**2.3** *In vitro* **disease modeling using pluripotent cells**

A major obstacle to disease research is the difficulty of acquiring diseased cells for study, usually because they are difficult to obtain from a living patient. For example, neurons are not easily obtained from a patient afflicted with Down syndrome, making detailed cell biology study of the neuronal basis for mental retardation impossible, and limiting our understanding of this disorder to more superficial behavioral neurological or postmortem pathological descriptions. However, a Down syndrome human ESC line as well as lines for other chromosomal trisomies have recently been derived (Biancotti et al., 2010), as has a human ESC line homozygous for Sickle Cell Disease (Pryzhkova et al., 2010). All were generated from embryos rejected by preimplantation genetic diagnosis (PGD) screens following *in vitro* fertilization (IVF). These lines allow cell culture study of diseased neurons,

ESCs are the prototypical pluripotent stem cell and thus the most thoroughly characterized. They can self-renew indefinitely and are effectively immortal in cell culture. Although they lack the self-organizing capabilities of the fertilized egg, they can form any tissue in the adult organism as demonstrated by two key studies: injecting ESCs into blastocysts gives rise to chimaeric animals with tissues contributed by the injected ESCs as well as the original ICM (Moustafa and Brinster, 1972); injection of ESCs into blastocysts that have been rendered tetraploid (four genome copies, and therefore genomically incapable of forming a complete organism) produces animals wholly derived from the injected ESCs (Eggan et al., 2002). The latter technique is possible because tetraploid blastocysts retain the structural organization of a normal blastocyst, and although the tetraploid ICM will inevitably die out or senesce (and be replaced by the injected cells), the trophoblast component retains its function despite tetraploidy since trophoblasts eventually fuse and become polyploid anyway upon embryonic implantation into the maternal uterus. These properties are shared with all pluripotent cells, EC cells injected into blastocysts can also give rise to chimaeric animals (Mintz and Illmensee, 1975). Because of this potential, ESCs very quickly became a

Modern reproduction techniques make it possible for a single ESC to give rise to a whole animal, greatly simplifying the process of genetically engineering animals. Previously, animals were bred extensively to isolate beneficial random mutations fertilized eggs were microinjected with DNA for random genomic integration (Gordon et al., 1980), or engineered animals were derived from nuclear-cloned somatic cells that had been engineered to the desired genotype; such a technique was used to generate cattle that lacked the prion protein and were thus made completely immune to bovine spongiform encephalopathy (BSE; mad cow disease, which transmits to humans as the variant Creutzfeldt-Jakob)(Richt et al., 2006). ESCs are easier to genetically engineer due to their infinite self-renewability, allowing a very small number of drug- or marker-selected cells to regenerate a whole culture or stable cell line. This technique has been used to generate a variety of mouse genetic models including sickle cell disease (Wu et al., 2006), thalassemia (Ciavatta et al., 1995), microcephaly (Pulvers et al., 2010), and T-cell lymphoma (Pechloff et

A major obstacle to disease research is the difficulty of acquiring diseased cells for study, usually because they are difficult to obtain from a living patient. For example, neurons are not easily obtained from a patient afflicted with Down syndrome, making detailed cell biology study of the neuronal basis for mental retardation impossible, and limiting our understanding of this disorder to more superficial behavioral neurological or postmortem pathological descriptions. However, a Down syndrome human ESC line as well as lines for other chromosomal trisomies have recently been derived (Biancotti et al., 2010), as has a human ESC line homozygous for Sickle Cell Disease (Pryzhkova et al., 2010). All were generated from embryos rejected by preimplantation genetic diagnosis (PGD) screens following *in vitro* fertilization (IVF). These lines allow cell culture study of diseased neurons,

**2. Applications of pluripotent stem cells** 

**2.2 ESCs in genetic engineering and animal disease modeling** 

al., 2010), as well as a p53-knockout rat for cancer research (Tong et al., 2010).

**2.3** *In vitro* **disease modeling using pluripotent cells** 

**2.1 Animals from pluripotent cells** 

focus of applied research.

or any other cell type, by differentiating diseased ESCs into any cell type of interest; however researchers are still limited by the small number of diseased human ESC lines available.

Cloned embryos can be derived from adult cells using somatic cell nuclear transfer (SCNT), a technique made famous by the cloning of the sheep Megan, Morag and Dolly in the 1990s (Wilmut et al., 1997). It has been proposed that new diseased human ESC lines can be derived using this technique to make cloned embryos from diseased patients, and then harvesting them to create novel diseased ESC lines for disease study. At the time of this writing, SCNT for this application (Therapeutic Cloning) is currently legal in the United States and the European Union, but its legal status in these states as well as elsewhere across the world has been subject to numerous prior and continuing legal challenges. Although several large organizations continue to research this technology, it has been supplanted in recent years by alternate techniques for deriving patient-specific pluripotent stem cells.

### **2.4 Therapeutic potential of pluripotent stem cells**

Pluripotent stem cells have been studied as, and shown great potential to be, a source of cell replacement therapies in a myriad of disease and injury models. Several human ESC lines have been differentiated into high-purity cardiomyocyte cultures that improve cardiac performance when transplanted into infracted rat hearts (Caspi et al., 2007). ESCs have also been differentiated into neural precursors and neurons including dopaminergic neurons which reverse the disease progression of Parkinsonian rats (Yang et al., 2008). In a model of spinal cord injury, ESC-derived oligodendrocytes transplanted into crushed rat spinal cords successfully restored locomotive function to the animals. Pancreatic beta cells, the insulinsecreting cells whose absence causes type I diabetes mellitus, have also been derived from ESCs and cure the diabetic phenotype of the mouse streptozotocin-induced model of diabetes upon transplantation (Kim et al., 2003). These are but a choice selection of the vast amount of scientific literature detailing the regenerative potential of ESCs.

At the time of this writing, two clinical trials are underway for ESC-based regenerative therapies in humans: an evaluation of human ESC-derived oligodendrocyte precursors to rescue neurologically complete spinal cord injury conducted by Geron Corporation, and ESC-derived retinal-pigmented epithelium for treatment of macular degeneration and Stargardt's macular dystrophy, which are major causes of blindness, conducted by Advanced Cell Technology Incorporated. A third proposed clinical trial is currently in the approval process between the Food and Drug Administration and applicant California Stem Cell Incorporated for ESC-derived motor neurons as a cure for type I spinal muscular atrophy, the leading genetic cause of infant mortality. These trials represent the first step in the direct evaluation of the therapeutic potential of pluripotent stem cells in human patients.

### **2.5 Pitfalls and obstacles to the use of ESCs**

Transplants of ESC-derived tissues and biological devices are just as subject to immune rejection as conventional organ transplants, even more so due to the limited selection of human ESC lines. Although the engineering of non-immunogenic ESCs has been the subject of many academic initiatives and company startups, ongoing clinical and preclinical research for ESC-therapies is focused, for the mean time, on immune-privileged regions of the body: specifically the brain, eye and spinal cord. A second scientific concern is the purity of ESC-derived transplants because of the hazard posed by contaminating undifferentiated ESCs that, if transplanted, can proliferate and form teratomas. The elimination of these leftover ESCs has been approached by several strategies: purification of differentiated cells by labeling and cell sorting (Pruszak et al., 2007), the engineering of special "suicide gene" containing ESCs (Schuldiner et al., 2003), and the treatment of cells to be transplanted with chemotherapeutics (Bieberich et al., 2004). The concomitant destruction of stem cells by anticancer therapies reflects the generalized similarity between stem cells and cancer cells [reviewed in (Reya et al., 2001)].

As many as seven human embryos are sacrificed for each new human ESC line derived (Thomson et al., 1998); while the ethics of this are philosophically subjective they have nonetheless given rise to numerous high-profile legal challenges to continued ESC research and funding. In addition, the patent on derivation of human ESC lines is held by the Wisconsin Alumni Research Foundation. Until its expiration in 2016, commercial users wishing to use Wisconsin ESC ("WiCell") technologies might also be required to pay a royalty.
