(piezo –actuated system) Development of porcine cloned embryos produced using fibroblast and porcine induced pluripotent stem cells as nuclei donor cell types Ndubuisi Samuel MACHEBE 1,2, 3*, Masaki HATA 1, Kohei ARIFUKU 1, Yurika NARUMIYA1, Yuki ITANI1, Kazuki OHATA1, Tomokazu FUKUDA4, Tetsuya TANI1 and Yoko KATO 1 1 Laboratory of Animal Reproduction, Kinki University, Nagamachi, Nara, Japan; 2 JSPS Postdoctoral Fellow for Foreign Researchers.; 3 Department of Animal Science, University of Nigeria, Nsukka; 4 Graduate School of Agriculture, Tohoku University, Sendai, Japan; *email: ndubuisi.machebe@unn.edu.ng ABSTRACT Developmental ability of porcine cloned embryos produced by nuclear transfer using fibroblast (Fib) and porcine induced pluripotent stem (piPS) cells as nuclei donors were investigated. Porcine ear skin fibroblast and piPS cells were used as nuclei donor cell types to produce cloned embryos by method of somatic cell nuclear transfer technique. Embryos produced by in-vitro fertilization (IVF) and parthenogenetic activation (PA) were used as controls. Cleavage rate was different between PA and IVF embryos whereas cleavage rate of piPS and fibroblast embryos compared favourably between both PA and IVF embryos. The proportion of embryos that developed to the blastocysts was higher for PA groups. However, the proportion of cloned blastocysts produced with piPS and fibroblast nuclei and those produced by IVF were not different. Average total cell numbers was the same between the experiment and control groups of embryos. Based on our findings, we conclude that there is no difference in in-vitro development rates of porcine cloned embryos produced by nuclear transfer with fibroblast and piPS cells as nuclei donor cell types. Supported by JSPS KAKENHI (26-04086). MATERIALS AND METHODS RESULTS IVF Blast Fibroblast PA piPSC piPSCs IVF Fig1. Blastocyst produced by SCNT Fig2. Hoechst 3342 staining of Blastocyst Blastocysts were washed in 30 ul drop DPBS and then stained in medium with Hoechst 3342 (1 µg/mL) for 5 min at room temperature. Then, the whole blastocysts were mounted in glycerol on a glass slide (Paul Marienfeld, Germany) and flattened under a coverslip . Digital photographs were obtained using an inverted microscope capable of UV illumination and equipped with excitation filters (460 nm for fluorescence). The cells in the images were counted. In-vitro maturation of porcine COC mNCSU37 FSH (1-3ug/ml), LH (0.6ug/ml), dbcAMP (1mM) Polar body mNCSU37 (20 hrs) (20 hrs) Pig Ovary Porcine COC MII oocyte Somatic Cell Nuclear Transfer (SCNT) Protocol P = 0.024 Fig. 3 Percentage of cleaved embryos 48 hrs after activation Cleavage rate was significantly higher for PA embryos and least for IVF embryos (P<0.05). Cleavage rate for porcine iPS and Fibroblast produced embryos were intermediate between PA and IVF embryos Exogenous Transcription Factors Oct4, Sox2, Klf4, c-Myc,Lin28, Nanog (OSKMLN) Lentiviral transduction Pig iPSC colony piPSCs Induction/ Maintenance medium DMEM/F12 + 15% KSR, porcine LIF, 5ng/ml bFGF, 0.25μM PD0325901, 0.75μM CHIR99021, 3μM GF-109203x (protein kinase C inhibitor), 0.25 μM Thiozovivin, 10μM b-mercaptoethanol, Nonessential amino acid SCNT (piezo –actuated system) Enucleation Pig FCs 120V /mm DC, 30usec, 0.28M Mannitol +0.01% PVA , 0.25mM CaCl2.2H20,: 0.1mM MgSO4.7H20 P = 0.001 Donor cells Fig. 4 Percentage of embryos that developed to the blastocyst after 168 hrs Blastocyst rate was higher for PA embryos compared to iPS, fibroblast cells and IVF embryos (P<0.05) . Blastocyst for iPS, fibroblast and IVF produced embryos were similar (P>0.05) 2 hrs mPZM5 5ug/ml CB 50nM TSA 40ug/ml Cycloheximide 22 hrs mPZM5 50nM TSA 39oC, 5% Co2 P = 0.581 Fig. 5 Total nuclei in blastocyst after 168 hrs The total number of nuclei in blastocyst produced were similar for all treatment (P >0.05) 144 hrs mPZM5 Pathenogenetic activation of MII oocytes Summary/Conclusion A precise embryo quality evaluation is of paramount importance to ascertain the quality of embryos produced in-vitro. The CLEAVAGE and BLASTOCYST RATES and NUCLEI NUMBER among other methods are used as an essential predictive factor for subsequent implantation and pregnancy rates, and these essential quality index for in-vitro produced embryos might differ according to cell types . For instance, it is worthwhile that good quality embryos should cleave early and have appropriate kinetic and good synchrony for division. Our findings showed that the percentage of cleavage, blastocyst rate and total nuclei number for blastocysts produced by SCNT using iPS and fibroblast cells as donor nuclei are similar. However, cleavage and blastocyst rate were higher in PA produced embryos compared with embryos produced by IVF. Based on our results, we therefore conclude that porcine blastocysts produced by reprogramming of iPS and fibroblast cells have similar developmental potential in-vitro. A follow-up study is on-going to ascertain the expression of some important genes in blastocyst produced by reprogramming of these donor cells Polar body *120V/mm DC, 30usec, (x2 pulse) *D-Mannitol (0.28M) +0.01% PVA, 0.25mM CaCl2.2H20, 0.1mM MgSO4.7H20 * mPZM5 *CB (5ug/ml) *(4 h) MII oocyte * mPZM 5 * (168h) 39 o C, 5% Co2 In-vitro fertilization (IVF) * Fresh high motile and viable boar spermatozoa was obtained by percoll density gradient centrifugation (50%, 80% percoll; 700 x g for 20 min) and the supernatant was removed. 20-30 eggs were cultured in 40 ul drop of PGMtac4 (144 hrs) after denuding and wash in PXM-Hepes * Sperm was diluted in PGMtac4 to 2 x 106 cells/ml * 50 ul of sperm suspension was added to 50 ul drop of fertilization media * 15-20 COCs was added in each drop after wash in PGMtac4 for 10-20 hrs at 39.0oC, 5% Co2, 5% O2, 90% N2.