Why Protect Biodiversity and Save Endangered Species?
Biodiversity on the planet is rapidly declining with the sharp increase in species extinction rates. As population numbers for many wildlife species continue to fall, research has shifted to applying molecular biology techniques in order to preserve the genetic presence of species on the planet even if the species itself is no longer able to survive. Ben-Nun and colleagues showed somatic cells of endangered species could undergo a reprogramming process to a state of pluripotency that is similar to the process used for human cells. These cells differentiate into cells of the three germ layers seen in embryonic development, however they do not require the extraction of embryonic tissue. Induced pluripotent stem cells (iPSCs) are attractive to conservation biology because of their potential use in the laboratory development of artificial gametes to be used for in-vitro production of viable embryos of endangered species. However, using iPSCs in the creation of gametes that eventually produce viable offspring after fertilization still requires extensive research before becoming a regular practice. The purpose of this review is to outline the production process of iPSCs for species and highlight the research that has come from the production of iPSCs of endangered species.
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Currently, the diversity of life on planet earth is declining rapidly, with over 27% of species considered as being threatened with extinction (Ceballos et al., 2015). The natural rate of species extinction has been accelerated at least over 100 times, by a conservative estimate, from human activity-both directly and indirectly (Ceballos et al., 2015). Along with the loss of interspecific diversity, drastic decreases in population size have negatively affected intraspecific diversity for endangered species, making sustainable population growth more difficult due to loss of genetic diversity (Verma and Verma, 2014). Decreasing the threats to wildlife is crucial in conservation efforts, however, there is less that can be done for species already impacted by the mass extinction.
One of the more recent resources for these species has been the application of stem cell technology. Stem cells are unique form because of their ability to specialize in other cell types and for their self-renewal capabilities. Advancements in biochemistry and molecular biology techniques have led to the ability of researchers to treat somatic cells to revert them to an inducible pluripotent state (Takahashi and Yamanaka, 2006). Thus, it is possible for stem cell technology to be used for research in fields where the use of embryonic stem cells -stem cells found in the inner cell mass of an early embryo (ESC) – (Thomson et al, 1998) is not feasible. Wildlife conservation is one of these fields because harvesting a viable embryo, embryonic tissue, or gametes from a critically endangered species, and destroying the blastocyst in the process, has many technical and ethical obstacles (Verma and Verma, 2014).
Although induced pluripotent stem cells (iPSCs) involve less ethical debate than embryonic stem cells, some of their theoretical applications in wildlife conservation have drawn a fair share of skeptics and critics. The ability to use iPSCs to produce gamete cells for critically endangered species has been proven to be true, however the viability of offspring produced from these cells is uncertain and there remains many questions surrounding the methodology of fertilization and development of the subsequent embryo (Saragusty et al, 2016). More controversial still is the idea of creating iPSCs derived from tissue samples of extinct species for the eventual goal of creating germ cell lines that could lead to the revival of these species. Many critics of de-extinction claim the technology has not advanced enough for this to be feasible, while others question the desire to reanimate extinct species and bring them into modern ecosystems (Cohen, 2014; Bennett et al., 2017). Other applications of iPSCs in wildlife conservation are less controversial and include using iPSCs as an assisted reproductive technology (ART) for endangered species (Verma and Verma, 2014). Additionally, iPSCs could also be used in the agricultural industry to create animal products for consumers as a method of climate conservation (Stanton et al, 2019).
This review will approach the topic by focusing on the Ben-Nun et al. (2011) paper where the authors were able to successfully generate iPSCs from cryopreserved (frozen) tissue samples of two critically endangered species. There will be discussions of the key elements of deriving iPSCs from different sources such as human tissue and cryopreserved animal tissue and cryopreservation of tissue samples. Then, it will outline differentiation if iPSCs into other cell types and the range of species for which iPSCs have been successfully produced. Furthermore, the review will highlight the applications of stem cell technology to the field of wildlife conservation.
Pluripotency in Stem Cells
The significance of stem cells in research lies largely in their differentiation abilities and self-renewal properties. In particular, embryonic stem cells (ESCs) and iPSCs are useful because they are pluripotent, meaning they differentiate to cells of the three germ layers that ultimately differentiate into specialized cells of an organism. This is significantly different than oligopotent or multipotent stem cells, which are more limited in their range of differentiation (Romito and Cobellis, 2015).
Pluripotency was previously thought to be unique to ESCs, indicating that there are specific cellular factors present (or silenced) in these cells that give them this character. Given that there are specific factors present in ESCs that relate to their pluripotent phenotype, Takahashi and Yamanaka (2006) hypothesized that these factors could be inserted into somatic cells to revert them to a dedifferentiated state, make them pluripotent. They assessed 24 factors found in ESCs for their ability to induce pluripotency by individually inserting the gene of each factor into an embryonic fibroblast (connective tissue cell) and culturing these fibroblasts. The genes were inserted into a gene known to be active in ESCs, Fbx15, along with genes for resistance to the antibiotic G418. This way, the researchers would know if the cultured cells were indeed pluripotent based on whether these cells could grow in the presence of G418 (pluripotent) or not (not pluripotent). After narrowing down the number of factors from 24 to 4 in fibroblasts, they inserted genes for the 4 factors, G418 resistance, and green fluorescent protein into fibroblasts from the mouse tail (somatic fibroblasts). Colonies that formed in the presence of G418 were used to establish iPSCs that were identical in appearance to ESCs (Figure 1A) and positive in expressing ESC markers (Figure 1B). These results both confirmed the identity of factors necessary for pluripotency in ESCs and showed that these factors could be overexpressed in somatic cells. Thus, pluripotency seen in ESCs can be induced in somatic cells.
After the success of creating iPSCs from mouse somatic cells, researchers quickly moved onto creating these pluripotent cell lines from human tissues. Takahashi and Yamanaka (2007) and Yu et al. (2007) both published research that outlined the specific genes that, when introduced and overexpressed in human somatic cells, would result in dedifferentiation of these cells into iPSCs. In both of these studies, two of the same genes induced pluripotency in both somatic mouse cells and somatic human cells (Oct4 and Sox2), however the conditions the cells were cultured were significant in the pluripotency of these cells (Takahashi and Yamanaka, 2007).
Generating iPSCs from Endangered Species from Cryopreserved Samples
Generating iPSCs from somatic cells of endangered species is slightly different from the process used in humans or mice. Unlike lab animal or human models, the somatic tissue sample is often from cryopreserved samples. Cryopreservation is a tissue preservation method that goes beyond the freezing of tissue, and indefinitely stores living cells by fixing them using cryopreserving and cryo-protecting agents at cryogenic temperatures reaching further below than -80°C (Bakhach, 2009). It has been successfully used in tissue storage for humans and animals alike. Successful cryopreservation methods result in the thawing of viable cells and methods variy by cell types within the same species (2009). This has been noted in non-human species as well, as seen in León-Quinto et al (2014) where researchers found different viability success between fetal and adult differentiated cells of the Iberian Lynx. They also outlined the techniques that increased the viability potential for fetal cells, which has applications in the use of cryopreservation for storing tissue samples of other big cats and mammals (2014).
The Zoological Society of San Diego is one of select group of organizations that have extensive, cryopreserved tissue samples of a large range of species. The “Frozen Zoo” cultures fibroblasts taken from tissue samples of animals in the zoo and at sanctuaries around the world. Ben-Nun et al (2011) used samples in the Frozen Zoo for their study of iPSC production from endangered species. The authors selected tissue samples of the drill primate (Mandrillus leucophaeus) and northern white rhino (Creototherium simum cottoni), both of which had critical conservation statuses, to create iPSC lines from cryopreserved tissue. The drill is a very highly endangered African primate species, vulnerable to the bush meat trade and habitat encroachment (Ben-Nun et al., 2011). At the time of publication, the northern white rhino population had 7 remaining individuals, however as of March 2018 the species has been declared effectively extinct because of the death of the last male, which left only two females in the population (Tunstall et al., 2018).
Researchers had previously shown that somatic cells could produce iPSCs by introducing reprogramming factors to induce pluripotency (Takahashi and Yamanaka, 2006; Takahashi and Yamanaka, 2007). Whether these factors could induce pluripotency in somatic cells of other species had not been determined. Ben-Nun et al. (2011) hypothesized that the reprogramming factors used by Takahashi and Yamanaka (2006, 2007) could induce pluripotency in somatic cells of the northern white rhino and the drill monkey. After thawing the cryopreserved fibroblasts, Ben-Nun et al (2011) tested their hypothesis by introducing the reprogramming factors to the drill and rhino cells using viruses carrying the human genes for the factors known to induce pluripotency: POU5F1 (the human equivalent of Oct4), SOX2, KLF4, and MYC. 4 iPSC lines were generated from the drill experiment and 3 from the rhino. The produced iPSC lines were similar in morphology to ESCs and pluripotent cells of related species. In the drill cells, researchers were able to measure an increase in expression in innate genes associated with pluripotency, an indication of success in the dedifferentiation process (Figure 2D). The state of being fully reprogrammed was confirmed by the decreased expression of the inserted genes, which is considered to be a key characteristic of cells that have been fully reprogrammed (Figure 2C and 3). These results showed that cryopreserved somatic cells of endangered primate and rhino species require the same transcription factors that reprogram human and mouse cells for reprogramming. Hence, dedifferentiating somatic cells of endangered species to a pluripotent state is possible and produces iPSCs for these species that can be further used in research or conservation.
Differentiating iPSCs into Other Cell Types
The success of reprogramming somatic cells to iPSCs in different species is remarkable, however the cells still need to be able to undergo differentiation to be useful in research and conservation applications. Cellular differentiation is when nonspecific cells specialize into a specific cell type. Unspecialized cells during development begin this differentiation process by aggregating together, forming the three primary germ layers: ectoderm, mesoderm, and endoderm (Romito and Cobellis, 2015). Differentiated cells express genes specific to their germ layer that differ from genes expressed by cells in the other germ layers and from pluripotent cells. In particular, cells of the ectoderm are associated with expression of the PAX6 gene, mesoderm cells express the MSX1 gene, endodermic cells show expression of the SOX17 gene, and pluripotent express the OCT4 gene (Ben-Nun et al, 2011).
Differentiation into cells of the primary germ layers is an important characteristic of iPSCs and ESCs both in their character and in their applications. The drill and rhino iPSCs Ben-Nun et al. (2011) produced were able to form embryo-like structures (termed embryoid bodies, EB) in-vitro, leading them to hypothesize that they would also differentiate into cells of each of the germ layers, a crucial characteristic these cells needed to have in order to be used in conservation efforts. To determine if the iPSCs they had produced could differentiate into germ layer cells, Ben-Nun et al (2011) evaluated the expression of genes specifically associated with each of the germ layers as well as the genes associated with the pluripotent state. They tested this by culturing two iPSC lines in different media that caused the cells to differentiate and form embryoid bodies. Then, researchers compared the expression profile of the genes associated with each of the primary germ layers and pluripotency in copy DNA (cDNA) from the EBs and the iPSCs using quantitative PCR (Figure 4A-B). The EBs were further tested for the presence of each specific germ layer and then chemically fixated before being washed with solution containing primary antibodies that would bind to specific cell markers for each germ layer. After washing away excess primary antibody, the cells were washed with another solution containing fluorescently tagged secondary antibodies that bound to a specific primary antibody (Figure 4C). The results showed that the EBs expressed higher levels of expression of the germ layer associated genes than the iPSCs. Also, the EBs did not contain any non-differentiated cells as seen in the absence of expression of the gene associated with pluripotency. The EBs showed complete differentiation of cells into each of the germ layers in-vitro. Therefore, iPSCs generated in the lab from cryopreserved samples are capable of differentiation as seen in ESCs.
Production of iPSCs from Different Species
With the success of creating iPSCs from rhino and drill fibroblasts, other researchers sought to determine whether it was possible to derive iPSCs from somatic tissues of other species. This process involved fine-tuning the process of deprogramming cells by species as the same exact process did not always yield the same levels of results across species. For Ben-Nun et al. (2011), this involved changing the virus vectors used during the retroviral reprogramming of the rhino somatic tissue because the rhino species was not susceptible to the virus used for the drill cells (2011). In the case of producing iPSCs of felid species, Verma and Verma (2014) realized there was an additional cellular factor required for the reprogramming of snow leopard somatic cells that was not necessary in reprogramming primate (human and non-human) or rhino cells (2012). These nuanced variations in the process across different species are important to know in order to successfully produce iPSCs that could be used in the conservation of a wide variety of species.
Interestingly, it was not always the case that the process of creating the iPSCs for different species required adjustments. The same cellular factors used by Takahashi and Yamanaka (2006, 2007) and Ben-Nun et al. (2011) have been successfully used in creating iPSCs for a variety of species, ranging from snow leopards (Verma and Verma, 2014) to Sumatran orangutans (Ramaswamy et al., 2015). In addition to mammal cells, these (human) cellular factors have been able to induce pluripotency in avian cells, creating avian iPSCs (Lu et al., 2012). These avian iPSCs are particularly useful for research because they exhibit useful characteristics under laboratory conditions not seen in avian ESCs (Lu et al., 2012). There is a gap in published research in the production of iPSCs for terrestrial ectotherm species (amphibians and reptiles) and it is therefore unknown whether these species are able to undergo somatic cell reprogramming (Mastromonaco et al., 2014). In any case, the ability of the same factors to induce pluripotency in mammal and non-mammalian avian species (endotherm species) shows possible conservation of these genes across evolutionary timelines (Lu et al., 2012).
iPSCs and Assisted Reproductive Technologies in Species Conservation
With the production of iPSCs, it is no longer necessary to obtain ESCs in order to have pluripotent stem cells of different species for studying. This has greatly reduced the ethical controversy and logistical challenges associated with obtaining and researching pluripotent cells of endangered species (Verma and Verma, 2014). Large stores of cryopreserved animal tissue samples across different classes of animals around the world have around for decades for purposes including research in genomics and developmental biology (Mastromonaco, 2014). These biobank stores can serve as sources of somatic tissue samples to produce iPSCs of many endangered species to be used in conservation efforts.
The development of assisted reproductive technologies (ART), which are laboratory procedures that involve handling of sperm, oocytes, and/or embryos for reproductive purposes (Verma and Verma, 2014), has significantly impacted reproduction potential for infertile and sub-fertile individuals. Using gamete cells of male and female donor (sperm and oocysts, respectively), fertilized embryos are able to be formed in the laboratory through a variety of processes such as in-vitro fertilization (IVF), artificial insemination (AI), somatic cell nuclear transfer (SCNT), and intra-cytoplasmic sperm injection (ICSI) (Verma and Verma, 2014). A combination of these techniques is often used in stimulating a pregnancy outside of fornication (Hildebrandt et al., 2018).
There are different kinds of gametes that can be used in assisted reproduction that classified as natural or artificial gametes. Natural gametes are obtained from fresh or cryopreserved gonad tissue or reproductive organs of the species in question. Artificial gametes are those that have been made in the lab via molecular manipulation of non-reproductive tissue (Saragusty et al., 2016). The latter includes the process of stimulating differentiation of iPSCs into gamete cells. This has been done using mouse iPSCs that were differentiated into precursor germ-like cells produced functional gametes when implanted in a mouse host (Hayashi et al., 2011; Hayashi et al., 2012). The process of artificial gamete production has not been attempted with the iPSCs generated from endangered species, partially due to limited knowledge regarding the species-specific requirements and processes of gametogenesis (Saragusty et al., 2016). However, iPSCs still demonstrate strong potential for being a source of gametes to be used in assisted reproduction for endangered species.
Using Stem Cell Technology in Conservation: The Northern White Rhino
The need for further advancements in ARTs – including enhanced incorporation of iPSC technology – is needed to preserve the genes of the northern white rhino (NWR) subspecies. The death of the last male in March 2018 meant there are now only two infertile females left in the population and extinction of the subspecies is eminent. Over the decades leading to the current population situation, low population numbers and compromised fertility of the individuals served to limit breeding opportunities for the species as the numbers declined (Tunstall et al., 2018). These two facts also limited the application of ARTs due to lack of useable gametes or surrogacy candidates for implanting an IVF-derived embryo (Tunstall et al., 2018). However, recent genomic comparisons of the NWR and southern white rhino (SWR) subspecies by Tunstall et al. (2018) have shown that the two subspecies are extremely genetically similar, a fact that could be useful in genetic conservation efforts of the NWR.
Producing an embryo with NWR genetic material has many associated challenges, such as the source of the gamete cells and ability for blastocysts to form in-vitro. The genetic similarity between the NWR and SWR subspecies is strong enough for researchers to hypothesize that SWR cells could be used in conservation efforts for the NWR (Tunstall et al., 2018). Based on this theory, Hildebrandt et al (2018) have hypothesized that SWR and NWR gametes could lead to successful creation of an in-vitro early embryo (blastocyst). To test this, researchers extracted SWR oocytes from living females by modifying techniques used to extract horse oocytes. The male gametes were obtained by electrically stimulating ejaculation from live males. The SWR sperm cells used in the experiment were fresh while the NWR sperm were from cryopreserved ejaculate. After maturing the oocyte in the lab, researchers used a process called intracytoplasmic sperm injection (ICSI) (manually injecting sperm into the oocyte) to fertilize the gametes and form a zygote (the product of the fusion of 2 gametes). The oocyte was injected with either SWR or NWR sperm to create a pure SWR fertilized zygote or hybrid SWR-NWR fertilized zygote, respectively. The results showed that SWR-NWR hybrid zygotes were successfully able to mature to blastocysts with inner cell mass structures that could serve as sources of embryonic stem cells (Figure 5). Thus, it is possible to use SWR cells to help preserve NWR genetic material by creating hybrid blastocysts in-vitro using assisted reproduction techniques in the laboratory. This experiment provides the basis for the next step of ARTs: the use of artificial gametes derived from iPSCs to form blastocysts and embryos in-vitro. In the future, this process could be repeated using artificial gametes produced from iPSCs in order to produce blastocysts.
The current extinction crisis calls for immediate action in worldwide wildlife conservation efforts in order to preserve the remaining biodiversity of the planet (Ceballos et al., 2015). Stem cell technology has the potential to be a game-changing tool in the field of conservation. By inducing pluripotency in adult somatic cells, researchers are able to study pluripotent cells without harvesting embryonic tissue (Takahashi and Yamanaka, 2006, 2007). Ben-Nun et al. (2011) showed that pluripotency could also be induced in the adult somatic cells of endangered species from cryopreserved samples. The iPSCs from endangered species could produce artificial gametes to be used in combination with assisted reproductive technologies to serve as a means of conserving the genome of endangered species for whom there are limited tissue samples (Figure 6).
There is still research that needs to be done before iPSCs are produced for gamete differentiation. The specific processes required for gamete differentiation of different animal types needs further research, which would require more research in animal developmental biology and reproductive biology (Saragusty et al., 2016). Furthermore, there is a wide gap in knowledge in the production of iPSCs in ectotherm species that needs to be addressed, especially considering that amphibians are considered one of the most threatened genera of animals on the planet (Mastromonaco et al., 2014). As with other conservation techniques, the need for more research with iPSCs is imperative for its successful use in saving endangered species. The refinement of this technology will one day see iPSCs as one of the most powerful tools in wildlife conservation.
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