Derek Toms

Epidermal Growth Factor-Expressing Lactococcus lactis Enhances Intestinal Development of Early-Weaned Pigs

Stress and incomplete gastrointestinal development in early-weaned piglets represent significant challenges in commercial swine farming. Orally ingested recombinant epidermal growth factor (EGF) has been shown to remain biologically active in the gastrointestinal tract as well as stimulate intestinal development, reducing the incidence of pathogen infection and diarrhea. We have previously shown that the food-grade bacterium Lactococcus lactis can be genetically altered to express biologically active EGF when fed to early-weaned mice. In this study, we assigned 8 pigs to each of 4 groups that were given EGF-expressing L. lactis (EGF-LL), empty vector-expressing L. lactis (EV-LL), recombinant human EGF, or unsupplemented bacterial media, all of which were delivered as 50-mL i.g. doses twice per day. All pigs were killed after 14 d to examine intestinal morphology. Pigs in the EGF-LL group had greater jejunal and duodenal villus heights (P < 0.0001) and intestinal length (P = 0.049) than pigs in the control group. Immunohistochemistry with antibodies against proliferating cell nuclear antigen (PCNA) revealed that the proliferation of intestinal cells was significantly greater in the EGF-LL group than in the control group. PCNA expression and intestinal length also were greater in the EV-LL group, which received L. lactis that did not express EGF, than in the control group (P = 0.049), further supporting the use of naturally occurring intestinal microbes as desirable vectors for recombinant protein delivery. Our data demonstrates the feasibility of delivering a growth factor using common probiotic bacteria to farm animals for commercial practice.

MicroRNA-378 regulates oocyte maturation via the suppression of aromatase in porcine cumulus cells

We sought to investigate whether miR-378 plays a role in cumulus cells and whether the manipulation of miRNA levels in cumulus cells influences oocyte maturation in vitro. Cumulus-oocyte complexes (COCs) from ovarian follicles had significantly lower levels of precursor and mature miR-378 in cumulus cells surrounding metaphase II (MII) oocytes than cumulus cells surrounding germinal vesicle (GV) oocytes, suggesting a possible role of miR-378 during COC maturation. Overexpression of miR-378 in cumulus cells impaired expansion and decreased expression of genes associated with expansion (HAS2, PTGS2) and oocyte maturation (CX43, ADAMTS1, PGR). Cumulus cell expression of miR-378 also suppressed oocyte progression from the GV to MII stage (from 54 ± 2.7 to 31 ± 5.1%), accompanied by a decrease of growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), zona pellucida 3 (ZP3), and CX37 in the oocytes. Subsequent in vitro fertilization resulted in fewer oocytes from COCs overexpressing miR-378 reaching the blastocyst stage (7.3 ± 0.7 vs. 16.6 ± 0.5%). miR-378 knockdown led to increased cumulus expansion and oocyte progression to MII, confirming a specific effect of miR-378 in suppressing COC maturation. Aromatase (CYP19A1) expression in cumulus cells was also inhibited by miR-378, leading to a significant decrease in estradiol production. The addition of estradiol to IVM culture medium reversed the effect of miR-378 on cumulus expansion and oocyte meiotic progression, suggesting that decreased estradiol production via suppression of aromatase may be one of the mechanisms by which miR-378 regulates the maturation of COCs. Our data suggest that miR-378 alters gene expression and function in cumulus cells and influences oocyte maturation, possibly via oocyte-cumulus interaction and paracrine regulation.

Midkine Promotes Proliferation of Primordial Germ Cells by Inhibiting the Expression of the Deleted in Azoospermia-Like Gene

Primordial germ cell (PGC) development is an area of research that is hampered by low cell numbers as well as difficulty in isolation. They are, however, required for the production of gametes and as such represent an important area of understanding that has widespread implications for fertility and reproductive technologies. Here we investigated the role of the heparin-binding growth factor midkine (MK) on PGC development, first using our established model of porcine stem cell-derived PGC-like cells and then confirming our findings in PGC. Our results show that MK has a mitogenic effect on PGC, mediated through an increased cell proliferation as well as decreased apoptosis. Upon further investigation, we found these effects concomitant with the decreased expression of the germ cell-specific gene deleted in azoospermia-like (DAZL). This decrease in DAZL expression, and consequent decreases in the meiosis-related genes SCP3 and DMC1, suggest a role for MK in preventing a shift in the PGC phenotype toward meiosis. MK instead increases activity of mitotic pathways in PGC, keeping them in a proliferative, less differentiated state. Lentiviral-mediated overexpression of DAZL further confirmed its role in promoting meiosis in and reducing proliferation of PGC. These effects were mitigated by the addition of MK, which was able to limit the effect of this DAZL overexpression. Furthermore, a loss-of-function study showed that a DAZL knockdown by small interfering RNA had the same effect as that induced by the addition of MK. Taken together, these data suggest that MK is able to maintain a proliferative PGC phenotype mediated by the suppression of DAZL in early germ cells.

The effects of glial cell line-derived neurotrophic factor on the in vitro matured porcine oocyte transcriptome

It is well documented that oocytes from small antral follicles are less competent than those derived from large follicles, and we have previously shown that glial cell line‐derived neurotrophic factor (GDNF) enhances developmental competence in oocytes from antral follicles. Exactly how GDNF effects this change and if it depends on the stage of oocyte development is currently unknown. The objective of this study was to examine the transcriptomic effects of follicle size and GDNF on the in vitro maturation of porcine oocytes. Microarray analysis uncovered differentially expressed transcripts among in vitro‐matured porcine oocytes from different‐size antral follicles, in the absence or presence of GDNF. Oocytes isolated from small follicles showed a lower state of maturation than those from large follicles, with several transcripts associated with meiotic arrest. Addition of GDNF to the culture media had effects that depended on the stage of the follicle from which the oocyte was isolated, with those from small follicles showing decreased expression of genes associated with acetyltransferase activity while those from large follicles showed decreased metabolic activity. In summary, our results revealed considerable differences between the transcriptomes of small‐ and large‐follicle‐derived oocytes. Furthermore, GDNF affects the developmental competence of oocytes in follicle‐stage dependent manner. Thus, improving our understanding of the requirements for successful in vitro maturation of porcine oocytes will inform current reproductive technologies, with implications for the future of animal and human health. Mol. Reprod. Dev. 2014.

Ovarian-Cell-Like Cells from Skin Stem Cells Restored Estradiol Production and Estrus Cycling in Ovariectomized Mice

Reduction of estradiol production and high serum concentrations of follicular stimulating hormone (FSH) are endocrine disorders associated with premature ovarian failure. Here, we report that transplantation of ovarian-like cells differentiated from stem cells restored endogenous serum estradiol levels. Stem cells were isolated from postnatal mouse skin and differentiated into ovarian-cell-like cells that are consistent with female germ, and ovarian follicle somatic cells. The ovarian-cell-like cells were transplanted into ovariectomized mice (Cell Trans), whereas control mice were subjected to bilateral ovariectomies without cell transplantation (OVX). Using vaginal cytology analysis, it was revealed that in 13 out of 19 Cell Trans mice, estrus cycles were restored around 8 weeks after cell transplantation and were maintained until 16 weeks post-transplantation, whereas in the OVX group, all mice were arrested at metestrus/diestrus of the estrus cycle. The uterine weight in the Cell Trans group was similar to sham operation mice (Sham OP), while severe uterine atrophy and a decreased uterine weight were observed in the OVX group. Histologically, ectopic follicle-like structures and blood vessels were found within and around the transplants. At 12-14 weeks after cell transplantation, mean serum estradiol level in Cell Trans mice (178.0±35 pg/mL) was comparable to that of the Sham OP group (188.9±29 pg/mL), whereas it was lower in the OVX group (59.0±4 pg/mL). Serum FSH concentration increased in the OVX group (1.62±0.32 ng/mL) compared with the Sham OP group (0.39±0.34 ng/mL). Cell Trans mice had a similar FSH level (0.94±0.23 ng/mL; P<0.05) to Sham OP mice. Our results suggest that ovarian somatic cells differentiated from stem cells are functional in vivo. In addition to providing insights into the function of ovarian somatic cells derived from stem cells, our study may offer potential therapeutic means for patients with hypo-estradiol levels like those encountered in premature ovarian failure.

Oxygenation in cell culture: Critical parameters for reproducibility are routinely not reported

Mammalian cell culture is foundational to biomedical research, and the reproducibility of research findings across the sciences is drawing increasing attention. While many components contribute to reproducibility, the reporting of factors that impact oxygen delivery in the general biomedical literature has the potential for both significant impact, and immediate improvement. The relationship between the oxygen consumption rate of cells and the diffusive delivery of oxygen through the overlying medium layer means parameters such as medium depth and cell type can cause significant differences in oxygenation for cultures nominally maintained under the same conditions. While oxygenation levels are widely understood to significantly impact the phenotype of cultured cells in the abstract, in practise the importance of the above parameters does not appear to be well recognized in the non-specialist research community. On analyzing two hundred articles from high-impact journals we find a large majority missing at least one key piece of information necessary to ensure consistency in replication. We propose that explicitly reporting these values should be a requirement for publication.

Endocrine Regulation in the Ovary by MicroRNA during the Estrous Cycle

Hormonal control of the estrous cycle that occurs in therian mammals is essential for the production of a functional egg. Supporting somatic cell types found within the ovary, such as granulosa and theca cells, respond to endocrine signals to support oocyte maturation and ovulation. Following the release of the egg, now available for fertilization, coordinated hormonal signaling between the mother and putative embryo are required for the establishment of pregnancy. If no conception occurs, both the ovary and uterus are “reset” in preparation for another cycle. The complex molecular changes that occur within cells in response to hormone signaling include a network of non-coding microRNAs (miRNAs) that posttranscriptionally regulate gene expression. They are thus able to fine-tune cellular responses to hormones and confer robustness in gene regulation. In this review, we outline the important roles established for miRNAs in regulating female reproductive hormone signaling during estrus, with a particular focus on signaling pathways in the ovary. Understanding this miRNA network can provide important insights to improving assisted reproductive technologies and may be useful in the diagnosis of female reproductive disorders.

Climbing the mountain: experimental design for the efficient optimization of stem cell bioprocessing

Abstract“To consult the statistician after an experiment is finished is often merely to ask him to conduct a post mortem examination. He can perhaps say what the experiment died of.” – R.A. Fisher While this idea is relevant across research scales, its importance becomes critical when dealing with the inherently large, complex and expensive process of preparing material for cell-based therapies (CBTs). Effective and economically viable CBTs will depend on the establishment of optimized protocols for the production of the necessary cell types. Our ability to do this will depend in turn on the capacity to efficiently search through a multi-dimensional problem space of possible protocols in a timely and cost-effective manner. In this review we discuss approaches to, and illustrate examples of the application of statistical design of experiments to stem cell bioprocess optimization.

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Effects of Enzyme Complex Supplementation to a Paddy-based Diet on Performance and Nutrient Digestibility of Meat-type Ducks

Paddy rice is rarely used as a feed because of its high fiber content. In this study, two experiments were conducted to study the effects of supplementing an enzyme complex consisting of xylanase, beta-glucanase and cellulase, to paddy-based diets on the performance and nutrient digestibility in meat-type ducks. In the both experiments, meat-type ducks (Cherry Valley) were randomly assigned to four treatments. Treatment 1 was a basal diet of corn-soybean; treatment 2 was a basal diet of corn-paddy-soybean; treatment 3, had enzyme complex added to the corn-paddy-soybean basal diet at levels of 0.5 g/kg diet; and treatment 4, had enzyme complex added to the corn-paddy-soybean diet at levels of 1.0 g/kg diet. The results showed that the enzyme complex increased the ADG, and decreased the ADFI and F/G significantly (p<0.05) in the ducks, and the ADFI for the ducks fed the corn-paddy-soybean diet showed no difference compared to the ducks fed corn-soybean diets at all stages of the experiment (p<0.05). When corn was partially replaced by paddy, the digestibility of CP and NDF was decreased and increased, respectively (p<0.05), and the level of enzyme complex had a significant effect on both CP and NDF digestibility (p<0.05). As for the AME, addition of enzyme complex increased it significantly (p<0.05), but both diet types and levels of enzyme complex had no effect (p>0.05). The outcome of this research indicates that the application of enzyme complex made up of xylanase, beta-glucanase, and cellulase, in the corn-paddy-soybean diet, can improve performance and nutrition digestibility in meat-type ducks.