Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices

BACKGROUND AND OBJECTIVES: Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. METHODS AND RESULTS: Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA-4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. CONCLUSIONS: Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.

Research progress in feeder layers of human embryonic stem cells / 国际生物医学工程杂志

Human embryonic stem cell (hES cells) lines can be derived from the inner cell mass (ICM) of preimplantation blastocysts.hES cells are commonly defined as undifferentiated pluripotent cells that can proliferate and have the capacity of both self-renewal and differentiation into one or more types of specialized cells.hES cells remain undifferentiated when culture on feeder layers,such as murine embryonic fibroblasts (MEFs).It is believed that various factors secreted from feeder layers are necessary to prevent hES cell from differentiation.In this review,we will summarize the advantages and disadvantages of various types of feeder cells by which a reference for future research will be provided.

Effect of Human Feeders on Growth of Human Embryonic Stem Cells / 中山大学学报(医学科学版)

[Objective] The aim was to choose the best feeder layer by observing the effects of various human feeders supporting human embryonic stem cells (hESCs), and to probe the correlation between the levels of basic fibroblast growth factor (bFGF) secreted by feeders and the growth of the hESCs. [Methods] The primary cells from various tissues were cultured, including foreskin, stromal endometrium, villus, adult fallopian tubal, fetal skin, fetal muscle and mouse embryonic fibroblasts (MEFs). The hESCs were transferred to various feeders, and then the best condition was probed, which was based on the feeder density and the time of mitomycin-C acting on the feeder. Comparing the characteristics of the hESCs, the best feeder was chosen of all kinds of feeders from various tissues that support the hESCs. The level of bFGF secreted by various feeders was detected using ELISA. [Results] All of tested feeders could support the hESCs growth for over 10 passages in the culture, especially the foreskin and the adult fallopian tubal. The density of feeders was related with the morphology and the differentiation rate of the hESCs. According to the characteristics of feeder, the feeder ranking was as follows: foreskin, stromal endometrium, villus, adult fallopian tubal, fetal skin and fetal muscle. Based on the characteristics of the hESCs, the order of feeders was: foreskin, adult fallopian tubal, stromal endometrium, villus, fetal muscle and fetal skin. The levels of bFGF (pg·10~(-5)·mL~(-1)) secreted by various feeders were as follows: adult fallopian tubal (13.23±3.39), foreskin (1.99±0.17), villus (1.40±0.17), fetal muscle (2.02 ±1.59), stromal endometrium (0.38±0.28), and fetal skin (0.29±0.29). [Conclusion] The foreskin and the adult fallopian tubal could support the hESCs better than others though all of them could;do, especially the, foreskin. The bFGF that secreted by the adult fallopian tubal was the highest of all. The correlation was not obvious .between the levels of bFGF secreted by feeders and the growth of the hESCs.

Influence of feeder layer on the expression of stem cell markers in cultured limbal corneal epithelial cells.

Background & objectives: The limbus is enriched with the stem cells of corneal epithelium. Auto- and allograft limbal transplantations are effective in restoring the corneal epithelium and inhibiting inflammation and neovascularization. Preserved human amniotic membrane (AM) is now widely used as a substrate for ocular surface reconstruction. The combination of limbal and AM transplantation has been shown to improve the surgical outcome in patients with total limbal stem cell deficiency (LSCD). The purpose of this study was to compare the expression of putative stem cell markers ATP binding cassette protein (ABCG2) and keratinocyte stem cell marker: p63 and differentiation markers. (connexin 43 and keratin 3 / keratin 12) on the limbal epithelial cells cultured over the denuded AM with and without the 3T3 murine fibroblast cells as feeder layer. Methods: Human limbal tissues obtained from the cadaveric donor eyes were cultured over the denuded human amniotic membrane in the presence of mitomycin C treated 3T3 fibroblasts and the cultured cells studied for the expression of ABCG2 and p63 by immunohistochemistry and Western blot. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was done on the cultured cells at varying intervals of time for expression of ABCG2, p63, connexin43 (Cnx43), and keratin 3 (K3) and keratin 12 (K12). Results: The growth rates were similar in both denuded AM and denuded AM + 3T3. The cells cultured over AM + 3T3 showed the expression of p63 and ABCG2 till 21 days of incubation by immunohistochemistry and Western blot. The expression of p63 and ABCG2 were retained till 21 days of incubation on the cells cultured over denuded AM + 3T3, whereas it was expressed only till day 8 on the cells cultured over the denuded membrane by semi quantitative RT-PCR. Cnx43 and K3/K12 were observed in both the conditions. Interpretation & conclusions: The limbal epithelial cells cultured in the presence of mitomycin C treated 3T3 feeder layer were able to maintain the expression of putative stem cell markers. Further in vitro studies using feeder layer will enable us to understand the factors, which play a role in maintaining the limbal stem cell niche.

Human dermal fibroblasts as a feeder layer promote the growth of human keratinocytes / 基础医学与临床

Objective To study the mechanism of dermal fibroblasts as a feeder layer to support the growth of human keratinocytes. Methods Human dermis fibroblasts were isolated and cultured and then treated with mitomycin-C. The expression of type Ⅰand type Ⅲ precollagen mRNA and relevant protein in feeder layer were examined by RT-PCR and Immunohistochemistry. KCs were cultured both on FB and NIH3T3 feed layer as control, the adhering numbers and the time of fusion were recorded. Results RT-PCR showed an increase of type Ⅰprecollagen mRNA in FB feeder layer as compared with that of normal fibroblasts (P

Effect of the Isolation Method of Mouse Inner Cell Mass, Types of Feeder Cells and Treatment Time of Mitomycin C on the Formation Rate of ICM Colony / 대한불임학회지

OBJECTIVE: This study was carried out to evaluate the effect of the isolation methods of inner cell mass from mouse blastocyst, types of feeder cells and treatment time of mitomycin C on the formation rate of ICM colony. METHODS: The inner cells were isolated by conventional immunosurgery, partial trophoblast dissection with syringe needles and whole blastocyst co-culture method. Commercially available STO and primary cultured mouse embryonic fibroblast (pMEF) feeder cells were used, and mitomycin C was treated for 1, 2 or 3 hours, respectively. The formation rate of ICM colony was observed after isolation of ICM and culture of ICM on the feeder cells for 7 days. RESULT: The ICM colony formation rate on STO were significantly higher in partial trophoblast dissection group (58%) than that in immunosurgery (12%) or whole blastocyst culture (16%) group (p<0.05). The formation rate on pMEF feeder layer was higher in partial trophoblast dissection (88%) and whole blastocyst culture (82%) group than that in immunosurgery (16%) group (p<0.05). When mitomycin C treated to pMEF for 2 hours, the formation rate of 88% was significantly higher than those of other conditions. CONCLUSIONS: Above results showed that the efficient isolation method of ICM from blastocyst was the partial trophoblast dissection and the appropriate treatment time of mitomycin C was 2 hours. However, the subculture of ICM colony and characterization of stem cells should be carried out to confirm the efficacy of the partial trophoblast dissection method.

Human Amniotic Fluid Cells Support Expansion Culture of Human Embryonic Stem Cells / 대한불임학회지

OBJECTIVE: This study was performed to evaluate the possibility of prolonged culture of human embryonic stem cells (hESC; SNUhES2) on human amniotic fluid cells (hAFC), which had been storaged after karyotyping. METHOD: The hAFC was prepared for feeder layer in the presence of Chang's medium and STO medium (90% DMEM, 10% FBS) at 37degrees C in a 5% CO2 in air atmosphere. Prior to use as a feeder layer, hAFC was mitotically inactivated by mitomycin C. The hESCs on hAFC were passaged mechanically every seven days with ES culture medium (80% DMEM/F12, 20% SR, bFGF). RESULTS: The hAFC feeder layer support the growth of undifferentiated state of SNUhES2 for at least 59 passages thus far. SNUhES2 colonies on hAFC feeder appeared slightly angular and flatter shape as compared with circular and thicker colonies observed with STO feeder layer and showed higher level with complete undifferentiation in seven days. Like hESC cultured on STO feeders, SNUhES2 grown on hAFC expressed normal karyotype, positive for alkaline phosphatase activity, high telomerase activity, Oct-4, SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81 and formed embryoid bodies (EBs). CONCLUSION: The hAFC supports undifferentiated growth of hESC. Therefore, these results may help to provide a clinically practicable method for expansion of hESC for cell therapies.

Research on the method of culture and identification of embryonic stem cells from human primordial germ cells / 中国免疫学杂志

Objective:To study the cooperated effect of Human Embryonic Lung Fibroblast(HELF) feeder layer and LIF in the culture of human Embryonic Stem(hES) cells and establish the method of identifying hES cells from Primordial Germ Cells(PGCs).Methods:Embryonic lungs were mechanically disaggregated,then cultured to establish HELF feeder layer.Gonadal ridges and mesenteries of embryos were mechanically disaggeregated,then cultured and passaged in vitro.Comparing the growth characters of hES cells in different conditions.To identify hES cells through their biological characteristics.Results:The coactions of HELF feeder layer and LIF play an important role in proliferation and undifferentiation of hES cells in vitro.High levels of AKP and telomerase activity are associated with hES cells. The cultrued cells have been continuously passaged for more than two months and found to be karyotypically normal and stable.When differentiating,they form embryoid bodies(EBs).Conclusion:To avoid indection of the heterogeneous protein,HELF feeder layer, in the presence of LIF,can be used in culture of hES cells;hES cells from PGCs can be identified through their biological characteristic. [

Growth and Characterization of the Uveal Melanoma in Vitro

This study was performed to observe the influence of the feeder layer on the culture of the human uveal melanoma cell. Two types of melanoma cells which were obtained from the patients with choroidal melanoma were implanted into the culture flask. The melanoma cell was seeded onto the culture dish covered with conjunctival fibroblast, Vero cell, and culture dish which did not contain the feeder layer respectively. The growth pattern of the melanoma cell was observed with phase contrast microscopy upto 30 days after seeding. There was no definite difference in growth pattern between each group. These results may indicate that the feeder layer is not an essential factor in the culture of uveal melanoma cell.

Biologic characters of human foreskin fibroblasts / 重庆医科大学学报

Objective:To study the biologic characters of human foreskin fibroblasts(HFF)to lay the foundation for a long-term culture for human embryonic germ cells in vitro.Methods:The method of separating HFF was investigated.By immunocytochemical method,MTT and flow cytometry,we investigated the morphology and the purity of HFF,study the cell growth curve and cell cycle of HFF,and the effects of different concentrations of mitomycin on growth of HFF at different times.Results:Epidermis and dermis were more easily separated after overnight digestion(12~16 h)in 0.25% Trypsin at 4℃,compared with incubation for 2 h in 5% CO2 at 37℃.Vimentin was strongly positive stained with the purity of 95 percent.There was no obvious stranded period.The rapid proliferation period was in 1~6 d of cell growth curve.The platform stage began at 7 d.Majority of the cells were in G1/G0 phases.The cell cycle corresponded to cell growth curve.Mitomycin C(MMC)inhibited the proliferation of HFF at 12.5 ?g/ml over 2.5 h of MMC treatment.Conclusion:Its biological characters showed that HFF can be used as feeder layer for in-vitro cultured human embryonic germ cells。

Study on the preparation of mouse embryonic stem cell feeder layer / 重庆医科大学学报

Objective:To establish a stable and effective system of mouse embryonic fibroblasts(MEFs) feeder layer in order to separate and culture mouse embryonic stem cells(ESC) in vitro.Methods:the MEFs were isolated from the mouse embryos at 13.5~14.5dpc(days post-coitus) through primary tissue digestion and tissue piece culture.The optional planting density of cell was determined according to the cellular growth curve detected by MTT and the morphology in the culture dish under inverted microscope.The optional concentration of mitomycin C and the time treating with mitomycin on MEFs were determined by MTT assay.Murine blastcysts(3.5dpc) were cultured on the feeder layer treated by mitomycin C and ESC growth was observed.Result:The method of tissue digestion and tissue piece culture is an efficient way to obtain primary MEFs;1?105~2?105/ml cells is an optional planting density;MEFs proliferation could be effeciently repressed after being treated with mitomycin C 10?g/ml for 3.5 hours or 20?g/ml for 2.5 hours,and the quantity of MEFs could maintain at least for 7 days.ES clone could be obtained from the mouse blastcysts(3.5dpc) after being cultured on the feeder layer.Conclusion:MEFs feeder layer could effectively support mouse embryonic stem cells to grow.