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KR20090008652A - Skin-derived multipotent adult stem cells having the ability to express oct-4 and methods for preparing the same - Google Patents

️Thu Jan 22 2009

Skin-derived multipotent adult stem cells having the ability to express oct-4 and methods for preparing the same Download PDF

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Publication number

KR20090008652A

KR20090008652A KR1020070071762A KR20070071762A KR20090008652A KR 20090008652 A KR20090008652 A KR 20090008652A KR 1020070071762 A KR1020070071762 A KR 1020070071762A KR 20070071762 A KR20070071762 A KR 20070071762A KR 20090008652 A KR20090008652 A KR 20090008652A Authority

South Korea

Prior art keywords

cells

stem cells

ssea

oct

isolated

Prior art date

2007-07-18

Application number

KR1020070071762A

Other languages

Korean (ko)

Other versions

KR101177869B1 (en

Inventor

이훈택

양지훈

심상우

Original Assignee

양지훈

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2007-07-18

Filing date

2007-07-18

Publication date

2009-01-22

2007-07-18 Application filed by 양지훈 filed Critical 양지훈

2007-07-18 Priority to KR1020070071762A priority Critical patent/KR101177869B1/en

2009-01-22 Publication of KR20090008652A publication Critical patent/KR20090008652A/en

2012-08-28 Application granted granted Critical

2012-08-28 Publication of KR101177869B1 publication Critical patent/KR101177869B1/en

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Abstract

본 발명은 Oct4 발현능을 가지는 피부 유래 다분화능 성체줄기세포 및 그의 제조 방법에 관한 것으로 보다 상세하게는 태아피부조직 유래 단핵세포 및 이미 충분히 분화된 성체조직에서 물리적으로 절개되고 이후에 절개부분이 회복되면서 형성되어진 스카(scar) 조직에서 분리한 단핵 세포를 BMP4를 함유하는 배지에서 배양하여 분리한 다분화능 성체줄기세포에 관한 것이다.The present invention relates to a skin-derived multipotent adult stem cell having Oct4 expression ability and a method for preparing the same, and more particularly, to physically dissected from fetal skin tissue-derived mononuclear cells and already sufficiently differentiated adult tissue, and then recovering the incision. The present invention relates to multipotent adult stem cells isolated by culturing mononuclear cells isolated from scar tissues formed while culturing in a medium containing BMP4.

본 발명에 따른 줄기세포는 성체 줄기세포임에도 불구하고 골형성 세포, 신경세포, 췌도세포 등로 분화할 수 있는 다분화능을 가지고 있어, 난치병 및 불치병의 치료에 유용하게 이용될 수 있다.Stem cells according to the present invention, despite being adult stem cells, have a multipotent ability to differentiate into osteogenic cells, neurons, pancreatic islet cells and the like, and can be usefully used for the treatment of incurable diseases and incurable diseases.

Description

옥트(Ｏｃｔ)-4 발현능을 가지는 피부 유래 다분화능 성체 줄기세포 및 그의 제조방법{Multipotent Adult Stem Cells Having an Ability of Oct4 Expression Derived from Skin and Method for Preparing the Same}Multipotent Adult Stem Cells Having an Ability of Oct4 Expression Derived from Skin and Method for Preparing the Same}

본 발명은 옥트(Oct)-4 발현능을 가지는 피부 유래 다분화능 성체 줄기세포 및 그의 제조방법에 관한 것으로 보다 상세하게는 피부유래 단핵세포 및 이미 한번 분화가 되었던 Scar tissue에서 분리한 단핵 세포를 BMP4 및/또는 LIF를 함유하는 혈청 또는 무혈청 배지에서 배양하여 분리한 다분화능 성체 줄기세포에 관한 것이다. The present invention relates to a skin-derived multipotent adult stem cell having an oct-4 expression ability and a method for preparing the same, and more specifically, to BMP4, a mononuclear cell isolated from skin-derived mononuclear cells and a scar tissue that has been previously differentiated. And / or pluripotent adult stem cells isolated by culturing in serum or serum-free medium containing LIF.

생명과학 및 의학의 발전에 따라 현대사회에서는 많은 질병들을 치료하는 것에 있어서 획기적인 치료방법을 개발해 내고 있다. 그러나 여전히 암을 비롯한 허혈성 괴사질환, 치매, 화상과 같은 난치병 및 불치병이 존재하고 있으며, 많은 연구에도 불구하고 그 해답을 찾지 못하고 있는 실정이다. 이러한 질병에 대한 대안으로 많은 연구자들은 대체요법이라는 해답을 찾아가고 있다. 그 가운데 줄기세포를 이용하여 세포치료를 통해 그 해답을 찾으려는 노력이 이어지고 있다.With the development of life sciences and medicine, modern society is developing breakthrough treatments for the treatment of many diseases. However, there are still cancers, incurable necrosis diseases, dementia, incurable diseases such as burns, and incurable diseases, and despite many studies, the answer is not found. As an alternative to these diseases, many researchers are looking for the answer to alternative therapies. Among them, efforts are being made to find the answer through cell therapy using stem cells.

세포치료법이란, 세포와 조직의 기능을 복원시키기 위하여 살아있는 자가, 동종, 또는 이종의 줄기세포를 체외에서 증식하고, 이를 선별하거나, 다른 방법으로 세포의 생물학적 특성을 변화시키는 방법 등을 총하여 질병을 치료 및 예방하는 것이다. 세포치료법은 환자 자신이나 타인 또는 다른 동물의 세체포를 체취하여 증식기키거나, 줄기세포를 원하는 세포로 분화시켜 치료에 이용하는 등 그 적용범위가 넓어서, 각종 난치병 및 불치병의 치료에 있어 무한한 가능성을 가지고 있다.Cell therapy involves the expansion of living autologous, allogeneic, or heterologous stem cells in vitro to restore the function of cells and tissues, selection of them, or other methods of altering the biological properties of the cells. Treatment and prevention. Cell therapy has a wide range of applications, including capturing and proliferating the capillary cells of patients, other people, or other animals, or using stem cells to differentiate them into desired cells, which have unlimited potential in the treatment of various incurable and incurable diseases. have.

골격의 근육모세포(skeletal myoblast), 내피 전구세포(endothelial progenitor cell) 또는 골수 줄기세포(bone marrow stem cell)를 경색이 일어난 심근(myocardium)으로 이식할 경우 심근의 기능이 개선된다는 것은 이미 보고된 바 있다(Menasche P. et al., J Am Coll Cardiol, 41:1078, 2003; Strauer B.E. et al., Circulation, 106:1913, 2002; 및 Stamm C. et al., Lancet, 361:45, 2003). 이러한 심근 기능의 개선은 줄기세포에서 분비된 동맥성 사이토카인(arteriogenic cytokine)에 의해 기계적 스캐폴드(mechanical scaffold)가 형성되거나, 허혈성 부위로 다른 유익한 세포(beneficial cell)들이 모여들기 때문이다 (Orlic D, Hill JM, Arai AE., Circ Res, 91:1092, 2002).Transplantation of skeletal myoblasts, endothelial progenitor cells, or bone marrow stem cells into myocardium has been reported to improve myocardial function. (Menasche P. et al., J Am Coll Cardiol, 41: 1078, 2003; Strauer BE et al., Circulation, 106: 1913, 2002; and Stamm C. et al., Lancet, 361: 45, 2003) . This improvement in myocardial function is due to the formation of a mechanical scaffold by arteriogenic cytokine secreted from stem cells, or the gathering of other beneficial cells into the ischemic site (Orlic D, Hill JM, Arai AE., Circ Res, 91: 1092, 2002).

한편, 줄기세포(stem cell)란 자기 복제 능력을 가지면서 두 개 이상의 세포로 분화하는 능력을 갖는 세포를 말하며, 만능줄기세포(totipotent stem cells), 전분화능 줄기세포(pluripotent stem cells), 다분화능 줄기세포(multipotent stemcells)로 분류할 수 있다.On the other hand, stem cells (stem cell) refers to a cell having the ability to differentiate into two or more cells while having a self-replicating ability, totipotent stem cells, pluripotent stem cells, multipotent It can be classified as multipotent stemcells.

만능 줄기세포(totipotent stem cells)는 하나의 완전한 개체로 발생해 나갈 수 있는 만능의 성질을 가진 세포로 난자와 정자의 수정 이후 8세포기까지의 세포 가 이러한 성질을 가지며 이 세포를 분리하여 자궁에 이식하면 하나의 완전한 개체로 발생해 나갈 수 있다.Pluripotent stem cells are pluripotent cells that can develop into a complete individual. Cells up to 8-cells after fertilization of eggs and sperm have these properties. Transplantation can result in one complete individual.

전분화능 줄기세포(pluripotent stem cells)는 외배엽, 중배엽, 내배엽층 유래의 다양한 세포와 조직으로 발생할 수 있는 세포로서, 수정 4-5일 후 나타나는 배반포(blastocyst)의 안쪽에 위치한 내세포괴(inner cell mass)에서 유래하며, 이를 배아 줄기세포라 하며 다양한 다른 조직 세포로 분화되지만 새로운 생명체를 형성하지는 못한다. Pluripotent stem cells are cells that can develop into a variety of cells and tissues derived from ectoderm, mesoderm, and endodermal layer. The inner cell mass located inside the blastocyst after 4-5 days of fertilization. It is called embryonic stem cells and differentiates into a variety of other tissue cells but does not form new life.

다분화능 줄기세포(multipotent stem cells)는 이 세포가 포함되어 있는 조직 및 기관에 특이적인 세포로만 분화할 수 있는 줄기세포로서, 태아기, 신생아기 및 성체기의 각 조직 및 장기의 성장과 발달은 물론 성체조직의 항상성 유지와 조직손상 시 재생을 유도하는 기능에 관여하고 있으며, 조직 특이적 다능성 세포들을 총칭하여 성체 줄기세포라고 한다.Multipotent stem cells are stem cells that can only differentiate into cells specific to the tissues and organs that contain them, as well as the growth and development of individual tissues and organs in the prenatal, neonatal, and adult phases. It is involved in maintaining homeostasis of adult tissues and inducing regeneration in case of tissue damage. Adult tissue-specific pluripotent cells are collectively called adult stem cells.

지금까지 알려진 제대혈 유래 성체 줄기세포로는 골세포나 골격근으로 분화 가능한 중간엽 줄기세포 (Lee, O.K. et al.,Blood, 103:1669, 2004; Gang, E.J. et al., BBRC, 321:102, 2004; Gang, E.J. et al., Stem Cell, 22:617, 2004), 심장세포로 분화가능한 심장줄기세포 (US 2004/0126879) 및 내피전구세포 (Yamamoto, K. et al., Arterio. Thromb. Vasc. Biol., 24:192, 2004) 등이 있다. 특히, 성체 줄기세포는 골수에 많이 포함되어 있는 것으로 알려져 있으며, 배아기의 중배엽에서 유래함으로써 뼈, 연골, 지방 등으로만 분화 가능한 것으로 인식되어 왔으나, 외배엽에서 유래하는 신경세포로도 분화할 수 있다는 사실이 밝혀져, 질환 치료에 있어 서 그 효용 범위가 더욱 넓어질 것으로 기대되고 있다.Umbilical cord blood-derived adult stem cells so far known are mesenchymal stem cells capable of differentiating into bone cells or skeletal muscle (Lee, OK et al., Blood, 103: 1669, 2004; Gang, EJ et al., BBRC, 321: 102, 2004; Gang, EJ et al., Stem Cell, 22: 617, 2004), heart stem cells capable of differentiating into heart cells (US 2004/0126879) and endothelial progenitor cells (Yamamoto, K. et al., Arterio. Thromb. Vasc. Biol., 24: 192, 2004). In particular, adult stem cells are known to be contained in the bone marrow a lot, and have been recognized as being able to differentiate only into bone, cartilage, fat, etc., derived from the mesoderm of the embryo, but can also differentiate into neurons derived from ectoderm. As a result, it is expected that its utility range will be further widened in the treatment of diseases.

그러나, 상기 줄기세포들은 중간엽 줄기세포에 의한 골세포나 골격근으로의 분화, 심장 줄기세포에 의한 심장세포로의 분화, 내피전구세포에 의한 혈관 내피세포로의 분화 등의 제한된 조직으로의 분화능만을 나타내어 진정한 다분화능 줄기세포라고 하기에는 곤란하다.However, the stem cells are only differentiated into limited tissues such as differentiation into bone cells or skeletal muscle by mesenchymal stem cells, differentiation into heart cells by cardiac stem cells, and differentiation into vascular endothelial cells by endothelial progenitor cells. It is difficult to say that it is a true multipotent stem cell.

이에 본 발명자들은 피부에서 분리한 단핵세포를 BMP4를 함유하는 배지를 이용하여 분리한 성체 줄기세포가 플라스틱 또는 유리 위에서 키웠을 경우 그 부착능이 우수하고, 배아 줄기세포의 특이적 마커로 인식되어 온 옥트(Oct)-4를 발현할 수 있고, 골형성 세포, 신경세포 등 다양한 조직으로 분화 할 수 있다는 것을 확인하고 본 발명을 완성하게 되었다. The present inventors have found that the adult stem cells isolated from the skin using a medium containing BMP4 are grown in plastic or glass, and have excellent adhesion, and have been recognized as specific markers of embryonic stem cells. The present invention was completed by confirming that (Oct) -4 can be expressed and can be differentiated into various tissues such as bone forming cells and neurons.

상기의 목적을 달성하기 위하여 본 발명은 피부 조직에서 분리한 단핵세포를 BMP4를 함유한 배지에서 배양한 다음 회수하는 것을 특징으로 하는, 다음과 같은 특성을 나타내는 성체 줄기세포의 제조방법을 제공한다:In order to achieve the above object, the present invention provides a method for producing adult stem cells having the following characteristics, characterized in that the mononuclear cells isolated from the skin tissue is cultured in a medium containing BMP4 and then recovered.

a) AP 염색에 양성인 세포가 나타남;a) cells positive for AP staining appear;

b) 옥트(Oct)-4 발현능을 가짐;b) has Oct-4 expression capacity;

c) 마우스 피부조직에서 분리한 경우에는 마우스 배아줄기 세포에서 특이적으로 발현되는 옥트(Oct)-4, SSEA-1 이 양성으로 발현되는 면역학적 특성을 나타냄;c) when isolated from mouse skin tissue, it exhibits immunological properties of positive expression of Oct-4 and SSEA-1 specifically expressed in mouse embryonic stem cells;

d) 인간 조직으로부터 분리한 경우에는 인간 줄기 세포에서 특이적으로 발현하는 옥트(Oct)-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 이 양성으로 발현되었으며, SSEA-1의 경우 음성으로 발현되는 면역학적 특성을 나타냄; 및 d) When isolated from human tissues, Oct-4, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, which are specifically expressed in human stem cells, were positively expressed. SSEA-1 exhibits negative immunological properties; And

e) 플라스틱에 부착되어 성장하며, 둥근형태 또는 선 형태의 형태학적 특성 을 나타낸다.e) attached to plastic and grown, showing round or linear morphological characteristics.

본 발명의 세포는 신경세포 또는 췌도세포로 분화하는 능력을 가지나 이에 한정되지 아니한다. The cells of the present invention have the ability to differentiate into neurons or islet cells, but are not limited thereto.

본 발명에 있어서 상기 배지는 BMP4 이외에 LIF를 더욱 포함하는 것이 바람직하다.In the present invention, the medium preferably further contains LIF in addition to BMP4.

본 발명의 일실시예에 있어서, BMP4 또는 LIF 함량은 피부 조직에서 분리한 단핵 세포에서 옥트(Oct)-4 발현능을 가지는 다분화능 성체 줄기세포로 유도할 수 있는 농도라면 어떠한 농도도 바람직하지만 각각 1ng/ml 이상∼100ng/ml 이하가 더욱 바람직하지만 이에 한정되지 아니한다.In one embodiment of the present invention, the BMP4 or LIF content may be any concentration as long as it can be induced to multipotent adult stem cells having Oct-4 expression in mononuclear cells isolated from skin tissue. 1 ng / ml or more and 100 ng / ml or less are more preferable, but are not limited thereto.

본 발명의 일실시예에 있어서 상기 방법은 일실시예에 있어서 상기 방법은 무혈청 또는 고순도의 동물혈청, 또는 인간 형청을 이용하는 것이 바람직하다.In one embodiment of the invention the method is in one embodiment the method is preferably using serum-free or high-purity animal serum, or human type serum.

본 발명에서 사용된 시스템에서 지지세포층 (feeder cell layer) 없이 콜로니 형태의 스피어를 형성하는데 있어 Knockout serum replacement (이하 KSR, Invitrogen)를 사용하여도 형성된 콜로니 형태의 스피어의 특성에 있어 변화나 이상이 없다.In the system used in the present invention, there is no change or abnormality in the characteristics of colony-shaped spheres formed by using Knockout serum replacement (hereinafter referred to as KSR, Invitrogen) in forming colony-shaped spheres without a feeder cell layer. .

또한 본 발명은 상기의 방법에 의하여 수득되고, 하기의 특성을 가지는 성체 줄기세포를 제공한다:The present invention also provides an adult stem cell obtained by the above method, and having the following characteristics:

a) AP 염색에 양성인 세포가 나타남;a) cells positive for AP staining appear;

b) 옥트(Oct)-4 발현능을 가짐;b) has Oct-4 expression capacity;

c) 마우스 피부조직에서 분리한 경우에는 마우스 배아줄기 세포에서 특이적 으로 발현되는 옥트(Oct)-4, SSEA-1 이 양성으로 발현되는 면역학적 특성을 나타냄;c) when isolated from mouse skin tissue, it exhibits immunological characteristics of positive expression of Oct-4 and SSEA-1, which are specifically expressed in mouse embryonic stem cells;

d) 인간 조직으로부터 분리한 경우에는 인간 줄기 세포에서 특이적으로 발현하는 옥트(Oct)-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 이 양성으로 발현되었으며, SSEA-1의 경우 음성으로 발현되는 면역학적 특성을 나타냄; d) When isolated from human tissues, Oct-4, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, which are specifically expressed in human stem cells, were positively expressed. SSEA-1 exhibits negative immunological properties;

e) 플라스틱에 부착되어 성장하며, 둥근형태 또는 선 형태의 형태학적 특성을 나타냄; 및e) attached to and grown on plastics, exhibiting morphological properties in the form of round or line; And

g) 신경 세포 또는 췌도 세포로 분화하는 능력을 가지고 있음.g) Has the ability to differentiate into nerve cells or pancreatic islet cells.

또한 본 발명은 BMP4 및/또는 LIF를 유효성분으로 포함하는 피부 조직에서 분리한 단핵 세포를 성체 줄기세포로 유도하는 조성물을 제공한다.In another aspect, the present invention provides a composition for inducing mononuclear cells isolated from skin tissue containing BMP4 and / or LIF as an active ingredient to adult stem cells.

여기서, 상기 성체 줄기세포는 Here, the adult stem cells

a) AP 염색에 양성인 세포가 나타남;a) cells positive for AP staining appear;

b) 옥트(Oct)-4 발현능을 가짐;b) has Oct-4 expression capacity;

e) 플라스틱에 부착되어 성장하며, 둥근형태 또는 선 형태의 형태학적 특성 을 나타내며, 본 발명의 세포는 신경 세포 또는 췌도 세포로 분화하는 능력을 가지는 것이 바람직하다.e) It is attached to plastic and grows, exhibits morphological characteristics in the form of round or linear form, and the cells of the present invention preferably have the ability to differentiate into neurons or pancreatic islet cells.

이하, 본 발명을 상세하게 설명한다.EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

본 발명의 성체 줄기세포가 발현하는 것으로 밝혀진 옥트(Oct)4는 배아 줄기세포(embryonic stem cell)에서 발현되는 전사인자(transcription factor)로서, 세포의 분화를 방지하는 역할을 담당하고, 세포의 자연적인 분화가 시작되면 사라지는 것으로, 전분화능 줄기세포의 마커로 알려져 있다 (Donovan, P. J., Nature Genet., 29:246, 2001; Pesce, M. and Scholer,H. R., Stem Cells, 19:271, 2001). 그러므로, 본 발명의 성체 줄기세포가 전분화능인 배아 줄기세포의 마커인 옥트(Oct)4를 발현한다는 상기 결과는 본 발명에 따른 성체 줄기세포가 전분화능이라는 것을 더욱 명확히 입증하는 것이다.Oct 4, which is found to express the adult stem cells of the present invention, is a transcription factor expressed in embryonic stem cells, and plays a role in preventing the differentiation of cells. Disappears when normal differentiation begins and is known as a marker of pluripotent stem cells (Donovan, PJ, Nature Genet., 29: 246, 2001; Pesce, M. and Scholer, HR, Stem Cells, 19: 271, 2001) . Therefore, the above result that the adult stem cell of the present invention expresses oct (Oct) 4, a marker of pluripotent embryonic stem cells, is more clearly proving that the adult stem cells according to the present invention are pluripotent.

또한 본 발명의 세포(ES 유사 세포)에서는 알칼리 포스파타제가 대량으로 발현된다. 이 알칼리 포스파타제의 발현은 시판되는 여러 가지의 알칼리 포스파타제 검출 키트를 이용함으로써 용이하게 측정할 수 있다. 이 검출 키트로서는, 예컨대 ALP 조직 염색 키트(Sigma사)나 벡터 레드 알칼리 포스파타제 기질 키트 I(후나코시) 등을 들 수 있다.In addition, alkaline phosphatase is expressed in large quantities in the cells of the present invention (ES-like cells). Expression of this alkaline phosphatase can be easily measured by using various commercially available alkaline phosphatase detection kits. As this detection kit, ALP tissue staining kit (Sigma Corporation), vector red alkali phosphatase substrate kit I (Funakoshi), etc. are mentioned, for example.

배양체의 형성은 세포를 비코트 배양 접시에서 LIF를 제외한 sphere 배지 [DMEM/F12 84%, Knockout serum replacement (Invitrogen) 15%, 1% Non-essential amino acid, 10uM β-mercaptoethanol]을 이용하여 24hr - 36hr 정도 배양하고, 현미경 하에서 세포가 응집하여 형성되는 구형체의 출현을 관찰함으로써 확인할 수 있었다.The formation of the cultures was carried out in 24hr-using sphere medium [DMEM / F12 84%, Knockout serum replacement (Invitrogen) 15%, 1% Non-essential amino acid, 10 uM β-mercaptoethanol] except LIF in non-coat culture dishes. It was confirmed by culturing for about 36 hours and observing the appearance of spherical bodies formed by aggregation of cells under a microscope.

또한 ES 세포를 비롯하여 다능성 줄기 세포의 분화를 동정하는 지표 중 하나로서, 분화 단계 특이적으로 발현량이 변화하는 세포 표면 항원의 검출을 들 수 있다. 본 발명의 세포는 SSEA(태아기 특이적 항원)-1 및 SSEA-4를 발현하고 있다.In addition, as one of the indicators for identifying the differentiation of pluripotent stem cells, including ES cells, the detection of cell surface antigens in which the expression level changes in differentiation stage is mentioned. The cells of the present invention express SSEA (fetal specific antigen) -1 and SSEA-4.

이 세포 표면 마커의 발현은 본 발명의 세포 캐릭터라이제이션 키트(Chemicon)를 이용한 면역 염색에 의해 평가할 수 있었다.Expression of this cell surface marker could be assessed by immunostaining using the cell characterization kit (Chemicon) of the present invention.

본 발명의 세포를 피더 세포나 LIF가 존재하지 않는 조건 하에서 배양하는 것에 의하면, 배양체를 경유하여 여러 가지의 세포로 자발적으로 분화한다. 이 ES 세포의 성질은 우선 상기 방법에 의해 배양체를 형성시키고, 그 후 젤라틴 코트 배양 접시에 배양체를 옮겨서, 1일간∼2일간 정도 배양을 행함으로써 관찰할 수 있다. 각 세포의 특징적인 형태로부터 신경양 세포, 지방양 세포, 또는 상피계 세포 등의 출현을 확인할 수 있다.By culturing the cells of the present invention under conditions where no feeder cells or LIF are present, they spontaneously differentiate into various cells via a culture. The properties of these ES cells can be observed by first forming a culture by the above method, then transferring the culture to a gelatin coat culture dish and culturing for one to two days. From the characteristic forms of each cell, the appearance of neuro sheep cells, fat sheep cells, epithelial cells, and the like can be confirmed.

또한 본 발명의 세포는 삼배엽(내배엽, 중배엽, 외배엽) 계열의 세포로 분화하는 능력을 갖는다. 이러한 세포의 성질은, 상기에 기재한 방법으로 형성시킨 배양체(심근양 세포가 출현한 배양체)로부터 RNA를 추출하고, 외배엽 계열의 세포(예컨대 신경 세포), 중배엽 계열의 세포(예컨대 심근 세포), 내배엽 계열의 세포(예컨대 간세포)의 각 마커 유전자의 발현을 RT-PCR 등의 방법을 이용하여 분석하거나 면역 염색을 함으로써 확인할 수 있다.In addition, the cells of the present invention has the ability to differentiate into cells of the trioderm (endoderm, mesoderm, ectoderm). The properties of these cells are obtained by extracting RNA from the culture (cultured cardiomyocyte cells) formed by the method described above, ectoderm-based cells (eg neurons), mesoderm-based cells (eg cardiomyocytes), Expression of each marker gene of endoderm-based cells (eg, hepatocytes) can be confirmed by analyzing by means of RT-PCR or immunostaining.

여기서 신경 세포 마커로서는 네스틴(Nestin), TUJ1(베타3-튜불린), MSI-1(무사시-1), GFAP(신경교 섬유성 산성 단백질 알파) 등을 들 수 있다. 또한 심근 세 포 마커로서는 CCC(심장 디히드로피리딘 감수성 칼슘 채널 단백질), ANF(심방 나트륨 배설 인자), KvLQT1 등을 들 수 있다. 또한 줄기 세포 마커로서는 ALB(알부민), TDO(트립토판 2,3-디옥시게나제), TAT(티로신 아미노트랜스퍼라제), G6P(글루코스-6-포스파타제) 등을 들 수 있다.Examples of the neuron markers include nestin, TUJ1 (beta3-tubulin), MSI-1 (musashi-1), and GFAP (neuroglial fibrous acidic protein alpha). Examples of myocardial cell markers include CCC (heart dihydropyridine sensitive calcium channel protein), ANF (atrial sodium excretory factor), KvLQT1, and the like. Examples of stem cell markers include ALB (albumin), TDO (tryptophan 2,3-dioxygenase), TAT (tyrosine aminotransferase), G6P (glucose-6-phosphatase), and the like.

BMP-4 단백질은 아미노산 서열이 BMP-2 단백질((U.S. Pat. No. 5,013,649; Groeneveld, E. H. J. ＆ Burger, E. H. Bone morphogenetic proteins in human bone regereration. 2000. Eur. J. Endocrinol. 142, 9-21)과 유사하며 현재까지 이들에 관한 연구도 주로 신생골의 형성과 관련된 것에 집중되어 왔고 실제로 인공뼈에 BMP-4를 첨가한 제품이 등장하여 상품화 단계에 이르고 있으며, 생체내 골 결손부에 가능한 고농도의 BMP-4를 전달하려는 시도는 현재도 계속 이루어지고 있다(U.S. Pat. No. 6,245,889). BMP-4 protein has an amino acid sequence of BMP-2 protein (US Pat. No. 5,013,649; Groeneveld, EHJ & Burger, EH Bone morphogenetic proteins in human bone regereration. 2000. Eur. J. Endocrinol. 142, 9-21) Similarly, the research on them has been focused mainly on the formation of new bones, and in fact, products with BMP-4 added to artificial bones have emerged in the commercialization stage, and high concentrations of BMPs are possible in bone defects in vivo. Attempts to deliver -4 continue to be made today (US Pat. No. 6,245,889).

그러나, 이러한 고전적인 BMP-4의 알려진 기능과는 별개로 1990년도 초반부 터 발생학에서 BMP-4의 기능에 대한 연구가 시작되어 1998년에는 백서에서 BMP-4와 FGF-8의 상호작용이 치아의 형태를 결정하는 주요인자라고 네이쳐(nature)지에 발표되었고, 이후에는 여러 종에서 발생의 초기단계에 배아의 향후 발생운명을 결정하는데 중요하게 관여한다고 알려져 있는데, 이는 배아에 대한 면역화학염색법이나 인사이투 하이브리다이제이션 실험에 의해 양성신호가 편측에 치우쳐 발현됨을 보임으로써 증명되었다.However, apart from these classically known functions of BMP-4, studies of BMP-4's function in embryology began in the early 1990s, and in 1998, the interaction of BMP-4 with FGF-8 in teeth It is a major determinant of morphology and has been published in Nature and has since been known to play an important role in determining the future fate of embryos at early stages of development in several species. It was proved by the redidation experiment that the positive signal is expressed on one side.

본 발명의 일 실시예에서 사용된 조건은 다음과 같다.The conditions used in one embodiment of the present invention are as follows.

피부조직으로부터 primary culture 배양시의 배양액의 조성은 ES-DMEM (DMEM84%, ES-FBS 15%, Non-essential amino acid 1%, 10uM β-mercaptoethanol) +10ng/ml BMP4, 부가적으로 10ng/ml LIFThe composition of the culture medium for primary culture from skin tissue was ES-DMEM (DMEM84%, ES-FBS 15%, Non-essential amino acid 1%, 10uM β-mercaptoethanol) + 10ng / ml BMP4, additionally 10ng / ml LIF

본 발명의 성체 줄기 세포 배양액의 조성은 STEM-F12 (DMEM/F12 84%, ES-FBS 15%, Non-essential amino acid 1%, 10uM β-mercaptoethanol) + 10ng/ml BMP4 부가적으로 10ng/ml LIFThe composition of the adult stem cell culture of the present invention is STEM-F12 (84% DMEM / F12, ES-FBS 15%, Non-essential amino acid 1%, 10uM β-mercaptoethanol) + 10ng / ml BMP4 additionally 10ng / ml LIF

본 발명의 세포의 배양 조건은 특별한 언급이 없는 한 37℃, 5% CO2, 95% O2이었다.Culture conditions of the cells of the present invention was 37 ℃, 5% CO2, 95% O2 unless otherwise specified.

또한 본 발명에서 사용된 항체는 특별한 언급이 없는 한 케미콘(Chemicon)사의 제품을 사용하였다.In addition, the antibody used in the present invention used a product of Chemicon (Chemicon) unless otherwise specified.

본 발명의 일 실시예에서 본 발명의 방법은 태아피부조직 또는 성인여성 복부의 제왕절개 후 생기는 흉터(Scar) 조직을 채취하여 antibiotics가 들어간 PBS로 충분히 세척한 다음, 지방을 제거 하고나서 조직을 잘게 자른 후 조직 자체에 ES cell 배양용 FBS처리를 하거나 또는 조직 및 세포배양용 코팅 접시에 극소량을 소적(micro-drop)하여 조직을 접시에 올리거나 또는 소적된 FBS에 올린 다음 상온(28℃)에서 약 1시간 가량 놓아두어서 조직과 접시 간에 coaggulation을 유도하여 고정되게 하였다. 이후 ES-DMEM(15% FBS, 1% Non-essential amino acid, 10uM β-mercaptoethanol)에 10ng/ml BMP4를 첨가한 다음 조직이 완전히 덮히지 않도록 접시에 넣고 최초 3일간 배양하면서 단핵세포들이 조직으로부터 빠져 나오는지(Outgrowth)를 확인하였다. 세포들이 조직에서 나오는것이 확인이 되면 ES-DMEM+10ng/ml BMP4를 조직이 떨어지지 않도록 천천히 첨가한 뒤 계속 배양하였 다. 약 7일 정도 배양 후 조직에서 나온 세포들이 바닥에 붙어있는것을 확인하고 나서 조직을 제거 하였다. 이후 세포들의 특성 유지함과 아울러 충분한 수의 세포확보를 위해 STEM-F12+10ng/ml BMP4로 배양액 교체 후 계속 배양하거나 계대배양을 실시하였다. In one embodiment of the present invention, the method of the present invention is to collect the scar tissue generated after caesarean section of fetal skin tissue or adult female abdomen, wash thoroughly with PBS containing antibiotics, and then remove the fat and finely tissue After cutting, the tissue is subjected to ES cell culture FBS treatment or micro-drops are applied to the tissue and cell culture coated dish, and the tissue is placed on the dish or placed on the dropped FBS, and then stored at room temperature (28 ° C). Leave for about 1 hour to induce coaggulation between tissue and dish to fix. Then, 10ng / ml BMP4 was added to ES-DMEM (15% FBS, 1% Non-essential amino acid, 10uM β-mercaptoethanol), and then plated so that the tissue was not completely covered. Outgrowth was checked. When the cells were confirmed to come out of the tissue, ES-DMEM + 10ng / ml BMP4 was added slowly so that the tissue does not fall, and continued incubation. After culturing for about 7 days, the cells were removed from the tissues after confirming that they were attached to the floor. Afterwards, in order to maintain the characteristics of the cells and secure a sufficient number of cells, the culture medium was replaced with STEM-F12 + 10ng / ml BMP4, and then cultured or passaged.

본 발명의 또 다른 일 실시예에서는 상기 배지 조건에 LIF 10ng/ml을 더욱 포함하여 상기와 같은 방법을 진행하였다.In another embodiment of the present invention further comprises 10ng / ml LIF in the medium conditions were carried out as described above.

본 발명은 BMP 4를 함유하는 배지를 이용하여 피부 유래 조직으로부터 옥트(Oct)4 발현능을 가지는 다분화능 성체 줄기세포를 분리하는 방법을 제공하는 효과가 있다. 본 발명에 따른 줄기세포는 성체 줄기세포임에도 불구하고 신경세포 또는 췌도 세포 등으로 분화할 수 있는 다분화능을 가지고 있어, 난치병 및 불치병의 치료에 유용하게 사용될 수 있다.The present invention has the effect of providing a method for separating multipotent adult stem cells having the ability to express Oct (Oct) 4 from the skin-derived tissue using a medium containing BMP 4. Stem cells according to the present invention, despite being adult stem cells, have a multipotent ability to differentiate into neurons or islet cells, etc., and thus can be usefully used for the treatment of incurable diseases and incurable diseases.

실시예 1: 인간의 태아 조직으로부터 유래한 세포의 배양 Example 1 Culture of Cells Derived from Human Fetal Tissue

계류유산으로 인해 적출되어지는 인간의 태아조직(Fetal tissue)을 얻어 in vitro에서 수술용 가위를 이용하여 잘게 자른 후(가로 세로 0.5mm) 각각의 조직을(5개) ES qualified FBS (이하 ES-FBS, Invitrogen) 처리를 하여 코팅된 플라스틱 용기에 고정시킨 뒤 배양하였다. 이때 첨가되는 배양액의 조건은 ES-DMEM [DMEM (Invitrogen) 84%, ES-FBS (Invitrogen) 15%, Non-essential amino acid (Invtrogen) 1%, 10uM β-mercaptoethanol(Invitrogen)] 배양액에 BMP4 10ng/ml (R&D systems)를 첨가하여 배양액을 만들며, 이는 마우스의 ES cell culture 시 사용되는 기본 배양액에 BMP4를 첨가하여 만든 것이 특징이다. Human fetal tissues extracted from mooring abortions were obtained and chopped in vitro using surgical scissors (0.5 mm horizontally) and each tissue (5 pieces) ES qualified FBS (hereinafter ES-). FBS, Invitrogen) was fixed in a coated plastic container and incubated. At this time, the conditions of the added medium were ES-DMEM [DMEM (Invitrogen) 84%, ES-FBS (Invitrogen) 15%, Non-essential amino acid (Invtrogen) 1%, 10uM β-mercaptoethanol (Invitrogen)] BMP4 10ng / ml (R & D systems) is added to make a culture, which is characterized by the addition of BMP4 to the basic culture used in ES cell culture of mice.

이러한 방법을 이용한 배양 후 7일 이후에는 각 조직을 떼어내고, 조직에서 나온 single cell 만을 DMEM/F12 (Invitrogen) 84%, ES-FBS 15%, Non-essential amino acid 1%, 10uM β-mercaptoethanol) 배지에 10ng/ml BMP4를 첨가하여 계속 배양하였다. 배양 후 7일이 지나면 각각의 플라스크 용기에 high density로 배양되게 되며, 이렇게 배양된 fibroblast 세포를 0.25% trypsin EDTA (Invitrogen)를 이용하여 떼어낸 다음 sphere 배지 [DMEM/F12 84%, Knockout serum replacement (Invitrogen) 15%, 1% Non-essential amino acid, 10uM β-mercaptoethanol]에 넣어 배양하였다. 이때 sphere 배양액은 세포 배양용 용기가 아닌 부유 배양용 플라스틱 용기를 사용하였다.After 7 days of incubation using this method, each tissue was detached, and only single cells from the tissues were treated with DMEM / F12 (Invitrogen) 84%, ES-FBS 15%, Non-essential amino acid 1%, 10uM β-mercaptoethanol). The culture was continued by adding 10ng / ml BMP4 to the medium. After 7 days of incubation, each flask was incubated with high density. The cultured fibroblast cells were removed using 0.25% trypsin EDTA (Invitrogen), followed by sphere medium [DMEM / F12 84%, Knockout serum replacement ( Invitrogen) 15%, 1% Non-essential amino acid, 10uM β-mercaptoethanol] were incubated. At this time, the sphere culture solution was used as a plastic container for suspension culture, not a container for cell culture.

이러한 배양액의 조건은 배양 후 24 시간 만에 각각의 세포들을 구형세포괴(sphere cell mass) 모양으로 뭉치게 만드는 것을 확인하였다.The conditions of these cultures were confirmed to aggregate individual cells into the shape of sphere cell mass within 24 hours after incubation.

실시예 2: 배지에 LIF를 더욱 포함하여 배양하는 인간의 태아 조직으로부터 유래한 세포의 배양 Example 2: Cultivation of cells derived from human fetal tissue cultured further comprising LIF in the medium

실시예 1의 배지 조건에 LIF 10ng/ml (Chemicon)를 포함하는 것을 제외하고는 실시예 1과 동일한 방법을 통하여 인간의 태아 조직으로부터 유래한 세포를 배양하였다.Cells derived from human fetal tissue were cultured in the same manner as in Example 1 except that LIF 10ng / ml (Chemicon) was included in the medium condition of Example 1.

실시예 3: 인간의 스카(scar) 조직으로부터 유래한 세포의 배양Example 3: Culture of Cells Derived from Human Scar Tissue

실시예 1 또는 실시예 2에서 인간의 태아조직(Fetal tissue) 대신에 제왕 절 개 후 다시 제왕절개 수술을 시도하는 30세에서 40세 사이의 Scar tissue 조직(Adult tissue)을 사용한 것을 제외하고는 실시예 1과 동일한 방법을 통하여 각각의 세포를 구형(sphere) 모양으로 뭉치게 만들어서 얻었다.Except that in Example 1 or Example 2, instead of human fetal tissue, Scar tissue was used between the ages of 30 and 40, which attempts to undergo cesarean section again after caesarean section. By the same method as in Example 1 was obtained by agglomerating each cell into a sphere (sphere) shape.

실시예 4: 피부 유래 다분화능 줄기세포의 신경세포로의 분화Example 4 Differentiation of Skin-derived Multipotent Stem Cells into Neurons

sphere 배지에 의해서 얻어진 스피어를 코트된 프라스틱 용기에 신경세포 분화를 위한 배지 DMEM/F12 84%, ES-FBS 15%, Non-essential amino acid 1%, 10uM β-mercaptoethanol) + 10ng/ml bFGF + 10ng/ml NGF + 10ng/ml EGF에 첨가한 후 3일동안 배양했다. 이러한 과정을 거쳐 신경세포로의 분화를 유도한 다음, 면역염색을 실시하였다(도 6). Spheres obtained by sphere medium were coated in a plastic container coated with a medium for neuronal differentiation DMEM / F12 84%, ES-FBS 15%, Non-essential amino acid 1%, 10uM β-mercaptoethanol) + 10ng / ml bFGF + 10ng It was added to / ml NGF + 10ng / ml EGF and incubated for 3 days. Through this process, differentiation into neurons was induced, followed by immunostaining (FIG. 6).

그 결과, 도 6에 나타난 바와 같이, 본 발명에 따른 인간 지방조직 유래 다분화능 줄기세포는 신경계 성상세포의 특이항원인 Nestin 과 Tuj-1 이 발현 되는 것을 확인하였으며, 도7과 같이 각각에서 얻어진 세포에서 RNA를 추출하여 RT-PCR을 한 결과 신경세포 특이물질인 MAP2(microtubule-associated protein2)와 NCAM, neuron filament-M 에 대하여 양성반응을 나타냈다.As a result, as shown in Figure 6, the human adipose tissue-derived multipotent stem cells according to the present invention was confirmed that the expression of Nestin and Tuj-1, the specific antigen of neural astrocytes, cells obtained in each as shown in FIG. RT-PCR was extracted from RNA and showed positive response to neuronal specific substance MAP2 (microtubule-associated protein2), NCAM and neuron filament-M.

실시예 5: 피부 유래 다분화능 줄기세포의 췌도세포로의 분화Example 5 Differentiation of Skin-derived Multipotent Stem Cells into Pancreatic Islet Cells

sphere 배지에 의해서 얻어진 스피어를 10mmol/L nicotinamide, 1mmol/L β-mercaptoethanol을 함유하고, 10% FBS을 첨가한 low glucose DMEM 배지에서 24시간동안 배양한 후, 10mmol/L nicotinamide, 1mmol/L β-mercaptoethanol 및 5% FBS를 첨가한 high glucose DMEM에 5일 동안 배양하여 인슐린 분비 췌장 베타세포로의 분화를 유도하였다. 분화유도 후 면역염색을 통해 확인한 결과, 도 9에서 나타난 바 와 같이, 세포 내에 insulin이 존재하였다. 인슐린 분비 췌장 베타세포에서 인슐린의 원형인 proinsulin이 생성되고, 이것은 인슐린과 C-peptide로 나누어지는 바, 상기 결과로부터 본 발명에 따른 피부 조직 유래 다분화능 줄기세포가 인슐린 분비 췌장 베타세포로 분화된 것을 확인할 수 있었다.The spheres obtained by sphere medium were incubated in low glucose DMEM medium containing 10 mmol / L nicotinamide and 1 mmol / L β-mercaptoethanol for 24 hours, and then 10 mmol / L nicotinamide and 1 mmol / L β- Differentiation into insulin-secreting pancreatic beta cells was induced by incubation for 5 days in high glucose DMEM containing mercaptoethanol and 5% FBS. As a result of immunostaining after differentiation induction, as shown in FIG. 9, insulin was present in the cells. Insulin-producing pancreatic beta cells produce proinsulin, the prototype of insulin, which is divided into insulin and C-peptide. From these results, the multipotent stem cells derived from skin tissues according to the present invention are differentiated into insulin-secreting pancreatic beta cells. I could confirm it.

실험예 1: 염색Experimental Example 1: Dyeing

실시예 1, 실시예 2, 및 실시예 3에서 얻어진 세포를 사용하여 ES colony에서 사용되는 염색 방법(Evans M, & Kaufman M.. (1981). Nature 292, 154, Morrison S.J. et al. (1997). Cell 88, 287-298)을 이용하여 염색하였으며, 본 발명의 실시예 1, 2 및 3에서 수득된 피부 유래 줄기세포 스피어를 코트되어 있는 배양용기에 하루를 배양하면 바닥에 스피어 형태를 유지한 체로 붙어 자라게 된다. 이렇게 붙어있는 스피어 세포를 PBS로 세 번 세척하고, 4% paraformaldehyde을 함유한 PBS로 30분간 고정하였다. PBS로 세 번 세척한 후, 0.1% Triton- X100을 함유한 PBS로 10분간 침투(permeabilization)시킨다. PBS로 세 번 세척한 후, 10% NGS로 1시간동안 반응시키고, 1차항체를 함유한 PBS에 하룻밤 동안 반응시킨다. PBS로 3회 세척하고, 2차항체로 암실에서 1시간동안 반응시켰다. PBS로 세 번 세척한 후, mounting하였다.Example 1, Example 2, and Example 3 and using the cells obtained by the dyeing method used in ES colony (Evans M, & Kaufman M .. (1981). Nature 292 , 154, Morrison SJ et al . (1997). Cell 88 , 287-298 ) and the skin-derived stem cell spheres obtained in Examples 1, 2, and 3 of the present invention were cultured on a coated culture vessel for one day and adhered to the bottom with a sieve keeping the spear shape. It grows up. The attached spheres were washed three times with PBS and fixed for 30 minutes with PBS containing 4% paraformaldehyde. After washing three times with PBS, it is permeabilized with PBS containing 0.1% Triton-X100 for 10 minutes. After washing three times with PBS, it was reacted with 10% NGS for 1 hour, and reacted overnight with PBS containing the primary antibody. Washed three times with PBS, and reacted with a secondary antibody in the dark for 1 hour. After washing three times with PBS, it was mounted.

그 결과, 다분화능 줄기세포 스피어는 신경 전구세포 및 췌도 전구세포의 마커라고 할 수 있는 Nestin, Tuj-1, Insulin, 미분화상태의 세포 마커라고 할 수 있는 Oct4 및 중간엽 줄기세포의 마커인 CD34, CD44, CD90에 대하여 양성반응을 나타내었다. ES colony에서 사용되는 염색 방법을 이용하여 염색하였으며, 인간 줄기세 포에 특이적으로 발현하는 옥트(Oct)-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 을 사용하여 염색하였으며, SSEA-1 은 발현하지 않는 것으로 확인하였다.As a result, multipotent stem cell spheres are nestin, Tuj-1, Insulin, which are markers of neural progenitor cells and islet progenitor cells, CD34, which is a marker of Oct4 and mesenchymal stem cells, which are markers of undifferentiated cells. CD44 and CD90 were positive. Staining was performed using the staining method used in ES colony. Oct-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, which are specifically expressed on human stem cells, were stained. Staining was performed, and SSEA-1 was confirmed not to be expressed.

또한 MSC maker 인 CD34, CD44, CD90 역시 발현하는 것을 확인하였다(도 8).In addition, it was confirmed that the MSC makers CD34, CD44, and CD90 were also expressed (FIG. 8).

실험예 2:RT-PCRExperimental Example 2: RT-PCR

본 발명의 방법에 의해 배양된 피부 줄기세포 유래의 신경세포 분화 유도 후 그 특성을 분석하기 위해 아래와 같은 신경 세포 특이 유전자 발현을 RT-PCR 방법을 통하여 비교해 보았다. The following neuronal cell-specific gene expression was compared by RT-PCR method to analyze its characteristics after induction of neuronal differentiation derived from skin stem cells cultured by the method of the present invention.

우선 신경세포 유도 배양액에 배양된 전체 RNA(total RNA)를 분리하였다. 각 세포를 피펫으로 분리하여 1.5㎖ 튜브로 옮기고, 시료 1g당 2-5㎖의 RNA 추출 완충용액[5 M 구아니디움 티오시아네이트(guanidium thiocyanate), 25 mM 소듐 시트레이트(sodium citrate, pH 7.0), 0.5% (w/v) 사코실(sarkosyl), 2 mM EDTA, 5% (w/v) - 머캡토에탄올 (mercaptoethanol)]을 처리하여 세게 흔들어 주었다. 0.5㎖ 2M 소듐 아세테이트(sodium acetate, pH 4.0), 5㎖ 페놀, 1㎖ 클로로포름:이소아밀알콜(v:v = 49:1)을 첨가하여 10초 동안 세게 흔들어 완전히 섞은 후, 4℃에서 15분간 방치하였다. 10,000×g, 4℃에서 원심분리하여 얻은 상층액을 새로운 50㎖ 튜브로 옮기고, 5㎖ 이소프로판올을 첨가하여 부드럽게 아래위로 흔들어서 섞었다.First, total RNA cultured in neuronal cell-derived culture medium was isolated. Pipette each cell into a 1.5 ml tube and transfer 2-5 ml RNA extract buffer [5 M guanidium thiocyanate, 25 mM sodium citrate, pH 7.0 per gram of sample]. ), 0.5% (w / v) sarkosyl, 2 mM EDTA, 5% (w / v)-mercaptoethanol] was shaken vigorously. Add 0.5 ml 2M sodium acetate (pH 4.0), 5 ml phenol, 1 ml chloroform: isoamyl alcohol (v: v = 49: 1), shake vigorously for 10 seconds and mix thoroughly for 15 minutes at 4 ° C. It was left. The supernatant obtained by centrifugation at 10,000 × g, 4 ° C. was transferred to a new 50 ml tube and mixed by shaking gently up and down with the addition of 5 ml isopropanol.

-20℃에서 최소 1시간 이상 방치한 후, 10,000×g, 4℃에서 원심분리하고 상층액을 제거하여 얻은 핵산 침전물을 1.4㎖ RNA 추출 완충용액에 녹였다. 1.4㎖의 이소프로판올을 첨가하고 -20℃에서 최소 1시간 이상 방치한 후, 10,000×g, 4℃ 에서 원심분리하여 얻은 핵산 침전물을 75% 에탄올로 세척하고 DEPC(diethylpyrocabonate)를 처리한 증류수에 녹여 RT-PCR(Reverse Transcription - Polymerase Chain Reaction) 실험에 사용하였다.After at least 1 hour at −20 ° C., the nucleic acid precipitate obtained by centrifugation at 10,000 × g and 4 ° C. and the supernatant was removed was dissolved in 1.4 ml RNA extraction buffer. After adding 1.4 ml of isopropanol and leaving at least at -20 ° C for at least 1 hour, the nucleic acid precipitate obtained by centrifugation at 10,000 × g, 4 ° C was washed with 75% ethanol and dissolved in DEPC (diethylpyrocabonate) -treated distilled water. -PCR (Reverse Transcription-Polymerase Chain Reaction) experiment was used.

ES 세포에서 분리한 1㎍의 전체 RNA를 주형으로 하고, 단일가닥 cDNA 합성 키트(1st strand cDNA synthesis kit,Roche)를 사용하여, 42℃에서 60분간 역전사 반응을 수행하였다. 이후 99℃에서 5분간 역전사효소를 불활성화시키고, 동일한 튜브에서 다음과 같은 프라이머(primer)가 첨가된 반응액에서 전체 반응물 5㎕를 주형으로 cDNA를 합성하였다.Using 1 μg of total RNA isolated from ES cells as a template, reverse transcription was performed at 42 ° C. for 60 minutes using a single strand cDNA synthesis kit (Roche). After inactivating the reverse transcriptase at 99 ° C. for 5 minutes, cDNA was synthesized using 5 μl of the total reactant as a template in the reaction solution to which the following primers were added in the same tube.

본 실험에 사용된 프라이머는The primers used in this experiment

MAP2 sense strand(서열번호 1), 5'-CTGTCCCTAGGTCAGCTTGC-3'MAP2 sense strand (SEQ ID NO: 1), 5'-CTGTCCCTAGGTCAGCTTGC-3 '

antisense strand(서열번호 2), 5'-GCATGGTGGCTCCCAATCTAT-3'; antisense strand (SEQ ID NO: 2), 5'-GCATGGTGGCTCCCAATCTAT-3 ';

NCAM sense strand(서열번호 3), 5'-CTCGGCCTTTGTGTTTCCAG-3'NCAM sense strand (SEQ ID NO: 3), 5'-CTCGGCCTTTGTGTTTCCAG-3 '

antisense strand(서열번호 4), 5'-TGGCAGGAGATGCCAAAGAT-3' antisense strand (SEQ ID NO: 4), 5'-TGGCAGGAGATGCCAAAGAT-3 '

NF-M sense strand(서열번호 5), 5'-CTTCAGCCAGTCCTCGTCCC-3'NF-M sense strand (SEQ ID NO: 5), 5'-CTTCAGCCAGTCCTCGTCCC-3 '

antisense strand(서열번호 6), 5'-TCCTCCAGGTGGTCCGAGTC-3' antisense strand (SEQ ID NO: 6), 5'-TCCTCCAGGTGGTCCGAGTC-3 '

beta-actin sense strand(서열번호 7), 5'-GTGGGGCGCCCCAGGCACCA-3'beta-actin sense strand (SEQ ID NO: 7), 5'-GTGGGGCGCCCCAGGCACCA-3 '

antisense strand(서열번호 8), 5'-CTCCTTAATGTCACGCACGATTTC-3'. 이 었다. antisense strand (SEQ ID NO: 8), 5'-CTCCTTAATGTCACGCACGATTTC-3 '. It was.

PCR 반응조건은 94℃에서 40초간 변성시키고, 61℃에서 40초간 중합(annealing)한 후, 72℃에서 40초 동안 확장시키는 반응을 35회 반복하도록 PCR을 수행하였다. PCR 생성물을 2% 아가로오스 겔에서 전기영동하여 DNA 밴드를 확인하고, 그 결과를 도 7에 나타내었다PCR reaction conditions were denatured at 94 ° C for 40 seconds, polymerized (annealing) at 61 ° C for 40 seconds, and PCR was performed to repeat the reaction for 35 times at 72 ° C for 40 seconds. PCR products were electrophoresed on 2% agarose gel to confirm DNA bands, and the results are shown in FIG. 7.

이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. therefore

본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

도 1은 본 발명에 따른 피부 유래 다분화능 성체 줄기세포의 모습을 나타낸 것이다. 8-13주경에 계류유산되어 적출된 태아에서 피부조직만을 회수하거나 선행제왕절개 후 두 번째 분만을 위한 제왕절개수술을 실시하는 30대 중반 여성의 하복부에서 흉터(Scar)조직을 회수하여 항생물질이 첨가된 PBS에서 충분히 세척한 뒤 조직을 잘게 잘라서 조직자체에 ES-FBS를 처리하여 세포배양접시에 올리거나 또는 세포배양접시 표면에 극소량의 ES-FBS를 소적하고 그 위에 잘게 자른 조직을 하나씩 올려서 상온에서 1시간 정도 놓아두면서 조직과 접시표면과의 coaggulation을 유발시켜서 고정이 되도록 한 뒤 ES-DMEM+10ng/ml BMP4를 조직이 완전히 덮히지 않을 정도로만 (5ml) 넣은 다음 배양을 실시하였다. 약 24-36시간 정도에서 조직으로부터 세포들이 나오기 시작하는것이 관찰되면 추가로 ES-DMEM+10ng/ml BMP4 5ml 정도를 더 넣고 계속 배양을 실시하였다. 이후 매 48시간 마다 배양액을 절반씩만 교체하면서 세포성장을 유도하였다. 배양을 시작한지 7일째 정도에 접시에서 조직을 제거하고 접시에 붙어서 성장중인 세포들은 배양액 교체를 하면서 계속 배양을 하거나 계대배양 (subculture)을 통해 대량배양을 실시하였다. 이때에는 ES-DMEM+10ng/ml BMP4을 그대로 사용하거나 또는 STEM-F12+10ng/ml BMP4 배양액을 사용하여서 5일에서 7일정도 배양하였다. Figure 1 shows the appearance of the skin-derived multipotent adult stem cells according to the present invention. Scar tissue was recovered from the lower abdomen of women in their mid-30s who had recovered only skin tissue from the fetuses that had been aborted at 8-13 weeks or had undergone cesarean section for the second delivery after the previous caesarean section. After sufficiently washing in the added PBS, the tissue is finely chopped and treated with ES-FBS on the tissue itself, and then placed on a cell culture dish, or a small amount of ES-FBS is dropped on the surface of the cell culture dish, and the chopped tissues are placed one by one on room temperature. Leave for about 1 hour in the coagulation between tissue and dish surface to induce fixation, and then ES-DMEM + 10ng / ml BMP4 was added only enough (5ml) the tissue was not completely covered and incubated. When cells began to emerge from the tissues in about 24-36 hours, 5 ml of ES-DMEM + 10ng / ml BMP4 was added and culture was continued. Thereafter, only half of the culture medium was changed every 48 hours to induce cell growth. About 7 days after the start of the culture, the tissues were removed from the dishes and the cells grown on the dishes were continuously cultured while replacing the culture medium or mass cultured by subculture. In this case, ES-DMEM + 10ng / ml BMP4 was used as it was or incubated for 5 to 7 days using STEM-F12 + 10ng / ml BMP4 culture.

도 2는 단일세포를 유리판(슬라이드 글라스) 위 혹은 코팅 되지 않은 플라스틱 배양접시에서 배양하면서 knockout serum replacement를 사용하여 콜로니 형태의 구형 세포괴 (sphere cell mass)을 만든 모습을 나타낸 것이다. 이를 위해 도 1 에서 보여진 대량배양된 피부 유래 다분화능 성체 줄기세포를 Sphere 배지 [DMEM/F12 (Invitrogen) 84%, Knockout serum replacement (Invitrogen)15%, 1% Non-essential amino acid (Invitrogen), 10uM β-mercaptoethanol (Invitrogen)]에 5×10⁵ 세포/ml 를 현탁시켜 3ml 정도를 비코트 페트리 접시 (SPL, Korea)에 접종하여 18-24시간 동안 배양하였다. 18-24시간 배양 후 현미경하에서 관찰을 통해 도 2에서 보이는 형태의 구형세포괴 (Sphere cell mass)를 관찰하였다.FIG. 2 shows the appearance of colony-shaped sphere cell mass using knockout serum replacement while culturing single cells on a glass plate (slide glass) or an uncoated plastic dish. To this end, the cultured skin-derived multipotent adult stem cells shown in FIG. 1 were prepared using Sphere medium [DMEM / F12 (Invitrogen) 84%, Knockout serum replacement (Invitrogen) 15%, 1% Non-essential amino acid (Invitrogen), 10 uM. [beta] -mercaptoethanol (Invitrogen)] was suspended in 5 × 10 ⁵ cells / ml and inoculated about 3ml in a non-coat Petri dish (SPL, Korea) and incubated for 18-24 hours. After culturing for 18-24 hours, the spherical cell mass of the form shown in FIG. 2 was observed through a microscope.

도 3은 도 2에서 만들어진 세포를 AP 염색을 통하여 염색해 본 결과이다.3 is a result of staining the cells made in Figure 2 through AP staining.

도 3의 A와 B는 Sphere 배지에 BMP4 만을 첨가한 배지에서 자란 콜로니 100X, 200X 사진이고, C와 D는 Sphere 배지에 LIF 만을 첨가한 배지에서 자란 콜로니 100X, 200X의 사진이며, E에서 H는 각각의 배지에서 만들어진 콜로니를 AP stain 을 한 사진 200X이다. 이 중에서 E는 BMP4와 LIF 를 제외한 Sphere 배지에서만 만들어진 콜로니, F는 BMP4 만을 넣어준 Sphere 배지에서 만들어진 콜로니, G: LIF 만을 넣어준 Sphere 배지에서 만들어진 콜로니, H는 BMP4+LIF 을 혼합하여 넣어준 Sphere 배지에서 만들어진 콜로니를 나타낸다.A and B of Figure 3 is a colony 100X, 200X photo grown in the medium added BMP4 to Sphere medium, C and D is a photo of colonies 100X, 200X grown in the medium added only LIF to Sphere medium, H in E Colonies made on each medium were photographed with AP stain 200X. Among these, E is colony made only in Sphere medium except BMP4 and LIF, F is colony made from Sphere medium with BMP4 only, G: colony made from Sphere medium with only LIF, H is mixed with BMP4 + LIF Indicates colonies made in the medium.

이러한 방법으로 유도된 각각의 콜로니들은 AP 염색을 했을 경우 BMP4만을 첨가 했을 경우 확연하게 AP 염색이 되었으나, LIF만을 첨가했을 경우에는 경향성만을 보였다. 그러나, 두 가지를 함께 첨가하였을 경우 그 발현 양상이 좋아진다는 것을 보여주고 있다. 이렇게 만들어진 스피어의 경우 도 4에서 볼 수 있는 것처럼 줄기세포의 특징을 발현하는 세포임을 확인할 수 있었다.Each colony induced by this method was obviously AP stained when only BMP4 was added when AP stained, but showed only tendency when only LIF was added. However, when the two are added together, the expression pattern is improved. In the case of the spear was made as shown in Figure 4 it was confirmed that the cells expressing the characteristics of the stem cells.

도 4는 면역형광 염색을 통한 피부유래 다분화능 성체 줄기세포의 특징발현을 나타내는 사진이다. 도 4a는 8주령 이상의 Adult mouse tail tissue에서 유래된 다분화능 성체 줄기세포를 나타내며, 이들 세포들은 도 1에서 설명된 방법을 통하여 만들어진 다분화능 성체 줄기세포들을 LIF와 BMP4가 첨가된 Sphere 배양액에 넣고 배양하여서 생기는 구형세포괴 또는 선형세포들이다. A는 Oct-4의 면역형광 염색 (blue: 핵, green: Oct-4), B는 SSEA 1의 면역형광 염색 (blue: 핵, green: SSEA1)을 나타내며;Figure 4 is a photograph showing the characteristic expression of skin-derived multipotent adult stem cells through immunofluorescence staining. FIG. 4A shows multipotent adult stem cells derived from adult mouse tail tissue of 8 weeks or older, and these cells were cultured by inserting the multipotent adult stem cells produced by the method described in FIG. 1 into a Sphere culture medium to which LIF and BMP4 were added. Spheroid cell mass or linear cells. A represents immunofluorescence staining (blue: nucleus, green: Oct-4) of Oct-4, B represents immunofluorescence staining (blue: nucleus, green: SSEA1) of SSEA 1;

도 4b는 인간 Human fetal skin에서 유래된 다분화능 성체 줄기세포를 보여주는 사진이다. 이들 세포들은 도 1에서 설명된 방법을 통하여 만들어진 다분화능 성체 줄기세포들을 LIF와 BMP4가 첨가된 Sphere 배양액에 넣고 배양하여서 생기는 구형세포괴들이다. A는 Oct-4의 면역형광 염색 (blue: 핵, green: Oct-4), B는 SSEA 3의 면역형광 염색 (blue: 핵, green: SSEA-3), C는 SSEA 4의 면역형광 염색 (blue: 핵, green: SSEA-4), D는 TRA-1-60의 면역형광 염색 (blue: 핵, green: TRA-1-60), E는 TRA-1-81의 면역형광 염색 (blue: 핵, green: TRA-1-81)를 나타낸다.Figure 4b is a photograph showing the multipotent adult stem cells derived from human Human fetal skin. These cells are spherical cell masses formed by culturing multipotent adult stem cells produced by the method described in FIG. 1 in a Sphere culture solution added with LIF and BMP4. A is immunofluorescence staining of Oct-4 (blue: nucleus, green: Oct-4), B is immunofluorescence staining of SSEA 3 (blue: nucleus, green: SSEA-3), C is immunofluorescence staining of SSEA 4 blue: nucleus, green: SSEA-4), D for immunofluorescence staining of TRA-1-60 (blue: nucleus, green: TRA-1-60), E for immunofluorescence staining of TRA-1-81 (blue: Nucleus, green: TRA-1-81).

도4c는 인간 성인 스카(scar) 조직에서 유래된 다분화능 성체 줄기세포를 보여주는 사진이다. 이들 세포들은 도 1에서 설명된 방법을 통하여 만들어진 다분화능 성체 줄기세포들을 LIF와 BMP4가 첨가된 Sphere 배양액에 넣고 배양하여서 생기는 구형세포괴 또는 선형세포들이다. A는 Oct4의 면역형광 염색 (blue: 핵, green: Oct-4), B는 SSEA 4의 면역형광 염색 (blue: 핵, green: SSEA-4)를 나타낸다.4C is a photograph showing multipotent adult stem cells derived from human adult scar tissue. These cells are globular cell masses or linear cells formed by culturing multipotent adult stem cells produced by the method described in FIG. 1 in a Sphere culture medium to which LIF and BMP4 are added. A represents immunofluorescence staining (blue: nucleus, green: Oct-4) of Oct4, B represents immunofluorescence staining (blue: nucleus, green: SSEA-4) of SSEA4.

도 5는 BMP4와 LIF를 첨가된 STEM-F12배지에서 성장시킨 다분화능 성체 줄기세포들을 이용하여 콜로니를 형성하지 않고서 STEM-F12 배지에 bFGF 와 EGF, NGF를 첨가하여 신경전구세포로 직접 분화시킨 모습을 나타낸 것이다. 이들 세포들은 도 1에서 설명된 방법을 통하여 만들어진 다분화능 성체 줄기세포들을 LIF와 BMP4가 제외하고 대신에 bFGF, EGF, 그리고 NGF를 첨가한 STEM-F12 배양액에서 유도된 신경전구세포의 모습이다. A는 CD44의 면역형광 염색 (blue: 핵, green: CD44), B는 Nestin의 면역형광 염색 (blue: 핵, green: Nestin)을 나타낸다.Figure 5 shows the direct differentiation of neural progenitor cells by adding bFGF, EGF and NGF to STEM-F12 medium without forming colonies using multipotent adult stem cells grown on STEM-F12 medium supplemented with BMP4 and LIF. It is shown. These cells show the appearance of neural progenitor cells derived from STEM-F12 culture medium in which multipotent adult stem cells made by the method described in FIG. 1 were added with bFGF, EGF, and NGF instead of LIF and BMP4. A represents immunofluorescence staining of CD44 (blue: nucleus, green: CD44), and B represents nesting immunofluorescence staining (blue: nucleus, green: Nestin).

도 6은 BMP4와 LIF를 첨가한 Sphere 배지를 이용하여 colony를 형성한 후 bFGF 와 EGF, NGF를 이용하여 신경전구세포로 분화시킨 모습을 나타낸 것이다. A는 Nestin의 면역형광 염색 (blue: 핵, green: Nestin 200X), B는 Tuj-1의 면역형광 염색 (blue: 핵, green: Tuj-1 200X), C는 Tuj-1의 면역형광 염색 (blue: 핵, green: Tuj-1 100X)을 나타낸다.FIG. 6 shows colony formation using Sphere medium added with BMP4 and LIF and then differentiated into neural progenitor cells using bFGF, EGF, and NGF. A is immunofluorescence staining of Nestin (blue: nucleus, green: Nestin 200X), B is immunofluorescence staining of Tuj-1 (blue: nucleus, green: Tuj-1 200X), C is immunofluorescence staining of Tuj-1 blue: nucleus, green: Tuj-1 100X).

도 7은 BMP4와 LIF를 첨가한 Sphere 배지를 이용하여 colony를 형성한 후에 bFGF 와 EGF, NGF를 이용하여 신경전구세포로 분화시킨 후 RT-PCR 을 통해 검증한 모습을 나타낸 것이다. 이들 세포들은 도 1에서 설명된 방법을 통하여 만들어진 다분화능 성체 줄기세포들을 LIF와 BMP4가 첨가된 Sphere 배양액에 넣고 배양하여서 구형세포괴를 형성하고 이후에 LIF와 BMP4 대신에 bFGF, EGF, 그리고 NGF를 첨가한 DMEM-F12 배양액에서 분화된 신경전구세포을 분자생물학 적인 방법인 RT-PCR을 통해 검증한 결과이다.FIG. 7 shows colony formation using Sphere medium added with BMP4 and LIF, followed by differentiation into neural progenitor cells using bFGF, EGF, and NGF, followed by RT-PCR. These cells were cultured by inserting multipotent adult stem cells prepared by the method described in FIG. 1 into a Sphere culture medium containing LIF and BMP4 to form a globular cell mass, and then adding bFGF, EGF, and NGF instead of LIF and BMP4. Neuroprogenitor cells differentiated in a DMEM-F12 culture were verified by RT-PCR, a molecular biological method.

도 8은 인간 성인 스카(scar) 조직에서 유래된 다분화능 성체 줄기세포를 보여주는 사진이다. 이들 세포들은 도 1에서 설명된 방법을 통하여 만들어진 다분화능 성체 줄기세포들을 LIF와 BMP4가 첨가된 Sphere 배양액에 넣고 배양하여서 생기는 구형세포괴들로, A와 B는 각각 MSC의 마커인 CD90 과 CD34의 면역형광 염색 (blue: 핵, green: CD90, CD34)을 나타낸다.8 is a photograph showing multipotent adult stem cells derived from human adult scar tissue. These cells are globular cell masses formed by culturing multipotent adult stem cells produced by the method described in FIG. 1 in a Sphere culture solution added with LIF and BMP4, and A and B are immunized by CD90 and CD34 markers, respectively. Fluorescent staining (blue: nucleus, green: CD90, CD34) is shown.

도 9은 BMP4와 LIF를 첨가한 Sphere 배지를 이용하여 colony를 형성한 후에 10mmol/L nicotinamide, 1mmol/L β-mercaptoethanol을 함유하고, 10% FBS을 첨가한 low glucose DMEM 배지에서 24시간동안 배양한 후, 10mmol/L nicotinamide, 1mmol/L β-mercaptoethanol 및 5% FBS를 첨가한 high glucose DMEM에 5일동안 배양하여 인슐린 분비 췌장 베타세포로의 분화를 유도하였다. A는 insulin 면역형광 염색 (blue: 핵, green: insulin).9 shows colonies using Sphere medium added with BMP4 and LIF, followed by incubation for 24 hours in low glucose DMEM medium containing 10 mmol / L nicotinamide and 1 mmol / L β-mercaptoethanol and 10% FBS. Thereafter, 10 mmol / L nicotinamide, 1 mmol / L β-mercaptoethanol and 5% FBS were incubated for 5 days in high glucose DMEM to induce differentiation into insulin secreting pancreatic beta cells. A is insulin immunofluorescent staining (blue: nuclear, green: insulin).

<110> YANG, Ji Hoon <120> Multipotent Adult Stem Cells Having an Ability of Oct4 Expression Derived from Skin and Method for Preparing the Same <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for MAP2 <400> 1 ctgtccctag gtcagcttgc 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for MAP2 <400> 2 gcatggtggc tcccaatcta t 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for NCAM <400> 3 ctcggccttt gtgtttccag 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for NCAM <400> 4 tggcaggaga tgccaaagat 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for NF-M <400> 5 cttcagccag tcctcgtccc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for NF-M <400> 6 tcctccaggt ggtccgagtc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for beta-actin <400> 7 gtggggcgcc ccaggcacca 20 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for beta-actin <400> 8 ctccttaatg tcacgcacga tttc 24 <110> YANG, Ji Hoon <120> Multipotent Adult Stem Cells Having an Ability of Oct4 Expression Derived from Skin and Method for Preparing the Same <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for MAP2 <400> 1 ctgtccctag gtcagcttgc 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for MAP2 <400> 2 gcatggtggc tcccaatcta t 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for NCAM <400> 3 ctcggccttt gtgtttccag 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for NCAM <400> 4 tggcaggaga tgccaaagat 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for NF-M <400> 5 cttcagccag tcctcgtccc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for NF-M <400> 6 tcctccaggt ggtccgagtc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sense primer for beta-actin <400> 7 gtggggcgcc ccaggcacca 20 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for beta-actin <400> 8 ctccttaatg tcacgcacga tttc 24

Claims (9)

피부 조직에서 분리한 단핵세포를 골 형성 단백질(BMP) 4를 포함하는 배지에서 배양한 다음 회수하는 것을 특징으로 하는, 다음과 같은 특성을 나타내는 성체 줄기세포의 제조방법:Method for producing adult stem cells having the following characteristics, characterized in that the mononuclear cells isolated from the skin tissue is cultured in a medium containing bone morphogenetic protein (BMP) 4 and recovered. a) 알칼리 포스파타제(AP) 염색에 양성인 세포가 나타남;a) cells positive for alkaline phosphatase (AP) staining appear; b) 옥트(Oct)-4 발현능을 가짐;b) has Oct-4 expression capacity; c) 마우스 피부조직에서 분리한 경우에는 마우스 배아줄기 세포에서 특이적으로 발현되는 옥트(Oct)-4 또는 SSEA-1이 양성으로 발현되는 면역학적 특성을 나타냄; 및 c) when isolated from mouse skin tissue, it exhibits immunological properties of positive expression of Oct-4 or SSEA-1 specifically expressed in mouse embryonic stem cells; And d) 인간 조직으로부터 분리한 경우에는 인간 줄기 세포에서 특이적으로 발현하는 옥트(Oct)-4, SSEA-3, SSEA-4, TRA-1-60 또는 TRA-1-81이 양성으로 발현되었으며, SSEA-1의 경우 음성으로 발현되는 면역학적 특성을 나타냄. d) when isolated from human tissues, positive expression of Oct-4, SSEA-3, SSEA-4, TRA-1-60 or TRA-1-81, which is specifically expressed in human stem cells, SSEA-1 shows negative immunological properties. 제 1항에 있어서, 상기 배지는 백혈병억제 인자(LIF)를 더욱 포함하는 것을 특징으로 하는 성체 줄기세포의 제조방법. The method of claim 1, wherein the medium further comprises leukemia inhibitory factor (LIF). 제1항 또는 제2항에 있어서, 상기 BMP4 또는 LIF 함량은 각각 1ng/ml∼ 100ng/ml 이하의 범위인 것을 특징으로 하는 방법.The method according to claim 1 or 2, wherein the BMP4 or LIF content is in the range of 1 ng / ml to 100 ng / ml or less, respectively. 제1항에 있어서 상기 방법은 배지에 혈청을 더욱 포함하는 것을 특징으로 하는 방법.The method of claim 1, wherein the method further comprises serum in the medium. 제 1항에 있어서, 상기 세포는 플라스틱에 부착되어 성장하며, 비코트 플라스틱에서는 둥근 형태 또는 선 형태의 형태학적 특성을 나타내는 것을 특징으로 하는 방법. The method of claim 1, wherein the cells adhere to the plastic and grow, wherein the non-coat plastic exhibits morphological properties in round or linear form. 제 1항에 있어서, 상기 세포는 신경세포 또는 췌도세포로 분화하는 능력을 가지는 것을 특징으로 하는 방법.The method of claim 1, wherein the cell has the ability to differentiate into neurons or pancreatic islets. 제1항 내지 제6항 중 어느 한 항의 방법에 의하여 수득되고, 하기의 특성을 가지는 성체 줄기세포:An adult stem cell obtained by the method of any one of claims 1 to 6 and having the following characteristics: a) AP 염색에 양성인 세포가 나타남;a) cells positive for AP staining appear; b) 옥트(Oct)-4 발현능을 가짐;b) has Oct-4 expression capacity; c) 마우스 피부조직에서 분리한 경우에는 마우스 배아줄기 세포에서 특이적으로 발현되는 옥트(Oct)-4 또는 SSEA-1 이 양성으로 발현되는 면역학적 특성을 나타냄;c) when isolated from mouse skin tissue, it exhibits immunological properties of positive expression of Oct-4 or SSEA-1 specifically expressed in mouse embryonic stem cells; d) 인간 조직으로부터 분리한 경우에는 인간 줄기 세포에서 특이적으로 발현하는 옥트(Oct)-4, SSEA-3, SSEA-4, TRA-1-60 또는 TRA-1-81 이 양성으로 발현되었으며, SSEA-1의 경우 음성으로 발현되는 면역학적 특성을 나타냄; d) when isolated from human tissues, positively expressed Oct-4, SSEA-3, SSEA-4, TRA-1-60 or TRA-1-81, which are specifically expressed in human stem cells, SSEA-1 exhibits negative immunological properties; e) 플라스틱에 부착되어 성장하며, 둥근형태 또는 선 형태의 형태학적 특성을 나타냄; 및e) attached to and grown on plastics, exhibiting morphological properties in the form of round or line; And g) 신경세포 또는 췌도세포로 분화하는 능력을 가짐.g) Has the ability to differentiate into neurons or pancreatic islets. BMP4를 유효성분으로 포함하는 피부 조직에서 분리한 단핵 세포를 성체 줄기세포로 유도하는 조성물:A composition for inducing mononuclear cells isolated from skin tissue containing BMP4 as an active ingredient into adult stem cells: 여기서, 상기 성체 줄기세포는 Here, the adult stem cells a) 알칼리 포스파타제(AP) 염색에 양성인 세포가 나타남;a) cells positive for alkaline phosphatase (AP) staining appear; b) 옥트(옥트(Oct))-4 발현능을 가짐;b) has the ability to express oct (Oct) -4; c) 마우스 피부조직에서 분리한 경우에는 마우스 배아줄기 세포에서 특이적으로 발현되는 옥트(Oct)-4, 또는 SSEA-1 이 양성으로 발현되는 면역학적 특성을 나타냄;c) when isolated from mouse skin tissue, exhibits immunological properties of Oct-4, or SSEA-1, which are specifically expressed in mouse embryonic stem cells; d) 인간 조직으로부터 분리한 경우에는 인간 줄기 세포에서 특이적으로 발현하는 옥트(Oct)-4, SSEA-3, SSEA-4, TRA-1-60, 또는 TRA-1-81 이 양성으로 발현되었으며, SSEA-1의 경우 음성으로 발현되는 면역학적 특성을 나타냄; 및 d) When isolated from human tissues, Oct-4, SSEA-3, SSEA-4, TRA-1-60, or TRA-1-81, which are specifically expressed in human stem cells, were positively expressed. , In the case of SSEA-1, exhibits immunological properties expressed negatively; And e) 플라스틱에 부착되어 성장하며, 둥근형태 또는 선 형태의 형태학적 특성을 나타냄; e) attached to and grown on plastics, exhibiting morphological properties in the form of round or line; 제 8항에 있어서, 상기 조성물은 백혈병억제 인자(LIF)를 더욱 포함하는 것을 특징으로 하는 조성물.The composition of claim 8, wherein the composition further comprises a leukemia inhibitory factor (LIF).

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