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STEM CELLS AND THEIR APPLICATIONS

Level of higher education:

Second (master's) Field of knowledge:

16 Chemical and bioengineering

Specialty: 162 Biotechnology and bioengineering Educational and professional program: Biotechnology and bioengineering

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MINISTRY OF EDUCATION AND SCIENCE OF UKRAINE

ODESSA NATIONAL UNIVERSITY NAMED AFTER I. I. MECHNYKOV

Department of microbiology, virology and biotechnology

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Tetyana V. Hudzenko, �PhD, Associate Professor of the Department of Microbiology, Virology and Biotechnology�Odessa Mechnikov National University �Odessa, Ukraine, 2, Dvoryanska str.�Tel: +38-0682593308�E mail: tgudzenko@ukr.net��

• Olena Horshkova
• Researcher of the Department of Microbiology, Virology and Biotechnology
• Odessa Mechnikov National University
• Odessa, Ukraine, 2, Dvoryanska str.
• Tel: +38-0682780205
• E mail: helen-good@ukr.net

• REFERENCES
• 1.Khawaja Husnain Haider Stem cells: From Potential to Promise (2021) https://doi.org/10.1007/978- 981-16-0301-3
• 2. Lee SM, Lee SD, Wang SZ, Sarkar D, Lee HM, Khan A, Bhati C, Sharma A, Kumaran V, Bruno D, Cotterell A, Levy MF. Effect of mesenchymal stem cell in liver regeneration and clinical applications. Hepatoma Res. (2021);7:53 http://dx.doi.org/10.20517/2394-5079.2021.07
• 3. Kulebyakin K, Tyurin-Kuzmin P, Efimenko A, Voloshin N, Kartoshkin A, Karagyaur M, Grigorieva O, Novoseletskaya E, Sysoeva V, Makarevich P and Tkachuk V (2021) Decreased Insulin Sensitivity in Telomerase-Immortalized Mesenchymal Stem Cells Affects Efficacy and Outcome of Adipogenic Differentiation in vitro. Front. Cell Dev. Biol. 9:662078. doi: 10.3389/fcell.2021.662078
• 4. Zakrzewski, W., Dobrzyński, M., Szymonowicz, M. et al. Stem cells: past, present, and future. Stem Cell Res Ther 10, 68 (2019). https://doi.org/10.1186/s13287-019-1165-5
• 5. Trokhymchuk I. M., Plyuta N. V., Logvinenko I. P., Sachuk R. M. Biotechnology with the basics of ecology: a textbook. - Kyiv: Publishing house "Kondor", 2019. - 304 p.
• 6.Negoro T., Takagaki Y., Okura H., Matsuyama A. Trends in clinical trials for articular cartilage repair by cell therapy. NPJ Regen Med. 2018. 3. P. 17.
• 7. Rybalchenko T.V., Kuznetsova G.M., Dzerzhinsky M.E., Rybalchenko V.K.. Mechanisms of cell differentiation, – Kyiv: VPC "Kyiv University", 2019. –399 p.
• 8. Mazurkevych A.Y., Kovpak V.V., Kovpak O.S. Cytogenetic analysis of rat adipose tissue cell culture at early passages // Ukrainian Journal of Veterinary Sciences. – 2017. – P. 159–167.
• 9. Eran Meshorer, Kathrin Plath. The Cell Biology of Stem Cells (2010). Springer, Boston, MAhttps://doi.org/10.1007/978-1-4419-7037-4.

16.Windley W, Teixeira F, Levin L, Sigurdsson A, Trope M. Disinfection of

immature teeth with a triple antibiotic paste. J Endod. 2005; 31: 439-43.https://goo.gl/GyfvG9

17.Ilancheran S, Moodley Y, Manuelpillai U. Human Fetal Membranes: A Source

of Stem Cells for Tissue Regeneration and Repair? Placenta. 2009; 30: 2-10. https://goo.gl/mNuHU3

18.Ying QL, Nichols J, Chambers I, Smith A. BMP Induction of Id Proteins Suppresses Differentiation and Sustains Embryonic Stem Cell Self-Renewal in Collaboration with STAT3. Cell. 2003; 115: 281-292. https://goo.gl/2Mg1HD

19.Lerou P. Embryonic stem cell derivation from human embryos. Methods Mol Biol. 2011; 767: 31-5. https://goo.gl/AGUhGe

20.Klimanskaya I, Chung Y, Becker S, Lu SJ, Lanza R. Human embryonic stem cell lines derived from single blastomeres. Nature. 2006; 444: 481-485.https://goo.gl/amBsRd

21.Zhang X, Huang, J. Integrative genome-wide approaches in embryonic stem cell research. Integr Biol (Camb). 2010; 2: 510-516. https://goo.gl/C6aMC5

22.Shamblott MJ, Axelman J, Littlefi eld JW, Blumenthal PD, Huggins GR, Cui Y, et al. Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proc Natl Acad Sci USA. 2001; 98: 113-118 https://goo.gl/GFyMNg

23. Kakinuma S, Nakauchi H, Watanabe M. Hepatic stem/ progenitor cells and

stem-cell transplantation for the treatment of liver disease. J Gastroenterology.

2009; 44: 167-172. https://goo.gl/1196in

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24. Rogers I, Casper RF. Umbilical cord blood stem cells. Best Pract Res Clin Obstet Gynaecol. 2004; 18: 893-908. https://goo.gl/uwrtZ9

25. Mitchell KE, Weiss ML, Mitchell BM, Martin P, Davis D, Morales L, et al. Matrix cells from Wharton’s jelly form neurons and glia. Stem Cells. 2003; 21: 50-60. https://goo.gl/c1xwjV

26. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res. 2007; 100: 1249-60. https://goo.gl/SvSKN7

27. Gao X, Song L, Shen K, Wang H, Niu W, Qin X. Transplantation of bone marrow derived cells promotes pancreatic islet repair in diabetic mice. Biochem Biophys Res Commun. 2008; 371: 132-137. https://goo.gl/26R2my

28. Deng ZL, Sharff KA, Tang N, Song WX, Luo J, Luo X, et al. Regulation of osteogenic differentiation during skeletal development. Front Biosci. 2008; 13: 2001-2021. https://goo.gl/6GPcYc17.

29.Zhang L, Chan C. Isolation and enrichment of rat mesenchymal stem cells

(MSCs) and separation of single-colony derived MSCs. J Vis Exp. 2010; 22:

1852. https://goo.gl/kggAuq

30. Kim HJ, Kim UJ, Vunjak-Novakovic G, Min BH, Kaplan DL. Infl uence of

macroporous protein scaffolds on bone tissue engineering from bone marrow

stem cells. Biomaterials. 2005; 26: 4442-4452. https://goo.gl/5k11we

31. Parekkadan B, Milwid JM. Mesenchymal stem cells as therapeutics. Annual

Review of Biomedical Engineering. 2010; 12: 87-117. https://goo.gl/La5Gv7

32. Xie CG, Wang JF, Xiang Y, Qiu LY, Jia BB, Wang LJ, et al. Cocultivation

of umbilical cord blood CD34+ cells with retro-transduced hMSCs leads

to effective amplifi cation of long-term culture-initiating cells. World J

Gastroenterol. 2006; 12: 393-402. https://goo.gl/9RZ2qN

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33. Imitola J. Prospects for Neural Stem Cell-Based Therapies for Neurological Diseases. Neurotherapeutics. 2007; 4: 701-714. https://goo.gl/wEVbm4

34. McDonald SAC, Graham TA, Humphries A, Wright NA, Preston SL, Brittan M and Direkze, NC. Stem Cells in the Gastrointestinal Tract Essentials of Stem Cell Biology (Second Edition). 2009; 36: 307-327.

34. Jia L, Zhou J, Peng S, Li J, Cao Y Duan E. Effects of Wnt3a on proliferation and differentiation of human epidermal stem cells. Biochem Biophys Res Commun. 2008; 368: 483-8. https://goo.gl/KTe2K624.

35.Verhulst S, Best J, van Grunsven LA, Laurent Dollé L. advances in hepatic

stem/progenitor cell biology. EXCLI J. 2015; 14: 33-47. https://goo.gl/ZJACFf

36. Alison MR, Choong C, Lim S. Application of liver stem cells for cell

therapy. Seminars in Cell & Developmental Biology. 2007; 18: 819-826.

https://goo.gl/cSXREq

37. Pagliuca FW, Millman JR, Gurtler M, Segel M, Dervort AV, Ryu JH, et al.

Generation of Functional Human Pancreatic b Cells In Vitro. Cell. 2014; 159:

428-439. https://goo.gl/FSw1Hq

38. Zulewski H, Abraham EJ, Gerlach MJ, Daniel PB, Moritz W, Müller B, et

al. Multipotential nestin positive stem cells isolated from adult pancreatic

islets differentiate ex vivo into pancreatic endocrine, exocrine and hepatic

phenotypes. Diabetes. 2001; 50: 521-533. https://goo.gl/tBy83f

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• The purpose of the course "Stem Cells and Their Applications" is to familiarize applicants with the theoretical foundations of life, differentiation, methods of obtaining and classification of stem cells and to provide a system of knowledge on the main areas of use of stem cells in medicine to future specialists - applicants for higher education in specialty 162 Biotechnology and Bioengineering.

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• Tasks:
• - to form in applicants a thorough systematic knowledge of the processes of life of stem cells;
• - to form methodological competencies regarding the main methods of obtaining, features of differentiation and classification of stem cells;
• - to form an idea of umbilical cord blood banks, the organization of its procurement, testing, typing, cryogenic storage in liquid nitrogen;
• - to form professional special competencies of fundamental knowledge of technologies in the treatment of leukemia and lymphoma, diabetes, Parkinson's disease, Alzheimer's disease, thromboembolic stroke, as well as in overcoming the consequences of chemotherapy;
• - to form professional special competencies on ethical issues of the use of embryonic stem cells and the prospects for their transplantation.

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MODULE I

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SOURCES OF OBTAINING, CHARACTERISTICS, FEATURES OF DIFFERENTIATION AND MODERN CLASSIFICATION OF STEM CELLS

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TOPIC 1. Subject, goal and objectives

of the course

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Goals, objectives, objects and methods of the course “Stem cells and their application”. History of the discovery of stem cells and the main directions of development of modern methods of their obtaining. Classification of stem cells. Embryonic and somatic own stem cells. Sources of stem cells in the body: bone marrow, peripheral blood, blood from the umbilical vein of newborns, etc. Division of stem cells into three main groups depending on the source of their receipt: embryonic, fetal and postnatal (stem cells of the adult organism).

Stem cells are primary cells capable of self-renewal by dividing and differentiating into specialized cell types.

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The term "stem cell" was introduced at the meeting of the Society of Hematologists on June 1, 1909 in Berlin by histologist O.O. Maksimov for the hematopoietic stem cells of the bone marrow. In the work "Lymphocyte as a common permanent cell of various blood elements in embryonic development and in the later life of mammals" he presented a unitary theory of hematopoiesis according to which all blood cells originate from a single progenitor cell.

Donnal Thomas performed the first human bone marrow transplant in 1959.

Alfred Knudtzon In 1974, he discovered that umbilical cord blood contains the same hematopoietic stem cells as bone marrow in a huge amount.

Mark Hedrick obtained stem cells from adipose tissue and established that the cell can give rise to various organs and tissues .

Ernest Armstrong McCulloch (27 April 1926 – 20 January 2011) was a University of Toronto cellular biologist, best known for demonstrating – with James Till – the existence of stem cells.

In 1988, hematology professor Elian Gluckman performed the world's first umbilical cord blood transplant from his newborn sister to a child with anemia in Paris.

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• 1992 rіk - First named collection of Stеm cells. Professor David Harris "about every episode" by freezing the Stеm cells of the umbilical cord blood of his first-year student.
• Today, David Harris is the director of the world's largest umbilical cord blood bank.

• 1998 - American biologist James Thomson saw embryonic cells for the first time.

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• 1996 - During the period from 1996 to 2004 there were 392 transplantations of autologous (human) stem cells. Thus, in 1996, cerebrovascular transplantation was very successful.

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• 1996 - It has been proven that it removes cancer cells, and also kills only those transplanted from the cerebrospinal fluid of a donor.

• 1997 - Over the past 10 years, 143 umbilical cord blood transplantations have been performed in 45 medical centers around the world.

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• 1998 - First transplantation of "name" cord blood cells from a girl with neuroblastoma (brain swelling). The total number of umbilical cord blood transplants exceeds 600.

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• American scientists James Thomson and John Becker were able to see human embryonic cord blood cells and remove their first lines.

• Born in 1998 - disintegrating method of growing Stеm cells in the nutrient medium.

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• 1999 - Science journal recognized the discovery of embryonic Stеm cells as the third most important concept in biology after the deciphering of the subsecular DNA helix and the Human Genome program.

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• 2001 - Published data on the feasibility of stagnation of umbilical cord blood cell transplantation in adult patients.

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• It is shown that adult hematopoietic and stromal cells of the human cerebrospinal fluid differentiate into cardiomyocytes and smooth cells.

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• 2003 - Journal of the National Academy of Sciences of the United States (PNAS USA) published information about those who, after 15 years of storing umbilical cord blood in rare nitrogen, save their blood biological properties.

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• Cryogenic preservation of Stеm cells began to be seen as “biological insurance.” The world's collection of Stеm cells , which are preserved in banks, has reached 72,000 copies.

• 2004 - total world collection of umbilical cord blood cells - 400,000 copies.

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• Over the past 20 years of successful grafting of Stеm cells , the method of observation and cultivation of mesenchymal Stеm cells from autologous cerebrospinal fluid has been developed and licensed.

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• The developed cultivation technique makes it possible to isolate the necessary number of Stеm cells with the required characteristics and their cell progeny in various organs and tissues.

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• When stored in a cryobank, cultures retain a high survival rate and high activity.

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• Today's Stеm cells are successfully cured in the treatment of important slack and swollen illnesses, heart disease, endocrine system, neurological illnesses, liver diseases, intestinal tract and lungs, illness of the musculoskeletal and musculoskeletal systems, illness of the skin.

• Chinese scientists are also working with stem cells.

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• Zhang Xinming, director of the Department of Biological Technologies of the Ministry of Science and Technology in Beijing (China), reported at a press conference on February 15, 2020 that preliminary results of research on cellular therapy for coronavirus infection indicate that this treatment method is safe and effective.

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• The group of Professor Wu Ji from the School of Biotechnology at Shanghai University managed to extract and isolate for 5 days female germ cells grown from the stem cells of adult mice.

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• Then the cells were incubated for six months and transplanted into the bodies of adult female mice. After the females went through the process of natural fertilization, in 80% of cases the "grown" eggs were successfully fertilized and the mice gave completely healthy offspring.

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• The results of the study suggest that eggs can be produced even in individuals that have undergone sterilization, after which the same individuals could give birth to offspring.

• A stem cell is a special type of the cell that has the ability to divide for unbounded periods of the time and to give rise to the mature and specialized cells that make up an organism. It considers a new strategy for repairing failing tissues, organs and may be in the future to provide solutions to many diseases.

• HIGHLIGHITS • STEM CELLS
• • Stem cells have unique characteristics, they are unspecialized cells; they can reproduce itself over and over again through asymmetric cell division.

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• • Th ere are diff erent kinds of stem cells which depend on their originality and/ or their potency.

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• • Stem cell therapy has generated incredible interest for repairing failing tissues and organs.

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• • Th ey seem to represent a future powerful tool in regenerative medicine.

• Stem cells have unparalleled characteristics. They are not specialized cells; they have the ability of self-replication and diff erentiation according to the suitable signal.

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• Stem cells can reproduce itself over and over again through asymmetric cell division, they can produce the newly produced off spring cells preserve the characteristics of the mother cell that has a diff erent potency and lineage potential, such as a committed progenitor that transiently amplifi es to make several off spring.

• Totipotency – the ability to form any of approximately 350 types of cells in the body (in mammals);
• Homing – the ability of stem cells, when introduced into the body, to find the area of ​​damage and fix there, performing the lost function;
• The factors that determine the uniqueness of stem cells are not in the nucleus, but in the cytoplasm. This is an excess of mRNA of all 3 thousand genes responsible for the early development of the embryo;
• The presence of telomerase activity.
• (With each replication, part of the telomeres is lost (Hayflick limit or bioclock).
• Stem, germ and tumor cells have telomerase activity, the ends of their chromosomes are superimposed, that is, these cells are able to undergo a potentially infinite number of cell divisions, they are immortal.

• CLASSIFICATION AND SOURCES OF STEM CELLS
• Stem cells can be classifi ed according to their origin into four broad types, from embryos;

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• from the fetus; from the infants and from the adult. Also, they can be classifi ed according to their potency.

STEM CELLS CLASSIFICATION ACCORDING TO THEIR ORIGIN

Embryonic Stem Cells (ESCs)�

• Embryonic stem cells: Embryonic stem cells are pluripotent, self-renewing cells that can be derived from both mouse or human blastocysts, they are taken from the very early stages of embryo development aft er 4-5 days aft er fertilization.

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• They can be stored in culture as undifferentiated cell lines and can be stimulated to differentiate into any cell line.

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• They can differentiate into endoderm, mesoderm, and ectoderm embryonic germ layers, and also any type of somatic cells. They, therefore, hold a great capacity in tissue regeneration therapy.

Embryonic Germ Stem Cells:�

• Embryonic Germ (EG) cells are taken from the later stages of the embryo development cells.

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• Th ey are derived from Primordial Germline Cells (PGCs) in the narrow-window timing.

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• The PGC-derived cells were pluripotent, although, it was not possible to demonstrate pluripotency by generating the formation of teratomas in mice

Fetal stem cells:

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• Fetal stem cells are primal cell types found in the organs of the fetuses. They are able to diff erentiate into two types of stem cells: pluripotent stem cells and hematopoietic stem cells.

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• Neural crest stem cells, fetal hematopoietic stem cells and pancreatic islet cells have been isolated in the fetuses.

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• Human fetal stem cells have been used by many people, children and adults that are suff erring from many of mankind’s most devastating diseases.

Umbilical cord stem cells: �

• Umbilical cord blood contains prevalent stem cells which diff er from those of bone marrow and adult peripheral blood.

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• Cord blood stem cells have shown to be multipotent as it being able to diff erentiate into neurons and liver cells .

Wharton’s jelly: �

• Wharton’s jelly, which is the umbilical cord matrix, is considered to be a source of mesenchymal stem cells.
• These cells express typical stem cell markers, can be propagated for long times and can be induced to diff erentiate in vitro into neurons.

Adult stem cell�

• Adult stem cells are any stem cells taken from mature tissue; they are found in the tissues of a fully developed child (whole embryo) or adult and can only produce a limited number of cell types.

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• They have limited potential as compared to the stem cells that derived from embryos and fetuses because of the stage of development of these cells.

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• They play a vital role in tissue repair, regeneration; and they are referred to their tissue origin. Bone marrow is an abundant source of adult stem cells .

Mesenchymal stem cells: �

• Mesenchymal Stem Cells (MSCs) are a diff erent population of cells with the potential to diff erentiate into various somatic lineages.

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• They were at fi rst described as adherent cells with a fi broblast-like appearance that can diff erentiate into osteocytes, chondrocytes, adipocytes, tenocytes and myocytes .
• MSCs can be isolated from the bone marrow and readily discreted from the hematopoietic stem cells due to their plastic adherence.

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• They are used in tissue engineering and regenerative medicine.
• They are character by long-storage without major loss of their potency.

Hematopoietic stem cells: �

• Hematopoietic stem cells are cells having the self-renewing potential and the capacity to give rise to diff erentiated cells of all hematopoietic lineages.
• Therefore, they transplanted for complete healing of hematologic disorders and aft er high-dose chemotherapy against malignant diseases.

Neural Stem Cells: �

• Neural stem cells are multipotent and self-replication cells, they are established in specialized molecular microenvironments in the adult mammalian brain.
• They can display the potential role in cellular therapy of the brain.

Gastrointestinal stem cells:�

• The stem cells of the gastrointestinal
• tract reside in a “niche” in the intestinal crypts and gastric glands.

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• The mechanism and the direction of the diff usion of this converted clone in the gastrointestinal mucosa are hotly disputed, and the central to this case is the position and nature of the gastrointestinal stem cells .

Epidermal stem cells: �

• The mammalian epidermis is a rapidly rejuvenating tissue that consists of three types of keratinocytes with varying diff erentiation potential: epidermal stem cells, Transiently Amplifi ed Cells (TA cells) and terminally diff erentiated cells.

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• The epidermal stem cells have free self-renewal power. They are establishing in the basal layer and remarkable in maintaining homeostasis and cellular regeneration of normal skin; wound healing and neoplasm formation, whereas TA cells, progeny of the epidermal stem cells, undergo terminal diff erentiation aft er 3–5 divisions. Aft er division, TA cells leave the basal layer and move through the suprabasal layers to the tissue surface, where they are periodically shed as squames.

Hepatic stem cells: �

• The liver has a strong regenerative capacity, utilizing diff erent modes of regeneration according to the type and extent of the injury. Mature liver cells can propagate to replace the damaged tissue permit the recovery of the parenchymal function.
• Chronic liver injury gives rise to a potential stem cell compartment which is located in the smallest branches of the intrahepatic biliary tree being activated, which called oval cell ductular reaction.
• These oval cells are derived from the canal of Hering, which amplifi es this biliary populations prior to these cells diff erentiate into hepatocytes. In the human liver, the organization of the biliary tree is diff erent, with the canal of hering extending to the proximate third of the lobule and so apparently requiring a name change from oval cells to hepatic progenitor cells.

Pancreatic stem cells: �

• Insulin-producing cells previously generated from pluripotent stem cells. The generation of these cells would provide a novel cell source for drug discovery and cell transplantation therapy in people suff ering from diabetes.
• Insulin-producing beta-cells turnover every 40-50 days by processes of apoptosis and the propagation and diff erentiation of the newly islet cells from progenitor epithelial cells, which are located in the pancreatic ducts.

TYPES OF STEM CELLS ACCORDING TO THEIR DIFFERENTIATION

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• Stem cells can be classifi ed according to their diff erentiation potential as a totipotent, pluripotent, multipotent, unipotent and oligopotent

Classification of stem cells based

on differentiation potential

Totipotent stem cells:

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• Totipotency means that it has the total potential to give rise to all types of cells. Totipotent is the capacity of a single cell to divide and diff erentiate into all cell types in an organism and produce fertile off spring. Oocytes and sperm are the best diff erentiated cells in our body and they are capable of formingany tissue in the body

Pluripotent stem cells: �

• Pluripotency is the ability of the cells to produce any type of cells in the organism. Th ey have been derivedfrom the mouse embryo. All are capable of diff erentiating into cells representative of a variety of adult tissue types in various assays, including embryoid body, teratoma, and some can contribute to mouse development in chimeras. There are many diff erences being recognized among pluripotent stem cell types, such as their morphology, gene expression profi les and growth factor requirements

Multipotent stem cells: �

• Multipotency means to those cells that can only give rise to cells of the tissue from which they are isolated

Unipotent stem cell:

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• Adult stem cells are found in the tissues of the adults they produce a limited number of cell types and can repair damaged tissue by replacing specialized cells. Because of their restricted lineage, they were thought to be either multipotent, with the ability to diff erentiate into a limited range of cells or unipotent, with the ability to produce only one cell type

Oligopotent stem cells:

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• Oligopotency means to those cells that can diff erentiate into only a few cell types, like lymphoid or myeloid stem cells

REFERENCES�

• 1. Cauffman G, De Rycke M, Sermon K, Liebaers I, Van de Velde H. Markers that defi ne stemness in ESC are unable to identify the totipotent cells in human preimplantation embryos. Oxford Journals Medicine Human Reproduction. 2009; 24: 63-70. https://goo.gl/kbJ7vA
• 2. Ralston A, Rossant J. The genetics of induced pluripotency. Reproduction J. 2010; 139: 35-44. https://goo.gl/ymdvmg
• 30. Pretson S L, Alison M R, Forbes S J, Direkze NC, Poulsom R, Wright NA. The new stem cell biology: something for everyone. Mol Pathol. 2003; 56:86-96. https://goo.gl/F7uzhC
• 3. Sage EK, Loebinger MR, Polak J and Janes SM. The role of bone marrowderived stem cells in lung regeneration and repair. Stem Book Cambridge (MA). Harvard Stem Cell Institute. 2008. https://goo.gl/B16fA1
• 4. Schöler HR. “The Potential of Stem Cells: An Inventory”. In Nikolaus
• Knoepffl er, Dagmar Schipanski, and Stefan Lorenz Sorgner. Human
• biotechnology as Social Challenge. Ashgate Publishing. 2007; 28.

• TOPIC 2. Characteristics of stem cells

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• Differentiation of multipotent embryonic stem cells into three different tissue types: endoderm, which gives rise to internal organs, mesoderm (connective tissue, muscles, circulatory system and bone tissue) and ectoderm (skin, sensory organs and nerve cells). Molecular basis of totipotency of embryonic stem cells (ESCs). Genome of zygote and ESCs, two main programs of embryogenesis: 1) gastrulation + organogenesis, 2) restricted maturation of differentiated cell lines. Sets of presynthesized mRNA classes of genes: early genes of ecto-, meso- and endoderm; set of mRNAs of homeotic (Nox) genes controlling segmentation and three-dimensional map of the embryo; five sets of mRNAs of totipotency genes. Factors of totipotency. Universal mechanism of totipotency of ESCs, MSCs and other types of stem cells: directed activation and subsequent import of mRNA into the nucleus. Differences in the potency of stem cells of different origins.

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• Embryonic stem cells. Histogenesis and regulation of pluripotency in the embryo Fertilization triggers the first cell division of the embryo.

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• In humans, at the 4–8 cell stage, the embryonic genome undergoes activation. As a result, the germinal vesicle (blastocyst) becomes compacted and its outer layer of trophectoderm is formed.

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• A small group of cells inside the blastocyst, called the internal mass of the blastocyst (IMB), is destined to form all the tissues of the body. From the VMB 4–5 days after fertilization, before implantation of the egg into the uterine wall, pluripotent SCs can be isolated, capable of forming all body tissues, as well as many, but not all, cells that support pregnancy. However, they are no longer capable of forming a new individual from themselves and this differs from totipotent cells. The latter, therefore, can be defined as cells capable, with appropriate maternal support, of forming a new individual. During development, such cells in mice persist only until the 8th cell stage of zygote division .

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Scheme of pre- and early post-implantation development of primates (A) Blastocyst stage of the embryo, showing the formation of the trophoblast (1) and the VMB (2); (B) Postimplantation day 9 of a rhesus monkey embryo. The formation of amnion (3), amniotic cavity (4), hypoblast (5) and epiblast (6) is shown; (C) Postimplantation day 10.5 of a human embryo. There is a characteristic covering of the blastocyst cavity with a network of extraembryonic endoderm (hypoblast cells (5); (D) 13th day of the rhesus macaque embryo. Formation of the primary streak (7) from the caudal part of the lower (tail) epiblast is noted, following the intensive development of extraembryonic mesoderm (8 The amniotic cavity (4) and the secondary yolk sac (9) are also visible.

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• In recent years, a number of interesting data have been obtained that clarify the molecular basis of the regulation of pluripotency in the embryo. A large group of genes essential for the development of pluripotent cells has been identified.

• This group includes genes for proteins that control totipotency of the ESC genome (Oct4, Otx1, Otx2), proteins that control totipotency and Embryonic stem cells pluripotency (Sox 1, Sox 2, Sox 3, Zic 1, Zic 2, Zic 3, Xash 3, Xbdx), proliferation signals (Wnt 1, Wnt 2), controllers of organ formation and markers for assessing differentiation efficiency (FoxD3, taube nuss, etc.)

• The key marker of pluripotent cells of an intact embryo and Embryonic stem cells is the Oct3/4 transcription factor. For Embryonic stem cells to remain undifferentiated, Oct3/4 expression must not decrease below a critical level.

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• However, expression of Oct3/4 alone is not sufficient to maintain this state of Embryonic stem cells. It was found that the Nanog transcription factor is essential for mouse Embryonic stem cells.

• As mouse ESCs differentiate, Nanog expression rapidly decreases, but if this gene is retained, for example by an innate promoter, mouse ESCs remain undifferentiated and can proliferate in a serum-free medium even in the absence of growth factors.

• As mouse ESCs differentiate, Nanog expression rapidly decreases, but if this gene is retained, for example by an innate promoter, mouse ESCs remain undifferentiated and can proliferate in a serum-free medium even in the absence of growth factors.
• Nanog is also detected in human ESCs, but at a lower level, and its function in these cells has not yet been sufficiently studied.

• There are 918 genes that are expressed more frequently in undifferentiated human ESCs than in differentiated descendants of these cells.

• Identified 92 genes, including Oct3/4 and Nanog, that were enriched in linear human ESCs. A specific gene, Esg 1, was discovered in mouse ESCs, which is exclusively associated with pluripotency

• An invaluable contribution to elucidating the mechanism of regulation of pluripotency was made by recent work in the field of epigenetic reprogramming by Japanese scientists Kazutoshi Takahashi from Kyoto University and Shiniya Yamanaka from the Japan Science and Technology Agency.

• Initially, these researchers selected 24 genes, the high expression of which is characteristic of ESCs, and introduced them into adult cells (fibroblasts) of mice using the method of viral transduction.
• As a result, the authors were able to show that the combination of only four genes Oct3/4, Sox2, c Myc and Klf4 is sufficient to form colonies that exhibit the morphology and growth characteristics characteristic of ESCs and express the corresponding genetic markers.

• Subcutaneous injection of the resulting cells into mice led to the formation of teratomas containing differentiated cells of all three germ layers, and there were 13 of them 14 injection into blastocysts made it possible to obtain chimeric mice.

• An unexpected result of the work on selection of a minimal combination of genes was the optional presence of Nanog in it.
• Introduction of the same genes (Oct3/4, Sox2, c Myc and Klf4) into adult human fibroblasts also makes it possible to obtain cells with pluripotent properties; these cells formed characteristic colonies, carried a set of surface antigens, expressed specific genes, and had high telomerase activity.
• Moreover, the resulting cells were able to differentiate into cells of all three germ layers not only during the formation of a teratoma, but also under conditions of normal growth in culture.

Phenotypic characteristics of ESCs

• Human ESCs are characterized by surface glycolipid stage-specific embryonic antigens 3 and 4 (SSEA 3 and SSEA 4), the functional significance of which is unknown, and high molecular weight glycoprotein antigens TRA 1 60 and TRA

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• Using PCR, the expression of transcription factors such as Oct3/4, FoxD3 is detected in ESCs , Rex 1 , etc., which are necessary for maintaining the undifferentiated phenotype and are of great importance in determining the early stages of embryogenesis and differentiation .

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• An additional characteristic of undifferentiated ESCs is the expression of high levels of alkaline phosphatase (ALP) on the cell surface.

• ESC differentiation is accompanied by a decrease in the levels of SSEA 3 and SSEA 4, an increase in the content of SSEA 1 and a decrease in the expression of OCT3/4 and alkaline phosphatase (ALP).

• None of the ESC antigens are unique to pluripotent cells.
• They are also found in other cells of the body .
• ESCs can proliferate in culture for many years and retain a normal karyotype

NANOG, SOX2, SALL4 and KRT18 expression throughout human preimplantation development and in a mature oocyte. Immunostaining

was performed using either a rabbit polyclonal IgG antibody against NANOG or SOX2, or a mouse monoclonal IgG1 antibody against SALL4 or

KRT18. As secondary antibodies, Alexa Fluor 647-conjugated donkey anti-rabbit IgGs, Alexa Fluor 488-conjugated goat anti-rabbit F(ab0)2 fragments

and donkey anti-mouse IgGs were used. Each image represents a section throughout the examined material. The arrowhead indicates staining in two

nuclei of the polar TE.

Summary of the expression of key stemness markers at the protein level in the nuclei of human oocytes and preimplantation embryos

These markers did not direct cells towards the ICM or TE lineage. A common expression of these markers is only guaranteed in hESC and in the ICM

of expanded blastocysts. (A) Mature oocyte, (B) Day 2 cleavage-stage embryo, (C) Day 3 cleavage-stage embryo, (D) compacted embryo, (E) early

blastocyst, (F) full blastocyst, (G) expanded blastocyst, (H) hESC. ICM, inner cell mass; TE, trophectoderm. White bars: no expression; colored bars:

expression in 100% of the samples tested unless the percentage of positive samples is given. *Expression was sporadically detected in a minority of cells.

NANOG (A), SOX2 (B), SALL4 (C) and KRT18 (D) expression in hESC colonies

and KRT18 expression in an early

differentiated colony (E)

Obtaining and cultivating �stem cells

Embryonic stem cells (ESCs) Еmbryonic germ cells (EGCs)

Embryonic germ cells (EGCs) are obtained from cells cultured in vitro the internal mass of the from primary germ cells isolated from the genital tubercles of fetuses of 5–7 weeks of gestation.

Obtaining and culturing embryonic stem cells

Embryonic stem cells (ESCs) are obtained from cells cultured in vitro the internal mass of the blastocyst (internal mass of the blastocyst - embryoblast) of the first 5–7 days after fertilization

Sources of pluripotent stem cells

• The optimal sources of human ESCs are blastocysts on the 5th day of development and primary germ cells isolated from the genital tubercles of 5–9 week embryos. It is from such cells that cloned lines of human ESCs originate. 15 ka H9.1, H9.2, etc.

Obtaining human ESC lines

• embryo

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• blastocyst

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• isolated embryoblast cells

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• dissociated embryonic cells

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• cloned embryonic cells

Obtaining human ESC lines

• Cells of the inner mass of the blastocyst are separated from the trophectoderm by immunosurgical manipulation, placed on a nutrient layer of fibroblasts and cultured in a medium containing fetal serum.

• More than 120 human ESC lines had been generated in this manner worldwide, of which 67 lines were included in the US National Institutes of Health registry.

• In recent years, standard conditions for culturing human ESCs have been developed, excluding the possibility of contamination with proteins of animal origin.
• 11 such human ESC lines are currently used for research purposes without mentioning all the data on their cultivation and differentiation potential

TOPIC 3. Mechanisms of stem cell commitment

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Sequential stepwise determination and commitment of the potencies of homogeneous cell groups - a divergent process. Periods of differentiation of embryonic cells. The importance of the microenvironment for the self-support of the stem cell population. Synthesis of special substances - chelons, which inhibit the intensity of reproduction of precursor cells and stem cells. Molecular markers of stem cells. Markers of hematopoietic stem cells (HSC). Markers of neural stem cells (NSC). Patterns of somatic cell differentiation. Types of cell populations. Cell population. Leblonowski cell populations: embryonic, static, growing and renewed. Scheme of development of a eukaryotic organism. Pluripotent cells. Cell clone. The concept of differon: stem cells, progenitor cells, mature cells that have reached the state of final (terminal) differentiation. Progenitor cells are committed, or semi-stem cells. Mature cells.

Mechanisms of stem cell commitment

• Commitment is a restriction of possible development paths due to determination. Commitment occurs in stages. Initially, the corresponding transformations of the genome concern large sections of it. Then they become more and more detailed, so first the most general properties of cells are determined, and then more specific ones.
• As is known, embryonic rudiments appear at the stage of gastrulation. The cells that make up their composition are not yet completely determined, so that from one rudiment there are cell aggregates that have different properties. Therefore, one embryonic rudiment can serve as a source of development for several tissues.

• There are four main periods of differentiation of embryonic cells:
• Ootypic
• Blastomeric
• Rudimentary
• Tissue differentiation
• Passing through these periods, embryonic cells form tissues (histogenesis).

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Methods of differentiating ESCs

Direct differentiation as a monolayer of extracellular matrix proteins.

Differentiation in coculture with stromal cells.

Formation of 3D spherical structures in suspension culture, called embryoid bodies (EBs).

Aggregation of ECs into three-dimensional EB structures has a general inductive effect and is often used as the first step to generate in vitro differentiation of many cell lines. Spontaneous differentiation induced by EB formation usually results in a small proportion of cells with any particular phenotype.

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• Factors influencing differentiation

1.Signaling pathways

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2.Microenvironment (niche factors)

• Extracellular matrix (ECM) – collagen; laminin; fibronectin
• Mechanical stimuli - stiffness; elasticity
• 3. Hormonal and chemical factors growth factors
• retinoic acid

4. Epigenetic modifications�DNA methylation �\histone modification�

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5. Genetic factors�

• The differentiation of ESCs depends largely on the activity of certain genes and genetic programs. Transcription factors such as Oct4, Sox2 and Nanog maintain the pluripotency of cells, while other genes are responsible for their transformation into specialized cell types. The gradual "switching off" of these genes under the influence of external signals leads to the launch of differentiation programs.

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FUNDAMENTALS OF CELL POPULATION KINETICS

• If one of the stem cells enters the differentiation pathway, then as a result of a sequential series of committed mitoses, first semi-stem cells and then differentiated cells with a specific function arise. The exit of a stem cell from the population serves as a signal for the division of another stem cell by the type of non-committed mitosis. The total number of stem cells is restored as a result. Under normal conditions of life, it remains approximately constant.

• The concept of differon
• The set of cells developing from one type of stem cells constitutes a stem differon. Often, different differons participate in the formation of tissue. Thus, the epidermis, in addition to keratinocytes, includes cells that develop into neutral ridges and have a different determination (melanocytes), as well as cells that develop by differentiation of blood stem cells, i.e., belong to the third differon (intraepidermal macrophages, or Langerhans cells).

Differon (histogenetic series) - a set of cell forms that make up a particular line of differentiation.

In the differon, the following are sequentially distinguished:

• stem cells,
• precursor cells,
• mature cells that have reached the state of final (terminal) differentiation.

Stem cells are a self-sustaining population of cells that can differentiate in several directions and form different cell types. Stem cells have high proliferative potential, but, as a rule, divide rarely.

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Progenitor cells. As they differentiate, their proliferative potential gradually decreases. The earliest stage of progenitor cells is distinguished: committed, or semi-stem, cells.

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Mature cells. The histogenetic series ends with them. The ability to proliferate completely disappears.

Stem cell niches

The SC niche is a microenvironment of regulatory connective cells and components anchored within it by adhesion. It is essential for stem cells to survive and maintain their stem potential.

The stem cell niche may contain cancer-associated fibroblasts, which can be formed due to the action of carcinogens. Thus, normal stem cells turn into cancer SCs, which are able to divide and transform into specific cancer cells and form cancerous tumors of various types.

An important discovery was that isolated ESCs or iPSCs from an animal with cancer, which were then cultured in special nutrient media, also later turned into cancer stem cells. This proves the direct responsibility of stem cells for the formation of cancer cells.

Molecular markers of stem cells

• Embryonic stem cell (ESC) markers. Classical ESC markers are alkaline phosphatase isozymes, the transcription factor Ost-4, high telomerase activity, and a number of cell surface markers, such as GSTM-2, TRA 1-60, SSEA-3, and SSEA-4, recognized by monoclonal antibodies to specific embryonic or tumor-determining antigens.

Hematopoietic stem cell (HSC) markers�

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• The cells that give rise to blood cells have markers on their surface called CD34, CD59, and Thy1, which can be used to identify them.

Neural stem cell (NSC) markers

• Molecular markers are known that allow the identification of both neural stem cells and successive phases of their development: nestin - for stem cells, vimentin - for progenitor cells, beta-tubulin - for neuroblasts, GFAP (glial fibrillary acidic protein) - for cells "moving" in the direction of glial development.

Differentiation patterns of�somatic cells�

• Types of cell populations. A cell population is a group of cells of one or more types that can be characterized in terms of space and time. This expansive understanding of the term can be applied to any cellular community - from the sum of all cells in the body throughout a person's life, from the zygote to, say, the cells of the corpus luteum of the ovary in one of the phases of the menstrual cycle. In other words, a differentiating criterion is needed to distinguish specific cell populations. The term cell population is used in at least two meanings: expansive and in relation to Leblon populations.

• The following cell communities are distinguished:
• Cell type - characterized by the actual expression (phenotype) or potential ability (sum of phenotypes) to express a specific spectrum of genes, which distinguishes this type of cell (erythroid, macrophage, etc.) from other types. This concept includes the concepts of cell phenotypes, as well as the plasticity and limits of the norm of the cell type. Cells with an identical set of genes allowed for expression (regardless of whether these genes are transcribed) belong to the same cell type. There are more than 200 cell types in the human body.

• Leblon cell populations. Based on the ability of cell renewal (including through proliferation), K. Leblon in 1964 distinguished four categories of cell populations: embryonic, static, growing and renewed. It should be noted that each of them is heterogeneous. The cells forming the population can be at different stages of differentiation, phases of the cell cycle, and in different functional states.

• A static population consists of a homogeneous group of cells that do not exhibit mitotic activity (for example, neurons).
• A growing population is characterized by the fact that the cells in it divide, mitotic activity gradually fades away.

• The renewal population is characterized by multiple mitoses and rapid cell death. In this case, the number of newly formed cells slightly exceeds cell loss (epidermis, intestinal epithelium, cells of the tissues of the internal environment). In neoplasia, cell production far exceeds cell death, which ensures rapid tumor growth.

• Embryonic population. The development of the human body begins with the fertilization of an egg and the formation of a zygote, which gives rise to the entire organism. A fertilized egg is totipotent, that is, it has unlimited potential in the sense that one of it is enough under appropriate conditions for the formation and development of a normal fetus. In the first hours after fertilization, it divides, forming identical totipotent cells, and any of them, when implanted in the woman's uterus, is capable of initiating fetal development.

• Cell clone - A group of cells originating from a single progenitor cell. The concept of a clone originated in immunology. When an antigen enters the body, one immunocompetent cell multiplies rapidly, and a large number of identical cells (clones) are formed, capable of synthesizing antibodies to this antigen. According to the clonal theory of development, embryonic structures are formed from a limited number of clones. Finally, tumors also develop as clones originating from a single transformed cell

TOPIC 4. Embryonic stem cells and adult stem cells.

• Features of embryonic stem cells. Sources of embryos: in vitro fertilization method, abortive material, embryos - cloning products, embryos, specially obtained isolation of embryonic stem cells, by mixing eggs and sperm. Embryonic pluripotent stem cells (ESC), which form the inner cell mass (ICM), or embryoblast at an early stage of embryo development. Hierarchy of embryonic stem cells. Fetal stem cells obtained from fetal material after abortion.
• Adult stem cells. Sources of stem cells in the adult body. Division of adult stem cells into three main groups: hematopoietic (blood-forming), multipotent mesenchymal (stromal) and tissue-specific progenitor cells, umbilical cord blood cells. Advantages of using umbilical cord blood in transplantation.
• Plasticity of hematopoietic stem cells and somatic embryonic stem cells. Pluripotent stem cells in the adult body.
• Stem cells of the nervous tissue. Hematopoietic stem cells (HSCs): capable of producing all blood cells. Muscle stem cells (MSCs). Olfactory stem cells of the nasal mucosa (OSCs). Liver stem cells (LSCs). Mobilization of donor and endogenous stem cells.

Expansion of embryonic� stem cells

• At one time, it was believed that the use of a substrate from mitotically inactivated mouse fibroblasts (the so-called “ feeder cells") is a prerequisite for the formation of ESC colonies in culture/
• Then it was found that mouse ESCs can proliferate in the absence of feeder cells if leukemia inhibitory factor (LIF) is added to the medium containing FBS. This cytokine supports murine ESCs through activation of the STAT3 protein complex.

• In a serum-free medium, LIF alone is not enough to prevent the differentiation of mouse ESCs into neural cells.
• That proliferation of murine ESCs in this environment could be supported by the addition of bone morphogenetic proteins (BMPs) to LIF.
• It is assumed that the effect of bone morphogenetic proteins on mouse ESCs is through the induction of differentiation inhibitor proteins and the suppression of extracellular receptor kinases (ERK) and p38 mitogen-activated protein kinases (MAPKs).

• LIF leukemia inhibitory factor (Leukaemia Inhibition Factor), a factor that maintains mouse ESCs in an undifferentiated state, is secreted by oviduct cells and increases at the blastocyst stage.
• In rodents, it contributes to the preservation of the viability of the embryonic diapause during embryonic diapause (maintenance of the embryo in the reproductive tract until the previous litter stops breastfeeding). During this stage, in the presence of LIF, a layer of extraembryonic endoderm is formed.

• All the major factors produced by feeder cells are not yet known.
• It is believed that these include various secreted substances; components that bind to cell membranes; extracellular matrix proteins.
• The role of interaction of specific cell receptors with ligands, signaling factors and other cell molecules is also unclear.

• Reported that long-term growth of human ESCs can support skin and muscle cells of 14-week-old fetuses.
• Іn addition to muscle and skin cells of human fetuses, used epithelial cells of the fallopian tubes removed from women during surgery or postnatal fibroblasts of the foreskin as a feeder.
• After long-term cultivation on these cells, ESCs continued to express markers of undifferentiated cells: alkaline phosphatase (ALP) , Oct3/4, SSEA 4, etc.

Pluripotency of ESCs

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• ESCs have a unique combination of functions, giving them the ability to unlimitedly proliferate in culture and differentiate, as suggested, into cells of any of the 250 specialized lineages of the body. Pluripotency, that is, the ability of ESCs to form all the cells of the embryo and adult, is initially noted during embryogenesis in the cells of the inner mass of the blastocyst and the subsequent epiblast. This ability is provided by external factors introduced into the growth medium from the outside or by the resulting feeder cells.

• Great progress in studying the molecular genetic basis of the pluripotent state became possible with the identification of a network of auto- and cross-acting control mechanisms mediated by three key transcription factors - Oct-3/4, Nanog and Sox2 . Each of these factors is required for pluripotency both in vivo and in vitro. Loss of Oct-4 or Nanog results in loss of pluripotency and spontaneous differentiation of cells into trophectoderm and primitive endoderm, respectively

• It was found that in ESCs of both one and another origin, many target genes are bound by combinations of Ost4, Nanog and Sox2, and the targets included both genes involved in transcription and inactive genes. When conducting an extended analysis of the network of transcription factors Oct4, Sox2, Nanog, c-Myc and Klf4 in mouse ESCs with the inclusion of proteins interacting with Oct4 or Nanog (Rex1, Dax1, Zpf 281 and Nac1), it was established that many promoters - ry were associated with a variety of common factors.

Stem cell biotechnology�

• Induced pluripotent stem cell (iPSC) method

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• Somatic cell nuclear transfer method

Embryonic stem cell method created by somatic cell nuclear transfer

Induced pluripotent stem cell (iPSC) method

1) - isolation and cultivation of donor cells

2) - transfection of stem cell-associated genes into donor cells using viral vectors.

3) - collection and cultivation of cells according to embryonic stem cell culture

4) - a small number of cells become iPS cells and generate embryonic-like colonies of stem cells.

TOPIC 5. Application of stem cells in medicine and scientific research

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• Types of stem cell transplantation: allogeneic, syngeneic, autologous, cord blood cell transplantation. Cell replacement therapy for Alzheimer's and Parkinson's diseases. Transplantation of embryonic blood stem cells - an alternative to bone marrow transplantation. Cell therapy. Restoration of the nervous system using stem cells - Parkinson's, Alzheimer's diseases, replacement of dead cells after thromboembolic stroke. Restoration of spinal cord damage using stem cells. Use of stem cells in orthopedics. Use of stem cells in ophthalmology. Treatment of diseases of the bone system. Treatment of tissue defects. Stem cells and diabetes. Main areas of use of stem cells in scientific research and oncological medical practice. Successes of modern therapy of malignant diseases. The use of hematopoietic stem cells in malignant and hereditary diseases of the bone marrow, application in the treatment of leukemia and lymphoma. The use of hematopoietic stem cells in overcoming the effects of chemotherapy. Gene therapy using ESC stem cells: legislation and bioethics. Gene therapy. Testing drugs on cell cultures.

• Prospects for the use of ESCs in biology and medicine
• ESCs as cells of choice in the treatment of human diseases

• Prospects for the use of ESCs in biology and medicine ESCs as cells of choice in the treatment of human diseases Due to the fact that the phenotypic characteristics of proliferating human ESCs remain unchanged after more than 20–30 passages, and these cells differ from most somatic cells in the absence or very low representation of major histocompatibility complex (MHC) class I and II antigens, the prospect of using human ESCs and EGCs as a source of cells for cell therapy attracts researchers in the field of experimental and clinical medicine.

• One of the decisive stages of such treatment is obtaining the desired cell type through ESC differentiation. But, although numerous key factors and stages of directed differentiation have been described, the nature of the complex culture system does not yet allow us to establish the exact direction of specific cell differentiation. Therefore, the decisive factor for the widespread use of human ESCs in clinical practice is the optimization of modern protocols and/or the development of new methods of precisely controlled differentiation.

• The most remarkable properties of ESCs—the ability for long-term self-renewal and the theoretically unlimited potential for proliferation and differentiation associated with the peculiarities of the phenotype of immature cells—continue to attract the attention of researchers.

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Differentiation of ESCs into cardiomyocytes

• Methods for differentiating cardiomyocytes from mouse ESCs have been well studied and make it possible to obtain cells from the atria, ventricles and sinus nodes. The original method for obtaining a suspension of cardiomyocytes from mouse ESCs was presented by Klug et al. The authors used a method of transduction of the gentamicin resistance gene (G418), triggered by a specific cardiomyocyte promoter.

• After transplantation into the heart of a mouse with cardiac dystrophy, these cardiomyocytes engrafted, grew, and functioned in situ. Other researchers have reported similar results based on studies of colonization of modified cells in the heart of mice with experimentally induced infarction.

• Adenoviral vectors were used to introduce reporter genes into mouse ESCs, the expression of which was triggered by the light chain gene of specific atrial myosin. Through this, it was possible to obtain a pure population of atrial cardiomyocytes

• For a long time, differentiation of human ESCs into cardiomyocytes was carried out according to protocols developed for mouse ESCs, in which mixed populations of cells were used . Further studies revealed that higher efficiency is achieved with transplantation of a specific type of cardiomyocytes.

Differentiation of ESCs into hematopoietic cells

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• The first reports of the formation of blood cells from mouse ESCs appeared almost 20 years ago, after the production of EBs from ESCs was first described. However, to achieve stable engraftment of ESC-derived SSCs in irradiated mice again required numerous experiments and considerable time

• Under these conditions, there may be some benefit from KS replacement treatment, but the latter must be accompanied by treatment targeting the pathophysiological process in the niches that need to be restored or replaced.

Differentiation of ESCs into CNS cells

• The formation of the nervous system is one of the earliest events in human development. Therefore, it is not surprising that neuronal tissue is found in EBs obtained from human ESCs / Mouse ESCs that differentiate in monolayer culture also form neuronal tissue cells

• Regenerating neurons must integrate correctly with existing developed neurons to establishing a functioning neural network. The ability of ESC-derived cell types to express embryonic phenotypes may predispose them to some intrinsic plasticity that should enable functional integration.

Studying embryogenesis using embryonic stem cells

• It has now been established that the initial driving forces of gastrulation and organogenesis during embryogenesis are embryonic induction and reversible epithelial-mesenchymal transitions. The target of these forces is not isolated pluripotent stem cells, but primitive primary tissues of the embryo

• Mechanisms of embryonic induction can be observed in ESC culture. Thus, the frequency of formation of cardiomyocytes from ESCs growing on a fibroblast feeder is 7–16%, while the addition of avian cardiogenic mesoderm/endoderm explant to the culture increases the formation of rhythmically contracting cardiomyocytes to 100%

• It is assumed that the main inducer of cardiomyocytes in such a culture is the signal-inducer Sparc, secreted by cells of the mesenchymal cardiogenic endoderm. In these cells, Sparc induces the expression of GATA-4, GATA-6, Nkx 2.5 and Mef2C. Sparc/BMP-2 inhibitors suppress cardiogenesis from ESCs.

• The pluripotency of the genome of “true” ESCs is controlled by the genes Oct3/4, Nanog, FoxD3, Rex1, Sox2, c-Myc and Klf4. During gastrulation and the formation of meso-, endo- and ectoderm, the expression of these genes decreases. “Pluripotency is a special status of an almost completely repressed cell genome”

• The only exceptions are embryonic germ cells, which develop according to a special program. Only primitive neuroectoderm cells, but not mesoderm or endoderm cells, could be reprogrammed to the status of “true” ESCs in vitro.

TOPIC 6. Ethical issues of using embryonic stem cells

• Biological difficulties in using stem cells in clinical practice: the problem of immunological rejection, the possibility of stem cell transformation into cancer.

• Medicine
• ESCs are used in cell therapy in the treatment of many diseases, such as: diabetes, Parkinsonism, Alzheimer's disease, which are poorly amenable to other types of therapy

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• Moral
• The embryo is considered the beginning of a new life, which already has its own genetic material, and therefore its use in experiments is unacceptable

Problems of immunological rejection of ESCs

• immune rejection of transplanted stem cells (SC)

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• graft-versus-host disease (GVHD)

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• reactivation of infection in the context of an altered immune status of the recipient.

The recipient's stem cells are recognized by the body as foreign material. The rejection process is influenced by the genetic aspects of the donor and is implemented through T-lymphocytes and antibodies

Immune rejection reaction

Graft-versus-host disease

Acute GVHD

Manifests up to 100 days after transplantation

Immune reaction of activated T-lymphocytes of the donor

gainst cells of the recipient. Manifests itself in skin forms (hyperbilirubinemia, hyperenzymemia and jaundice).

The risk of development increases with: use of a cell preparation from an unrelated donor, donor that does not match the blood group, with a significant age of the donor or recipient, if the donor is a woman who has had several pregnancies.

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Chronic GVHD

Manifests after 100 days from transplantation in the

form of lesions of the skin, eyes and oral cavity

Activation of infections

• It is the result of changes in the donor's immune system after stem cell transplantation. An important example is the development of cytomegalovirus (CMV) infection. Clinical manifestations include pneumonia and gastroenteritis.

Lung cells infected with CMV

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• REFERENCES
• Avilion A. A., Nicolis S. K., Pevny L. H. еt al. Multipotent cell lineages in early mouse develop- ment depend on Sox2 function // Genes Dev.— 2003. — Vol. 17. — P. 126–140.

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• Nakano T., Kodama H., Honjo T. In vitro development of primitive and definitive erythrocytes from different precursors //Science. — 1996. — Vol. 272. — P. 722–724.

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• Shen M. M. and Leder P. Leukemia inhibitory factor is expressed by the preimplantation uter- us and selectively blocks primitive ectoderm formation in vitro //Proc. Natl. Acad. Sci. — 1992. — Vol. 89. — P. 8240–8244.

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• Tanaka T. S., Kunath T., Kimber W. L. и др. Gene expression profilling of embryo-derived stem cells reveals genes associated with pluripotence and lineage specificity // Genome Res.— 2002. — Vol. 12. — P. 1921–1928.