Publications by authors named "Koula Sourris"

14 Publications

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Human yolk sac-like haematopoiesis generates , and/or dependent blood and -positive endothelium.

Development 2020 10 29;147(20). Epub 2020 Oct 29.

Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia

The genetic regulatory network controlling early fate choices during human blood cell development are not well understood. We used human pluripotent stem cell reporter lines to track the development of endothelial and haematopoietic populations in an model of human yolk-sac development. We identified SOX17CD34CD43 endothelial cells at day 2 of blast colony development, as a haemangioblast-like branch point from which SOX17CD34CD43 blood cells and SOX17CD34CD43 endothelium subsequently arose. Most human blood cell development was dependent on RUNX1. Deletion of only permitted a single wave of yolk sac-like primitive erythropoiesis, but no yolk sac myelopoiesis or aorta-gonad-mesonephros (AGM)-like haematopoiesis. Blocking GFI1 and/or GFI1B activity with a small molecule inhibitor abrogated all blood cell development, even in cell lines with an intact gene. Together, our data define the hierarchical requirements for RUNX1, GFI1 and/or GFI1B during early human haematopoiesis arising from a yolk sac-like SOX17-negative haemogenic endothelial intermediate.
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http://dx.doi.org/10.1242/dev.193037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648599PMC
October 2020

Generation of seven iPSC lines from peripheral blood mononuclear cells suitable to investigate Autism Spectrum Disorder.

Stem Cell Res 2019 08 1;39:101516. Epub 2019 Aug 1.

Bruce Lefroy Centre for Genetic Health Research, Murdoch Children's Research Institute, Melbourne, Victoria 3052, Australia; Department of Paediatrics, University of Melbourne, Parkville, Australia. Electronic address:

We have generated and characterized seven human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) from a single family, including unaffected and affected individuals clinically diagnosed with Autism Spectrum Disorder (ASD). The reprogramming of the PBMCs was performed using non-integrative Sendai virus containing the reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC. All iPSC lines exhibited a normal karyotype and pluripotency was validated by immunofluorescence, flow cytometry and their ability to differentiate into the three embryonic germ layers. These iPSC lines are a valuable resource to study the molecular mechanisms underlying ASD.
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http://dx.doi.org/10.1016/j.scr.2019.101516DOI Listing
August 2019

Generation of iPSC lines from peripheral blood mononuclear cells from 5 healthy adults.

Stem Cell Res 2019 01 27;34:101380. Epub 2018 Dec 27.

Murdoch Children's Research Institute, The Royal Children's Hospital, Melbourne, Victoria 3052, Australia; Department of Paediatrics, The University of Melbourne, Melbourne, Victoria 3052, Australia; Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria 3800, Australia.

We describe the generation and characterization of 5 human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) of healthy adult individuals. The PBMCs were reprogrammed using non-integrating Sendai viruses containing the reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC. The iPSC lines exhibited a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. These iPSC lines can be used as controls in studying disease mechanisms.
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http://dx.doi.org/10.1016/j.scr.2018.101380DOI Listing
January 2019

GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives.

Stem Cell Reports 2016 09 1;7(3):518-526. Epub 2016 Sep 1.

Murdoch Childrens Research Institute, Flemington Road, Parkville, VIC 3052, Australia; Department of Paediatrics, University of Melbourne, Parkville, VIC 3050, Australia; Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3800, Australia. Electronic address:

The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that β-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.
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http://dx.doi.org/10.1016/j.stemcr.2016.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5032031PMC
September 2016

Derivation of endothelial cells from human embryonic stem cells in fully defined medium enables identification of lysophosphatidic acid and platelet activating factor as regulators of eNOS localization.

Stem Cell Res 2013 Jan 22;10(1):103-17. Epub 2012 Oct 22.

Monash Immunology and Stem Cell Laboratories (MISCL), Monash University, Clayton, Victoria, Australia.

The limited availability of human vascular endothelial cells (ECs) hampers research into EC function whilst the lack of precisely defined culture conditions for this cell type presents problems for addressing basic questions surrounding EC physiology. We aimed to generate endothelial progenitors from human pluripotent stem cells to facilitate the study of human EC physiology, using a defined serum-free protocol. Human embryonic stem cells (hESC-ECs) differentiated under serum-free conditions generated CD34(+)KDR(+) endothelial progenitor cells after 6days that could be further expanded in the presence of vascular endothelial growth factor (VEGF). The resultant EC population expressed CD31 and TIE2/TEK, took up acetylated low-density lipoprotein (LDL) and up-regulated expression of ICAM-1, PAI-1 and ET-1 following treatment with TNFα. Immunofluorescence studies indicated that a key mediator of vascular tone, endothelial nitric oxide synthase (eNOS), was localised to a perinuclear compartment of hESC-ECs, in contrast with the pan-cellular distribution of this enzyme within human umbilical vein ECs (HUVECs). Further investigation revealed that that the serum-associated lipids, lysophosphatidic acid (LPA) and platelet activating factor (PAF), were the key molecules that affected eNOS localisation in hESC-ECs cultures. These studies illustrate the feasibility of EC generation from hESCs and the utility of these cells for investigating environmental cues that impact on EC phenotype. We have demonstrated a hitherto unrecognized role for LPA and PAF in the regulation of eNOS subcellular localization.
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http://dx.doi.org/10.1016/j.scr.2012.10.003DOI Listing
January 2013

Pdgfrα and Flk1 are direct target genes of Mixl1 in differentiating embryonic stem cells.

Stem Cell Res 2012 Mar 8;8(2):165-79. Epub 2011 Oct 8.

Differentiation and Transcription Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, 3002, Australia.

The Mixl1 homeodomain protein plays a key role in mesendoderm patterning during embryogenesis, but its target genes remain to be identified. We compared gene expression in differentiating heterozygous Mixl1(GFP/w) and homozygous null Mixl1(GFP/Hygro) mouse embryonic stem cells to identify potential downstream transcriptional targets of Mixl1. Candidate Mixl1 regulated genes whose expression was reduced in GFP+ cells isolated from differentiating Mixl1(GFP/Hygro) embryoid bodies included Pdgfrα and Flk1. Mixl1 bound to ATTA sequences located in the Pdgfrα and Flk1 promoters and chromatin immunoprecipitation assays confirmed Mixl1 occupancy of these promoters in vivo. Furthermore, Mixl1 transactivated the Pdgfrα and Flk1 promoters through ATTA sequences in a DNA binding dependent manner. These data support the hypothesis that Mixl1 directly regulates Pdgfrα and Flk1 gene expression and strengthens the position of Mixl1 as a key regulator of mesendoderm development during mammalian gastrulation.
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http://dx.doi.org/10.1016/j.scr.2011.09.007DOI Listing
March 2012

Generation of human embryonic stem cell reporter knock-in lines by homologous recombination.

Curr Protoc Stem Cell Biol 2009 Nov;Chapter 5:Unit 5B.1 1.1-34

Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Australia.

This unit describes a series of technical procedures to form clonal human embryonic stem cell (hESC) lines that are genetically modified by homologous recombination. To develop a reporter knock-in hESC line, a vector is configured to contain a reporter gene adjacent to a positive selection cassette. These core elements are flanked by homologous sequences that, following electroporation into hESCs, promote the integration of the vector into the appropriate genomic locus. The positive selection cassette facilitates the enrichment and isolation of genetically modified hESC colonies that are then screened by PCR to identify correctly targeted lines. The selection cassette, flanked by loxP sites, is subsequently excised from the positively targeted hESCs via the transient expression of Cre recombinase. This is necessary because the continued presence of the cassette may interfere with the regulation of the reporter or neighboring genes. Finally, these genetically modified hESCs are clonally isolated using single-cell deposition flow cytometry. Reporter knock-in hESC lines are valuable tools that allow easy and rapid identification and isolation of specific hESC derivatives.
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http://dx.doi.org/10.1002/9780470151808.sc05b01s11DOI Listing
November 2009

Expansion of human embryonic stem cells in vitro.

Curr Protoc Stem Cell Biol 2008 May;Chapter 1:Unit 1C.1.1-1C.1.7

Monash University, Clayton, Victoria.

This unit describes a protocol for the large-scale expansion of karyotypically normal human embryonic stem cells (hESCs). hESCs can be maintained indefinitely as dense colonies that are mechanically cut into pieces, which are subsequently transferred to fresh organ culture dishes seeded with primary mouse embryonic fibroblasts (MEFs). hESCs can also be enzymatically passaged (bulk culture); however, over time, this style of culturing may lead to the acquisition of chromosomal abnormalities. Nevertheless, enzymatic passaging can be used for short periods (up to 25 passages) without the appearance of cells with abnormal karyotypes.
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http://dx.doi.org/10.1002/9780470151808.sc01c01s5DOI Listing
May 2008

Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors.

Blood 2008 Feb 21;111(4):1876-84. Epub 2007 Nov 21.

Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria, Australia.

Differentiating human embryonic stem cells (HESCs) represent an experimental platform for establishing the relationships between the earliest lineages that emerge during human development. Here we report the targeted insertion in HESCs of sequences encoding green fluorescent protein (GFP) into the locus of MIXL1, a gene transiently expressed in the primitive streak during embryogenesis.(1,2) GFP fluorescence in MIXL1(GFP/)(w) HESCs differentiated in the presence of BMP4 reported the expression of MIXL1, permitting the identification of viable human primitive streak-like cells. The use of GFP as a reporter for MIXL1 combined with cell surface staining for platelet-derived growth factor receptor alpha (PDGFRalpha) enabled the isolation of a cell population that was highly enriched in primitive hematopoietic precursors, the earliest derivatives of the primitive streak. These experiments demonstrate the utility of MIXL1(GFP/w) HESCs for analyzing the previously inaccessible events surrounding the development of human primitive streak-like cells and their subsequent commitment to hematopoiesis.
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http://dx.doi.org/10.1182/blood-2007-06-093609DOI Listing
February 2008

A method for genetic modification of human embryonic stem cells using electroporation.

Nat Protoc 2007 ;2(4):792-6

Monash Immunology and Stem Cell Laboratories, STRIP 1, West Ring Road, Monash University Campus, Clayton, Victoria 3800, Australia.

The ability to genetically modify human embryonic stem cells (HESCs) will be critical for their widespread use as a tool for understanding fundamental aspects of human biology and pathology and for their development as a platform for pharmaceutical discovery. Here, we describe a method for the genetic modification of HESCs using electroporation, the preferred method for introduction of DNA into cells in which the desired outcome is gene targeting. This report provides methods for cell amplification, electroporation, colony selection and screening. The protocol we describe has been tested on four different HESC lines, and takes approximately 4 weeks from electroporation to PCR screening of G418-resistant clones.
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http://dx.doi.org/10.1038/nprot.2007.105DOI Listing
June 2007

Mixl1 and oct4 proteins are transiently co-expressed in differentiating mouse and human embryonic stem cells.

Stem Cells Dev 2005 Dec;14(6):656-63

Monash Immunology and Stem Cell Laboratories (MISCL), Monash University, Clayton, Victoria 3800, Australia.

Embryonic stem cells (ESCs) have the capacity to form all the tissues in the body and hence, directed differentiation of ESCs along specific lineages represents a means to generate therapeutically useful cell types. It has been postulated that, during in vitro differentiation, ES cells sequentially pass through similar developmental stages as cells in the embryo. The availability of reagents that identify these stages would facilitate the monitoring and optimization of ESC differentiation. One key stage, the development of endodermal and mesodermal precursors in the early embryo, is marked by the transient expression of the transcription factor, Mixl1 and the stem cell gene, Oct4. In order to identify corresponding cells during ESC differentiation, we generated monoclonal antibodies to the Mixl1 protein that robustly detected both mouse and human proteins. Intracellular flow cytometry was used to show that approximately 90% of differentiating mouse ESCs transiently co-expressed Oct4 and Mixl1 proteins and that a subset of differentiating human ES cells also coexpressed MIXL1 and OCT4 proteins. These experiments have demonstrated for the first time by protein expression that both human and mouse ESCs passed through developmental stages during in vitro differentiation that corresponded to those observed in early mammalian development. Furthermore, these studies confirmed that anti-Mixl1 antibodies are a valuable reagent for monitoring ESC differentiation and will facilitate the efficient generation of clinically relevant cell types.
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http://dx.doi.org/10.1089/scd.2005.14.656DOI Listing
December 2005

The hESC line Envy expresses high levels of GFP in all differentiated progeny.

Nat Methods 2005 Apr 23;2(4):259-60. Epub 2005 Mar 23.

Monash Immunology and Stem Cell Laboratories, STRIP 1, West Ring Road, Monash University Campus, Clayton, Victoria, 3800, Australia.

Human embryonic stem cells (hESCs) have been advanced as a potential source of cells for use in cell replacement therapies. The ability to identify hESCs and their differentiated progeny readily in transplantation experiments will facilitate the analysis of hESC potential and function in vivo. We have generated a hESC line designated 'Envy', in which robust levels of green fluorescent protein (GFP) are expressed in stem cells and all differentiated progeny.
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http://dx.doi.org/10.1038/nmeth748DOI Listing
April 2005

The primitive streak gene Mixl1 is required for efficient haematopoiesis and BMP4-induced ventral mesoderm patterning in differentiating ES cells.

Development 2005 Mar 26;132(5):873-84. Epub 2005 Jan 26.

Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, VIC 3800, Australia.

The homeobox gene Mixl1 is expressed in the primitive streak of the gastrulating embryo, and marks cells destined to form mesoderm and endoderm. The role of Mixl1 in development of haematopoietic mesoderm was investigated by analysing the differentiation of ES cells in which GFP was targeted to one (Mixl1(GFP/w)) or both (Mixl1(GFP/GFP)) alleles of the Mixl1 locus. In either case, GFP was transiently expressed, with over 80% of cells in day 4 embryoid bodies (EBs) being GFP(+). Up to 45% of Mixl1(GFP/w) day 4 EB cells co-expressed GFP and the haemangioblast marker FLK1, and this doubly-positive population was enriched for blast colony forming cells (BL-CFCs). Mixl1-null ES cells, however, displayed a haematopoietic defect characterised by reduced and delayed Flk1 expression and a decrease in the frequency of haematopoietic CFCs. These data indicated that Mixl1 was required for efficient differentiation of cells from the primitive streak stage to blood. Differentiation of ES cells under serum-free conditions demonstrated that induction of Mixl1- and Flk1-expressing haematopoietic mesoderm required medium supplemented with BMP4 or activin A. In conclusion, this study has revealed an important role for Mixl1 in haematopoietic development and demonstrates the utility of the Mixl1(GFP/w) ES cells for evaluating growth factors influencing mesendodermal differentiation.
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http://dx.doi.org/10.1242/dev.01657DOI Listing
March 2005

Mixl1 is required for axial mesendoderm morphogenesis and patterning in the murine embryo.

Development 2002 Aug;129(15):3597-608

The Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Vic 3050, Australia.

In Xenopus, the Mix/Bix family of homeobox genes has been implicated in mesendoderm development. Mixl1 is the only known murine member of this family. To examine the role of Mixl1 in murine embryogenesis, we used gene targeting to create mice bearing a null mutation of Mixl1. Homozygous Mixl1 mutant embryos can be distinguished from their littermates by a marked thickening of the primitive streak. By the early somite stage, embryonic development is arrested, with the formation of abnormal head folds, foreshortened body axis, absence of heart tube and gut, deficient paraxial mesoderm, and an enlarged midline tissue mass that replaces the notochord. Development of extra-embryonic structures is generally normal except that the allantois is often disproportionately large for the size of the mutant embryo. In chimeras, Mixl1(-/-) mutant cells can contribute to all embryonic structures, with the exception of the hindgut, suggesting that Mixl1 activity is most crucial for endodermal differentiation. Mixl1 is therefore required for the morphogenesis of axial mesoderm, the heart and the gut during embryogenesis.
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August 2002