• Molecules and Cells
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• v.37 no.6
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• pp.473-479
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• 2014

Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost-effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.65-72
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• 2020

Fish ovarian germline stem cells (OGSCs) that have the abilities to self-renew and differentiate into functional gametes can be used in various researches and applications. A main issue to be solved for effective utilization of fish OGSCs is the development of their stable in vitro culture condition, but only few researches about fish OGSC culture have been reported so far. In this study, in order to find the clues to develop the culture condition for OGSCs from Japanese medaka (Oryzias latipes), we tried to establish somatic cell lines as a candidate for the feeder cells and evaluated its supporting effects on the culture of ovarian cell populations from O. latipes. As the results, the somatic cell lines could be established only from the embryonic tissues among three tissues derived from embryos, fins and ovaries. Three embryonic cell lines were tested as a feeder cell for the culture of ovarian cell population and all three cell lines induced cell aggregation formation of the cultured ovarian cells whereas the feeder-free condition did not. Furthermore, a significant cellular proliferation was observed in the ovarian cells cultured on two of three cell lines. As a trial to increase the capacity of the cell lines as a feeder cell that supports the proliferation of the cultured ovarian cells, we subsequently established a stable line that expresses the foreign O. latipes fibroblast growth factor 2 (FGF2) from an embryonic cell line and evaluated its effectiveness as a feeder cell. The ovarian cells cultured on FGF2 expressing feeder cells still formed cell aggregates but did not show a significant increase in cellular proliferation compared to those cultured on non-transformed feeder cells. The results from this study will provide the fundamental information for in vitro culture of medaka OGSCs.

• Development and Reproduction
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• v.19 no.3
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• pp.119-126
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• 2015

The suitable feeder cell layer is important for culture of embryonic stem (ES) cells. In this study, we investigated the effect of two kinds of the feeder cell, MEF cells and STO cells, layer to mouse ES (mES) cell culture for maintenance of stemness. We compare the colony formations, alkaline phosphatase (AP) activities, expression of pluripotency marker genes and proteins of D3 cell colonies cultured on MEF feeder cell layer (D3/MEF) or STO cell layers (D3/STO) compared to feeder free condition (D3/-) as a control group. Although there were no differences to colony formations and AP activities, interestingly, the transcripts level of pluripotency marker genes, Pou5f1 and Nanog were highly expressed in D3/MEF (79 and 93) than D3/STO (61and 77) or D3/- (65 and 81). Also, pluripotency marker proteins, NANOG and SOX-2, were more synthesized in D3/MEF ($72.8{\pm}7.69$ and $81.2{\pm}3.56$) than D3/STO ($32.0{\pm}4.30$ and $56.0{\pm}4.90$) or D3/- ($55.0{\pm}4.64$ and $62.0{\pm}6.20$). These results suggest that MEF feeder cell layer is more suitable to mES cell culture.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.291-294
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• 2012

Embryonic stem cell classically cultured on feeder layer with FBS contained ES medium. Feeder-free mouse ES cell culture systems are essential to avoid the possible contamination of nonES cells. First we determined the difference between ES cell and MEF by Oct4 population. We demonstrate to culture and to induce differentiation on feeder free condition using a commercially available mouse ES cell lines.

• Molecules and Cells
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• v.41 no.7
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• pp.631-638
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• 2018

Spermatogonial stem cells (SSCs) derived from mouse testis are unipotent in regard of spermatogenesis. Our previous study demonstrated that SSCs can be fully reprogrammed into pluripotent stem cells, so called germline-derived pluripotent stem cells (gPS cells), on feeder cells (mouse embryonic fibroblasts), which supports SSC proliferation and induction of pluripotency. Because of an uncontrollable microenvironment caused by interactions with feeder cells, feeder-based SSC reprogramming is not suitable for elucidation of the self-reprogramming mechanism by which SSCs are converted into pluripotent stem cells. Recently, we have established a Matrigel-based SSC expansion culture system that allows longterm SSC proliferation without mouse embryonic fibroblast support. In this study, we developed a new feeder-free SSC self-reprogramming protocol based on the Matrigel-based culture system. The gPS cells generated using a feeder-free reprogramming system showed pluripotency at the molecular and cellular levels. The differentiation potential of gPS cells was confirmed in vitro and in vivo. Our study shows for the first time that the induction of SSC pluripotency can be achieved without feeder cells. The newly developed feeder-free self-reprogramming system could be a useful tool to reveal the mechanism by which unipotent cells are self-reprogrammed into pluripotent stem cells.

• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.81-88
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• 1998

The Southeast Asian javanica rice variety Tinawen was investigated for efficient protoplast culture and plant regeneration from cell suspension-derived protoplasts using a feeder cell culture method. Feeder cells of both Lolium multiflorum and Oryza ridleyi, either alone, or in combination, were employed and plants were regenerated from protoplast-derived colonies on several plant regeneration media. Dehydration of protoplast-derived colonies was also investigated as a means of enhancing plant regeneration. In the presence of L. multiflorum or O. ridleyi feeder cells, the protoplast plating efficiency ranged from 0.09% to 1.48%, depending on the feeder cell type and the age of the cell suspension. L. multiflorum feeder cells induced approximately 6-fold higher plating efficiency compared with those of O. ridleyi. The plant regeneration frequencies were 19.3-31.7% with L. multiflorum, 13.0-18.0% with O. ridleyi and 18.0-22.0% with a mixture of both in various plant regeneration media when protoplast-derived colonies were dehydrated, while for the non-dehydrated colonies, the values were 2.0-7.0%, 3.0-5.0% and 0-4.0%, respectively. Flow cytometric analysis of 34 protoplast-derived plants showed that the majority of plants were diploids and only 2 plants were tetraploids. The plants which were transferred to glasshouse were fertile.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.86-93
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• 2020

The establishment of porcine embryonic stem cells (ESCs) from porcine somatic cell nuclear transfer (SCNT) blastocysts is influenced by in vitro culture day of porcine reconstructed embryo and feeder cell type. Therefore, the objective of the present study was to determine the optimal in vitro culture period for reconstructed porcine SCNT embryos and mouse embryonic fibroblast (MEF) feeder cell type for enhancing colony formation efficiency from the inner cell mass (ICM) of porcine SCNT blastocysts and their outgrowth. As the results, porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days showed significantly increased efficiency in the formation of colonies, compared to those for 7 days. Moreover, MEF feeder cells derived from outbred ICR mice showed numerically the highest efficiency of colony formation in blastocysts produced through in vitro culture of porcine SCNT embryos for 8 days and porcine ESCs with typical ESC morphology were maintained more successfully over Passage 2 on outbred ICR mice-derived MEF feeder cells than on MEF feeder cells derived from inbred C57BL/6 and hybrid B6CBAF1 mice. Overall, the harmonization of porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days and MEF feeder cells derived from outbred ICR mice will greatly contribute to the successful establishment of ESCs derived from porcine SCNT blastocysts.

• Proceedings of the KIEE Conference
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• 2008.10c
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• pp.161-163
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• 2008

The paper deals with a inverter control scheme to apply feeder flow control in the hybrid system consisted of a photovoltaic system and a fuel-cell system. The inverter operation modes and a feeder control reference is changed by changing of the loads. Simulation results using the PSCAD/EMTDC are presented to establish a inverter control method for a Feeder flow control mode.

• Development and Reproduction
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• v.12 no.2
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• pp.141-149
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• 2008

This study was conducted to develop an efficient cryopreservation method of human embryonic stem (ES) cells using vitrification. In an initial experiment, sub-clumps of human ES cells (CHA-hES3 and CHA-hES4) were vitrified using grids after incubation with STO feeder cells for 1 or 16 h (Groups 1-1 and 1-2, respectively). After storage for $2{\sim}4$ months, thawed clumps were re-plated on a fresh feeder layer. The survival rates of warmed CHA-hES3 and CHA-hES4 cells of Group 1-2 were significantly higher than those of the corresponding Group 1-1 cells. In the second experiment, human ES cells were vitrified after incubation with feeder or feeder-conditioned medium (Groups 2-1 to -7). Relative mRNA expression of BM proteins and survival rates were increased following incubation of ES cells with fresh feeder cells for 16 h. In conclusion, increasing of tight adhesion between ES cells by extended incubation with feeder could reduce cryoinjury after vitrifying/warming.

• Korean Journal of Plant Tissue Culture
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• v.24 no.5
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• pp.295-303
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• 1997

To investigate the response on feeder cell cultures, protoplasts isolated from cell suspensions initiated from mature seed scutellum-derived callus of the Japonica rice variety Taipei 309, were cultured on filter membranes under various conditions. The effects of various factors, such as gelling agents, feeder cell and protoplast densities, species of feeder cells and heat shock treatment, have been investigated to improve protoplast plating efficiencies on filter membranes. Maximum protoplast plating efficiencies were obtained when protoplasts were cultured on KPR medium semi-solidified with Sea Plaque agarose at a density of $5\;\times\;10^{5}\;ml^{-1}$ protoplasts in the presence of Lolium multiflorum as feeder cells (0.5 ml pcv per 10 ml of protoplast culture medium). Pre-culture heat shock treatments for 1 min. and 5 min. to the protoplasts did not give any appreciable increase on the plating efficiency of protoplasts in the presence of feeder cells. Maltose-supplemented medium was superior for plant regeneration from protoplast-derived colonies compared with medium containing only sucrose. The plants were transferred to the glasshouse, flowered and were fertile.