Cloning and characterizing embryonic stem cell lines derived from New Zealand White Rabbit embryos

本研究目的為探討紐西蘭白兔胚幹細胞（rabbit embryonic stem cells, rES cells）株之建立、培養與其細胞特性之分析。試驗一以STO（SIM mouse embryo-derived thioquanine and ouabain resistant）為飼養層細胞時，以全囊胚與免疫手術法分離內細胞群（inner cell mass, ICM）皆無法建立rES cell lines。而在MEF（mouse embryonic fibroblasts）飼養層上，其建立效率則顯著提高 (0% vs 24%)。在MEF飼養層條件下，白血病抑制因子（leukemia inhibitory factor, LIF）之添加能進一步提升rES cells建立之效率至57%。細胞株經由免疫螢光染色、西方吸漬法（Western blot）與反轉錄聚合酶反應（RT-PCR）偵測後，皆表現胚幹細胞特有多能性標誌（pluripotency marker），包括鹼性磷酸酶（alkaline phosphatase, AP）、Oct4、TRA-1-60、TRA-1-81、Nanog與Sox-2。在體外誘導分化形成類胚體（embryoid bodies, EBs）後，也發現三胚層細胞之特有標誌（MAP2、Desmin與GATA4），顯示此些細胞株具有分化之多能性。試驗二觀察單獨或共同添加LIF與纖維母細胞生長因子（basic fibroblast growth factor 2, bFGF2）對rES cells生長之影響並探討其訊息傳遞路徑。 結果顯示，rES cells 在MEF飼養層上，經由bFGF2訊息傳遞路徑 (MAPK/ERK and PI3K/AKT) 維持其不分化狀態。rES在LIF 與 bFGF2共同添加時較單獨添加LIF或 bFGF2條件之多能性標誌表現量高。以藥物抑制STAT3、MEK/ERK 與AKT之磷酸化，會造成rES cells失去自我更新能力，顯示bFGF2訊息傳遞路徑與LIF傳導路徑影響 rES cells生長與自我更新能力。試驗三以蛋白質體學方法比較纖維母細胞、與來自受精胚（f-rES）及孤雌激活胚（p-rES）之胚幹細胞蛋白質表現之差異，並進行蛋白質之鑑定。結果顯示，在三種不同細胞之間有100個蛋白質點呈現差異性。在這些蛋白質點中，91%成功鑑定出為63種已知蛋白質，這些已知蛋白質有14%是細胞核蛋白、13%屬於細胞骨架、8%屬粒線體、8%為內質網與57%存在於細胞質等相關蛋白。本研究有效率建立表現多能性標誌之rES cell lines並維持其於未分化狀態。LIF 和bFGF2可協同維持rES cell lines之多能性且表現與體細胞所缺乏之特異蛋白。未來研究將集中於誘導rES cells分化以及利用基因晶片來偵測其基因表現。The purposes of this study were to examine technical details in deriving and maintaining rabbit embryonic stem (rES) cell lines and to analyze their characteristics. In Experiment 1, when SIM mouse embryo-derived thioquanine and ouabain resistant (STO) cells were used as feeder cells, no rES cell lines were established using either intact blastocysts or inner cell mass (ICMs). On the mouse embryonic fibroblasts (MEF) feeder, rES cell lines were efficiently (24%) derived. Addition of leukemia inhibitory factor (LIF) to the cells cultured on the MEF feeders further increased the derivation efficiency (57%) of rES cells. Most of the rES cell lines expressed alkaline phosphotase (AP), SSEA-4, Oct-4, TRA-1-60 and TRA-1-81. Western-blot or RT-PCR analysis also confirmed the expression of Oct-4, Nanog, and Sox-2. When induced to form embryoid bodies (EBs) in vitro, the rES cells generated EBs with three germ layers expressing the marker genes including MAP2, Desmin and GATA4, respectively. In Experiment 2, we investigated the individual and combined effects of LIF and basic fibroblast growth factor 2 (bFGF2) on deriving and maintaining rES cell lines. First, when grown on MEF feeders, rES cell lines can be established and prevented from differentiation via bFGF2 (MAPK/ERK and PI3K/AKT) signaling. When both LIF and bFGF2 supplemented, rES cells acquired the highest expression of pluripotency markers than those supplemented solely with LIF or bFGF2. Induced dephosphorylation of STAT3, MEK/ERK and AKT by specific inhibitors suppressed their activities and caused remarkable losses of self-renewal capacity of rES cells. Experiment 3 aimed to determine the proteomics profiles of the fertilized embryo-derived and parthenote-derived ES cells, designated as f-rES and p-rES cells, respectively, and fibroblasts. Collectively, the expression levels of 100 spots differed significantly among these three cell types (P<0.05). Of those differentially expressed spots, 91% were identified and represented 63 distinct proteins. The proteins with known identities were mostly located in cytoplasmic compartments as cytoskeletal, mitochondrial, endoplasmic reticulum, and cytosolic proteins (13%, 8%, 8% and 57%, respectively) and nuclear (14%). We conclude that rES cell lines can be efficiently cloned using our current protocols and these ES cells express pluripotent stem cell makers and remain undifferentiation. LIF and FGF cooperation synergistically support stemness of rabbit ES cells and the expression of some novel key proteins distinguishes rabbit ES cells from their somatic counterpart. Further investigations will be focused on differentiation of rES cells and global screening of their gene expression profiles by microarrays which would invite more in-depth studies towards rabbit ES cell applications.Page ACKNOWLEDGMENTS--------------------------------------------------------------- i ABSTRACT (in Chinese)---------------------------------------------------------------- iii ABSTRACT (in English)---------------------------------------------------------------- v TABLE OF CONTENTS---------------------------------------------------------------- vii LIST OF TABLES------------------------------------------------------------------------ x LIST OF FIGURES----------------------------------------------------------------------- xi CHAPTER 1: Literature review-------------------------------------------------------------------------- 1 1. Abstract------------------------------------------------------------------------- 2 2. Introduction to embryonic stem (ES) cells--------------------------------- 3 3. Isolation of rES cell lines---------------------------------------------------- 7 3.1. Culture conditions for derivation and maintenance of rES cell lines----------------------------------------------------------------------- 7 3.2. Expression profiles of pluripotency genes and protein markers- 12 4. Rabbit models for biomedical research------------------------------------ 14 4.1. Retinal cells for eye diseases ----------------------------------------- 16 4.2. Rabbit ES cells for the treatment of cardiovascular diseases---- 18 4.3. Induction of ES cells into insulin-producing cells for the treatment of diabetes---------------------------------------------------- 20 4.4. Rabbit model for the study of tuberculosis ------------------------- 21 4.5. Rabbit model for the study of Alzheimer''s disease----------------- 22 4.6. Rabbit model for the study of fulminant hepatic failure---------- 23 4.7. Peyronie's disease----------------------------------------------------- 24 5. Conclusion remarks----------------------------------------------------------- 25 Page CHAPTER 2: Study 1: Culture and characterization of embryonic stem cell lines isolated from New Zealand White rabbits-------------------------------------------- 26 1. Abstract------------------------------------------------------------------------- 27 2. Introduction ------------------------------------------------------------------- 28 3. Material and method---------------------------------------------------------- 30 4. Results ------------------------------------------------------------------------- 38 5. Discussion---------------------------------------------------------------------- 48 CHAPTER 3: Study 2: LIF and FGF support stemness and self-renewal of rabbit embryonic stem cells derived from fertilized embryos--------------------------------- 52 1. Abstract------------------------------------------------------------------------- 53 2. Introduction ------------------------------------------------------------------- 54 3. Material and method---------------------------------------------------------- 57 4. Results ------------------------------------------------------------------------- 64 5. Discussion---------------------------------------------------------------------- 84 CHAPTER 4: Study 3: Proteomic analyses of rabbit embryonic stem cells derived from fertilized embryos and parthenotes------------------------------------------ 89 1. Abstract------------------------------------------------------------------------- 90 2. Introduction ------------------------------------------------------------------- 91 3. Material and method---------------------------------------------------------- 93 4. Results ------------------------------------------------------------------------- 101 5. Discussion---------------------------------------------------------------------- 120 CONCLUSIONS-------------------------------------------------------------------------- 125 APPENDICES Table 1 of the supplemental data----------------------------------------------- 128 Table 2 of the supplemental data----------------------------------------------- 134 Preparation of feeder cells ------------------------------------------------------ 139 Preparation rES cell culture medium----------------------------------------- 143 Page Karyotype Analysis-------------------------------------------------------------- 146 Alkaline phosphatase (AP) staining------------------------------------------- 148 Immunocytochemistry----------------------------------------------------------- 150 Western Blot---------------------------------------------------------------------- 152 REFERENCES---------------------------------------------------------------------------- 15

Partial replacement of commercial fish meal with Amazon sailfin catfish Pterygoplichthys pardalis meal in diets for juvenile Mekong giant catfish Pangasianodon gigas

Aquatic resources in Thailand have been affected by the introduction of Amazon sailfin catfish, Pterygoplichthys pardalis. This species has had a negative impact on native fish and has not been caught and consumed widely. At present there is debate as to whether it will be best to remove them from natural water sources or find some way to gain benefits from the presence of this species in Thailand. This research was conducted to evaluate the possibility of replacing standard fish meal with P. pardalis meal in the diets of juvenile Mekong giant catfish, Pangasianodon gigas. Five diets with replacement levels of 0% (control), 25%, 50%, 75% and 100% were tested for 90 days. The results showed that the weight gain (WG), average daily growth rate (ADG), specific growth rate (SGR), feed conversion rate (FCR), feed efficiency (FE) and protein efficiency ratio (PER) were not significantly different when compared with the control group. In none of the groups mortality occurred. Moreover, serum biochemistry indices such as levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose, albumin, globulin, protein, triglyceride and cholesterol were not significantly different from the control group (ANOVA, p >  0.05). Based on our results, we conclude that commercial fish meal can be replaced with P. pardalis meal to make up 100% of the diet of P. gigas without producing any adverse effects on growth performance, feed utilization and serum biochemical indices. Keywords: Aquaculture, Fish meal replacement, Formulated feed, Growth performance, Serum biochemistr

Nucleus, Cytoskeleton, and Mitogen-Activated Protein Kinase p38 Dynamics during In Vitro Maturation of Porcine Oocytes

The mitogen-activated kinase (MAPK) p38, a member of the MAPK subfamily, is conserved in all mammalian cells and plays important roles in response to various physiologic cues, including mitogens and heat shock. In the present study, MAPK p38 protein expression in porcine oocytes was analyzed during in vitro maturation (IVM) by Western blotting and immunocytochemistry. The levels of p-p38 or activated p38 and p38 expression were at the lowest in the germinal vesicle (GV) stage oocyte, gradually rising at the germinal vesicle breakdown (GVBD) and then reaching a plateau throughout the IVM culture (p &lt; 0.05). Similarly, the expression level of total p38 was also lower in the GV oocyte than in the oocyte of other meiotic stages and uprising after GVBD and remained high until the metaphase III (MII) stage (p &lt; 0.05). In the GV stage, phosphorylated p38 (p-p38) was initially detectable in the ooplasm and subsequently became clear around the nucleus and localized in the ooplasm at GVBD (18 h post-culture). During the metaphase I (MI) and metaphase II (MII) stages, p-p38 was evenly distributed throughout the ooplasm after IVM for 30 or 42 h. We found that the subcellular localization increased in p-p38 expression throughout oocyte maturation (p &lt; 0.05) and that dynamic reorganization of the cytoskeleton, including microfilaments and microtubules, was progressively changed during the course of meiotic maturation which was likely to be associated with the activation or networking of p38 with other proteins in supporting oocyte development. In conclusion, the alteration of p38 activation is essential for the regulation of porcine oocyte maturation, accompanied by the progressive reorganization and redistribution of the cytoskeleton and MAPK p38, respectively, in the ooplasm

LIF and FGF Cooperatively Support Stemness of Rabbit Embryonic Stem Cells Derived from Parthenogenetically Activated Embryos

We investigated the individual and combined effects of leukemia inhibitory factor (LIF) and basic fibroblast growth factor 2 (bFGF2) on the derivation and maintenance of rabbit embryonic stem cell lines isolated from parthenogenetic activated embryos (p-rES). First, we demonstrated that p-rES cell lines can be prevented from differentiation via LIF (STAT3) and bFGF2 (MEK-ERK1/2 and PI3K-AKT) signaling on MEF feeders. High levels of ERK1/2 and AKT activities were crucial for maintaining p-rES cells in an undifferentiated state. Although the p-rES cells under the influence of LIF (500, 1000, and 2000 U/mL) or bFGF2 (5, 10, and 20 ng/mL) alone showed enhanced expression in the pluripotency markers, the highest levels of marker expressions coincided with the simultaneous presence of LIF (1000U/mL) and bFGF2 (10 ng/mL). The phosphorylation status of LIF and bFGF2 downstream signaling molecules including STAT3, ERK, and AKT was also intensively involved in the maintenance of p-rES cell proliferation and self-renewal. Induced dephosphorylation of STAT3, ERK1/2, and AKT by specific inhibitors caused remarkable losses of self-renewal capacity of p-rES cells. We conclude that bFGF2 and LIF by itself are self-sufficient in maintaining the state of undifferentiation and self-renewal of rabbit p-ES cells, yet are most effective when acting concomitantly

Successful induction of antisera against rabbit embryos for isolation of the ICM and putative embryonic stem cells

In Expt 1, goat antisera against rabbit blastocysts were induced using spleen cell injection and skin-graft for immunosurgical isolation of ICM cells. Goats received rabbit spleen cell suspension (4 x 10(8) cells/ml) intravenously once a week for three consecutive weeks, plus an additional dose (boost injection) 10 days after the third injection, or a piece of rabbit skin (3 x 3 cm) transplantation. Blood samples were collected starting from the day after the last cell injection for 21 days. Serum was separated, heat inactivated and stored in frozen condition before titre analysis. Results showed that the anti sera/antibodies derived by spleen cell injection reached their peak titre 7 days after the last cell injection, compared with 5 days by the skin-grafted group. In Expt 2, morphologically normal blastocysts were collected for isolating ICMs immunosurgically or for direct culture of zona-free whole blastocysts. In both methods, ICM cells started attaching to the feeder layer and outgrowing from the centre portion of the cells on day 3 after the onset of culture. ICM outgrowths increased in size during days 4-5, and most cells differentiated morphologically after day 6. One colony derived from isolated ICM developed into morphologically ES-like cells expressing alkaline phosphatase activity. Our results indicated that both skin-grafting and spleen cell injection were effective inducing antisera against rabbit embryonic cells. More studies are required to optimize the culture system for rabbit ES cells

Proteomic Profiling of Rabbit Embryonic Stem Cells Derived from Parthenotes and Fertilized Embryos

<div><p>Rabbit embryonic stem (rES) cells can be derived from various sources of embryos. However, understanding of the gene expression profile, which distincts embryonic stem (ES) cells from other cell types, is still extremely limited. In this study, we compared the protein profiles of three independent lines of rabbit cells, i.e., fibroblasts, fertilized embryo-derived stem (f-rES) cells, and parthenote-derived ES (p-rES) cells. Proteomic analyses were performed using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Collectively, the expression levels of 100 out of 284 protein spots differed significantly among these three cell types (<i>p<0.05</i>). Of those differentially expressed spots, 91% were identified in the protein database and represented 63 distinct proteins. Proteins with known identities are mainly localized in the cytoplasmic compartments (48%), nucleus (14%), and cytoskeletal machineries (13%). These proteins were majorly involved in biological functions of energy and metabolic pathways (25%), cell growth and maintenance (25%), signal transduction (14%), and protein metabolisms (10%). When protein expression levels among cell types were compared, six proteins associated with a variety of cellular activities, including structural constituents of the cytoskeleton (tubulins), structural molecule (KRT8), catalytic molecules (α-enolase), receptor complex scaffold (14-3-3 protein sigma), microfilament motor proteins (Myosin-9), and heat shock protein (HSP60), were found highly expressed in p-rES cells. Two proteins related to HSP activity and structural constituent of cytoskeleton in f-rES cells, and one structural molecule activity protein in fibroblasts showed significantly higher expression levels (<i>p<0.05</i>). Marker protein expressions in f-rES and p-rES cells were further confirmed by Western blotting and immunocytochemical staining. This study demonstrated unique proteomic profiles of the three rabbit cell types and revealed some novel proteins differentially expressed between f-rES and p-rES cells. These analyses provide insights into rES cell biology and would invite more in-depth studies toward rES cell applications.</p></div

Analyses of expressions of pluripotency related gene in rabbit embryonic stem cells.

<p>(A) Western blot analyses of Oct4 and Nanog expressions in rabbit fibroblast, f-rES, and p-rES cells. Note that both f-rES and p-rES cell lines expressed all the pluripotency markers. Beta-actin is served as a loading control. (B) Immunocytochemical analyses of marker expressions of the three cell types (fibroblast, f-rES, and p-rES cells). The rES cell line expressed the markers recognized by antibodies against Oct4, Nanog, TRA-1-60, TRA-1-81, and SSEA-4. The nucleus is labeled by DAPI, and negative control is only stained with secondary antibody without primary antibody. Scale bar = 100 µm.</p