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

Captive breeding and embryonic development of honey gourami, Colisa sota (Ham.-Buch.)

Honey Gourami, Colisa sota, has high ornamental as well as food value. The natural resources of this species are gradually declining, due to destruction of its habitat, over fishing for aquarium trade and human consumption. The fish was bred in captivity under controlled environment. It laid about 200-400 eggs in bubble nest built by the male. Hatching started within 28-30hrs. after egg laying. The hatchlings became free swimming by 3rd to 4th day of hatching. The male showed territoriality and parental care by guarding the eggs and hatchlings. The larval survival was 30-35%. The breeding behavior, embryonic and post embryonic development of the fish were studied.

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,

Impairments in embryonic genome activation in rhesus monkey somatic cell nuclear transfer embryos

Somatic cell nuclear transfer (SCNT) is a remarkable process in which a somatic cell nucleus is acted upon by the ooplasm via mechanisms that today remain unknown. Here we show the developmental competence (% blastocyst) of embryos derived from SCNT (21%)

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

Embryonic and larval development of Mystus gulio (Ham.)

Mystus gulio eggs are strongly adhesive and contain relatively small yolk (0.75-1.0 mm). The egg envelop is thick and transparent. First cleavage (two cells), four cells, eight cells, sixteen cells and multi cells stages were found 20, 25, 35-40, 60 and 70 minutes after fertilization, respectively. The morula stage was visualized within 1.5 h after fertilization. The heart beat visible and the circulatory system commenced after 16 h of fertilization. Embryos hatched 18-20h after activation of egg. The newly hatched larva measured 2.82±0.03 mm in length and 0.32±0.06 mg in weight. The yolk sac was fully absorbed by the third day though larvae commenced exogenous feeding even before completion of yolk absorption. A 5-day old post larva began wandering in search of food. Ten-day old post larvae endowed with eight branched rays in dorsal fin and seven in caudal fin. Fifteen-day old post larvae had the pectm:al spine become stout though the embryonic fin folds had to be disappeared. The length of fingerlings ranged from 25-30 mm after 30 days, and their external features were just like those of an adult except that they were not sexually matured.

The study of biological characteristics and heavy metal determination of Cd, Pb, Fe, V, Ni in the muscle, liver and gonad tissues of kingfish Scomberomorus commerson in the coastal waters of the Hormozagan Province (Persian Gulf)

Biological studies and heavy metal (Ni, v, Fe, Pb,Cd) determination in liver, gonad and muscle of Scomberomorus commerson were carried out from Oct 2006 to Sept 2007 in Hormozgan coastal waters. 599 Samples were gutted for reproduction and nutrition studies, fork length and weight were measured to nearest cm and g respectively. Meanwhile 40 samples were also investigated for heavy metal studies. All specimens were collected from two major landing sites (Bandar Lengeh & Bandar Abbas). Minimums & maximum fork length & weight were 29, 128 cm & 235 and 15350 g respectively.Isometric growth was shown according to our study and b was estimated 2.9 (overall), 2.91(male) & 2.89 (female). The average relative gut length was 0.52± 0.007 and it was determined that S. commerson is a carnivorous. More than 99 percent of gut content was different teleost fishes. Gastro somatic index had two peaks in Nov & Jan (before spawning) and with a decreased trend in July, the spawning period. Occurrence of empty stomach was estimated % 65.77. Maximum amount of condition factor was in Dec. Spawning season was started from June. The average of Absolute & relative fecundity (to weight unit) was 1217149±179315 and 178.2±15.58 respectively. Lm50% was estimated 75 cm for females. Sex ratio was 0.97: 1 (male: female). Chi- Square test showed no significant difference (p>0.05). Maximum amount of hepatosomatic index was estimated in March.Metal concentrations were determined using either Flame Atomic Absorption Spectroscopy (for Fe) or Graphite Furnace Atomic Absorption Spectroscopy (for Pb,Cd,Ni and V). The mean concentration (μg/g dry weight)of Pb,Cd,Ni,V and Fe in the liver were 0.0309, 0.0268, 0.0672, 0.0077, 2.5159 in the gonad 0.0440 ,0.0295, 0.1096, 0.0000, 1.4449 and in the muscle 0.0244, 0.0324, 0.0656, 0.0128, 1.6138 respectively. The maximum metal concentrations were below the maximum permissible limits for human consumption recommended by the USEPA, WHO and the UK. The results of Kendall's Tau-b correlation coefficient were as follows: The Liver tissue: There were significant positive linear relationships between accumulation of V, Fe, and Pb with Fork length, Pb and Fe with weight, GSI with Pb, Cd, V and 109 Fe, and a negative linear relationships between HSI with accumulation of V and Fe, Fork length, weight and GSI. The Gonad tissue: There were significant positive linear relationships between GSI with accumulation of Pb, Cd, Fe, Fork length and weight, a negative linear relationship between HSI with Fork length, weight and GSI. The Muscle tissue: There were significant positive linear relationships between accumulations of V, Fe with Fork length and weight factors and as well as GSI with Cd, V, Fe, Pb, Fork length and weight,a negative linear relationship between HSI with Fork length, weight, Cd, Fe and GSI. The results of Mann-Whitney U tests (P≤0.05) show that there were significant differences between summer and autumn from heavy metal contents in the studied tissues point of view. The only exceptions were for Ni in the liver, gonads and muscle and as well as there were significant differences between male and female from heavy metal contents in the studied tissues. The only exceptions were for Pb in muscle, Ni in liver, gonad and muscle, V in muscle, and Cd and Fe in gonads.

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.