Sue Mei Lim
Monash University
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Publication
Featured researches published by Sue Mei Lim.
Cell | 2012
Jose M. Polo; Endre Anderssen; Ryan M. Walsh; Benjamin A. Schwarz; Christian M. Nefzger; Sue Mei Lim; Marti Borkent; Effie Apostolou; Sara Alaei; Jennifer Cloutier; Ori Bar-Nur; Sihem Cheloufi; Matthias Stadtfeld; Maria E. Figueroa; Daisy Robinton; Sridaran Natesan; Ari Melnick; Jinfang Zhu; Sridhar Ramaswamy
Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming.
Nature Methods | 2011
David A. Elliott; Stefan R. Braam; Katerina Koutsis; Elizabeth S. Ng; Robert Alexander Jenny; Ebba L. Lagerqvist; Christine Biben; Tanya Hatzistavrou; Claire E. Hirst; Qing C. Yu; Rhys J.P. Skelton; Dorien Ward-van Oostwaard; Sue Mei Lim; Ouda Khammy; Xueling Li; Susan M. Hawes; Richard P. Davis; Adam L Goulburn; Robert Passier; Owen W.J. Prall; John M. Haynes; Colin W. Pouton; David M. Kaye; Andrew G. Elefanty; Edouard G. Stanley
NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5eGFP/w hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP+ cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.
Nature Protocols | 2007
Magdaline Costa; Mirella Dottori; Koula Sourris; Pegah Jamshidi; Tanya Hatzistavrou; Richard P. Davis; Lisa Azzola; Steven A. Jackson; Sue Mei Lim; Martin F. Pera; Andrew G. Elefanty; Edouard G. Stanley
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.
Stem Cells | 2011
Adam L Goulburn; Darym Alden; Richard P. Davis; Suzanne J. Micallef; Elizabeth S. Ng; Qing Cissy Yu; Sue Mei Lim; Chew-Li Soh; David A. Elliott; Tanya Hatzistavrou; Justin L Bourke; Bradley Watmuff; Richard J. Lang; John M. Haynes; Colin W. Pouton; Antonietta Giudice; Alan Trounson; Stewart A. Anderson; Edouard G. Stanley; Andrew G. Elefanty
We have used homologous recombination in human embryonic stem cells (hESCs) to insert sequences encoding green fluorescent protein (GFP) into the NKX2.1 locus, a gene required for normal development of the basal forebrain. Generation of NKX2.1‐GFP+ cells was dependent on the concentration, timing, and duration of retinoic acid treatment during differentiation. NKX2.1‐GFP+ progenitors expressed genes characteristic of the basal forebrain, including SHH, DLX1, LHX6, and OLIG2. Time course analysis revealed that NKX2.1‐GFP+ cells could upregulate FOXG1 expression, implying the existence of a novel pathway for the generation of telencephalic neural derivatives. Further maturation of NKX2.1‐GFP+ cells gave rise to γ‐aminobutyric acid‐, tyrosine hydroxylase‐, and somatostatin‐expressing neurons as well as to platelet‐derived growth factor receptor α‐positive oligodendrocyte precursors. These studies highlight the diversity of cell types that can be generated from human NKX2.1+ progenitors and demonstrate the utility of NKX2.1GFP/w hESCs for investigating human forebrain development and neuronal differentiation. STEM CELLS 2011;29:462–473
Diabetologia | 2012
Suzanne J. Micallef; Xueling Li; J. V. Schiesser; Claire E. Hirst; Qing Cissy Yu; Sue Mei Lim; M. C. Nostro; David A. Elliott; Farida Sarangi; Leonard C. Harrison; Gordon Keller; Andrew G. Elefanty; Edouard G. Stanley
Aims/hypothesisWe aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS+) cells derived in vitro.MethodsHomologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS+ cells.ResultsDifferentiation of INSGFP/w hESCs using published protocols demonstrated that all GFP+ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP+ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INSGFP/w hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP+ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP+ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS+ cells.Conclusions / interpretationINSGFP/w hESCs are a valuable tool for investigating the nature of early INS+ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS+ cells from differentiating hESCs.
PLOS ONE | 2011
Lloyd Pereira; Michael S. Wong; Sue Mei Lim; Alexandra Sides; Edouard G. Stanley; Andrew G. Elefanty
Mixl1 is a homeodomain transcription factor required for mesoderm and endoderm patterning during mammalian embryogenesis. Despite its crucial function in development, co-factors that modulate the activity of Mixl1 remain poorly defined. Here we report that Mixl1 interacts physically and functionally with the T-box protein Brachyury and related members of the T-box family of transcription factors. Transcriptional and protein analyses demonstrated overlapping expression of Mixl1 and Brachyury during embryonic stem cell differentiation. In vitro protein interaction studies showed that the Mixl1 with Brachyury associated via their DNA-binding domains and gel shift assays revealed that the Brachyury T-box domain bound to Mixl1-DNA complexes. Furthermore, luciferase reporter experiments indicated that association of Mixl1 with Brachyury and related T-box factors inhibited the transactivating potential of Mixl1 on the Gsc and Pdgfrα promoters. Our results indicate that the activity of Mixl1 can be modulated by protein-protein interactions and that T-box factors can function as negative regulators of Mixl1 activity.
Stem Cells | 2009
Sue Mei Lim; Lloyd Pereira; Michael Sebastian Wong; Claire E. Hirst; Benjamin Eugene Van Vranken; Marjorie Pick; Alan Trounson; Andrew G. Elefanty; Edouard G. Stanley
The Mixl1 gene encodes a homeodomain transcription factor that is required for normal mesoderm and endoderm development in the mouse. We have examined the consequences of enforced Mixl1 expression during mouse embryonic stem cell (ESC) differentiation. We show that three independently derived ESC lines constitutively expressing Mixl1 (Mixl1C ESCs) differentiate into embryoid bodies (EBs) containing a higher proportion of E‐cadherin (E‐Cad)+ cells. Our analysis also shows that this differentiation occurs at the expense of hematopoietic mesoderm differentiation, with Mixl1C ESCs expressing only low levels of Flk1 and failing to develop hemoglobinized cells. Immunohistochemistry and immunofluorescence studies revealed that Mixl1C EBs have extensive areas containing cells with an epithelial morphology that express E‐Cad, FoxA2, and Sox17, consistent with enhanced endoderm formation. Luciferase reporter transfection experiments indicate that Mixl1 can transactivate the Gsc, Sox17, and E‐Cad promoters, supporting the hypothesis that Mixl1 has a direct role in definitive endoderm formation. Taken together, these studies suggest that high levels of Mixl1 preferentially allocate cells to the endoderm during ESC differentiation. STEM CELLS 2009;27:363–474
Developmental Biology | 2012
Lloyd Pereira; Michael S. Wong; Sue Mei Lim; Edouard G. Stanley; Andrew G. Elefanty
The Mix/Bix family of paired-like homeobox genes encode evolutionarily conserved, sequence specific, DNA-binding transcription factors that have been implicated in the co-ordination of gene expression, axis formation and cell fate determination during gastrulation in vertebrates. When mutated, these genes give rise to dramatic phenotypes in amphibians, zebrafish and mice, that can be traced back to defects in the formation and specification of mesoderm and endoderm. We review here the biochemical properties of the Mix/Bix proteins and summarise genetic, molecular and embryological studies of Mix/Bix function in mesendoderm development. We emphasise recent data generated using embryonic stem cell differentiation systems that have provided important new insights into Mix/Bix function and the biological roles of these proteins in regulating the earliest phases of vertebrate development.
Stem Cell Research | 2013
Magdaline Costa; Koula Sourris; Sue Mei Lim; Qing C. Yu; Claire E. Hirst; Helena C. Parkington; Vanta J. Jokubaitis; Anthony E. Dear; Hong B. Liu; Suzanne J. Micallef; Kathy Koutsis; Andrew G. Elefanty; Edouard G. Stanley
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.
Immunology and Cell Biology | 2015
Jaber Firas; E Xiaodong Liu; Sue Mei Lim; Jose M. Polo
Cellular reprogramming refers to the conversion of one cell type into another by altering its epigenetic marks. This can be achieved by three different methods: somatic cell nuclear transfer, cell fusion and transcription factor (TF)‐mediated reprogramming. TF‐mediated reprogramming can occur through several means, either reverting backwards to a pluripotent state before redifferentiating to a new cell type (otherwise known as induced pluripotency), by transdifferentiating directly into a new cell type (bypassing the intermediate pluripotent stage), or, by using the induced pluripotency pathway without reaching the pluripotent state. The possibility of reprogramming any cell type of interest not only sheds new insights on cellular plasticity, but also provides a novel use of this technology across several platforms, most notably in cellular replacement therapies, disease modelling and drug screening. This review will focus on the different ways of implementing TF‐mediated reprogramming, their associated epigenetic changes and its therapeutic potential.