Fig. 5: Evaluation of stem cell qualities of Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl iPSCs.

a Colony formation image of Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl iPSCs after digestion with Accutase on days 3 and 5. The day 3 (i–iii), and the day 5 (iv–vi) show. Okinawa rail iPSCs (i and iv), Japanese ptarmigan iPSCs (ii and v), Blakiston’s fish owl (iii and vi) iPSCs. b Cell numbers of the three endangered avian-derived iPSCs after digestion with Accutase on day 5. Yellow bar: Okinawa rail iPSCs; green bar: Japanese ptarmigan iPSCs; and purple bar: Blakiston’s fish owl iPSCs. n = 6. *P < 0.05. Centerlines of box plots indicate medians; box limits indicate the 25th and 75th percentiles. c Alkaline phosphatase staining of Blakiston’s fish owl iPSCs on day 7. This image show the iPSC colonies of digestion with Accutase and Y27632 minus (i), digestion with Accutase and Y27632 plus (ii), and mechanical passage (iii). d Enumeration of cell number of Blakiston’s fish owl iPSCs on day 7. Centerlines of box plots indicate medians; box limits indicate the 25th and 75th percentiles. n = 4. *P < 0.05. e Mitochondrial staining of iPSCs. MitoTracker staining (i–v) and TMRE staining (vi–x) of mouse (i and vi), chicken (ii and vii), Okinawa rail (iii and viii), Japanese ptarmigan (iv and ix), and Blakiston’s fish owl (v and x) shows. The bars represent 200 μm. f Detection of MitoTracker or TMRE density in iPSCs. Blue bar: mouse iPSCs; red bar: chick iPSCs; yellow bar: Okinawa rail iPSCs; green bar: Japanese ptarmigan iPSCs; purple bar: Blakiston fish owl iPSCs. (n = 6). Centerlines of box plots indicate medians; box limits indicate the 25th and 75th percentiles.* P < 0.05. g Alkaline phosphatase-positive colony formation of Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl iPSCs after 2-deoxyglucose (2DG) treatment (0 mM, 2 mM, 4 mM). Okinawa rail iPSCs (i–iii), Japanese ptarmigan iPSCs (iv–vi), and Blakiston fish owl iPSCs (vii–ix) show. 0 mM 2DG (i, iv, and vii), 2 mM 2DG medium (ii, v, and viii), and 4 mM 2DG medium (iii, vi, and ix). h Cell counting of the Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl iPSCs treated with 2DG. Centerlines of box plots indicate medians; box limits indicate the 25th and 75th percentiles. n = 6 (Okinawa rail and Blakiston’s fish owl), n = 4 (Japanese ptarmigan). *P < 0.05.