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Conceptualization of human skin cell subpopulation states

The article is concerned with the study of the dynamics of states of subpopulations of human skin cells based on the results of few patented inventions in biology and cell medicine. The variety of potential states of subpopulations is substantiated with the methods of conceptual analysis. The article presents the results of this analysis demonstrating the need for profound changes in diagnostic and therapeutic approaches in dermatology as a scientific discipline and practice. The possibility of obtaining the skin cytoimmunogram enables us to study the phenomenology of the cell subpopulations states dynamics by conceptual methods. Extending of these conceptual differences gives us the access to a wide range of research objectives in der- matology, based on the distinctions of the phenotypes of skin cells subpopulations called phenotypic der- matology. Conceptualization of the phenomenology of states of subpopulations of skin cells and its dynam- ics reveals classes of potential situations that are still not distinguishable in dermatology. Further extension of the conceptual distinctions between the states of the subpopulations of skin cells and its dynamics, the unfolding of the generic structures of this dynamics will make it possible to deduce hidden circumstances of skin pathologies to an explicit level up to now. These opportunities give an expansive character to the generation of objectives of theoretical research that can not be put in any other way. Thus, the deduced problems become the basis for setting new, increasingly “subtle” experiments and the generator of the re- quirements for the development of the experimental base of dermatology.

Key words: skin cells, conceptual analysis, phenotypic dermatology

Trends in the development of science clearly demonstrate that its achievements are associated not only with new technologies of research activity, but also with progressive types of comprehension of the results obtained. This is dictated by the high demand not so much for individual scientific discoveries and their involvement in practice, as for the generation of classes of discoveries that form the front of solutions to the current problems of medicine.

Thus, in domestic and foreign medicine, the demand for scientific developments that accelerate wound healing of the skin has remained relevant for a long time [1-5]. Proceeding from the notion of wounds, the main task of wound healing has always been considered as restoration of skin functionality without scar complications [5, 6].

However, the complexity and polyfunctionality of the skin as an organ of the immune system, with a variety of cell types, their relations and cooperation, and, most importantly, their adhesion into a structural conglomerate, did not allow to make stimulation effects in wound healing targeted, which could be the basis for the wound process management [2].

An approach to overcoming this practical quandary opened up in the course of a number of studies on the properties and functional states of skin cell subpopulations performed by a group of Tyumen scientists in 2010-2017. The possibility of these studies, as well as the possibility to strictly distinguish the cell subpopulations themselves, arose in connection with the invention of a way to separate skin cells while preserving their viability. The comprehension of the results of these studies triggered the need to go out to mobilize methods of conceptual analysis [7], which was the goal of the research. This challenge was also facilitated by the fact that the development of immunology reveals new properties of subpopulations arising from the various relationships that skin cells enter into, guiding researchers along a path of careful study of the different types of set structures formed by skin cells and their features as causes of the symptoms of certain skin diseases [8].

The history of dermatology and cell biology "remembers" a lot of attempts to separate the skin cell substrate to obtain a suspension and study the phenotype of the cells included in it. However, the method of obtaining a viable heterogeneous skin cell population was patented only in 2012.

The results presented by the authors showed the possibility to obtain cell suspension with viability in native samples of up to 99.8%, and after cryopreservation of samples up to 87.0%. [9]. The discovery was recognized as promising for the development of dermatology, and the resolution of the 10th International Conference of Immunologists of the Urals recommended the patent for implementation in the public health system.

Skin cell subpopulations are in different functional states, which is reflected in skin ability to act as a barrier between human external and internal environments [10]. Being a two-component tissue system formed by epidermis and dermis [11], skin is represented by a number of cell subpopulations with strict functional purpose of the functions performed [12]. The dynamics of the functional state of the skin, ensured by the constant renewal of the composition of recirculating lymphocytes between regional lymph nodes, blood and skin, thus creating immune homeostasis, causes a difficulty in studying the properties of cell subpopulations, but also opens up the possibility to comprehend immune mechanisms, including in inflammatory conditions [13-15]. At the same time, resident cells are known to play a role in skin immune homeostasis rather than circulating cells [16, 17].

Knowing that keratinocytes constitute more than 90% of the cells of the upper skin layer, the epidermis [18], and dermal cells are overwhelmingly represented by fibroblasts (fibrocytes), mast cells, monocytes (macrophages), endothelial cells, dendritic cells and lymphocytes [11], of which 90% are T-lymphocytes located in the upper layers of the dermis and epidermis and 10% are B-lymphocytes present in the middle and deep layers of the dermis [19, 20], sets of cell differentiation markers were determined which best characterize the dynamics of skin cell subpopulation states: CD49f+ keratinocytes, of which CD49f+HLA-DR+ activated; CD45-CD14-CD44+ fibroblasts (fibrocytes), of which CD45-CD14-CD44+CD80+ activated; CD249+ mast cells, of which CD249+CD63+ activated; CD45+CD14+ monocytes (macrophages), of which CD45+CD14+HLA-DR+ activated intraepidermal macrophages CD207+, including activated CD207+CD80+, CD207+HLA-DR+, CD207+CD80+HLA-DR+; endothelial cells CD146+, including activated CD146+CD34+, CD146+HLA-DR+, CD146+CD54+, CD146+CD54+HLA-DR+; epithelial stem cells CD34+CD45dim; lymphocytic population: CD45+CD3+ T-lymphocytes, CD45+CD3+CD4+CD8- T-helpers, CD45+CD3+CD4-CD8+ T-suppressors, CD45+CD3-CD19+ B-lymphocytes, CD45+CD3-CD16+CD56+ NK-lymphocytes. This allowed to improve the method to the possibility of obtaining an assessment of skin cell functions of individual subpopulations [21].

Called skin cytoimmunogram, the method made it possible to measure activity of native skin cell subpopulations under healthy and pathological conditions, to evaluate function and degree of skin cell response in response to any external and internal environment influences.

In the perspective of large-scale application it will allow to create a sex-and-age register of skin condition, which will make it possible, in its turn, to objectively assess the dynamics of skin diseases, to select drugs individually, to control the effectiveness of external medications, to form criteria of age-related skin changes [22], to testify objectively about the current status of patient's local immunity and can become a substantial basis for treatment and preventive programs in dermatology [23].

Having become the first invasive technique evaluating not only the structural state of skin cells, but also determining the function of its subpopulations without loss of their viability, skin cytoimmunogram demonstrated the possibility to identify the object of purposeful influence in the mechanism of wound healing, which, according to a number of researchers, seems to be an extremely urgent task [24].

Thus, the hypothesis of possible stimulation of reparative potencies in the skin by introducing a substance of natural origin, predictably influencing the mechanism of granulation tissue formation, which is known to be an obligatory stage for subsequent epithelization and formation of aesthetic result, was realized with the possibility of skin cytoimmunogram application in investigation of CD34+CD45dim phenotype cells, which are fundamental in epithelization implementation [25] and their stimulation by a substance of xenogenic origin.

A lyophilized extract of 3-9 days of gestation chicken embryo cells at a concentration of 0.75×106 to 1.25×106 cells of CD34+CD45dim phenotype in 1 ml of hydroxyethylcellulose-based gel-forming biopolymer was proposed to be the active substance. It was found experimentally that the suspension should contain the indicated concentration of CD34+CD45dim phenotype cells to obtain a distinct effect. Attempting to reduce the concentration of CD34+CD45dim phenotype cells below 0.75×106/ml significantly reduced the regenerative effect. In turn, increasing the concentration above 1.25×106/ml did not result in a significant increase in the efficacy of the agent. In addition, interacting with the receptor field of the wound, having a broad spectrum of antimicrobial action, showing activity against Gram-positive and Gram-negative bacteria, Candida fungi and dermatophytes, the substance was predicted to stimulate the migration and proliferation of fibroblasts, keratinocytes, endothelial and other cells actively involved in wound healing, promoting epithelialization, restoration of tissue elasticity, normalization of orientation and maturation of collagen fibers, preventing pathological scarring [26]. The use of this substance was confirmed by the Protocol of tests № 594-12P/7-NT of 24.12.2012 of the testing laboratory No ROSS RU.0001.21AV65 of 13.07.2011 and the declaration of conformity of the Customs Union No TS RU D-RU.AL14.V.03012 of 25.12.2012 (Technical regulation of the Customs Union "On safety of perfume and cosmetic products" (TR TS 009/2011).

The Cellgel invention that followed - an extract of chicken embryo cells adsorbed on a gel-forming biopolymer, allowed significant progress in the study of the issue of controlled wound healing, providing a significant (several times) reduction in treatment time, effective closure of the tissue defect with restoration of function, without scar formation, due to increased reparative activity of skin cells with the CD34+CD45dim phenotype [27]. Developed with targeting in mind, it has demonstrated high efficacy of wound healing in skin lesions of various etiologies. The production of Cellgel wound healing agent was innovative, the success of its application was guaranteed, its cost-effectiveness was justified, and its social orientation was unconditional [28].

Thus, the skin cytoimmunogram as a method of cell separation allowed a methodologically strict approach to the invention of the Cellgel wound healing agent, which became a winner of the All-Russian contest of the "100 Best Products of Russia" Program and subsequently became the basis for further scientific research [29-31].

New questions arise for the practice of dermatology due to the new possibilities of skin research:

  • How to make individualized diagnoses of skin diseases, accelerating the treatment process, if the distinctions between phenotypes of cell subpopulations are only empirical?
  • How to accelerate the theoretical investigation of skin diseases ahead of practice, if problem statements and associated results are based only on a large body of empirical data from patient examinations and therefore depend on the development of instrumental tools for disease investigation?
  • How can we use the new possibilities of skin conditions research to raise the level of development of dermatology, which still relies on traditional methods of a mostly symptomatic nature?
  • Answers to these and similar questions lead to an understanding of a number of theoretical problems in modern dermatology:

There is a lack of an exhaustive understanding of the full diversity of cell subpopulations whose condition may influence the development of skin diseases. Reliance on empirical phenomenology is important, but extremely inert;
There is a lack of insight into the diversity of possible phenotypes of cell subpopulations. The notion of this diversity and the distinction of all possible phenotypes could lead to a wide range of new research tasks, which science is developing;
The relationships between skin cell states and pathologies have not been established for the diversity of possible phenotypes of cell subpopulations.
A solution to these problems may follow in the course of a conceptual analysis of the phenomenology of the dynamics of skin cell subpopulation states. This implies formation of generic and species notions of the opened subject area based on the notion of subpopulations as specific structures of heterogeneous sets [32]. In these structures, the numbers of elements express the volumes of concepts, and the structures themselves express their contents. It is supposed that conceptual analysis will allow to set tasks for the study of medical and biological regularities between various phenomena, i.e., it will lead to the "mechanisms" of a new type of dermatology.

The representations about skin properties derived from skin cytoimmunograms can be summarized in the following statements:

  • The variety of cell types that are provably involved in the creation of certain described skin condition phenomena is limited, there are twelve of them (K=12). Moreover, five of them are of the same type (lymphocytes);
  • Each cell possesses a certain set of features which are created by membrane molecules on its surface. A limited number of functional characteristics is taken - twenty-four for each cell type. Moreover, one of these attributes characterizes a specific cell species. It is possible that with the development of scientific methods of research other signs will be discovered;
  • Living cells are always active. But their activity is manifested in different ways and affects skin condition in different ways. The activity of a cell is a manifestation of its particular function in the structure of a particular skin puncture biopsy. If a cell does not show any function, we can say that its activity is zero, although it is alive. It is accepted to consider that in this case the cell is passive;
  • On manifestation (activation) in a cell of a certain function in composition of the skin each combination of features of the cell manifested in some way is indicative;
  • The cell traits are detected with the help of markers - differentiation clusters (CD). Each marker acts as an unambiguous indicator of one or another manifested cell trait;
  • The number of markers is increasing with the development of im- munology and cellular medicine and their indicative component is being refined. However, a limited composition of markers is applied in each research act of experiment. Of course, this affects the accuracy of recognition of the variety of cell traits and the variety of cell types;
  • The number of potentially possible functions of each cell is determined by combinations of all its traits;
  • The same cell species can exhibit either one function or several at once;
  • Cells are grouped into subpopulations. In each subpopulation, a specific proportion of cells of each species with their different functions arise. All theoretically conceivable subpopulations are admissible, since there are no restrictions on their diversity;
  • A subpopulation with a specific composition of cells, each with a specific composition of functions, forms a subpopulation phenotype;
  • Each subpopulation phenotype uniquely corresponds to a specific immunological skin condition expressed in a clinical or physical symptom.

These statements provide the basis for the generation of a series of concepts, each of which defines a class of possible phenomena of scientific interest. In what follows, with brief justification, only those concepts that define static skin phenomena are presented. They can be regarded as instantaneous "slices" of the dynamics of its states. The concepts are given in the mathematical apparatus of genera of structures, which is involved in conceptual methods [33].

Conceptualization of skin cells

Diversity of skin cell functions" concept
Base sets:
X1 is the set of features of any species cell that are experimentally detected by special markers.

Genus relation: D1 ∈ B(X1). Here "B" is the sign of the operation of generating a set from all subsets of the original set (booleans).
The type of this structure: "The set of all subsets of a cell's traits".

Since every cell of every species can exhibit any combination of traits, this structure establishes a complete variety of all combinations of traits that only a skin cell of any species can have. Given that each combination of features of a cell indicates the activity of its particular function, this structure simultaneously expresses the complete variety of functions that cells can activate, irrespective of their species.

If the number of features of each cell is equal to P and one of them is used to determine the species of the cell itself, the variety of combinations of features and, consequently, the functions of cells can reach the number 2(P-1)-1. Here it is taken into account that if a cell does not exhibit any features (empty subset of features - ∅), it is functionally passive. Taking into account the known number of features of a cell of each species (P=24) it turns out that the maximal power of hypothetical variety of functions of one cell of any species (F) is more than 8 million (specifically: F=8388607). Moreover, each of these functions can be distinguished.

This first result of conceptualization allows to theoretically deduce (define) each function of a cell, investigating its influence on health state; to set tasks to find effective ways to detect functions; to look for ways to activate each function or to suppress it. At present, of all the variety of skin cell functions, a tiny fraction is dermatologically accounted for. It is possible that some potential functions will never manifest or will manifest in some unique situations. But, in the absence of substantiated statements to that effect, the occurrence of unfamiliar functional skin cell phenomena cannot be ruled out.

The Concept of Species Cells with Functions

Base sets:
X1 - the set of features of any species cell, which are experimentally detected by special markers;
X2 - the set of skin cell species. These are all those distinct from each other cells that participate in the creation of skin health phenomena.

The genus relation has the following form: D2 ∈ X2 × IN(X1).
Here "×" is the sign of the Cartesian product of sets.

The type of this structure is: "The set of cell species and the set of their functions".
This structure defines all situations in which it is necessary to distinguish specific species cells with all their functions.

Rather, this concept is useful for setting narrow theoretical goals in dermatology. Thus, the consideration of cells with their full set of functions may allow us to investigate the peculiarities of the action of all the functions of each species cell. It may be possible to answer the question of whether the fact that the same function appears in different species cells at the same time will change the effect on the skin. Assuming that the number of species cells is K=12, and the number of functions of each species cell is F=8,388,607, the diversity of "species cells with their functions" (S) compositions is S=R×F=100663284. Of course, it will take many years to empirically study properties of all these situations. But, the space for theoretical research is open.

However, real situations in which a particular skin puncture biopsy reveals different numbers of species cell compositions are not yet expressible here. In this sense, a concept with a different generic structure should be recognized as more constructive and meaningfully rich.


The concept "Variety of Species Cells with Sets of Their Functions".

The basic sets of this concept:
X2 - the set of skin cell species;
X3 - set of cell functions.
The generic relation has the following form: D3 ∈ IN(X2 × IN(X3)).
The type of this structure is: "The set of subsets of species cells together with the set of subsets of their functions".

It expresses all situations where, in a particular puncture skin biopsy, one can consider separately from each other such subpopulations in which cells of all species (or only a few) are assembled together with their manifested functions. Such situations resulting from the evolution of skin cell states can be appropriately called "subpopulation phenotypes". Their diversity is great, for example:

  • Single cell types and their functions. These are all those subpopulation phenotypes that are defined by the previous genus of the structure.
  • Phenotypes of subpopulations formed from several cell types (not all) at one time together with their different functions;
  • Phenotypes of subpopulations formed by cells of all species, with each cell species exhibiting one or two of its functions;
  • Phenotypes of subpopulations formed by cells of all species, with each cell species exhibiting all its functions simultaneously.

This concept opens up the possibility to distinguish phenotypes with complete/incomplete sets of cell species with all different activities of functions, including those in which all cell species exhibit all their functions simultaneously. Based on it, new classes of problems can already be set to investigate health/disease situations with different compositions of functions in complete sets of cell species and other situations. But, these conceptual constructions do not yet take into account the circumstances when in the skin portion there turns out to be some certain proportion of cells of each species with specific (different functions). It would be another concept that expresses the full diversity of phenotypes of subpopulations of cells. At the same time unusual theoretically possible situations can be expressed. For example, when there is a complete diversity of cells of all species, there will be cells with different sets of functions among cells of the same species.

This will force researchers to answer a series of questions about what are the properties of such phenotypes of cell subpopulations and how it is related to the pathologies that are the subject of dermatology as a science of skin and its diseases.

The genetically determined process of formation of a variety of specialized cell phenotypes reflecting their function is the result of coordinated expression of a certain set of genes resulting in cell differentiation that changes their functions, morphology and metabolic activity and creates preconditions for their high specialization and selective action, which in the end is realized in an enormous variety of clinical phenomena of the same skin diseases seen by dermatologists.

Deepening these conceptual distinctions allows us to reach a broad class of research tasks in dermatology that relies on distinguishing phenotypes of subpopulations of skin cells - phenotypic dermatology. Based on the conceptually possible variety of situations that phenotypic dermatology must master, the main types of theoretical and experimental search tasks are as follows:

  • The tasks of identifying the full diversity of skin cells of each species;
  • Tasks of identifying all possible functions of skin cells of each known species;
  • Tasks of identifying the influence of possible functions of cells of each species on skin conditions;
  • To identify possible combined potential functions for all cells of each species at the same time;
  • Tasks to identify the joint influence of possible functions of cells of each species on skin conditions;
  • Tasks of identifying the joint influence of possible functions of cells of all species simultaneously on skin conditions;
  • To identify ways to activate/suppress the functions of cells of each species;
  • Tasks to identify the relationships between the abilities of cells to activate their functions in coexistence;
  • Tasks of identifying the effects of concentrations of cells of each species on skin conditions;
  • Tasks of identifying concentrations of species cells with diverse activated functions on skin conditions;
  • Tasks of identifying properties of transitions between states of populations of cells with diverse functional capabilities;
  • Tasks of identifying ways of purposeful change (control) of transitions between states of cell populations.
  • Obviously, behind each type of such problems there is a problem of recognition of particular properties of components of complex skin cell morphology [34]. Removal of these problems by both theoretical and experimental immunological methods can raise dermatology to qualitatively new levels.

An example of a practical solution to one of the named problems of phenotypic dermatology is the development of an amplifier of reparative potencies. Thus, guided by the thesis that healing processes obey the same principles as proliferation and apoptosis and are regulated through cell-cell and cell-matrix signals [35], similar studies data were obtained on the dose-dependent effect of Cellgel on cell viability and apoptosis processes in culture, which led to the conclusion that a deeper study of immune mechanisms of cell death regulation by skin cytoimmunogram, can contribute to increasing the effectiveness of Cellgel wound healing agent when it is used in vitro. A condition for the appearance of this effect is the activation of specific functions of skin cells of each species, the way of which must be found.

Taking into account the results of conceptualization this problem was formulated as follows:

Among the full diversity of phenotypes of subpopulations of species cells with sets of their functions given by the conceptual scheme D3, find a phenotype that is defined by the following term: TR1 = {d ∈ D3 (Pr1d ∈ X2)}
Develop a technical device capable of activating the cell functions of this set.
The relevance of solving this problem is determined by the Russian Federation Government Decree and the Ministry of Economic Development Order on ensuring state quality control over the research of perfumes and cosmetics for the procedure of conformity assessment to the requirements of the technical regulations of the Customs Union "On Cosmetic Products Safety" (TR TS 009/2011).
(Federal law of 28.12.2013 No 412-FZ (ed. from 23.06.2014) "On accreditation in the national accreditation system").

The solution to this problem was the invention of the skin cell reparative potency amplifier [36]. On the one hand, this invention is a response to the need to introduce expert systems into medical practice [37]. On the other hand, it is the first of many possible steps towards a therapeutic tactic, in which, for example, wound healing becomes a controlled process with a given requirement for its result. His utility model relates to biotechnology in cellular medicine, namely devices for dealing with human skin cells, creating a practical convenience of in vitro activation and introduction of activated skin cells into the wound. This invention opens a number of solutions for so-called "targeted delivery" of drugs [38], the prospects of which in practical dermatology have been outlined by a number of Russian and foreign authors [39,40]. The main purpose of the amplifier is achieved by providing limited contact of the Cellgel active ingredient with the skin activated cells prepared by the patented method - skin cytoimmunogram and isolated from the external environment in a separate container, representing a divided viable heterogeneous cell population in the form of suspension.

Amplifier of reparative potencies is the first practical step in the direction of phenotypic dermatology, whose success will be based on conceptually strict distinction of properties of various phenotypes of skin cell subpopulations and targeted regulation of these properties (activation, suppression, enhancement, modification, etc.) taking into account individual characteristics of patients.


The results of immunological studies of skin cell subpopulations reveal a picture to which modern dermatology is not yet clearly related. But, the translation into effective remedies of the first useful phenomena of this picture already indicates that a new history is beginning in this scientific discipline.

The conceptualization of the phenomenology of skin cell subpopulation states and its dynamics reveals classes of potential situations that have not yet been distinguished in dermatology. This circumstance explains the fact that the gigantic variety of skin diseases that practitioners deal with have not yet acquired their own names, much less unique treatment approaches.

Further deepening of conceptual distinctions between the states of skin cell subpopulations and their dynamics, deployment of generic structures of these dynamics will make it possible to bring the hitherto hidden circumstances of skin pathologies to the explicit level. These possibilities give an expansive character to the generation of tasks of theoretical research, which cannot be set in any other way. Thus, the derived tasks become the basis for setting new, increasingly "subtle" experiments and the generator of requirements for the development of the experimental base of dermatology.

On the basis of the synthesis of experimental and conceptual developments, it seems possible to change the pattern (paradigm) of approaches to the diagnosis and treatment of skin diseases.

The near future of this shift is already evident in a series of successive inventions creating a frontline of practice-oriented innovations and discoveries. One of them is the demonstrated amplifier of reparative potencies, which will be followed by others.


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