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Articles by Seon-Mi Park in JoVE

 JoVE Biology

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

1Center for Regenerative Medicine (CReM), Boston University School of Medicine, 2Department of Hematology, Children's Hospital of Philadelphia, 3Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia


JoVE 4327

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Other articles by Seon-Mi Park on PubMed

Eu10Mn6Sb13: a New Ternary Rare-Earth Transition-metal Zintl Phase

A new transition-metal-containing Zintl compound, Eu(10)Mn(6)Sb(13), was prepared by a high-temperature Sn-flux synthesis. The structure was determined by single-crystal X-ray diffraction. Eu(10)Mn(6)Sb(13) crystallizes in the monoclinic space group C2/m with a = 15.1791(6) A, b = 19.1919(7) A, c = 12.2679(4) A, beta = 108.078(1)*, Z = 4 (R1 = 0.0410, wR2 = 0.0920), and T = 90(2) K. The structure of Eu(10)Mn(6)Sb(13) is composed of double layers of Mn-centered tetrahedra separated by Eu(2+) cations. The double layers are composed of edge- and corner-sharing Mn-centered tetrahedra which form cavities occupied by Eu(2+) cations and [Sb(2)](4-) dumbbells. Linear [Sb(3)](5-) trimers bridging two tetrahedra across the cavity are also present. Bulk susceptibility data indicate paramagnetic behavior with a ferromagnetic component present below 60 K. Temperature-dependent electrical resistivity measurements show semiconducting behavior above 60 K (E(a)() = 0.115(2) eV), a large and unusually sharp maximum in the resistivity at approximately 40 K, and metallic behavior below 40 K. (151)Eu Mössbauer spectra confirm that the europium is divalent with an average isomer shift of -11.2(1) mm/s at 100 K; the spectra obtained below 40 K reveal magnetic ordering of six of the seven europium sublattices and, at 4.2 K, complete ordering of the seven europium sublattices.

Sr2MnSb2: a New Ternary Transition Metal Zintl Phase

A transition metal-containing Zintl phase, Sr2MnSb2, was prepared from a stoichiometric combination reaction of the elements in Sn flux, and its structure was determined by single-crystal X-ray diffraction methods. The compound crystallizes in the orthorhombic space group Pnma with a = 15.936(3) A, b = 14.498(3) A, c = 8.2646(17) A, and Z = 12. The structure of Sr2MnSb2 is composed of corrugated layers of corner- and edge-shared MnSb4 tetrahedra. The basic building unit of the layer is a [Mn3Sb6]12- cluster composed of three edge-shared MnSb4 tetrahedra. These trinuclear clusters share four Sb vertexes to form a layer with cavities. Sr2+ cations are located at the inter- and intralayer space. Magnetic susceptibility measurements indicate an effective magnetic moment of 4.85 mu(B), a value smaller than what would be expected from Mn2+ ions.

Embryotrophic Effects of Ethylenediaminetetraacetic Acid and Hemoglobin on in Vitro Porcine Embryos Development

This study investigated the embryotrophic effects of ethylenediaminetetraacetic acid (EDTA) and hemoglobin (Hb) on porcine preimplantation embryo development. Porcine embryos produced by in vitro maturation/fertilization were cultured for 6 days in modified North Carolina State University-23 medium (mNCSU-23) supplemented with EDTA and/or Hb. In Exp. 1, culturing porcine zygotes with 100 microM EDTA significantly increased cleavage frequencies (85.3%) at 48 h post insemination and the number of inner cell mass (ICM) (9.6+/-5.5) compared to the control (7.0+/-2.8). However, 100 microM EDTA did not improve blastocyst formation compared to 0, 1 or 10 microM EDTA. In Exp. 2, in vitro fertilized oocytes were cultured with 0, 1 or 10 microg/ml Hb. Culturing with Hb did not promote porcine embryo development, but significantly increased the cell numbers of blastocysts in 1 microg/ml Hb compared to 0 or 10 microg/ml Hb. In Exp. 3, culturing embryos with 100 microM EDTA+1 microg/ml Hb significantly improved frequencies of cleavage, blastocyst formation, and total cell numbers in blastocysts compared to the control. Moreover, 100 microM EDTA, 1 microg/ml Hb and their combination reduced reactive oxygen species (ROS) accumulation and decreased the incidence of apoptosis. In conclusion, the present study clearly demonstrated that the combining treatment of EDTA and Hb improved IVF porcine embryo development.

Crystal Structure of Fatty Acid-CoA Racemase from Mycobacterium Tuberculosis H37Rv

Effect of Potassium Simplex Optimization Medium and NCSU23 Supplemented with Beta-mercaptoethanol and Amino Acids of in Vitro Fertilized Porcine Embryos

The present study was conducted to examine the comparative efficacy of potassium simplex optimization medium (KSOM) and North Carolina State University (NCSU)-23 medium supplemented with beta-mercaptoethanol (beta-ME) and amino acids (AA) on the developmental competence of porcine in vitro fertilized (IVF) embryos. Four experiments were conducted. KSOM and NCSU-23 medium were used to culture porcine parthenogenetic (Exp. 1) and IVF (Exp. 2) embryos. KSOM and NCSU-23 were equally effective in supporting porcine parthenogenetic and IVF embryo development from the 1-cell stage to blastocysts. The NCSU-23 medium (Exp. 3) and KSOM (Exp. 4) were supplemented with amino acid (AA; 5 microl/ml non-essential amino acids + 10 microl/ml essential amino acids) and/or 10 microM beta-mercaptoethanol (beta-ME). The quality of blastocysts from Exp. 3 and 4 was evaluated by counting the number of total cells and determining the ratio of the inner cell mass (ICM) to trophoectoderm (TE) cells. Supplementing with AA and beta-ME or beta-ME alone in NCSU-23 produced significant (p<0.05) differences in terms of rate of cleavage to the 2- to 4- cell (80.8 to 85.4% vs. 73.6%) and blastocyst (30.4 to 30.5 vs. 23.5%) stages and the number of TE (51.4 to 53.8 vs. 35.8) and total cells (67.2 to 71.2 to 48.8) over the control group. On the other hand, supplementing KSOM with AA and/or beta-ME produced significant (p<0.05) differences in terms of rate of cleavage to the 2- to 4-cell (78.8% vs. 67.7%) and morula (57.8% vs. 46.3%) stages and the number of ICM (18.6 to 19.2 vs. 11.6) and total cells (62.8 to 70.6 vs. 42.8) over control group. In conclusion, our study demonstrates that both KSOM and NCSU-23 medium supplemented with AA and beta-ME and/or only beta-ME alone are superior to normal KSOM and NCSU-23 for porcine IVF embryo culture in terms of embryo developmental competence and quality.

Analysis of Nuclear Reprogramming in Cloned Miniature Pig Embryos by Expression of Oct-4 and Oct-4 Related Genes

Xenotransplantation is a rapidly expanding field of research and cloned miniature pigs have been considered as a model animal for it. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several kinds of aberrant development. A possible explanation for the developmental failure of SCNT embryos is insufficient reprogramming of the somatic cell nucleus by the oocyte. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and embryonic germ cell nuclei with Oct-4 and Oct-4 related genes (Ndp5211, Dppa2, Dppa3, and Dppa5), which are important for embryonic development, Hand1 and GATA-4, which are important for placental development, as molecular markers using RT-PCR. The Oct-4 expression level was significantly lower (P<0.05) in cloned hatched blastocysts derived from fibroblasts and many of fibroblast-derived clones failed to reactivate at least one of the tested genes, while most of the germ cell clones and control embryos correctly expressed these genes. In conclusion, our results suggest that the reprogramming of fibroblast-derived cloned embryos is highly aberrant and this improper reprogramming could be one reason of the early lethality and post-implantation anomalies of somatic cell-derived clones.

Temporal Effects of Alpha-tocopherol and L-ascorbic Acid on in Vitro Fertilized Porcine Embryo Development

The susceptibility of embryos to reactive oxygen species (ROS) varies in different stages of embryo development. The present study evaluated temporal effects of alpha-tocopherol and L-ascorbic acid on the porcine embryo development, and investigated whether a single or twice supplements of these two antioxidants at a divided concentrations favors the embryo development. In order to determine temporal effects of alpha-tocopherol and/or L-ascorbic acid, 100 microM alpha-tocopherol or 200 microM L-ascorbic acid were supplemented to the North Carolina State University (NCSU)-23 embryo culture media at 0, 48, 96 and 120 h of culture. In another set of experiments, the concentration was divided into two equal halves, i.e., 50 microM alpha-tocopherol and 100 microM L-ascorbic acid, and supplemented twice at 0 and 48, 0 and 96, or 48 and 96 h of culture. Supplementing culture media with 100 microM alpha-tocopherol for the entire culture period of 168 h or starting from the 48 h of culture yielded higher blastocyst percentage compared with the control or starting from the 96 or 120 h of culture. L-Ascorbic acid (200 microM) alone or together with alpha-tocopherol (100 microM) with a single supplement did not affect the frequency of blastocyst formation or number of cells in blastocyst. L-ascorbic acid with a divided supplements yielded higher blastocyst percentage compared with the control. No synergistic effect was observed on embryo development at a single supplement of these antioxidants. Although, at divided supplements higher blastocyst percentage was observed compared with control group, no further beneficial effect was observed compared with alpha-tocopherol or L-ascorbic acid alone. Our results demonstrated that the embryotrophic effects of alpha-tocopherol and/or L-ascorbic acid, in terms of frequency of blastocyst formation and number of cells in blastocyst, depends on the concentration and supplementation timing.

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.

Effects of Insulin-transferrin-selenium in Defined and Porcine Follicular Fluid Supplemented IVM Media on Porcine IVF and SCNT Embryo Production

Insulin-transferrin-selenium (ITS) together has been used in different in vitro maturation system to support in vitro maturation of oocytes. The present study was designed to evaluate the effects of ITS in defined (0.1% PVA) and porcine follicular fluid (10% pFF) supplemented IVM media on the developmental competence of porcine oocytes. Three combinations of ITS, 10 mg/L insulin (Ins), 5.5mg/L transferrin (Tf) and 5 microg/L selenium (Se), 20mg/L Ins, 11 mg/L Tf and 10 microg/L Se, and 30 mg/L Ins, 16.5 mg/L TF and 15 microg/L Se, were used. The data were analyzed by one-way ANOVA and Tukey was used as the post hoc test. Both in the defined and pFF supplemented media, higher concentration of intracellular glutathione was observed in presence of ITS (4.6-4.8, and 6.9-7.1 picomole/oocyte for defined and pFF groups, respectively) compared to the respective control (2.1 and 4.3 picomole/oocyte for defined and pFF group, respectively). ITS decreased polyspermy and increased male pronucleus formation in both the defined and pFF supplemented medium. There was no difference in different treatment groups. The highest frequency of blastocyst formation rate and number of cells in blastocyst following IVF and SCNT was observed in pFF+ITS group (p<0.05). In conclusion, ITS addition during IVM improved the developmental competence of porcine oocytes in both the defined and pFF supplemented groups. Thus, we recommended to supplement porcine IVM medium with 10 microg/mL insulin, 5.5 microg/mL transferrin and 5 microg/mL selenium.

The Analysis of Chromatin Remodeling and the Staining for DNA Methylation and Histone Acetylation Do Not Provide Definitive Indicators of the Developmental Ability of Inter-species Cloned Embryos

The restricted supply of oocytes in the domestic dog limits the development of reproductive biotechnologies in this species. Inter-species somatic cell nuclear transfer could be an alternative for cloning animals whose oocytes are difficult to obtain. In this study, the possibility of cloning dog embryos using pig oocytes was investigated by evaluating nuclear remodeling. Chromatin remodeling, assessed by premature chromosome condensation, pseudo-pronuclei formation, DNA methylation and histone acetylation, along with the developmental ability was compared between intra- and inter-species cloned embryos. The incidence of premature chromosome condensation was significantly higher in intra-species cloned embryos relative to inter-species cloned embryos (87.2% vs. 61.7%; P<0.05), but comparable pseudo-pronuclei formation was observed in both (85.3% vs. 75.8%). None of the inter-species cloned embryos developed beyond the 8-cell stage while 18.3% of intra-species cloned embryos developed to the blastocyst stage. The relative level of both DNA methylation and histone acetylation was similar between intra- and inter-species cloned embryos at all times examined. These results suggest that although partial chromatin remodeling occurs, further investigation is needed to be able to use pig oocytes as recipient oocytes in dog cloning.

Anti-apoptotic Effect of Melatonin on Preimplantation Development of Porcine Parthenogenetic Embryos

In the present study, we investigated the effect of melatonin on the preimplantation development of porcine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos. Parthenogenetic embryos were cultured in mNCSU-23 supplemented with various concentrations of melatonin for 7 days. The results revealed that 100 pM was the optimal concentration, which resulted in significantly increased cleavage and blastocyst formation rates. Additionally, 100 pM melatonin provided the highest increase in total cell number of blastocysts. Therefore, the subsequent experiments were performed with 100 pM melatonin. ROS level in 2-8 cell stage embryos in the presence or absence of melatonin was evaluated. Embryos cultured with melatonin showed significantly decreased ROS. Blastocysts cultured with melatonin for 7 days were analyzed by the TUNEL assay. It was observed that melatonin not only increased (P < 0.05) the total cell number but also decreased (P < 0.05) the rate of apoptotic nuclei. Blastocysts cultured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl and Bax, and of pluripotency marker gene Oct-4 by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher (1.7-fold) compared to the control while the expression of Bax was significantly decreased relative to the control (0.7-fold) (P < 0.05). Moreover, the expression of Oct-4 was 1.7-fold higher than the control. These results indicated that melatonin had beneficial effects on the development of porcine parthenogenetic embryos. Based on the findings of parthenogenetic embryos, we investigated the effect of melatonin on the development of porcine SCNT embryos. The results also demonstrated increased cleavage and blastocyst formation rates, and the total cell numbers in blastocysts were significantly higher when the embryos were cultured with melatonin. Therefore, these data suggested that melatonin may have important implications for improving porcine preimplantation SCNT embryo development.

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