Current Gene Therapy - Volume 13, Issue 2, 2013

The unlimited proliferation capacity of embryonic stem cells (ESCs) combined with their pluripotent differentiation potential in various lineages raised great interest in both the scientific community and the public at large with hope for future prospects of regenerative medicine. However, since ESCs are derived from human embryos, their use is associated with 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 forced expression of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. This first demonstration attracted public attention and opened a new field of stem cells research with both cognitive – such as disease modeling - and therapeutic prospects. This pioneer work just received the 2012 Nobel Prize in Physiology or Medicine. Many methods have been reported since 2006, for the generation of induced pluripotent stem (iPS) cells. Most strategies currently under use are based on gene delivery via gamma-retroviral or lentiviral vectors; some experiments have also been successful using plasmids or transposons- based systems and few with adenovirus. However, most experiments involve integration in the host cell genome with an identified risk for insertional mutagenesis and oncogenic transformation. To circumvent such risks which are deemed incompatible with therapeutic prospects, significant progress has been made with transgene-free reprogramming methods based on e.g.: sendai virus or direct mRNA or protein delivery to achieve conversion of adult cells into iPS. In this review we aim to cover current knowledge relating to both delivery systems and combinations of inducing factors including chemicals which are used to generate human iPS cells. Finally, genetic instability resulting from the reprogramming process is also being considered as a safety bottleneck for future clinical translation and stem cell-therapy prospects based on iPS.

Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are two types of pluripotent stem cells that hold great promise for biomedical research and medical applications. iPSCs were initially favorably compared to ESCs. 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 reports suggest that iPSCs might be characterized by higher occurrence of epigenetic and genetic aberrations than ESCs as a consequence of the reprogramming process. We focus here on the DNA integrity of pluripotent stem cells and examine the 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 a mutation level similar to that of the parental cells, suggesting that “genome-friendly” reprogramming techniques can be developed. The issue of iPSC DNA integrity clearly highlights the crucial need of guidelines to define the acceptable level of genomic integrity of pluripotent stem cells for biomedical applications. We discuss here the main issues that such guidelines should address.

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. These cells can be expanded and differentiated to produce a potentially limitless supply of the affected cell type, which can then be used as a tool to improve understanding of disease mechanisms and test therapeutic interventions. This process requires high levels of scrutiny and validation at every stage, but international standards for the characterisation of pluripotent cells and their progeny have yet to be established. Here we discuss the current state of the art with regard to modelling diseases affecting the ectodermal, mesodermal and endodermal lineages, focussing on studies which have demonstrated a disease phenotype in the tissue of interest. We also discuss the utility of pluripotent cell technology for the modelling of cancer and infectious disease. Finally, we spell out the technical and scientific challenges which must be addressed if the field is to deliver on its potential and produce improved patient outcomes in the clinic.

The fundamental inaccessibility of the human neural cell types affected by neurological disorders prevents their isolation for in vitro studies of disease mechanisms or for drug screening efforts. Pluripotent stem cells represent a new interesting way to generate models of human neurological disorders, explore the physiopathological mechanisms and develop new therapeutic strategies. Disease-specific human embryonic stem cells were the first source of material to be used to study certain disease states. The recent demonstration that human somatic cells, such as fibroblasts or blood cells, can be genetically converted to induced pluripotent stem cells (hiPSCs) together with the continuous improvement of methods to differentiate these cells into disease-affected neuronal subtypes opens new perspectives to model and understand a large number of human pathologies. This review focuses on the opportunities concerning the use disease-specific human pluripotent stem cells as well as the different challenges that still need to be overcome. We also discuss the recent improvements in the genetic manipulation of human pluripotent stem cells and the consequences of these on disease modeling and drug screening for neurological diseases.

The liver is affected by many types of diseases, including metabolic disorders and acute liver failure. Orthotopic liver transplantation (OLT) is currently the only effective treatment for life-threatening liver diseases but transplantation of 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 of high quality adult hepatocytes, their cryopreservation and their absence of proliferation in culture. Liver is also a key organ to assess the pharmacokinetics and toxicology of xenobiotics and for drug discovery, but appropriate cell culture systems are lacking. All these problems have highlighted the need to explore other sources of cells such as stem cells that could 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 confirmed but they display no telomerase activity. The recent discovery that human induced pluripotent stem cells can be generated from somatic cells has renewed hopes for regenerative medicine and in vitro disease modelling, as these cells are easily accessible. We review here the present progresses, limits and challenges for the generation of functional hepatocytes from human pluripotent stem cells in view of their potential use in regenerative medicine and drug discovery.

Cardiac diseases are the major causes of morbidity and mortality in the world. Cardiomyocyte death is a common consequence of many types of heart diseases and is usually irreversible. Scar tissues formed by cardiac fibroblasts serve compensatory roles for the injured heart but eventually weaken cardiac function and result in life-threatening heart failures. Unfortunately, adult human hearts have limited regenerative capacities. In the past decades, many interventional approaches have been taken in an attempt to restore functional cardiomyocytes in an injured heart. Promising advances have 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 infarcted hearts. Although many of these studies are at early preliminary stages, the plausibility of applying cardiac reprogramming on patients for regenerative purposes is exciting, and may lead to numerous novel research directions in the field. This review will discuss the history, recent advances and challenges of cellular reprogramming, specifically in the field of cardiac regeneration.

Induced pluripotent stem cells (iPSc) are a scientific and medical frontier. Application of reprogrammed somatic cells for clinical trials is in its dawn period; advances in research with animal and human iPSc are paving the way for 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. A recent clinical trial showing a degree of visual restoration via a subretinal electronic prosthesis implies that even a severely degenerate retina may have the capacity for repair after cell replacement through potential plasticity of the visual system. Successful differentiation of neural retina from iPSc and the recent generation of an optic cup from human ESc invitro increase the feasibility of generating an expandable and clinically suitable source of cells for human clinical trials. In this review we shall present recent studies that have propelled the field forward and discuss challenges in utilizing iPS cell derived retinal cells as reliable models for clinical therapies and as a source for clinical cell transplantation treatment for patients suffering from genetic retinal disease.

Emx2 encodes for a transcription factor controlling several aspects of cerebral cortex development. Its overexpression promotes self-renewal of young cortico-cerebral precursors, it promotes neuronal rather than gliogenic fates and it 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 delivered to 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 neuronally committed precursors, resulting in an appreciable expansion of the neuronogenic precursors pool. This method makes Emx2 overexpression for purposes of brain repair a more feasible goal, avoiding the drawbacks of exogenous gene copies introduction. Interestingly, the two genomic enhancers targeted by these pri-miRNAs were discovered to be naturally transcribed. Their expression profile suggests their possible involvement in regulation of Emx2 transcription.