at clinically relevant concentrations by establishing an in vitro model based on human induced pluripotent stem cells (iPSCs)-derived neural progenitor cells (NPCs). Notably, ketamine increased the proliferation of NPCs independent of the NMDA receptor, while transcriptome analysis revealed significant upregulation of insulin-like growth factor 2 (IGF2) and p11, a member of the S100 EF-hand protein family, which are both implicated in the pathophysiology of depression, 24 h after ketamine treatment. Ketamine (1 µM) was able to increase cyclic adenosine monophosphate (cAMP) signaling in NPCs within 15 min and cell proliferation, while ketamine-induced IGF2 expression was reduced after PKA inhibition with cAMPS-Rp. Furthermore, 24 h post-administration of ketamine (15 mg/kg) in vivo confirmed phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the subgranular zone (SGZ) of the hippocampus in C57BL/6 mice. In conclusion, ketamine promotes the proliferation of NPCs presumably by involving cAMP-IGF2 signaling.

Acid-Sensitive Ion Channels Are Expressed in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are potential sources for cardiac regeneration and drug development. hiPSC-CMs express all the cardiac ion channels and the unique cardiac Ca2+-signaling phenotype. In this study, we tested for expression of acid sensing ion channels (ASICs) in spontaneously beating cardiomyocytes derived from three different hiPSC lines (IMR-90, iPSC-K3, and Ukki011-A). Rapid application of solutions buffered at pH 6.7, 6.0, or 5.0 triggered rapidly activating and slowly inactivating voltage-independent inward current that reversed at voltages positive to ENa, was suppressed by 5 µM amiloride and withdrawal of [Na+]o, like neuronal ASIC currents. ASIC currents were expressed at much lower percentages and densities in undifferentiated hiPSC and in dermal fibroblasts. ASIC1 mRNA and protein were measured in first 60 days but decreased in 100 days postdifferentiation hiPSC cultures. Hyperacidification (pH 5 and 6) also triggered large Ca2+ transients in intact hiPSC-CMs that were neither ruthenium red nor amiloride-sensitive, but were absent in whole cell-clamped hiPSC-CMs. Neither ASIC1 current nor its protein was detected in rat adult cardiomyocytes, but hyperacidification did activate smaller and slowly activating currents with drug sensitivity similar to TRPV channels. Considering ASIC expression in developing but not adult myocardium, a role in heart development is likely.

Impurity of stem cell graft by murine embryonic fibroblasts - implications for cell-based therapy of the central nervous system.

Stem cells have been demonstrated to possess a therapeutic potential in experimental models of various central nervous system disorders, including stroke. The types of implanted cells appear to play a crucial role. Previously, groups of the stem cell network NRW implemented a feeder-based cell line within the scope of their projects, examining the implantation of stem cells after ischemic stroke and traumatic brain injury. Retrospective evaluation indicated the presence of spindle-shaped cells in several grafts implanted in injured animals, which indicated potential contamination by co-cultured feeder cells (murine embryonic fibroblasts - MEFs). Because feeder-based cell lines have been previously exposed to a justified criticism with regard to contamination by animal glycans, we aimed to evaluate the effects of stem cell/MEF co-transplantation. MEFs accounted for 5.3 ± 2.8% of all cells in the primary FACS-evaluated co-culture. Depending on the culture conditions and subsequent purification procedure, the MEF-fraction ranged from 0.9 to 9.9% of the cell suspensions in vitro. MEF survival and related formation of extracellular substances in vivo were observed after implantation into the uninjured rat brain. Impurity of the stem cell graft by MEFs interferes with translational strategies, which represents a threat to the potential recipient and may affect the graft microenvironment. The implications of these findings are critically discussed.

Conserved TCP domain of Sas-4/CPAP is essential for pericentriolar material tethering during centrosome biogenesis.

Pericentriolar material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4's tethering role, we determined the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of ß-sheets fold. This unique feature of the TCP domain suggests that it could provide an "extended surface-like" platform to tether the Sas-4-PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9-10th ß-strands (ß9-10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 ß9-10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the ß9-10 surface in mediating protein-protein interactions for efficient Sas-4-PCM scaffold centriole tethering. Hence, we provide possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication.

Interactions of human embryonic stem cell-derived neural progenitors with an electrospun nanofibrillar surface in vitro.

Stem cell technology combined with nano-scaffold surfaces provides a new tool for better induction involved in cell lineage differentiations and therefore for central nervous system repair. This study was undertaken to investigate appropriate neural cell-substrate interactions. Neural progenitors (NPs) were established from human embryonic stem cells (hESCs), as a first step, using an adherent system and a defined medium supplemented with a combination of factors. Next, the behavior of hESC-derived NPs (hESC-NPs) was evaluated on a synthetic, randomly oriented, three-dimensional nanofibrillar matrix composed of electrospun polyamide nanofibers (Ultra-Web™) using a variety of experimental approaches. We have demonstrated that homogenous, expandable, and self-renewable NPs can be easily generated from hESCs; they can express related markers Nestin, Sox1, and Pax6; and they can undergo multipotency differentiation to neurons and glials. Functionally, NPs cultured on nanofibers demonstrated an increase in the rate of migration, proliferation, morphology, and neurite length when compared with NPs cultured on two-dimensional culture surfaces. The results suggest that topographical features of the extracellular matrix of the cell environment have paved the way for a better understanding of human neuronal development, thus allowing for future clinical applications.

Epigenetic analysis of human embryonic carcinoma cells during retinoic acid-induced neural differentiation.

Differentiation of stem cells from a pluripotent to a committed state involves global changes in genome expression patterns, critically determined by chromatin structure and interactions of chromatin-binding proteins. The dynamics of chromatin structure are tightly regulated by multiple epigenetic mechanisms such as histone modifications and the incorporation of histone variants. In the current work, we induced neural differentiation of a human embryonal carcinoma stem cell line, NTERA2/NT2, by retinoic acid (RA) treatment, primarily according to two different methods of adherent cell culture (rosette formation) and suspension cell culture (EB formation) conditions, and histone modifications and variations were compared through these processes. Western blot analysis of histone extracts showed significant changes in the acetylation and methylation patterns of histone H3, and expression level of the histone variant H2A.Z, after RA treatment in both protocols. Using chromatin immunoprecipitation (ChIP) coupled with real-time PCR, it was shown that these epigenetic changes occurred on the regulatory regions of 4 marker genes (Oct4, Nanog, Nestin, and Pax6) in a culture condition dependent manner. This report demonstrates the dynamic interplay of histone modification and variation in regulating the gene expression profile, during stem cell differentiation and under different culture conditions.

Dehydroepiandrosterone stimulates neurogenesis in mouse embryonal carcinoma cell- and human embryonic stem cell-derived neural progenitors and induces dopaminergic neurons.

To evaluate the effect of dehydroepiandrosterone (DHEA) as a neurosteroid on the rate of neurogenesis, neural survival, and proliferation of pluripotent stem cell-derived neurons, we have added DHEA to mouse P19 embryonal carcinoma cell- and human embryonic stem cell-derived neural progenitors (ECC- and ESC-NPs). In ECC-derived NPs, flow cytometric analysis of nestin and Tuj1-positive cells revealed that the percentages of these cells increased significantly for the markers following DHEA treatment of the cells. Moreover, the percentages of tyrosine hydroxylase (TH)-positive cells, the marker of dopaminergic neurons, significantly increased in the presence of DHEA. The expression of neural-specific genes such as Mash1, Pax6, Tuj1, and TH was also detected by RT-PCR analysis. BrdU incorporation and estrogen receptor (EsR) were found to be increased after DHEA induction. Moreover, apoptosis was significantly decreased after DHEA treatment. DHEA effect was also confirmed on human ESC-NPs by the enhancement of Tuj1- and TH-immunofluorescent-positive cells and TH and Nurr1 transcripts, as detected by quantitative RT-PCR. In conclusion, these results have presented evidence that DHEA was able to induce neurogenesis in mouse ECC and human ESC-NPs. This observation was related to the division of NPs and the reduction of apoptosis. Moreover, DHEA has dopaminergic potential in the cells of both orders. This provides a better insight into the differentiation and maintenance of neural cells and treatment of a wide variety of neurological diseases such as Alzheimer's and Parkinson's by stem cells.

Neurogenic and mitotic effects of dehydroepiandrosterone on neuronal-competent marrow mesenchymal stem cells.

To establish whether dehydroepiandrosterone (DHEA) as a neurosteroid could enhance the rate of neuronal differentiation in neuronal-competent bone marrow mesenchymal stem cells (BM-MSCs), we added DHEA before and after plating the neurosphere-like aggregates. Flow cytometric analysis of Tubulin-III and Tau positive cells revealed that the percentages of these cells were increased significantly for the two markers following DHEA treatment at both stages. Moreover, Western blot analysis revealed that Tubulin-III protein was strongly induced by DHEA. The expression of neuronal specific genes such as Isl-1, Tubulin III, Pax6 and Nestin was also detected by RT-PCR analysis as well as BrdU incorporation and found to have increased significantly after DHEA induction. In conclusion, these results provide evidence that DHEA can affect neuronal-competent MSCs in inducing the expression of a comprehensive set of genes and proteins that define neuronal cells. DHEA was also able to induce the division of neuronal-competent MSCs, thereby increasing the number of cells with major neuronal characteristics. To our knowledge, this is the first report which shows that DHEA can induce the division and differentiation of MSCs into neurons in vitro and should provide an improved basis for new treatments using MSCs of a wide variety of neurological diseases.

Neural differentiation from human embryonic stem cells in a defined adherent culture condition.

Understanding how to direct human embryonic stem cells (hESCs) toward a specific lineage pathway and generate appropriate cell types robustly is very important, not only for the study of developmental biology but also for potentially using these cells to treat human diseases. In this study, hESCs were differentiated to the neural lineage in defined adherent culture by retinoic acid and basic fibroblast growth factor. Our protocol seems to recapitulate the early steps of nervous system development in vivo in that undifferentiated hESCs organized into rosettes and then neural tube-like structures are formed. Differentiating cells expressed neuroectodermal and mature neuron markers during neural plate and tube formation and maturation, as shown by reverse transcriptase-PCR. More than 90% of differentiated cells expressed additional neuron-specific antigens (i.e., tubulin-III, MAP-2, synaptophysin and neurofilament protein). Ultrastructural analysis of differentiating neural tube-like structures in three dimensional collagen scaffolds showed an ependymal-like layer and neural structure with typical synapses. These results provide a simple and relatively defined system for differentiation of hESCs to neural lineages, particularly neurons with typical cellular, molecular and ultrastuctureal markers. The culture of neural precursor cells in a collagen scaffold may provide a new approach for the repair of spinal cord injury.