Cell Reprogramming for Regenerative Medicine (1)
Article by Anne B.C. Cherry and George Q. Daley Reprogramming Cellular Identity for Regenerative Medicine
published in the journal Cell (148, March 16, 2012).
Translation (with abbreviations): Evgenia Ryabtseva
Cell differentiation: from ESC to iPSC
Despite the fact that the process of development of the organism leads in the direction of limiting the differentiation capabilities of cells, the results of recent studies have demonstrated the possibility of transformation of cells of one type into another, which opens up huge prospects in the field of biomedical research, including modeling diseases using stem cells isolated from a patient.
This article examines evidence in favor of the fact that due to the significant elimination of the effects of environmental factors and epigenetic changes that occur during reprogramming, in the short term, patient-specific models of diseases with a strong genetic component with a high degree of penetrance (probability of phenotypic manifestation in the population) are the most informative. The possibilities of applying new cell reprogramming capabilities in biomedicine are also discussed.
As the zygote divides and becomes a complex organism, the cells inevitably differentiate. The expression of genes within a single genome naturally changes under the influence of a complex set of factors until various differentiation sprouts are formed and the fate of tissues is determined. The ability of a multicellular organism to create a variety of cell types based on a single genome provides it with the ability to adapt and thrive in different environmental conditions. Evolutionarily earlier unicellular organisms lack this ability. Despite the fact that some complex organisms, such as salamanders, can regenerate large fragments of the body by dedifferentiating cells, most multicellular organisms lose this ability at the end of embryogenesis.
As a result of evolution, mammalian cells that have passed the full cycle of natural development and differentiation and have reached their final specialized state lose the ability to self-renew and inevitably enter the phase of physiological aging. Mutations of genes involved in the functioning of mechanisms that ensure that cells belong to a certain type, their stability and physiological aging increase the likelihood of their malignancy. For example, the acquisition by granulocyte-macrophage progenitor cells of the ability to self-renew means their transformation into leukemic stem cells (Krivtsov et al., 2006). Pathological conditions that contribute to the acquisition by cells of the ability to change their affiliation can similarly lead to the development of different types of cancer.
Currently, scientists are studying the mechanisms that ensure the transformation of differentiated cells into cells of another type (metaplasia) or into less differentiated cells (dysplasia). Existing data indicate that changes in the epigenome and gene expression lead to such transformations, which, in turn, provides a favorable ground for the appearance of mutations that contribute to malignant transformation (Kang et al., 2003; Nardone et al., 2007; Herfs et al., 2009).
Changing the ownership of cells in the laboratoryThe process of cell differentiation during the development of the organism is described in detail during in vivo studies, however, answers to some questions are easier to obtain under strictly controlled conditions of tissue culture in vitro.
Human embryonic cells (EC) isolated from the internal cell mass of blastocysts were first successfully obtained less than 15 years ago by a group of scientists from the University of Wisconsin working under the leadership of Thomson (Thomson et al., 1998). The uniqueness of these pluripotent cells lies in their ability to divide indefinitely and retain the ability to differentiate into cells of three embryonic sprouts. Obtaining these cells gave the specialists great hopes about studying the mechanisms of development of the human body and the progression of various diseases, as well as the possibility of replacing the affected tissues with new ones.
The tissue belonging of cells is a reflection of their epigenomes, which can be changed as a result of many internal processes, including normal development, metaplasia and dysplasia. Experiments on direct differentiation, transdifferentiation and reprogramming can also be carried out with them.
However, this task is extremely difficult. Mouse embryonic cells have been widely used in research work for more than three decades, but so far attempts to transform them into functionally complete blood cells, pancreas and highly specialized neurons have been only partially successful. Despite this, biologists are confident that embryonic cells can be differentiated into cells of different types. The difficulty lies in the development of protocols that would provide cultivation conditions that repeat in the smallest detail the conditions in which the natural development of the embryo takes place.
During in vitro differentiation, stem cells induce the formation of aggregates with a predictable structure (embryonic bodies) that repeats the organization of the embryo. Growth factors that selectively guide the differentiation of pluripotent cells have also been identified. The aim of these approaches is to reproduce epigenetic changes occurring during embryogenesis in order to obtain tissues similar to those formed as a result of embryonic development. Researchers have managed to develop protocols that allow the transformation of human embryonic stem cells into cells of many types, including motor neurons, retinal epithelial cells and hematopoiesis progenitor cells (Wichterle et al., 2002; Klimanskaya et al., 2004; Ng et al., 2005). These protocols confirmed the general opinion that the effect of differentiation factors should repeat the natural changes occurring during the development of the organism.
However, this paradigm was refuted in 2006, when Takahashi and Yamanaka published the results of experiments in which they managed to "unnaturally" transform mature mouse fibroblasts into so-called induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006).
Scientists have been able to return differentiated cells to the embryonic state before, but the method of somatic cell nucleus transfer used for this is inefficient, time-consuming and poorly understood (Rideout et al., 2001). Yamanaka's approach, on the contrary, is based on the use of several known factors that allow radically changing the affiliation of cells. This revolutionary research led to a revolution in cell biology and provided specialists with a powerful new strategy for biomedical research and regenerative medicine.
The pluripotency of mouse iPSCs has been confirmed by a variety of tests, including gene expression analysis and epigenome profiling, as well as the formation of chimeras and tetraploid embryos (Okita et al., 2007; Zhao et al., 2009). Shortly after the first publication appeared, scientists from many laboratories managed to reproduce the results of reprogramming on human cells (Yu et al., 2007; Takahashi et al., 2007; Park et al., 2008b). Analysis of gene expression and epigenome confirmed that the resulting iPSCs are much closer to embryonic stem cells than to the cells of the tissue from which they were isolated (Hawkins et al., 2010; Doi et al., 2009; Kim et al., 2010; Polo et al., 2010). This confirmed that the transduction of four transcription factors (Oct4, Sox2, Klf4 and c-Myc) really reverses the development of mammalian cells.
The original publication devoted to the reprogramming of iPSCs inspired scientists to various attempts to manipulate their differentiation. Ectopic expression of transcription factors is currently being considered as a potential method of direct transformation (transdifferentiation) into cells of another type – for example, the production of neurons and cardiomyocytes from fibroblasts and insulin-producing beta cells from exocrine cells (Zhou et al., 2008; Vierbuchen et al., 2010; Ieda et al., 2010; Efe et al., 2011). Researchers continue to publish protocols for the transformation of pluripotent cells into cells of various types, more efficient production of stem cells from somatic cells, as well as the transformation of differentiated cells from one type to another.
This diversity was the result of a radical change in ideas about the possibilities of changing the affiliation of cells in vitro (Fig. 1). The recent awareness of cell plasticity has made this area of research one of the most interesting areas of modern stem cell biology. Potential areas of application of iPSCs are the creation of disease models, drug screening and cell therapy. The introduction of cell therapy methods into practice is still on the horizon, while the use of reprogramming technology for disease modeling and drug testing has already begun.
Figure 1. The variability of cellular affiliation under in vivo and in vitro conditions.
The study of human diseases can be carried out on a variety of platforms, from epidemiology to animal models and cell cultures. All these approaches provide different types of information and each of them has its own limitations. New platforms for the study of diseases do not arise so often, so the appearance of pluripotent stem cells is a very significant event.
Researchers hope that the differentiation of a patient's own iPSCs into cells responsible for the development of a particular disease will become a powerful new tool that will allow us to prove existing or formulate new hypotheses about the etiology of diseases (Maehr et al., 2009). For diseases in which the toxicity of the drugs used for the heart or kidneys is a factor limiting the effectiveness of treatment, stem cell-based models will allow experimentally assessing and reducing the toxicity associated with treatment. Finally, the cell models obtained from iPSC with disease-specific phenotypes can be used for screening of potential agents capable of influencing the molecular mechanisms of disease pathogenesis in vitro and subsequent testing on living organisms.
Continuation: Modeling of diseases in vitroPortal "Eternal youth" http://vechnayamolodost.ru