Publications by authors named "Paul J Verma"

46 Publications

Targeting epigenetic nuclear reprogramming in aggregated cloned equine embryos.

Reprod Fertil Dev 2019 Jan;31(12):1885-1893

Laboratorio de Biotecnología Animal, Facultad de Agronomia, Universidad de Buenos Aires, Av. San Martin 4453, C1417DSE, Ciudad Autónoma de Buenos Aires, Argentina; and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2290, C1425FQB, Ciudad Autónoma de Buenos Aires, Argentina; and Corresponding author. Email:

Epigenetic perturbations during the reprogramming process have been described as the primary cause of the low efficiency of somatic cell nuclear transfer (SCNT). In this study, we tested three strategies targeting nuclear reprogramming to investigate effects on equine SCNT. First, we evaluated the effect of treating somatic cells with chetomin, a fungal secondary metabolite reported to inhibit the trimethylation on histone 3 lysine 9 (H3K9 me3). Second, caffeine was added to the culture medium during the enucleation of oocytes and before activation of reconstructed embryos as a protein phosphatase inhibitor to improve nuclear reprogramming. Third, we tested the effects of the histone deacetylase inhibitor trichostatin A (TSA) added during both activation and early embryo culture. Although none of these treatments significantly improved the developmental rates of the invitro aggregated cloned equine embryos, the first equine cloned foal born in Australia was produced with somatic cells treated with chetomin. The present study describes the use of chetomin, caffeine and TSA for the first time in horses, serving as a starting point for the establishment of future protocols to target epigenetic reprogramming for improving the efficiency of equine cloning. Cloning is an expensive and inefficient process, but has gained particular interest in the equine industry. In this study we explored different strategies to improve cloning efficiency and produced the first cloned foal born in Australia. Our data serve as a starting point for the establishment of future protocols for improving equine cloning efficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1071/RD19239DOI Listing
January 2019

GSK3 inhibition, but not epigenetic remodeling, mediates efficient derivation of germline embryonic stem cells from nonobese diabetic mice.

Stem Cell Res 2018 08 21;31:5-10. Epub 2018 Jun 21.

Turretfield Research Centre, South Australian Research & Development Institute, Rosedale, SA 5350, Australia; Material Science and Engineering, Monash University, Clayton, VIC 3800, Australia. Electronic address:

The nonobese diabetic (NOD) mouse strain is a predominant animal model of type 1 diabetes. However, this mouse strain is considered to be non-permissive for embryonic stem cell (ESC) derivation using conventional methods. We examined small molecule inhibition of glycogen synthase kinase 3 (GSK3) to block spontaneous cell differentiation and promote pluripotency persistence. Here we show a single pharmacological GSK3 inhibitor, 6-bromoindirubin-3'-oxime (BIO), in combination with leukemia inhibition factor (LIF), promoted generation of stable NOD ESC lines at >80% efficiency. Significantly, expansion of the established NOD ESC lines no longer required treatment with BIO. These NOD ESC lines contributed to chimeric mice and transmitted to germline progeny that spontaneously developed diabetes. By contrast, 5-aza-2'-deoxycytidine (AZA), a small molecule inhibitor of DNA methylation, and trichostatin A (TSA) and valproic acid (VPA), small molecule inhibitors of histone deacetylase, could not promote generation of NOD ESCs by epigenetic remodeling. These combined findings provide strategic insights for imposing pluripotency in cells isolated from a non-permissive strain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.scr.2018.06.001DOI Listing
August 2018

Brain urea increase is an early Huntington's disease pathogenic event observed in a prodromal transgenic sheep model and HD cases.

Proc Natl Acad Sci U S A 2017 12 11;114(52):E11293-E11302. Epub 2017 Dec 11.

Centre for Brain Research, School of Biological Sciences, The University of Auckland, Auckland 1010, New Zealand;

The neurodegenerative disorder Huntington's disease (HD) is typically characterized by extensive loss of striatal neurons and the midlife onset of debilitating and progressive chorea, dementia, and psychological disturbance. HD is caused by a CAG repeat expansion in the () gene, translating to an elongated glutamine tract in the huntingtin protein. The pathogenic mechanism resulting in cell dysfunction and death beyond the causative mutation is not well defined. To further delineate the early molecular events in HD, we performed RNA-sequencing (RNA-seq) on striatal tissue from a cohort of 5-y-old -line sheep expressing a human CAG-expansion cDNA transgene. Our HD sheep are a prodromal model and exhibit minimal pathology and no detectable neuronal loss. We identified significantly increased levels of the urea transporter in the striatum, along with other important osmotic regulators. Further investigation revealed elevated levels of the metabolite urea in the striatum and cerebellum, consistent with our recently published observation of increased urea in postmortem human brain from HD cases. Extending that finding, we demonstrate that postmortem human brain urea levels are elevated in a larger cohort of HD cases, including those with low-level neuropathology (Vonsattel grade 0/1). This elevation indicates increased protein catabolism, possibly as an alternate energy source given the generalized metabolic defect in HD. Increased urea and ammonia levels due to dysregulation of the urea cycle are known to cause neurologic impairment. Taken together, our findings indicate that aberrant urea metabolism could be the primary biochemical disruption initiating neuropathogenesis in HD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1711243115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748180PMC
December 2017

Alzheimer's disease markers in the aged sheep (Ovis aries).

Neurobiol Aging 2017 10 24;58:112-119. Epub 2017 Jun 24.

The Neurogenetics Group, School of Biological Sciences, University of Auckland, Auckland, New Zealand. Electronic address:

This study reports the identification and characterization of markers of Alzheimer's disease (AD) in aged sheep (Ovis aries) as a preliminary step toward making a genetically modified large animal model of AD. Importantly, the sequences of key proteins involved in AD pathogenesis are highly conserved between sheep and human. The processing of the amyloid-β (Aβ) protein is conserved between sheep and human, and sheep Aβ/Aβ ratios in cerebrospinal fluid (CSF) are also very similar to human. In addition, total tau and neurofilament light levels in CSF are comparable with those found in human. The presence of neurofibrillary tangles in aged sheep brain has previously been established; here, we report for the first time that plaques, the other pathologic hallmark of AD, are also present in the aged sheep brain. In summary, the biological machinery to generate the key neuropathologic features of AD is conserved between the human and sheep, making the sheep a good candidate for future genetic manipulation to accelerate the condition for use in pathophysiological discovery and therapeutic testing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neurobiolaging.2017.06.020DOI Listing
October 2017

Inhibition of JAK-STAT ERK/MAPK and Glycogen Synthase Kinase-3 Induces a Change in Gene Expression Profile of Bovine Induced Pluripotent Stem Cells.

Stem Cells Int 2016 6;2016:5127984. Epub 2016 Jan 6.

Department of Materials Engineering, Monash Institute of Medical Engineering, Faculty of Engineering, Monash University, Clayton, VIC 3168, Australia; South Australian Research & Development Institute (SARDI), Turretfield Research Centre, Rosedale, SA 5350, Australia.

Pluripotent stem cells (PSCs) fall in two states, one highly undifferentiated, the naïve state, and the primed state, characterized by the inability to contribute to germinal lineage. Several reports have demonstrated that these states can be modified by changes to the cell culture conditions. With the advent of nuclear reprogramming, bovine induced pluripotent stem cells (biPSCs) have been generated. These cells represent examples of a transient-intermediate state of pluripotency with remarkable characteristics and biotechnological potential. Herein, we generated and characterized biPSC. Next, we evaluated different culture conditions for the ability to affect the expression of the set of core pluripotent transcription factors in biPSC. It was found that the use of 6-bromoindirubin-3-oxime and Sc1 inhibitors alone or in combination with 5-AzaC induced significantly higher levels of expression of endogenous REX1, OCT4, NANOG, and SOX2. Furthermore, LIF increased the levels of expression of OCT4 and REX1, compared with those cultured with LIF + bFGF. By contrast, bFGF decreased the levels of expression for both REX1 and OCT4. These results demonstrate that the biPSC gene expression profile is malleable by modification of the cell culture conditions well after nuclear reprogramming, and the culture conditions may determine their differentiation potential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2016/5127984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4736581PMC
February 2016

Bone morphogenetic protein 4 and retinoic acid trigger bovine VASA homolog expression in differentiating bovine induced pluripotent stem cells.

Mol Reprod Dev 2016 Feb 25;83(2):149-61. Epub 2016 Jan 25.

South Australian Research and Development Institute (SARDI), Turretfield Research Centre, Rosedale, SA, Australia.

Primordial germ cells (PGCs) are the earliest identifiable and completely committed progenitors of female and male gametes. They are obvious targets for genome editing because they assure the transmission of desirable or introduced traits to future generations. PGCs are established at the earliest stages of embryo development and are difficult to propagate in vitro--two characteristics that pose a problem for their practical application. One alternative method to enrich for PGCs in vitro is to differentiate them from pluripotent stem cells derived from adult tissues. Here, we establish a reporter system for germ cell identification in bovine pluripotent stem cells based on green fluorescent protein expression driven by the minimal essential promoter of the bovine Vasa homolog (BVH) gene, whose regulatory elements were identified by orthologous modelling of regulatory units. We then evaluated the potential of bovine induced pluripotent stem cell (biPSC) lines carrying the reporter construct to differentiate toward the germ cell lineage. Our results showed that biPSCs undergo differentiation as embryoid bodies, and a fraction of the differentiating cells expressed BVH. The rate of differentiation towards BVH-positive cells increased up to tenfold in the presence of bone morphogenetic protein 4 or retinoic acid. Finally, we determined that the expression of key PGC genes, such as BVH or SOX2, can be modified by pre-differentiation cell culture conditions, although this increase is not necessarily mirrored by an increase in the rate of differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/mrd.22607DOI Listing
February 2016

Synthetic mRNA Reprogramming of Human Fibroblast Cells.

Methods Mol Biol 2015 ;1330:17-28

Stem Cell and Genetic Engineering Group, Department of Materials Engineering, Faculty of Engineering, Monash University, Clayton, VIC, 3800, Australia.

Reprogramming of somatic cells, such as skin fibroblasts, to pluripotency was first achieved by forced expression of four transcription factors using integrating retroviral or lentiviral vectors, which result in integration of exogenous DNA into cellular genome and present a formidable barrier to therapeutic application of induced pluripotent stem cells (iPSCs). To facilitate the translation of iPSC technology to clinical practice, mRNA reprogramming method that generates transgene-free iPSCs is a safe and efficient method, eliminating bio-containment concerns associated with viral vectors, as well as the need for weeks of screening of cells to confirm that viral material has been completely eliminated during cell passaging.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-2848-4_2DOI Listing
September 2016

Integration-Free Human Induced Pluripotent Stem Cells From Type 1 Diabetes Patient Skin Fibroblasts Show Increased Abundance of Pancreas-Specific microRNAs.

Cell Med 2014 Nov 2;7(1):15-24. Epub 2014 May 2.

Cell Reprogramming and Stem Cells, Monash Institute of Medical Research, Monash University, Clayton, VIC, Australia; ¶Turretfield Research Centre, South Australian Research and Development Institute, Rosedale SA 3050, Australia.

Type 1 diabetes (T1D) is a disease that is typically associated with multigenetic changes as well as environmental triggers. Disease-specific induced pluripotent stem cells (iPSCs) are preferable cell sources to study T1D, as they are derived from patient cells and therefore capture the disease genotype in a stem cell line. The purpose of this study was to generate integration-free iPSCs from adult skin fibroblasts with T1D. iPSCs were generated by transfection of synthetic mRNAs encoding transcription factors OCT4, SOX2, KLF4, c-MYC, and LIN28. Phase-contrast microscopy, immunocytochemistry, karyotyping, bisulfite genomic sequencing, reverse transcription-polymerase chain reaction, and teratoma formation assay were used to determine reprogramming efficiency, pluripotency, and differentiation potential. Following 18 consecutive days of synthetic mRNA transfections, the T1D patient skin fibroblasts underwent morphological changes, and the aggregated clumps exhibited a human embryonic stem cell (ESC)-like morphology with a high nucleus/cytoplasm ratio. Highly efficient generation of iPSCs was achieved using the mRNA reprogramming approach. The disease-specific iPSCs expressed pluripotency markers, maintained a normal karyotype, and formed teratomas containing tissues representative of the three germ layers when injected into immune-deficient mice. Of interest, the iPSCs showed upregulations of pancreas-specific microRNAs, compared with parental fibroblasts. These data indicate that T1D patient skin fibroblasts can be reprogrammed to pluripotency using a synthetic mRNA approach. These cells can serve as a useful tool for the identification of genes that are involved in autoimmune reactions as well as generating patient-matched β-cells for cell-based therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3727/215517914X681785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4733842PMC
November 2014

Embryonic stem cell-somatic cell fusion and postfusion enucleation.

Methods Mol Biol 2015 ;1222:61-9

Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia,

Embryonic stem (ES) cells are able to reprogram somatic cells following cell fusion. The resulting cell hybrids have been shown to have similar properties to pluripotent cells. It has also been shown that transcriptional changes can occur in a heterokaryon, without nuclear hybridization. However it is unclear whether these changes can be sustained following removal of the dominant ES nucleus. In this chapter, methods are described for the cell fusion of mouse tetraploid ES cells with somatic cells and enrichment of the resulting heterokaryons. We next describe the conditions for the differential removal of the ES cell nucleus, allowing for the recovery of somatic cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-1594-1_5DOI Listing
June 2015

Generation of induced pluripotent stem cells from mouse adipose tissue.

Methods Mol Biol 2014 ;1194:253-70

Research Department of Haematology, UCL Cancer Institute, University College London, London, WC1E 6BT, UK.

The discovery that embryonic stem (ES) cell-like cells can be generated by simply over-expressing four key genes in adult somatic cells has changed the face of regenerative medicine. These induced pluripotent stem (iPS) cells have a wide range of potential uses from drug testing and in vitro disease modeling to personalized cell therapies for patients. However, prior to the realization of their potential, many issues need to be considered. One of these is the low-efficiency formation of iPSC. It has been extensively demonstrated that the somatic cell type can greatly influence reprogramming outcomes. We have shown that adipose tissue-derived cells (ADCs) can be easily isolated from adult animals and can be reprogrammed to a pluripotent state with high efficiency. Here, we describe a protocol for the high-efficiency derivation of ADCs and their subsequent use to generate mouse iPSC using Oct4, Sox2, Klf4, and cMyc retroviral vectors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-1215-5_14DOI Listing
March 2015

Isolation and handling of mouse embryonic fibroblasts.

Methods Mol Biol 2014 ;1194:247-52

Stem Cells and Reprogramming Group, Biological Engineering Laboratories, Faculty of Engineering, Monash University, Clayton, VIC, 3800, Australia.

Primary mouse embryonic fibroblasts (MEFs) are the most commonly used feeder layers that help to support growth and maintain pluripotency of embryonic stem cells (ESC) in long-term culture. Feeders provide substrates/nutrients that are essential to maintain pluripotency and prevent spontaneous differentiation of ESC. Since embryonic fibroblasts stop dividing after a few passages, care must be taken to isolate them freshly. Here, we provide a protocol to derive MEFs and describe the method to inactivate the cells using mitomycin C treatment. The protocol also describes freezing, thawing, and passaging of MEFs. This basic protocol works well in our laboratory. However, it can be modified and adapted according to any user's particular requirement.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-1215-5_13DOI Listing
March 2015

Optimization of agitation speed in spinner flask for microcarrier structural integrity and expansion of induced pluripotent stem cells.

Cytotechnology 2016 Jan 26;68(1):45-59. Epub 2014 Jul 26.

Division of Biological Engineering, Monash University, Melbourne, VIC, Australia.

In recent times, the study and use of induced pluripotent stem cells (iPSC) have become important in order to avoid the ethical issues surrounding the use of embryonic stem cells. Therapeutic, industrial and research based use of iPSC requires large quantities of cells generated in vitro. Mammalian cells, including pluripotent stem cells, have been expanded using 3D culture, however current limitations have not been overcome to allow a uniform, optimized platform for dynamic culture of pluripotent stem cells to be achieved. In the current work, we have expanded mouse iPSC in a spinner flask using Cytodex 3 microcarriers. We have looked at the effect of agitation on the microcarrier survival and optimized an agitation speed that supports bead suspension and iPS cell expansion without any bead breakage. Under the optimized conditions, the mouse iPSC were able to maintain their growth, pluripotency and differentiation capability. We demonstrate that microcarrier survival and iPS cell expansion in a spinner flask are reliant on a very narrow range of spin rates, highlighting the need for precise control of such set ups and the need for improved design of more robust systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10616-014-9750-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4698264PMC
January 2016

C-terminal region of MAP7 domain containing protein 3 (MAP7D3) promotes microtubule polymerization by binding at the C-terminal tail of tubulin.

PLoS One 2014 13;9(6):e99539. Epub 2014 Jun 13.

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India.

MAP7 domain containing protein 3 (MAP7D3), a newly identified microtubule associated protein, has been shown to promote microtubule assembly and stability. Its microtubule binding region has been reported to consist of two coiled coil motifs located at the N-terminus. It possesses a MAP7 domain near the C-terminus and belongs to the microtubule associated protein 7 (MAP7) family. The MAP7 domain of MAP7 protein has been shown to bind to kinesin-1; however, the role of MAP7 domain in MAP7D3 remains unknown. Based on the bioinformatics analysis of MAP7D3, we hypothesized that the MAP7 domain of MAP7D3 may have microtubule binding activity. Indeed, we found that MAP7 domain of MAP7D3 bound to microtubules as well as enhanced the assembly of microtubules in vitro. Interestingly, a longer fragment MDCT that contained the MAP7 domain (MD) with the C-terminal tail (CT) of the protein promoted microtubule polymerization to a greater extent than MD and CT individually. MDCT stabilized microtubules against dilution induced disassembly. MDCT bound to reconstituted microtubules with an apparent dissociation constant of 3.0 ± 0.5 µM. An immunostaining experiment showed that MDCT localized along the length of the preassembled microtubules. Competition experiments with tau indicated that MDCT shares its binding site on microtubules with tau. Further, we present evidence indicating that MDCT binds to the C-terminal tail of tubulin. In addition, MDCT could bind to tubulin in HeLa cell extract. Here, we report a microtubule binding region in the C-terminal region of MAP7D3 that may have a role in regulating microtubule assembly dynamics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099539PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4057234PMC
October 2015

Cardiogenesis of embryonic stem cells with liquid marble micro-bioreactor.

Adv Healthc Mater 2015 Jan 12;4(1):77-86. Epub 2014 May 12.

Division of Biological Engineering, Monash University, VIC, 3800, Australia; Department of Mechanical & Aerospace Engineering, Monash University, VIC, 3800, Australia.

A liquid marble micro-bioreactor is prepared by placing a drop of murine embryonic stem cell (ESC) (Oct4B2-ESC) suspension onto a polytetrafluoroethylene (PTFE) particle bed. The Oct4B2-ESC aggregates to form embryoid bodies (EBs) with relatively uniform size and shape in a liquid marble within 3 d. For the first time, the feasibility of differentiating ESC into cardiac lineages within liquid marbles is being investigated. Without the addition of growth factors, suspended EBs from liquid marbles express various precardiac mesoderm markers including Flk-1, Gata4, and Nkx2.5. Some of the suspended EBs exhibit spontaneous contraction. These results indicate that the liquid marble provides a suitable microenvironment to induce EB formation and spontaneous cardiac mesoderm differentiation. Some of the EBs are subsequently plated onto gelatin-coated tissue culture dishes. Plated EBs express mature cardiac markers atrial myosin light chain 2a (MLC2a) and ventricular myosin light chain (MLC2v), and the cardiac structural marker α-actinin. More than 60% of the plated EBs exhibit spontaneous contraction and express mature cardiomyocyte marker cardiac troponin T (cTnT), indicating that these EBs have differentiated into functional cardiomyocytes. Together, these results demonstrate that the liquid-marble technique is an easily employed, cost effective, and efficient approach to generate EBs and facilitating their cardiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adhm.201400138DOI Listing
January 2015

Functional evaluation of ES-somatic cell hybrids in vitro and in vivo.

Cell Reprogram 2014 Jun 2;16(3):167-74. Epub 2014 May 2.

1 Monash Institute of Medical Research, Monash University , Clayton VIC, 3168, Australia .

Embryonic stem cells (ESCs) have previously been reported to reprogram somatic cells following fusion. The resulting ES-somatic cell hybrids have been shown to adopt the transcriptional profile of ESCs, suggesting that the pluripotent program is dominant. ES-somatic cell hybrids have most characteristics of pluripotent cells in vitro; however, it remains unclear whether the somatic genome is an active partner in the hybrid cells or simply retained predominately as silent cargo. Furthermore, the functional properties of ES-somatic cell hybrids in vivo have been limited to studies on their contribution to teratomas and developing embryos/chimeras. The extent of their pluripotency remains largely unclear. Here we determined that the somatic genome is actively transcribed by generating ES-somatic cell hybrids using Rag2-deficient ESCs fused to autologous wild-type somatic cells. Rag2 expression was detected during in vitro differentiation, suggesting that the somatic genome follows the correct temporal cues during differentiation. Furthermore, ES-somatic cell hybrids maintain their tetraploid state following 4 weeks of differentiation in vivo and are immune tolerated when transferred into matched individuals. The ES-somatic cell hybrids can efficiently differentiate into hematopoietic precursors in both myeloid and lymphoid lineages in vitro, suggesting that the somatic genome is actively transcribed following cell fusion based reprogramming. However, the ES-somatic cell hybrids showed an altered hematopoietic potential following in vitro differentiation and were unable to show hematopoietic engraftment in a mouse model.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/cell.2013.0079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4030652PMC
June 2014

Gelatin-PMVE/MA composite scaffold promotes expansion of embryonic stem cells.

Mater Sci Eng C Mater Biol Appl 2014 Apr 7;37:184-94. Epub 2014 Jan 7.

Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai, India.

We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2-1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin-PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.msec.2013.12.033DOI Listing
April 2014

Effect of oxygen on cardiac differentiation in mouse iPS cells: role of hypoxia inducible factor-1 and Wnt/beta-catenin signaling.

PLoS One 2013 12;8(11):e80280. Epub 2013 Nov 12.

Heart Failure Research Group, Baker IDI Heart and Diabetes Institute, Melbourne, Australia.

Background: Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes.

Objective: We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS) cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha) and the canonical Wnt pathway in this process.

Methods: Embryoid bodies (EBs) derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture.

Results: At 14 days of differentiation, 59 ± 2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway.

Conclusion: Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080280PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827186PMC
July 2014

Ansamitocin P3 depolymerizes microtubules and induces apoptosis by binding to tubulin at the vinblastine site.

PLoS One 2013 4;8(10):e75182. Epub 2013 Oct 4.

IITB-Monash Research Academy, Indian Institute of Technology Bombay, Mumbai, India.

Maytansinoid conjugates are currently under different phases of clinical trials and have been showing promising activity for various types of cancers. In this study, we have elucidated the mechanism of action of ansamitocin P3, a structural analogue of maytansine for its anticancer activity. Ansamitocin P3 potently inhibited the proliferation of MCF-7, HeLa, EMT-6/AR1 and MDA-MB-231 cells in culture with a half-maximal inhibitory concentration of 20±3, 50±0.5, 140±17, and 150±1.1 pM, respectively. Ansamitocin P3 strongly depolymerized both interphase and mitotic microtubules and perturbed chromosome segregation at its proliferation inhibitory concentration range. Treatment of ansamitocin P3 activated spindle checkpoint surveillance proteins, Mad2 and BubR1 and blocked the cells in mitotic phase of the cell cycle. Subsequently, cells underwent apoptosis via p53 mediated apoptotic pathway. Further, ansamitocin P3 was found to bind to purified tubulin in vitro with a dissociation constant (Kd) of 1.3±0.7 µM. The binding of ansamitocin P3 induced conformational changes in tubulin. A docking analysis suggested that ansamitocin P3 may bind partially to vinblastine binding site on tubulin in two different positions. The analysis indicated that the binding of ansamitocin P3 to tubulin is stabilized by hydrogen bonds. In addition, weak interactions such as halogen-oxygen interactions may also contribute to the binding of ansamitocin P3 to tubulin. The study provided a significant insight in understanding the antiproliferative mechanism of action of ansamitocin P3.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075182PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790769PMC
July 2014

Induction of pluripotency.

Adv Exp Med Biol 2013 ;786:5-25

Reprogramming and Stem Cell Laboratory, Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, 27-31 Wright St., Clayton, VIC, 3168, Australia.

The molecular and phenotypic irreversibility of mammalian cell differentiation was a fundamental principle of developmental biology at least until the 1980s, despite numerous reports dating back to the 1950s of the induction of pluripotency in amphibian cells by nuclear transfer (NT). Landmark reports in the 1980s and 1990s in sheep progressively challenged this dogmatic assumption; firstly, embryonic development of reconstructed embryos comprising whole (donor) blastomeres fused to enucleated oocytes, and famously, the cloning of Dolly from a terminally differentiated cell. Thus, the intrinsic ability of oocyte-derived factors to reverse the differentiated phenotype was confirmed. The concomitant elucidation of methods for human embryonic stem cell isolation and cultivation presented opportunities for therapeutic cell replacement strategies, particularly through NT of patient nuclei to enucleated oocytes for subsequent isolation of patient-specific (autologous), pluripotent cells from the resulting blastocysts. Associated logistical limitations of working with human oocytes, in addition to ethical and moral objections prompted exploration of alternative approaches to generate autologous stem cells for therapy, utilizing the full repertoire of factors characteristic of pluripotency, primarily through cell fusion and use of pluripotent cell extracts. Stunningly, in 2006, Japanese scientists described somatic cell reprogramming through delivery of four key factors (identified through a deductive approach from 24 candidate genes). Although less efficient than previous approaches, much of current stem cell research adopts this focused approach to cell reprogramming and (autologous) cell therapy. This chapter is a quasi-historical commentary of the various aforementioned approaches for the induction of pluripotency in lineage-committed cells, and introduces transcriptional and epigenetic changes occurring during reprogramming.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-94-007-6621-1_2DOI Listing
September 2013

Design and screening of a glial cell-specific, cell penetrating peptide for therapeutic applications in multiple sclerosis.

PLoS One 2012 25;7(9):e45501. Epub 2012 Sep 25.

Reprogramming and Stem Cell Laboratory, Centre for Reproduction & Development, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia.

Multiple Sclerosis (MS) is an autoimmune, neurodegenerative disease of the central nervous system (CNS) characterized by demyelination through glial cell loss. Current and proposed therapeutic strategies to arrest demyelination and/or promote further remyelination include: (i) modulation of the host immune system; and/or (ii) transplantation of myelinating/stem or progenitor cells to the circulation or sites of injury. However, significant drawbacks are inherent with both approaches. Cell penetrating peptides (CPP) are short amino acid sequences with an intrinsic ability to translocate across plasma membranes, and theoretically represent an attractive vector for delivery of therapeutic peptides or nanoparticles to glia to promote cell survival or remyelination. The CPPs described to date are commonly non-selective in the cell types they transduce, limiting their therapeutic application in vivo. Here, we describe a theoretical framework for design of a novel CPP sequence that selectively transduces human glial cells (excluding non-glial cell types), and conduct preliminary screens of purified, recombinant CPPs with immature and matured human oligodendrocytes and astrocytes, and two non-glial cell types. A candidate peptide, termed TD2.2, consistently transduced glial cells, was significantly more effective at transducing immature oligodendrocytes than matured progeny, and was virtually incapable of transducing two non-glial cell types: (i) human neural cells and (ii) human dermal fibroblasts. Time-lapse confocal microscopy confirms trafficking of TD2.2 (fused to EGFP) to mature oligodendrocytes 3-6 hours after protein application in vitro. We propose selectivity of TD2.2 for glial cells represents a new therapeutic strategy for the treatment of glial-related disease, such as MS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045501PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458069PMC
April 2013

Cryopreservation and long-term maintenance of bovine embryo-derived cell lines.

Reprod Fertil Dev 2013 ;25(4):707-18

Centre for Reproduction and Development, Monash Institute of Medical Research, Clayton, Vic. 3168, Australia.

The aim of this study was to develop methods for cryopreservation and long-term maintenance of putative bovine embryonic stem cells (ESCs). Putative bovine ESC (bESC) lines (n=3) isolated in conventional medium were used to compare slow-freezing and vitrification. After warming, vitrified cells (96.9%) demonstrated significantly (P<0.05) better survival than frozen-thawed cells (81.5%) and formed significantly more colonies with good morphology (vitrification: 93/93, 100.0%; slow-freezing: 74/106, 69.81%; P<0.05). The effect of inhibitors of differentiation (PD184352, SU5402, CHIR99021) on ESC maintenance was assessed on putative bESC lines established in N2B27-3i medium (n=8) or conventional medium (n=1) after culture over 30 passages (>240 days). All cell lines expressed ALP, SSEA1, SSEA4, OCT4, REX1 and SSEA1. OCT4 expression was confirmed by relative real-time PCR and was upregulated in early passages of putative bESCs cultured in N2B27-3i (2.9±0.89-fold higher at Passage (P) 2-4), whereas the converse was observed later (P22-26; 2.2±0.1-fold increase in conventional medium). Putative bESC lines isolated in N2B27-3i medium (n=3) or conventional medium (n=1) were vitrified at P18 and, after warming, were cultured for a further 12 passages. These cells survived vitrification and expressed OCT4, REX1, SSEA1, ALP, SSEA1 and SSEA4. These results demonstrate that putative bESC lines that express pluripotent markers can be cultured long term and retain expression of pluripotent markers after vitrification.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1071/RD12018DOI Listing
November 2013

Temporal Requirements of cMyc Protein for Reprogramming Mouse Fibroblasts.

Stem Cells Int 2012 26;2012:541014. Epub 2012 Apr 26.

Cell Reprogramming and Stem Cells Laboratory, Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, VIC 3168, Australia.

Exogenous expression of Oct4, Sox2, Klf4, and cMyc forces mammalian somatic cells to adopt molecular and phenotypic characteristics of embryonic stem cells, commencing with the required suppression of lineage-associated genes (e.g., Thy1 in mouse). Although omitting cMyc from the reprogramming cocktail minimizes risks of uncontrolled proliferation, its exclusion results in fold reductions in reprogramming efficiency. Thus, the feasibility of substituting cMyc transgene with (non-integrative) recombinant "pTAT-mcMyc" protein delivery was assessed, without compromising reprogramming efficiency or the pluripotent phenotype. Purification and delivery of semisoluble/particulate pTAT-mcMyc maintained Oct4-GFP(+) colony formation (i.e., reprogramming efficiency) whilst supporting pluripotency by various criteria. Differential repression of Thy1 by pTAT-mcMyc ± Oct4, Sox2, and Klf4 (OSK) suggested differential (and non-additive) mechanisms of repression. Extending these findings, attempts to enhance reprogramming efficiency through a staggered approach (prerepression of Thy1) failed to improve reprogramming efficiency. We consider protein delivery a useful tool to decipher temporal/molecular events characterizing somatic cell reprogramming.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2012/541014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3350996PMC
August 2012

The state of the art for pluripotent stem cells derivation in domestic ungulates.

Theriogenology 2012 Nov 11;78(8):1749-62. Epub 2012 May 11.

Monash Institute for Medical Research, Clayton, Monash University, Victoria, Australia.

Since the successful isolation, characterization and long-term culture of embryonic stem cells (ESCs) from mice in the early 1980s and from humans a decade later, considerable effort has been made to establish ESCs lines from livestock. The derivation of validated ESCs lines is a necessary step if the generation of economically relevant transgenic animals is to be achieved. However, this is still elusive, as the isolation of true ESCs lines for livestock has not been accomplished to date. It has been demonstrated that by forced expression of a defined set of transcription factors, it is possible to reprogram somatic cells to cells that closely resemble an ES-like state. These cells were termed induced pluripotent stem cells (iPSCs). We introduce the basic concepts relating to stem cell biology and give an overview of the various attempts to isolate and generate pluripotent stem cells (PSCs) from species relevant to livestock production. Further, we point out the issues to be addressed and hurdles to be overcome to realize the promise of stem cells in agriculture.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.theriogenology.2012.03.031DOI Listing
November 2012

Induction of pluripotency in adult equine fibroblasts without c-MYC.

Stem Cells Int 2012 19;2012:429160. Epub 2012 Mar 19.

Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, Clayton, VIC 3800, Australia.

Despite tremendous efforts on isolation of pluripotent equine embryonic stem (ES) cells, to date there are few reports about successful isolation of ESCs and no report of in vivo differentiation of this important companion species. We report the induction of pluripotency in adult equine fibroblasts via retroviral transduction with three transcription factors using OCT4, SOX2, and KLF4 in the absence of c-MYC. The cell lines were maintained beyond 27 passages (more than 11 months) and characterized. The equine iPS (EiPS) cells stained positive for alkaline phosphatase by histochemical staining and expressed OCT4, NANOG, SSEA1, and SSEA4. Gene expression analysis of the cells showed the expression of OCT4, SOX2 NANOG, and STAT3. The cell lines retained a euploid chromosome count of 64 after long-term culture cryopreservation. The EiPS demonstrated differentiation capacity for the three embryonic germ layers both in vitro by embryoid bodies (EBs) formation and in vivo by teratoma formation. In conclusion, we report the derivation of iPS cells from equine adult fibroblasts and long-term maintenance using either of the three reprogramming factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2012/429160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3328202PMC
July 2012

Comparative study on the therapeutic potential of neurally differentiated stem cells in a mouse model of multiple sclerosis.

PLoS One 2012 13;7(4):e35093. Epub 2012 Apr 13.

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

Background: Transplantation of neural stem cells (NSCs) is a promising novel approach to the treatment of neuroinflammatory diseases such as multiple sclerosis (MS). NSCs can be derived from primary central nervous system (CNS) tissue or obtained by neural differentiation of embryonic stem (ES) cells, the latter having the advantage of readily providing an unlimited number of cells for therapeutic purposes. Using a mouse model of MS, we evaluated the therapeutic potential of NSCs derived from ES cells by two different neural differentiation protocols that utilized adherent culture conditions and compared their effect to primary NSCs derived from the subventricular zone (SVZ).

Methodology/principal Findings: The proliferation and secretion of pro-inflammatory cytokines by antigen-stimulated splenocytes was reduced in the presence of SVZ-NSCs, while ES cell-derived NSCs exerted differential immunosuppressive effects. Surprisingly, intravenously injected NSCs displayed no significant therapeutic impact on clinical and pathological disease outcomes in mice with experimental autoimmune encephalomyelitis (EAE) induced by recombinant myelin oligodendrocyte glycoprotein, independent of the cell source. Studies tracking the biodistribution of transplanted ES cell-derived NSCs revealed that these cells were unable to traffic to the CNS or peripheral lymphoid tissues, consistent with the lack of cell surface homing molecules. Attenuation of peripheral immune responses could only be achieved through multiple high doses of NSCs administered intraperitoneally, which led to some neuroprotective effects within the CNS.

Conclusion/significance: Systemic transplantation of these NSCs does not have a major influence on the clinical course of rMOG-induced EAE. Improving the efficiency at which NSCs home to inflammatory sites may enhance their therapeutic potential in this model of CNS autoimmunity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035093PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3325988PMC
August 2012

The effects of nuclear reprogramming on mitochondrial DNA replication.

Stem Cell Rev Rep 2013 Feb;9(1):1-15

Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria, 3168, Australia.

Undifferentiated mouse embryonic stem cells (ESCs) possess low numbers of mitochondrial DNA (mtDNA), which encodes key subunits associated with the generation of ATP through oxidative phosphorylation (OXPHOS). As ESCs differentiate, mtDNA copy number is regulated by the nuclear-encoded mtDNA replication factors, which initiate a major replication event on Day 6 of differentiation. Here, we examined mtDNA replication events in somatic cells reprogrammed to pluripotency, namely somatic cell-ES (SC-ES), somatic cell nuclear transfer ES (NT-ES) and induced pluripotent stem (iPS) cells, all at low-passage. MtDNA copy number in undifferentiated iPS cells was similar to ESCs whilst SC-ES and NT-ES cells had significantly increased levels, which correlated positively and negatively with Nanog and Sox2 expression, respectively. During pluripotency and differentiation, the expression of the mtDNA-specific replication factors, PolgA and Peo1, were differentially expressed in iPS and SC-ES cells when compared to ESCs. Throughout differentiation, reprogrammed somatic cells were unable to accumulate mtDNA copy number, characteristic of ESCs, especially on Day 6. In addition, iPS and SC-ES cells were also unable to regulate ATP content in a manner similar to differentiating ESCs prior to Day 14. The treatment of reprogrammed somatic cells with an inhibitor of de novo DNA methylation, 5-Azacytidine, prior to differentiation enabled iPS cells, but not SC-ES and NT-ES cells, to accumulate mtDNA copies per cell in a manner similar to ESCs. These data demonstrate that the reprogramming process disrupts the regulation of mtDNA replication during pluripotency but this can be re-established through the use of epigenetic modifiers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12015-011-9318-7DOI Listing
February 2013

Generation and characterization of reprogrammed sheep induced pluripotent stem cells.

Theriogenology 2012 Jan 29;77(2):338-46.e1. Epub 2011 Sep 29.

Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, Clayton, VIC 3168, Australia.

Embryonic stem cells (ESCs) from domestic species have numerous potential applications in agricultural and biomedical sciences; however, despite intensive efforts, derivation of ESCs from sheep remains elusive. The objective was to derive sheep induced pluripotent stem cells (iPSCs), as an alternative pluripotent cell type to ESCs, from sheep fibroblasts by ectopic expression of heterologous transcription factors OCT4, SOX2, KLF4, and cMYC. Sheep fibroblasts were infected with pantropic retroviruses coding the four transcription factors and reprogrammed to pluripotency at a rate of 0.002%. The sheep iPSCs (siPSCs) reactivated endogenous OCT4 and SOX2 genes assessed by qRT-PCR and immuno-cytochemistry, retained normal karyotyping, and more importantly, concurrently silenced all exogenous transgenes. The siPSCs were enzymatically dissociated to single cells, making them amenable to efficient transfection and fluorescent-activated cell sorting techniques. Further, the siPSCs differentiated in vitro to form embryoid bodies, and in vivo to form robust teratomas, containing cells representative of the three germ layers. Moreover, when injected into diploid or tetraploid sheep embryos, siPSCs contributed to the inner cell mass of resulting blastocysts, suggesting true pluripotential. These reprogrammed siPSCs may constitute a robust pluripotent alternative to elusive sheep ESCs, with great potential for use in agriculture and pharmaceutical biotechnology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.theriogenology.2011.08.006DOI Listing
January 2012

Cellular reprogramming of somatic cells.

Indian J Exp Biol 2011 Jun;49(6):409-15

Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton VIC 3168, Australia.

The process of 'cell reprogramming' can be achieved by somatic cell nuclear transfer, cell fusion with embryonic stem cells, exposure to stem cell extracts, or by inducing pluripotentcy mediated by defined factors giving rise to what are termed induced pluripotent stem cells. More recently, the fate of a somatic cell can be directly induced to uptake other cell fates, termed lineage-specific reprogramming, without the need to de-differentiate the cells to a pluripotent state. In this review we will describe the different methods of reprogramming somatic cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
June 2011

Generation of stable pluripotent stem cells from NOD mouse tail-tip fibroblasts.

Diabetes 2011 May 4;60(5):1393-8. Epub 2011 Apr 4.

Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia.

Objective: The NOD mouse strain has been widely used to investigate the pathology and genetic susceptibility for type 1 diabetes. Induced pluripotent stem cells (iPSCs) derived from this unique mouse strain would enable new strategies for investigating type 1 diabetes pathogenesis and potential therapeutic targets. The objective of this study was to determine whether somatic fibroblasts from NOD mice could be reprogrammed to become iPSCs, providing an alternative source of stem cells for the production of genetically modified NOD cells and mice.

Research Design And Methods: Adult tail-tip fibroblasts from male NOD mice were reprogrammed by retroviral transduction of the coding sequences of three transcription factors, OCT4, SOX2, and KLF4, in combination with a histone deacetylase inhibitor, valproic acid.

Results: Eighteen NOD iPSC lines were generated, and three of these cell lines were further characterized. All three cell lines exhibited silencing of the three reprogramming transgenes and reactivation of endogenous pluripotent markers (OCT4, SOX2, NANOG, REX1, and SSEA1). These NOD iPSCs readily differentiated in vitro to form embryoid bodies and in vivo by teratoma formation in immunodeficient mice. Moreover, NOD iPSCs were successfully transfected with a reporter transgene and were capable of contributing to the inner cell mass of C57BL/6 blastocysts, leading to the generation of a chimeric mouse.

Conclusions: Adult tail-tip fibroblasts from NOD mice can be reprogrammed, without constitutive ectopic expression of transcription factors, to produce iPSCs that exhibit classic mouse embryonic stem cell (ESC) features. These NOD iPSCs can be maintained and propagated under normal ESC culture conditions to produce genetically altered cell lines, differentiated cells, and chimeric mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db10-1540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3292312PMC
May 2011

Generation of clinically relevant "induced pluripotent stem" (iPS) cells.

J Stem Cells 2011 ;6(3):109-27

Reprogramming and Stem Cell Laboratory, Monash Institute of Medical Research, Monash University, Clayton, VIC, Australia.

Proviral expression of early development genes Oct4 and Sox2, in concert with cMyc and Klf4 or Nanog and Lin28, can induce differentiated cells to adopt morphological and functional characteristics of pluripotency indistinguishable from embryonic stem cells. Termed induced pluripotent stem (iPS) cells, in mice the pluripotency of these cells was confirmed by altered gene/surface antigen expression, remodeling of the epigenome, ability to contribute to embryonic lineages following blastocyst injection and commitment to all three germ layers in teratomas and liveborn chimeras. Importantly, in vitro directed differentiation of iPS cells yield cells capable of treating mouse models of humanized disease. Despite these impressive results, iPS cell conversion is frustratingly inefficient. Also, the unpredictable and random mutagenesis imposed on the host cell genome, inherent with integrative viral methodologies, continues to hamper use of these cells in a therapeutic setting. This has initiated exploration of non-integrating strategies for generating iPS cells. Here, we review mechanisms that drive conversion of somatic cells to iPS cells and the strategies adopted to circumvent integrative viral strategies. Finally, we discuss practical, ethical and legal considerations that require addressing before iPS cells can realize their potential as patient-specific cells for treatment of degenerative disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
January 2013