• 식물조직배양학회지
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• 제25권2호
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• pp.81-88
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• 1998

Southeast Asian javanica 벼 품종 Tinawen의 진탕 배양세포로부터 나출된 원형질체의 효과적인 배양과 식물체 재분화가 조사되었다. Lolium multiforum과 Oryza ridleyi의 진탕 배양세포들을 feeder cell로 사용했고 여러가지 재분화 배지를 이용하여 원형질체로부터 유도된 colony들을 재분화 시켰으며, 또한 식물체 재분화율을 높히기 위해 원형질체로 부터 유도된 colony들을 dehydration 시켜 재분화율을 조사하였다. L multiflorum 또는 O. ridleyi의 진탕 배양세포들을 feeder cell로 사용했을 때 원형질체의 평판효율은 feeder cell type과 age에 따라 차이가 났지만 0.09%에서 1.48% 범위로 나타났고, L. multiflorum을 feeder cell로 사용했을 때가 O. ridleyi cell을 사용했을때 보다 6배 높게 원형질체 평판효율을 얻었다. Feeder cell로 L. multiflorum을 사용하여 배양된 원형질채로부터 유도된 colony들을 dehydration 시킨 경우는 19.3-31.7%, O. ridleyi을 사용한 경우는 13.0-18.0%, 또한 이들 두 진탕 배양세포들을 혼합한 것을 사용한 경우는 18.0-22.0%의 식물체 재분화율을 얻은 반면에, dehydration을 시키지 않았을 때는 각각 2.0-7.0%, 3.0-5.0%, 0-4.0%의 재분화율을 얻었다. 원형 질체에서 재분화된 식물체의 flow cytometry를 이용한 배수성 분석 결과 대부분의 식물체가 이배체로 나타난 반면, 단지 34개중 두 식물체에서만이 4배체로 나타났다. 재분화된 식물체들은 온실에 옮겨 기른 결과 정상적인 임성을 나타내었다.

• Clinical and Experimental Reproductive Medicine
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• 제32권2호
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• pp.133-147
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• 2005

Objectives: This study was carried out to establish human embryonic stem cells derived from frozen-thawed embryos using mouse embryonic fibroblasts (mEFs), human fetal skin and muscle fibroblasts as feeder cells, and to identify the characteristic of embryonic stem cells. Methods: When primary mEFs, human fetal skin and muscle fibroblasts were prepared, passaging on 4 days from replating could have effective trypsinization and clear feeder layers. Eight of 23 frozenthawed 4~8 cell stage embryos donated from consenting couples developed to blastocysts. Inner cell mass (ICM) was isolated by immunosurgery. ICM was co-cultured on mEFs, human fetal skin or muscle fibroblasts. The ICM colonies grown on mEFs, human fetal skin or muscle fibroblasts were tested the expression of stage specific embryonic antigen-3, -4 (SSEA-3, -4), octamer binding transcription factor-4 mRNA (Oct-4) and alkaline phosphatase surface marker. Results: We obtained 1 ICM colony from 2 ICM co-cultured on mEFs as feeder cells and did not obtain any ICM colony from 6 ICM clumps co-cultured on human fetal skin or muscle fibroblasts. The colony formed on mEFs could be passaged 30 times every 5 days with sustaining undifferentiated colony appearance. When the colonies cultured on mEFs were grown on human fetal skin or muscle fibroblasts, the colonies could be passaged 15 times every 9 days with sustaining undifferentiated colony appearance. The colonies grown on mEFs and human fetal fibroblasts expressed SSEA-4 and alkaline phosphatase surface markers and positive for the expression of Oct-4 by reverse transcription-polymerase chain reaction (RT-PCR). The produced embryoid body differentiated spontaneously to neural progenitorlike cells, neuron-like cells and beating cardiomyocyte-like cells, and frozen-thawed embryonic stem cells displayed normal 46,XX karyotype. Conclusions: The human embryonic stem cells can be established by using mEFs and human fetal fibroblasts produced in laboratory as feeder cells.

• 한국발생생물학회지:발생과생식
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• 제20권1호
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• pp.63-71
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• 2016

Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feeder layers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KO-SR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xeno-free conditions for clinical grade hESCs culture will be useful data in future clinical studies.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.77-77
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• 2003

Coculture of HSC with bone marrow-derived mesenchymal stem cells (BM-MSCs) is one of used methods to increase cell numbers before transplant to the patients. However, because of difficulties to purify HSCs after coculture with BM-MSCs, it needs to develop a method to overcome the problem. In the present study, we have examined whether a culture insert placed over a feeder layer might support the expansion of HSCs within the insert. $CD34^+/ $ cells isolated from the umbilical cord blood by using midiMACS were divided into three groups. A group of 1 $\times$ $10^5$ cells were grown on a culture insert without feeder layer (Direct). The same number of HSCs was directly cocultured with BM-MSCs (Contact). The third group was placed onto an insert below which BM-MSCs were grown (Insert). To distinguish feeder cells from HSCs, BM-MSCs was pre-labeled fluorescently with PKH26 and 1 $\times$ $10^5$ cells were seeded in the culture dishes. After culture for 13 days, the expansion factor (x) of HSCs that were grown without feeder layer (Direct) was $26.6 \pm 8.4.$ In contrast, the number of HSCs directly cocultured with feeder layer was 59.6 $\pm$ 0.5 and that of HSCs cultured onto an insert was $46.9 \pm 8.4.$ The percentage of BM-MSCs cells remained being fluorescent was $97.9 \pm 0.3%$ after culture. Immune-phenotypically large proportion of cultured cells were founded to be differentiated into myeloid/monocyte progenitor cells. The ability of BM-MSCs, fetal lung, cartilage and brain tissue cells to support ex vivo expansion of HSCs was also examined using the insert. After 11 days of coculture with each of these cells, the expansion factor of HSCs was 15.0, 39.0, 32.0 and 24.0, respectively. Based upon these observations, it is concluded that the coculture method using insert is very effective to support ex vivo expansion of HSCs and to eliminate the contamination of other cells used to coculture wth HSCs.

• 한국수정란이식학회지
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• 제26권2호
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• pp.111-115
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• 2011

In general, zona pellucida (ZP) of the blastocyst has to be removed first, then either isolated the inner cell mass (ICM) or ZP-removed whole blastocyst, which is then cultured on the feeder layer to induce ICM outgrowth for the generation of embryonic stem cells (ESC). However, it is unclear whether ICM isolation before seeding on feeder layer is beneficial or not because the interaction between ICM and trophoblasts may affect cellular growth and/or pluripotency during the culture on the feeder. In the present study, two ZP removal methods (mechanically by splitting with a 28-gauge needle versus chemically by the treatment of acid-Tyrode's solution) and two ICM isolation methods (ZP-free whole blastocyst seeding versus mechanical isolation of ICM) were evaluated for the efficient isolation and culture of putative parthenogenetic bovine ESC. The number of maintained outgrown colonies was counted in each experimental group. As the result, mechanical removal of ZP with a needle and followed by whole ZP-free blastocyst seeding on feeder cells tended to attach more on the feeder layer and resulted in more outgrown colonies with its simple and less time-costing benefits. Currently we are generating ESC lines in HanWoo cattle by using this method for initial outgrowth of the parthenogenetic bovine blastocysts.

• Clinical and Experimental Reproductive Medicine
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• 제31권4호
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• pp.261-271
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• 2004

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 $37^{circ}C$ in a 5% $CO_2$ 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.

• 한국발생생물학회지:발생과생식
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• 제16권4호
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• pp.353-361
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• 2012

Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery. However, human ES cells are difficult to culture and maintain at a large scale, which is one of the most serious obstacles in human ES cell research. Culture of human ES cells on MEF cells after disassociation with accutase has previously been demonstrated by other research groups. Here, we confirmed that human ES cells (H9) can maintain stem cell properties when the cells are passaged as single cells under a feeder-free culture condition. Accutase-dissociated human ES cells showed normal karyotype, stem cell marker expression, and morphology. We prepared frozen stocks during the culture period, thawed two of the human ES cell stocks, and analyzed the cells after culture with the same method. Although the cells revealed normal expression of stem cell marker genes, they had abnormal karyotypes. Therefore, we suggest that accutase-dissociated single cells can be usefully expanded in a feeder-free condition but chromosomal modification should be considered in the culture after freeze-thawing.

• Asian-Australasian Journal of Animal Sciences
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• 제23권9호
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• pp.1152-1158
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• 2010

In differentiating human embryonic stem (d-hES) cells there are a number of types of cells which may secrete various nutrients and helpful materials for pre-implantation embryonic development. This study examined whether the d-hES could function as a feeder cell in vitro to support mouse embryonic development. By RT-PCR analysis, the d-hES cells revealed high expression of three germ-layered differentiation markers while having markedly reduced expression of stem cell markers. Also, in d-hES cells, LIF expression in embryo implantation-related material was confirmed at a similar level to undifferentiated ES cells. When mouse 2PN embryos were cultured in control M16 medium, co-culture control CR1aa medium or co-cultured with d-hES cells, their blastocyst development rate at embryonic day 4 (83.9%) were significantly better in the d-hES cell group than in the CR1aa group (66.0%), while not better than in the M16 group (90.7%)(p<0.05). However, at embryonic days 5 and 6, embryo hatching and hatched-out rates of the dhES cell group (53.6 and 48.2%, respectively) were superior to those of the M16 group (40.7 and 40.7%, respectively). At embryonic day 4, blastocysts of the d-hES cell group were transferred into pseudo-pregnant recipients, and pregnancy rate (75.0%) was very high compared to the other groups (M16, 57.1%; CR1aa, 37.5%). In addition, embryo implantation (55.9%) and live fetus rate (38.2%) of the d-hES cell group were also better than those of the other groups (M16, 36.7 and 18.3%, respectively; CR1aa, 23.2 and 8.7%, respectively). These results demonstrated that d-hES cells can be used as a feeder cell for enhancing in vitro and in vivo developmental potential of mouse pre-implantation embryos.

• 한국가축번식학회지
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• 제18권4호
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• pp.299-307
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• 1995

전능성을 지닌 우 수정란 세포배양기술 체계 확립은 가축육종에 중요한 의의를 지닌다. 이러한 체계는 1) 핵치환에 의한 다수의 클론동물 생산에 대한 기전, 2) 형질전환세포를 선발하기 위한 marker의 사용으로 효과적인 유전자 전이체계와 3) site specific 유전자 전이에 대한 기전 또는 homologous DNA서열 재조합에 의한 결손 등에 대한 기전을 이해하는데 이용될 수 있다. 우 수정란세포의 배양은 배반포 내부 세포괴, 상실배와 16∼20세포기로부터 확립하였다. 이들 모든 세포들은 생쥐 배아간 세포형태와 유사하였으며 배양시 분화와 증식에서 다능성을 나타내었다. 배양체계는 미세소적이나 배양용기, 우 도는 설치류 섬유아 세포주를 단기간 배양 또는 장기간 배양방법을 이용하였다. 유사분열시 요구되는 배양체계나 배양액 그리고 분화 억제에 대한 괄목할만한 장점은 아직 밝혀지지 않고 있다. 현재 16∼20 세포기의 배양세포의 전능성에 대해서는 알려져 있지 않다. 배양된 ICM세포 전능성은 27일간 배양한 ICM 세포로부터 4마리의 산자 생산에 의해 입증되었다.

• Journal of Microbiology and Biotechnology
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• 제32권1호
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• pp.126-140
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• 2022

Stem cells can be applied usefully in basic research and clinical field due to their differentiation and self-renewal capacity. The aim of this study was to establish an effective novel therapeutic cellular source and create its molecular expression profile map to elucidate the possible therapeutic mechanism and signaling pathway. We successfully obtained a mesenchymal stem cell population from human embryonic stem cells (hESCs) cultured on chemically defined feeder-free conditions and treated with connective tissue growth factor (CTGF) and performed the expressive proteomic approach to elucidate the molecular basis. We further selected 12 differentially expressed proteins in CTGF-induced hESC-derived mesenchymal stem cells (C-hESC-MSCs), which were found to be involved in the metabolic process, immune response, cell signaling, and cell proliferation, as compared to bone marrow derived-MSCs(BM-MSCs). Moreover, these up-regulated proteins were potentially related to the Wnt/β-catenin pathway. These results suggest that C-hESC-MSCs are a highly proliferative cell population, which can interact with the Wnt/β-catenin signaling pathway; thus, due to the upregulated cell survival ability or downregulated apoptosis effects of C-hESC-MSCs, these can be used as an unlimited cellular source in the cell therapy field for a higher therapeutic potential. Overall, the study provided valuable insights into the molecular functioning of hESC derivatives as a valuable cellular source.