Cell Reprogramming for Regenerative Medicine (3)

Genomics, epigenomics and external factors

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Contribution of genetics, environment and epigenetics to the development of diseases with complex etiologyDespite the fact that identical twins have identical genomes, it is not uncommon for one of them to develop a disease with a complex etiology during his life, for example, schizophrenia, type 2 diabetes or Parkinson's disease.

Traditionally, such phenotypic variability is considered as the sum of genetic variability and variability of environmental factors (Visscher et al., 2008). However, this view confuses the effects of environmental factors at a particular time and long-term epigenetic changes, without taking into account epigenetic changes resulting from stochastic processes or genetic predisposition to epigenome variability. The inability to distinguish the results of random environmental influences from random epigenetic changes may be the reason for the inability to identify and analyze the phenotypes of disease models in vitro. Therefore, when modeling a disease, a more informative approach is to assume that the cellular phenotypes of the disease are formed under the influence of three types of differently inherited factors:

• Genetic predisposition. Variations in the DNA sequence can increase or decrease the likelihood of a particular cellular phenotype. DNA sequences are completely and accurately transmitted to daughter cells during division.
• Environmental impacts (non-epigenetic). Environmental conditions (nutrient deficiency or exposure to chemicals) at any given time affect the condition and function of cells. These conditions are external factors and are not transmitted to daughter cells.
• Epigenetic effects. These stable and highly likely gene expression changes transmitted to daughter cells result from (1) environmental factors, (2) stochastic epigenetic changes, and (3) genetic predisposition to such changes.

With this in mind, human diseases can be considered as the total result of genetic, external and epigenetic factors (Petronis, 2006). The cause of the development of some rare diseases is only one of these effects, whereas many common diseases are caused by a complete triad of causal factors. All this should be taken into account when modeling diseases using iPSC. Examples of rare monofactor pathologies are described below, after which more common multifactorial diseases are discussed.

Genetic diseasesMonofactor diseases include conditions in which a single gene is responsible for the presence, absence or severity of a particular phenotype.

The gene variants that cause such pathologies include CAG repeats in Huntington's disease, mutations in the genes of blood clotting factors in hemophilia and altered forms of ion channels in hereditary long QT syndrome. There are also multifactorial diseases caused by a combination of certain genetic variants, regardless of external and epigenetic effects, as well as pathologies developing as a result of somatic mutation. For diseases caused by a violation of the DNA sequence, iPSCs created from cells with this mutation will be carriers of the causal genotype. When differentiating such cells, the resulting target cells will have a disease-causing genetic defect.

Diseases caused by environmental exposureSuch diseases occur directly as a result of environmental factors.

A good example is a burn caused by boiling water on the skin. In such situations, epigenetic and genetic factors and, accordingly, heritability are excluded. Comparison of iPSCs obtained from the cells of a patient and a healthy person will not provide any information about the pathophysiology of the burn.

Epigenetic diseasesEpigenetic diseases include diseases whose phenotype is due to the epigenetic state of the genome.

The cause of their development may be abnormal expression of a single gene, as in rare X-chromosome and Prader-Willi syndromes, or more common global regulatory disorders, in which countless stochastic and epigenetic changes caused by external factors form a predisposition to the disease (Feinberg et al., 2010). Regardless of the cause of epigenetic changes, in a number of rare conditions, the observed phenotype is 100% due to epigenetic variations. Such phenomena include anomalies of neural tube formation during global DNA hypomethylation. Such epigenetic changes are usually "erased" during cell reprogramming, so modeling of the corresponding states using iPSCs is most likely impossible.

If the disease does not fall into any of the listed categories, its phenotype is the total result of a triad of variations: genetic, epigenetic and environmental. A good example is the result of exposure to solar ultraviolet radiation, in which the human genotype, which causes pigmentation of his skin, provides resistance or predisposition to burns. In addition, exposure to ultraviolet radiation induces the activity of genes that produce the pigment melanin, which protects the skin from sunburn. And finally, the intensity and duration of exposure affects the severity of the burn. When modeling a sunburn by reprogramming cells for physiologically adequate reproduction of the phenotype, all three factors must be taken into account.

The value of modeling diseases with stem cellsPatient-specific disease modeling is most informative for diseases with significant genetic components

When somatic cells are reprogrammed into pluripotent cells, their epigenetic history is erased, and standardized cultivation conditions normalize the results of environmental factors.

When working with iPSC lines, only one of the three factors of the development of complex diseases described above – genetic variability - can be adequately reproduced (Figure 3).

Figure 3. Adequacy of modeling of patient-specific and induced diseases using iPSC

To date, scientists have already obtained quite a lot of cell lines, for the creation of which somatic cells were isolated from patients, reprogrammed them into iPSCs, which, in turn, differentiated into target cells affected by the analyzed disease. At the same time, in some cases, it was possible to identify pronounced differences between the cells of patient-specific and control lines, while in other cases, such differences were not detected. The only multifactorial disease for which the corresponding cellular phenotype was obtained was schizophrenia, the heritability of which reaches 80% (Cardno et al., 1999). All other analyzed cellular phenotypes relate to genetic diseases, such as congenital dyskeratosis, brittle X chromosome syndrome, familial Parkinson's disease and others.

Apparently, the value of patient-specific disease models created with the help of iPSCs is directly proportional to the degree of heritability of pathology. As for multifactorial diseases, the etiology of which combines three components (genetic, epigenetic and environmental), the bet is that the cells isolated from patients with a highly penetrant hereditary predisposition to the disease will allow to study the pathological mechanisms of its development.

Modeling of diseases with significant epigenetic and environmental components with the help of stem cells requires experimental induction of the diseaseDespite the fact that reprogramming eliminates epigenetic and environmental causes of the disease, modeling using stem cells can also be useful in the study of non-genetic diseases.

In cases where the causes of the disease are relatively well known, its induction can be carried out directly in culture. For example, iPSCs can be differentiated into melanocytes and keratinocytes forming an epidermal model that can be used to study sunburn (Lin and Fisher, 2007).

Creating such models for diseases with obscure etiology is a much more difficult task, implying the reproduction of environmental conditions, epigenetic changes and other factors potentially leading to the appearance of the desired phenotype.

What's in the future?In addition to the currently developed methods of disease modeling and drug screening, the possibility of converting cells of one type into another gives clinicians hope for the emergence of new therapeutic opportunities.

At the heart of a huge number of diseases is the absence of any important cells or proteins, so the hope for their cure by replacing the missing component rests on cellular engineering and regenerative medicine.

This can be achieved using two fundamental approaches: (1) the creation of iPSCs and their differentiation into target cells for subsequent administration to the patient, or (2) the direct transformation of healthy cells into missing or damaged cells in order to start the regeneration process inside the body.

The first approach is in many ways similar to the methods currently used for modeling diseases, while the second requires manipulations on the tissue belonging of cells in the conditions of the body. Scientists also managed to achieve encouraging results when trying to convert exocrine cells of the pancreas into insulin-secreting beta cells using viral vectors expressing the necessary genes (Zhou et al., 2008). Unlike the introduction of iPSC, the direct conversion method is not associated with the risk of preserving residual pluripotent cells in the body. However, its use is associated with other difficulties: the need for targeted delivery of therapeutic agents and prevention of the appearance of partially transformed and malignant cells.

In addition to experimental and possibly therapeutic tools, manipulations on the tissue belonging of cells can provide specialists with new methods for studying pathologies such as metaplasia, neoplasia and developmental disorders. In fact, the reprogramming factors proposed by Yamanaka – Oct4, Sox2, KLF4 and Myc – are powerful oncogenes, and the reprogramming process is an analogue of the dedifferentiation that cells of some tumors presumably undergo. Therefore, the study of these mechanisms can help to understand the mechanisms of cell malignancy.

ConclusionThe development of a method for reprogramming cells and the realization that the cellular structure is plastic and can be manipulated, led to the emergence of bold ideas among researchers about new approaches to studying the mechanisms of disease development, as well as the development of new drugs and methods of cell therapy for a number of diseases.

However, many diseases are based on significant epigenetic and environmental components, so it is obvious that in the foreseeable future, patient-specific disease models created with the help of stem cells are the most informative for pathologies with a highly penetrant genetic etiology. However, the first wave of models should help specialists understand which aspects of pathophysiology can and cannot be studied on such cellular models. The use of pluripotent stem cells to model diseases with insignificant genetic components may make sense, but it is undoubtedly associated with serious difficulties. One of the tasks in this case is to reproduce the influence of epigenetics and the external environment in such a way that a complex of relevant genetic and non-genetic factors ensures that an adequate phenotype of the disease is obtained in vitro. Given the speed of development of modern science and the appearance of publications describing iPSC-based models of diseases, in the near future specialists should formulate principles that will bring research work closer to clinical practice.

The list of references is given in a separate file.

Portal "Eternal youth" http://vechnayamolodost.ru15.06.2012