Additional reprogramming focused on optimizing the protocol and was successful in generating iPSCs from seven additional NWR fibroblast cell lines, as well as two SWRs. These individuals' fibroblasts were plated on 0.1% gelatin in 50:50 medium at a range of approximate cell densities from 4,800 to 10,600 cells/cm2 instead of the initial protocol recommendation of 21,000–32,000 cells/cm2. This lower cell density is similar to published densities used for retroviral reprogramming in horse [22,27] and the previous retroviral reprogramming in endangered species [24], as well as the updated protocol from Thermo Fisher Scientific.

Wells were chosen at ∼50% confluency, the lower end of the initially recommended range of 50%–80% confluency. Cell density was too low to reliably count, so cell number was estimated based upon 36 h of growth (∼1.5 doublings) and infected with the recommended MOI of KOS-5, MYC-5, and KLF4-3. Additional wells were transduced with 1.5 times (7.5 - 7.5 - 4.5) and 2 times (10 - 10 - 6) the recommended MOI. See Table 1 for successful combinations of cell density and MOI for each rhinoceros.

Northern White Rhinoceros and Southern White Rhinoceros Fibroblast Cell Lines Used in These Experiments and the Resulting Induced Pluripotent Stem Cell Clones

Those individuals with estimated dates of birth (est) were wild caught. SD-WAP refers to the San Diego Zoo Safari Park, Escondido, CA. Zoo Dvůr Králové is in the Czech Republic (Štefánikova 1029, 544 01 Dvůr Králové nad Labem, Czechia). Chromosome complement was provided with the cell line from the Frozen Zoo® and is used for comparison to the derived induced pluripotent stem cell lines. Italicized clones were derived from alternate MOIs and/or initial plating density, also indicated by italics.

MOI, multiplicity of infection; NA, not applicable; NWR, northern white rhinoceros; SB, studbook; SD-WAP, San Diego Zoo Safari Park; SWR, southern white rhinoceros.

The medium was changed according to the protocol. In brief, fibroblast medium (50:50) was changed within 24 h post-transduction and every other day until replating. The cells were replated using TrypLE™ Select (Gibco) 1–2 days earlier than recommended. Cells for the feeder-free condition were replated on day 5 post-transduction at a density of 4,300 cells/cm2, and cells on MEFs were replated on day 6 post-transduction at a density of 360 cells/cm2. All MEF cultures were changed to KB Medium, and feeder-free cultures were changed to mTeSR1 (Stem Cell Technologies) 24 h later and subsequently fed every day.

Multiple clones were picked between days 16 and 23 post-transduction, with the most successful lines picked between day 16 and 19. Overall, clones picked from feeder layers were more successful, resulting in more clones and fewer losses to differentiation. All clones were expanded until at least passage 10 and then cryopreserved in 10% dimethyl sulfoxide (Sigma) and 90% KOSR using a CoolCell® LX control rate freezing container (BioCision) at −80°C for a minimum of 4 h before being transferred to nitrogen vapor holding.

The difference in growth rate of the initial fibroblast cells between the initial and optimized reprogramming efforts is most likely caused by the lack of 50:50 medium used during the initial reprogramming.

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