Woo Suk Hwang

Patient-Specific Embryonic Stem Cells Derived from Human SCNT Blastocysts

Patient-specific, immune-matched human embryonic stem cells (hESCs) are anticipated to be of great biomedical importance for studies of disease and development and to advance clinical deliberations regarding stem cell transplantation. Eleven hESC lines were established by somatic cell nuclear transfer (SCNT) of skin cells from patients with disease or injury into donated oocytes. These lines, nuclear transfer (NT)–hESCs, grown on human feeders from the same NT donor or from genetically unrelated individuals, were established at high rates, regardless of NT donor sex or age. NT-hESCs were pluripotent, chromosomally normal, and matched the NT patients DNA. The major histocompatibility complex identity of each NT-hESC when compared to the patients own showed immunological compatibility, which is important for eventual transplantation. With the generation of these NT-hESCs, evaluations of genetic and epigenetic stability can be made. Additional work remains to be done regarding the development of reliable directed differentiation and the elimination of remaining animal components. Before clinical use of these cells can occur, preclinical evidence is required to prove that transplantation of differentiated NT-hESCs can be safe, effective, and tolerated.

Dogs cloned from adult somatic cells

Several mammals — including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats — have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.

The Beneficial Effects of Insulin and Metformin on In Vitro Developmental Potential of Porcine Oocytes and Embryos

Abstract To investigate whether insulin and/or metformin improve the developmental competence of porcine oocytes and embryos, insulin (100 ng/ml) and/or metformin (10−5 M) were supplemented during in vitro maturation (IVM) and/or in vitro culture (IVC) periods. Insulin (33 to 34% vs. 21%) or insulin plus metformin (27 to 39% vs. 21%) significantly (P < 0.01) increased the rates of blastocyst formation, whereas metformin alone had no effect when added during the first half (0–22 h), the latter half (22–44 h), or the entire (0–44 h) maturation period. No additional effect of insulin plus metformin on increasing blastocyst formation was observed compared to insulin alone. When supplemented during IVC, insulin (34% vs. 23%) or insulin plus metformin (35% vs. 23%) significantly (P < 0.05) increased the rates of blastocyst formation, whereas metformin alone had no effect. Compared to insulin alone, no additional effect of insulin plus metformin on increasing blastocyst formation was observed. When added during the entire IVM and IVC, insulin (40% vs. 24%) or insulin plus metformin (52% vs. 21%) significantly increased the rates of blastocyst formation, whereas metformin alone had no effect. In addition, the effect of insulin was enhanced when supplemented with metformin compared to insulin alone (52% vs. 40%). After IVM, oocyte glutathione (GSH) content and tyrosine kinase activity were measured. Insulin significantly (P < 0.01) increased oocyte GSH content (6.2 pmol vs. 4.3 pmol) and metformin significantly (P < 0.01) enhanced the action of insulin on GSH content (7.3 pmol vs. 6.2 pmol) and tyrosine kinase activity (1.9 arbitrary units [AU] vs. 1.5 AU) when compared to insulin alone. In conclusion, insulin increased the developmental potential of porcine oocytes and embryos, and metformin enhanced the action of insulin when supplemented during the entire IVM and IVC. The effects of insulin and metformin were associated with oocyte GSH content and tyrosine kinase activity.

Beneficial effects of brain-derived neurotropic factor on in vitro maturation of porcine oocytes

In an effort to improve the quality of in vitro produced porcine embryos, we investigated the effect of brain-derived neurotropic factor (BDNF), a neurotropin family member, on in vitro maturation (IVM) of porcine oocytes. The expression of BDNF and truncated isoforms of its receptor, tyrosine kinase B (TrkB), and p75 common neurotropin receptor was detected in both follicular cells and metaphase-I stage oocytes by RT-PCR. However, mRNA of full-length TrkB was not found in oocytes although it was detected in follicular cells. The expression pattern of BDNF and TrkB was confirmed by immunohistochemistry. Supplementation with BDNF (30 ng/ml) during IVM significantly (P < 0.05) increased the first polar body extrusion and glutathione levels in oocytes, whereas the effect of BDNF on nuclear maturation was diminished when gonadotropin and epidermal growth factor (EGF) were added to the culture media. However, treatment with BDNF (30 ng/ml) along with EGF (10 ng/ml) in the presence of gonadotropin significantly (P < 0.05) increased the developmental competence of oocytes to the blastocyst stage after both in vitro fertilization (IVF; 29.1% when compared with control, 15.6%) and somatic cell nuclear transfer (SCNT; 13.6% when compared with control, 3%). This appeared to reflect a stimulatory interaction between BDNF and EGF to enhance the cytoplasmic maturation of oocytes to support successful preimplantation development. In conclusion, BDNFenhanced nuclearand cytoplasmic maturation of oocytes by autocrine and/or paracrine signals. Also, when used together with EGF, BDNF increased the developmental potency of embryos after IVF and SCNT, demonstrating an improved in vitro production protocol for porcine oocytes.

Effect of glycosaminoglycans on the preimplantation development of embryos derived from in vitro fertilization and somatic cell nuclear transfer

The purpose of this study was to evaluate the effect of glycosaminoglycans (GAGs) added to the culture medium on the developmental competence of bovine embryos derived from in vitro fertilization (IVF) and from somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were either inseminated with 1 x 10(6) spermatozoa mL(-1) or enucleated and reconstructed with bovine adult ear fibroblasts by SCNT. The embryos were then cultured in modified synthetic oviduct fluid (mSOF) containing 8 mg mL(-1) bovine serum albumin (BSA) (control mSOF) or control mSOF supplemented with various GAGs (hyaluronic acid, heparin or chondroitin sulfate) in a dose-dependent manner (0.1, 0.5 or 1.0 mg mL(-1)). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos, 8-16-cell embryos and blastocysts. The mean cell number of flattened blastocysts stained with 5 microM bisbenzimide on Day 8 was counted. The percentage of blastocyst formation (IVF and SCNT embryos) from cleaved embryos was significantly higher (P < 0.05) in control mSOF supplemented with 0.5 mg mL(-1) hyaluronic acid (45% and 47%), heparin (40% and 47%) or chondroitin sulfate (38% and 44%) compared with control mSOF (30-31% and 30-33%). When compared with the efficacy of 0.5 mg mL(-1) GAGs, no significant differences were observed in the developmental competence of both IVF and SCNT embryos. Supplementing control mSOF with 0.5 mg mL(-1) GAGs had no effect on the cell number of IVF embryos. In contrast, supplementing 0.5 mg mL(-1) of hyaluronic acid, heparin or chondroitin sulfate to control mSOF significantly (P < 0.05) increased the numbers of total cells (93-98 v. 88 cells) and trophectoderm (TE) cells (64-66 v. 55 cells), and decreased the inner cell mass (ICM) to TE cell ratio (48.2-49.8 v. 61.3) in SCNT blastocysts compared with embryos in control mSOF. In conclusion, supplementation of culture media with GAGs may improve the development of bovine IVM-IVF and SCNT embryos to the blastocyst stage. The GAGs increased the quality of blastocysts by increasing total cell numbers in the SCNT embryos.

Establishment and characterization of endothelial cell lines from the aorta of miniature pig for the study of xenotransplantation

Pig endothelial cells are the first cells to interact with human immune components after organ xenotransplantation, which is a procedure currently considered to be the best treatment option for end‐stage organ failure. It is, therefore, essential to study the mechanisms of molecular interaction between pig endothelial cells and human immune components, in order to overcome xenograft rejection. The aim of this study was to establish immortalized pig aortic endothelial cell lines, in order to facilitate future in vitro studies of human anti‐pig immune responses. Endothelial cell lines were established following the transfection of primary endothelial cells isolated from the aortas of the Minnesota miniature pig with plasmid pRNS‐1 carrying genes for neomycin resistance and the SV40 large T antigen. The immortalized cell lines showed a relatively rapid doubling time (17.6 h) and the endothelial cell phenotype, as indicated by the formation of typical cobblestone monolayers and by the constitutive expression of PECAM‐1 and the von Willebrand factor. Flow cytometric analysis demonstrated the constitutive expression of SLA class I and CD86, whereas the expression of E‐selectin and SLA class II was only induced after stimulation with human TNF‐α and pig IFN‐γ, respectively. On the other hand, no CD80 expression was detected in the primary cells or cell lines in the presence or absence of either human TNF‐α or pig IFN‐γ. A vigorous human T cell proliferation against these cell lines was observed in the mixed lymphocyte—endothelial cell culture. These results suggest that pig endothelial cells, immortalized by the introduction of SV40 T, retain their original characteristics, except for the acquired property of immortalization, and that they may be useful for future in vitro studies of xenogeneic human anti‐pig immune responses.

Birth of Beagle dogs by somatic cell nuclear transfer.

The present study was undertaken to evaluate two enucleation methods for somatic cell nuclear transfer (SCNT), and to standardize the optimum number of embryos for transfer to each recipient for canines. Oocytes retrieved from outbreed dogs were reconstructed with adult somatic cells from a male Beagle dog. A total of 134 or 267 oocytes were enucleated either by aspiration or squeezing method, fused with two DC pulses of 1.75 kV/cm for 15 micros electrical stimulation, chemically activated after 1h of fusion using 10 microM calcium ionophore for 4 min and cultured 4h in 1.9 mM 6-dimethylaminopurine. Finally, 103 or 214 embryos for aspiration or squeezing method were transferred to 6 or 11 naturally synchronized recipients, respectively. A total of 53, 317 and 342 embryos were transferred to 7, 17 and 12 recipients for the group of 4-10, 11-25 and 26-40 embryos, respectively. There was no difference between fusion rate (76.87% vs. 80.15%), full term pregnancy rate (16.66% vs. 27.27%) and percent of live puppies born (0.97% vs. 1.87%) for aspiration and squeezing method (P>0.05). Production efficiency of cloned dogs was significantly affected by the number of embryos transferred to each recipient. No pregnancy was established for the group of 4-10 embryos (n=7) and 26-40 embryos (n=12) while pregnancy was detected in 23.53% recipients received a group of 11-25 embryos (n=17). Among them, five (1.76%) live puppies were born (P<0.05). These data show an increase in the overall efficiency of SCNT in canine species.

Cloning Missy: Obtaining Multiple Offspring of a Specific Canine Genotype by Somatic Cell Nuclear Transfer

The present study was undertaken to evaluate two activation methods for somatic cell nuclear transfer (SCNT), namely, fusion and simultaneous activation (FSA, fusion medium contains calcium), versus fusion followed by chemical activation (F+CA, fusion medium does not contain calcium), and to evaluate the effects of parity of recipient dogs on the success of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells collected from an 11-year-old female dog named Missy. In the FSA method, oocytes were fused and activated at the same time using two DC pulses of 1.75 kV/cm for 15 microsec. In the F+CA method, oocytes were fused with two DC pulses of 1.75 kV/cm for 15 microsec, and then activated 1 h after fusion by 10 microM calcium ionophore for 4 m and cultured for 4 h in 1.9 mM 6-dimethylaminopurine for postactivation. Activation method had a significant impact on the production efficiency of cloned dogs. There was a significant difference in full-term pregnancy rate and percentage of live puppies between the two methods (6.3% and 38.5% for FSA and F+CA, respectively). In our study, four out of five live offspring produced by F+CA survived versus FSA, which did not result in any surviving puppies. Overall, as few as 14 dogs and 54 reconstructed embryos were needed to produce a cloned puppy. In addition, the parity of recipient bitches had no effect on the success of SCNT in canine species. Both the nullipara and multipara bitches produced live puppies following SCNT-ET.

X-linked gene transcription patterns in female and male in vivo, in vitro and cloned porcine individual blastocysts.

To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF), and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average) were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes) but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT) process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process, which may eventually lead to problems with embryonic or placental defects.

Production of cloned dogs by decreasing the interval between fusion and activation during somatic cell nuclear transfer.

To improve the efficiency of somatic cell nuclear transfer (SCNT) in dogs, we evaluated whether or not the interval between fusion and activation affects the success rate of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells from a male or female Golden Retriever. In total, 151 and 225 reconstructed oocytes were transferred to 9 and 14 naturally synchronized surrogates for male and female donor cells, respectively. Chromosomal morphology was evaluated in 12 oocytes held for an interval of 2 hr between fusion and activation and 14 oocytes held for an interval of 4 hr. Three hundred seventy‐six and 288 embryos were transferred to 23 and 16 surrogates for the 2 and 4 hr interval groups, respectively. Both the male (two pregnant surrogates gave birth to three puppies) and female (one pregnant surrogate gave birth to one puppy) donor cells gave birth to live puppies (Pu2009>u20090.05). In the 2 hr group, significantly more reconstructed oocytes showed condensed, metaphase‐like chromosomes compared to the 4 hr group (Pu2009<u20090.05). A significantly higher pregnancy rate and a greater number of live born puppies were observed in the 2 hr group (13.0% and 1.1%, respectively) compared to the 4 hr group (0%) (Pu2009<u20090.05). In total, three surrogate dogs carried pregnancies to term and four puppies were born. These results demonstrate that decreasing the interval between fusion and activation increases the success rate of clone production and pregnancy. These results may increase the overall efficiency of SCNT in the canine family. Mol. Reprod. Dev. 76: 483–489, 2009.