• 식물조직배양학회지
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• 제24권3호
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• pp.167-173
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• 1997

The embryogenic callus (EC), from which somatic embryos could be induced, was compared with nonembryogenic callus(NE) to study the origin and features of totipotent cell in water dropwort (Oenanthe stolonifera DC). To induce and maintain of EC and the NE, meristematic stem and immature floret were inoculated in MS media supplemented with 1 mg/L 2,4-D, and with 2.5 mg/L NAA and 5mg/L BA, respectively, The EC was not induced from the NE even after subculturing in MS medium supplemented with 1 mg/L 2,4-D. Plantlets were not regenerated from the NE in hormone-free medium. In histochemical comparison of the EC with the NE by light microscopy, the EC had smaller cells in size, dense cytoplasm, and more starch granules of cells compared to the NE cells. The cell from the EC, as observed by transmission electron microscopy, had smaller vaculoes, well developed ribosomes, mitochondria, and endoplasmic reticulum, whereas the cells from the NE had larger vacuoles and underdeveloped organelles. In protein pattern from NE, EC and Somatic embryo (SE), as analyzed by SDS polyacrylamide gel electrophoresis, different proteins specific for tissue were observed: 17 and 28 KD for NE, 50, 52, 57, 66, 68 KD for EC and 20 KD for SE. DNA polymorphism was also observed between EC and NE as analyzed by RAPD (randomly amplified polymorphic DNA) method. The origin of totipotent stem cell and the relationship between irreversible genomic change arose in differentiation and the loss of totipotency in plant were discussed.

• 식물조직배양학회지
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• 제27권6호
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• pp.423-428
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• 2000

In 1997 when cloned sheep Dolly and soon after Polly were born, it had become head-line news because in the former the nucleus that gave rise to the lamb came from cells of six-year-old adult sheep and in the latter case a foreign gene was inserted into the donor nucleus to make the cloned sheep produce human protein, factor IX, in e milk. In the last few years, once the realm of science fiction, cloned mammals especially in livestock have become almost commonplace. What the press accounts often fail to convey, however, is that behind every success lie hundreds of failures. Many of the nuclear-transferred egg cells fail to undergo normal cell divisions. Even when an embryo does successfully implant in the womb, pregnancy often ends in miscarriage. A significant fraction of the animals that are born die shortly after birth and some of those that survived have serious developmental abnormalities. Efficiency remains at less than one % out of some hundred attempts to clone an animal. These facts show that something is fundamentally wrong and enormous hurdles must be overcome before cloning becomes practical. Cloning researchers now tent to put aside their effort to create live animals in order to probe the fundamental questions on cell biology including stem cells, the questions of whether the hereditary material in the nucleus of each cell remains intact throughout development, and how transferred nucleus is reprogrammed exactly like the zygotic nucleus. Stem cells are defined as those cells which can divide to produce a daughter cell like themselves (self-renewal) as well as a daughter cell that will give rise to specific differentiated cells (cell-differentiation). Multicellular organisms are formed from a single totipotent stem cell commonly called fertilized egg or zygote. As this cell and its progeny undergo cell divisions the potency of the stem cells in each tissue and organ become gradually restricted in the order of totipotent, pluripotent, and multipotent. The differentiation potential of multipotent stem cells in each tissue has been thought to be limited to cell lineages present in the organ from which they were derived. Recent studies, however, revealed that multipotent stem cells derived from adult tissues have much wider differentiation potential than was previously thought. These cells can differentiate into developmentally unrelated cell types, such as nerve stem cell into blood cells or muscle stem cell into brain cells. Neural stem cells isolated from the adult forebrain were recently shown to be capable of repopulating the hematopoietic system and produce blood cells in irradiated condition. In plants although the term$\boxDr$ stem cell$\boxUl$is not used, some cells in the second layer of tunica at the apical meristem of shoot, some nucellar cells surrounding the embryo sac, and initial cells of adventive buds are considered to be equivalent to the totipotent stem cells of mammals. The telomere ends of linear eukaryotic chromosomes cannot be replicated because the RNA primer at the end of a completed lagging strand cannot be replaced with DNA, causing 5' end gap. A chromosome would be shortened by the length of RNA primer with every cycle of DNA replication and cell division. Essential genes located near the ends of chromosomes would inevitably be deleted by end-shortening, thereby killing the descendants of the original cells. Telomeric DNA has an unusual sequence consisting of up to 1,000 or more tandem repeat of a simple sequence. For example, chromosome of mammal including human has the repeating telomeric sequence of TTAGGG and that of higher plant is TTTAGGG. This non-genic tandem repeat prevents the death of cell despite the continued shortening of chromosome length. In contrast with the somatic cells germ line cells have the mechanism to fill-up the 5' end gap of telomere, thus maintaining the original length of chromosome. Cem line cells exhibit active enzyme telomerase which functions to maintain the stable length of telomere. Some of the cloned animals are reported prematurely getting old. It has to be ascertained whether the multipotent stem cells in the tissues of adult mammals have the original telomeres or shortened telomeres.

• Asian-Australasian Journal of Animal Sciences
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• 제16권8호
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• pp.1102-1107
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• 2003

The ES cell can provide a useful system for studying differentiation and development in vitro and a powerful tool for producing transgenic animalds. To investigate the culture condition of chicken embryonic stem (CES) cells which can retain their multipotentiality or totipotency, three kinds of feeder layer cells, SNL cells, primary mice embryonic fibroblasts (PMEF) cells and primary chicken embryonic fibroblasts (PCEF) cells, were used as the feeder cells in media of DMEM supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and stem cell factor (SCF) for co-culture with blastoderm cells from stage X embryos of chicken. The alkaline phosphatase (AKP) test, differentiation experiment in vitro and chimeric chicken production were carried out. The results showed that culture on feeder layer of PMEF yielded high quality CES cell colonies. The typical CES cells clone shape revealed as follows: nested aggregation (clone) with clear edge and round surface as well as close arrangement within the clone. Strong alkaline phosphatase (AKP) reactive cells were observed in the fourth passage cells. On the other hand, the fourth passage CES cells could differentiate into various cells in the absence of feeder layer cells and LIF in vitro. The third and fourth passage cells were injected into the subgerminal cavity of recipient embryos at stage X. Of 269 Hailan embryos injected with CES cells of Shouguang Chickens, 8.2% (22/269) survived to hatching, 5 feather chimeras had been produced. This suggests that an effective culture system established in this study can promote the growth of CES cells and maintain them in the state of undifferentiated and development, which lays a solid foundation for the application of CES cells and may provide an alternative tool for genetic modification of chickens.

• Journal of Plant Biotechnology
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• 제33권3호
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• pp.195-207
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• 2006

Stem cells are the primordial, initial cells which usually divide asymmetrically giving rise to on the one hand self-renewals and on the other hand progenitor cells with potential for differentiation. Zygote (fertilized egg), with totipotency, deserves the top-ranking stem cell - he totipotent stem cell (TSC). Both the ICM (inner cell mass) taken from the 6 days-old human blastocyst and ESC (embryonic stem cell) derived from the in vitro cultured ICM have slightly less potency for differentiation than the zygote, and are termed pluripotent stem cells. Stem cells in the tissues and organs of fetus, infant, and adult have highly reduced potency and committed to produce only progenitor cells for particular tissues. These tissue-specific stem cells are called multipotent stem cells. These tissue-specific/committed multipotent stem cells, when placed in altered environment other than their original niche, can yield cells characteristic of the altered environment. These findings are certainly of potential interest from the clinical, therapeutic perspective. The controversial terminology 'somatic stem cell plasticity' coined by the stem cell community seems to have been proved true. Followings are some of the recent knowledges related to the stem cell. Just as the tissues of our body have their own multipotent stem cells, cancerous tumor has undifferentiated cells known as cancer stem cell (CSC). Each time CSC cleaves, it makes two daughter cells with different fate. One is endowed with immortality, the remarkable ability to divide indefinitely, while the other progeny cell divides occasionally but lives forever. In the cancer tumor, CSC is minority being as few as 3-5% of the tumor mass but it is the culprit behind the tumor-malignancy, metastasis, and recurrence of cancer. CSC is like a master print. As long as the original exists, copies can be made and the disease can persist. If the CSC is destroyed, cancer tumor can't grow. In the decades-long cancer therapy, efforts were focused on the reducing of the bulk of cancerous growth. How cancer therapy is changing to destroy the origin of tumor, the CSC. The next generation of treatments should be to recognize and target the root cause of cancerous growth, the CSC, rather than the reducing of the bulk of tumor, Now the strategy is to find a way to identify and isolate the stem cells. The surfaces of normal as well as the cancer stem cells are studded with proteins. In leukaemia stem cell, for example, protein CD 34 is identified. In the new treatment of cancer disease it is needed to look for protein unique to the CSC. Blocking the stem cell's source of nutrients might be another effective strategy. The mystery of sternness of stem cells has begun to be deciphered. ESC can replicate indefinitely and yet retains the potential to turn into any kind of differentiated cells. Polycomb group protein such as Suz 12 repress most of the regulatory genes which, activated, are turned to be developmental genes. These protein molecules keep the ESC in an undifferentiated state. Many of the regulator genes silenced by polycomb proteins are also occupied by such ESC transcription factors as Oct 4, Sox 2, and Nanog. Both polycomb and transcription factor proteins seem to cooperate to keep the ESC in an undifferentiated state, pluripotent, and self-renewable. A normal prion protein (PrP) is found throughout the body from blood to the brain. Prion diseases such as mad cow disease (bovine spongiform encephalopathy) are caused when a normal prion protein misfolds to give rise to PrP$^{SC}$ and assault brain tissue. Why has human body kept such a deadly and enigmatic protein? Although our body has preserved the prion protein, prion diseases are of rare occurrence. Deadly prion diseases have been intensively studied, but normal prion problems are not. Very few facts on the benefit of prion proteins have been known so far. It was found that PrP was hugely expressed on the stem cell surface of bone marrow and on the cells of neural progenitor, PrP seems to have some function in cell maturation and facilitate the division of stem cells and their self-renewal. PrP also might help guide the decision of neural progenitor cell to become a neuron.

• Journal of Yeungnam Medical Science
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• 제15권1호
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• pp.36-46
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• 1998

췌장의 고형 유두상 상피종양의 임상 병리학적 특징을 분석하고 면역조직화학적 및 전자현미경적 소견을 바탕으로 종양세포의 기원을 살펴보고자 본 연구를 시행하였다. 1990년부터 1996년까지 영남대학교 의과대학 부속병원에서 체장의 고형 유두상 상피종양으로 진단받은 8례를 대상으로 임상병리학적 특정의 분석과 면역조직화학적 및 전자현미경적 검색을 시행하였다. 8례 모두 여성이었고 연령은 21세에서 54세 사이였고 평균연령은 34세였다. 종양의 위치는 미부(4례), 체부-미부(2례), 체부(1례), 두부(1례)에 각각 발생하였다. 육안적으로 고형성 부위, 출혈과 낭성 변화가 혼합된 피막을 가진 종괴로 평균 크기는 9.3cm였고, 조직학적으로 원형 내지 다각형의 균일한 종양세포가 판상 혹은 유두상 배열을 보였다. 변역조직화학 염색에서 8례(100%)가 ${\alpha}1$-antitrypsin에 양성이었고, 7례(87.5%)가 cytokeratin, 7례 (87.5%)가 progesterone 수용체, 6례(75%)가 vimentin, 그리고 1례 (12.5%)가 synaptophysin에 각각 양성이었다. Estrogen 수용체에 대하여는 전예가 음성 반응을 보였다. 전자현미경 소견상 종양세포의 세포질에는 미토콘드리아가 풍부하였고 효소원 과립과 환충판이 관찰되었고 인접한 세포들 사이에 소관 구조를 닮은 간극이 관찰되어 췌장의 고형 유두상 상피종양은 전능 간세포(totipotent stem cell)에서 기원할 것으로 생각된다.

• 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.

• 한국가축번식학회지
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• 제19권1호
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• pp.55-63
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• 1995

The putative totipotency germ cells has a relative abundance of alkaline phosphatases. Thus, histological staining of AP activity offers a new route to isolate totipotent cells and also provides insights into culture systems of these cells. Furthermore, the AP staining technique is simple and fast, requires only the napthol AS/MS substrate in combination with trapping diazonium salts such as fast red or fast blue. However, our unexpected finding was that AP staining of mouse ES cells were detected in the undifferentiaed epiblast-derived cells as well as several types of differentiating cells. This findings are different from results of Talbot et al. (1993) reported usefulness of the AP staining and implies that histological staining of AP may not by useful to determine undifferentiaed state or totipotency of ES cells. Thus, we have investigated the patterns of AP expression by RT-PCR in order to identify a marker of undifferentiated ES/primordial germ (PG) cells. In RT-PCR analysis, embryonic (E)-AP was detected only in undifferentiated ES cells, but intestinal(I)-AP was not detected in all of the examined ES and PG cells. In addition, nonspecific (NS)-AP wasdetected in undifferentiated PG cell from day 7, 5 to 13 of gestation. Histological activity of AP in ES cells was completely suppressed by addition of L-phenylalanine (Phe), L-homoarginine (Har), and L-phenylalanylglycylglycine (PheGlyGly) as an inhibitor, but RT-PCR showed the same results as in the absence of an inhibitors. Our findings suggested that expression of E-AP and NS-AP may use as a marker to determine the undifferentiated status in ES and PG cells.