304 RHYTHMIC BEATING OF HEART MUSCLE GENERATED FROM GOAT EMBRYONIC STEM CELLS

Abstract

The aim of the present study was to isolate, culture, and characterise goat embryonic stem cell (ESC)-like cells from in vitro fertilized goat embryos. Oocytes were collected from slaughterhouse goat ovaries, and IVF was performed with standard protocol. A total of 60.48% cleavage, 24.01% morulae, 11.35% blastocyst, and 3.4% hatched blastocyst were obtained. Goat ESC-like cells were isolated from individual blastomere cells of early embryos after 2 mg mL^–1 of pronase treatment and inner cell mass (ICM) of blastocyst. The ICM were isolated mechanically from 80 expanded blastocysts and 20 hatched blastocysts and enzymatically from 45 expanded blastocysts and 20 hatched blastocysts. The primary colony formation was obtained mechanically from expanded blastocysts (66%) and hatched blastocysts (90%) and enzymatically from expanded blastocysts (30%) and hatched blastocysts (73%). The ICM were cultured on 10 μg mL^–1 of mitomycin-C inactivated fetal fibroblast feeder layer and with leukemia inhibitory factor (LIF) without feeder layer. Embryonic stem cells were cultured and characterised by immunofluorescence of surface markers such as alkaline phosphatase, Oct-4, SSEA-1, SSEA-3, SSEA-4 and intracellular molecular markers Oct-4, Sox2, Nanog. The primary colony formation was significantly higher when hatched blastocysts (90%) were used for ICM than when expanded blastocysts (66%), morulae (15%), and single blastomere (10%) were used. Five goat ESC lines were produced, which were maintained undifferentiated on a feeder layer in ESC medium containing LIF up to 5th, 7th, 10th, 20th, and 22nd passages. Three goat ESC lines were also produced in ESC medium containing LIF without feeder layer and maintained undifferentiated up to 5th, 10th, and 12th passages. All the cell lines expressed alkaline phophatase and OCT-4, at 5th, 10th, 15th, and 20th passages. The ESC of passage 20 were used for embryoid body formation for 3 days. The embryoid bodies were cultured to induce differentiation in medium contain activin-A, fibroblast growth factor-2, and BMP-4. The embryoid bodies were analysed with molecular markers such as Gata, BMP4, and Nestin and were found positive. The cardiac tissues were also observed to be positive with cardiac-specific molecular markers such as α actinin, troponin, and α-myosin heavy chain and its histology. The rhythmic beating was found after 30 days of culture, and the beating was still continuing after 42 days. These goat ESC were cryopreserved into LN₂ for a long period of time for further use in the future. It could be concluded that goat ESC were maintained undifferentiated up to 22nd passage with feeder layer and 12th passages without feeder layer using LIF only. Cardiomyocyte rhythmic beating of heart muscle was generated from goat ESC. To the best of our knowledge, this is the first time beating has been observed in goat ESC cardiomyocytes in the world. The authors have not found any report on animals such as cattle, buffalo, sheep, and pig, except mouse, monkey, and human being.