Germ cells production from established goat embryonic stem cells and - PDF document

1 Germ cells production from established goat embryonic stem cells and iPS cells Dhruba Malakar*, Hruda Nanda Malik and Dinesh Kumar Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, India *Corresponding author. Tel:+91-9416741839; fax: +91-184 2250042 E-mail address: dhrubamalakar@gmail.com Abstract: Objective/Background: World trend of fertility rate was declined drastically. In vitro derivation of both germ cells from embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) can be an alternative technique. Here we are presenting how ESC and iPSC are differentiating into germ cells like cells using goat as an animal model with this objective. The aim of the present study was to produce in vitro germ cells like cells from IVF ES cells, parthenogenetically produced ES cells and iPSC. Material & Methods: Embryonic stem cells were generated by IVF and parthenogenetically activated blastocyst. Oocytes were collected from the ovaries of slaughtered goats and matured in TCM- 199 supplemented with FSH, LH and β -estradiol as maturation factors. Matured oocytes were in vitro fertilized with capacitated goat spermatozoa and cultured in RVCL medium. For parthenogenesis, matured oocytes were activated with 5 µM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h and cultured in RVCL medium for blastocyst. Inner cell masses were isolated from blastocysts and cultured for ES cells on 10 µg/ml mitomycin-C treated feeder layer. The iPS cells were produced using transcription factors of Nanog, Oct4, Sox2 and Lin28 with lentiviruses in goat adult fibroblasts cells. Selected iPS cells were characterized using different methods. Characterization of ES cells and iPS cells was carried with Alkaline phosphatase staining, intracellular markers of Oct4, Nanog, sox2 and surface markers of SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. The embryoid bodies of goat ES cells were spontaneous differentiated to neuron, fibroblast, muscle cells and directed differentiation to rhythmic beating of cardiomyocytes. Cardiomyocyte was characterized with α -actinin, C-Troponin and Gata4 genes expressed in

2 RT-PCR and immunohistochemistry. The present study was experimentally established goat ES cells and further subcultured to 22 passages and cryopreserved these cells. Directed differentiation of ES cells into germ cell like cells: Goat ES cell colonies were cultured for embryoid bodies. These embryoid bodies were cultured in ES cell medium supplemented with retinoic acid and BMP-4. The differentiated ES cells for germ cell like cells were characterized with germ cell marker genes like VASA, STELLA and PUM1 immunostaining and Western Blotting of differentiated ES cells of these genes were expressed in the present study. Results: The VASA, STELLA and PUM1 germ cell specific marker genes were expressed in germ cells of directed differentiated ES cells and genes were 891bp, 365bp and 822bp respectively. Immunostaining germ cell like cells of differentiated goat ES cells with VASA primary antibody was already shown positive sign. VASA, STELLA and PUM1 germ cell specific marker proteins were also identified in Western blotting as these proteins expressed in the differentiated germ cells of directed differentiating embryonic stem cells. Here we mentioned that within one month, we will able to complete our rest of the research work. Conclusion : VASA, STELLA and PUM1 germ cell markers genes were expressed in directed differentiating ES cells. The proteins of these genes have already expressed in immunohistochemistry and western blotting in the differentiated ES cells and iPS cells. Characterization of germ cells from IVF, parthenogenetic ES cells and iPS cells were obtained optimistic result. Key words: Cardiomyocyte, Embryonic stem cell, Germ cell, Goat, iPSC, VASA Introduction: The ability to generate functional haploid germ cells acts as a yard stick to measure reproductive performance of mammals. One of the most common causes of male infertility is abnormal germ cell differentiation leading to azoospermia or oligospermia. In females, the most common cause of infertility involves ovulatory dysfunction. World trend of fertility rate declined gradually is shown in the figure below. Elucidating the molecular mechanisms involved in establishing the oocyte reserve and formation of spermatogonial stem cells is challenging because these events are completed before birth. In vitro derivation of both germ cells and matured functional gametes from

3 embryonic stem cell (Esc) and induced pluripotent stem cell (iPSc) can be an alternative technique to meet upto these challenges. Here we are presenting how embryonic stem cells and iPSc are differentiating into germ cells like cells using goat as an animal model. Male germ cells derived from ESCs and IPSc can be engrafted in host testes to produce viable sperm which helps in understanding of molecular mechanism of spermatogenesis and possibly provide new treatments for male infertility. A limitless supply of eggs derived from ESC and iPSc can have a radical impact on medicine. An ability to efficiently develop ESC and iPSc derived oocytes for nuclear transfer studies would be a significant advance and may provide a limitless source of oocytes. If these oocytes provide all the cues necessary to allow reprogramming of donor nuclei and successfully develop to the blastocyst stage, patient-specific ESC and iPSc lines resulting from nuclear transfer could be created, helping circumvent the major obstacle of donor oocyte availability in the construction of patient-specific ESC lines by nuclear transfer. Establishment of iPS cell line from adult goat fibroblast cells as a model for production of germ cells. Embryonic stem (ES) cells derived from inner cell mass of mammalian blastocysts grow rapidly and infinitely having the ability to differentiate into all types of cells (Evans and Kaufman, 1981; Martin, 1981). These properties of ES cells are maintained by symmetrical self-renewal, producing two identical stem cell daughters upon cell division (Burdon et al., 2002). The generation of pluripotent cells from differentiated adult cells has vast therapeutic implications, particularly in the context of in vitro disease modelling, pharmaceutical screening, and cellular replacement therapies. In addition, the ability to revert somatic cells to an embryonic state provides a unique tool to dissect the molecular events that permit the conversion of one cell type to another.

4 Thus, the direct generation of pluripotent cells without the use of embryonic material has been deemed a more suitable approach that lends itself well to mechanistic analysis and has fewer ethical implications. The direct reprogramming of somatic cells to pluripotency was accomplished in 2006, when Takahashi and Yamanaka converted adult mouse fibroblasts to iPSCs through ectopic expression of a selected group of transcription factors. Subsequent reports optimized this technique, demonstrating that iPSCs were indeed highly similar to ESCs when tested across a rigorous set of assays (Maherali et al., 2007; Okita et al., 2007; Wernig et al., 2007). In 2007, direct reprogramming was achieved in human cells (Takahashi et al., 2007b; Yu et al., 2007), providing an invaluable contribution to the field of regenerative medicine. Materials and Methods: In vitro production of goat embryos Blastocysts were produced following in vitro maturation, fertilization, culture and vitrification procedures, as described by Pawar et al, (2009). Briefly, oocytes collected from slaughterhouse were matured in TCM 199 (HEPES modified), containing 10 µg/ml luteinizing hormone (LH), 5 µg/ml follicle stimulating hormone (FSH), 1 µg/ml oestradiol- 17β, 50 µg/ml sodium pyruvate, 3.5 µg/ml L -glutamine, 50 µg/ml gentamicin, 5.5 mg/ml glucose, 3 mg/ml Bovine Serum Albumin (BSA) and 10% FCS (Malakar and Majumdar, 2005). After in vitro fertilization with fresh semen, the blastocyst and hatched blastocysts were cultured with a medium containing TCM 199 (HEPES modification), 30 µg/ml sodium pyruvate, 100 µg/ml L-glutamine, 50 µg/ml gentamicin, 10 µl/ml essential amino acids, 5 µl/ml non-essential amino acids (NEAA), 10 mg/ml BSA (Fraction-V), 10% FCS and 50 mM cysteamine for 8 days. Parthenogenetic activated goat embryos: Parthenogenetic embryos were produced with different methods such as electrical stimulus, the use of chemical agents such as Ca2+ ionophore, ethanol, strontium chloride, phorbol ester, thimerosal and phospholipase zeta (Ross et al., 2008) have been successfully used to activate bovine parthenotes. The present study was conducted by chemical activation of oocytes with the aid of a Ca ionophore and 6-DMAP. In vitro matured oocytes were activated with 5 µM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h. The putative