Homologous feeder cells support undifferentiated growth and pluripotency in monkey embryonic stem cells

In the present study, five homologous feeder cell lines were developed for the culture and maintenance of rhesus monkey embryonic stem cells (rESCs). Monkey ear skin fibroblasts (MESFs), monkey oviductal fibroblasts (MOFs), monkey follicular granulosa fibroblast-like (MFG) cells, monkey follicular granulosa epithelium-like (MFGE) cells, and clonally derived fibroblasts from MESF (CMESFs) were established and compared with the ability of mouse embryonic fibroblasts (MEFs) to support rESC growth. MESF, MOF, MFG, and CMESF cells, but not MFGE cells, were as good as or better than MEFs in supporting undifferentiated growth while maintaining the differentiation potential of the rESCs. In an effort to understand the unique properties of supportive feeder cells, expression levels for a number of candidate genes were examined. MOF, MESF, and MEF cells highly expressed leukemia inhibitory factor, ciliary neurotrophic factor, basic fibroblast growth factor, stem cell factor, transforming growth factor PI, bone morphogenetic protein 4, and WNT3A, whereas WNT2, WNT4, and WNT5A were downregulated, compared with MFGE cells. Additionally, all monkey feeder cell lines expressed Dkk1 and LRP6, antagonists of the WNT signaling pathway, but not WNT1, WNT8B, or Dkk2. rESCs grown on homologous feeders maintained normal karyotypes, displayed the characteristics of ESCs, including morphology, alkaline phosphatase, Oct4, the cell surface markers stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor-related antigen (TRA)-1-60, and TRA-1-81, and formed cystic embryoid bodies in vitro that included differentiated cells representing the three major germ layers. These results indicate that the four homologous feeder cell lines can be used to support the undifferentiated growth and maintenance of pluripotency in rESCs.

Generation and characterization of rabbit embryonic stem cells

We described the derivation of four stable pluripotent rabbit embryonic stem cell ( ESC) lines, one ( RF) from blastocysts fertilized in vivo and cultured in vitro and three ( RP01, RP02, and RP03) from parthenogenetic blastocysts. These ESC lines have be

Effects of control of C/N ratio by low-cost carbohydrate addition on water quality and pond ecology in freshwater prawn Macrobrachium rosenbergii post-larvae nursing system

An experiment was conducted to evaluate the effects of control of carbon/nitrogen ratio (C/N ratio) by addition of low cost carbohydrate to the water column on water quality and pond ecology in freshwater prawn Macrobrachium rosenbergii post-larvae nursing system. In this experiment, two level of dietary protein 20% and 35% without carbohydrate addition (‘P20' and ‘P35') and with carbohydrate addition (‘P20+CH' and ‘P35+CH') were compared in small ponds of 40 m² area stocked with 20 post-larvae (0.021 ± 0.001g) per m² . Maize flour was used as low cost carbohydrate and applied to the water column followed by the first feeding during the day. The addition of carbohydrate significantly reduced (p< 0.05) ammonia-nitrogen (NH sub(3)-N) and nitrite-nitrogen (NO sub(2) - N) of water in P20 + CH and P35 + CH treatments. It significantly increased (p< 0.05) the total heterotrophic bacteria (THB) population both in water and sediment. Fifty nine genera of plankton were identified belonging to the Bacillariophyceae (11), Chlorophyceae (21), Cyanophyceae (7), Dinophyceae (1), Rotifera (7) and Crustacea (9) without any significant difference (p>0.05) of total phytoplankton and zooplankton among the treatments. Survival rate of prawn was significantly lowest (p<0.05) in P20 and no significant difference (p>0.05) was observed between P20+CH and P35 treatments. Control of C/N ratio by the addition of low-cost carbohydrate to the pond water column benefited the freshwater prawn nursing practices in three ways (1) increased heterotrophic bacterial growth supplying bacterial protein augment the prawn post-larvae growth performances, (2) reduced demand for supplemental feed protein and subsequent reduction in feed cost and (3) reduced toxic NH sub(3)-N and NO sub(2)-N levels in pond nursing system.

Dissecting Signaling Pathways That Govern Self-renewal of Rabbit Embryonic Stem Cells

The pluripotency and self-renewal of embryonic stem cells (ESC) are regulated by a variety of cytokines/growth factors with some species differences. We reported previously that rabbit ESC (rESC) are more similar to primate ESC than to mouse ESC. However,

Neural progenitors derived from monkey embryonic stem cells in a simple monoculture system

A simple monoculture system. combined with a chemically defined medium containing hepatocyte growth factor (HGF) and G5 supplement, was used to induce rhesus monkey embryonic stem cells (rESC) directly into neuroepithelial (NE) cells. Under these conditio

Dietary protein and energy interations: an approach to optimizing dietary protein to energy ratio in walking catfish, Clarias batrachus

An 8 weeks feeding trial was conducted in a static indoor rearing system to investigate protein to energy ratio (PIE ratio) in walking catfish Clarias batrachus. Six fishmeal based diets of two protein levels (25 and 35%), each with three lipid levels (5, 10 and 15%) resulted in P/E ratios ranging from 13.57 to 21.97 mg protein kJˉ¹ gross energy (GE) were fed to 50 fish in triplicate. Fish were fed 6% of their body weight three times per day adjusted fortnightly. Significantly higher (p<0.05) growth rates in terms of weight gain, % weight gain and specific growth rate (SGR) were evident in fish fed with higher protein diet. The highest growth rate was found by fish fed 35% protein, 17.06 kJˉ¹GE with a P/E ratio of 20.55 mg protein kJˉ¹GE. Significantly better (p<0.05) feed conversion ratio (FCR) was also evident in fish fed with higher protein diet and best FCR was found by fish fed 35% protein, 10% lipid, 17.06 kJˉ¹GE with a P/E ratio of 20.55 mg protein kJˉ¹GE. Significantly indifferent (p>0.05) values of protein utilisation were found in between the both (higher and lower) protein diets. Higher lipid deposition (p<0.05) in whole body was observed with increasing dietary lipid level at each protein diet and as higher (p<0.05) for the lower protein diets. The study reveals that C. batrachus performed best the diet containing 35%, 17.06 kJ gˉ¹ and 20.55 mg protein kJ gˉ¹ GE protein, gross energy and P/E ratio respectively.

Optimum dietary carbohydrate to lipid ratio in stinging catfish, Heteropneustes fossilis (Bloch, 1792)

A feeding trial of 8 weeks was conducted in a static indoor rearing system to investigate the optimum carbohydrate to lipid ratio (CHO:L ratio) in stinging catfish, Heteropneustes fossilis. Five iso-nitrogenous (35% crude protein) and iso-energetic (17.06 kJ gˉ¹ gross energy (GE)) fish meal based diets with varying carbohydrate to lipid (CHO:L g/g) ratios of 0.60, 0.98, 1.53, 2.29 and 3.44 for diets 1-5, were tested, respectively. The diets containing a fixed protein to energy ratio (P:E ratio) of 20.50-mg protein kJˉ¹ GE were fed to triplicate groups of 40 fish (per 70-L tank). Fish were fed 5% of their body weight per day adjusted fortnightly. Diet 1, containing 10% carbohydrate and 17% lipids with a CHO:L ratio of 0.60 produced the poorest (p<0.05) growth rates, feed and protein efficiency. Increasing carbohydrate content in the diets to 26% concomitant with a reduction in lipid content to 11% with a CHO:L ration of 2.29 of diet 5 significantly improved (p<0.05) growth rates, feed and protein efficiency. But did not differ with diet 4, containing CHO:L ratio 2.29. A further increase in dietary carbohydrate up to 31% and a decrease in lipids levels to 9% with a CHO:L ratio ranging from 2.29 to 3.44 (diet 4-5) did not significantly improve the fish performance. Apparent net protein utilisation (ANPU) of fish fed diet 5 was higher (p<0.05) than for diets 1 and 2 but did not differ from diets 3 and 4. Higher lipid deposition (p<0.05) in whole body was observed with decreasing dietary CHO:L ratios as increasing lipid levels. Whole body protein of fish fed varying CHO:L diets did not show any discernible changes among the dietary treatments. This study revealed that H. fossilis can perform equally well on diets containing carbohydrate ranging from 26 to 31%, with 9 to 11% lipid or at CHO:L g/g ratio of 2.29-3.44.

Comparative Analysis of Five Different Homologous Feeder Cell Lines in the Ability to Support Rhesus Embryonic Stem Cells

我们以前的研究建立了五株猕猴饲养层细胞系来支持猕猴胚胎干细胞(rESCs)的生长:一岁猴耳皮肤成纤维细胞(MESFs)、两岁猴输卵管成纤维细胞(MOFs)、成年猴卵泡颗粒成纤维样细胞(MFGs)、成年猴卵泡颗粒上皮样细胞(MFGEs),以及MESFs的克隆成纤维细胞(CMESFs).我们发现MESFs、CMESFs、MOFs和MFGs,而不足MFGEs支持猕猴胚胎干细胞(rESCs,rhesus embryonic stem cells)的生长.通过半定量PCR的方法,我们在支持性的饲养层细胞中检测到了一些基因的高表达.在本研究中,我们运用Affymetrix公司的GeneChip Rhesus Macaque Genome Array芯片来研究这五株同源饲养层的表达谱,希望发现哪些细胞因子和信号通路在维持rESCs中起到重要作用.结果表明,除MFGE外,包括GREM2、bFGF,、KITLG,、DKK3、GREM1、AREG、SERPINF1和LTBF1等八个基因的mRNA在支持性的饲养层细胞中高表达.本研究结果提示,很多信号通路在支持rESCs的未分化生长和多潜能性方面可能起到了冗余的作用.

Serum-free culture of rhesus monkey embryonic stem cells

Previous studies have shown that the maintenance and proliferation of undifferentiated rhesus monkey embryonic stem (rES) cells requires medium supplemented with fetal bovine serum (FBS). Due to the uncharacterized composition and variation in serum nature, the present study aimed to replace the serum-containing medium with a serum-free medium in the rES cell culture. The results showed that after the initial 48-h culture in the routinely used serum-containing medium, rES cells can grow and proliferate for a prolonged period in the serum-free medium composed of DMEM supplemented with a cocktail of BSA, IGF-1, TGF-alpha, bFGF, aFGF, estradiol, and progesterone. rES cells cultured in the serum-free medium maintained high level of alkaline phosphatase activity and OCT4 level. There was no indication of differentiation as judged by the marker gene expression of all three embryonic germ layers and trophoblast. In addition, serum-free culture would not affect the passage capacity and differentiation potential of rES cells. This work will facilitate the future study of induced differentiation of rES cells and other applications.

Feeder layer- and serum-free culture of rhesus monkey embryonic stem cells

The common culture system of rhesus monkey embryonic stem (rES) cells depends largely on feeder cells and serum, which limits the research and application of rES cells. This study reports a feeder layer-free and serum-free system for culture of rES cells.