Insulin increases epiblast cell number of in vitro cultured mouse embryos via the PI3K/GSK3/p53 pathway

High-quality embryos give rise to embryonic stem cells (ESCs) at greater efficiencies than poor-quality embryos. However, most embryos available for human ESC derivation are of a reduced quality as a result of culture in relatively simple media up to 10 years earlier, before cryopreservation, or before compaction. In the present study, we used a mouse model to determine whether a culture with insulin from the 8-cell stage could increase the number of ESC progenitor epiblast cells in blastocysts, as well as endeavor to determine the molecular mechanism of the insulin's effect. Culture in media containing 1.7 ρM insulin increased epiblast cell number (determined by Oct4 and Nanog co-expression), and proportion in day 6 blastocysts. The inhibition of phosphoinositide 3 kinase (PI3K) (via LY294002), an early second messenger of the insulin receptor, blocked this effect. The inhibition of glycogen synthase kinase 3 (GSK3) or p53, 2 s messengers inactivated by insulin signaling (via CT99021 or pifithrin-α, respectively), increased epiblast cell numbers. When active, GSK3 and p53 block the transcription of Nanog, which is important for maintaining pluripotency. A simultaneous inhibition of GSK3 and p53 had no synergistic effects on epiblast cell number. The induced activation of GSK3 and p53, via the inhibition of proteins responsible for their inactivation (PKA via H-89 and SIRT-1 via nicotinamide, respectively), blocked the insulin's effect on the epiblast.From our findings, we conclude that insulin increases epiblast cell number via the activation of PI3K, which ultimately inactivates GSK3 and p53. Furthermore, we suggest that the inclusion of insulin in culture media could be used as a strategy for increasing the efficiency with which the ESC lines can be derived from cultured embryos.Jared M. Campbell, Mark B. Nottle, Ivan Vassiliev, Megan Mitchell, and Michelle Lan

Targeting gene expression to endothelium in transgenic animals: A comparison of the human ICAM-2, PECAM-1 and endoglin promoters

It is highly likely that successful pig-to-human xenotransplantation of vascularized organs will require genetic modification of the donor pig, and in particular of donor vascular endothelium. Promoters are generally tested in transgenic mice before generating transgenic pigs. Several promoters have been used to drive endothelial cell-specific expression in mice but none have yet been tested in pigs. We compared the promoters of three human genes that are predominantly expressed in vascular endothelium: intercellular adhesion molecule 2 (ICAM-2), platelet endothelial cell adhesion molecule 1 (PECAM-1) and endoglin. Expression of human complement regulatory proteins (hCRPs), directed by each of the promoters in mice, was largely restricted to vascular endothelium and leukocyte subpopulations. However, expression from the PECAM-1 promoter was weak in liver and non-uniform in the small vessels of heart, kidney, and lung. Conversely, expression from the endoglin promoter was consistently strong in the small vessels of these organs but was absent in larger vessels. The ICAM-2 promoter, which produced strong and uniform endothelial expression in all organs examined, was therefore used to generate hCRP transgenic pigs. Leukocytes from 57 pigs containing at least one intact transgene were tested for transgene expression by flow cytometry. Forty-seven of these transgenic pigs were further analyzed by immunohistochemical staining of liver biopsies, and 18 by staining of heart and kidney sections. Only two of the pigs showed expression, which appeared to be restricted to vascular endothelium in heart and kidney but was markedly weaker than in transgenic mice produced with the same batch of DNA. Thus, in this case, promoter performance in mice and pigs was not equivalent. The weak expression driven by the human ICAM-2 promoter in pigs relative to mice suggests the need for additional regulatory elements to achieve species-specific gene expression in pigs.This work was supported in part by grants from Ministerio de Ciencia y Tecnologia and Comunidad de Madrid to CB.Peer Reviewe

Use of Insulin to Increase Epiblast Cell Number: Towards a New Approach for Improving ESC Isolation from Human Embryos

Human embryos donated for embryonic stem cell (ESC) derivation have often been cryopreserved for 5–10 years. As a consequence, many of these embryos have been cultured in media now known to affect embryo viability and the number of ESC progenitor epiblast cells. Historically, these conditions supported only low levels of blastocyst development necessitating their transfer or cryopreservation at the 4–8-cell stage. As such, these embryos are donated at the cleavage stage and require further culture to the blastocyst stage before hESC derivation can be attempted. These are generally of poor quality, and, consequently, the efficiency of hESC derivation is low. Recent work using a mouse model has shown that the culture of embryos from the cleavage stage with insulin to day 6 increases the blastocyst epiblast cell number, which in turn increases the number of pluripotent cells in outgrowths following plating, and results in an increased capacity to give rise to ESCs. These findings suggest that culture with insulin may provide a strategy to improve the efficiency with which hESCs are derived from embryos donated at the cleavage stage

Survival of porcine delipated oocytes and embryos after cryopreservation by freezing or vitrification

The present study examined whether delipated porcine oocytes and embryos at various stages of development can be cryopreserved by conventional slow cooling or vitrification. Most (93%) of the 27 delipated morulae developed to blastocysts after freezing with 1.5 M propanediol + 0.1 M sucrose. Late morulae and early blastocysts delipated at 2-4 cell stage and cultured in vitro survived freezing either with 1.5 M glycerol + 0.25 M sucrose (10/18, 56%) or 1.8 M ethylene glycol + 0.25 M sucrose (14/19, 74%). Delipated 2-4 cell stage embryos and oocytes could be cryopreserved by vitrification with 40% ethylene glycol, 1 M sucrose and 20% fetal calf serum. Half (7/14) of the vitrified, delipated embryos developed to blastocysts after thawing. Of 48 delipated oocytes, 27 (56%) maintained an intact outline of the ooplasm after vitrification and underwent subzonal sperm injection. Fertilization was confirmed in 12 (25%) of these oocytes and 3 (6%) developed to morula stage. This study also aimed at developing a non-invasive method for cryopreserving porcine embryos after reducing their cytoplasmic lipid content without micromanipulation. Morulae and early blastocysts were centrifuged in the presence of cytochalasin B and cryoprotectants and then frozen immediately. More than half (14/24, 58%) of the centrifuged morulae developed to blastocycts after freezing with 1.5 M propanediol + 0.1 M sucrose. Greater than 70% of centrifuged early blastocysts survived freezing either with 1.5 M propanediol (30/31, 97%), 1.5 M glycerol (22/29, 76%) or 1.8 M ethylene glycol (21/29, 72%). These results demonstrated that delipation (lipid removal) from porcine oocytes and embryos at various stages enables their cryopreservation. A new insight into the development of a non-invasive method for cryopreserving porcine embryos was also provided

Transgenic perspectives in xenotransplantation, 2001

Mark B. Nottle; Anthony J. F. D'Apice; Peter J. Cowan; Andrew C. Boquest; Sharon J. Harrison; Christopher G. Grupe

Rhesus monkeys and baboons develop clotting factor VIII inhibitors in response to porcine endothelial cells or islets

BackgroundXenotransplantation of porcine organs holds promise of solving the human organ donor shortage. The use of α-1,3-galactosyltransferase knockout (GTKO) pig donors mitigates hyperacute rejection, while delayed rejection is currently precipitated by potent immune and hemostatic complications. Previous analysis by our laboratory suggests that clotting factor VIII (FVIII) inhibitors might be elicited by the structurally restricted xenoantibody response which occurs after transplantation of either pig GTKO/hCD55/hCD59/hHT transgenic neonatal islet cell clusters or GTKO endothelial cells.MethodsA recombinant xenoantibody was generated using sequences from baboons demonstrating an active xenoantibody response at day 28 after GTKO/hCD55/hCD59/hHT transgenic pig neonatal islet cell cluster transplantation. Rhesus monkeys were immunized with GTKO pig endothelial cells to stimulate an anti-non-Gal xenoantibody response. Serum was collected at days 0 and 7 after immunization. A two-stage chromogenic assay was used to measure FVIII cofactor activity and identify antibodies which inhibit FVIII function. Molecular modeling and molecular dynamics simulations were used to predict antibody structure and the residues which contribute to antibody-FVIII interactions. Competition ELISA was used to verify predictions at the domain structural level.ResultsAntibodies that inhibit recombinant human FVIII function are elicited after non-human primates are transplanted with either GTKO pig neonatal islet cell clusters or endothelial cells. There is an apparent increase in inhibitor titer by 15 Bethesda units (Bu) after transplant, where an increase greater than 5 Bu can indicate pathology in humans. Furthermore, competition ELISA verifies the computer modeled prediction that the recombinant xenoantibody, H66K12, binds the C1 domain of FVIII.ConclusionsThe development of FVIII inhibitors is a novel illustration of the potential impact the humoral immune response can have on coagulative dysfunction in xenotransplantation. However, the contribution of these antibodies to rejection pathology requires further evaluation because "normal" coagulation parameters after successful xenotransplantation are not fully understood

Sustained function of genetically modified porcine lungs in an ex vivo model of pulmonary xenotransplantation

BACKGROUND Xenotransplantation could provide a solution to the donor shortage that is currently the major barrier to solid-organ transplantation. The ability to breed pigs with multiple genetic modifications provides a unique opportunity to explore the immunologic challenges of pulmonary xenotransplantation. METHODS Explanted lungs from wild-type and 3 groups of genetically modified pigs were studied: (i) α1,3-galactosyltransferase gene knockout (GTKO); (ii) GTKO pigs expressing the human complementary regulatory proteins CD55 and CD59 (GTKO/CD55-59); and (iii) GTKO pigs expressing both CD55-59 and CD39 (GTKO/CD55-59/CD39). The physiologic, immunologic and histologic properties of porcine lungs were evaluated on an ex vivo rig after perfusion with human blood. Results Lungs from genetically modified pigs demonstrated stable pulmonary vascular resistance and better oxygenation of the perfusate, and survived longer than wild-type lungs. Physiologic function was inversely correlated with the degree of platelet sequestration into the xenograft. Despite superior physiologic profiles, lungs from genetically modified pigs still showed evidence of intravascular thrombosis and coagulopathy after perfusion with human blood. CONCLUSIONS The ability to breed pigs with multiple genetic modifications, and to evaluate lung physiology and histology in real-time on an ex vivo rig, represent significant advances toward better understanding the challenges inherent to pulmonary xenotransplantation.Glen P. Westall, Browyn J. Levvey, Evelyn Salvaris, Julian Gooi, Sylvana Marasco, Frank Rosenfeldt, Chris Egan, Robin McEgan, Mark Mennen, Prue Russell, Simon C. Robson, Mark B. Nottle, Karen M. Dwyer, Greg I. Snell, Peter J. Cowa