By: Una Choi
Background: Animal Chimeras
Chimeras prefigure prominently in classical and modern mythology; creatures ranging from the Greek chimera, a monster bearing lion, goat, and serpent anatomy, to the modernized hippogriffs found in popular fantasy fiction today have captured imaginations for centuries.
Biologically, animal chimeras are organisms containing two disparate genomes. Natural chimeras are commonly seen through mechanisms like fetal microchimerism, a phenomenon describing the retention of fetal cells in the mother’s body for months and even years after pregnancy (1). Chimera here refers to the deliberate transplantation of human stem cells into nonhuman animal embryos. Human stem cells were first successfully derived from adult tissue cells in 2007 (2). Pluripotent stem cells (PSC) differentiate into any cell type found in the original organism. Although initial attempts to trigger the differentiation of these stem cells into therapeutic tissues involved in vitro exposure to various chemicals, the difficulty of achieving the precise environment required for successful differentiation has led to a recent trend of transplanting stem cells of one species into embryos of another.
Diabetes and Rat-Mouse Chimeras
Dr. Hiromitsu Nakauchi of Stanford utilized rat-mouse chimeras to reverse successfully diabetes in mice (2). Dr. Nakauchi rendered the Pdx1 gene associated with pancreas development in rats non-functional and injected these same rat embryos with mouse stem cells, forcing the rats to develop its pancreas with pure mouse cells (2). The mice recipients originated from the same inbred strain as the donor mice, so they did not reject the transplanted organs (3). In constructing a pancreas derived almost solely from donor mouse cells, Dr. Nakauchi decreased risk of tissue rejection, thus enhancing the likelihood of a successful transplantation of the donor-derived tissue into the donor animal.
These mouse-derived pancreases were not solely constructed from mouse cells; indeed, 10% were composed of rat cells, as the rat supplied the blood vessels. These blood vessels, however, were rapidly replaced when the pancreases were transplanted in the mice (3).
This successful growth and implantation suggests a possible treatment for Type 1 diabetes, an autoimmune condition associated with the destruction of pancreatic beta cells (3). When Dr. Nakauchi transplanted these formed islets into diabetic mice, the mice-derived islets normalized the hosts’ blood glucose levels for over 370 days without immunosuppression, revealing the therapeutic potential of cross-species implantation (3).
Similar attempts to knock out completely the genes associated with the development of a particular organ in a developing animal have proven successful with the murine pancreas, heart and eye.
Mice and rats, however, differ drastically from humans. Consequently, several researchers are focusing on pigs as potential sites for human organ development. Pigs’ organs are similar in size to that of humans, and their metabolism also closely resembles human metabolism (1).
While researchers have successfully blocked the generation of the murine pancreas, heart, and eye, efforts to create pigs incapable of developing the organ of interest have proven unsuccessful. Organs like the pancreas, which stems from a single kind of progenitor cell, will be more easily constructed than complex organs like the heart.
In a 2017 paper published in Cell, Wu et al. found that naïve human PSCs, which have an unlimited self-renewal capacity, successfully engrafted into pre-implantation pig blastocysts (4). This transplantation, however, failed to contribute significantly to normal embryonic development in the pigs. The same group then injected human PSCs into cattle blastocysts. Both naïve and intermediate human PSCs survived and integrated into the cattle. Similarly, when human PSCs were injected into pig blastocysts, the blastocysts retained the human cells. These human PSCs selectively incorporated into the inner cell mass (ICM), marking the first step into successful incorporation of the donor cells into the host (4).
When the human PSCs were later injected into pig embryos, 50 of the 67 embryos exhibiting retained human PSCs were morphologically underdeveloped. In addition, this formation of interspecies chimeras was highly inefficient, revealing the persisting challenges to constructing viable pig-human chimeras (4).
The incorporation of porcine, bovine, and equine biological heart valves into human patients and the use of insulin derived from porcine pancreas are widely accepted medical tools (1). The production of a human-derived organ in a pig or other nonhuman animal, however, has been met with significant controversy.
The accidental incorporation of human cells in non-target locations in the host animal can result in ethical consequences; if human-derived cells are significantly utilized in the development of the non-human brain or the reproductive organs, the test animal may be considered excessively humanized. A 2013 study from the University of Rochester Medical Center reported that mice injected with human brain cells exhibited enhanced synaptic plasticity and learning (2). These human glial progenitor cells outcompeted their murine counterparts, resulting in white matter largely derived from humans.
These concerns of unwanted integration have resulted in the prohibition of human stem cell transplantation into monkey early embryos, as the evolutionary closeness may result in an increased susceptibility of monkey brains to human cell-catalyzed alteration (2).
In response to the above concerns, the National Institutes of Health (N.I.H.) instituted a 2015 moratorium on the use of public funds to incorporate human cells into animal embryos. This ban is still in place at the time of this article’s writing. These efforts have delayed current chimera research dependent on public funds; Dr. Nakauchi’s pancreas experiment is the result of eight to nine years of work and a 2014 relocation from Tokyo to Stanford due to Japanese regulations.
Scientists have pointed to new molecular techniques to address some of the more common ethical concerns. CRISPR-Cas9, a popular gene editing technique, might be used to direct implanted human cells to target organs in the embryo, thus preventing the accidental incorporation of human cells into the brain and reproductive tissues (2). The injected human stem cells could also be modified to include ‘suicide genes’ activated upon neural differentiation, ensuring the elimination of any human-derived differentiated brain cells (2).
In addition, primate cells divide more slowly than non-primate cells; primate neural progenitor cells go through more cell divisions (5). A sow’s gestation period, for example, is around 3 months, representing far shorter development period than that of humans (1). Transplanted human neural progenitor cells in chimeras would only be able to achieve the same high threshold of cell divisions if they were able to somehow sense the shortened development window and divide more rapidly. This scenario, however, is unlikely, as previous studies of human/mouse blood stem cell xenografts suggest the host regulates human stem cell proliferation; the likelihood of an accidental integration of human stem cells into the murine brain and a subsequent development of human cognitive capacities is slim.
The discovery that human-derived stem cells were utilized in the development of tissues in pigs holds promising implications for the field of organ transplants. Around 76,000 people in the United States await transplants. 2 In Europe, over 60,000 people are on the organ transplant waiting list. Attempts to grow human-derived organs in pigs and other animals could thus address the organ shortage.
Because the organs generated in the developing embryos are derived from donor cells, transplantation of those organs into the donor or organisms closely resembling the donor can decrease risk of organ rejection. This reduces the need for immunosuppressant drugs commonly used with organ transplants today.
The ability to generate human-derived tissues in non-human embryos allows scientists to study human cell development outside of human embryos. 5
Una Choi ’19 is a sophomore in Kirkland House studying Molecular and Cellular Biology and Economics.
 Bourret, R. et al. J. Stem Cell Res. Ther. 2016, 7, 1-7.
 Wade, N. New prospects for growing human replacement organs in animals. NYTimes
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 Kilsoo, J. et al. Stem Cells Dev. 2012, 21, 2642-2655.
 Wu, J. et al. Cell 2017, 168, 473-486.
 Karpowicz, P. et al. Nature 2004, 10, 331-335.