Rapid Derivation of Cloning-Competent Cells from Peripheral Blood Advances Conservation Biobanking
Laura Knecht, Anthony Mastracci, Riley Harbert, Fatima Charles, Laura L Kuperman-Yao, Tyler Miyawaki, Gregory L Gedman, Kathleen Morrill Pirovich, Jason Herrick, Linda Lay, Alba Ledesma, Steve Metzler, Dennis Milutinovitch, James B Papizan, Parker Pennington, Lexie Russell, Kerry Ryan, Kanokwan Srirattana, Kaitlin Steiger, Darya A Tourzani, Kelcey Walker, Rafael V Sampaio, Sven Bocklandt, Ben Lamm, Matt James, Shawn Walker, Beth Shapiro, James KehlerAbstract
Establishing viable cell lines from endangered species is essential for conservation, yet traditional fibroblast derivation from skin biopsies faces challenges including contamination risk and extended culture timelines. Here, we demonstrate that endothelial progenitor cells (EPCs) and pericytes isolated from peripheral blood represent robust alternatives to fibroblasts for biobanking. Compared to canid fibroblasts, canid blood-derived cells exhibit 2–3 fold faster doubling rates (15–20 hours versus ~35 hours for fibroblasts) and reduced time to banked cell lines (1.5–2 weeks versus 3–4 weeks for fibroblasts). Proteomic profiling of 32 canonical markers confirmed EPCs and pericytes represent distinct populations with lineage-specific molecular signatures. Optical genome mapping demonstrated equivalent genomic stability across cell types with no detectable structural variants or aneuploidies. Finally, interspecific somatic cell nuclear transfer (iSCNT) experiments confirmed both EPCs and pericytes generate viable canid embryos with efficiency meeting or exceeding fibroblasts. As a proof of concept for conservation cloning, iSCNT embryos made with gray wolf blood-derived cells had a 15% implantation rate following embryo transfer, and resulted in six viable fetuses. These findings support integrating blood-derived cell banking into conservation programs, which enables opportunistic genetic preservation during standard management activities and expands options for genetic rescue through assisted reproductive technologies.