C.J. Gartley

Derivation and Characterization of Canine Embryonic Stem Cell Lines with In Vitro and In Vivo Differentiation Potential

Embryonic stem cells (ESCs) represent permanent cell lines that can be maintained in an undifferentiated state. In an environment that induces differentiation, they form derivatives of the three embryonic germ layers: mesoderm, ectoderm, and endoderm. These characteristics give ESCs great potential for both basic research and clinical applications in the areas of regenerative medicine and tissue engineering. The establishment of ESCs from large animals that model human diseases is of significant importance. We describe the derivation of permanent canine cell lines from preimplantation‐stage embryos. Similar to human ESCs, canine ESCs expressed OCT3/4, NANOG, SOX2, SSEA‐3, SSEA‐4, TRA‐1–60, TRA‐1–81, and alkaline phosphatase, whereas they expressed very low levels of SSEA‐1. They maintained a normal karyotype and morphology typical of undifferentiated ESCs after multiple in vitro passages and rounds of cryopreservation. Plating cells in the absence of a feeder layer, either in attachment or suspension culture, resulted in the formation of embryoid bodies and their differentiation to multiple cell types. In vivo, canine ESCs gave rise to teratomas comprising cell types of all three embryonic germ layers. These cells represent the first pluripotent canine ESC lines with both in vitro and in vivo differentiation potential and offer the exciting possibility of testing the efficacy and safety of ESC‐based therapies in large animal models of human disease. STEM CELLS 2009;27:329–340

Canine spermatozoa — cryopreservation and evaluation of gamete interaction

Abstract Successful cryopreservation of spermatozoa for genome banking or transport for articifial insemination (AI) in domestic and nondomestic Canidae depends on understanding the species-specific conditions required. Experiment 1 was designed to evaluate the post-thaw effects of 5 cooling rates on percentages of motile cells, progressive motility, morphology and acrosomal integrity of domestic dog spermatozoa. Semen samples (n = 18) extended in glycerol-Tris egg yolk buffer were cooled to 0°C, drawn into 0.5-ml straws and subjected to A) cooling at 0.5 °C/min to − 20 °C and at 2 °C/min from −20°C to −70°C, B) cooling at 12 °C/min, C) cooling at 28°C/min, D) cooling at 99 °C/min, and E) cooling at 214 °C/min. Samples were thawed by placing straws into 70 °C water for 6 sec. The contents were poured into microcentrifuge tubes, kept at 0 °C, and evaluated at 0, 2, 4 and 6 h. Overall, the smallest decline in the proportion of motile spermatozoa and in the progressive motility score was with the 12 and 28 °C/min freezing rates, and the greatest with the 214 °C/min rate. There were no differences in morphology of spermatozoa among the protocols, but the proportions of viable spermatozoa and of undamaged acrosomes were highest for the 12 and 28 °C/min rates. In Experiment 2 the capacity of fresh spermatozoa to penetrate fresh, cooled or salt-stored canine oocytes in vitro was evaluated. Canine oocytes harvested from ovaries were either immediately placed into TALP (fresh ova), or into hypertonic salt solution (saltstored ova): for cooled oocytes the ovaries were stored in PBS with BSA at 5 °C overnight before the ova were harvested. Oocytes and spermatozoa were co-incubated for 24 h, stained with Hoechst 33258 and examined under fluorescent light at x 400. Penetration and attachment of spermatozoa to the zona did not differ significantly between fresh and cooled oocytes (73 and 65%; 4.1 and 2.9 sperm/ovum, respectively), but cooled ova tended to show fewer attached spermatozoa. Salt-stored oocytes showed the lowest number of spermatozoa penetrating or attaching to the zona (0.7 sperm/ovum; P

Characterization of canine embryonic stem cell lines derived from different niche microenvironments.

Embryo-derived stem cells hold enormous potential for producing cell-based transplantation therapies, allowing high-throughput drug screening and delineating early embryonic development. However, potential clinical applications must first be tested for safety and efficacy in preclinical animal models. Due to physiological and genetic parity to humans, the domestic dog is widely used as a clinically relevant animal model for cardiovascular, neurodegenerative, orthopedic, and oncologic diseases. Therefore, we established numerous putative canine embryonic stem cell (cESC) lines by immunodissection of the inner cell mass (ICM), which we termed OVC.ID.1-23, and by explant outgrowths from whole canine blastocysts, named OVC.EX.1-16. All characterized lines were immunopositive for OCT4, SOX2, NANOG, SSEA-3, and SSEA-4; displayed high telomerase and alkaline phosphatase (ALP) activities; and were maintained in this state up to 37 passages ( approximately 160 days). Colonies from OVC.EX lines showed classic domed hESC-like morphology surrounded by a ring of fibroblast-like cells, whereas all OVC.ID lines exhibited a mixed cell colony of tightly packed cESCs surrounded by a GATA6+/CDX2- hypoblast-derived support layer. Spontaneous serum-only differentiation without feeder layers demonstrated a strong lineage selection associated with the colony niche type, and not the isolation method. Upon differentiation, cESC lines formed embryoid bodies (EB) comprised of cells representative of all germinal layers, and differentiated into cell types of each layer. Canine ESC lines such as these have the potential to identify differences between embryonic stem cell line derivations, and to develop or to test cell-based transplantation therapies in the dog before attempting human clinical trials.

Further development of a transcervical technique for artificial insemination in sheep using previously frozen semen

A transcervical technique (the Guelph System for transcervical AI) was used to inseminate 2060 ewes on 65 farms (average 31 ewes, range 5 to 107) in Ontario, Canada, from October 1990 to September 1992, using previously frozen semen. Estrus was synchronized using progestagen pessaries and PMSG with median inseminations done at 54 h from pessary removal. Maiden ewes were not included. Only ewes in which the cervix could be penetrated were inseminated with 150 million spermatozoa per insemination. A total of 1809 were penetrated and inseminated (penetration rate 87.8%). Success of penetration increased from 76.3% in the first 500 ewes to 97.9% in the last 500 (P=0.01). Cervical penetration was more successful in ewes in the accelerated lambing program (92.3%, average 3.1 mo since the previous lambing) than those in the annual lambing program (82.4%, average 7.0 mo since the previous lambing; P=0.06). The lambing rate for ewes bred during the combined traditional breeding seasons (Fall of 1990, 1991, 1992) was 50.7% compared to 24.4% for ewes bred at other periods (P=0.00001). The average time required for handling and insemination decreased from 8.62 min in the first 500 ewes to 3.62 min in the last 500 ewes. The Guelph System for Transcervical AI was found to be successful for cervical penetration in most ewes. Penetration success was affected by period since the last lambing and by inseminator experience. The lambing rate was higher for ewes bred during the traditional Fall breeding seasons than during other times of the year.

In Utero Injection of α-L-Iduronidase-Carrying Retrovirus in Canine Mucopolysaccharidosis Type I: Infection of Multiple Tissues and Neonatal Gene Expression

Canine alpha-L-iduronidase (alpha-ID) deficiency is caused by a single base pair mutation in the alpha-ID gene, resulting in no enzyme activity in homozygous affected pups. The disease clinically resembles human mucopolysaccharidosis type I (MPSI). We used the canine MPSI model system to address the efficacy of a new retroviral vector, MND-MFG, containing the human alpha-ID cDNA (MND-MFG-alpha-ID) for direct in utero gene delivery to MPSI cells. In vitro, the MND-MFG-alpha-ID vector showed high-level, long-term expression of the transgene in both canine and human alpha-ID-deficient fibroblasts. The effectiveness of this vector for in utero gene transfer and expression in multiple tissues was assessed by injecting viral supernatants into MPSI fetuses and evaluating transduction efficiency and enzyme expression at various times after birth. Transduction of a spectrum of cell types and tissues was observed in all seven live-born pups and in one stillborn pup. Although enzyme activity was not detected in adult tissues from the seven surviving pups, significant alpha-ID enzyme activity was detected in both the liver and kidney of the deceased pup. Our combined gene delivery vector and in utero transfer approach, while encouraging in terms of overall gene transfer efficiency to multiple tissues and successful short-term gene expression, was unable to meet the important requirement of sustained in vivo gene expression.

Endocrine and behavioral events of estrous cyclicity and synchronization in wood bison ( Bison bison athabascae )

Abstract This study was designed to noninvasively characterize estrous cycles and evaluate the effectiveness of estrous cycle synchronization protocols in wood bison. Daily urine and fecal samples and behavioral observations were collected over 2 breeding seasons from a captive herd of 18 adult, female wood bison. All samples were analyzed by enzymeimmunoassay for pregnanediol-glucuronide (PdG: urine) or progesterone (P4: feces). In Year 1, animals were divided into 3 treatment groups: 1) natural estrous controls, n = 6; 2) PG, 2 injections of cloprostenol, 11 d apart, n = 6; 3) SMB, Syncro-Mate-B implants left in place for 9 d with an estradiol valerate injection at the time of implantation, n = 6. In Year 2, control animals remained the same and synchronization regimens were repeated, but with each animal receiving alternate treatments. In both years, 11 d after treatment cessation, females were subjected to ultrasound examination to evaluate ovarian activity. Combined endocrine and behavioral data from natural estrous cycles demonstrated seasonal effects, with mean cycle lengths of 20.8 ± 0.3, 21.5 ± 0.5 and 21.1 ± 0.4 d, based on behaviors, PdG and P4 respectively. Females which demonstrated behavioral estrus did so within 4 (SMB, 11 12 ) or 5 (PG, 10 12 ) days after treatment. The SMB-treated animals exhibited estrous behavior within a 3-d span, with 55% exhibiting estrus 3 d after treatment ended, while the onset of estrus in PG-treated animals occurred over 4 d, with only 40% occurring on a single day. At the second estrus, 77% of SMB-treated animals and 74% of PG-animals demonstrated estrous behavior within 4 d, while at the third estrus, 88 and 90% of treated animals showed estrous behavior within 3 or 4 d, respectively. Ultrasound assessment revealed that only 50% ( 12 24 ) of treated animals possessed corpora lutea (CL), 67% in PG-treated females and 33% in SMB-animals. The total number of CL tended to be greater in PG- (10) than in SMB-(5) treated animals. Females with no CL demonstrated attenuated progestin profiles compared to those with ovulations. Additionally, a total of 28 follicles was detected, with a greater proportion (P

Adoptive transfer of genetically modified human hematopoietic stem cells into preimmune canine fetuses

To develop a surrogate model system for assaying gene transfer into human hematopoietic stem cells (HSCs) with in vivo repopulating potential, we injected human marrow cells transduced with a reporter retroviral vector in long-term marrow cultures (LTMCs), into the yolk sacs of preimmune canine fetuses. Of eight mid-gestation fetuses injected through the exteriorized uterine wall and under ultrasound guidance, seven were born alive. One puppy died in the neonatal period accidentally. The remaining six puppies are all healthy at 31 months of age. There was no evidence for graft-versus-host disease or any untoward effects of in utero adoptive transfer of transduced human LTMC cells. All puppies were chimeras. Human cells, detected by fluorescence in situ hybridization, were present in blood, declining from 38% to 0.05% between 10 and 44 weeks after birth. Corresponding numbers for marrow were from 20% to 0.05%. Human cells were also detected in assays of hematopoietic cell progenitors and in stimulated blood cultures. All six puppies were positive for the presence of proviral DNA at various time-points after birth. In three dogs, provirus was detected up to 41 weeks after birth in blood or marrow, and in one dog up to 49 weeks in blood. These data support the further development of this large-animal model system for studies of human hematopoiesis.

Hormonal control of estrouscyclicity and attempted superovulation in wood bison (Bison bison athabascae)

The wood bison (Bison bison athabascae) is a threatened Canadian species that has faced extinction twice in the last 100 yr. Development of assisted reproductive technologies could help ensure the long-term propagation and genetic management of this species. The objectives of this study were to refine estrus synchronization techniques and evaluate superovulatory responses after FSH or eCG administration. In Experiment 1, females were fitted with Syncro-mate B (SMB) implants for 9 d and received an injection of either estradiol valerate (E2V; n = 9) or cloprostenol (PGF; n = 9) at implant insertion (Day-9). In Experiment 2, estrus was synchronized with SMB implants and a PGF injection of Day-9, and superovulation was attempted on Day-2 with either 2500 IU eCG (n = 5) or 400 mg Folltropin-V (n = 5). In each experiment, biosin were examined daily for estrual behavior. Ultrasonography was used during the luteal phase to detect ovulation and assess ovarian status; feces were analyzed by ELISA for immunoreactive progestogens (P) to study ovarian endocrine responses. In Experiment 1, a closer synchrony of estrus was observed between Days 2 to 4 among the PGF-treated (77.8%) than the E2V-treated (66.7%) females. Corpora lutea (CL) were detected in 55% of E2V- and PGF-treated females. In Experiment 2, neither treatment successfully induced superovulation, with only a single female per treatment producing > or = 1 CL. In both experiments, progestogen profiles were similar for each treatment (P < 0.05).

Assessing reproductive patterns and disorders in free-ranging dogs in Jodhpur, India to optimize a population control program.

The objectives were to test the hypothesis that estrus and pregnancy are seasonal in free-ranging female dogs (>3 mo old) in Jodhpur, India, and to determine litter size, and the prevalence of fetal resorption in this population. The prevalence of estrus and pregnancy was determined in 5400 free-ranging bitches (trapped and released) at the time of ovariohysterectomy. In a separate study, the uteri and ovaries of 246 free-ranging bitches were examined to determine litter size and fetal resorption. The bitches exhibited seasonal estrus and pregnancy (P < 0.00001), with a higher percentage of bitches in estrus or pregnant during the late monsoon season (September to November) compared to the other three seasons. The mean litter size based on embryo/fetal counts was 4.6 (95% CI = 4.0-5.3; n = 40) and based upon placental site counts was 4.4 (95% CI = 3.9-4.8; n = 105). Prevalence of fetal resorption was 32.6% (95% CI = 20.5-47.5; n = 43) with a mean of 2.8 resorptions per litter in those with at least one resorption (95% CI = 1.8-3.8; n = 14). This was the first study to estimate previous litter size of non-pregnant, free-ranging dogs based upon placental sites. Litter size data from this study will be used in a population demographic model to predict the long-term impact of animal birth control (ABC) on the free-ranging dog population in Jodhpur. Increasing the efforts to surgically sterilize bitches prior to the time of year of peak pregnancy or whelping will help maximize the impact of an ABC program on the Jodhpur free-ranging dog population.

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.