Embryonic stem cells – a cure for old age (3)
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III. DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS INTO CELLS OF CERTAIN TYPESThe most promising feature of CESCS is their ability to give rise to tissue-specific progenitor cells capable of differentiation into specialized postmitotic cells suitable for use in cell therapy.
Moreover, CESCS, due to their ability to infinite division, represent an almost inexhaustible source of specific cell populations. The purpose of the current research is to search for and improve methods for directing the differentiation of CESCS to obtain pure homogeneous populations of cells of certain types suitable for replacing damaged cells or for stimulating the full functioning of surrounding cells. In the following sections, we will describe several examples of the differentiation of CESCS into tissue-specific cells, in particular, with the potential for use in the treatment of age-related diseases.
A. Endodermal cells obtained from cESCEndoderm derivatives include cells forming the lungs, liver, pancreas, bladder, pharynx, thyroid gland, parathyroid gland and gastrointestinal tract.
The first stage of creating endodermal cells is the formation of a definitive endoderm. D’Amour et al.  demonstrated that selective induction of definitive endoderm formation can be achieved by adding high concentrations of activin A at certain stages of differentiation under the condition of low serum content in the medium. The action of activin A is similar to the action of Nodal ligand activating a signaling mechanism mediated by transforming growth factor beta (TGFß). The role of activin A in inducing the formation of a definitive endoderm is enhanced in the presence of additional factors, such as Wnt3a  and Noggin , or with simultaneous suppression of the signaling mechanism mediated by phosphoinositide-3-kinase .
Induction of definitive endoderm formation from cESC can lead to the appearance of specific populations of progenitor cells, including precursors of pancreatic islet beta cells, hepatocytes or alveolar epithelial cells. The ultimate goal of creating these cells is to treat diabetes, as well as liver and lung diseases, respectively. The most successful examples available today include the creation of precursors of pancreatic islet beta cells, carried out by Kroon et al. (Kroon et al.)  through the sequential action of activin A and Wnt3A on the cESC, followed by the introduction of keratinocyte growth factor or fibroblast growth factor-7 into the medium to induce the formation of primitive digestive tubes. After that, retinoic acid, cyclopamine and Noggin are added to the culture, which provides inhibition of the signaling mechanisms mediated by the Sonic Hedgehog (Shh) and TGFß protein, inducing differentiation of cells of the posterior surface of the anterior intestine, which are the source of pancreatic progenitor cells. These cells are cultured to produce pancreatic endoderm cells. After implantation in immunodeficient mice, such cells acquired histological and structural characteristics of beta cells of pancreatic islets and demonstrated the ability to produce insulin for at least 100 days. These results were received with great enthusiasm, as they demonstrated the possibility of curing diabetes. Type 2 diabetes is the most common variant of the disease, which affects more than 25% of the U.S. population aged 65 years and older .
Hepatocytes are also obtained after differentiation of cESC into a definitive endoderm [35, 36]. A population of highly stable functional hepatocytes was obtained by sequentially using a medium with a low serum content, a matrix of type I collagen and hepatocyte differentiation factors, including fibroblast growth factor, BMP4, hepatocyte growth factor, oncostatin M and dexamethasone . These cells expressed known markers of mature liver cells, demonstrated functions corresponding to their status and, when administered to mice with liver damage, integrated and differentiated into mature liver cells. The ability to differentiate into liver cells can be useful in the treatment of a number of diseases of this organ, often developing in elderly people, such as cirrhosis, hepatic cell carcinoma and diabetes-associated chronic liver diseases .
The possibility of using cESC for the treatment of lung injuries has also been the subject of research. An important achievement in the development of a method of directed differentiation into lung cells was made by Wang et al. (Wang et al.) [38, 39]. In this work, genetically modified CESCS carrying reporter genes specific to lung cells under the control of promoters of tissue-specific genes, such as surfactant protein C, aquaporin-5 and T1-alpha, formed pure populations of alveolar epithelial cells of type I and II. When administered to mice with acute lung injuries, these cells demonstrated adequate functional properties, including the ability to gas exchange and improvement of the damage zone at the tissue level.
B. Mesodermal cells obtained from cESCThe direction of cESC differentiation into the mesoderm requires activation of a signaling mechanism mediated by transforming growth factor-beta and can be carried out by step-by-step dose-dependent addition of activin A, BMP4 and growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) .
Mesoderm derivatives were also successfully obtained by spontaneous differentiation of CESCS through the stage of formation of CHET without the initial direction of their differentiation into the mesoderm. Stable differentiation of CESCS into hematopoietic stem cells, giving rise to all types of blood cells and components of the immune system, was achieved by using a serum-free medium by forming a CHET rotation . Specific hematopoietic cells, such as functional dendritic cells, were successfully obtained from cESC by spontaneous formation of CHET in a serum-free medium and addition of BMP4 at certain time stages . Hematopoietic progenitor cells that transform into functional T-lymphocytes and natural killers that specifically affect human tumor cells both in vitro and in vivo were obtained from CESCS cultured together with stromal cells . Thus, the possibility of differentiating CESCS into hematopoietic cells opens up prospects for improving existing therapeutic approaches to cancer treatment that require blood cell transplantation, as well as immunotherapy methods involving the induction of antigen-specific immune reactions .
Cardiomyocytes, which are another important mesoderm derivative from a therapeutic point of view, were successfully obtained from cESC using several methods . Under certain conditions of cultivation, CESCS can spontaneously differentiate into cardiomyocytes through the stage of formation of CHET. Morphological, molecular and electrophysiological properties of the cardiomycites obtained in this case are similar to the properties of adult cardiomyocytes , while they demonstrate quantifiable reactions to physiological stimuli similar to the reactions of atrial tissue, ventricles, as well as the focus of automatism/conduction of the heart [47-50]. Cardiomyocytes were also obtained by directed differentiation using the action of activin A and BMP4 on a dense monolayer of cESC. During in vivo transplantation, these cells successfully formed functional cardiomyocytes . In another study, additional additives to the nutrient medium, including vascular endothelial growth factor, Wnt inhibitor and Dickkopf homologue (DKK1), were used to stimulate the differentiation of CHET into cardiomyocytes, after which bFGF was introduced into the medium . The success of these studies was assessed by measuring the expression level of proteins specific to mature heart cells, such as cardiac troponin T, atrial myosin light chain-2, and cardiac muscle transcription factors Tbx5 and Tbx20. Several groups of researchers have created reporter lines of CESCS specific to cardiac tissue, which can be used to test various differentiation strategies [49, 53] and create specific subtypes of cardiomyocytes .
Judging by the results of the test for the formation of teratomas, the differentiation of CESCS can be easily directed towards connective tissues, such as bone or cartilage (Fig. 3). Therefore, CESCS can be a valuable source of cells suitable for connective tissue replacement therapy in diseases such as osteoarthritis and osteoporosis, characterized by destruction of articular cartilage and pathological fractures caused by low bone density, respectively. The most successful and effective protocols for directing the differentiation of CESCS into chondrocytes imply the use of three-dimensional cultivation systems, which is achieved by sowing CESCS in high density, which leads to the formation of a dense clot, or by introducing cells into a synthetic three-dimensional matrix. Such systems enable the exchange of signals between undifferentiated cescs and mature chondrocytes, which stimulates stable homogeneous chondrogenic differentiation. For example, the creation of a unicellular suspension of dissociated CHET cultured as high-density micromass in the presence of BMP2 promotes the formation of chondrocytes . Co-cultivation of cESC with primary chondrocytes or in the presence of osteogenic additives and polymer matrices made it possible to obtain cells similar to cartilaginous or osteogenic [55, 56]. More recently, the use of three-dimensional cultivation systems, which do not imply the use of a feeder layer, has made it possible to obtain multipotent precursor cells of connective tissue from cESC, forming cartilage-like structures. Implantation of these cartilaginous structures differentiated in vitro to injured immunodeficient mice restored the mobility of the ankle joints of animals, which is possible only in the presence of an intact Achilles tendon . Moreover, there is evidence that cell transplantation promotes the growth and repair of damaged tissues also by stimulating endogenous cells .
C. Ectodermal derivatives of cESCThe dominant mechanism of differentiation of cESC cultures leads to the formation of ectoderm, which gives rise to cells of the nervous system and epidermis.
The neural progenitor cells obtained from cESC are characterized by the formation of rosette-like structures formed in the presence of FGF2 or EGF by spontaneous differentiation during the outgrowth of cESC or after seeding of CHET on the surface of the adhesive substrate [59, 60]. These "neural sockets" have become a marker of nerve progenitor cells obtained from cESC, capable, when exposed to appropriate stimuli, of differentiation into a wide range of nerve cells. The purpose of many studies is to develop methods for stimulating the formation of neural sockets to create enriched populations of specific types of nerve cells. One example is the use of stromal cell lines  that produce ectodermal signaling factors necessary for the induction of nerve cell formation and contribute to the formation of neuronal sockets [62, 63].
The rejection of FGF2 and EGF and the use of other factors can lead to the differentiation of neuronal sockets into specific subtypes of nerve cells. Neural roller stem cells obtained from neuronal sockets can differentiate, with the addition of BDNF, GDNF, NGF and dbcAMP, into peripheral sympathetic and sensitive neurons or, in the presence of CNTF, neuregulin-1-beta and dbcAMP, into Schwann cells . Neuroglial cells, such as oligodendrocytes, are formed by the addition of B27, thyroid hormone, retinoic acid, FGF2, epidermal growth factor and insulin . In addition, FGF8 and Shh induce the differentiation of progenitor nerve cells derived from cESC into dopaminergic neurons , whereas the addition of Shh and retinoic acid induces their differentiation into motor neurons . Recently, functional cholinergic neurons of the basal parts of the brain have been obtained from CESCS through the formation of neurospheres and subsequent addition of Shh, FGF8 and BMP9 or overexpression of LHX8 and GBX9 .
The possibility of differentiating CESCS into nerve cells and other cells of the nervous system has aroused great interest, since such cells can be used for drug testing or for cell replacement therapy of a number of neurodegenerative diseases. In particular, the successful cultivation of dopaminergic neurons, especially those belonging to the midbrain-forming subtype, can potentially be used to treat Parkinson's disease, characterized by progressive death and impaired functioning of these neurons. Cholinergic neurons obtained from cESC can also be used for the treatment of Alzheimer's disease, in which degeneration of cholinergic neurons of the basal parts of the brain leads to disability-causing cognitive function disorders. However, cESC can be used without resorting to substitution therapy methods. During a clinical study in which autologous fibroblasts programmed for the expression of human NGF were implanted into the forebrain of patients with moderate Alzheimer's disease, a significant decrease in the rate of deterioration of cognitive function was observed . Thus, theoretically, genetically modified precursor nerve cells obtained from cESC, capable of quickly taking root and producing protein products of therapeutic genes, such as NGF, can be used to prevent degeneration of cholinergic neurons of the basal parts of the brain.
Similarly, motor neurons obtained from cESC can be used in the therapy of amyotrophic lateral sclerosis (ALS), characterized by progressive loss of motor neurons of the cerebral cortex and brainstem, as well as the spinal cord. The results of the study of ALS models also indicate that auxiliary cells of the nervous tissue, such as oligodendrocytes and Schwann cells, can contribute to the development of this disease [70, 71]. Therefore, the possibility of differentiating CESCS into both neurons and other types of cells of the central nervous system looks like a promising therapeutic approach.
The effectiveness of transplantation of oligodendrocytes obtained from cESC for the treatment of spinal cord injuries is currently being studied as part of the first clinical trial using cESC, which has received approval from the US Food and Drug Administration . Spinal cord injuries lead to rapid loss of oligodendrocytes, which leads to demyelination and death of neurons. As part of a clinical trial sponsored by Geron Corporation, purified oligodendrocyte progenitor cells are injected into the spinal cord of paralyzed patients within two weeks after injury. This is the first clinical study focused on the safety of such therapy, but it is expected that progenitor cells will undergo irreversible differentiation into oligodendrocytes and begin to produce myelin, which isolates the membranes of nerve cells and is necessary for the effective transmission of nerve impulses. If this study succeeds in achieving successful integration and functioning of oligodendrocytes, its results may form the basis for new methods of treating ALS, manifested by demyelination of motor neurons.
Retinal pigment epithelial cells (PES) are another specific type of cells derived from the neuroectoderm. PES cells support the functioning of retinal nerve cells by phagocytosis and renewal of the rhodopsin pigment of the external segments of photoreceptors. According to recently published data, the differentiation of CESCS into PES cells can be induced by the presence of nicotinamide and activin A in the serum-free medium . After transplantation into an animal model of macular degeneration – a disease caused by impaired functioning and death of PES cells – pigmented cells obtained from cESC acquire morphological and functional characteristics of PES cells. These data formed the basis for the second and third clinical trials using CESCS, which were approved by the US Food and Drug Administration and sponsored by Advanced Cell Technology. As part of these studies, PES cells obtained from cESC will be implanted directly into the collapsing retina of patients with Stargardt macular degeneration – a juvenile form of the disease – or with a dry form of age-related macular degeneration in order to restore visual acuity. The beginning of these three clinical trials is the frontier of transferring the results of research work with cESC into the practice of treating degenerative diseases, and specialists in the fields of stem cell biology and geriatric medicine are looking forward to the results with great impatience.
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