Papers by Julianna Kobolak
Cellular reprogramming, 2010
Pluripotent stem cells would have great potential in cell therapies and drug development when gen... more Pluripotent stem cells would have great potential in cell therapies and drug development when genetically matched with the patient; thus, histocompatible cells could be used in transplantation therapy or as a source of patient-specific cells for drug testing. Pluripotent embryonic stem cells (ESCs)-generated via somatic cell nuclear transfer (SCNT) or parthenogenesis (pESC)-are potential sources of histocompatible cells and tissues for transplantation. Earlier studies used the piezoelectric microinjection (PEM) technique for nuclear transfer (NT) in mouse. No specific studies examined zona-free (ZF) NT as an alternative NT method to generate genetically matched ESCs of a nuclear donor. In this study, we compared the efficiency of nuclear transfer-derived ESC (ntESC) line establishment from ZF-NT, ZF-parthenogenetic (PGA), and ZF-fertilized embryos with that of the PEM-NT method. Different nuclei donor cells [cumulus, ESC, and mouse embryonic fibroblast (MEF)] were used and the effic...
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Cellular reprogramming, 2012
Embryonic stem cells derived from nuclear transfer embryos (ntESCs) are particularly valuable for... more Embryonic stem cells derived from nuclear transfer embryos (ntESCs) are particularly valuable for regenerative medicine, as they are a patient-specific and histocompatible cell source for the treatment of varying diseases. However, currently, little is known about their cellular and molecular profile. In the present study, in a mouse model different donor cell-derived ntESCs from various genetic backgrounds were compared with reference ESCs and analyzed comprehensively at the cellular level. A number of pluripotency marker genes were compared by flow cytometry and immunocytochemistry analysis. Significant differences at the protein level were observed for POU5F1, SOX2, FGF4, NANOG, and SSEA-1. However, such differences had no effect on in vitro cell differentiation and cell fate: derivatives of the three germ layers were detected in all ntESC lines. The neural and cardiac in vitro differentiation revealed minor differences between the cell lines, both at the mRNA and protein level. ...
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Stem Cells International, 2016
Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are trad... more Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers, PAX6 and NESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers GFAP and aquaporin 4 (AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes.
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Cytokine, 2011
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Methods, 2015
Mesenchymal stem cells (MSCs) are multipotent stem cells. Although they were originally identifie... more Mesenchymal stem cells (MSCs) are multipotent stem cells. Although they were originally identified in bone marrow and described as 'marrow stromal cells', they have since been identified in many other anatomical locations in the body. MSCs can be isolated from bone marrow, adipose tissue, umbilical cord and other tissues but the richest tissue source of MSCs is fat. Since they are adherent to plastic, they may be expanded in vitro. MSCs have a distinct morphology and express a specific set of CD (cluster of differentiation) molecules. The phenotypic pattern for the identification of MSCs cells requires expression of CD73, CD90, and CD105 and lack of CD34, CD45, and HLA-DR antigens. Under appropriate micro-environmental conditions MSCs can proliferate and give rise to other cell types. Therefore, they are ideally suited for the treatment of systemic inflammatory and autoimmune conditions. They have also been implicated as key players in regenerating injured tissue following injury and trauma. MSC populations isolated from adipose tissue may also contain regulatory T (Treg) cells, which have the capacity for modulating the immune system. The immunoregulatory and regenerative properties of MSCs make them ideal for use as therapeutic agents in vivo. In this paper we review the literature on the identification, phenotypic characterization and biological properties of MSCs and discuss their potential for applications in cell therapy and regenerative medicine. We also discuss strategies for biomaterial micro-engineering of the stem cell niche.
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Reproduction, Fertility and Development, 2007
ABSTRACT
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BMC cancer, 2015
The cancer stem cell (CSC) hypothesis suggests that only a subpopulation of cells within a tumour... more The cancer stem cell (CSC) hypothesis suggests that only a subpopulation of cells within a tumour is responsible for the initiation and progression of neoplasia. The original and best evidence for the existence of CSCs came from advances in the field of haematological malignancies. Thus far, putative CSCs have been isolated from various solid and non-solid tumours and shown to possess self-renewal, differentiation, and cancer regeneration properties. Although research in the field is progressing extremely fast, proof of concept for the CSC hypothesis is still lacking and key questions remain unanswered, e.g. the cell of origin for these cells. Nevertheless, it is undisputed that neoplastic transformation is associated with genetic and epigenetic alterations of normal cells, and a better understanding of these complex processes is of utmost importance for developing new anti-cancer therapies. In the present review, we discuss the CSC hypothesis with special emphasis on age-associated...
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Cellular reprogramming, 2010
Pluripotent stem cells would have great potential in cell therapies and drug development when gen... more Pluripotent stem cells would have great potential in cell therapies and drug development when genetically matched with the patient; thus, histocompatible cells could be used in transplantation therapy or as a source of patient-specific cells for drug testing. Pluripotent embryonic stem cells (ESCs)-generated via somatic cell nuclear transfer (SCNT) or parthenogenesis (pESC)-are potential sources of histocompatible cells and tissues for transplantation. Earlier studies used the piezoelectric microinjection (PEM) technique for nuclear transfer (NT) in mouse. No specific studies examined zona-free (ZF) NT as an alternative NT method to generate genetically matched ESCs of a nuclear donor. In this study, we compared the efficiency of nuclear transfer-derived ESC (ntESC) line establishment from ZF-NT, ZF-parthenogenetic (PGA), and ZF-fertilized embryos with that of the PEM-NT method. Different nuclei donor cells [cumulus, ESC, and mouse embryonic fibroblast (MEF)] were used and the effic...
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Reproduction, Fertility and Development, 2008
ABSTRACT
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Cellular reprogramming, 2012
In a mouse model nuclear transfer embryo-derived embryonic stem cell lines (ntESCs) of various ge... more In a mouse model nuclear transfer embryo-derived embryonic stem cell lines (ntESCs) of various genetic backgrounds and donor cell types were compared with reference ESCs and analyzed comprehensively at molecular level as a second part of a larger study. Expression profiles of ntESCs established by different NT-methods (piezoelectric microinjection or zona-free) were indistinguishable. However, expression profiling analyses identified differentially regulated genes between reference ESCs and ntESCs from different genetic backgrounds. A number of pluripotency and stemness marker genes significantly differed at the mRNA level between the cell lines. However, cluster and lineage analyses revealed that such differences had no effect on cell differentiation and cell fate. Regardless of the donor cell type, gene expression profiles of ntESCs were more similar to each other than to their counterpart fertilized embryo-derived ESCs of the same genotype. Overall, the results indicated that exp...
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Reproduction, Fertility and Development, 2006
ABSTRACT
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World Journal of Stem Cells, 2010
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Reproduction, Fertility and Development, 2006
ABSTRACT
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Molecular Reproduction and Development, 1997
The transient expression of reporter gene constructs in embryos provides a powerful tool to chara... more The transient expression of reporter gene constructs in embryos provides a powerful tool to characterise cis-acting transcriptional elements of the genes involved in development. In the present study, we have analysed the expression pattern of several muscle-specific and ubiquitous regulatory sequences in microinjected zebrafish embryos. By using a fast and reproducible coinjection strategy, the mosaic expression of lacZ reporter gene was monitored in wholemount embryos injected with sequences containing putative enhancer elements and a carp myosin heavy chain promoter/lacZ reporter construct. We have found that a 0.9-kb myosin heavy chain (MyHC) proximal promoter containing several putative myogenic regulatory factors (MRF) binding sites is sufficient to restrict lacZ expression to the skeletal muscle fibres of prim-6 stage zebrafish embryos. Expression of a rat-derived foetal myosin light chain enhancer (MyLC) and different fragments of a carp beta-actin regulatory region together with the MyHC promoter were compared by accumulating the type, number and spatial distribution of beta-galactosidase-expressing cells on an expression map. beta-galactosidase activity increased similarly whether the MyLC enhancer was ligated to the promoter/ reporter construct directly or when coinjected as a separate fragment whilst skeletal muscle specificity was retained. The coinjection of two different forms of the beta-actin regulatory elements also showed a marked effect on the MyHC promoter activity. The coinjection of putative enhancers with minimal promoter constructs and subsequent analysis of the transient expression pattern in the developing embryos provides a rapid and simple technique to identify cis acting activator elements of genes expressed in the vertebrate embryo.
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DNA Sequence, 2008
The product of the POUSF1 gene, Oct4, plays an important role both in embryonic development and i... more The product of the POUSF1 gene, Oct4, plays an important role both in embryonic development and in the self-renewal and differentiation of totipotent cells. To understand the function of Oct4 in rabbit ES cells, we cloned and sequenced the rabbit POU5F1 gene, as well as the cDNA encoded by the gene. The Oct4 cDNA contains a 1083 bp ORF encoding a 360 aa protein and a 241 bp 3' UTR sequence. Oct4 mRNA was expressed at a high level in rabbit ES cells and was barely detectable in the adult spleen, kidney, brain and muscle tissues. The POU5F1 gene is approximately 6 kb in length and includes five exons and four introns. Gene organization is similar to that of the mouse, human and bovine orthologs. Sequencing of the gene revealed an 82% (mouse), 90% (human) and 89% (bovine) overall identity at the protein level. The rabbit POUSF1 gene was mapped to chromosome 12q1.1 by PCR amplification of DNA from two putative POU5F1-containing BAC clones, which were previously mapped to chromosome 12q1.1. The cloning of the rabbit POU5F1 gene will facilitate studies on its roles in rabbit embryogenesis and ES cells.
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Journal of Proteomics, 2013
Mouse embryonic stem cells (mESC) and mouse epiblast stem cells (mEpiSC) share similar pluripoten... more Mouse embryonic stem cells (mESC) and mouse epiblast stem cells (mEpiSC) share similar pluripotency factors like NANOG or POU5F1, however, their state of pluripotency differs significantly. mESC and mEpiSC can be derived from embryos generated by fertilization (FT) or by somatic cell nuclear transfer (NT). In this study we performed a 4-plex iTRAQ LC-MS/MS based approach, facilitating the multiplexed comparison of the four indicated types of stem cells. From four replicates of each cell type, 1650 proteins were quantified. 234 non redundant proteins with significant abundance alterations between FT/NT-mESC and FT/NT-mEpiSC, and 44 between FT and NT derived cells were detected. Bioinformatic analysis revealed that several pluripotency associated proteins, among them POU5F1, DNMT3L, TIF1B, and proteins involved in DNA repair like MSH2 and MSH6, are more abundant in mESC compared to mEpiSC. The abundance level of these proteins is not affected by the mode of embryo generation, whereas several cytoskeleton proteins show a higher abundance in NT-mESC compared to FT-mESC. In addition, a number of cytoskeletal proteins are enriched in mEpiSC, e.g., myosins, filamins and intermediate filament proteins, reflecting the progressed differentiation state of epiblast derived versus inner cell mass derived murine pluripotent stem cells. This study aims to get new insights in the pluripotency state of stem cells and to deepen the knowledge of early cell differentiation. In an iTRAQ MS approach, we quantitatively compared proteomes of inner cell mass derived stem cells (mESC) with epiblast derived stem cells (mEpiSC). These stem cell types are derived from embryos of different developmental stages, and therefore vary considerably in their state of pluripotency and reflect different stages of early differentiation. The proteins which show significant abundance differences between the two stem cell lines represent (i) promising targets to further decipher molecular processes during early embryo development and (ii) useful molecular markers to monitor early differentiation events of stem cells by targeted approaches.
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Inflammation Research, 2003
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Cellular reprogramming, 2012
Embryonic stem cells derived from nuclear transfer embryos (ntESCs) are particularly valuable for... more Embryonic stem cells derived from nuclear transfer embryos (ntESCs) are particularly valuable for regenerative medicine, as they are a patient-specific and histocompatible cell source for the treatment of varying diseases. However, currently, little is known about their cellular and molecular profile. In the present study, in a mouse model different donor cell-derived ntESCs from various genetic backgrounds were compared with reference ESCs and analyzed comprehensively at the cellular level. A number of pluripotency marker genes were compared by flow cytometry and immunocytochemistry analysis. Significant differences at the protein level were observed for POU5F1, SOX2, FGF4, NANOG, and SSEA-1. However, such differences had no effect on in vitro cell differentiation and cell fate: derivatives of the three germ layers were detected in all ntESC lines. The neural and cardiac in vitro differentiation revealed minor differences between the cell lines, both at the mRNA and protein level. ...
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Cytokine, 2011
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Papers by Julianna Kobolak