Differentiation of Human-Induced Pluripotent Stem Cell-Derived Endocrine Progenitors to Islet-like Cells Using a Dialysis Suspension Culture System

The production of functional islet-like cells from human-induced pluripotent stem cells (hiPSCs) is a promising strategy for the therapeutic use and disease modeling for type 1 diabetes. However, the production cost of islet-like cells is extremely high due to the use of expensive growth factors for differentiation. In a conventional culture method, growth factors and beneficial autocrine factors remaining in the culture medium are removed along with toxic metabolites during the medium change, and it limits the efficient utilization of those factors. In this study, we demonstrated that the dialysis suspension culture system is possible to reduce the usage of growth factors to one-third in the differentiation of hiPSC-derived endocrine progenitor cells to islet-like cells by reducing the medium change frequency with the refinement of the culture medium. Furthermore, the expression levels of hormone-secretion-related genes and the efficiency of differentiation were improved with the dialysis suspension culture system, possibly due to the retaining of autocrine factors. In addition, we confirmed several improvements required for the further study of the dialysis culture system. These findings showed the promising possibility of the dialysis suspension culture system for the low-cost production of islet-like cells.

The chemokine CXCL14 mediates platelet function and migration via direct interaction with CXCR4

Aims Beyond classical roles in thrombosis and haemostasis, it becomes increasingly clear that platelets contribute as key players to inflammatory processes. The involvement of platelets in these processes is often mediated through a variety of platelet-derived chemokines which are released upon activation and act as paracrine and autocrine factors. In this study, we investigate CXCL14, a newly described platelet chemokine and its role in thrombus formation as well as monocyte and platelet migration. In addition, we examine the chemokine receptor CXCR4 as a possible receptor for CXCL14 on platelets. Furthermore, with the use of artificially generated platelets derived from induced pluripotent stem cells (iPSC), we investigate the importance of CXCR4 for CXCL14-mediated platelet functions. Methods and results In this study, we showed that CXCL14 deficient platelets reveal reduced thrombus formation under flow compared with wild-type platelets using a standardized flow chamber. Addition of recombinant CXCL14 normalized platelet-dependent thrombus formation on collagen. Furthermore, we found that CXCL14 is a chemoattractant for platelets and mediates migration via CXCR4. CXCL14 promotes platelet migration of platelets through the receptor CXCR4 as evidenced by murine CXCR4-deficient platelets and human iPSC-derived cultured platelets deficient in CXCR4. We found that CXCL14 directly interacts with the CXCR4 as verified by immunoprecipitation and confocal microscopy. Conclusions Our results reveal CXCL14 as a novel platelet-derived chemokine that is involved in thrombus formation and platelet migration. Furthermore, we identified CXCR4 as principal receptor for CXCL14, an interaction promoting platelet migration.

Comparison of In Vitro and In Situ Methods for Studying Lipolysis

Lipolysis is a highly regulated process and is controlled by nervous system, hormones, and paracrine/autocrine factors. Dysregulation of lipolysis is associated with some pathophysiological conditions including diabetes, metabolic syndrome, and obesity. Nowadays, special attention isthereforepaid to study lipolysis using different experimental models. This review summarizes the current experimental methods for studying lipolysis. Culture of preadipocyte cell lines, use of differentiated stroma-vascular cells, primary culture of adipocyte, organ culture of adipose tissue, and microdialysis technique are the most widely used techniques to study lipolysis. The advantages and limitations of using these methods are discussed.

Estrogens and Spermiogenesis: New Insights from Type 1 Cannabinoid Receptor Knockout Mice

Spermatogenesis is a complex mechanism which allows the production of male gametes; it consists of mitotic, meiotic, and differentiation phases. Spermiogenesis is the terminal differentiation process during which haploid round spermatids undergo several biochemical and morphological changes, including extensive remodelling of chromatin and nuclear shape. Spermiogenesis is under control of endocrine, paracrine, and autocrine factors, like gonadotropins and testosterone. More recently, emerging pieces of evidence are suggesting that, among these factors, estrogens may have a role. To date, this is a matter of debate and concern because of the agonistic and antagonistic estrogenic effects that environmental chemicals may have on animal and human with damaging outcome on fertility. In this review, we summarize data which fuel this debate, with a particular attention to our recent results, obtained using type 1 cannabinoid receptor knockout male mice as animal model.

141 BOVINE EMBRYO CULTURE IN THE MODIFIED WELL OF THE WELL SYSTEM INCREASES BLASTOCYST RATE BUT LOWERS BLASTOCYST QUALITY

Individual culture of cattle embryos in serum-containing medium has a detrimental effect on blastocyst development. As we previously demonstrated, this effect is not caused by the individual culture itself but by some component present in serum (Heras et al. AETE Proc. 2012). The high blastocyst rates achieved with group culture in serum-containing medium can be explained by the neutralizing effect of the produced autocrine factors. Autocrine factors produced by individually cultured embryos cannot neutralize the detrimental component due to its low amount and their diffusion in the high culture volume. Using a modified well of the well (mWOW; Vajta et al. 2000 Mol. Rep. Dev.), where embryos are singly cultured in a small well, the effect of the autocrine factors produced by individual embryos can be maximized. We hypothesized that the use of mWOW for individual embryo culture in serum-containing medium, can maximize the neutralizing effect of the autocrine factors, increasing both blastocyst rate and quality. Bovine oocytes (n = 1143, 3 replicates) were matured in TCM-199 with 20% fetal bovine serum (FBS). Presumptive zygotes were cultured in groups (Gr; 25 embryos/50-µL drops), individually (Ind; 20-µL drops), or in mWOW (20-µL drops) in SOFaa with 5% FBS. Blastocyst evaluation occurred 7 and 8 days post-insemination (dpi). Hatching rate is the proportion of hatching/hatched blastocysts out of total 8-dpi blastocysts. Blastocysts were collected at 8 dpi for differential apoptotic staining, evaluating total cell number (TCN), inner cell mass (ICM) ratio, and apoptosis (ACR; Wydooghe et al. 2011 Anal. Biol.). Developmental data were analyzed using binary logistic regression; data concerning blastocyst quality were analyzed using linear mixed model analysis. Since ACR was not normally distributed, a logarithmic transformation was performed. Differences at P < 0.05 were considered significant. Regarding blastocyst development, all groups were significantly different both at 7 dpi (Gr 33.9%, Ind 10.7%, mWOW 19.6%) and at 8 dpi (Gr 37.3%, Ind 14.0%, mWOW 29.1%). Hatching rates were significantly higher in Gr (27.9%) compared to Ind (4.0%) and mWOW (4.1%). Concerning blastocyst quality, TCN was significantly higher (P < 0.001) in Gr (233.23 ± 8.39), no differences between Ind (143.77 ± 6.32) and mWOW (125.11 ± 4.30) were observed. No differences in ICM ratio were observed (Gr 0.41 ± 0.01, Ind 0.30 ± 0.02, mWOW 0.37 ± 0.02). ACR was significantly different between all groups (Gr 0.03 ± 0.002, Ind 0.04 ± 0.003, mWOW 0.08 ± 0.005). In conclusion, the mWOW had a positive effect on blastocyst quantity, increasing blastocyst rates compared to individual culture, but still lower than in group culture. Surprisingly, mWOW had a negative effect on blastocyst quality, producing the highest ACR and the lowest TCN. This negative effect can be caused by toxic products arising from the manufacture (although the wells were rigorously flushed), or by the longer manipulation time needed. This work was funded by FWO G.0210.09N and IWT g111438.

234 PREVALENCE OF MEMBRANE-DAMAGED CELLS IN BOVINE EMBRYOS AFTER CULTURE TO THE BLASTOCYST STAGE IN DEFINED MEDIUM

Autocrine factors are believed to be the reason why embryos cultured in a group show superior development compared with a single culture in many species. A widespread concern with group culture is that degenerating embryos may have a negative effect on the other embryos. Previously, we were able to identify some proteins in embryo-conditioned medium by using tandem mass spectrometry, predominantly stress-related proteins. Our hypothesis was that poor-quality embryos with membrane-damaged cells passively released these identified proteins into the medium. Therefore, we wanted to investigate what percentage of membrane-damaged cells occurred in an average population of Day 8 bovine embryos. Bovine presumed zygotes (n = 606) were cultured individually in 20-µL drops of modified SOF supplemented with insulin, transferrin, and selenium and 0.1 mg mL–1 of polyvinylpyrrolidone. At 8 days post-insemination, blastocyst development was evaluated, and subsequently, all embryos were stained with Hoechst/propidium iodide. In this way, we were able to detect the total cell number (TCN) and the percentage of membrane-damaged cells (MDC ratio) because propidium iodide will stain only the nuclei of membrane-damaged cells pink. Embryo-conditioned medium was collected individually and kept at –80°C until further analysis. At 8 days post-insemination, a blastocyst rate of 27.5% was observed. After determination of the TCN per embryo, we divided the embryos in two groups: 207 embryos had a TCN of more than 64 cells (normal development; range: 64 to 313 cells) and 399 embryos had a TCN of fewer than 64 cells (arrested development; range: 1 to 61 cells). In the first group, 76.3% (138 embryos) had an MDC ratio of less than 10%; however, only 11.1% (23 embryos) showed no membrane-damaged cells at all. In the group with arrested development, only 17.8% (71 embryos) of the embryos had an MDC ratio of less than 10%, and only 13.5% (54 embryos) had no membrane-damaged cells. Of the delayed embryos, 48.6% (194 embryos) showed an MDC ratio of more than 50%, and 34.5% (138 embryos) displayed even 100% of membrane-damaged cells. In conclusion, by use of Hoechst/propidium iodide staining, we were able to detect the TCN and MDC ratio of the embryos. We observed that only a small percentage of the embryos had an MDC ratio of 0%, even if the embryos had more than 64 cells. Almost half of the arrested embryos with fewer than 64 cells had a MDC ratio of more than 50%, indicating that poor-quality embryos consist predominantly of membrane-damaged cells that can passively release proteins into the medium, with a possible negative effect on the surrounding embryos in group culture. It must be noted, however, that for logistical reasons, these results were obtained in a single culture. Further research will be done to identify the candidate autocrine factors and proteins released into the medium by poor-quality embryos by analysing embryo-conditioned medium pooled according to the results of the Hoechst/propidium iodide staining.

Autocrine regulation of biliary pathology by activated cholangiocytes

The bile duct system of the liver is lined by epithelial cells (i.e., cholangiocytes) that respond to a large number of neuroendocrine factors through alterations in their proliferative activities and the subsequent modification of the microenvironment. As such, activation of biliary proliferation compensates for the loss of cholangiocytes due to apoptosis and slows the progression of toxic injury and cholestasis. Over the course of the last three decades, much progress has been made in identifying the factors that trigger the biliary epithelium to remodel and grow. Because a large number of autocrine factors have recently been identified as relevant clinical targets, a compiled review of their contributions and function in cholestatic liver diseases would be beneficial. In this context, it is important to define the specific processes triggered by autocrine factors that promote cholangiocytes to proliferate, activate neighboring cells, and ultimately lead to extracellular matrix deposition. In this review, we discuss the role of each of the known autocrine factors with particular emphasis on proliferation and fibrogenesis. Because many of these molecules interact with one another throughout the progression of liver fibrosis, a model speculating their involvement in the progression of cholestatic liver disease is also presented.