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Hoseok Song

Disease Research and Drug Development Models Based on Genetically Altered Human Embryonic Stem Cells

Abstract: Mouse models have become an indispensable tool to study the pathogenesis of human diseases. However, due to the apparent cellular and physiological differences between mouse and human, mouse models often fail to faithfully recapitulate certain defects observed in human patients. In addition, these differences contribute to the dilemma in drug development that many therapeutic strategies work well in mouse models but do poorly in human patients. Therefore, it has become increasingly important to develop additional physiologically relevant human disease models for mechanistic studies and drug development. With the unlimited self-renewal capability and the pluripotency to differentiate into all cell types in the body, human embryonic stem cells (hESCs) with causative genetic mutations as well as their differentiated derivatives represent the much needed human disease models for studies on disease mechanisms and for drug development. Here we summarize recent progresses in developing hESCs into human disease models.

Since the successful derivation of human embryonic stem cells (hESCs) from the inner cell mass of the early stage (blastocyst) of human embryos (Thomson et al., 1998), significant progresses have been achieved in generating hESCs with causative genetic mutations in various human diseases. In this context, over three dozen lines of hESCs with genetic mutations have been derived from blastocysts embedded with genetic disorders, and more recently, through genetic modification of existing hESC lines. Continuous studies of these genetically modified hESCs and their derivatives lend support to realizing their potential in modeling human diseases.

hESCs Derived from Embryos with Genetic Disorders

During the process of preimplantation genetic diagnosis that is used to prevent transmission of genetic disorders of high-risk parents to offspring, human blastocysts diagnosed of harboring genetic abnormalities have been donated for research, and from which dozens of hESC lines have been derived (Sermon et al., 2009). These genetic abnormalities include monogenic mutations, inherited chromosomal abnormalities, and aneuploidy such as trisomy 21. The established hESC lines with causative genetic abnormalities include the potential disease models for neurofibromatosis type 1, cystic fibrosis, Becker muscular dystrophy, Huntington’s disease, Emery-Dreifuss muscular dystrophy, spinal muscular atrophy, ocular albinism, facio-scapulohumeral dystrophy, spinocerebellar ataxia type 7, and osteogenesis imperfecta type 1 (Sermon et al., 2009). While the established hESCs offer a great opportunity to study human diseases caused by complex genetic alterations, one drawback for these mutant hESCs to be developed into human disease models is the lack of isogenic wild-type control for mechanistic studies.

Somatic Cell Nuclear Transfer (SCNT)

SCNT is a highly challenging technology for creating hESCs that have the same genetic background as the donor. Briefly, the nucleus of a somatic cell derived from a donor is retrieved and injected into an enucleated egg, which is then stimulated to divide multiple times in culture to form a blastocyst. The blastocyst can be used to derive hESCs with the same genetic material as the donor. Therefore, SCNT, also called therapeutic cloning, enables the production of hESCs that will be immunologically tolerated by the donor. SCNT has been successfully performed in various species including primate and human, raising the possibility to develop patient-specific hESCs for personalized therapy (Andrew et al., 2008; Byrne et al., 2007). However, due to the extreme technical difficulty and ethical concerns for the requirement of human eggs associated with SCNT, there has been no report of successful generation of patient-specific hESCs with this technology.

Genetic Manipulation of hESCs

If the tremendous progresses achieved with mouse genetics can be a guide, genetic manipulation of hESCs will greatly facilitate our effort to study human gene functions and develop hESC-based disease models. However, genetic manipulation of hESCs through homologous recombination has been challenging due to their inefficient clonal expansion and their heterogeneous genomic DNA that reduces the frequency of homologous recombination. The plasmid-based conventional targeting vector, which works well in the isogenic inbred mouse ESCs, fails to achieve efficient homologous recombination in hESCs. Accordingly, there have been few reports of successful generation of homozygous mutant hESCs using the conventional gene targeting approach. Two recent advances, however, have helped to resolve this technical bottleneck.

Zinc-finger Nuclease (ZFN) Induced Homologous Recombination in hESCs

ZNF has been successfully used in the genome editing of plant, zebra, and rat (Hitoshi and Makoto, 2009). ZFN is a fusion protein composed of the FokI nuclease domain and the engineered C2H2 zinc-finger domain designed to bind specifically to a sequence within the intended target locus. The dimmerization of the nuclease domains activates the nuclease, leading to the introduction of a DNA double-stranded break into the targeted locus that can promote homologous recombination. Recent studies have shown that ZNF-mediated genome editing significantly increases the efficiency of homologous recombination at both active and silenced genetic loci in hESCs (Hockemeyer et al., 2009; Zou et al., 2009). While promising, several technical issues need to be addressed before this approach can be widely adapted for genetic manipulation of hESCs. First, the success of this approach depends on the design of zinc-finger motifs that can specifically bind to the endogenous genomic sequences around the intended target loci. This is a time-consuming task since the engineered zinc-finger motif needs to be experimentally optimized for the binding specificity in cells, which remains to be validated with vigorous genomic analysis such as ChIP-Seq technology. The potential off-target effects of the engineered ZFNs could induce unwanted genetic changes in hESCs. In addition, the efficiency of ZFN-mediated genome editing for generating homozygous mutant hESCs remains to be established, especially at the highly polymorphic genetic loci.

Bacterial Artificial Chromosome (BAC) Based Gene Targeting in hESCs

Previous studies in mouse ESCs have shown that larger homologous arms can increase the frequency of homologous recombination (Yang and Seed, 2003). BAC vector is capable of stably propagating large segments of human genomic DNA (150 kb-200kb) and the efficient editing of BAC genomic DNA through recombineering technology in bacteria (Copeland et al., 2001). Consistent with the notion that the larger homologous arms can increase the frequency of homologous recombination, recent studies demonstrate that BAC-based targeting vectors can achieve high frequency of homologous recombination in hESCs independent of genetic background and gene loci (Song et al., 2010). In addition to being able to significantly increase the frequency of homologous recombination in hESCs, the BAC based strategy has other advantages over the conventional gene targeting strategy. For example, while the conventional gene targeting vector needs to be generated with genomic DNA isogenic to the hESCs, the BAC targeting vector obtained from the commercial sources is suitable for hESCs of various genetic backgrounds. In addition, unlike the conventional gene targeting vectors that often involve time-consuming and technically difficult cloning steps, the BAC recombineering technique allows rapid genetic modification of the genomic DNA in bacteria. One potential drawback for the BAC-based targeting vector is that it is difficult to confirm the homologous recombination event due to the large size of the homologous arms. This problem has been partially resolved by shortening one of the homologous arms to allow the confirmation of homologous recombination event by Southern blotting as is usually done for the conventional gene targeting (Song et al., 2010).

Employing this BAC-based strategy, sequential disruption of both alleles of the ATM (Ataxia telangiectasia mutated) or p53 gene in hESCs has been achieved with high efficiency, leading to the generation of ATM-/- and p53-/- hESCs as potential models for the two major human genetic instability syndromes (Song et al., 2010). ATM is a protein kinase mutated in the human genetic instability syndrome Ataxia-telangiectasia (A-T) characterized by multi-systemic and cellular defects, including neuronal degeneration, abolished germ cell development, immunodeficiency, increased cancer risk, accelerated telomere shortening, and radiosensitivity (Shiloh, 1995). While ATM-deficient mice recapitulate many A-T systemic and cellular defects, they fail to develop A-T related neuronal defects and accelerated telomere shortening (Barlow et al., 1996; Elson et al., 1996; Xu et al., 1996; Xu and Baltimore, 1996). The findings that ATM-/- hESCs and their differentiated derivatives recapitulate the A-T defects including the accelerated telomere shortening validate the usefulness of ATM-/- hESCs as a disease model for A-T (Song et al., 2010).

p53 is the most commonly mutated tumor suppressor in human cancers and is also mutated in the human genetic instability syndrome Li-Frameni syndrome (Stiewe, 2007). As the guardian of the genome, p53 is required to maintain genomic stability in both somatic cells and stem cells (Zhao and Xu, 2010). While p53-/- mice, which would die mostly of thymic lymphomas or sarcomas (Donehower et al., 1992; Jacks et al., 1994), have been used extensively in cancer research, they do not fully recapitulate the tumorigenesis in human patients who primarily develop tumors of epithelial origin when p53 is inactivated. p53-/- hESCs and their derivatives recapitulate the defects observed in p53-deficient human cells such as the impaired G2/M checkpoint (Sancar et al., 2004; Song et al., 2010; Song et al., 2007). In addition, these findings indicate an important role of p53 in maintaining genomic stability in human ESCs (Lin et al., 2005; Song et al., 2010). In summary, these findings demonstrate the potential of BAC-based technology for the development of genetically modified hESCs into relevant models for human diseases.

hESCs Versus Induced Pluripotent Stem Cells (iPSCs) as Disease Models

The recent groundbreaking discovery that somatic cells can be reprogrammed into the induced pluripotent stem cells (iPSCs) with defined reprogramming factors has provided a potentially useful methodology for the development of human disease models (Takahashi and Yamanaka, 2006). For example, iPSCs have been established from fibroblasts derived from patients with amyotrophic lateral sclerosis (ALS), spinal muscular atrophy, and Parkinson’s disease (Dimos et al., 2008; Ebert et al., 2009; Soldner et al., 2009). While iPSC technology is ideal to model human diseases with complex genetic traits, several bottlenecks need to be addressed to facilitate its application. For example, recent findings indicate that the reprogramming factors have oncogenic potential and the reprogramming efficiency is greatly increased by the inactivation of critical tumor suppressors such as p53 and Arf (Deng and Xu, 2009). Therefore, while it is known that hESCs are genetically stable when cultured properly, the genomic stability and tumorigenic potential of iPSCs remain to be vigorously examined (Deng and Xu, 2009). For the genetically modified hESCs, the parental hESCs can be used as the isogenic positive controls. However, iPSCs lack such isogenic controls. In addition, somatic cells derived from human patients could have already harbored undefined genetic alterations that might impact on the disease phenotypes. This is particularly true in modeling human genetic instability syndromes because the adult somatic cells derived from such patients will have accumulated extensive genetic alterations. In summary, further development of both iPSC technology and genetic manipulation of hESCs will help to realize the great potential of developing pluripotent stem cells into relevant human disease models for mechanistic studies and drug development.


This work was supported by a grant from California Institute of Regenerative Medicine to Y.X. (RC1-148).

(Corresponding author: Yang Xu, Ph.D., Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.)


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[Discovery Medicine, 9(49):500-503, June 2010.]

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