IX. EMBRYONIC STEM CELL-BASED THERAPIES
Currently, no ES cell-based therapies are on going in humans. Only allogeneic or matched donor-derived adult stem cells have been employed in human cell-grafting therapies, the best examples of which are bone marrow transplantations for the treatment of leukemia after myeloablative therapies. The availability of human ES cells, however, represents an extraordinary opportunity for cell transplantation that may be applicable to a wide range of human ailments. Three properties make ES cells relative to adult stem cells very attractive for replacement therapies (158). 1) Human ES cells can be grown indefinitely in culture. 2) ES cells can be genetically manipulated, and loss of function genes (e.g., CTFR) can theoretically be repaired by the introduction of transgenes into ES cells either by random transgenesis or through gene targeting. Importantly, homologous recombination could be used to correct specific genetic mutations that would not lead to random mutations in tumor-promoting genes. 3) Numerous differentiation protocols have already been established that permit the generation of almost any cell type, either through the use of established culture conditions or when coupled with genetic manipulations. In theory, hES cells could be applied to a wide range of human ailments, but the proof of principle has largely come from the use of mouse ES cells.
A. Animal Models for Cell Therapy
Mouse and human ES-derived progeny have been analyzed in various animal models of human diseases (see Table 6), and some examples are discussed with respect to cardiovascular and neurorepair and for the treatment of diabetes.
1. ES cells for cardiac repair
As described earlier, cardiac-restricted promoters have been used to select cardiomyocytes from differentiating ES cells (115, 192, 229, 237). Loren Field's group (192) published the first therapeutic demonstration of mouse ES cell derivatives. In this study, purified (99.6%) cardiomyocytes were injected into the ventricular myocardium of adult dystrophic (mdx) mice and were found to be present in the grafts for at least 7 wk after implantation, without tumor formation (192). Min et al. (231) subsequently reported improved left ventricular function in postinfarcted rats after transplantation of "beating cells" derived from ES cells. The engrafted cardiomyocytes expressed sarcomeric -actin, -myosin heavy chain, and troponin I and were rod-shaped with typical striations, suggesting differentiation into mature cardiomyocytes. ES cell-derived cardiomyocytes thus expressed myofilament proteins and were able to form "normal coupling units" with endogenous cardiomyocytes. It was unclear from these studies if the transplanted cardiomyocytes coupled normally with endogenous cells, or whether any beneficial effects of cell integration were long-term.
These results validate the potential use of ES-derived cardiomyocytes for cardiac therapy; however, experiments to generate 100% pure and stable cardiac grafts have not proven successful (see sect. IVB). Recent studies from our laboratories have also indicated that culture conditions can significantly affect the quality of the cardiac tissues generated from ES cells. We have found that changes in plating conditions can lead to ES-derived cardiac cells that are immature, arrhythmic, show signs of cell stress, and are predisposed to cell death via a p53-mediated cascade (unpublished data). These findings underscore the role of regulated cell-cell and cell-ECM interactions and the need to establish histotypic culture conditions for the generation of suitable cardiac grafts from in vitro-differentiated ES cells. The generation of cardiomyocytes growing in three-dimensional aggregates may offer an alternative.
2. ES cells used for the in vitro formation of vascular structures
Recently, human ES cells have been employed to isolate endothelial cells. During differentiation, transcripts characteristic for endothelial cells were detected, including GATA-2, PECAM1, Flk1, and VE-cadherin. PECAM1 antibodies were used to isolate endothelial cells from the 13-day EBs after enzymatic dissociation. The isolated PECAM1+ cells were seeded onto highly porous PLLA/PLGA biodegradable polymer scaffolds (280), and the sponges were implanted subcutaneously into SCID mice (208). After development in SCID mice as well as after in vitro differentiation in Matrigel, microvessels developed from these hES cells. Similarly, rhesus monkey ES cells differentiated into endothelial cells and when introduced into a Matrigel plug and implanted subcutaneously into mice formed intact vessels and recruited new endothelial cell growth in vivo (184).
3. ES cells for neurorepair
The successful generation of apparently "normal" neural cell types from in vitro differentiated ES cells has naturally led to intense interest in their potential use to repair or limit the damage associated with infarct or neurodegenerative diseases. Brustle et al. (55) first demonstrated that ES cell-derived neural cells could survive, respond to environmental signals, and exhibit aspects of region-specific differentiation when introduced into developing mouse brain. They showed in a later study, and following in vitro generation of precursors for oligodendrocytes and astrocytes, that these transplanted cells interact with host neurons and myelinate the axons in brain and spinal cords in a rat model of human dysmyelinating Pelizaeus-Merzbacher disease (54). The resulting remyelination of axons led to a recovery of the pathological phenotype in the animals.
In transplantation experiments in which dissociated neural progenitors have been introduced to appropriate sites, mouse ES cells have also been found to differentiate into dopaminergic neurons, and to promote partial recovery in a rat model of Parkinson's disease (191). The efficient generation of midbrain dopaminergic neurons from human ES cells opens the possibility to test their therapeutic effects in animal models (see Ref. 266). mES-derived GABAergic neurons were found to survive after transplantation into a rat model of Huntington's disease (95), while oligodendrocytes myelinated host axons after transplantation that could partially restore function in rodents with spinal cord injury (211, 225). These experiments provide a clear indication that mES cells can serve as a valuable source of specific neuronal and glial cells for transplantation (see Fig. 9 and Table 6). It remains to be seen whether transplanted hES-derived neural cells can persist and function over long periods. This issue has been highlighted by a clinical study, in which the transplantation of neural cells derived from fetal brain to Parkinson's patients showed no significant benefit; moreover, 2 years after surgery, some treated patients developed persistent dyskinesia (128). Nonetheless, this study provided important information about the ability of dopaminergic neurons to survive in humans.
4. ES cells for the treatment of diabetes
As described in section IVC, mES cells differentiate into functional isletlike cells that are able to rescue experimentally induced diabetes in mouse models (38, 162, 207); however, similar strategies must be established with hES cells. Efficient selection methods with pancreatic lineage-specific promoters will be necessary to overcome current limitations, such as tumor formation of grafts and low (therapeutically irrelevant) insulin levels. In parallel with a common "lineage-selection" strategy, selection of differentiated pancreatic cells expressing glycolipids or other cell surface markers of pancreatic -cells [i.e., A2B5 (105), 3G5 (276), IC2 (53)] might be feasible; however, it has to be shown that the composition of cell surface markers of ES-derived pancreatic cells is similar to those expressed in islets. The engineering of pancreatic islets in vitro clearly requires a further maturation of ES-derived cell clusters. Insulin-producing -cells depend on specific signals from nonpancreatic cells: cell-to-cell interactions and characteristic "biosociology" are necessary for tissue-specific function of -cells (272). This could be achieved by histotypic culture systems (38) that are additionally supported by vascularization. Finally, the maturation process could be enhanced by delivery of specific pancreatic transcription factors or developmental control genes in a "gain-of-function" approach.
Although the data presented so far illustrate the capacity of both mouse and human ES cells to differentiate into therapeutically useful cell types, it is still unclear whether hES-derived progeny would function normally in the body especially with respect to long-term functionality.
B. Therapeutic Cloning
Cloning is defined as the production of a set of individuals with the same genotype. This occurs naturally by asexual reproduction in hydra, sea anemone, planarians, and annelids (407), but cloning is also possible with mammalian somatic cells, as illustrated by the birth of Dolly the sheep in 1997 (392). Two forms of cloning are generally described from adult somatic cells: reproductive and therapeutic. Therapeutic cloning utilizes nuclear transfer techniques (238) to produce pluripotent ES cells with the genome of the nucleus of origin. Specifically the nucleus of an adult donor cell is introduced into an enucleated donor oocyte to generate a cloned embryo. The somatic cell nucleus, at a low frequency and depending on the donor cell type, may regain its pluripotentiality to initiate the earliest stages of embryonic development. If these cells are transferred to the uterus of a female recipient, then the developing embryo would have the potential to grow into an infant, in a process known as "reproductive cloning," i.e., Dolly. If the developing mass of cells is however left in culture, ES cells can be isolated from the inner cell mass of a developing blastocyst. The ES cells derived in this manner are genetically identical to the donor cells, except for the mitochondrial genome, and can be induced to differentiate into replacement cells for transplantation. This process is commonly referred to as "therapeutic cloning." Importantly, the differentiated cells generated in this manner are autologous, thus eliminating the problem of immuno-incompatibility and the requirement for immunosuppression (see Fig. 10). Moreover, ES cells provide a renewable source for replacement cells.
As pointed out earlier, ES cells are amenable to genetic manipulation. When combined with therapeutic cloning, ES cell derivatives offer the potential for both gene- and cell-based therapies. A demonstration of this potential was published in 2002 (298). Nuclear transfer (nt) ES cells were produced from tail tip cells of immunodeficient mice, homozygous for a knockout mutation in the "recombination activating gene" 2 (RAG2 mice). These mice lack mature B and T cells. By homologous recombination, this genetic mutation was "cured," and the targeted ntES cells were differentiated in vitro into EBs and into hematopoietic precursor cells by expressing HoxB4. When reintroduced into irradiated Rag2-deficient animals, these precursor cells partially repopulated the deficient immune system, and functional B and T cells were detected in these mice. Unexpectedly, the initial attempts at engraftment with these cells failed, because of an increase in natural killer cells. Immunosuppression was therefore required to rescue this phenotype. This experiment, however, serves as a proof of principle therapy where nuclear transfer was combined with gene therapy to treat a genetic disorder. More recently, Barberi et al. (25) showed that transplantation of ntES cell-derived dopaminergic neurons could correct the phenotype of a mouse model of Parkinson's disease.
Based on these findings, therapeutic cloning in combination with established hES cell protocols could offer a means to obtain autologous cells for the treatment of a variety of diseases. Proof of principle of this strategy has been reported. The application of the somatic cell nuclear transfer (SCNT) technology using human oocytes and cumulus (nucleus donor) cells resulted in the derivation of a pluripotent ES cell line from a cloned human blastocyst (165). After continuous proliferation for more than 70 passages, SCNT-hES cells maintained a normal karyotype, were genetically identical to the somatic nuclear donor cells, and showed differentiation capabilities in vivo (teratoma formation) and in vitro (165).
However, Mombaerts (234) has suggested that it will be prohibitively expensive to pursue this approach until the efficiency of nuclear transfer is improved or an alternative source of human oocytes can be found. The recent report of ES cell-derived oocytes suggests that the latter may be feasible (164), and if so, some of the ethical problems associated with therapeutic cloning may be overcome. Another possibility would be the reprogramming of adult somatic nuclei by fusion with hES cells (96). Before these principles can be applied clinically, it will be necessary to minimize epigenetic reprogramming of ES cells in culture, determine the genetic consequences of using aged nuclei, evaluate the effects of oocyte-derived mitochondrial proteins in somatic cells obtained by nuclear transfer, and reconcile the potential immunological rejection of these cells (158, 407). It is therefore important to continue research in this field to determine the potential of these therapies for humans.