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Incomplete Transgene Silencing Identified as the Downfall of iPSCs

“Comparative Analysis of Targeted Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) and Human Embryonic Stem Cells Reveals Variability Associated With Incomplete Transgene Silencing in Retrovirally Derived hiPSC Lines”

From Stem Cells Translational Medicine

Multiple studies have been published on the comparison of human induced pluripotent stem cells (iPSCs) to human embryonic stem cells (hESCs) to analyse whether iPSCs are a suitable replacement for hESCs in a therapeutic context. However, many of these studies have focused on the undifferentiated phenotype, non-directed differentiation (embryoid bodies or teratomas), or their differentiation toward a single specific lineage (Boulting et al, Hu et al, Osafune et al and Zhang et al). However now, in a study from the laboratory of Timo Otonkoski from the University of Helsinki, Finland, published in Stem Cells Translational Medicine, researchers have taken advantage of protocols optimized for endoderm, mesoderm, and ectoderm differentiation, to make a mass comparison of differentiation efficiency.   Interestingly, no systematic differences were observed, with the main difference arising in one cell line with incomplete transgene silencing (Toivonen, Ojala and Hyysalo et al).

Step Forward in Modelling Aggregation Based Disease

“Modeling Human Protein Aggregation Cardiomyopathy Using Murine Induced Pluripotent Stem Cells”

From Stem Cells Translational Medicine

Mutations in the heat shock protein αB-crystallin (CryAB) are linked to skeletal and cardiac myopathies (Rajasekaran et al, Vicart et al and Wang et al) with one specific mutation (R120G) also leading to the formation of large cytoplasmic aggregates (Bova et al and Maloyan et al). To model this mutation, researchers from the laboratory of Ivor Benjamin at the Departments of Internal Medicine and Biochemistry, Salt Lake City, Utah, USA have taken advantage of an existing transgenic R120G mouse model to generate induced pluripotent stem cells (iPSCs) and differentiate them into cardiomyocytes. From here they show that these disease specific iPSCs recapitulate both key hallmarks of this mutation found in animal models and patients, CryAB protein aggregation and cellular hypertrophy, thereby presenting an attractive cell model system for further research (Limphong and Zhang et al).

hESC-derived cells for Cranial Tissue Regeneration

“Derivation of Multiple Cranial Tissues and Isolation of Lens Epithelium-Like Cells From Human Embryonic Stem Cells”

From Stem Cells Translational Medicine

Cells from the neural plate border (NPB) are known to give rise to neural crest cell (NC) and cranial placode (CP) progenitors (Streit) aided by signalling from the neural plate, the non-neural epithelium, and the underlying mesoderm (Litsiou et al, McCabe and Bronner-Fraser and Patthey et al). While NC cells have been studied after derivation and isolation from human embryonic stem cells (ESCs) (Chimge and Bayarsaihan), less is understood about CP derivatives in humans (Qiu et al and Yang et al), which give rise to lens, olfactory, hypophyseal, otic, epibranchial, trigeminal, lateral line fate in vivo. Now, in a study published in Stem Cells Translational Medicine, Mengarelli and Tiziano from Monash University, Victoria, Australia have studied hESC-derived NC-like cells, demonstrating their heterogeneity and have isolated and purified a CP derivative; proliferative lens epithelium-like cells which are capable of forming lentoid bodies.

Integration-Free iPSCs made Easy

Original article from STEM CELLS

"An Efficient Nonviral Method to Generate Integration-Free Human-Induced Pluripotent Stem Cells from Cord Blood and Peripheral Blood Cells"

Induced pluripotent stem cell (iPSCs) production is a labour intensive process which has yet to identify a preferred cell source or protocol for their induction suitable for their transition into a therapeutic context. However, some progress has been made. As a cell source for reprogramming, cord blood cells and peripheral blood mononuclear cells (PMNCs) could be useful due to the ease of their collection and the large amounts of cells with different human leukocyte antigen haplotypes already collected by cord blood banks. iPSCs have been generated from such cells already (Chou et al, Loh et al, Seki et al and Yu et al); however each of these reports has associated problems. Initially retroviral methods of iPSC production were used (Loh et al), although the protocol was relatively labour-intensive and places a major burden on the donor for cell provision. Then non-integrating Sendai viruses were used as a vector (Seki et al), although these infectious viruses are hard to handle and modify. Lastly, episomal plasmids, which use components of the Epstein-Barr virus, OriP and EBNA1, were used for the deviation of non-integrating iPSCs (Yu et al and Chou et al); however the authors of this study suggest that this method is inefficient. Now, in a study in Stem Cells, researchers from the laboratory of Shinya Yamanaka at the Department of Reprogramming Science, Center for iPS Cell Research and Application (CiRA), Kyoto University, Japan report on their novel protocol in which they demonstrate highly efficient iPSC induction production from PMNCs using a modified combination of previously described plasmids (Okita et al).

 

iPSCs Studies Uncover Down Syndrome Developmental Defect

Original article from STEM CELLS

"Integration-Free Induced Pluripotent Stem Cells Model Genetic and Neural Developmental Features of Down Syndrome Etiology"

Patients with Down syndrome (DS), the most frequent cause of human congenital mental retardation (Parker et al), exhibit severe aberrations in their nervous system (see paper for extensive references) but the unavailability of foetal brain tissue to study has hampered research into the early development of brain phenotypes. While mouse models show limited use due to the observed non-equivalence of mouse DS and human DS (O'Doherty et al, Reeves, Watase and Zoghbi, and Yu et al), induced pluripotent stem cells (iPSCs) have the potential for creating useful developmental models of DS. Now, in a study published in Stem Cells, researchers from the laboratory of Ernst J. Wolvetang at the Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Australia have created non-viral DS iPSCs allowing the study of their development into distinct cell types and additionally they provide proof of concept for screening corrective therapeutics(Briggs et al).

 

New human growth factor for stem cell culture discovered

March 2013 - Researchers at the cancer and stem cell development company Minerva Biotechnologies have announced that a newly discovered natural human growth factor, called NM23-H1, can maintain the pluripotency of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSC) in a culture system that is free of feeder cells, conditioned media, or additional exogenous cytokines or growth factors.1 The most common method utilised to maintain and expand hESCs has been the culture of hESCs alongside a supporting layer of fibroblast feeder cells (most often of animal origin) in the presence of basic fibroblast growth factor (bFGF) and other poorly characterised media components that can show batch-to-batch variability. Defined media formulations for hESC/hiPSC culture are already on the market and in vogue (for example mTeSR and StemPro), but these are complex, containing up to 18 components, and still require the presence of extracellular matrix proteins for stem cell adhesion and growth. Another defined formulation, E8, provides a simpler alternative, but works best under hypoxic conditions.2 An additional concern is the huge amount of bFGF these defined formulations contain, up to 25 fold greater than levels used for normal stem cell culture, which may adversely affect their differentiation potential. 

Corrected iPSCs as Therapy for Muscular Atrophy

 “Genetic Correction of Human Induced Pluripotent Stem Cells from Patients with Spinal Muscular Atrophy”

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder which is associated with the selective degeneration of spinal cord motor neurons (Crawford and Pardo). It is understood to be caused by a genetic defect in the survival motor neuron 1 (SMN1) gene causing an individual to rely on the protein produced from a paralogous gene; SMN2 (Lefebvre et al 1995 and Coovert et al).    SMN1 and 2 differ by a 6 base-pair sequence in exon 7 which leads to the production of high proportions of an unstable, truncated protein (Lorson et al) which is correlated to disease severity (Lefebvre et al 1997). Recently, the differentiation of induced pluripotent stem cells (iPSCs) from SMA patients into motor neurons has been demonstrated (Ebert et al), and the potential combination of this with ex vivo correction of the SMN1 gene could prove to be a potentially interesting therapeutic avenue of exploration. Now, researchers from the laboratory of Giacomo P. Comi at the Dino Ferrari Centre, University of Milan, Italy have successfully generated iPSCs from SMA patients (SMA-iPSCs) using non-integrating episomal vectors combined with targeted gene conversion of SMN2 into an SMN1-like gene. Subsequently differentiated motor neurons showed no disease-associated phenotype and their transplantation into an SMA mouse model extended the life span of the animals and improved the disease phenotype (Corti et al).

Investigating Therapies in Disease-Specific iPSCs

"Modeling and Rescue of the Vascular Phenotype of Williams-Beuren Syndrome in Patient Induced Pluripotent Stem Cells"

Williams-Beuren syndrome (WBS) is associated with abnormal arterial narrowing caused by a hemizygous microdeletion involving 26–28 genes, including Elastin (ELN), affecting 1:10,000 individuals. Cardiovascular complications are the major cause of death with only a 46% chance of event-free survival at 20 years (Wessel et al and Kececioglu et al). ELN remains the primary gene responsible for the cardiovascular phenotype (Li et al), through vascular smooth muscle overgrowth in the vessel wall (Akhtar et al) causing arterial stiffening and narrowing (Kim et al, Mochizuki et al and Urban et al). Now, in a study in Stem Cells Translational Medicine, researchers from the laboratory of James Ellis and Seema Mital from the Hospital for Sick Children, Toronto, Canada have generated induced pluripotent stem cell (iPSC) lines from WBS patients and have differentiated these cells to SMCs to allow assessment of potential therapeutic compounds Two potential therapeutic agents were identified (Kinnear et al).

Early Reprogramming Unveiled

"Facilitators and Impediments of the Pluripotency Reprogramming Factors' Initial Engagement with the Genome"

Each passing month brings more in depth analysis of the mechanisms lying behind the induction of pluripotency from somatic cells. We currently know that only a small percentage of cells become reprogrammed into induced pluripotent stem cells (iPSCs) (Plath and Lowry) after transduction with DNA-binding transcription factors such as OCT4 (O), SOX2 (S), KLF4 (K) and MYC (M) (together OSKM), although the initial interactions of these factors with the host DNA-histone landscape is relatively unknown. Factors such as MYC are thought to accelerate the reprogramming process (Nakagawa et al and Wernig et al) either through relaxing the chromatin environment or by promoting cell growth and survival, therefore perhaps allowing more chances for stochastic events to occur (Hanna et al and Knoepfler). Now in a study published in Cell researchers from the laboratory of Kenneth S. Zaret at the Smilow Center for Translational Research, Perelman School of Medicine, University of Pennsylvania, USA have shown that the reprogramming factors bind to the genome in human fibroblasts in a unique manner. Factors bind to distal promoter elements of genes required for reprogramming in fibroblasts, while MYC cooperates with OCT4, SOX2 and KLF4 to allow binding to closed chromatin domains to overcome heterochromatic features of the somatic cell genome in order that reprogramming to pluripotency can occur (Soufi et al).

Neural Differentiation Goes SMAD(7)

"SMAD7 Directly Converts Human Embryonic Stem Cells to Telencephalic Fate by a Default Mechanism"

Neural induction (Spermann and Mangold) is generally understood to be controlled through the inhibition of transforming growth factor (TGF) β signaling. Signalling is controlled through ligand-dependent dimerization and activation of type I and type II serine-threonine kinase receptors which, in turn, induce signaling either by the canonical pathway, via receptor-associated SMAD proteins (R-SMADs), or the noncanonical pathway (Poorgholi Belverdi et al and Taylor and Wrana). Among the many inhibitors of TGFβ signaling, SMAD7 is known to be a potent, cell autonomous inhibitor that functions downstream of receptor activation and can act as a potent neural inducer when overexpressed in Xenopus (Casellas and Brivanlou and Yan et al). However, in comparison with other members of the SMAD family (see original article for extended information and references), its exact role during human development remains to be elucidated. Now, researchers from the laboratory of Ali H. Brivanlou at The Rockefeller University, New York, USA, in a study published in Stem Cells, have revealed the role of SMAD7 in neural induction from human embryonic stem cells (hESCs), finding that it has the ability to directly convert hESCs from a pluripotent state to a telencephalic (anterior forebrain) fate (Ozair et al).

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