@article{lim_koh_thomas_breen_olby_2017, title={Evaluation of gene expression and DNA copy number profiles of adipose tissue-derived stromal cells and consecutive neurosphere-like cells generated from dogs with naturally occurring spinal cord injury}, volume={78}, ISSN={0002-9645}, url={http://dx.doi.org/10.2460/ajvr.78.3.371}, DOI={10.2460/ajvr.78.3.371}, abstractNote={Abstract
OBJECTIVE To evaluate gene expression and DNA copy number in adipose tissue-derived stromal cells (ADSCs) and in ADSC-derived neurosphere-like cell clusters (ADSC-NSCs) generated from tissues of chronically paraplegic dogs.
ANIMALS 14 client-owned paraplegic dogs.
PROCEDURES Dorsal subcutaneous adipose tissue (< 1 cm3) was collected under general anesthesia; ADSCs were isolated and cultured. Third-passage ADSCs were cultured in neural cell induction medium to generate ADSC-NSCs. Relative gene expression of mesenchymal cell surface marker CD90 and neural progenitor marker nestin was assessed in ADSCs and ADSC-NSCs from 3 dogs by quantitative real-time PCR assay; expression of these and various neural lineage genes was evaluated for the same dogs by reverse transcription PCR assay. Percentages of cells expressing CD90, nestin, glial fibrillary acidic protein (GFAP), and tubulin β 3 class III (TUJ1) proteins were determined by flow cytometry for all dogs. The DNA copy number stability (in samples from 6 dogs) and neural cell differentiation (14 dogs) were assessed with array-comparative genomic hybridization analysis and immunocytochemical evaluation, respectively.
RESULTS ADSCs and ADSC-NSCs expressed neural cell progenitor and differentiation markers; GFAP and microtubule-associated protein 2 were expressed by ADSC-NSCs but not ADSCs. Relative gene expression of CD90 and nestin was subjectively higher in ADSC-NSCs than in ADSCs. Percentages of ADSC-NSCs expressing nestin, GFAP, and TUJ1 proteins were substantially higher than those of ADSCs. Cells expressing neuronal and glial markers were generated from ADSC-NSCs and had no DNA copy number instability detectable by the methods used.
CONCLUSIONS AND CLINICAL RELEVANCE Results suggested ADSCs can potentially be a safe and clinically relevant autologous source for canine neural progenitor cells. Further research is needed to verify these findings.}, number={3}, journal={American Journal of Veterinary Research}, publisher={American Veterinary Medical Association (AVMA)}, author={Lim, Ji-Hey and Koh, Sehwon and Thomas, Rachael and Breen, Matthew and Olby, Natasha J.}, year={2017}, month={Mar}, pages={371–380} }
 @misc{koh_piedrahita_2014, title={From "ES-like" cells to induced pluripotent stem cells: A historical perspective in domestic animals}, volume={81}, ISSN={["1879-3231"]}, DOI={10.1016/j.theriogenology.2013.09.009}, abstractNote={Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provide great potential as cell sources for gene editing to generate genetically modified animals, as well as in the field of regenerative medicine. Stable, long-term ESCs have been established in laboratory mouse and rat; however, isolation of true pluripotent ESCs in domesticated animals such as pigs and dogs have been less successful. Initially, domesticated animal pluripotent cell lines were referred to as "embryonic stem-like" cells owing to their similar morphologic characteristics to mouse ESCs, but accompanied by a limited ability to proliferate in vitro in an undifferentiated state. That is, they shared some but not all the characteristics of true ESCs. More recently, advances in reprogramming using exogenous transcription factors, combined with the utilization of small chemical inhibitors of key biochemical pathways, have led to the isolation of iPSCs. In this review, we provide a historical perspective of the isolation of various types of pluripotent stem cells in domesticated animals. In addition, we summarize the latest progress and limitations in the derivation and application of iPSCs.}, number={1}, journal={THERIOGENOLOGY}, author={Koh, Sehwon and Piedrahita, Jorge A.}, year={2014}, month={Jan}, pages={103–111} }
 @article{koh_thomas_tsai_bischoff_lim_breen_olby_piedrahita_2013, title={Growth Requirements and Chromosomal Instability of Induced Pluripotent Stem Cells Generated from Adult Canine Fibroblasts}, volume={22}, ISSN={1547-3287 1557-8534}, url={http://dx.doi.org/10.1089/scd.2012.0393}, DOI={10.1089/scd.2012.0393}, abstractNote={In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing 4 transcription factors, Oct3/4, Klf4, Sox2, and c-Myc (OKSM). Here, we report the derivation of induced pluripotent stem cells (iPSCs) from adult canine fibroblasts by retroviral OKSM transduction. The isolated canine iPSCs (ciPSCs) were expanded in 3 different culture media [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), or FGF2 plus LIF]. Cells cultured in both FGF2 and LIF expressed pluripotency markers [POU5F1 (OCT4), SOX2, NANOG, and LIN28] and embryonic stem cell (ESC)-specific genes (PODXL, DPPA5, FGF5, REX1, and LAMP1) and showed strong levels of alkaline phosphatase expression. In vitro differentiation by formation of embryoid bodies and by directed differentiation generated cell derivatives of all 3 germ layers as confirmed by mRNA and protein expression. In vivo, the ciPSCs created solid tumors, which failed to reach epithelial structure formation, but expressed markers for all 3 germ layers. Array comparative genomic hybridization and chromosomal fluorescence in situ hybridization analyses revealed that while retroviral transduction per se did not result in significant DNA copy number imbalance, there was evidence for the emergence of low-level aneuploidy during prolonged culture or tumor formation. In summary, we were able to derive ciPSCs from adult fibroblasts by using 4 transcription factors. The isolated iPSCs have similar characteristics to ESCs from other species, but the exact cellular mechanisms behind their unique co-dependency on both FGF2 and LIF are still unknown.}, number={6}, journal={Stem Cells and Development}, publisher={Mary Ann Liebert Inc}, author={Koh, Sehwon and Thomas, Rachael and Tsai, Shengdar and Bischoff, Steve and Lim, Ji-Hey and Breen, Matthew and Olby, Natasha J. and Piedrahita, Jorge A.}, year={2013}, month={Mar}, pages={951–963} }
 @article{lim_koh_olby_piedrahita_mariani_2012, title={Isolation and characterization of neural progenitor cells from adult canine brains}, volume={73}, ISSN={0002-9645}, url={http://dx.doi.org/10.2460/ajvr.73.12.1963}, DOI={10.2460/ajvr.73.12.1963}, abstractNote={Abstract
Objective—To isolate and characterize neural stem and progenitor cell populations in the brain of adult dogs.
Animals—7 healthy adult dogs.
Procedures—Dogs (age, 10 to 60 months) were euthanized for reasons unrelated to the study. The subventricular zone (SVZ) adjacent to the lateral ventricles and subgranular zone (SGZ) of the hippocampus were isolated and used to generate single cell suspensions for nonadherent culture. The resulting primary neurospheres were serially passaged to assess self-renewal capacity. Neurospheres were differentiated by the withdrawal of growth factors and the addition of serum. Differentiated and undifferentiated neurospheres were analyzed via reverse transcriptase PCR assay or immunocytochemical staining for markers of pluripotency and neural lineage.
Results—Neurospheres were generated from the SVZ and SGZ in all dogs. The SVZ generated more primary neurospheres than did the SGZ. Serial passage was successful, although few neurospheres could be generated after the fifth passage. Undifferentiated neurospheres were positive for SOX2, nestin, and glial fibrillary acidic protein (GFAP) and negative for OCT4 and NANOG. After differentiation, GFAP, neuronal class III β-tubulin, and 2′, 3′-cyclic nucleotide 3′-phosphodiesterase–positive progeny were noted migrating out of the neurospheres.
Conclusions and Clinical Relevance—Results suggested the persistence of SOX2-positive, nestin-positive, GFAP-positive, OCT4-negative, and NANOG-negative neural progenitor cells in the SVZ and SGZ regions of mature canine brains, which are capable of producing multiple cell lineages. This study may serve as a basis for future studies investigating the role of these cells in various disease processes, such as neoplasia, or for regenerative purposes.}, number={12}, journal={American Journal of Veterinary Research}, publisher={American Veterinary Medical Association (AVMA)}, author={Lim, Ji-Hey and Koh, Sehwon and Olby, Natasha J. and Piedrahita, Jorge and Mariani, Christopher L.}, year={2012}, month={Dec}, pages={1963–1968} }
 @article{koh_lee_wang_cabot_machaty_2009, title={STIM1 regulates store-operated Ca2+ entry in oocytes}, volume={330}, ISSN={["0012-1606"]}, DOI={10.1016/j.ydbio.2009.04.007}, abstractNote={The single transmembrane-spanning Ca2+-binding protein, STIM1, has been proposed to function as a Ca2+ sensor that links the endoplasmic reticulum to the activation of store-operated Ca2+ channels. In this study, the presence, subcellular localization and function of STIM1 in store-operated Ca2+ entry in oocytes was investigated using the pig as a model. Cloning and sequence analysis revealed the presence of porcine STIM1 with a coding sequence of 2058 bp. In oocytes with full cytoplasmic Ca2+ stores, STIM1 was localized predominantly in the inner cytoplasm as indicated by immunocytochemistry or overexpression of human STIM1 conjugated to the yellow fluorescent protein. Depletion of the Ca2+ stores was associated with redistribution of STIM1 along the plasma membrane. Increasing STIM1 expression resulted in enhanced Ca2+ influx after store depletion and subsequent Ca2+ add-back; the influx was inhibited when the oocytes were pretreated with lanthanum, a specific inhibitor of store-operated Ca2+ channels. When STIM1 expression was suppressed using siRNAs, there was no change in cytosolic free Ca2+ levels in the store-depleted oocytes after Ca2+ add-back. The findings suggest that in oocytes, STIM1 serves as a sensor of Ca2+ store content that after store depletion moves to the plasma membrane to stimulate store-operated Ca2+ entry.}, number={2}, journal={DEVELOPMENTAL BIOLOGY}, author={Koh, Sehwon and Lee, Kiho and Wang, Chunmin and Cabot, Ryan A. and Machaty, Zoltan}, year={2009}, month={Jun}, pages={368–376} }