Xue F.,Renova Life, Inc. |
Ma Y.,Yale University |
Chen Y.E.,University of Michigan |
Zhang J.,University of Michigan |
And 9 more authors.
Cellular Reprogramming | Year: 2012
The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs. © Copyright 2012, Mary Ann Liebert, Inc. 2012.
Chen C.H.,National Taiwan University |
Chen C.H.,Animal Technology Institute Taiwan |
Du F.,Nanjing Normal University |
Du F.,Renova Life, Inc. |
And 13 more authors.
Theriogenology | Year: 2013
The first successful rabbit SCNT was achieved more than one decade ago, yet rabbits remain one of the most difficult species to clone. The present study was designed to evaluate the effects of two histone deacetylase inhibitors (HDACis), namely trichostatin A (TSA) and scriptaid (SCP), on cloning efficiency in rabbits. The in vitro development, acetylation levels of histone H4 lysine 5 (H4K5), and octamer-binding transcription factor 4 (Oct-4) expression patterns of cloned embryos were systemically examined after various HDACi treatments. Supplementation of TSA (50 nM) or SCP (250 nM) in the culture medium for 6 hours improved blastocyst development rates of cloned embryos compared with the treatment without HDACi. The combined treatment with TSA (50 nM) and SCP (250 nM) further enhanced morula (58.6%) and blastocyst (49.4%) rates in vitro. More importantly, compared with single HDACi treatments, embryos with the combined treatment had a higher level of H4K5 and an increased total cell number (203.7 ± 14.4 vs. 158.9 ± 9.0 or 162.1 ± 8.2; P < 0.05) with a better Oct-4 expression pattern in hatching blastocysts, indicating substantially improved embryo quality. This was apparently the first report regarding Oct-4 expression in cloned rabbit embryos. We inferred that most cloned rabbit embryos had an aberrant inner cell mass (ICM) structure accompanied with abnormal spatial distribution of Oct-4 signals. This study demonstrated a synergistic effect of TSA and SCP treatments on cloned rabbit embryos, which might be useful to improve cloning efficiency in rabbits. © 2013 Elsevier Inc.
Chen C.-H.,National Taiwan University |
Xu J.,Renova Life, Inc. |
Chang W.-F.,National Taiwan University |
Liu C.-C.,National Taiwan University |
And 5 more authors.
Reproductive BioMedicine Online | Year: 2012
This study documents the spatial and temporal distribution of Oct-4, Cdx-2 and acetylated H4K5 (H4K5ac) by immunocytochemistry staining using in-vivo-derived rabbit embryos at different stages: day-3 compact morulae, day-4 early blastocysts, day-4 expanded blastocysts, day-5 blastocysts, day-6 blastocysts and day-7 blastocysts. The Oct-4 signal was stronger in the inner cell mass (ICM)/epiblast cells than in the trophectoderm (TE) cells in all blastocyst stages except day-4 expanded blastocysts, where the signal was similarly weak in both the ICM and TE cells. The Cdx-2 signal was first detected in a small number of TE cells of day-4 early blastocysts, and became evident in the TE cells exclusively afterwards. A consistently strong H4K5ac signal was observed in the TE cells in all blastocyst stages examined. In particular, this signal was stronger in the TE than in the ICM cells in day-4 early blastocysts, day-4 expanded blastocysts and day-5 blastocysts. Double staining of H4K5ac with either Oct-4 or Cdx-2 on embryos at different blastocyst stages confirmed these findings. This work suggests that day 4 is a critical timing for lineage formation in rabbit embryos. A combination of Oct-4, Cdx-2 and H4K5ac can be used as biomarkers to identify different lineage cells in rabbit blastocysts. © 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Chen C.-H.,National Taiwan University |
Chang W.-F.,National Taiwan University |
Liu C.-C.,National Taiwan University |
Su H.-Y.,National Taiwan University |
And 8 more authors.
Reproductive BioMedicine Online | Year: 2012
Rabbit is a unique species to study human embryology; however, there are limited reports on the key transcription factors and epigenetic events of rabbit embryos. This study examined the Oct-4 and acetylated H4K5 (H4K5ac) patterns in rabbit embryos using immunochemistry staining. The average intensity of the Oct-4 signal in the nuclei of the whole embryo spiked upon fertilization, then decreased until the 8-cell stage and increased afterwards until the compact morula (CM) stage. It decreased thereafter from the CM stage to the early blastocyst (EB) stage, with a minimum at the expanded blastocyst (EXPB) stage and came back to a level similar to that of the CM-stage embryos in the hatching blastocysts (HB). The Oct-4 signal was observed in both the inner cell mass (ICM) and the trophectoderm (TE) cells of blastocysts. The average H4K5ac signal intensity of the whole embryo increased upon fertilization, started to decrease at the 4-cell stage, reached a minimum at the 8-cell stage, increased again at the EXPB stage and peaked at the HB stage. While TE cells maintained similar levels of H4K5ac throughout the blastocyst stages, ICM cells of HB showed higher levels of H4K5ac than those of EB and EXPB. Understanding key genetic and epigenetic events during early embryo development will help to identify factors contributing to embryo losses and consequently improve embryo survival rates. As a preferred laboratory species for many human disease studies such as atherosclerosis, rabbit is also a pioneer species in the development of several embryo biotechnologies, such as IVF, transgenesis, animal cloning, embryo cryopreservation and embryonic stem cells. However, there are limited reports on key transcription factors and epigenetic events of rabbit embryos. In the present study, we documented the temporal and spatial distribution of Oct-4 protein and H4K5 acetylation during early embryo development using the immunostaining approach. We also compared the patterns of these two important biomarkers between the inner cell mass (ICM) and the trophectoderm (TE) cells in blastocyst-stage embryos. Our findings suggest that a combination of Oct-4, H4K5ac and possibly other biomarkers such as Cdx-2 is needed to accurately identify different lineages of cells in morula and blastocyst stage rabbit embryos. Importantly, we revealed a novel wave of Oct-4 intensity change in the ICM cells of rabbit blastocysts. The signal was high at the early blastocyst stage, reached a minimum at the expanded blastocyst stage and returned to a high level at the hatching blastocyst stage. We hypothesize that the signal may have reflected the regulation of Oct-4 through enhancer switching and therefore may be related to cell lineage formation in rabbit embryos. These findings enrich our understanding on key genetic and epigenetic programming events during early embryo development in rabbits. © 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
PubMed | ARSIAL, Renova Life, Inc., Lannuo Biotechnologies Wuxi Inc. and Nanjing Normal University
Type: Journal Article | Journal: Cryobiology | Year: 2015
The aim of this study was to assess modified droplet vitrification (MDV) for the cryopreservation of early developmental mouse embryos. Mouse embryos were equilibrated in holding solution for 3 min followed by immersion in vitrification solution for 30-45 s, and then three embryos per 3-L vitrification droplet were directly dropped into liquid nitrogen. Vitrified embryos were warmed to examine their developmental potential both in vitro and in vivo. The results demonstrated that MDV vitrified and warmed embryos had a survival rate of 98.1-99.6% (P>0.05); however, blastocyst development post warming and culture in vitro demonstrated that vitrified 4-celled, 8-celled, 16-celled, morulae, and blastocyst embryos had significant higher developmental potentials (94.7-99.5%) than those from zygotes (9.2%) and 2-celled embryos (85.7%) (P<0.05). Compared to CryoLoop and CryoTech vitrification, MDV showed similar results with regards to rates of survival, blastocyst development, but with the higher hatching rate (76.1% vs. 64.0-67.3%) (P<0.05). Cryopreservation by MDV resulted in a similar blastocyst developmental potential in 4-celled and 16 celled embryos from ICR (94.7-99.5%), C57BL/6J (94.7-96.4%), and their crossbred F1 strain (97.9-98.9%) (P>0.05). After embryo transfer of vitrified ICR embryos from 4-celled, 16-celled, morulae and blastocyst stage, 40.7-43.7% of the embryos developed into live offspring (P>0.05), but MDV vitrification resulted in the highest birth rate (43.8%) compared to CryoLoop (38.3%) and CryoTech (35.4%) (P<0.05), when 4-celled mouse embryos were used for vitrification. Our study clearly demonstrated that MDV is the most efficient vitrification to cryopreserve embryos at least 4-celled and advanced stages, which can be used to preserve important mouse genomes from different strains and different developmental stages.
PubMed | ARSIAL, Renova Life, Inc., Instituto Superiore Of Sanita, Lannuo Biotechnologies Wuxi Inc. and Nanjing Normal University
Type: Journal Article | Journal: Reproduction in domestic animals = Zuchthygiene | Year: 2016
The aim of this study was to determine whether vitrification is an effective method when used for Japanese Black Cattle (Wagyu) in vivo-derived embryos, collected following a superovulation treatment and embryo transfer (MOET) programme. In vivo-derived morula and blastocysts collected on day 7 after artificial insemination, were vitrified using a modified droplet vitrification (MDV) procedure and subsequently warmed for transfer (ET) into synchronized recipients. Fresh embryos, and embryos cryopreserved using a standardized slow freezing procedure (direct thaw/direct transfer, DT) served as ET controls. Two different follicle-stimulating hormone (FSH) sources, Folltropin() Canada (FSH BAH, 24 donors) and a brand prepared by the Chinese Academy of Science (FSH CAS, 16 donors), were compared in a series of superovulation outcomes following well-established FSH administration protocols. Following data analysis, the total number of ovulations recorded at the time of embryo flushing (10.5 vs 8.5; p = 0.28) and the total number of transferable embryos (6.2 vs 5.1; p = 0.52) were similar between the two FSH sources. ET for MDV (39.7%, n = 78), DT (35.2%, n = 71) and fresh controls (47.1%, n = 34) resulted in similar pregnancy rates (p > 0.05). When MDV was used, a higher pregnancy rate (42.6%) resulted from the transfer of vitrified morulae, when compared to the DT counterparts (24.3%), (p = 0.05). Transfer of vitrified morulae resulted also in higher pregnancy rate, when compared to the transfer of vitrified blastocysts (42.6% vs. 29.4%; p < 0.05). Transfer of DT blastocysts resulted in higher pregnancy rate than morulae, similarly cryopreserved (47.1% vs. 24.3%, p < 0.05). In conclusion, MDV is an effective alternative methodology for cryopreservation of in vivo-derived embryos. This study gives also indication that, compared to vitrified blastocysts, MDV of morula stage embryos results in higher pregnancy rates following warming and transfer into synchronized recipients.
Lei L.,Northwest University, China |
Li L.,University of Maryland University College |
Du F.,Renova Life, Inc. |
Du F.,Nanjing Normal University |
And 3 more authors.
Molecular Reproduction and Development | Year: 2013
NANOG is an essential transcription factor involved in the proliferation and maintenance of embryonic stem cells (ESC) and reprogramming of somatic cells to a pluripotent state. Oct4 and Nanog promoter-driven enhanced green fluorescent protein (EGFP) reporters have been employed for establishing lines of induced pluripotent stem cells (iPSC) from mouse, human, and pig. In ruminants, including cattle, in which no fully validated ESC lines have been established, iPSC generated by reprogramming somatic cells to an ESC-like state may prove useful in the production of genetically modified livestock. In this study, utility of the bovine NANOG reporter was tested for use with cattle. Seven proximal bovine NANOG promoter fragments of different size were fused to the LUC gene, and were tested in mouse ESC lines using a dual-luciferase assay. Three of the bovine NANOG promoters, 315bp (-134/+181), 446bp (-265/+181), and 1,100bp (-919/+181), were fused to a nuclear localized signal EGFP reporter gene. The fidelity of these constructs was analyzed by transfection into mouse ESC and bovine fetal fibroblasts (bFFs), and subsequent reprogramming of the bFF. Fusion of the transgenic bFF with human teratocarcinoma (NTERA2) cells induced nuclear expression of the EGFP reporter. Similarly, bFF-derived somatic cell nuclear transfer (SCNT) embryos expressed EGFP in a stage- and location-appropriate manner. Following reprogramming of transgenic bFFs for 10 days with an Oct4-Sox2-Klf4-cMyc vector, iPSC expressed EGFP and alkaline phosphatase. These results indicate that NANOG reporters can be used to monitor nuclear reprogramming of bFFs and to distinguish cell allocation in SCNT-derived embryos. © 2013 Wiley Periodicals, Inc.
PubMed | University of Michigan, Renova Life, Inc., 3 Lannuo Biotechnologies Wuxi Inc. and Nanjing Normal University
Type: Journal Article | Journal: Cellular reprogramming | Year: 2015
The rabbit is a useful animal model for regenerative medicine. We previously developed pluripotent rabbit embryonic stem cell (rbESC) lines using fresh embryos. We also successfully cryopreserved rabbit embryos by vitrification. In the present work, we combined these two technologies to derive rbESCs using vitrified-thawed (V/T) embryos. We demonstrate that V/T blastocysts (BLs) can be used to derive pluripotent rbESCs with efficiencies comparable to those using fresh BLs. These ESCs are undistinguishable from the ones derived from fresh embryos. We tested the developmental capacity of rbESCs derived from V/T embryos by BL injection experiments and produced chimeric kits. Our work adds cryopreservation to the toolbox of rabbit stem cell research and applications and will greatly expand the available research materials for regenerative medicine in a clinically relevant animal model.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.13M | Year: 2010
DESCRIPTION (provided by applicant): The rabbit is a good animal model for the study of many human diseases because of its anatomical, genetic and biochemical similarities to the human. It is also used for drug screening, production of antibody and production of therapeutic proteins. The rabbit is large enough to provide adequate quantities of tissue for experimental work, produce sufficient amount of antibody or therapeutic proteins, and yet it is small enough to be economical for most laboratories. To date, pronuclear microinjection remains the primary method to produce transgenic rabbits. There are no germline competent rabbit embryonic stem (rbES) cell lines available. Pronuclear microinjection, however, is of low efficiency, results in random integration, and cannot produce gene targeted transgenic animals. The purpose of this SBIR project is to establish germline competent rbES cell lines for the production of gene targeted transgenic rabbits. In Phase I we established rabbit embryonic fibroblasts as feeder layers, synthesized recombinant rabbit leukemia inhibitory (LIF) factor, and derived putative rbES cells. In Phase II, we will work to validate if these rbES cells can contribute to germ line cells after blastocyst injection and we will work to produce gene targeted transgenic rabbits from these cells. Specific Aims in Phase II project include: (1) derive new rbES cell lines with inhibitors of differentiation; (2) evaluate germline competency of rbES cells; and (3) produce gene targeted transgenic rabbits. The success of this project will establish the first germline competent ES cell lines in a non-murine species, improve the transgenic efficiency in rabbits, and make the production of gene targeted rabbits possible. PUBLIC HEALTH RELEVANCE: The rabbit is a good animal model for the study of many human diseases because of its anatomical, genetic and biochemical similarities to the human. It is also used for drug screening, production of antibody and production of therapeutic proteins. The main reason that rabbit has not been in these fields is that there have been no established germline competent rabbit embryonic stem (rbES) cell lines. The success of this project will establish the first germline competent ES cell lines in a non-murine species, improve the transgenic efficiency in rabbits, and make the production of gene targeted rabbits possible.
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 155.99K | Year: 2013
DESCRIPTION (provided by applicant): Project Summary We propose to develop a non-invasive method to predict mammalian oocyte quality by analyzing the surrounding cumulus cells' gene expression patterns (cGEP). The result of the cGEP assay---a cGEP score---can be used to predict embryo quality and ultimately the embryo transfer (ET) results. This technology, if success, will for the first time, provide a quantitative measurement of the in vitro fertilized IVF) embryos. It will help users to non-invasively identify the best embryo for transfer. Single embryo transfer is expected to decrease the frequencies of multiple gestations (i.e., increase the frequencies of singletons) in human IVF practices. Multiple gestations are associated with low birth weight babies, preterm births and maternal complications. While the increasing use of Assisted Reproductive Technology (ART) over the past two decades has helped thousands of infertile couples to have children, almost one third of ART pregnancies are twins or higherorder multiple gestations; 51% of all ART neonates are the products of multiple gestations, a frequency 15- to 20-fold greater than with spontaneous conceptions. In many IVF and intracytoplasmic sperm injection (ICSI) procedures, more than one embryo is transferred to ensure satisfactory pregnancy results. This is largely due to the inability to accurately select good embryos by morphology examinations alone, the major judgment method in most IVF clinics. In fact, up to 40% of embryos with normal morphology have abnormal chromosomes. Such practices (transferring multiple embryos) contribute greatly to the high percentage of multiple gestations in ART. The ultimate goal of the present project is to develop a non-invasive method to predict oocyte and embryo quality in human IVF practices, in a 24-48 h turnover time. With this technology, when oocytes are collected and subjected to IVF procedures, the cumulus cells from each individual oocyte can be used for cumulus gene expression pattern (cGEP) assay. A cGEP score will be generated for each oocyte; consequently, each embryo generated from this IVF session will have a corresponding cGEP score. The cGEP score will assist users to make wise decisions on how and when to transfer the embryos. To our knowledge, this application represents one of the first commercial attempts to develop a non-invasive technology to predict human embryo quality. Cumulus cells are abundant and are considered to be disposable byproducts of oocyte retrieval for IVF treatment. Collection and analysis of cumulus cells is non-invasive and does not harm the oocyte. Biomarkers from cumulus cells can be used for diagnosis of infertility or reproductive toxicity. They can be used to improve the efficiency and efficacy of infertility treatmentsby identifying the most viable oocytes for fertilization in vitro, as well as exclusion of bad embryos for transfer. This will reduce the ned for and side effects associated with hyperstimulation of ovarian development during IVF. By limiting the numberof oocytes needed for fertilization, fewer extra embryos will be generated, which avoids the ethical and social complications of handling and disposing of these extra embryos. It will also reduce the number of embryos transferred, thus reducing the risk associated with high-order multiple gestations. Multiple gestations are associated with low birth weight babies, preterm births and maternal complications 1. Reducing the frequencies of multiple gestations would not only reduce these clinical complicationsof the mothers and the newborns, but also reduce the medical costs associated with them. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: We propose to develop a non-invasive method to predict mammalian oocyte quality by analyzing the surrounding cumulus cells' gene expression patterns (cGEP). This technology will help users to non-invasively identify the best embryo for transfer. Single embryo transfer is expected to decrease the frequencies of multiple gestations (i.e., increase the frequenciesof singletons) in human IVF practices.