Colossal Biosciences creates technologies for species restoration, critically endangered species protection, and the repopulation of critical ecosystems that support the continuation of life on Earth. The Texas-based company is the first to apply CRISPR technology for the purposes of species de-extinction.

Now, the company has announced the clearance of one of the major milestones in its woolly mammoth project by successfully reprogramming elephant iPSCs (induced pluripotent stem cells).

Eriona Hysolli, PhD, head of biological sciences at Colossal Biosciences

“In the past, a multitude of attempts to generate elephant iPSCs have not been fruitful. Elephants are a very special species and we have only just begun to scratch the surface of their fundamental biology,” shared Eriona Hysolli, PhD, head of biological sciences at Colossal Biosciences.

The Yamanaka factors, first elucidated in 2006, are transcription factors that reprogram cells to induce pluripotent stem cells. They have been used to derive iPSCs in mice and other species such as humans, horses, pigs, cattle, rabbits, monkeys, apes, big cats, rhinos, and avian species iPSCs. Although the reprogramming protocol is relatively universal across species, species-specific tweaks are necessary. However, elephant iPSCs have remained elusive.

“Elephants might get the ‘hardest to reprogram’ prize,” noted George Church, PhD, professor at Harvard Medical School and Colossal’s co-founder, “but learning how to do it anyway will help many other studies, especially on endangered species. This milestone gives us insights into developmental biology and the balance between senescence and cancer. It opens the door for obtaining gametes and other cell types without surgery on precious animals. It opens the door to establishing connections between genes and traits for both modern and extinct relatives—including resistance to environmental extremes and pathogens.”

Using chemical-based induction media first, followed by the addition of transcription factors Oct4, Sox2, Klf4, Myc +/- Nanog and Lin28, and p53 pathway suppression, the team has achieved the most successful reprogramming of elephant iPSCs yet.

iPSCs [Colossal Biosciences]
Asian elephant iPSC colonies stained for pluripotency factors OCT4 (Magenta) and SOX2 (green), nuclear DNA Hoechst (blue), and cytoskeletal protein actin (red). [Colossal Biosciences]
The approach differs from other more standard reprogramming protocols for other species due in part to the complexities of the TP53 pathway in elephants as their genome contains up to 19 copies of TP53 retrogenes. TP53 is a core gene utilized by the cell to carefully regulate its growth so as not to become cancerous. Additionally, reprogramming, which in itself is quite long and inefficient for higher mammal species, takes longer for elephants. But, the successful iPSC cells now express multiple core pluripotency factors and can differentiate into the three germ layers that later give rise to each cell type in the body.

These newly reprogrammed iPSC cells have been validated through immunostaining, PCR of pluripotency and differentiation markers, transcriptomics analysis, embryoid bodies, and teratoma formation. In addition, these cells can be multiplex-edited and differentiated to study cold adaptation traits like woolly hair growth and fat storage in cellular and organoid models.

This work has been posted to bioRxiv and submitted to a peer review journal.

More work needs to be done, the company noted, but this announcement marks the first successful steps. The mammoth stem cell team is now focused on further maturing these cells, and pursuing additional iPSC generation strategies that have so far also been successful.

“We are most excited to use the cells we have developed to grow elephant gametes in a dish. While elephants have been a challenging species, this has been an incredibly unique opportunity with so much to learn and share now and in the near future,” shared Evan Appleton, PhD, the team lead at Colossal.

The team is also working to establish a mechanism that can explain why elephant cell reprogramming has been challenging. Doing so is critical to deriving iPSCs faster, achieving more advanced tri-lineage differentiation, particularly in vitro gametogenesis, which is crucial to test the full potential of the iPSCs.

Once the iPSCs can be used to establish a model for synthetic elephant embryos, it will also be integral to understanding the long and complex elephant (and by association mammoth) development and gestation cycle. This will be critical to Colossals’ re-wilding efforts which rely heavily on leveraging ex utero development for species preservation and restoration. All of these scientific developments hold extension possibilities across the field of developmental biology which have ramifications far beyond the current Colossal projects.

“This collaboration has been a true pleasure and a colossal accelerant for our challenging project,” noted Church.

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