Current Gene Therapy (v.13, #2)

Technological Overview of iPS Induction from Human Adult Somatic Cells by Emilie Bayart, Odile Cohen-Haguenauer (73-92).
The unlimited proliferation capacity of embryonic stem cells (ESCs) combined with their pluripotent differentiationpotential in various lineages raised great interest in both the scientific community and the public at large with hopefor future prospects of regenerative medicine. However, since ESCs are derived from human embryos, their use is associatedwith significant ethical issues preventing broad studies and therapeutic applications. To get around this bottleneck,Takahashi and Yamanaka have recently achieved the conversion of adult somatic cells into ES-like cells via the forcedexpression of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. This first demonstration attracted public attentionand opened a new field of stem cells research with both cognitive

Embryonic Stem Cells or Induced Pluripotent Stem Cells? A DNA Integrity Perspective by Qiang Bai, Romain Desprat, Bernard Klein, Jean-Marc Lemaitre, John De Vos (93-98).
Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are two types of pluripotent stem cellsthat hold great promise for biomedical research and medical applications. iPSCs were initially favorably compared toESCs. This view was first based on ethical arguments (the generation of iPSCs does not require the destruction of an embryo)and on immunological reasons (it is easier to derive patient HLA-matched iPSCs than ESCs). However, several reportssuggest that iPSCs might be characterized by higher occurrence of epigenetic and genetic aberrations than ESCs as aconsequence of the reprogramming process. We focus here on the DNA integrity of pluripotent stem cells and examinethe three main sources of genomic abnormalities in iPSCs: (1) genomic variety of the parental cells, (2) cell reprogramming,and (3) in vitro cell culture. Recent reports claim that it is possible to generate mouse or human iPSC lines with amutation level similar to that of the parental cells, suggesting that genome-friendly reprogramming techniques can bedeveloped. The issue of iPSC DNA integrity clearly highlights the crucial need of guidelines to define the acceptable levelof genomic integrity of pluripotent stem cells for biomedical applications. We discuss here the main issues that suchguidelines should address.

Modelling Human Disease with Pluripotent Stem Cells by Richard Siller, Sebastian Greenhough, In-Hyun Park, Gareth J. Sullivan (99-110).
Recent progress in the field of cellular reprogramming has opened up the doors to a new era of disease modelling,as pluripotent stem cells representing a myriad of genetic diseases can now be produced from patient tissue. Thesecells can be expanded and differentiated to produce a potentially limitless supply of the affected cell type, which can thenbe used as a tool to improve understanding of disease mechanisms and test therapeutic interventions. This process requireshigh levels of scrutiny and validation at every stage, but international standards for the characterisation of pluripotent cellsand their progeny have yet to be established. Here we discuss the current state of the art with regard to modelling diseasesaffecting the ectodermal, mesodermal and endodermal lineages, focussing on studies which have demonstrated a diseasephenotype in the tissue of interest. We also discuss the utility of pluripotent cell technology for the modelling of cancerand infectious disease. Finally, we spell out the technical and scientific challenges which must be addressed if the field isto deliver on its potential and produce improved patient outcomes in the clinic.

Genetically-Modified Human Pluripotent Stem Cells: New Hopes for the Understanding and the Treatment of Neurological Diseases? by Stephane Nedelec, Brigitte Onteniente, Marc Peschanski, Cecile Martinat (111-119).
The fundamental inaccessibility of the human neural cell types affected by neurological disorders prevents theirisolation for in vitro studies of disease mechanisms or for drug screening efforts. Pluripotent stem cells represent a newinteresting way to generate models of human neurological disorders, explore the physiopathological mechanisms and developnew therapeutic strategies. Disease-specific human embryonic stem cells were the first source of material to be usedto study certain disease states. The recent demonstration that human somatic cells, such as fibroblasts or blood cells, canbe genetically converted to induced pluripotent stem cells (hiPSCs) together with the continuous improvement of methodsto differentiate these cells into disease-affected neuronal subtypes opens new perspectives to model and understand a largenumber of human pathologies. This review focuses on the opportunities concerning the use disease-specific human pluripotentstem cells as well as the different challenges that still need to be overcome. We also discuss the recent improvementsin the genetic manipulation of human pluripotent stem cells and the consequences of these on disease modeling anddrug screening for neurological diseases.

Human Pluripotent Stem Cells for Modelling Human Liver Diseases and Cell Therapy by Noushin Dianat, Clara Steichen, Ludovic Vallier, Anne Weber, Anne Dubart-Kupperschmitt (120-132).
The liver is affected by many types of diseases, including metabolic disorders and acute liver failure. Orthotopicliver transplantation (OLT) is currently the only effective treatment for life-threatening liver diseases but transplantationof allogeneic hepatocytes has now become an alternative as it is less invasive than OLT and can be performed repeatedly.However, this approach is hampered by the shortage of organ donors, and the problems related to the isolation ofhigh quality adult hepatocytes, their cryopreservation and their absence of proliferation in culture. Liver is also a key organto assess the pharmacokinetics and toxicology of xenobiotics and for drug discovery, but appropriate cell culture systemsare lacking. All these problems have highlighted the need to explore other sources of cells such as stem cells thatcould be isolated, expanded to yield sufficiently large populations and then induced to differentiate into functional hepatocytes.The presence of a niche of facultative progenitor and stem cells in the normal liver has recently been confirmedbut they display no telomerase activity. The recent discovery that human induced pluripotent stem cells can be generatedfrom somatic cells has renewed hopes for regenerative medicine and in vitro disease modelling, as these cells are easilyaccessible. We review here the present progresses, limits and challenges for the generation of functional hepatocytes fromhuman pluripotent stem cells in view of their potential use in regenerative medicine and drug discovery.

Direct Somatic Cell Reprogramming: Treatment of Cardiac Diseases by Chuner Guo, Kishan Patel, Li Qian (133-138).
Cardiac diseases are the major causes of morbidity and mortality in the world. Cardiomyocyte death is a commonconsequence of many types of heart diseases and is usually irreversible. Scar tissues formed by cardiac fibroblastsserve compensatory roles for the injured heart but eventually weaken cardiac function and result in life-threatening heartfailures. Unfortunately, adult human hearts have limited regenerative capacities. In the past decades, many interventionalapproaches have been taken in an attempt to restore functional cardiomyocytes in an injured heart. Promising advanceshave been made in directly reprogramming mouse fibroblasts into cardiomyocyte-like cells both in vitro and in vivo. Recently,several different methods have been reported, including the use of transcription factors and microRNAs. In addition,two in vivo studies showed heart function improvements with delivery of reprogramming factors in mouse infarctedhearts. Although many of these studies are at early preliminary stages, the plausibility of applying cardiac reprogrammingon patients for regenerative purposes is exciting, and may lead to numerous novel research directions in the field. This reviewwill discuss the history, recent advances and challenges of cellular reprogramming, specifically in the field of cardiacregeneration.

Induced pluripotent stem cells (iPSc) are a scientific and medical frontier. Application of reprogrammed somaticcells for clinical trials is in its dawn period; advances in research with animal and human iPSc are paving the wayfor retinal therapies with the ongoing development of safe animal cell transplantation studies and characterization of patient-specific and disease-specific human iPSc. The retina is an optimal model for investigation of neural regeneration;amongst other advantageous attributes, it is the most accessible part of the CNS for surgery and outcome monitoring. Arecent clinical trial showing a degree of visual restoration via a subretinal electronic prosthesis implies that even a severelydegenerate retina may have the capacity for repair after cell replacement through potential plasticity of the visualsystem. Successful differentiation of neural retina from iPSc and the recent generation of an optic cup from human ESc invitroincrease the feasibility of generating an expandable and clinically suitable source of cells for human clinical trials. Inthis review we shall present recent studies that have propelled the field forward and discuss challenges in utilizing iPS cellderived retinal cells as reliable models for clinical therapies and as a source for clinical cell transplantation treatment forpatients suffering from genetic retinal disease.

Promotion of Cortico-Cerebral Precursors Expansion by Artificial pri-miRNAs Targeted Against the Emx2 Locus by Assunta Diodato, Moira Pinzan, Marilena Granzotto, Antonello Mallamaci (152-161).
Emx2 encodes for a transcription factor controlling several aspects of cerebral cortex development. Its overexpressionpromotes self-renewal of young cortico-cerebral precursors, it promotes neuronal rather than gliogenic fates andit protects neuronal progenitors from cell death. These are all key activities for purposes of gene-promoted brain repair.

Artificial pri-miRNAs targeting non-coding cis-active modules and/or conserved sequences of the Emx2 locus were deliveredto embryonic cortico-cerebral precursors, by lentiviral vectors. A subset of these pri-miRNAs upregulated Emx2,possibly stimulating its transcription. That led to enhanced self-renewal, delayed differentiation and reduced death of neuronallycommitted precursors, resulting in an appreciable expansion of the neuronogenic precursors pool. This methodmakes Emx2 overexpression for purposes of brain repair a more feasible goal, avoiding the drawbacks of exogenous genecopies introduction.

Interestingly, the two genomic enhancers targeted by these pri-miRNAs were discovered to be naturally transcribed. Theirexpression profile suggests their possible involvement in regulation of Emx2 transcription.