Melinda Pirity - Academia.edu (original) (raw)

Papers by Melinda Pirity

Blood–brain barrier (BBB) models derived from human stem cells are powerful tools to improve our ... more Blood–brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of cerebrovascular diseases and to facilitate drug devel- opment for the human brain. Yet providing stem cell–derived endothelial cells with the right signaling cues to acquire BBB characteristics while also retaining their vas- cular identity remains challenging. Here, we show that the simultaneous activation of cyclic AMP and Wnt/β-catenin signaling and inhibition of the TGF-β pathway in endothelial cells robustly induce BBB properties in vitro. To target this interaction, we present a small-molecule cocktail named cARLA, which synergistically enhances barrier tightness in a range of BBB models across species. Mechanistically, we reveal that the three pathways converge on Wnt/β-catenin signaling to mediate the effect of cARLA via the tight junction protein claudin-5. We demonstrate that cARLA shifts the gene expressional profile of human stem cell–derived endothelial cells toward the in vivo brain endothelial signature, with a higher glycocalyx density and efflux pump activity, lower rates of endocytosis, and a characteristic endothelial response to proinflammatory cytokines. Finally, we illustrate how cARLA can improve the predictive value of human BBB models regarding the brain penetration of drugs and targeted nanoparticles. Due to its synergistic effect, high reproducibility, and ease of use, cARLA has the potential to advance drug development for the human brain by improving BBB models across laboratories.

Transgenic Research, 2010

Scientific Reports

We have previously reported that RING1 and YY1 binding protein (RYBP) is important for central ne... more We have previously reported that RING1 and YY1 binding protein (RYBP) is important for central nervous system development in mice and that Rybp null mutant (Rybp−/−) mouse embryonic stem (ES) cells form more progenitors and less terminally differentiated neural cells than the wild type cells in vitro. Accelerated progenitor formation coincided with a high level of Pax6 expression in the Rybp−/− neural cultures. Since Pax6 is a retinoic acid (RA) inducible gene, we have analyzed whether altered RA signaling contributes to the accelerated progenitor formation and impaired differentiation ability of the Rybp−/− cells. Results suggested that elevated Pax6 expression was driven by the increased activity of the RA signaling pathway in the Rybp−/− neural cultures. RYBP was able to repress Pax6 through its P1 promoter. The repression was further attenuated when RING1, a core member of ncPRC1s was also present. According to this, RYBP and PAX6 were rarely localized in the same wild type cell...

Molecular and cellular biology, 2005

The Rybp/DEDAF protein has been implicated in both transcriptional regulation and apoptotic signa... more The Rybp/DEDAF protein has been implicated in both transcriptional regulation and apoptotic signaling, but its precise molecular function is unclear. To determine the physiological role of Rybp, we analyzed its expression during mouse development and generated mice carrying a targeted deletion of Rybp using homologous recombination in embryonic stem cells. Rybp was found to be broadly expressed during embryogenesis and was particularly abundant in extraembryonic tissues, including trophoblast giant cells. Consistent with this result, rybp homozygous null embryos exhibited lethality at the early postimplantation stage. At this time, Rybp was essential for survival of the embryo, for the establishment of functional extraembryonic structures, and for the execution of full decidualization. Through the use of a chimeric approach, the embryonic lethal phenotype was circumvented and a role for Rybp in central nervous system development was uncovered. Specifically, the presence of Rybp-defi...

Biotechnology Letters, 2011

Embryoid body (EB) formation is a common intermediate during in vitro differentiation of pluripot... more Embryoid body (EB) formation is a common intermediate during in vitro differentiation of pluripotent stem cells into specialized cell types. We have optimized the slow-turning, lateral vessel (STLV) for large scale and homogenous EB production from mouse embryonic stem cells. The effects of inoculating different cell numbers, time of EB adherence to gelatin-coated dishes, and rotation speed for optimal EB formation and cardiac differentiation were investigated. Using 3x10 5 cells/ml, 10 rpm rotary speed and plating of EBs onto gelatin-coated surfaces three days after culture, were the best parameters for optimal size and EB quality on consequent cardiac differentiation. These optimized parameters enrich cardiac differentiation in ES cells when using the STLV method.

Experimental Cell Research, 2014

One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific c... more One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific cells which can be used to obtain multiple types of differentiated cells as disease models. Minimally or non-integrating methods to deliver the reprogramming genes are considered to be the best but they may be inefficient. Lentiviral delivery is currently among the most efficient methods but it integrates transgenes into the genome, which may affect the behavior of the iPSC if integration occurs into an important locus. Here we designed a polycistronic lentiviral construct containing four pluripotency genes with an EGFP selection marker. The cassette was excisable with the Cre-loxP system making possible the removal of the integrated transgenes from the genome. Mouse embryonic fibroblasts were reprogrammed using this viral system, rapidly resulting in large number of iPSC colonies. Based on the lowest EGFP expression level, one parental line was chosen for excision. Introduction of the Cre recombinase resulted in transgene-free iPSC subclones. The effect of the transgenes was assessed by comparing the parental iPSC with two of its transgene-free subclones. Both excised and non-excised iPSCs expressed standard pluripotency markers. The subclones obtained after Cre recombination were capable of differentiation in vitro, in contrast to the parental, non-excised cells and formed germ-line competent chimeras in vivo.

Reproduction, Fertility and Development, 2012

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate to all the s... more Pluripotent stem cells have the capacity to divide indefinitely and to differentiate to all the somatic tissues. They can be genetically manipulated in vitro by knocking in and out genes, therefore they serve as an excellent tool for gene-function studies and for the generation of models for human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, several attempts have been made to generate pluripotent stem cells from other species as it would help us to understand the differences and similarities of signaling pathways involved in pluripotency and differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved among different species. This review gives an overlook of embryonic and induced pluripotent stem cell (iPSCs) research in the rabbit which is one of the most relevant non-rodent species for animal models. To date, several lines of putative ESCs and iPSCs have been described in ...

Experimental Cell Research, 2012

Induced pluripotent stem (iPS) cell technology involves reprogramming somatic cells to a pluripot... more Induced pluripotent stem (iPS) cell technology involves reprogramming somatic cells to a pluripotent state. The original technology used to produce these cells requires viral gene transduction and results in the permanent integration of exogenous genes into the genome. This can lead to the development of abnormalities in the derived iPS cells. Here, we report that non-viral transfection of a Sleeping Beauty (SB) transposon containing the coding sequences Oct3/4 (Pouf1), Sox-2, Klf-4 and c-Myc (OSKM) linked with 2A peptides, can reprogram mouse fibroblasts. We have established reprogrammed mouse cell lines from three different genetic backgrounds: (1) ICR-outbred, (2) C57BL/6-inbred and (3) F1-hybrid (C57BL/6 x DBA/2J), with parallel robust expression of all exogenous (Oct3/4, Sox-2, Klf-4, and c-Myc) and endogenous (e.g. Oct3/4 and Nanog) pluripotency genes. The iPS cell lines exhibited characteristics typical for undifferentiated embryonic stem (ES) cell lines: ES cell-like morphology, alkaline phosphatase (ALP) positivity and gene expression pattern (shown by reverse transcription PCR, and immunofluorescence of ES cell markers-e.g. Oct3/4, SSEA1, Nanog). Furthermore, cells were able to form embryoid bodies (EBs), to beat rhythmically, and express cardiac (assayed by immunofluorescence, e.g. cardiac Troponin T, desmin) and neuronal (assayed by immunofluorescence e.g. nestin, Tuj1) markers. The in vitro differentiation potential was found to be the highest in the ICR-derived iPS lines (ICR-iPS). Interestingly, the ICR-iPS lines had even higher differentiation potential than the ICR-ES cell lines: the rate of EBs forming rhythmically beating cardiomyocytes was 4% in ICR-ES and 79% in ICR-iPS cells, respectively. In vivo, the ICR and F1 hybrid iPS cells formed chimeras and one of the iPS cells from the F1 hybrid background transmitted to the germline. Our results suggest that iPS technology may be useful for generating pluripotent stem cells from genetic backgrounds of which good quality ES cell generation is difficult. These studies provide insights into viral-free iPS technology and may contribute towards defining future cell-based therapies, drug-screening methods and production of transgenic animals using genetically modified iPS cells.

BMC Molecular Biology, 2009

Background The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is cruci... more Background The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs). The objective of this study was to identify and to analyse the promoter region of rabbit POU5F1 gene; furthermore to examine its expression pattern in preimplantation stage rabbit embryos. Results The upstream region of rabbit POU5F1 was subcloned sequenced and four highly conserved promoter regions (CR1-4) were identified. The highest degree of similarity on sequence level was found among the conserved domains between rabbit and human. Among the enhancers the proximal enhancer region (PE-1A) exhibited the highest degree of homology (96.4%). Furthermore, the CR4 regulator domain containing the distal enhancer (DE-2A) was responsible for stem cell-specific expression. Also, BAC library screen revealed the existence of a processed pseudogene of rabbit POU5F1...

We have previously established that epigenetic regulator RING1 and YY1 binding protein (RYBP) is ... more We have previously established that epigenetic regulator RING1 and YY1 binding protein (RYBP) is required for the contractility of embryonic stem (ES) cell derived cardiomyocytes (CMCs), suggesting its essential role in contractility. In order to investigate the underlying molecular events of this phenotype, we compared the transcriptomic profile of the wild type and Rybp null mutant ES cells and CMCs differentiated from these cell lines. We identified genes related to ion homeostasis, cell adhesion and sarcomeric organization affected in the Rybp null mutant CMCs, by using hierarchical gene clustering and Gene Ontology analysis. We have also demonstrated that the amount of RYBP is drastically reduced in the terminally differentiated wild type CMCs whilst it is broadly expressed in the early phase of differentiation when progenitors form. We also describe that RYBP is important for the proper expression of key cardiac transcription factors including Mesp1, Shh and Mef2c. These findi...

Genes

Separation of germline cells from somatic lineages is one of the earliest decisions of embryogene... more Separation of germline cells from somatic lineages is one of the earliest decisions of embryogenesis. Genes expressed in germline cells include apoptotic and meiotic factors, which are not transcribed in the soma normally, but a number of testis-specific genes are active in numerous cancer types. During germ cell development, germ-cell-specific genes can be regulated by specific transcription factors, retinoic acid signaling and multimeric protein complexes. Non-canonical polycomb repressive complexes, like ncPRC1.6, play a critical role in the regulation of the activity of germ-cell-specific genes. RING1 and YY1 binding protein (RYBP) is one of the core members of the ncPRC1.6. Surprisingly, the role of Rybp in germ cell differentiation has not been defined yet. This review is focusing on the possible role of Rybp in this process. By analyzing whole-genome transcriptome alterations of the Rybp-/- embryonic stem (ES) cells and correlating this data with experimentally identified bin...

Epigenomes

Originally two types of Polycomb Repressive Complexes (PRCs) were described, canonical PRC1 (cPRC... more Originally two types of Polycomb Repressive Complexes (PRCs) were described, canonical PRC1 (cPRC1) and PRC2. Recently, a versatile set of complexes were identified and brought up several dilemmas in PRC mediated repression. These new class of complexes were named as non-canonical PRC1s (ncPRC1s). Both cPRC1s and ncPRC1s contain Ring finger protein (RING1, RNF2) and Polycomb group ring finger catalytic (PCGF) core, but in ncPRCs, RING and YY1 binding protein (RYBP), or YY1 associated factor 2 (YAF2), replaces the Chromobox (CBX) and Polyhomeotic (PHC) subunits found in cPRC1s. Additionally, ncPRC1 subunits can associate with versatile accessory proteins, which determine their functional specificity. Homozygous null mutations of the ncPRC members in mice are often lethal or cause infertility, which underlines their essential functions in mammalian development. In this review, we summarize the mouse knockout phenotypes of subunits of the six major ncPRCs. We highlight several aspects of their discovery from fly to mice and emerging role in target recognition, embryogenesis and cell-fate decision making. We gathered data from stem cell mediated in vitro differentiation assays and genetically engineered mouse models. Accumulating evidence suggests that ncPRC1s play profound role in mammalian embryogenesis by regulating gene expression during lineage specification of pluripotent stem cells.

Methods in Cell Biology, Feb 1, 1998

Embryonic stem (ES) cells have afforded a means of directly modifying the mouse genome in vitro a... more Embryonic stem (ES) cells have afforded a means of directly modifying the mouse genome in vitro and then introducing such changes directly into animals. The advent of this technology has made the mouse the mammal of choice for mutagenesis approaches used in the study of embryonic development and disease conditions. This chapter deals with the maintenance and modification of these pluripotent cell lines and describes the routes that can be taken for their efficient introduction to the in vivo environment.

Stem Cells International, 2016

Rybp (Ring1 and Yy1 Binding Protein) is a transcriptional regulator and member of the noncanonica... more Rybp (Ring1 and Yy1 Binding Protein) is a transcriptional regulator and member of the noncanonical polycomb repressive complex 1 with essential role in early embryonic development. We have previously described that alteration of Rybp dosage in mouse models induced striking neural tube defects (NTDs), exencephaly, and disorganized neurocortex. In this study we further investigated the role of Rybp in neural differentiation by utilising wild type (rybp+/+) andrybp nullmutant (rybp-/-) embryonic stem cells (ESCs) and tried to uncover underlying molecular events that are responsible for the observed phenotypic changes. We found thatrybp nullmutant ESCs formed less matured neurons, astrocytes, and oligodendrocytes from existing progenitors than wild type cells. Furthermore, lack ofrybpcoincided with altered gene expression of key neural markers including Pax6 and Plagl1 pinpointing a possible transcriptional circuit among these genes.

Stem Cells and Development, 2015

Ring1 and Yy1 Binding Protein (Rybp) has been implicated in transcriptional regulation, apoptotic... more Ring1 and Yy1 Binding Protein (Rybp) has been implicated in transcriptional regulation, apoptotic signaling and as a member of the polycomb repressive complex 1 has important function in regulating pluripotency and differentiation of embryonic stem cells. Earlier, we have proven that Rybp plays essential role in mouse embryonic and central nervous system development. This work identifies Rybp, as a critical regulator of heart development. Rybp is readily detectable in the developing mouse heart from day 8.5 of embryonic development. Prominent Rybp expression persists during all embryonic stages and Rybp marks differentiated cell types of the heart. By utilizing rybp null embryonic stem cells (ESCs) in an in vitro cardiac differentiation assay we found that rybp null ESCs do not form rhythmically beating cardiomyocytes. Gene expression profiles revealed a down-regulation of terminal cardiac and upregulation of germ line specific markers in the rybp null cardiomyocytes. Furthermore, transcriptome analysis uncovered a number of novel candidate target genes regulated by Rybp. Among these are several important in cardiac development and contractility such as Plagl1, Isl1,

Experimental Neurology, 2015

This is the peer reviewed version of the following article: Grafted murine induced pluripotent st... more This is the peer reviewed version of the following article: Grafted murine induced pluripotent stem cells prevent death of injured rat motoneurons otherwise destined to die.

Molecular genetics and metabolism, Jan 30, 2015

POLYCOMB group (PCG) proteins belong to the family of epigenetic regulators of genes playing impo... more POLYCOMB group (PCG) proteins belong to the family of epigenetic regulators of genes playing important roles in differentiation and development. Mutants of PcG genes were isolated first in the fruit fly, Drosophila melanogaster, resulting in spectacular segmental transformations due to the ectopic expression of homeotic genes. Homologs of Drosophila PcG genes were also identified in plants and in vertebrates and subsequent experiments revealed the general role of PCG proteins in the maintenance of the repressed state of chromatin through cell divisions. The past decades of gene targeting experiments have allowed us to make significant strides towards understanding how the network of PCG proteins influences multiple aspects of cellular fate determination during development. Being involved in the transmission of specific expression profiles of different cell lineages, PCG proteins were found to control wide spectra of unrelated epigenetic processes in vertebrates, such as stem cell pl...

Cellular reprogramming, 2012

Mouse embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells can be used as models ... more Mouse embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells can be used as models of neuronal differentiation for the investigation of mammalian neurogenesis, pharmacological testing, and development of cell-based therapies. Recently, mouse iPS cell lines have been generated by Sleeping Beauty (SB) transposon-mediated transgenesis (SB-iPS). In this study, we determined for the first time the differentiation potential of mouse SB-iPS cells to form neuronal progenitor cells (NPCs) and neurons. Undifferentiated SB-iPS and ES cells were aggregated into embryoid bodies (EBs) and cultured in neuronal differentiation medium supplemented with 5 μM all-trans retinoic acid. Thereafter, EBs were dissociated and plated to observe further neuronal differentiation. Samples were fixed on days 10 and 14 for immunocytochemistry staining using the NPC markers Pax6 and Nestin and the neuron marker βIII-tubulin/Tuj1. Nestin-labeled cells were analyzed further by flow cytometry. Our resul...

Anticancer research

Hyperthermia is used in the treatment of some human malignancies. Thermotolerance may interfere w... more Hyperthermia is used in the treatment of some human malignancies. Thermotolerance may interfere with the efficacy of hyperthermic treatment, and thermotolerant cells may also display an enhanced resistance to some anticancer drugs. We have earlier isolated stable heat-resistant rat hepatoma variants and examined whether heat resistance influenced the drug sensitivity of the cells. Heat-resistant variants were isolated by ten repeated cycles of heat exposure at 45 degrees C for 80 min. Highly multidrug-resistant variants were isolated by stepwise selection with colchicine. The heat-resistant variants became moderately multidrug resistant. This resistance was further increased by stepwise selection with colchicine (highly multidrug resistant variants). The levels of P-glycoprotein were elevated both in moderately and highly drug resistant variants. Decreased retention of antitumor drugs was observed in the multidrug resistant variants, verapamil increased doxorubicin retention signifi...

Methods in cell biology, 1998