Synaptically-competent neurons derived from canine embryonic stem cells by lineage selection with EGF and noggin

Pluripotent stem cell lines have been generated in several domestic animal species; however, these lines traditionally show poor self-renewal and differentiation. Using canine embryonic stem cell (cESC) lines previously shown to have sufficient self-renewal capacity and potency, we generated and compared canine neural stem cell (cNSC) lines derived by lineage selection with epidermal growth factor (EGF) or Noggin along the neural default differentiation pathway, or by directed differentiation with retinoic acid (RA)-induced floating sphere assay. Lineage selection produced large populations of SOX2+ neural stem/progenitor cell populations and neuronal derivatives while directed differentiation produced few and improper neuronal derivatives. Primary canine neural lines were generated from fetal tissue and used as a positive control for differentiation and electrophysiology. Differentiation of EGF- and Noggin-directed cNSC lines in N2B27 with low-dose growth factors (BDNF/NT-3 or PDGFαα) produced phenotypes equivalent to primary canine neural cells including 3CB2+ radial progenitors, MOSP+ glia restricted precursors, VIM+/GFAP+ astrocytes, and TUBB3+/MAP2+/NFH+/SYN+ neurons. Conversely, induction with RA and neuronal differentiation produced inadequate putative neurons for further study, even though appropriate neuronal gene expression profiles were observed by RT-PCR (including Nestin, TUBB3, PSD95, STX1A, SYNPR, MAP2). Co-culture of cESC-derived neurons with primary canine fetal cells on canine astrocytes was used to test functional maturity of putative neurons. Canine ESC-derived neurons received functional GABAA- and AMPA-receptor mediated synaptic input, but only when co-cultured with primary neurons. This study presents established neural stem/progenitor cell populations and functional neural derivatives in the dog, providing the proof-of-concept required to translate stem cell transplantation strategies into a clinically relevant animal model. © 2011 Wilcox et al

Skin Lipoma in an Arabian Leopard (Panthera paradus nimr) Acta Scientiae Veterinariae

ABSTRACT Background: The Arabian leopard (Panthera paradus nimr) is the largest living felid in the arid Arabian Peninsula and classifi ed on the IUCN red list as critically endangered. Unlike felids, neoplasia prevalence in canids such as benign lipoma and malignant liposarcoma has been long and well documented. Only until recently a plethora of reports emerged demonstrating that neoplasia occurrence in wild exotic felids is prevailed more than expected. Soft tissue tumors arise from fatty cells form either a benign lipoma or a dangerously malignant liposarcoma. Alarming though, such cellular transformation might endanger the life of an already endangered animal. Case: An intact Arabian male leopard living in captivity at the Oman wildlife animal breeding center (N23.70 E58.09 A5.80 m) aged approximately 18 years and weight 31 kg was admitted to the veterinary clinic for semen collection and routine physical examination. The animal was identifi ed with two large adjacent subcutaneous masses on the upper rear left limb, clinically resembling that of a lipomatosis. Only one large tissue mass was surgically excised from the base with no incident of bleeding. Gross examination revealed a soft, smooth, rubbery, homogeneous, lack of internal fl uid and whitish color lobule. Morphometry measurement of the mass shows that the weight, diameter, circumference, thickness and surface area were 3.6 gm, 2.6 cm, 10.2 cm, 3 cm and 17.8 cm 3 respectively. On visual examination, neither mucin fl uid nor mucosal ulcerations were detected. Microscopically, dark discrete spots were observed on the anterior central and periphery of the mass surface outgrowth. Moreover, histopathological diagnoses with haematoxylin and eosin (HE), masson fontana (MF) and elastic verrhof van giesson (EVG) revealed normal nuclear and non-granular cytoplasm resembling that of a fatty cell originating from a fat adipose tissue. Adipocytes had reasonable amounts of cytoplasm and well defi ned borders. The nuclei were round to oval shape and no cells were found to be multinucleated. No evidence of high nuclear cytoplasmic ratio was observed. Few lymphocytes and plasma cells were present with no visible lymphatic vessels. Taken together, the lesion was diagnosed as a lobulated soft mass resembling that of an adipose tissue, specifi cally a benign neoplastic lipoma. Discussion: To date not a single report describes maladies in big cats from arid regions. This is the fi rst study to demonstrate the occurrence of neoplasia in a wild felid namely; the Arabian leopard. Additionally, while recent reports have shown neoplasia occurrence in the Panthera subspecies in tropical, polar and temperate zones, this is the fi rst report to manifest the disease in an arid region. The increase in neoplasm frequency in exotic felids is a concerning fact as numerous members of the Panthera family including the Arabian leopard are classifi ed by the IUCN as endangered or critically endangered species. With less than 200 animals in the wild, only 14 founder individuals in captivity and an aged female population the occurrence of lipoma tumors in the Arabian leopard is a worrisome sign. Taken together, the data suggests the rise of uncommon diseases in carnivores and ubiquitously around different climate zones of the world. Thus highlights the importance of routine physical examinations, investing substantially in diagnostic equipment and healthcare endowment in captive exotic felids

Synaptically-Competent Neurons Derived from Canine Embryonic Stem Cells by Lineage Selection with EGF and Noggin

Pluripotent stem cell lines have been generated in several domestic animal species; however, these lines traditionally show poor self-renewal and differentiation. Using canine embryonic stem cell (cESC) lines previously shown to have sufficient self-renewal capacity and potency, we generated and compared canine neural stem cell (cNSC) lines derived by lineage selection with epidermal growth factor (EGF) or Noggin along the neural default differentiation pathway, or by directed differentiation with retinoic acid (RA)-induced floating sphere assay. Lineage selection produced large populations of SOX2+ neural stem/progenitor cell populations and neuronal derivatives while directed differentiation produced few and improper neuronal derivatives. Primary canine neural lines were generated from fetal tissue and used as a positive control for differentiation and electrophysiology. Differentiation of EGF- and Noggin-directed cNSC lines in N2B27 with low-dose growth factors (BDNF/NT-3 or PDGFαα) produced phenotypes equivalent to primary canine neural cells including 3CB2+ radial progenitors, MOSP+ glia restricted precursors, VIM+/GFAP+ astrocytes, and TUBB3+/MAP2+/NFH+/SYN+ neurons. Conversely, induction with RA and neuronal differentiation produced inadequate putative neurons for further study, even though appropriate neuronal gene expression profiles were observed by RT-PCR (including Nestin, TUBB3, PSD95, STX1A, SYNPR, MAP2). Co-culture of cESC-derived neurons with primary canine fetal cells on canine astrocytes was used to test functional maturity of putative neurons. Canine ESC-derived neurons received functional GABAA- and AMPA-receptor mediated synaptic input, but only when co-cultured with primary neurons. This study presents established neural stem/progenitor cell populations and functional neural derivatives in the dog, providing the proof-of-concept required to translate stem cell transplantation strategies into a clinically relevant animal model