Embryonic stem cells – a cure for old age (2)

(Continued. The beginning of the article is here.)

II. HUMAN EMBRYONIC STEM CELLS:
SOURCES OF PRODUCTION, MAINTENANCE OF VIABILITY AND BASIC PROPERTIES

Human embryonic stem cells (CESCS) are pluripotent stem cells obtained at different stages of embryonic development. CESCS have a unique ability to unlimited proliferation and differentiation into cells of all types of tissues. The unlimited potential of CESCS makes them particularly attractive for use in clinical practice. In particular, the ability of cESC to regenerate may be the key to the success of the treatment of age-related diseases, the characteristic of which, as discussed in the previous section, is progressive dysfunction and/or death of somatic cells.

Table 1.
Methods of differentiation of CESCS into certain types of cells
for use in the treatment of age-related diseasesClinical application

A. Sources and allocation of CHESKUsually, cESC is isolated by microsurgical removal of the internal cell mass (ECM) from the preimplantation embryo at the blastocyst stage (Fig. 1). The cells of the ECM are pluripotent, that is, they have the ability to differentiate into the extraembryonic endoderm and three germ layers forming all the tissues of the embryo: ectoderm, mesoderm and endoderm.

Under certain conditions, these cells can proliferate in vitro, maintaining an undifferentiated state and pluripotency for an indefinitely long time. In order to create cell lines, CESCS were also isolated from single blastomeres of 4- or 8-cell embryos [5-8], 16-cell morula [9, 10] and VCM parthenogenetic embryos. A single blastomere has a high degree of totipotency and is capable of giving rise to an entire embryo. Therefore, the CESCS obtained from blastomeres make it possible to circumvent the ethical contradictions associated with the use of CESCS, since theoretically the removal of a single blastomere should not deprive the remaining blastomeres of the ability to form a normal embryo. Similarly, parthenogenetic embryos formed as a result of artificial insemination of donor eggs [11-14] are acceptable sources of cESC, since in this case the need for the creation and subsequent destruction of viable embryos is excluded. Moreover, CESCS isolated from parthenotes are particularly attractive material, since they are homozygous for alleles of human lymphocytic antigen (HLA) antigens, which can prevent the development of an immunological rejection reaction when using CESCS for transplantation (discussed further). Since the first isolation of pluripotent CESCS from VCM, performed in 1998 by Thomson and colleagues [15], hundreds of cESC lines have been created from cells isolated from various embryonic sources, currently used worldwide for fundamental and clinical research. In the USA, more than 80 cESC lines are used that meet state requirements [16], some of them are currently used in clinical trials.

B. Cellular and molecular characteristics of pluripotent CESCSPluripotent CESCS have specific morphological and molecular properties characteristic of most cESC lines.

The morphological parameters of a single cESC include an enlarged nucleus and distinguishable nucleoli. In culture, proliferating CESCS form compact colonies consisting of round–shaped cells (Fig. 2). In the process of differentiation, these colonies lose their compact morphology, while spread-out cells appear at the colony boundaries - differentiated cells migrating from the colony. Spontaneous differentiation of CESCS in culture can be prevented by regular addition of fresh nutrient medium [17].

Figure 2. Typical undifferentiated and differentiated CESCS in culture.
(A) A compact colony of proliferating pluripotent CESCS can be observed when cultured in an appropriate medium on a feeder layer of mouse embryonic fibroblasts.
(B) Floating human embryoid bodies (CHET) appear 3 days after induction of differentiation.
(C) Differentiating cells, including cardiomyocytes, appear inside adhesive cultures 48 hours after seeding the CHET into a gelatin-coated culture plate. One division of the scale = 25 microns.

As a result of the work of the International Stem Cell Initiative, a consortium in which researchers from more than 15 countries participated, a panel of markers identified for 59 independently obtained lines of pluripotent CESCS was created [18]. These markers have found application in the routine assessment of the characteristics of pluripotent cescs. These include genes whose known functions are to maintain pluripotency or other developmental processes, such as Nanog, POU-domain of Class 5 homeodomain-1-containing protein (POU5F/OCT4), teratocarcinoma-like growth factor-1 (TDGF1), DNA (cytosine-5)-methyltransferase-3-beta (DNMT3ß), A-receptor-beta-3 gamma-aminobutyric acid (GABRB3) and growth and differentiation factor-3 (GDF4). Pluripotent cescs also express a number of surface markers, such as stadium-specific embryonic antigens-3 and -4 (SSEA-3, SSEA-4), as well as keratinosulfate (TRA-1-60, TRA-1-81, GDTM2 and GCT343) and protein antigens (CD9 and Thy1). Moreover, CESCS can also be identified based on the expression of alkaline phosphatase, stem cell factor (SCF/c-Kit ligand) and Class I HLA proteins. Currently, work is underway to compile a profile of microRNA expression in pluripotent cescs. Despite the fact that a thorough study of miRNA expression has not yet been carried out, several studies have identified a number of microRNA clusters, the expressed expression of which is characteristic of cESC lines. Some of them are known for their participation in maintaining the pluripotent state of the cESC [19, 20]. These include miRNA-92b, miRNA-302 cluster, miRNA-200c, miRNA-368 and miRNA-154* clusters, miRNA-371, miRNA-372, miRNA-373*, miRNA-373 and miRNA-515 cluster [21, 22]. Pluripotent cescs also have distinctive epigenetic characteristics. In general, the structure of the cESC chromatin is an open conformation that allows transcription factors to penetrate into it and regulate gene expression levels [23]. In addition, the DNA methylation profiles of CESCS differ from the methylation profiles of other cell types. A distinctive feature is the reduced levels of methylation of CpG dinucleotides, especially characteristic of the promoter regions of genes responsible for pluripotency, such as OCT4 and Nanog [24]. These unique epigenetic features of CESCS are necessary to maintain their pluripotency status and, accordingly, are suitable for use as markers of undifferentiated Cescs.

C. Determination of pluripotency of cESCCESCS are capable of differentiation into cells forming three germ leaves.

This fact can be verified using generally accepted in vivo and in vitro techniques commonly used to confirm the pluripotency of cESC lines. The most commonly used in vivo method involves the induction of teratoma formation after transplantation of undifferentiated CESCS to immunodeficient mice [25-28]. Teratomas are benign tumors consisting of tissue structures formed from all three embryonic germ sheets (Fig. 3).

Figure 3. Teratoma formation is a method for assessing the ability of CESCS to differentiate in vivo.
Proliferating cESC cultures were used to form teratomas by implanting them into the renal capsule using traditional methods [25-28].
(A) An explanted teratoma.
(B-F) To identify embryonic tissues, teratoma sections are stained with hematoxylin and eosin. The photo shows characteristic tissues belonging to all three embryonic leaves, including mesoderm (B,C), endoderm (D) and ectoderm (E,F).
(B) Incipient renal tubules and glomeruli immersed in primitive renal epithelium.
(C) Cartilage surrounded by a capsule of compacted mesenchyme.
(D) Glandular intestinal structure.
(E) Incipient neural tube.
(F) Primitive squamous epithelium.
The division of the scale on micrographs is 100 microns.

The results of the analysis of teratomas formed from the established CESCS can be used to determine their ability to differentiate. The ability of CESCS to differentiate into cells of various types can also be tested in vitro by forming embryoid bodies (ET). ET are spherical colonies of non-cohesive differentiating cescs containing populations of cells characteristic of all three embryonic germ sheets. Under suitable conditions, ET can differentiate into cells of certain types. As will be described below, the formation of ET is usually used as an intermediate step in the directed differentiation of CESCS into tissue-specific cell populations.

Table 2. Standardization and quality control of cESC for use in clinical practiceRequirement

Continuation: differentiation of human embryonic stem cells into cells of certain types.

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19.12.2011