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 ...

University of Calgary

The aim of this study was to determine the impact of barium sulfate on remodeling and regeneration in standard tibial defects in rabbits treated with the Norian skeletal repair system (SRS). Two formulations of SRS (with and without barium sulfate) were injected into the medullary canal of the tibia of New Zealand white rabbits. Animals were sacrificed at 6 weeks, 6 months, 1 year, and 2 years. Over the 2-year duration of the study, standard SRS and SRS with barium sulfate appeared to be biocompatible and osteoconductive with no evidence of either inflammation or fibrous tissue around the implant materials or at the bone-material interfaces. This outcome underscores the osteophilic property of the SRS. A difference we observed between the standard SRS and the SRS with barium sulfate was the appearance of acellular material contiguous to the SRS with barium sulfate. Energy dispersive X-ray spectroscopy (EDX) analysis was conducted and confirmed that the acellular material was barium sulfate. Pathological examination of additional tissues including regional lymph nodes revealed neither dissemination of calcium phosphate nor barium sulfate. We concluded that the residual barium sulfate detected by EDX was localized to the intramedullary canal of the tibia.

Bone healing response to an injectable calcium phosphate cement with enhanced radiopacity.

Allograft tissues are used in over one million musculoskeletal procedures per year. Consequently, it is crucial tissue banks use procedures to militate against allograft associated bacterial and viral infections. Recent studies have identified an important pathogen inactivation technology for musculoskeletal allografts that utilizes high-dose gamma irradiation (50 kGy) under controlled conditions. A total dose of 50 kGy assures that the current standard for medical devices for a microbial sterility assurance level of 10(- 6) is met. Furthermore, the pathogen inactivation technology results in a greater than four log inactivation of enveloped and nonenveloped viruses. Efficacious clinical outcome from musculoskeletal allografts exposed to this innovative sterilization procedure will require that there is no performance decrement in the allograft's biological properties. Therefore, to validate this objective, we executed a study focusing on remodeling and osteoconduction of bone allografts treated with a high dose of gamma irradiation (50 kGy), radioprotectants and well-defined operating parameters of temperature and water content. A rabbit calvarial model was used to test the hypothesis that remodeling and osteoconduction of allogeneic bone treated with the new pathogen inactivation technology would be equivalent to nontreated allogeneic bone. Results indicated treated bone allografts were comparable to nontreated allografts. We conclude, therefore, that based on this outcome and other reports, that high doses of gamma irradiation under optimized conditions designed to reduce free radical damage to tissue will provide safer allografts.

Response of bone subjected to optimized high dose irradiation.

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.

Epidermal growth factor-expressing Lactococcus lactis enhances intestinal development of early-weaned pigs.

The study of primordial germ cell development in vivo is hampered by their low numbers and inaccessibility. Recent research has shown the ability of embryonic and adult stem cells to differentiate into primordial germ cells and more mature gametes and this generation of germ cells in vitro may be an attractive model for their study. One of the biggest challenges facing in vitro differentiation of stem cells into primordial germ cells is the lack of markers to clearly distinguish the two. As both cell types originate early in embryonic development they share many pluripotent markers such as OCT4, VASA, FRAGILIS, and NANOG. Genome wide microarray profiling has been used to identify transcriptome patterns unique to primordial germ cells. A more thorough analysis of the temporal and quantitative expression of a panel of genes may be more robust in distinguishing these two cell populations. MicroRNAs, short RNA molecules that have been shown to regulate translation through interactions with mRNA transcripts, have also recently come under investigation for the role they may play in pluripotency. Attempts to elucidate key microRNAs responsible for both stem cell and primordial germ cell characteristics have recently been undertaken. Unique microRNAs, either individually or as global profiles, may also help to distinguish differentiated primordial germ cells from stem cells in vitro. This review will examine gene expression and microRNA signatures in stem cells and germ cells as ways to distinguish these closely related cell types.

Stem cells and germ cells: microRNA and gene expression signatures.

L&#39;&eacute;tude du d&eacute;veloppement des cellules germinales primordiales in vivo est entrav&eacute;e par leurs faibles effectifs et l&#39;inaccessibilit&eacute;. Recherches r&eacute;centes ont d&eacute;montr&eacute; la capacit&eacute; des cellules souches embryonnaires et adultes &agrave; se diff&eacute;rencier en cellules germinales primordiales et des gam&egrave;tes plus matures et cette g&eacute;n&eacute;ration de cellules germinales in vitro peuvent &ecirc;tre un mod&egrave;le attrayant pour leur &eacute;tude. Un des plus grands d&eacute;fis la diff&eacute;renciation in vitro des cellules souches en cellules germinales primordiales est l&#39;absence de marqueurs de distinguer clairement les deux. Comme les deux types de cellules sont originaires au d&eacute;but du d&eacute;veloppement embryonnaire, ils partagent de nombreux marqueurs pluripotentes comme OCT4, VASA, FRAGILIS et NANOG. Profilage de microarray large du g&eacute;nome a &eacute;t&eacute; utilis&eacute; pour d&eacute;finir les mod&egrave;les de transcriptome uniques de cellules germinales primordiales. Une analyse plus approfondie de l&#39;expression temporelle et quantitative d&#39;un groupe de g&egrave;nes peut &ecirc;tre plus robuste pour distinguer les populations de ces deux cellules. Micro-ARN, des mol&eacute;cules d&#39;ARN courts qui auraient d&ucirc; &ecirc;tre divulgu&eacute;s pour r&eacute;glementer la traduction gr&acirc;ce &agrave; des interactions avec la transcription de l&#39;ARNm, est venus r&eacute;cemment vis&eacute; par l&#39;enqu&ecirc;te pour le r&ocirc;le qu&#39;ils peuvent jouer dans la pluripotence. Tentatives d&#39;&eacute;lucider les microARN cl&eacute;s responsables de cellules souches et des caract&eacute;ristiques de cellules germinales primordiales ont r&eacute;cemment &eacute;t&eacute; entrepris. MicroARN unique, soit individuellement ou sous forme de profils globales, peut &eacute;galement aide &agrave; distinguer distingue les cellules germinales primordiales in vitro des cellules souches. Cet examen examinera les signatures expression et micro-ARN de g&egrave;ne dans les cellules souches et des cellules germinales comme moyens de distinguer ces types de cellules &eacute;troitement apparent&eacute;es.

Cellules souches et des cellules germinales : microARN et g&egrave;ne signatures d&#39;expression.

Lo studio dello sviluppo delle cellule germinali primordiali in vivo &egrave; ostacolato dai loro numeri bassi e inaccessibilit&agrave;. Recenti ricerche hanno dimostrato la capacit&agrave; delle cellule staminali adulte ed embrionali di differenziarsi in cellule germinali primordiali e gameti pi&ugrave; maturi e questa generazione di cellule germinali in vitro pu&ograve; essere un modello attraente per il loro studio. Una delle pi&ugrave; grandi sfide per la differenziazione in vitro di cellule staminali in cellule germinali primordiali &egrave; la mancanza di marcatori di distinguere chiaramente i due. Come entrambi i tipi di cellule provengono presto nello sviluppo embrionale che condividono molti marcatori pluripotenti come OCT4, VASA, FRAGILIS e NANOG. Genome wide microarray profiling &egrave; stato usato per identificare i modelli transcriptome unici a cellule germinali primordiali. Un&#39;analisi pi&ugrave; approfondita dell&#39;espressione temporale e quantitativa di un pannello di geni pu&ograve; essere pi&ugrave; affidabile nel distinguere queste popolazioni di due cellule. MicroRNA, brevi molecole di RNA che hanno dimostrati di regolare traduzione attraverso interazioni con trascritti di mRNA, anche recentemente &egrave; venuti sotto inchiesta per il ruolo che possono giocare in pluripotenza. Recentemente sono stati intrapresi tentativi per delucidare la chiave microRNA responsabile sia delle cellule staminali e le caratteristiche delle cellule germinali primordiali. Unico microRNA, sia individualmente o come profili globali, pu&ograve; anche aiuto per distinguere differenziato le cellule germinali primordiali dalle cellule staminali in vitro. Questa recensione esaminer&agrave; le firme espressione e microRNA genica in cellule staminali e cellule germinali come modi per distinguere questi tipi di cellule strettamente correlati.

Cellule staminali e le cellule germinali: le firme di espressione genica e microRNA.

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.

Midkine promotes proliferation of primordial germ cells by inhibiting the expression of the deleted in azoospermia-like gene.

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.

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

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.

Ovarian-cell-like cells from skin stem cells restored estradiol production and estrus cycling in ovariectomized mice.

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.

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

In developing ovarian follicles, the progesterone receptor (PGR) is essential for mediating transcription of key factors that coordinate cellular functions including follicular remodeling. With recent investigations examining the role of microRNA (miRNA) in regulating ovarian function we used a lentiviral approach to over express miR-378 in cultured primary porcine granulosa cells to study the role this miRNA may play in granulosa cell development. We revealed that miR-378-3p decreased protein levels and mRNA levels of PGR via targeting its 3'UTR. We observed that this regulation of PGR by miR-378-3p resulted in a corresponding decrease in gene transcripts of ADAMTS1, CTSL1, and PPARG, all known to be regulated by PGR and important for follicular maturation and remodeling. Our study provides the first evidence for post-transcriptional regulation of PGR and further elucidates the role of miR-378-3p in the ovary.

Progesterone receptor expression in granulosa cells is suppressed by microRNA-378-3p.

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.

Derek Toms

Department of Comparative Biology and Experimental Medicine

University of Calgary

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

Derek Toms

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Comparative Biology and Experimental Medicine

University of Calgary

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

Department of Comparative Biology and Experimental Medicine