mbryonic stem (ES) cells are regarded with great promise for future treatment of congenital disease. These cells are derived from a very early embryo called a “blastocyst.” The well-known capacity of these cells to differentiate into a broad spectrum of cell types makes them highly attractive for their use in the emerging era of regenerative medicine, as they can potentially regenerate every tissue of the body. In addition to tissue regeneration, we have identified a novel function of ES cells: they are capable of secreting healing factors. This set of defined factors corrects a mutant environment that otherwise is predisposed to develop a congenital disease. (Science. 2004 306(5694): 247-5; Nat Clin Pract Cardiovasc Med. 2006 Mar:S14-7).
The approach that we use is to inject normal mouse ES cells into early mouse embryos that harbor a mutation. The embryos that do not receive ES cell treatment develop a disease that recapitulates a human disease. For example, our first strain of mice that we studied is called “Id knockout” mice. These mice recapitulate the “thin myocardial syndrome,” as they die in utero due to severe cardiac abnormalities, including a marked thinning of the myocardium that prevents the hearts from pumping blood throughout the fetuses. As the ES cells are derived from blastocysts, injection of normal ES cells into mutant blastocysts results in a mutual recognition without rejection. The normal ES cells are incorporated in the mutant embryo in a “salt and pepper” pattern. As a result of the injection of ES cells, the developing animal will be composed of “wild type” or normal cells (derived from the ES cells) and mutant cells (derived from the blastocysts). These mice are termed “chimeras” or “mosaic” mice. In this case, the chimeras are symbolized WT/Id (wild type component derived from the ES cells and Id knockout component derived from the blastocysts).
Rescue of congenital heart disease
For our rescue studies of congenital heart disease (Id knockout embryos), we selected chimeras with a low percentage of ES cells, for example 10-20 %. The Id knockout embryos die in utero, but the introduction of the ES cells allowed the resultant chimeric embryos to survive to term, and in some cases beyond birth, even until adulthood. When we examined the hearts from the rescued animals, they were indistinguishable from those of normal animals, even though the rescued hearts contained 80% of mutant cells on average (Fig. 1). Thus, 20 % of tissue derived from wild type ES cells sufficed to prevent disease from occurring. To understand the molecular mechanisms involved in the rescue, we examined the profiles of gene expression in the rescued hearts, and compared them with normal hearts. Surprisingly, the mutant component of the heart contained a normal profile. Thus, the normal ES cells normalized the function of the neighbor mutant cells. We went on to identify two factors, one long-range called “IGF1” and the other short-range called “Wnt5a”. These two factors are emitted by the ES cells and induce corrections in the mutant component of the heart. The long-range factor can travel long distances through the circulation. In fact, we injected IGF1 in the pregnant mice harboring the Id knockout embryos. This small molecule crossed the placenta to partially correct disease and to extend survival of the Id knockout fetuses. As a result of maternal injection of recombinant IGF1, the Id knockout embryos survived to term, with partially corrected hearts. The short-range factor travels only short-distances, and could only exert the function when the ES cells are in direct contact (cell-to-cell communication) with mutant tissue, for example in the chimeric embryos. Thus, the ES cells emit healing factors. The message of this rescue experiment is that the ES cells can eventually be replaced by the factors that are responsible for their therapeutic effects.
Rescue of muscular dystrophy
Another disease that we are interested in preventing is muscular dystrophy. There is currently no cure for this devastating disease. The challenge with this model is that the mutation affects the presence of a structural protein called dystrophin, and therefore 20% of chimerism might not suffice to restore structural stability to the whole muscle (the main structure affected by the absence of dystrophin), and thus to correct disease. Because the muscle is interconnected by fibers composed by many cells that fuse together, we reasoned that, facilitated by the existence of the process of fiber fusion, the protein dystrophin would spread throughout the fibers. We injected wild type ES cells this time into dystrophin deficient blastocysts (called mdx blastocysts) and allowed the resultant animals to go to term and to become adults. We selected chimeric mice containing 20% of muscle derived from the normal ES cells and 80% of the muscle derived from the mdx blastocysts (without dystrophin). These chimeras are symbolized WT/mdx. To our surprise, the muscle had no signs of muscular dystrophy, as the fibers were not necrotic or regenerative (Fig. 2). Moreover, molecular markers of muscular dystrophy were absent in the chimeras. Most of the fibers contained dystrophin, despite that the animals contained only 20% of incorporated ES cells. Thus, the structural protein dystrophin spread throughout the muscle. Surprisingly, some portions of the muscle that were not made out of ES-derived cells and therefore did not contain dystrophin were normal as well (Fig. 2D). This novel observation is still under study, and we reason that the fibers that do not contain dystrophin are stabilized by the presence of neighbor fibers that do contain dystrophin, perhaps again, by emission of healing molecules. We are currently attempting to identify molecules that may bridge the information between the dystrophin plus and the dystrophin minus muscle fibers. Importantly, a fraction of ES cells incorporated into the mdx muscle during early development suffices to rescue muscular dystrophy.
New generation of stem cells
Can this approach be applied to patients? The generation of human chimeras is certainly not the purpose of our study. Rather, the experiments are aimed at identifying novel mechanisms of disease correction. We exploit the ability of the ES cells to produce defined factors that may ultimately be used to treat congenital diseases. The identification of corrective molecules derived from the ES cells will have tremendous implications for the treatment of human disease, as it may be possible in the future to replace the ES cells with a “pill” of healing factors. Recently, a new generation of stem cells has emerged. These are somatic cells (from the skin for example) subjected to dedifferentiation by the addition of four defined factors. These cells, named induced pluripotent stem cells (iPS cells), resemble ES cells in many aspects. The iPS cells have the potential to differentiate into a wide range of cell types. Importantly, the iPS cells can generate chimeras when injected into early blastocysts. One fundamental difference between ES cells and iPS cells is that the generation of the latter does not implicate the destruction of embryos, and thus, there are no ethical concerns associated with using the iPS cells. It would be intriguing to test the corrective behavior of the iPS cells in our mouse models of human disease, for example, whether the iPS cells secrete healing factors, and if the factors are similar to those secreted by the ES cells (i.e. IGF1, Wnt5a). We have obtained mouse iPS cells, and hope to address this question in the near future.
Diego Fraidenraich received his PhD degree from the University of Buenos Aires, Argentina, and trained at New York University and at Memorial Sloan-Kettering Cancer Center. He is currently assistant professor in the Department of Cell Biology and Molecular Medicine at UMDNJ-New Jersey Medical School. His work is supported by the National Institutes of Health, the New Jersey Commission on Science and Technology, the American Heart Association and the Muscular Dystrophy Association.