Biology subjects

Kehler, J.

Publications and source records attributed to Kehler, J..

4 recordsLinked to original sources

bioRxiv2025

Rapid Derivation of Cloning-Competent Cells from Peripheral Blood Advances Conservation Biobanking

I.Establishing viable cell lines from endangered species is essential for conservation, yet traditional fibroblast derivation from skin biopsies faces challenges including variable success rates, contamination risk, and extended culture timelines. We demonstrate that endothelial progenitor cells (EPCs) and pericytes isolated from peripheral blood represent superior alternatives for biobanking across three mammalian genera (Canis, Bison, and Equus). Blood-derived cells exhibited 2-3 fold faster doubling rates (15-20 hours versus >35 hours for fibroblasts) and reduced time to generate banked lines from 3-4 weeks to 1.5-2 weeks. 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 all cell types with no detectable structural variants or aneuploidies. Critically, interspecific somatic cell nuclear transfer (iSCNT) experiments confirmed both EPCs and pericytes generate viable embryos with efficiency meeting or exceeding fibroblasts. Gray wolf blood-derived cells produced six viable fetuses with 15% implantation rate, while bison EPCs showed higher blastocyst formation (7%) than fibroblasts (3%) from the same individual. Blood collection during routine veterinary procedures offers minimally invasive sampling with reduced contamination compared to skin biopsies. These findings support integrating blood-derived cell banking into conservation programs, enabling opportunistic genetic preservation during standard management activities and expanding options for genetic rescue through assisted reproductive technologies.

cell biology↗

bioRxiv2025

Multiplex-edited mice recapitulate woolly mammoth hair phenotypes

The woolly mammoth (Mammuthus primigenius) possessed a thick woolly coat and other cold-adaptive traits that enabled survival in harsh arctic environments. Current de-extinction efforts focus on genetically modifying the closely related Asian elephant to express woolly mammoth traits. In this study, we establish a multiplex-edited mouse model with modifications in genes associated with hair morphology and lipid metabolism, enabling insights into traits involved in developing woolly hair textures. Our optimized workflows achieved high editing efficiencies and produced genetically modified mice with simultaneous editing of up to seven different genes. Selected modifications include loss-of-function mutations in Fgf5, Tgm3, and Fam83g, among others. The resulting mice display exaggerated hair phenotypes including curly, textured coats, and golden-brown hair. This study advances methods of rapid establishment of complex genetic models. These approaches inform de-extinction efforts and research involving the genetic basis of mammalian hair development.

genetics↗

bioRxiv2024

DERIVATION OF ELEPHANT INDUCED PLURIPOTENT STEM CELLS

The crisis of biodiversity loss in the anthropogenic era requires new tools for studying non-model organisms. Elephants, for example, are both an endangered species and excellent models studying complex phenotypes like size, social behavior, and longevity, but they remain severely understudied. Here we report the first derivation of elephant (Elephas maximus) induced pluripotent stem cells (emiPSCs) achieved via a two-step process of chemical-media induction and colony selection, followed by overexpression of elephant transcription factors OCT4, SOX2, KLF4, MYC {+/-} NANOG and LIN28A, and modulation of the TP53 pathway. Since the seminal discovery of reprogramming by Shinya Yamanaka, iPSCs from many species including the functionally extinct northern white rhinocerous have been reported, but emiPSCs have remained elusive. While for multiple species the reprogramming protocol was adopted with little changes compared to model organisms like mouse and human, our emiPSC protocol requires a longer timeline and inhibition of TP53 expansion genes that are hypothesized to confer unique cancer resistance in elephants. iPSCs unlock tremendous potential to explore cell fate determination, cell and tissue development, cell therapies, drug screening, disease modeling, cancer development, gametogenesis and beyond to further our understanding of this iconic megafauna. This study opens new frontiers in advanced non-model organism cellular models for genetic rescue and conservation.

cell biology↗

bioRxiv2023

Cross-pathway integration of cAMP signals through cGMP and calcium-regulated phosphodiesterases in mouse striatal cholinergic interneurons.

Acetylcholine plays a key role in striatal function, yet the intricate dynamics of cyclic nucleotide signaling which govern the firing properties of cholinergic interneurons (ChINs) have remained elusive. Since phosphodiesterases determine the dynamics of cyclic nucleotides, in this study, we used FRET biosensors and pharmacological compounds to examine phosphodiesterase activity in ChINs in mouse brain slices. Intriguingly, these neurons displayed strikingly low levels and slow cAMP responsiveness compared to medium-sized spiny neurons (MSNs). Our experiments revealed that PDE1, PDE3 and PDE4 are important regulators of cAMP level in ChINs. Notably, the induction of cGMP production by nitric oxide (NO) donors increases cAMP by inhibiting PDE3 - a mechanism hitherto unexplored in neuronal context. Furthermore, the activation of NMDA or metabotropic glutamate receptors increases intracellular calcium, consequently activating PDE1 and thereby decreasing both cAMP and cGMP. This interplay of phosphodiesterases enables the control of cAMP by the neuromodulatory influences of glutamate and NO. Remarkably, the NO/cGMP signal results in different effects: NO enhances cAMP in ChINs by inhibiting PDE3, whereas it reduces cAMP levels in MSNs by activating PDE2A. These findings underscore the specificity of intracellular signaling in ChINs compared to MSNs and show how the NO-cGMP pathway affects these various neuronal types differently. These observations have significant implications for understanding the regulation of the striatal network and the integration of dopaminergic signals and suggest innovative therapeutic strategies for addressing basal ganglia disorders with unmet medical need.

neuroscience↗