Three essential factors for tissue regeneration are cell, protein and scaffold. In this chapter, we will discuss the types and characteristics of stem cells, the most important cell source.
Stem cells can differentiate into various cell types and can be divided to produce more stem cells. Stem cells can be defined by three properties: 1) self-renewal, 2) clonality, and 3) differentiation into various cell types. Stem cells can be divided into embryonic stem cells (ESC) and adult stem cells (ASC), depending on the cell acquisition process and differentiation capacity. Stem cells can also be divided into unipotency, which can be differentiated to only one type of cell, multipotency, which can be differentiated to multiple types of cells, and pluripotency, which can differentiate to every type of cells.
ESCs are pluripotency, while ASCs are unipotency or multipotency. Since the first derivation of ESC from mouse blastocyst in 1981, it has been researched in various fields, until achieving the first derivation of human ESC (hESC) by Thomson et al. in 1998. hESCs are expected to become a new cell source for the treatment of refractory diseases, because they are favorable for in-vitro proliferation and can differentiate all type of cells that consists human body. It has been reported that approximately 400 types of hESC have been successfully derivated around the world. ASCs are extracted from each organ after complete development and can differentiated to a cell with tissue-specific function.
Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) are the examples. Recently introduced stem cell is induced pluripotent stem cell (iPSC), which is artificially derived from a somatic cell by inducing dedifferentiation through a virus which delivers genes that interferes with the maintenance of undifferentiation. Like ESCs, iPSCs are capable of differentiating to every type of cells. A lot of studies are currently under way for derivation of pluripotent stem cells by dedifferentiationfor individualized treatment.
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Definition of hESC
hESCs are cells derived from the inner cell mass of an embryo before implantation. hESCs should have self-renewal capacity for in-vitro proliferation while maintaining undifferentiated state and pluripotency for differentiation to every type of cells consisting the human body. Since hESCs can differentiate to various types of cells, such as nerve cells, myocytes and somatic cells, under certain condition, they are expected to become a good cell course for the treatment of diseases not curable with current medicine and be used as a model for studies on early development of a human body and for development of various new drugs.
Because hESCs are cultured with supporting cells, which express receptors that contribute to maintaining undifferentiated state, hESCs are vulnerable to immunorejection to foreign pathogen and protein. To reduce such risks, studies are attempting to establish methods for derivation of stem cells that can be used in the clinical setting by developing a culture without mouse-derived supporting cells or culture fluid without animal-derived ingredients.
Characteristics of hESC
1. Origin of hESC
As described earlier, hESCs are derived from the inner cell mass of blastocyst after fertilization and before implantation. The fertilized embryo continues cleavage until reaching morula, which later becomes a blastocyst with a big blastocoele at the center. Cells in the blastocyst differentiated to inner cell mass, which will become an embryo in the future, or to trophoblastic cells, which will form placenta and amnion. Cells in the inner cell mass are pluripotent cells, which will be isolated and cultured for derivation of ESCs with pluripotency.
2. Cell biology of hESC
(1) Microstructure
hESCs grow in colony, not as a single cell. Under in-vitro culture, thousands of single cells grow in the form of circular single layer. At the center are actively differentiating cells, and differentiated cells move to the outer boundary of the colony, growing the colony. Undifferentiated hESCs appear on transmission electron microscope as having very large nuclei compared to the cytoplasm, unapparent boundary of cell membrane, and free ribosome inside the cytoplasm. Small mitochondria, the characteristic of an immature cell, can be observed, but not cell organelle, such as rough endoplasmic reticulum and Golgi’s complex, or lipid droplets. Unlike the microstructure of undifferentiated stem cells, differentiated cells clearly shows Golgi’s complex connected with small secretory vesicles. Rough endoplasmic reticulum can be also seen actively synthesizing secretory proteins. Lipid droplets and large mitochondria are also observed.
According to the microstructure of hESCs observed by transmission electron microscope, undifferentiated hESCs maintain relatively simple structure during proliferation, while differentiated cells contain all types of cell organelles found in adult somatic cells.
(2) Expression of transcripts for maintaining undifferentiated state and controlling of pluripotency
Undifferentiated hESCs are known to show a specific pattern of gene expression different from somatic cells. When hESCs are compared with other types of cells by long-term undifferentiated state and self-renewal, hESCs have various types of signal transductions activated. Genes associated with DNA repair, protein structure, ubiquitin system, and antitoxicity are also frequently expressed. These phenomena are similar to the transcription pattern of cells in stressful environment.
A lot of studies also compared gene expression of mouse ESC with human ESC. Common molecular mechanism of genes associated with the signal transduction of FGF, TFGβ and Wnt, which are commonly expressed in both hESC and mouse ESC, contributes to the maintenance of both stem cells. Genes associated with metabolism, ribosome protein and cytoskeleton are expressed in both stem cells. There were also reports suggesting that ribosome associated genes are expressed a lot due to the rapid proliferation of human ESCs. On the other hand, the expression of LIF, which is known to be involved in maintaining undifferentiated state of mouse ESC, was different between the two stem cells, supporting the fact that LIF is not involved in the maintenance of undifferentiated state in human ESC.
(3) Cell cycle
Human ESCs are characterized by active self-renewal, which is associated with specific cell cycle of hESCs compared to other somatic cells. Among the cell cycle, mouse ESCs have short G1 phase, while human ESCs also have short G1 phase but very high rate of cells remaining in S phase. In this case, the cell cycle lacks resting phase, alternating between DNA replication (S phase) and chromosome segregation (M phase). This pattern continues only during when human ESCs are in undifferentiated state and starts to include resting phase when the human ESC starts to differentiate to a certain type of a cell. It has been reported that the basic mechanism of cell cycle is similar between mouse and human ESCs, but human ESCs have slower progression of a cell cycle than mouse ESCs. As for cyclin and cyclin-dependent kinase which are associated with cell cycle control, CDK4 and cyclin D2 gene, which are associated with G1 phase, are expressed more abundantly, suggesting that the cell cycle of hESCs are more closely related to the control of G1 phase.
Production of hESC
1. Isolation of inner cell mass from blastocyst
The condition of blastocyst is the most important for derivation of hESC from surplus embryos. From thawing to blastocyst, cultured embryos are divided into healthy blastocyst with distinct inner cell mass, blastocyst with definite boundary with trophoblastic cells and small inner cell mass, or blastocyst without definite boundary and unapparent inner cell mass.
Inner cell mass is isolated from healthy, swollen blastocyst by immunosurgical method and cultured on supporting cells. When the boundary of inner cell mass is not clear, the transparent zone is removed and the inner cell mass is placed directly on the supporting cells.
48 hours after placing the inner cell mass on the supporting cells, the cell mass is observed by with a microscope to confirm whether it is attached well to the supporting cells. After a week to 10 days, the cell mass is cut to small pieces with a glass pipette, placed on new supporting cells, subcultured at a certain interval for derivation of stem cells.
2. Culture process
Established hESCs are placed on new supporting cells every 5-7 days for subculture. The culture liquid is replaced from 48 hours after placing hESC fragments to new supporting cells for 5-7 days of culture. For subculture, both mechanical methods where the hESCs are cut to small pieces with a glass pipette and detaching hESCs using enzyme can be used. When the mechanical subculture method is used, differentiated and undifferentiated cells can be transferred separately, and hESC colony of identical size can be obtained.
When enzyme is used, whether Oct3/4, surface antigen SSEA4 and Tra-1-60/81 are expressed, alkaline phosphatase activation and high telomerase activity can be confirmed. In addition, whether the cell maintains normal karyotype is confirmed by karyotyping and the formation of embryoid bodies that are known to contain all three-germ-layer stem cells or teratoma formation after injection in immunideficient mouse are analyzed as well. DNA fingerprinting for cell line identification is performed to confirm STR locus, as well as the analysis to find any infection by different Mycoplasma species, which is known to happen frequently during in-vitro culture.
3. Derivation of hESC from single blastomere
Typical derivation of hESC creates ethical problem because a surplus embryo needs to be damaged to isolate the inner cell mass. Considering the fact that each blastomere in the early phase of embryo has identical genetic properties and can develop normally when one blastomere is separated from the others, a study reported establishing ESCs from single blastomere. The hESC established from single blastomere expressed the markers of pluripotency, such as Oct4 and SSEA4. This study found that stem cell could be established from single blastomere, suggesting new possibility of establishing hESC without damaging embryos.
Properties and Differentiation of hESC
1. Properties of hESC
Various analyses are conducted to confirm whether established hESC has all the properties required for ESC. Property analyses are also performed at certain interval (e.g. every 10-15 weeks) to confirm whether the established hESC maintains the properties of hESC during lengthy in-vitro culture. Among the analyses to be performed for ESC are the expression of markers, such as Oct3/4, surface antigen SSEA4 and Tra-1-60/81, which are known to be expressed at a high rate in undifferentiated cells, the activation of alkaline phosphatase, and high telomerase activity. In addition, whether the cell maintains normal karyotype is confirmed by karyotyping and the formation of embryoid bodies that are known to contain all three-germ-layer stem cells or teratoma formation after injection in an immunideficient mouse are analyzed as well. DNA fingerprinting for cell line identification is performed to confirm STR locus, as well as the analysis to find any infection by different mycoplasma species, which is known to happen frequently during in-vitro culture.
2. Differentiation of hESC
A lot of studies are investigating methods to differentiate pluripotent hESC to certain types of cells, including myocyte, insulin-secreting cells and nerve cells, which can be directly used for the treatment of refractory diseases. Differentiation to myocyte has been investigated in various ways, and there have been reports of inducing differentiation of hESC to cardiac mesoderm by inhibiting Notch signal transduction.
The authors inhibited Notch signal by treating γ-secretase inhibitor(GSI) to undifferentiated hESCs and obtained pulsating myocyte after 2 weeks of culture. The pulsating myocytes were found to express myocyte-specific markers and the presence of a calcium channel was confirmed by coculture with mouse myocytes and electrophysiological analysis. Another study reported differentiation of hESC to myocyte using myocyte inducer BMP-2 and long-term cultured embryoid bodies. Differentiation to an insulin-secreting cell is also currently underway. There was a study reporting successful introduction of PDX-1, a transcript that controls differentiation to insulin-secretory cell, to hESC by means of proteins. Using protein delivery method, this study suggested a new gene delivery method available for hESC, which is difficult to manipulate the gene compared to other cells, and the study also presented a new method for inducing differentiation in hESC by achieving successful differentiation to insulin-secretory cell.
In addition, a study reported maintaining the expression of PDX-1 transcript using betacellulin and nicotinamide and inducing differentiation to pancreatic cells. As for differentiation to nerve cells, a study reported successful differentiation of early hESC to dopamine-producing nerve cells. Spherical neuronal precursor was used to produce dopamine-producing nerve cells with short duration of differentiation and very high yield, the cell was transplanted to a mouse model of Parkinson’s disease. The animal transplanted with dopamine-producing nerve cells derived from hESC showed improvement from the behavioral test. ASC will be discussed in the next chapter.
- To be continued -
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