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Background: ATP-binding cassette (ABC) transporters have key roles in various physiological functions as well as providing chemical defense and stress tolerance in human tissues. In this study, we have examined the expression pattern of all ABC proteins in pluripotent human embryonic stem cells (hESCs) and in their differentiated progenies. We paid special attention to the cellular expression and localization of multidrug transporter ABC proteins., Methods: Stem cell differentiation was carried out without chemical induction or cell sorting, and specialized cell types were separated mechanically. Cellular features regarding pluripotency and tissue identity, as well as ABC transporter expression were studied by flow cytomtery, immuno-microscopy, and qPCR-based low-density arrays., Results: Pluripotent hESCs and differentiated cell types (cardiomyocytes, neuronal cells, and mesenchymal stem cells) were distinguished by morphology, immunostaining markers, and selected mRNA expression patterns. We found that the mRNA expression levels of the 48 human ABC proteins also clearly distinguished the pluripotent and the respective differentiated cell types. When multidrug and lipid transporter ABC protein expression was examined by using well characterized specific antibodies by flow cytometry and confocal microscopy, the protein expression data corresponded well to the mRNA expression results. Moreover, the cellular localization of these important human ABC transporter proteins could be established in the pluripotent and differentiated hESC derived samples., Conclusions: These studies provide valuable information regarding ABC protein expression in human stem cells and their differentiated offspring. The results may also help to obtain further information concerning the specialized cellular functions of selected ABC transporters., (© 2014 Clinical Cytometry Society.)

Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Maturation of microRNAs (miRNAs) begins by the "Microprocessor" complex, containing the Drosha endonuclease and its partner protein, "DiGeorge Syndrome Critical Region 8" (DGCR8). Although the main function of the two proteins is to coordinate the first step of precursor miRNAs formation, several studies revealed their miRNA-independent functions in other RNA-related pathways (e.g., in snoRNA decay) or, for the DGCR8, the role in tissue development. To investigate the specific roles of DGCR8 in various cellular pathways, we previously established a human embryonic stem-cell (hESC) line carrying a monoallelic DGCR8 mutation by using the CRISPR-Cas9 system. In this study, we genetically characterized single-cell originated progenies of the cell line and showed that DGCR8 heterozygous mutation results in only a modest effect on the mRNA level but a significant decrease at the protein level. Self-renewal and trilineage differentiation capacity of these hESCs were not affected by the mutation. However, partial disturbance of the Microprocessor function could be revealed in pri-miRNA processing along the human chromosome 19 miRNA cluster in several clones. With all these studies, we can demonstrate that the mutant hESC line is a good model to study not only miRNA-related but also other "noncanonical" functions of the DGCR8 protein. [ABSTRACT FROM AUTHOR]

One of the most promising applications of human pluripotent stem cells is their utilization for human-based pharmacological models. Despite the fact that membrane transporters expressed in the liver play pivotal role in various hepatic functions, thus far only little attention was devoted to the membrane transporter composition of the stem cell-derived liver models. In the present work, we have differentiated HUES9, a human embryonic stem cell line, toward the hepatic lineage, and monitored the expression levels of numerous differentiation marker and liver transporter genes with special focus on ABC transporters. In addition, the effect of bile acid treatment and polarizing culturing conditions on hepatic maturation has been assessed. We found that most transporter genes crucial for hepatic functions are markedly induced during hepatic differentiation; however, as regards the transporter composition the end-stage cells still exhibited dual, hepatocyte and cholangiocyte character. Although the bile acid treatment and sandwich culturing only slightly influenced the gene expressions, the stimulated cell polarization resulted in formation of bile canaliculi and proper localization of transporters. Our results point to the importance of membrane transporters in human stem cell-derived hepatic models and demonstrate the relevance of cell polarization in generation of applicable cellular models with correctly localized transporters. On the basis of our observations we suggest that conventional criteria for the evaluation of the quality of stem cell-derived hepatocyte-like cells ought to be augmented with additional elements, such as polarized and functional expression of hepatic transporters. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cells provide new tools for developmental and pharmacological studies as well as for regenerative medicine applications. Calcium homeostasis and ligand-dependent calcium signaling are key components of major cellular responses, including cell proliferation, differentiation or apoptosis. Interestingly, these phenomena have not been characterized in detail as yet in pluripotent human cell sates. Here we review the methods applicable for studying both short- and long-term calcium responses, focusing on the expression of fluorescent calcium indicator proteins and imaging methods as applied in pluripotent human stem cells. We discuss the potential regulatory pathways involving calcium responses in hPS cells and compare these to the implicated pathways in mouse PS cells. A recent development in the stem cell field is the recognition of so called “naïve” states, resembling the earliest potential forms of stem cells during development, as well as the “fuzzy” stem cells, which may be alternative forms of pluripotent cell types, therefore we also discuss the potential role of calcium homeostasis in these PS cell types. [ABSTRACT FROM AUTHOR]

ABCG2 is a plasma membrane multidrug transporter with an established role in the cancer drug-resistance phenotype. This protein is expressed in a variety of tissues, including several types of stem cell. Although ABCG2 is not essential for life, knock-out mice were found to be hypersensitive to xenobiotics and had reduced levels of the side population of hematopoietic stem cells. Previously we have shown that ABCG2 is present in human embryonic stem cell (hESC) lines, with a heterogeneous expression pattern. In this study we examined this heterogeneity, and investigated whether it is related to stress responses in hESCs. We did not find any difference between expression of pluripotency markers in ABCG2-positive and negative hESCs; however, ABCG2-expressing cells had a higher growth rate after cell separation. We found that some harmful conditions (physical stress, drugs, and UV light exposure) are tolerated much better in the presence of ABCG2 protein. This property can be explained by the transporter function which eliminates potential toxic metabolites accumulated during stress conditions. In contrast, mild oxidative stress in hESCs caused rapid internalization of ABCG2, indicating that some environmental factors may induce removal of this transporter from the plasma membrane. On the basis of these results we suggest that a dynamic balance of ABCG2 expression at the population level has the advantage of enabling prompt response to changes in the cellular environment. Such actively maintained heterogeneity might be of evolutionary benefit in protecting special cell types, including pluripotent stem cells. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) are promising tools to model complex neurological or psychiatric diseases, including schizophrenia. Multiple studies have compared patient-derived and healthy control NPCs derived from iPSCs in order to investigate cellular phenotypes of this disease, although the establishment, stabilization, and directed differentiation of iPSC lines are rather expensive and time-demanding. However, interrupted reprogramming by omitting the stabilization of iPSCs may allow for the generation of a plastic stage of the cells and thus provide a shortcut to derive NPSCs directly from tissue samples. Here, we demonstrate a method to generate shortcut NPCs (sNPCs) from blood mononuclear cells and present a detailed comparison of these sNPCs with NPCs obtained from the same blood samples through stable iPSC clones and a subsequent neural differentiation (classical NPCs—cNPCs). Peripheral blood cells were obtained from a schizophrenia patient and his two healthy parents (a case–parent trio), while a further umbilical cord blood sample was obtained from the cord of a healthy new-born. The expression of stage-specific markers in sNPCs and cNPCs were compared both at the protein and RNA levels. We also performed functional tests to investigate Wnt and glutamate signaling and the oxidative stress, as these pathways have been suggested to play important roles in the pathophysiology of schizophrenia. We found similar responses in the two types of NPCs, suggesting that the shortcut procedure provides sNPCs, allowing an efficient screening of disease-related phenotypes. [ABSTRACT FROM AUTHOR]

Background: De novo mutations (DNMs) have been implicated in the etiology of schizophrenia (SZ), a chronic debilitating psychiatric disorder characterized by hallucinations, delusions, cognitive dysfunction, and decreased community functioning. Several DNMs have been identified by examining SZ cases and their unaffected parents; however, in most cases, the biological significance of these mutations remains elusive. To overcome this limitation, we have developed an approach of using induced pluripotent stem cell (iPSC) lines from each member of a SZ case-parent trio, in order to investigate the effects of DNMs in cellular progenies of interest, particularly in dentate gyrus neuronal progenitors. Methods: We identified a male SZ patient characterized by early disease onset and negative symptoms, who is a carrier of 3 non-synonymous DNMs in genes LRRC7, KHSRP, and KIR2DL1. iPSC lines were generated from his and his parents' peripheral blood mononuclear cells using Sendai virus-based reprogramming and differentiated into neuronal progenitor cells (NPCs) and hippocampal dentate gyrus granule cells. We used RNASeq to explore transcriptomic differences and calcium (Ca2+) imaging, cell proliferation, migration, oxidative stress, and mitochondrial assays to characterize the investigated NPC lines. Results: NPCs derived from the SZ patient exhibited transcriptomic differences related to Wnt signaling, neuronal differentiation, axonal guidance and synaptic function, and decreased Ca2+ reactivity to glutamate. Moreover, we could observe increased cellular proliferation and alterations in mitochondrial quantity and morphology. Conclusions: The approach of reprograming case-parent trios represents an opportunity for investigating the molecular effects of disease-causing mutations and comparing these in cell lines with reduced variation in genetic background. Our results are indicative of a partial overlap between schizophrenia and autism-related phenotypes in the investigated family. Limitations: Our study investigated only one family; therefore, the generalizability of findings is limited. We could not derive iPSCs from two other siblings to test for possible genetic effects in the family that are not driven by DNMs. The transcriptomic and functional assays were limited to the NPC stage, although these variables should also be investigated at the mature neuronal stage. [ABSTRACT FROM AUTHOR]

Cardiac cell lines and primary cells are widely used in cardiovascular research. Despite increasing number of publications using these models, comparative characterization of these cell lines has not been performed, therefore, their limitations are undetermined. We aimed to compare cardiac cell lines to primary cardiomyocytes and to mature cardiac tissues in a systematic manner. Cardiac cell lines (H9C2, AC16, HL-1) were differentiated with widely used protocols. Left ventricular tissue, neonatal primary cardiomyocytes, and human induced pluripotent stem cell-derived cardiomyocytes served as reference tissue or cells. RNA expression of cardiac markers (e.g. Tnnt2 , Ryr2) was markedly lower in cell lines compared to references. Differentiation induced increase in cardiac- and decrease in embryonic markers however, the overall transcriptomic profile and annotation to relevant biological processes showed consistently less pronounced cardiac phenotype in all cell lines in comparison to the corresponding references. Immunocytochemistry confirmed low expressions of structural protein sarcomeric alpha-actinin, troponin I and caveolin-3 in cell lines. Susceptibility of cell lines to sI/R injury in terms of viability as well as mitochondrial polarization differed from the primary cells irrespective of their degree of differentiation. Expression patterns of cardiomyocyte markers and whole transcriptomic profile, as well as response to sI/R, and to hypertrophic stimuli indicate low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless their differentiation. Low resemblance of cell lines to mature adult cardiac tissue limits their potential use. Low translational value should be taken into account while choosing a particular cell line to model cardiomyocytes. [Display omitted] • Immortalized cardiac cell lines show low resemblance to mature cardiac cells. • Organized sarcomeric structure is detectable in HL-1 cells but not in H9C2 or AC16. • Differentiation of AC16 or H9C2 induces minor changes towards cardiac phenotype. • Functional tests reveal the restricted responsiveness of all the cardiac cell lines. • This is the first systematic study showing major limitations of cardiac cell lines. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the biological significance of DNMs in SCZ by combining induced pluripotent stem cell (IPSC) based disease modeling and CRISPR based genome editing. B Discussion: b The combination of CRISPR based genome editing and induced pluripotent stem cell-based disease modeling can shed light on the molecular disease pathway underlying schizophrenia. [Extracted from the article]

Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation. [ABSTRACT FROM AUTHOR]

Induced Pluripotent Stem Cells (IPSCs) have the capability to differentiate into any kind of cell types, such as Neuronal Progenitor Cells (NPCs), and then to mature neurons. By this new approach we can investigate neuronal differentiation processes or the role of specific genes using genome editing techniques (e.g., ZFN, TALEN, or CRISPR). For example, the neuron-specific roles of the disrupted in schizophrenia 1 (DISC1) can be studied in a simple, Petri-dish based model. The DISC1gene has been intensively studied over the last decades after the description of a balanced translocation of t(1;11)(q42.1;q14.3) in a multigenerational Scottish family with several cases of psychosis and affective disorders, followed by the identification of a frameshift DISC1 mutation in an American family affected with schizophrenia. Previous molecular biological experiments have shown that DISC1 scaffold protein can affect neuronal proliferation and migration, and synapse maintenance. Animal model findings also pointed to its role in hippocampal neurogenesis, which is a crucial neuronal mechanism affected in several psychiatric disorders. We aimed to characterize the possible roles of DISC1 in vitro neuronal cell culture. We applied an isogenic IPSC line in a model of hippocampal dentate gyrus neurogenesis, given that impaired hippocampal neurogenesis has been implicated in the pathogenesis of both schizophrenia and affective disorders. IPSC line XCL1 was engineered using zinc finger nuclease technology to produce an isogenic DISC1 exon-2 biallelic knockout cell line (XCL1 DISC1 exon-2 KO/KO). The locus of the genetic engineering was selected because of the frameshift mutation. The isogenic IPSC pair was characterized in terms of pluripotency and spontaneous differentiation potential. Following this, IPSC lines were differentiated into neuronal lineages using established protocols to generate hippocampal dentate gyrus granule cells. The dentate gyrus protocol is founded on dual SMAD inhibition, Wnt- and SHH inhibition, after generating free-floated embryoid bodies from pluripotent stem cells, which results in forebrain NPCs. We have successfully derived and characterized NPCs and then matured neurons from human IPSC XCL1 and deletion pair, which were differentiated according to the DG PROX1 protocol. There was no significant difference at the spontaneous differentiation potential between the isogenic IPSC XCL1 lines. The immunofluorescence staining showed that NPCs express Nestin and Sox2. This intermediate cell type can be further differentiated into PROX1 and MAP2 positive functional neurons which have dendritic spines. Till now we have not found significant differences between wild type and knock out cell lines based on induced outgrowth measurement and structure of ICC. We have found some alteration in Ca-dynamics between the two cell lines, however these measurements need further investigation. The generated neuronal progenitor cells and mature neurons carrying biallelic knockout of the DISC1 gene were amenable for assays comparing the isogenic pairs by immunofluorescence microscopy, and transcriptomics. Functional studies, such as calcium-imaging assays, single cell patch-clamp electrophysiology and real-time live cell visualization of induced neurite outgrowth are under way to further. These investigations may help to explore the role of DISC1 in hippocampal neurogenesis and the etiology of psychiatric disorders. [ABSTRACT FROM AUTHOR]