Publications by authors named "Kuldip S Sidhu"

19 Publications

  • Page 1 of 1

Derivation, propagation, and characterization of neuroprogenitors from pluripotent stem cells (hESCs and hiPSCs).

Methods Mol Biol 2012 ;873:237-46

Stem Cell Laboratory, Faculty of Medicine, School of Psychiatry, University of New South Wales, Randwick, NSW, Australia.

The differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) towards functional neurons particularly hold great potential for the cell-based replacement therapy in neurodegenerative diseases. Here, we describe a stepwise differentiation protocol that mimics the early stage of neural development in human to promote the generation of neuroprogenitors at a high yield. Both the hESCs and hiPSCs are initially cultured in an optimized feeder-free condition, which offer an efficient formation of aggregates. To specify the neuroectodermal specification, these aggregates are differentiated in a defined neural induction medium to develop into neural rosettes-like structures. The rosettes are expanded into free-floating sphere and can be further propagated or developed into variety of neuronal subtypes.
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http://dx.doi.org/10.1007/978-1-61779-794-1_15DOI Listing
August 2012

Suppression of NANOG induces efficient differentiation of human embryonic stem cells to pancreatic endoderm.

Pancreas 2012 Jan;41(1):54-64

Diabetes Transplant Unit, Prince of Wales Hospital & The University of New South Wales, Australia.

Objective: A challenge in using human embryonic stem cells (hESCs) as the source of surrogate β cells is the establishment of methods that could effectively direct their differentiation into functional β cells. The aim of this study was to assess the effect of NANOG gene suppression in differentiating hESCs as a mean of increasing the efficiency with which endoderm-derived pancreatic cells could be generated.

Methods: A homogenous cell population with stable suppression of NANOG was generated in hESC ENVY line using plasmid-based siRNA approach. Pancreatic differentiation was undertaken according to the ontology-based in vitro selection protocol and followed by transplantation into immunodeficiency mice to mature in vivo.

Results: We observed up-regulation of definitive endoderm genes, which expand the role of NANOG in blocking definitive endoderm differentiation. The ontology-based differentiation protocol resulted in increased expression of markers essential for pancreatic epithelium development. Transplantation of these cells further revealed a homogenous pancreatic exocrine-like morphology that stained positively for amylase.

Conclusions: The suppression of NANOG displayed an effective differentiation toward endoderm and pancreatic progenitors. Investigation of the factors required for endocrine formation combined with a prolonged in vivo culturing could be further used to increase the ratio of endocrine-exocrine cells fate.
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http://dx.doi.org/10.1097/MPA.0b013e31822362e4DOI Listing
January 2012

Amyloid precursor proteins, neural differentiation of pluripotent stem cells and its relevance to Alzheimer's disease.

Stem Cells Dev 2012 May 18;21(7):997-1006. Epub 2012 Jan 18.

Faculty of Medicine, School of Psychiatry, University of New South Wales, Sydney, NSW, Australia.

Alzheimer's disease (AD) is a leading cause of age-related dementia that is characterized by an extensive loss of neurons and synaptic transmission. The pathological hallmarks of AD are neurofibrillary tangles and deposition of β-amyloid (Aβ) plaques. Previous research has investigated how Aβ fragments disrupt synaptic mechanisms in the vulnerable regions of the brain. There is a tremendous potential for stem cell technology to extend upon this research, not only in terms of developing therapeutic applications, but also in modeling AD. Indeed, the advent of induced pluripotent stem cell technology has opened up exciting new avenues for generating patient and disease-specific cell lines from somatic cells that may be used to model AD. Amyloid precursor protein (APP) is a key protein in neuronal development and this article reviews the role of APP in AD. Stem cell technology offers the opportunity to make use of APP in the directed differentiation of induced pluripotent stem cells into functional neurons, a process that may help generate a model of AD and thereby facilitate an understanding of the mechanisms underlying this disease.
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http://dx.doi.org/10.1089/scd.2011.0564DOI Listing
May 2012

Human induced pluripotent stem cells derived under feeder-free conditions display unique cell cycle and DNA replication gene profiles.

Stem Cells Dev 2012 Jan 1;21(2):206-16. Epub 2011 Jun 1.

Stem Cell Laboratory, School of Psychiatry, University of New South Wales, Sydney, Australia.

Use of animal feeder layers and serum containing media in the derivation and propagation of induced pluripotent stem cells (iPSCs) can hinder clinical translation, because of the presence of xeno-material/pathogens. A defined and standardized system would be ideal for generating a homogenous population of iPSCs, which closely resembles human embryonic stem cells (hESCs). This article presents a novel and extensive comparison between in-house produced iPSCs and hESCs under "feeder" and "feeder-free" conditions, using transcriptomic genome-wide microarray analysis. We generated a list of pluripotency-associated and bivalent domain-containing genes by meta-analysis to measure qualitatively the degree of reprogramming in feeder-free derived iPSCs, in which both profiles displayed similar levels of gene expression as in hESCs. Gene ontology analysis showed that feeder-free iPSCs have enriched terms belonging to DNA repair/replication and cell cycle, which are signature to pluripotent cells. Transcriptomic data combined with directed differentiation assays, indicated that variability among iPSC lines is minimized when using a feeder-free cultural system, which may serve as a platform for further developing regenerative medicine compliant human iPSCs.
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http://dx.doi.org/10.1089/scd.2010.0440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258437PMC
January 2012

New approaches for the generation of induced pluripotent stem cells.

Authors:
Kuldip S Sidhu

Expert Opin Biol Ther 2011 May 12;11(5):569-79. Epub 2011 Feb 12.

Stem Cell Lab, University of New South Wales, School of Psychiatry, Faculty of Medicine, 402-408 Wallace Wurth Building, High Street, Randwick, NSW 2152, Australia .

Introduction: The advent of induced pluripotent stem cell (iPSC) technology has opened up new vistas to generate patient-specific pluripotent stem cells from somatic cells. During the last 5 years, the iPSCs produced from a variety of somatic cell sources are found to be very similar, if not identical to embryonic stem cells. Invariably these cells are produced by viral transduction of four transcriptional factors that renders these cells unfit for therapeutic purposes.

Areas Covered: This review discusses current developments emphasising on new and improved methods of generating iPSCs, including minimal or no genetic modifications via excisable lentiviral and transposon vectors or through repeated application of transient plasmid, episomal and adenovirus vectors. Recent use of small molecules, synthetic mRNA and microRNAs is also reviewed.

Expert Opinion: iPSC technology is emerging as an unprecedented opportunity in biomedical research, disease modeling, drug discovery and regenerative medicine. However, to harness the full potential of this technology, a number of issues that need to be resolved pertaining to iPSC safety, stability, culture variability, their comparison with ES cells, the reprogramming mechanisms and better ways to direct a specific reprogramming process including lineage specifications.
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http://dx.doi.org/10.1517/14712598.2011.558837DOI Listing
May 2011

A combined epigenetic and non-genetic approach for reprogramming human somatic cells.

PLoS One 2010 Aug 19;5(8):e12297. Epub 2010 Aug 19.

Faculty of Medicine, University of New South Wales, Sydney, Australia.

Reprogramming of somatic cells to different extents has been reported using different methods. However, this is normally accompanied by the use of exogenous materials, and the overall reprogramming efficiency has been low. Chemicals and small molecules have been used to improve the reprogramming process during somatic cell nuclear transfer (SCNT) and induced pluripotent stem (iPS) cell generation. We report here the first application of a combined epigenetic and non-genetic approach for reprogramming somatic cells, i.e., DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, and human embryonic stem cell (hESC) extracts. When somatic cells were pretreated with these inhibitors before exposure to hESC (MEL1) extracts, morphological analysis revealed a higher rate of hESC-like colony formation than without pretreatment. Quantitative PCR (qPCR) demonstrated that pluripotency genes were upregulated when compared to those of somatic cells or treated with hESC extracts alone. Overall changes in methylation and acetylation levels of pretreated somatic cells suggests that epigenetic states of the cells have an effect on reprogramming efficiency induced by hESC extracts. KnockOutserum replacement (KOSR) medium (KO-SR) played a positive role in inducing expression of the pluripotency genes. hESC extracts could be an alternative approach to reprogram somatic cells without introducing exogenous materials. The epigenetic pre-treatment of somatic cells could be used to improve the efficiency of reprogramming process. Under differentiation conditions, the reprogrammed cells exhibited differentiation ability into neurons suggesting that, although fully reprogramming was not achieved, the cells could be transdifferentiated after reprogramming.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0012297PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924394PMC
August 2010

Derivation of a new human embryonic stem cell line, Endeavour-2, and its characterization.

In Vitro Cell Dev Biol Anim 2010 Apr 23;46(3-4):269-75. Epub 2010 Feb 23.

Diabetes Transplant Unit, Prince of Wales Hospital & University of New South Wales, Sydney, NSW, Australia.

Here, we describe the derivation of a novel human embryonic stem cell (hESC) line, Endeavour-2 (E-2), propagated on human fetal fibroblasts (HFF) in a serum-replacement media. The inner cell mass (ICM) was manually dissected from the blastocyst without using immunodissection and, therefore, antibodies from animal sources. A total of 20 embryos were thawed and cultured, eight embryos were hatched, and five ICMs were obtained. They were transferred onto HFF used as feeder layer, and one colony representing the initial cell proliferation of a new hESC line, E-2, was obtained. The newly emerged hESC colony was passaged first by physical dissection and subsequently by enzymatic dissociation. E-2 has been in culture for over 6 months and has been shown to possess typical features of a pluripotent hESC line including expression of stem cell surface markers (SSEA4, TRA-160, and integrin alpha-6), intracellular alkaline phosphatase, and pluripotency gene markers, OCT4 and NANOG. This hESC line shows lineage-specific differentiation into various representative cell types expressing markers characteristic of the three somatic germ layers under both in vitro and in vivo conditions. E-2 line shows a normal karyotype (46 XX) and has been successfully cryopreserved and thawed several times using slow-freezing procedures. E-2 adds to the repertoire of existing hESC lines for research and development purposes in the field of regenerative medicine.
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http://dx.doi.org/10.1007/s11626-010-9280-8DOI Listing
April 2010

Derivation of male germ cell-like lineage from human fetal bone marrow stem cells.

Reprod Biomed Online 2009 Jul;19(1):99-105

Shaanxi Key Lab for Agriculture Molecular Biotechnology Centre,Shaanxi Centre of Stem Cells Engineering & Technology, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China.

Mesenchymal stem cells derived from bone marrow are a well characterized population of adult stem cells that can be maintained and propagated in culture for a long time with the capacity to form a variety of cell types. Reports have shown that murine and human embryonic stem cells can differentiate into primordial germ cells and then to early gametes. Evidence has indicated that some adult stem cells also have the potential to differentiate into germ cells. Currently, there are no reports on directed differentiation of human mesenchymal stem cells into germ cells. This study investigated the ability of retinoic acid and testicular extracts to induce human bone marrow stem cells (hBMSC) to differentiate into male germ cells. It was found that a small population of hBMSC seem to transdifferentiate into male germ cell-like cells. These cells expressed early germ cell markers OCT4, STELLA, NANOG and VASA, and male germ-ceil-specific markers such as DAZL, TH2, c-kit, beta(1)-integrin, ACR, PRMl, FSHR, STRA8 and SCP3, as analysed by reverse transcription-polymerase chain reaction and immunohistochemistry. These results demonstrated that hBMSC may differentiate into male germ cells and the same could be used as a potential source of cells for reproductive toxicological studies.
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http://dx.doi.org/10.1016/s1472-6483(10)60052-1DOI Listing
July 2009

Derivation and propagation of hESC under a therapeutic environment.

Curr Protoc Stem Cell Biol 2008 Jul;Chapter 1:Unit 1A.4

Diabetes Transplant Unit, The Prince of Wales Hospital and The University of New South Wales, New South Wales, Australia.

The pluripotent nature of human embryonic stem cells (hESC) makes them very attractive as a source of various cell types that could be used therapeutically in regenerative medicine. However, eliminating all sources of contamination, animal-derived or human cell-derived, during hESC derivation and propagation is necessary before hESC derivatives can be used clinically. Although there is continuing progress toward this goal, none of the methods to date to produce hESC lines under good manufacturing practices (GMP) has been published. The long-term success for GMP compliance depends critically on maintaining and implementing a stringent quality control system which is also dictated by the regulatory authorities in different countries. In this unit, an approach is described based upon the experience of this author and others towards achieving clinical-grade hESC lines systematically involving all the steps from start to finish under GMP environment. This unit provides a basic layout for GMP set up to achieve quality controls, a step-by-step guide to producing new hESC lines under defined conditions, and standard operating procedures used to achieve this outcome.
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http://dx.doi.org/10.1002/9780470151808.sc01a04s6DOI Listing
July 2008

Neural precursors from canine skin: a new direction for testing autologous cell replacement in the brain.

Stem Cells Dev 2008 Dec;17(6):1087-94

School of Psychiatry, The University of New South Wales, Sydney, Australia.

Recent work indicates that neural progenitors can be isolated from the skin of rodents and humans. The persistence of these cells in accessible adult tissue raises the possibility of their exploitation for research and therapeutic purposes. This study reports on the derivation, culture, and characterization of homogenous canine skin-derived neuroprecursor cells (SKiNPs) from mature animals. Canine tissue was used because naturalistic brain diseases in community-dwelling dogs are emerging as ecologically sound models for a range of neurological conditions. Adult SKiNPs were initially isolated as neurospheres and then cultured for 10-15 passages in an adherent monolayer assay. Serumfree expansion conditions contained B-27, 20 ng/mL EGF, and 40 ng/mL bFGF. Gene expressions by PCR indicated expression of nestin, CD133, NCAM, and FGF2R, but not GFAP. Highly uniform expression of nestin (76 +/- 8.3%), NCAM (84 +/- 3.3%), betaIII-tubulin (96 +/- 4.3%), and CD133 (68 +/- 13.5%) was also observed. Directed differentiation of SKiNPs in the presence of serum induced betaIIItubulin, NSE, NCAM, and MAP2 in >90% of differentiated cells by immunophenotype analysis. Our culture system rapidly induces canine skin cells into neural precursors, maintains nestin expression in more than 75% of proliferating cells, and generates an almost universal neuronal-like phenotype after 7 days of in vitro differentiation. Their biological characteristics are suggestive of transiently amplifying fate-restricted neuroprecursors rather than true neural stem cells. This system may be an effective alternative for autologous neurorestorative cell replacement in canine models for further translational research.
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http://dx.doi.org/10.1089/scd.2008.0008DOI Listing
December 2008

Derivation of a new human embryonic stem cell line, endeavour-1, and its clonal propagation.

Stem Cells Dev 2008 Feb;17(1):41-51

Stem Cell Laboratory, Faculty of Medicine, University of New South Wales, Randwick, Australia 2052.

Here we describe the derivation of a novel human embryonic stem (hES) cell line, Endeavour-1 (E1), its four new clonal lines (E1C1, E1C2, E1C3, E1C4), and their characterization. E1 and its clonal lines are propagated on human fetal fibroblasts (HFFs) derived and grown in a largely serum-free medium. Seven inner cell masses were isolated from 34 donated human embryos (27 survived), and one new hES cell line was obtained. E1 has been in culture for over 1 year and possesses all the typical features of stem cells, i.e., expression of stem cell surface markers (stage-specific embryonic antigens SSEA-3 and SSEA-4, and tumor recognition antigens TRA-1-60 and TRA-1-81), staining for alkaline phosphatase, and the presence of the pluripotent gene marker (nanog). This line shows pluripotency both under in vitro and in vivo conditions. E1 has a normal karyotype (46XX). Using our optimized procedure for cloning, four new clonal lines were derived from E1: E1C1, E1C2, E1C3, and E1C4. These clonal lines show normal characteristics: karyotype of that of the parent line (46XX) except for E1C3, which showed reciprocal translocation involving chromosomes 15 and 17; stem cell surface markers SSEA-4, TRA-1-60, and TRA-1-81; and gene expression for pluripotency (Nanog). All of these clonal lines formed embryoid bodies (EBs) in suspension cultures. After seeding, the EBs differentiated, forming cell lineages derived from all three germ layers as indicated by immunolocalization for the ectodermal marker beta-III tubulin, the mesodermal marker CD34, and the endodermal marker alpha-fetoprotein (AFP). There were subtle differences in the expression of these markers between clones. These clonal lines showed pluripotency in vivo. E1 and its clonal lines can differentiate to definitive endoderm after treatment with activin A, and, as indicated by expression of SOX17, FOXa2, and GATA-4 by RT-PCR, there are some subtle differences between these clonal lines. This may help in selecting clonal lines for specific lineage specification and for developing future cell therapy for various diseases.
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http://dx.doi.org/10.1089/scd.2007.0055DOI Listing
February 2008

Current concepts in reprogramming somatic cells to pluripotent state.

Curr Stem Cell Res Ther 2008 Jan;3(1):66-74

Stem Cell Laboratory, Faculty of Medicine, The University of New South Wales, Randwick, 2031, NSW, Australia.

Recently considerable interests have been roused in nuclear reprogramming by somatic cell nuclear transfer using an egg cytoplasm and/or by other means, such as fusion, cell extracts treatment and genes transfections. However, the very mechanism of reprogramming still remains elusive. Epigenetic modifications, which play a significant role in normal mammalian development in vivo is also involved in the process of reprogramming in vitro. The latter shares some of the other features observed in nuclear reprogramming in vivo. In this review, we discuss the main epigenetic changes involved in nuclear reprogramming and currently available approaches to achieve nuclear reprogramming in vitro and its future prospects.
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http://dx.doi.org/10.2174/157488808783489480DOI Listing
January 2008

Stem-cell therapy for diabetes cure: how close are we?

Curr Stem Cell Res Ther 2006 Sep;1(3):425-36

Stem Cells and Diabetes Section, Lab # 10, National Centre for Cell Science, Ganeshkhind Road, Pune 411007, India.

Transplantation of insulin-producing cells offers a promising therapy to treat diabetes. However, due to the limited number of donor islet cells available, researchers are looking for different sources of pancreatic islet progenitor or stem cells. A stem cell with extensive proliferative ability may provide a valuable source of islet progenitor cells. Several studies have demonstrated that a progenitor/stem-cell population can be expanded in vitro to generate large numbers of islet progenitor cells. However, efficient and directed differentiation of these cells to an endocrine pancreatic lineage has been difficult to achieve. We discuss here various pancreatic islet stem cells that we and others have obtained from embryonic, fetal or adult human tissues. We review the progress that has been achieved with pancreatic islet progenitor cell differentiation in the last 2 decades and discuss how close we are to translate this research to the clinics.
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http://dx.doi.org/10.2174/157488806778226830DOI Listing
September 2006

Epigenetic modifications of embryonic stem cells: current trends and relevance in developing regenerative medicine.

Stem Cells Cloning 2008 Nov 17;1:11-21. Epub 2008 Nov 17.

Stem Cell Lab, Faculty of Medicine, School of Psychiatry, University of New South Wales, Sydney, NSW, Australia.

Epigenetics is a growing field not only in the area of cancer research but recently in stem cells including human embryonic stem cell (hESC) research. The hallmark of profiling epigenetic changes in stem cells lies in maintaining pluripotency or multipotency and in attaining lineage specifications that are relevant for regenerative medicine. Epigenetic modifications including DNA methylation, histone acetylation and methylation, play important roles in regulating gene expressions. Other epigenetic modifications include X chromosome silencing, genomic stability and imprinting and mammalian development. This review attempts to elucidate the mechanism(s) behind epigenetic modifications and review techniques scientists use for identifying each modification. We also discuss some of the trends of epigenetic modifications in the fields of directed differentiation of embryonic stem cells and de-differentiation of somatic cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3781683PMC
http://dx.doi.org/10.2147/sccaa.s3566DOI Listing
November 2008

Derivation of Motor Neurons from three Clonal Human Embryonic Stem Cell Lines.

Curr Neurovasc Res 2006 Nov;3(4):281-8

Stem Cell Division, Diabetes Transplant Unit, Prince of Wales Hospital and University of New South Wale, Australia.

Human embryonic stem cells (hESC) demonstrate a remarkable proliferative and developmental potential and thus have huge therapeutic potential. To direct the differentiation of hESC to a specific lineage of high purity for cell transplantation is highly desirable. Here we describe a modified in vitro procedure to direct differentiation of three clonal hESC lines, hES 3.1, hES 3.2 and hES 3.3 efficiently to spinal motor neurons by using various differentiation factors namely retinoic acid (RA), sonic hedgehog (Shh), bone morphogenetic protein-2 (BMP-2) and Wnt3A. The highest number of motor neurons (58.0 +/- 7.6%) were obtained by an early treatment of embryoid bodies with a combination of RA + Shh from all the clonal hESC lines combined. The hES 3.1 line, however, produced relatively more motor neurons (69.5 +/- 11.8%) compared to other two hES clones, 3.2 (52.4 +/- 13.1%) and 3.3 (52.3 +/- 15.5%). Immunolocalisation studies revealed the expression of neuronal specific marker, beta omega-tubulin and motor neuron specific marker, HB9/HLXB9 in all the three hESC clones after 45 days of differentiation. The RT-PCR analyses showed the presence of the neuron-specific genes. This modified differentiation protocol provides a mean of obtaining an enriched population of motor neurons from hESC for possible use in studies of lineage development, drug discovery and also as a potential cell therapy for motor neuron disease.
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http://dx.doi.org/10.2174/156720206778792902DOI Listing
November 2006

Transgenic human fetal fibroblasts as feeder layer for human embryonic stem cell lineage selection.

Stem Cells Dev 2006 Oct;15(5):741-7

Stem Cell Division, Diabetes Transplant Unit, Prince of Wales Hospital and University of New South Wales, NSW 2031, Australia.

Successful gene targeting in human embryonic stem (hES) cells requires the use of primary fibroblast feeder layers, which assist in the maintenance of the pluripotent state of hES cells. Such feeder layers must also survive any further selection strategy for hES cells. Here we report the production of a novel transgenic human fetal fibroblast (tHFF) as a feeder layer that is resistant to puromycin and can be used for gene targeting and selection of positive clones in hES cells. tHFFs survive under a wide range of puromycin concentrations (0.5-2 microg/ml) and also supports the undifferentiated growth of hES cells. We have demonstrated here that tHFFs are suitable for selecting Envy-hES cells that were transfected with a green fluorescent protein-small interfering RNA (GFP-siRNA) plasmid construct to induce GFP gene down-regulation. The later studies were designed to isolate and propagate stably knockdown cells. tHFFs thus can be used for targeting other genes that would serve as a model to select and understand the differentiation process in hES cells.
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http://dx.doi.org/10.1089/scd.2006.15.741DOI Listing
October 2006

Differentiation of encapsulated embryonic stem cells after transplantation.

Transplantation 2006 Nov;82(9):1175-84

Diabetes Transplant Unit, Prince of Wales Hospital, and The University of New South Wales, Sydney, Australia.

Background: Embryonic stem cells (ESC) when transplanted into recipients with different major histocompatibility antigens may be rejected, especially as cells differentiate and expression of these antigens increases. One method to prevent rejection is to place the developing ESC in microcapsules. It is currently unknown what effect encapsulation has on the ability of ESC to differentiate.

Methods: Human ESC (hESC; hES03 line) and mouse ESC (mESC; R1 line) were encapsulated in 2.2% barium alginate and transplanted intraperitoneally in SCID and BALB/c mice respectively. Cell morphology, viability, and gene characterization were assessed after retrieving the capsules up to four weeks from SCID mice and three months from BALB/c mice.

Results: Encapsulation prevented hESC and mESC from forming teratomas up to four weeks and three months, respectively. mESC but not hESC formed aggregates within the capsules, which remained free of fibrosis. Some but not all the transplanted encapsulated hESC differentiated towards all three lineages, but more so towards an endodermal lineage as shown by increased expression of alpha fetoprotein. This was similar to what occurred when encapsulated and non-encapsulated hESC were cultured in vitro for two weeks. In contrast to the hESC, transplanted encapsulated mESC differentiated mostly towards an ectodermal lineage as shown by increased expression of nestin and glial fibrillary acidic protein. In vitro, encapsulated and nonencapsulated mESC also began to differentiate, but not down any specific lineage.

Conclusions: Encapsulated ESC do differentiate, although along multiple pathways, both when transplanted and maintained in culture, just as nonencapsulated ESC do when removed from their feeder layer.
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http://dx.doi.org/10.1097/01.tp.0000239518.23354.64DOI Listing
November 2006

Derivation of three clones from human embryonic stem cell lines by FACS sorting and their characterization.

Stem Cells Dev 2006 Feb;15(1):61-9

Stem Cell Group, Diabetes Transplant Unit, Prince of Wales Hospital and University of New South Wales, Sydney, NSW 2031 Australia.

Here we describe the first report of three human embryonic stem cell (hESC) clones, hES 3.1, 3.2, and 3.3, derived from the parent line hES3 by sorting of single-cell preparations by flow cytometry. The viability of single-cell preparations before and after cell sorting remained >98%. The hESC were selected by size gating and forward-angle light scatter and were dispersed directly as single cell/ well into 96-well plates containing human fetal fibroblasts as feeder layers. Single stem cell dispersion into 96-well plates was confirmed by using cells from a hES3 line that constitutively expressed green fluorescence protein (eGFP) under similar conditions of flow cytometry. Three clones were obtained from the parent line hES3 -- hES3.1, 3.2, and 3.3 -- and they have been in continuous culture for more than 1 year. The cloning efficiency was less than <0.5%. These hESC clones show normal stem cell characteristics, such as undifferentiated growth, high nucleocytoplasmic ratio, the same karyotype as that of the parent line (46 XX), stem cell surface markers (i.e., SSEA3, SSEA4, OCT4, TRA-1-60, and TRA-1-81), and gene expression for pluripotency (Oct-4 and nanog). They all formed embryoid bodies in suspension cultures, and after seeding in culture plates they showed pluripotency in vitro by forming cell lineages derived from all three germ layers as indicated by expression of the ectodermal marker nestin, the mesodermal marker renin, and the endodermal markers alpha-fetoprotein and GATA6. All clones showed normal expression of alkaline phosphatase activity, a marker of in vitro pluripotency. When hESC clones (1-2 x 10(6) total) were injected into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice under the kidney capsule, all formed teratomas within 6-8 weeks. Analysis of the stem cell surface marker TRA-1-160 by flow cytometry showed nonsignificant (p < 0.05) differences between the clones and the parent line. The clones also differed in their expression of genes, with only one, hES 3.2, expressing the endodermal markers, i.e., alpha-fetoprotein and GATA6. The ability to produce clones from a parent hESC line rapidly by FACS sorting will help provide a homogeneous population of cells for achieving uniformed lineage specifications for future transplantation therapies and biomedical research.
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http://dx.doi.org/10.1089/scd.2006.15.61DOI Listing
February 2006

Growth and differentiation of embryoid bodies derived from human embryonic stem cells: effect of glucose and basic fibroblast growth factor.

Biol Reprod 2005 Dec 3;73(6):1147-56. Epub 2005 Aug 3.

Diabetes Transplant Unit, Prince of Wales Hospital and The University of New South Wales, Sydney, New South Wales 2031, Australia.

Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). This differentiation occurs following suspension culturing of EBs in media containing a high (25 mM) glucose concentration. Although high-glucose-containing media is used for maintenance and proliferation of ES cells, it has not been demonstrated whether this is a necessary requirement for EB development. To address this, we examined the growth and differentiation of EBs established in 0-mM, 5.5-mM (physiological), and 25-mM (high) glucose concentrations, through morphometric analysis and examination of gene and protein expression. The effect on EB development of supplementation with basic fibroblast growth factor (FGF2) was also studied. We report that the greatest rate of EB growth occurs in 5.5 mM glucose media. A morphological study of EBs over 104 days duration under glucose-containing conditions demonstrated the development of all three major embryonic cell types. The difference from normal human development was obvious in the lack of rostrocaudal control by the notochord. In the latest stages of development, the main tissue observed appeared to be cartilage and cells of a mesodermal lineage. We conclude that physiological glucose concentrations are suitable for the culturing of EBs, that the addition of FGF2 enhances the temporal expression of genes including POU5F1, nestin, FOXA2, ONECUT1, NEUROD1, PAX6, and insulin, and that EBs can be cultured in vitro for long periods, allowing for further examination of developmental processes.
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http://dx.doi.org/10.1095/biolreprod.104.036673DOI Listing
December 2005
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