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Stem cell research is a complex and dynamic field with an ever-present possibility for introduction into conservation efforts. There is a high likelihood that stem cells and targeted stem cell therapies, utilizing induced pluripotent stem cells (iSPCs), will provide treatment and possible cures for cancerous and degenerative diseases and, conserve genetic material within avian species. Although the financial aspects of stem cell research have given rise to major regulation of the field, studies conducted by Katayama, M., Fukuda, T., Kaneko, and the time-capsule team at the National Institute of Environmental Studies found novel iSPCs for three endangered species of bird in Japan which could provide an important resource for future conservation efforts. 

Stem cell research can be differentiated into subcategories from where specific stem cells are derived: Induced pluripotent stem cells (iPSCs) are autologous or allogeneic material and are derived from somatic cells; Embryonic Stem Cells (ESCs) are solely allogenic and are derived from the inner cell masses of blastocyst; and Spermatogonial Stem Cells (SSCs) consist of both autologous or allogeneic material. Although ESC and SSC stem cells are credible for research, they do not have high degrees of proliferation when unisolated. iPSC cells provide the most likely candidate for conservation efforts as they are created by transforming somatic cells (which are differentiated) into pluripotent stem cells. This process is completed by forcing the expression of specific genes within a process known as somatic cell nuclear transfer. Trans-acting elements, or transcription factors, are proteins responsible for initiating transcription that bind to promoter regions of a cell. Since all cells contain the organism’s genome, differentiation (the process in which immature stem cells become matured cells with specific functions) within cells is caused by the introduction of specific transcription factors that initiate or terminate the transcription of a particular gene. Therefore, microbiologists hypothesized that if a genome were exposed to specific transcription factors, it could express the undifferentiated characteristics of a stem cell. Shinya Yamanaka created the correct conditions and provided the transcription factors necessary to induce pluripotency in somatic cells through this forced expression in 2006. 

Across the globe, hundreds of avian species are endangered, and humans pose a massive threat to their survival.  In Japan — where Katayama, M., Fukuda, T., Kaneko, and the time-capsule team created new iSPCs to protect three endangered bird species — 13 avian species are categorized as critically endangered (Japan - Species | CEPF, n.d.). With the preservation of biodiversity as an important goal for many conservationists, genetic preservation of threatened species using integrated stem cell technologies provides a bank of genetic information that allows differentiation into specific cellular types. Ultimately, scientific advancement in this field could save numerous avian species from extinction and help them reestablish healthy wild populations. Providing researchers with a way to access and differentiate any given bird cell allows for capabilities beyond genetic banking. 

To generate the iSPCs derived from the endangered bird species in the study — the Okinawa rail (Hypotaenidia okinawae), Japanese ptarmigan (Lagopus muta japonica), and Blakiston’s fish owl (Bubo blakistoni) —, the team first exposed the cells targeted for induction (fibroblasts from the bird species that had been cryopreserved in liquid nitrogen for 8-12 years) to PB-R6F, a group of transcription factors (modified Oct3/4 (M3O), Sox2, Klf4, c-Myc, Lin28, and Nanog) known to establish iPSC’s for chickens. PB-R6F was successful for the Okinawa rail, Japanese ptarmigan, and the control subjects of chicken and mouse fibroblasts, yet it did not cause Blakiston’s fish-owl iPSC colonies to appear. To solve this problem, the team modified the polycistronic single all-in-one vector (PB-TAD-7F) to include modified Oct3/4 (M3O), Sox2, Klf4, c-Myc, Klf2, Lin28, and Nanog, resulting in the establishment of the iPSC colonies at a 5% or lower success rate using the liposuction method. A vector map of PB-TAD-7F is shown below.  

 With the addition of hygromycin, the experiment was repeated creating a much higher number of primary colonies of all species. The colonies' morphologies differed — the Japanese ptarmigan was three-dimensional and the Blakiston’s fish owl was flat, yet the experiment yielded Stanley passages iPSCs more than 20 times. With these iPSCs, a wide array of tests were run, including the effect of inhibitors, catalysts, and other variables. 

    The results of this experiment demonstrated the genetic differences between the iPSCs within the three bird species. These iPSC colonies (shown below) are the first ever established for the Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl.