**2.1 ES cells**

The culture of ES cells from mouse blastocysts was first reported by Evans et al. in 1981 and herein opened a new era of research in gene targeting to model human

**99**

*Innovations in Human Stem Cell Research: A Holy Grail for Regenerative Medicine*

diseases [3]. Based on this technology, Martin Evans, Mario Capecchi, and Oliver Smithies shared the 2007 Nobel Prize for their discoveries of principles for introducing specific gene modifications in mice by the use of ES cells. In 1998, Thomson and collaborators reported the derivation of ES cell lines from human blastocysts produced by in vitro fertilization (IVF) [4]. As these human ES cells are immortal and can give rise to all cell types in the body, they are invaluable tools to dissect the molecular and genetic events in human development and also allow drug testing in a wide range of diseases (**Figure 1**). Moreover, human ES cells provide an unlimited source to derive cells and tissues that could be used as cell-based therapies to treat degenerative diseases such as heart disease and type 1 diabetes (**Figure 1**). Indeed, detailed differentiation protocols have been developed to derive specific mature cells from human ES cells, for example, dopaminergic neurons, cardiomyocytes, β cells, and keratinocytes as therapies for Parkinson's disease, myocardial infarction, type I diabetes, and skin disease, respectively. However, as the derivation of ES cells involves destruction of human embryos, even though the embryos were produced by IVF and would otherwise be discarded, it nevertheless has raised ethical concerns and elicited controversial debates on the use of human ES cells [5]. Another hurdle for the therapeutic use of ES-derived cells is that they can only be used as allogeneic cells; thus they are subject to immune-based rejection in

The 2012 Nobel Prize in Physiology or Medicine was awarded to two scientists, Sir John B. Gurdon and Shinya Yamanaka, PhD, whose stem cell research was separated by over four decades but both demonstrated that the developmental clock of a specialized cell can be turned back to an immature (pluripotent) state. In John B. Gurdon's classic experiment in 1962, he replaced the nucleus in an egg cell of a frog with the nucleus from a mature intestinal cell [7]. The modified egg cell subsequently developed into a normal tadpole. Using the same principle of Gurdon's work which is known as somatic cell nuclear transfer (SCNT), the first mammalian clone "Dolly the Sheep" was born in 1996, following transfer of the nucleus from a mammary gland into an enucleated sheep egg [8]. Therefore, factors presented in mature, metaphase II-arrested oocyte cytoplasm are capable of reprogramming somatic cell nuclei to an undifferentiated state. This unique ability of oocytes has led to an important application of utilizing SCNT in therapeutic cloning to generate ES cells (ESCs) (NT-ESCs) for

Forty-four years after Gurdon's discovery, Shinya Yamanaka took a different approach and transduced the differentiated mammalian cells, i.e., fibroblasts with a cocktail of reprogramming factors (Oct3/4, Sox2, Klf4, and c-Myc) using retroviruses (**Figure 1C**) [9]. About 30 days after transduction, induced pluripotent stem cells (iPSCs) with properties similar to ES cells were generated at an efficiency of ~0.02% [9]. After the initial discovery of Yamanaka's reprogramming techniques, several other reprogramming methods, such as transduction with nonintegrating viruses (adenovirus and Sendai virus) or transfection with protein, mRNA, Piggy Bac element, minicircle vectors, and episomal plasmids, have been developed to produce iPSCs without integration of exogenous genes to the genome of recipient cells (reviewed in [10]). iPSCs can also be generated from more easily accessible cell

Therefore, the PSCs produced by SCNT (NT-ESCs) and forced expression of reprogramming transcription factors (iPSCs) are both alternatives to ESCs in regenerative medicine. Epigenetic and transcriptomic comparisons between

*DOI: http://dx.doi.org/10.5772/intechopen.88790*

their recipients [6].

**2.2 PSCs derived from reprogramming**

regenerative medicine (**Figure 1B**).

sources such as blood [11].

*Innovations in Human Stem Cell Research: A Holy Grail for Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.88790*

diseases [3]. Based on this technology, Martin Evans, Mario Capecchi, and Oliver Smithies shared the 2007 Nobel Prize for their discoveries of principles for introducing specific gene modifications in mice by the use of ES cells. In 1998, Thomson and collaborators reported the derivation of ES cell lines from human blastocysts produced by in vitro fertilization (IVF) [4]. As these human ES cells are immortal and can give rise to all cell types in the body, they are invaluable tools to dissect the molecular and genetic events in human development and also allow drug testing in a wide range of diseases (**Figure 1**). Moreover, human ES cells provide an unlimited source to derive cells and tissues that could be used as cell-based therapies to treat degenerative diseases such as heart disease and type 1 diabetes (**Figure 1**). Indeed, detailed differentiation protocols have been developed to derive specific mature cells from human ES cells, for example, dopaminergic neurons, cardiomyocytes, β cells, and keratinocytes as therapies for Parkinson's disease, myocardial infarction, type I diabetes, and skin disease, respectively. However, as the derivation of ES cells involves destruction of human embryos, even though the embryos were produced by IVF and would otherwise be discarded, it nevertheless has raised ethical concerns and elicited controversial debates on the use of human ES cells [5]. Another hurdle for the therapeutic use of ES-derived cells is that they can only be used as allogeneic cells; thus they are subject to immune-based rejection in their recipients [6].

### **2.2 PSCs derived from reprogramming**

The 2012 Nobel Prize in Physiology or Medicine was awarded to two scientists, Sir John B. Gurdon and Shinya Yamanaka, PhD, whose stem cell research was separated by over four decades but both demonstrated that the developmental clock of a specialized cell can be turned back to an immature (pluripotent) state. In John B. Gurdon's classic experiment in 1962, he replaced the nucleus in an egg cell of a frog with the nucleus from a mature intestinal cell [7]. The modified egg cell subsequently developed into a normal tadpole. Using the same principle of Gurdon's work which is known as somatic cell nuclear transfer (SCNT), the first mammalian clone "Dolly the Sheep" was born in 1996, following transfer of the nucleus from a mammary gland into an enucleated sheep egg [8]. Therefore, factors presented in mature, metaphase II-arrested oocyte cytoplasm are capable of reprogramming somatic cell nuclei to an undifferentiated state. This unique ability of oocytes has led to an important application of utilizing SCNT in therapeutic cloning to generate ES cells (ESCs) (NT-ESCs) for regenerative medicine (**Figure 1B**).

Forty-four years after Gurdon's discovery, Shinya Yamanaka took a different approach and transduced the differentiated mammalian cells, i.e., fibroblasts with a cocktail of reprogramming factors (Oct3/4, Sox2, Klf4, and c-Myc) using retroviruses (**Figure 1C**) [9]. About 30 days after transduction, induced pluripotent stem cells (iPSCs) with properties similar to ES cells were generated at an efficiency of ~0.02% [9]. After the initial discovery of Yamanaka's reprogramming techniques, several other reprogramming methods, such as transduction with nonintegrating viruses (adenovirus and Sendai virus) or transfection with protein, mRNA, Piggy Bac element, minicircle vectors, and episomal plasmids, have been developed to produce iPSCs without integration of exogenous genes to the genome of recipient cells (reviewed in [10]). iPSCs can also be generated from more easily accessible cell sources such as blood [11].

Therefore, the PSCs produced by SCNT (NT-ESCs) and forced expression of reprogramming transcription factors (iPSCs) are both alternatives to ESCs in regenerative medicine. Epigenetic and transcriptomic comparisons between

*Innovations in Cell Research and Therapy*

cells (PSCs) in the ICM are the source for embryonic stem (ES) cells [3]. Apart from PSCs, there are tissue-specific stem cells residing in several organs such as the skin, gut, blood, and brain, also referred to as "adult" or "somatic" stem cells with self-renewal and multipotent differentiation capacity that are responsible for most

*Derivation of pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) from fertilization of oocytes and sperm (A), nuclear transfer ESCs (NT-ESCs) from somatic cell nuclear transfer (SCNT) of somatic cell nuclei into denucleated oocytes (B), and induced pluripotent stem cells from forced expression of Yamanaka transcription factors in somatic cells (C). All the three types of PSCs can be propagated extensively in vitro and undergo directed differentiation into any cell type of the body, which can be utilized in disease modeling and drug screening and developed as cell-based therapies. As generation of ESCs involve disruption of the embryos, they can only be used as an allogenic source, while NT-ESCs and iPSCs can be developed in both allogenic and* 

The culture of ES cells from mouse blastocysts was first reported by Evans et al. in 1981 and herein opened a new era of research in gene targeting to model human

regenerative activities throughout the life of the organism.

**2. Pluripotent stem cells (PSCs)**

**98**

**2.1 ES cells**

**Figure 1.**

*autologous settings.*

isogenic iPSCs and NT-ESCs have demonstrated that NT-ESCs more closely resemble bona fide ESCs derived from fertilized embryos [12, 13]. Moreover, a most important difference between iPSCs and NT-ESCs is the source of mitochondrial DNA (mtDNA). The mtDNA in NT-ESCs is of an oocyte germline origin, while in iPSCs is of a parental somatic origin. Due to the random nature of somatic mtDNA mutations, the frequency of mtDNA defects in iPSCs has been demonstrated to increase with the age of somatic cells [14]. Thus, NT-ESCs, carrying mutation-free mtDNA and closely resembling ESCs, represent an invaluable stem cell source for regenerative medicine. However, as the derivation of NT-ESCs requires donor oocytes, which are more technically challenging than iPSCs and are also subject to ethical and/or legal restrictions, the majority of current PSC research has been carried out using iPSCs.
