KR101980453B1 - Composition For Promoting Production of Stem Cell-derived Exosomes - Google Patents
Composition For Promoting Production of Stem Cell-derived Exosomes Download PDFInfo
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- KR101980453B1 KR101980453B1 KR1020170162194A KR20170162194A KR101980453B1 KR 101980453 B1 KR101980453 B1 KR 101980453B1 KR 1020170162194 A KR1020170162194 A KR 1020170162194A KR 20170162194 A KR20170162194 A KR 20170162194A KR 101980453 B1 KR101980453 B1 KR 101980453B1
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- stem cells
- exosomes
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- pioglitazone
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Abstract
Description
본 발명은 피오글리타존(pioglitazone), 메트포르민(metformin) 및 AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)로 이루어진 군으로부터 선택된 1 이상의 물질을 포함하는 줄기세포 유래 엑소좀 생성 촉진용 조성물 및 이를 이용한 줄기세포 유래 엑소좀의 생산 방법에 관한 것이다.The present invention is a composition for promoting stem cell-derived exosomes production composition comprising one or more substances selected from the group consisting of pioglitazone (pioglitazone), metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) and stem cells derived from the same It relates to a method for producing exosomes.
미세소포체 (Extracellular Vesicles)는 Microvesicles, Exosome 등을 포함하는 30~1000 nm 크기의 구형 지질이중층(lipid-bilayer)으로 구성된 소포체(vesicle)이다. 엑소좀의 지질이중층은 기원 세포(공여세포)와 같은 인지질 이중막 구조로 되어 있으며, 세포가 세포외로 분비하는 물질의 구성체로 세포-세포간의 커뮤니케이션 및 세포성 면역 중재 등의 기능적인 역할을 수행하는 것으로 알려져 있다. 엑소좀은 기원 세포(공여세포) 특유의 생물학적 기능을 반영하는 세포특이적 구성 성분을 함유하며, 인지질, mRNA, miRNA 외에도 다양한 수용성 단백질, 외재성 단백질 및 막관통 단백질 성분 등을 포함한다. 엑소좀의 마커 단백질로는 CD63, CD81 등이 잘 알려져 있으며, 여기에는 주로 EGFR과 같은 세포 표면의 수용체, 신호전달에 관여하는 분자, 세포의 부착(adhesion)에 관여하는 단백질, hsp(heat shock protein), 소포체 형성과 관련하는 Alix 등 단백질들이 포함되는 것으로 알려져 있다. 이러한 엑소좀은 비만세포, 림프구, 성상세포, 혈소판, 신경세포, 내피세포, 상피세포 등 모든 동물 세포에서 배출되며 혈액, 소변, 점액, 타액, 담즙액, 복수액, 뇌척수액 등의 다양한 체액에서 발견된다. 엑소좀은 뇌혈관 장벽(Blood-Brain Barrier, BBB)도 통과할 수 있으며, 표피 세포와 내피세포의 세포막 투과가 가능할 정도로 선택적 투과성이 높아 특정 약물의 나노캐리어(nanocarrier)인 DDS(drug delivery system) 개발에도 활용되고 있다.Extracellular Vesicles are vesicles composed of spherical lipid-bilayers of 30-1000 nm in size, including Microvesicles, Exosomes, and the like. The lipid bilayer of exosomes has the same phospholipid bilayer structure as cells of origin (donor cells), and is a construct of substances secreted by the cells extracellularly, and performs a functional role such as cell-cell communication and cellular immune mediation. It is known. Exosomes contain cell-specific components that reflect the biological function peculiar to the cells of origin (donor cells), and include various soluble proteins, exogenous proteins, and transmembrane protein components in addition to phospholipids, mRNAs and miRNAs. The marker proteins of exosomes are well known, such as CD63 and CD81, mainly including receptors on cell surfaces such as EGFR, molecules involved in signal transduction, proteins involved in cell adhesion, and heat shock proteins. And Alix, which are involved in endoplasmic reticulum formation. These exosomes are released from all animal cells such as mast cells, lymphocytes, astrocytes, platelets, nerve cells, endothelial cells, epithelial cells, and found in various body fluids such as blood, urine, mucus, saliva, bile, ascites, and cerebrospinal fluid. do. Exosomes can also pass through the blood-brain barrier (BBB), and have high selective permeability to allow cell membrane penetration of epidermal and endothelial cells, and thus, a drug delivery system (DDS), a nanocarrier for certain drugs. It is also used for development.
중간엽 줄기세포에서 분비하는 엑소좀 및 미세소포체(microvesicle)는 세포-세포간 커뮤니케이션(cell-to-cell communication)에 관여하며 줄기세포가 가지는 재생의학적인 치료 효능을 보인다고 알려져 있다. 줄기세포를 체내에 이식한 후에 장기간의 생존 없이 세포에서 분비되는 파라크린 인자(paracrine factors)에 트로픽 효과(trophic effect)를 가져오는 것이 알려져 있고, 이러한 인자에는 성장인자(growth factor), 케모카인(chemokine), 사이토카인(cytokine) 등과 같은 저분자가 엑소좀과 같은 세포외 소포체(extracellular vesicle)에 의하여 분비되며, 이러한 엑소좀은 줄기세포에서 유래한다. 따라서 엑소좀은 줄기세포의 특성을 규명하고 이의 치료적 효능을 평가하는데 활용되고 있다. Exosomes and microvesicles secreted from mesenchymal stem cells are known to be involved in cell-to-cell communication and show regenerative medical efficacy of stem cells. It is known to have a trophic effect on paracrine factors secreted by cells without prolonged survival after transplanting stem cells into the body, and these factors include growth factors and chemokines. Low molecules such as cytokines are secreted by extracellular vesicles such as exosomes, which are derived from stem cells. Therefore, exosomes are used to characterize stem cells and evaluate their therapeutic efficacy.
최근에는 중간엽 줄기세포 자체를 사용하지 않고 중간엽 줄기세포가 분비하는 엑소좀을 이용하여 다양한 질환의 치료효과에 대한 연구가 활발하게 진행 중이며, 학계 및 산업계에서는 이를 통해 기존의 줄기세포 치료법의 한계를 극복할 수 있는 새로운 대안이 될 수 있을 것으로 예상한다. 이러한 엑소좀을 상업적으로 이용하기 위해서는 다량의, 그리고 양질의 엑소좀이 필요하다. 그러나 현재 줄기세포로부터 얻을 수 있는 엑소좀의 양은 매우 소량에 불과하고, 줄기세포 유래 엑소좀의 생산량을 증가시킬 수 있는 물질에 대한 개발도 아직까지 미비한 실정이다. 이에 따라 줄기세포 유래 엑소좀의 생산을 촉진하는 신규한 방법 및 물질 개발에 대한 필요성이 제기되었다. Recently, studies on the therapeutic effects of various diseases using exosomes secreted by mesenchymal stem cells, rather than mesenchymal stem cells themselves, are being actively conducted. It is expected to be a new alternative to overcome this problem. Commercial use of these exosomes requires large amounts of high quality exosomes. However, the amount of exosomes currently available from stem cells is very small, and the development of a substance capable of increasing the production of stem cell-derived exosomes is still insufficient. Accordingly, there is a need for the development of new methods and materials for promoting the production of stem cell-derived exosomes.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.
본 발명자들은 줄기세포 유래 엑소좀의 생산을 촉진하는 방법 및 물질을 개발하고자 예의 연구 노력하였다. 그 결과 피오글리타존, 메트포르민, 및 AICAR를 줄기세포에 전처리하는 경우에 줄기세포 유래 엑소좀의 수 뿐만 아니라, 엑소좀 내 단백질 및 RNA의 함량이 크게 증가하는 것을 확인하고, 본 발명을 완성하게 되었다. The present inventors made diligent research efforts to develop methods and materials for promoting the production of stem cell-derived exosomes. As a result, when pre-treatment of pioglitazone, metformin, and AICAR to stem cells, not only the number of stem cell-derived exosomes, but also a significant increase in the content of proteins and RNA in the exosomes was completed.
따라서, 본 발명의 목적은 피오글리타존(pioglitazone), 메트포르민(metformin) 및 AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)로 이루어진 군으로부터 선택된 1 이상의 물질을 포함하는 줄기세포 유래 엑소좀 생성 촉진용 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a composition for promoting stem cell-derived exosome generation comprising at least one substance selected from the group consisting of pioglitazone, piformlitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide). It is.
본 발명의 다른 목적은 피오글리타존(pioglitazone), 메트포르민(metformin) 및 AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)로 이루어진 군으로부터 선택된 1 이상의 물질로 전처리된 줄기세포에서 유래한 엑소좀을 제공하는 것이다. Another object of the present invention is to provide exosomes derived from stem cells pretreated with one or more substances selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide).
본 발명의 또 다른 목적은 상기 줄기세포 유래 엑소좀 생성 촉진용 조성물을 포함하는 줄기세포 치료제를 제공하는 것이다.Still another object of the present invention is to provide a stem cell therapeutic agent comprising the composition for promoting stem cell-derived exosome generation.
본 발명의 또 다른 목적은 상기줄기세포에서 유래한 엑소좀을 포함하는 줄기세포 치료제를 제공하는 것이다. Still another object of the present invention is to provide a stem cell therapeutic agent comprising an exosome derived from the stem cells.
본 발명의 또 다른 목적은 피오글리타존(pioglitazone), 메트포르민(metformin) 및 AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)로 이루어진 군으로부터 선택된 1 이상의 물질을 포함하는 세포배양 배지에 줄기세포를 배양하는 단계를 포함하는 줄기세포 유래 엑소좀의 생산방법을 제공하는 것이다. Another object of the present invention is the step of culturing the stem cells in a cell culture medium containing one or more substances selected from the group consisting of pioglitazone (pioglitazone), metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) It provides a method for producing a stem cell-derived exosomes comprising.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 피오글리타존(pioglitazone), 메트포르민(metformin) 및 AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)로 이루어진 군으로부터 선택된 1 이상의 물질을 포함하는 줄기세포 유래 엑소좀 생성 촉진용 조성물을 제공한다.According to an aspect of the present invention, the present invention promotes the generation of stem cell-derived exosomes comprising at least one substance selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) It provides a composition for.
본 발명의 상기 조성물의 구성성분 중 하나인 피오글리타존은 티아졸리디네디온(thiazolidinedione, TZD) 계열의 혈당 강하 효과를 가진 당뇨병 치료약이다. 혈당 강하 효과가 있으나 심혈관 질환에 대한 효과는 통계적으로 유의성 있는 결과가 보고되지는 않았다.Pioglitazone, which is one of the components of the composition of the present invention, is a diabetic drug having a hypoglycemic effect of thiazolidinedione (TZD) family. It has a hypoglycemic effect, but the effect on cardiovascular disease has not been reported statistically significant.
본 발명의 상기 조성물의 구성성분 중 다른 하나인 메트포르민(metformin)은 바이구아니드계(biguanides) 경구용 당뇨병치료제이다. 메트포르민 또한 혈당 개선효과가 있으나 심혈관질환을 예방하는 효과는 증거가 아직 제한적이다. 작용기전의 하나로 간에서 AMP-activated protein kinase (AMPK)를 활성화함으로써 포도당신생합성을 막고, 세포에 포도당이 흡수되는 것을 촉진하고, 대사증후군을 억제한다. 이러한 메트포르민의 작용기전은 정확하게 밝혀지지는 않았으나, 1) 미토콘드리아에서 일어나는 세포호흡을 억제하고 2) AMPK를 활성화시키며, 3) 글루카곤에 의해 유도되는 cAMP(Cyclic adenosine monophosphate)의 농도 상승을 막아 결과적으로 PKA(Protein kinase A)의 활성을 억제하고 4) 장내의 정상세균총(gut flora)에도 영향을 준다고 알려져 있다.Metformin, another component of the composition of the present invention, is a biguanides oral diabetes treatment. Metformin also improves blood sugar, but there is still limited evidence to prevent cardiovascular disease. One of the mechanisms of action is the activation of AMP-activated protein kinase (AMPK) in the liver, which prevents glucose biosynthesis, promotes glucose uptake into cells, and inhibits metabolic syndrome. The mechanism of action of metformin has not been accurately determined, but 1) it inhibits cell respiration in mitochondria, 2) activates AMPK, and 3) prevents the increase of glucagon-induced concentration of cAMP (Cyclic adenosine monophosphate). It is known to inhibit the activity of protein kinase A and to affect gut flora in the gut.
본 발명의 상기 조성물의 성분 중 또 다른 하나인 AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)는 이노신 모노포스페이트(inosine monophosphate)의 생성과정의 중간체이다. AICAR는 AMP의 유사체로서 AMP-의존적 단백질 키나아제(AMPK)를 자극하는 것으로 알려져 있으며, 심장 허혈성 손상을 치료 및 보호하는 용도로서 임상적으로 사용되어 왔다.Another component of the composition of the present invention AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) is an intermediate in the production of inosine monophosphate (inosine monophosphate). AICAR is known to stimulate AMP-dependent protein kinase (AMPK) as an analog of AMP and has been used clinically for the treatment and protection of cardiac ischemic injury.
본 발명에 따른 상기 조성물은 상술한 성분 이외에 공지된 엑소좀 생성 촉진 물질을 더 포함할 수 있다.The composition according to the present invention may further include a known exosome production promoting material in addition to the above-mentioned components.
본 명세서에서 용어 “엑소좀(Exosome)은 세포 유래성 소포체로, 거의 모든 진핵 생물의 체액에 존재한다. 엑소좀의 직경은 30-100 nm 정도이며, 이는 LDL 단백질보다는 크지만, 적혈구보다는 훨씬 작다. 엑소좀은 다중소포체(multivesicular bodies)가 세포막과 융합될 때 세포로부터 방출되거나, 세포막으로부터 곧바로 방출된다. 엑소좀이 응고, 세포간 신호전달 등과 같은 중요하면서도 특화된 기능을 수행한다는 점은 잘 알려져 있다. As used herein, the term “Exosome” is a cell-derived endoplasmic reticulum, which is present in the body fluids of almost all eukaryotic organisms. The diameter of the exosomes is on the order of 30-100 nm, which is larger than the LDL protein but much smaller than the red blood cells. Exosomes are released from cells when the multivesicular bodies are fused with the cell membrane, or directly from the cell membrane. It is well known that exosomes perform important and specialized functions such as coagulation and intercellular signaling.
본 명세서에서 용어 "줄기세포"는 미분화된 세포로서 자기 복제 능력을 가지면서 두 개 이상의 서로 다른 종류의 세포로 분화하는 능력을 갖는 세포를 말한다. 본 발명의 줄기세포는 자가 또는 동종 유래 줄기세포일 수 있으며, 인간 및 비인간 포유류를 포함한 임의 유형의 동물 유래일 수 있고, 상기 줄기세포가 성체로부터 유래된 것이든 배아로부터 유래된 것이든 이에 한정되지 않는다. 본 발명의 줄기세포는 배아 줄기세포, 유도만능줄기세포 또는 성체 줄기세포를 포함하며, 구체적으로는 유도만능 줄기세포 또는 성체 줄기세포이다. As used herein, the term “stem cell” refers to a cell that has the ability to differentiate into two or more different types of cells while having self-replicating ability as an undifferentiated cell. Stem cells of the present invention may be autologous or allogeneic stem cells, may be from any type of animal, including humans and non-human mammals, and is not limited to whether the stem cells are derived from adults or embryos Do not. Stem cells of the present invention include embryonic stem cells, induced pluripotent stem cells or adult stem cells, specifically, induced pluripotent stem cells or adult stem cells.
본 명세서에서 용어, '배아줄기세포'란, 수정란이 모체의 자궁에 착상하기 직전인 포배기 배아에서 내세포괴(inner cell mass)를 추출하여 체외에서 배양한 것으로서, 개체의 모든 조직의 세포로 분화할 수 있는 다능성(多能性, pluripotent)이거나 전능성(全能性, totipotent)이 있는 자가재생산능(self-renewal)을 갖는 세포를 의미하며, 넓은 의미로는 배아줄기세포로부터 유래한 배아체(embryoid bodies)도 포함한다. 본 발명의 배아줄기세포로는 인간, 원숭이, 돼지, 말, 소, 양, 개, 고양이, 생쥐, 토끼 등의 모든 유래의 배아줄기세포를 포함하나, 바람직하게는 인간 유래의 배아줄기세포이다.As used herein, the term 'embryonic stem cell' is a cultured in vitro by extracting the inner cell mass from the blastocyst embryo just before the fertilized egg implants in the mother's womb, and will differentiate into cells of all tissues of the individual. It refers to a cell having a pluripotent or pluripotent or self-renewal capable of being pluripotent, and in a broad sense, an embryoid derived from embryonic stem cells. bodies). Embryonic stem cells of the present invention include embryonic stem cells of all derived from humans, monkeys, pigs, horses, cattle, sheep, dogs, cats, mice, rabbits, etc., preferably human embryonic stem cells.
본 명세서에서 용어, '유도만능줄기세포'란, 분화된 세포들로부터 인위적인 역분화 과정을 통해 다능성 분화능을 가지도록 유도된 세포들을 일컫는 말로서 역분화줄기세포 (iPSC: induced pluripotent stem cells)이라고도 한다. 인위적인 역분화 과정은 레트로바이러스 및 렌티바이러스를 이용한 바이러스-매개 또는 비바이러스성 벡터 이용, 단백질 및 세포 추출물 등을 이용하는 비바이러스-매개 역분화 인자의 도입에 의해 수행되거나, 줄기세포 추출물, 화합물 등에 의한 역분화 과정을 포함한다. 유도만능줄기세포는 배아줄기세포와 거의 같은 특성을 가지며, 구체적으로는 비슷한 세포 모양을 보여주고, 유전자, 단백질 발현 패턴이 유사하며, in vitro 및 in vivo에서 전분화능을 가지고, 테라토마 (teratoma)를 형성하며, 생쥐의 배반포 (blastocyst)에 삽입시켰을 때, 키메라 (chimera) 생쥐를 형성하고, 유전자의 생식선 전이 (germline transmission)가 가능하다. 본 발명의 유도만능줄기세포로는 인간, 원숭이, 돼지, 말, 소, 양, 개, 고양이, 생쥐, 토끼 등의 모든 유래의 유도만능줄기 세포를 포함하나, 바람직하게는 인간 유래의 유도만능줄기세포이다.As used herein, the term 'induced pluripotent stem cells' refers to cells induced to have pluripotent differentiation ability through artificial dedifferentiation from differentiated cells and is also referred to as induced pluripotent stem cells (iPSC). . Artificial dedifferentiation process is performed by the use of virus-mediated or non-viral vectors with retroviruses and lentiviruses, introduction of non-virus-mediated dedifferentiation factors using protein and cell extracts, or by stem cell extracts, compounds, etc. Includes reverse differentiation process. Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically, have similar cell morphology, similar gene and protein expression patterns, and have pluripotency in vitro and in vivo. When inserted into the blastocyst of the mouse, chimera mice are formed, and germline transmission of the gene is possible. Induced pluripotent stem cells of the present invention include all induced pluripotent stem cells derived from humans, monkeys, pigs, horses, cattle, sheep, dogs, cats, mice, rabbits, etc., but preferably induced pluripotent stems derived from humans It is a cell.
상기 성체 줄기세포는 인간 또는 동물의 다양한 조직 기원의 줄기세포(예컨대 조혈모세포, 유선 줄기세포, 장 줄기세포, 혈관내피 줄기세포, 신경 줄기세포, 후각신경 줄기세포, 정소 줄기세포 등)와, 인간 또는 동물 조직 유래 중간엽 줄기세포(mesenchymal stromal cell), 인간 또는 동물의 다양한 조직 기원의 유도만능줄기세포로부터 유래된 중간엽 줄기세포, 다분화능 줄기세포 등일 수 있으며, 구체적으로는 인간 또는 동물 조직 유래의 중간엽 줄기세포 또는 유도만능줄기세포 유래의 중간엽 줄기세포이나, 이에 한정되지 않는다. 상기 중간엽 줄기세포는 제대, 제대혈, 골수, 지방, 근육, 신경, 피부, 양막 또는 태반 등으로부터 유래된 중간엽 줄기세포일 수 있으며, 이에 한정되지 않는다.The adult stem cells are stem cells of various tissues of human or animal origin (for example, hematopoietic stem cells, mammary stem cells, intestinal stem cells, vascular endothelial stem cells, neural stem cells, olfactory nerve stem cells, testicular stem cells, etc.) and human Or mesenchymal stromal cells derived from animal tissues, mesenchymal stem cells derived from induced pluripotent stem cells of various tissue origins of humans or animals, multipotent stem cells, and the like, specifically, derived from human or animal tissues. Mesenchymal stem cells derived from mesenchymal stem cells or induced pluripotent stem cells, but are not limited thereto. The mesenchymal stem cells may be mesenchymal stem cells derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane or placenta, and the like.
본 명세서에서 “생성 촉진”이란 대조군에 비하여 특정물질의 생산, 생성, 방출 등이 동일한 시간 내에서 증가되는 것을 말한다. 구체적으로 본 발명에서는 줄기세포로부터 엑소좀이 생산되는 수량과, 엑소좀 내 단백질, RNA 등의 함량이 증가되는 것을 의미한다. In the present specification, "promoting production" refers to an increase in production, production, release, etc. of a specific substance compared to the control within the same time. Specifically, the present invention means that the amount of exosomes produced from stem cells, and the amount of protein, RNA, etc. in the exosomes is increased.
본 발명의 일 구현예에 따르면, 본 발명의 조성물은 배지조성물로서 줄기세포 배양에 사용될 수 있고, 생체 내(in vivo) 엑소좀 생성 촉진용도의 약제학적 조성물로서 줄기세포와 함께 생체 내로 병행 투여되거나, 선행 또는 후행적으로 투여되어 줄기세포의 in vivo 효능을 강화할 수 있다. 상기 줄기세포의 in vivo 효능 강화란, 관절염이나 신경병증 등 특정 질환에 대한 치료목적(in vivo 효능)을 가진 줄기세포의 효능이 줄기세포가 방출하는 엑소좀에 의해 매개됨을 전제로, 본 발명의 상기 조성물을 줄기세포 투여와 함께 투여시 줄기세포로부터 엑소좀의 생성이 촉진되는 결과 줄기세포의 질환 치료 효과(in vivo 효능)를 강화시키는 용도를 의미한다. According to one embodiment of the present invention, the composition of the present invention can be used in culture of stem cells as a medium composition, and administered in parallel with the stem cells as a pharmaceutical composition for promoting in vivo exosome production Prior or subsequent administration can be used to enhance the in vivo efficacy of stem cells. Enhancing the in vivo efficacy of the stem cells, on the premise that the efficacy of stem cells having a therapeutic purpose (in vivo efficacy) for specific diseases such as arthritis or neuropathy is mediated by the exosomes released by the stem cells, When the composition is administered with stem cell administration, it means that the production of exosomes from the stem cells is promoted, thereby enhancing the therapeutic effect (in vivo efficacy) of stem cells.
본 발명의 상기 조성물이 약제학적 조성물인 경우, 본 발명의 약제학적 조성물에 포함될 수 있는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences(19th ed., 1995)에 상세히 기재되어 있다.When the composition of the present invention is a pharmaceutical composition, pharmaceutically acceptable carriers that may be included in the pharmaceutical composition of the present invention are conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, Starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, Magnesium stearate, mineral oil, and the like, but is not limited thereto. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약제학적 조성물은 경구 및 비경구로 투여할 수 있고, 예컨대 정맥 내 주입, 피하 주입, 근육 주입, 복강 주입, 국소 투여, 비강 내 투여, 폐내 투여, 직장내 투여, 경막 내 투여, 안구 투여, 피부 투여 및 경피 투여 등으로 투여할 수 있다. The pharmaceutical compositions of the present invention may be administered orally and parenterally, such as intravenous, subcutaneous, intramuscular, intraperitoneal, topical, intranasal, pulmonary, rectal, intradural, ocular. , Dermal administration and transdermal administration.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다. 본 발명의 구체적인 구현예에 따르면, 본 발명의 약제학적 조성물의 1일 투여량은 0.0001-1000 ㎎/㎏이다. Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a specific embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001-1000 mg / kg.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화 함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 산제, 좌제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media or in the form of extracts, powders, suppositories, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
본 발명에서 "배지조성물"는 생체 외(in vitro)에서 세포의 성장과 생존 및 증식에 필요한 필수성분을 포함하는 조성물을 의미하는 것으로, 당해 분야에서 통상적으로 사용되는 줄기세포 배양용 배지를 모두 포함하며, 예를 들어 DMEM(Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM(α-Minimal essential Medium), G-MEM(Glasgow's Minimal Essential Medium), IMDM(Isocove's Modified Dulbecco's Medium), KnockOut DMEM 등의 상업적으로 제조된 배지 또는 인위적으로 합성한 배지를 이용할 수 있으나, 이에 한정되지 않는다. 본 발명의 배지조성물은 일반적으로 탄소원, 질소원 및 미량원소 성분을 포함하며, 아미노산, 항생제 등을 더 포함할 수 있다.In the present invention, "medium composition" refers to a composition containing essential components necessary for the growth, survival and proliferation of cells in vitro, and includes all of the stem cell culture medium commonly used in the art. For example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM / Commercial such as F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMOD (Isocove's Modified Dulbecco's Medium), KnockOut DMEM Media prepared by or artificially synthesized may be used, but is not limited thereto. The medium composition of the present invention generally includes a carbon source, a nitrogen source, and a trace element component, and may further include an amino acid, an antibiotic, and the like.
본 발명의 상기 배지조성물은 본 발명의 구성성분[피오글리타존(pioglitazone), 메트포르민(metformin), AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)]을 종래의 줄기세포 배양용 배지 내에 첨가하여 제조될 수 있다.The medium composition of the present invention can be prepared by adding the components of the present invention (pioglitazone, metformin, AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide)) in a conventional stem cell culture medium. .
본 발명의 구체적인 구현예에 따르면, 상기 피오글리타존은 배지 내에 0.01~100 μM, 0.01~10 μM, 0.01~5 μM, 0.1~50 μM, 0.1~25 μM, 0.1~10 μM의 농도로 첨가될 수 있으며, 보다 구체적으로는 0.1~10 μM, 1~10 μM, 1~8 μM, 1~5μM, 3~5 μM, 가장 구체적으로는 4 μM의 농도로 첨가될 수 있으나, 이에 한정되는 것은 아니다.According to a specific embodiment of the present invention, the pioglitazone may be added at a concentration of 0.01 to 100 μM, 0.01 to 10 μM, 0.01 to 5 μM, 0.1 to 50 μM, 0.1 to 25 μM, 0.1 to 10 μM in the medium. For example, the concentration may be 0.1-10 μM, 1-10 μM, 1-8 μM, 1-5 μM, 3-5 μM, and most specifically 4 μM, but is not limited thereto.
본 발명의 다른 구체적인 구현예에 따르면, 상기 메트포르민(metformin)은 배지 내에 0.01~100 mM, 0.01~10 mM, 0.01~5 mM, 0.1~50 mM, 0.1~25 mM, 0.1~10 mM의 농도로 첨가될 수 있으며, 보다 구체적으로는 0.1~10 mM, 1~10 mM, 1~8 mM, 1~5 mM, 3~5 mM, 가장 구체적으로는 4 mM의 농도로 첨가될 수 있으나, 이에 한정되는 것은 아니다. According to another specific embodiment of the present invention, the metformin is metformin at a concentration of 0.01-100 mM, 0.01-10 mM, 0.01-5 mM, 0.1-50 mM, 0.1-25 mM, 0.1-10 mM. It may be added, more specifically, it may be added in a concentration of 0.1 ~ 10 mM, 1 ~ 10 mM, 1 ~ 8 mM, 1 ~ 5 mM, 3 ~ 5 mM, most specifically 4 mM, but is not limited thereto. It doesn't happen.
본 발명의 또 다른 구체적인 구현예에 따르면, 상기 AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)는 배지 내에 1~1000 μM, 1~800 μM, 1~700 μM, 1~600 μM, 1~500 μM, 1~400 μM, 1~300 μM, 1~200 μM, 1~100 μM, 10~1000 μM, 10~800 μM, 10~700 μM, 10~600 μM, 10~500 μM, 10~400 μM, 10~300 μM, 10~200 μM, 10~100 μM, 50~500 μM, 50~400 μM, 50~300 μM, 50~200 μM, 50~100 μM의 농도로 첨가될 수 있고, 가장 구체적으로는 100 1~1000 μM, 1~800 μM, 1~700 μM, 1~600 μM, 1~500 μM, 1~400 μM, 1~300 μM, 1~200 μM, 1~100 μM의 농도로 첨가될 수 있으나, 이에 한정되는 것은 아니다.According to another specific embodiment of the present invention, the AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) is 1 ~ 1000 μM, 1 ~ 800 μM, 1 ~ 700 μM, 1 ~ 600 μM, 1 ~ 500 μM in the medium , 1-400 μM, 1-300 μM, 1-200 μM, 1-100 μM, 10-1000 μM, 10-800 μM, 10-700 μM, 10-600 μM, 10-500 μM, 10-400 μM , 10-300 μM, 10-200 μM, 10-100 μM, 50-500 μM, 50-400 μM, 50-300 μM, 50-200 μM, 50-100 μM, can be added at the most specific 100-1000 μM, 1-800 μM, 1-700 μM, 1-600 μM, 1-500 μM, 1-400 μM, 1-300 μM, 1-200 μM, 1-100 μM It may be added, but is not limited thereto.
본 발명의 일 양태에 따르면, 본 발명은 피오글리타존(pioglitazone), 메트포르민(metformin) 및 AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)로 이루어진 군으로부터 선택된 1 이상의 물질로 전처리된 줄기세포에서 유래한 엑소좀을 제공한다. According to one aspect of the invention, the invention is exosomes derived from stem cells pretreated with at least one substance selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) To provide.
본 발명의 다른 일 양태에 따르면, 본 발명은 다음 단계를 포함하는 줄기세포 유래 엑소좀의 생산 방법을 제공한다:According to another aspect of the present invention, the present invention provides a method for producing stem cell-derived exosomes comprising the following steps:
(a) 피오글리타존(pioglitazone), 메트포르민(metformin) 및 AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)로 이루어진 군으로부터 선택된 1 이상의 물질을 포함하는 세포배양 배지에서 줄기세포를 배양하는 단계.(a) culturing the stem cells in a cell culture medium comprising at least one substance selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide).
본 발명의 일 구현예에 따르면, 상기 생산 방법은 다음 단계를 더 포함할 수 있다: (b) 배양된 줄기세포를 세척한 후 세포배양 배지에서 추가로 배양하는 단계 및 (c) 상기 세포배양 배지로부터 엑소좀을 분리하는 단계.According to one embodiment of the invention, the production method may further comprise the following steps: (b) washing the cultured stem cells and then further culturing in cell culture medium and (c) the cell culture medium Separating the exosomes from the.
상기 (a) 단계는 줄기세포를 피오글리타존(pioglitazone), 메트포르민(metformin) 및 AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)로 이루어진 군으로부터 선택된 1 이상의 물질로 전처리하여 줄기세포에 자극을 가하는 과정이다. 본 과정에 의해 줄기세포는 상기 물질로 전처리 되지 않은 줄기세포에 비하여 더 많은 수의 엑소좀을 생산하고, 엑소좀 내 단백질 및 RNA의 함량도 증가된다. In the step (a), the stem cells are pretreated with one or more substances selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) to stimulate the stem cells. By this process, stem cells produce a greater number of exosomes than stem cells not pretreated with the substance, and the content of proteins and RNA in exosomes is also increased.
본 발명의 구체적인 구현예에 있어서, 상기 (b) 단계의 세포배양 배지는 엑소좀이 제거된 우태아혈청(Fetal bovine serum)을 추가적으로 포함한다. 상기 세포배양 배지에 엑소좀을 제거한 FBS를 사용하는 이유는 일반적으로 사용하는 FBS에는 소 혈청유래의 엑소좀이 매우 많이 포함되어 있기 때문에, 줄기세포가 분비하는 엑소좀 외에 FBS에서 유래된 엑소좀이 혼입되는 것을 방지하기 위함이다.In a specific embodiment of the present invention, the cell culture medium of step (b) further comprises fetal bovine serum from which exosomes have been removed. The reason for using FBS from which exosomes are removed in the cell culture medium is that exosomes derived from FBS, in addition to exosomes secreted by stem cells, are generally used because FBS contains a large amount of exosomes derived from bovine serum. This is to prevent mixing.
상기 (b) 단계는 본 발명에 따른 상기 물질로 전처리 된 줄기세포를 세척하여 상기 전처리 물질을 제거하고, 새로운 세포배양 배지에서 배양함으로써 줄기세포로부터 엑소좀의 분비 또는 생산을 유도하는 과정이다. 상기 (a) 단계의 줄기세포 전처리 물질(피오글리타존, 메트포르민, AICAR)은 상기 (b) 단계의 세척과정에 의해 제거되고, 세척 이후의 줄기세포, 줄기세포에서 생산된 엑소좀 또는 배양배지 내에 잔류하지 않는다. 따라서 상기 (a) 단계에서 본 발명의 줄기세포 전처리 물질(피오글리타존, 메트포르민, AICAR)로 처리된 줄기세포와 줄기세포에서 생산된 엑소좀 및 배양배지는 상술한 전처리 물질의 잔류에 의한 영향 없이 후속적 연구 또는 질병의 치료 목적으로 유용하게 사용될 수 있다. Step (b) is a process of inducing secretion or production of exosomes from stem cells by washing the stem cells pretreated with the material according to the present invention to remove the pretreatment material and culturing in a new cell culture medium. The stem cell pretreatment material (pioglitazone, metformin, AICAR) of step (a) is removed by the washing process of step (b) and does not remain in the exosomes or culture medium produced from the stem cells, stem cells after washing. Do not. Therefore, in step (a), exosomes and culture media produced from stem cells and stem cells treated with the stem cell pretreatment material of the present invention (pioglitazone, metformin, AICAR) are not subsequently affected by the residue of the pretreatment material. It can be usefully used for research or treatment of diseases.
본 발명의 구체적인 구현예에 따르면, 상기 (b) 단계의 추가 배양은 12시간 내지 120 시간, 24시간 내지 96시간, 48시간 내지 96시간, 또는 60시간 내지 84시간, 가장 구체적으로는 72시간 동안 배양될 수 있으나, 이에 한정되지 않는다. According to a specific embodiment of the present invention, the further culture of step (b) is 12 hours to 120 hours, 24 hours to 96 hours, 48 hours to 96 hours, or 60 hours to 84 hours, most specifically 72 hours It may be cultured, but is not limited thereto.
또한, 본 발명의 다른 구현예에 있어서, 상기 (c) 단계의 세포배양 배지로부터 엑소좀을 분리하는 단계는 상기 (b) 단계에서 추가배양한 줄기세포의 배양 배지를 200-400 x g에서 5 내지 20분간 원심분리하여 남아있는 세포와 세포 잔여물을 제거한 뒤, 상층액을 취하여 9,000-12,000 x g로 60-80분간 고속원심분리한 후, 다시 상층액을 취하여 90,000-120,000 x g로 80-100분간 원심분리하고 상층액을 제거함으로써 하층에 남아 있는 엑소좀을 얻을 수 있다. 본 발명의 특정 구현예에 따르면, 중간엽줄기세포 배양 배지를 수거하여 300 x g에서 10분간 원심분리하여 남아있는 세포와 세포잔여물을 제거하고, 상층액을 취하여 0.22 μm 필터를 이용하여 여과한 다음, 고속원심분리기(high speed centrifuge)를 이용하여 10,000 x g, 4 ℃에서 70분간 원심분리한다. 원심분리 된 상층액을 다시 취하여 초원심분리기 (ultracentrifuge)를 이용하여 100,000 x g, 4 ℃에서 90분간 원심분리하여 상층액을 제거함으로써 하층에 남아있는 엑소좀을 분리하였다.In addition, in another embodiment of the present invention, the step of separating the exosomes from the cell culture medium of the step (c) is the culture medium of the stem cells further cultured in the step (b) from 5 to 5 to 200-400 xg After centrifugation for 20 minutes to remove the remaining cells and cell residues, the supernatant was taken and centrifuged at 9,000-12,000 xg for 60-80 minutes, then the supernatant was again centrifuged at 90,000-120,000 xg for 80-100 minutes. The exosomes remaining in the lower layer can be obtained by separating and removing the supernatant. According to a specific embodiment of the present invention, the mesenchymal stem cell culture medium is collected and centrifuged at 300 xg for 10 minutes to remove remaining cells and cell residues, and the supernatant is taken and filtered using a 0.22 μm filter. , Centrifuge for 70 minutes at 10,000 xg, 4 ℃ using a high speed centrifuge. The supernatant was centrifuged again and centrifuged at 100,000 x g, 4 ° C. for 90 minutes using an ultracentrifuge to separate the exosomes remaining in the lower layer.
하기 실시예에서 입증된 바와 같이, 본 발명에 따른 상기 전처리 물질로 줄기세포를 전처리 하는 경우 전처리하지 않은 줄기세포와 비교하여 생산되는 엑소좀의 수량 뿐만 아니라 엑소좀 내 단백질 및 RNA의 함량이 증가된다. 따라서, 본 발명의 엑소좀 생성 촉진용 조성물로 전처리된 줄기세포에서 유래한 엑소좀은 종래의 일반적인 줄기세포 또는 본 발명의 엑소좀 생성 촉진용 조성물로 전처리 되지 않은 줄기세포로부터 유래한 엑소좀과 달리 단백질 함량 및 RNA의 함량이 월등하게 향상되었다는 점에서 실질적으로 상이하다. 본 발명의 줄기세포 유래 엑소좀 생성 촉진용 조성물은 추가적인 연구가 필요하겠으나, 단순히 줄기세포가 생성하는 엑소좀의 숫자만을 증가시키는 것이 아니라 엑소좀 내 존재하는 단백질과 RNA의 함량 또한 증가시키므로 우수한 기능성을 가진 엑소좀을 대량으로 생산할 수 있는 것으로 추정된다.As demonstrated in the following examples, the pretreatment of the stem cells with the pretreatment material according to the present invention increases the amount of protein and RNA in the exosomes as well as the amount of exosomes produced in comparison to the stem cells not pretreated. . Therefore, exosomes derived from stem cells pretreated with the composition for promoting exosome production of the present invention, unlike the exosomes derived from conventional stem cells or stem cells not pretreated with the composition for promoting exosome production of the present invention. Substantially different in that the protein content and RNA content are significantly improved. Stem cell-derived exosome production promoting composition of the present invention will require additional research, but not only increase the number of exosomes produced by stem cells, but also increases the content of proteins and RNA present in the exosomes excellent functionality It is estimated that they can produce a large amount of exosomes.
또한, 본 발명의 일 양태에 따르면, 본 발명은 상술한 피오글리타존(pioglitazone), 메트포르민(metformin) 및 AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)로 이루어진 군으로부터 선택된 1 이상의 물질 또는 이를 포함하는 조성물 및 줄기세포를 포함하는 줄기세포 치료제를 제공한다. In addition, according to an aspect of the present invention, the present invention is a pioglitazone (pioglitazone), metformin (metformin) and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) at least one selected from the group consisting of or a composition comprising the same and It provides a stem cell therapy comprising stem cells.
본 발명에서 상기 '세포 치료제'란 세포와 조직의 기능을 복원시키기 위하여 살아있는 자가(autologous), 동종(allogenic), 이종(xenogenic) 세포를 체외에서 증식ㆍ선별하거나 여타한 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 치료, 진단 및 예방의 목적으로 사용되는 의약품을 말한다. 이러한 세포치료제는 크게 두 가지로 분류할 수 있으며 그 첫 번째는 조직재생, 장기기능 회복 또는 면역세포의 기능조절을 위한 "줄기세포 치료제"이며, 두 번째는 생체 내 면역반응의 억제 혹은 면역반응의 항진 등 면역반응 조절을 위한 “면역세포 치료제”이다. 본 발명의 조성물 및 이의 구성성분은 줄기세포 유래 엑소좀의 생성을 촉진하며, 이를 통해 줄기세포의 치료적 효능을 강화시키는 효과를 가지므로, 본 명세서 내에서 “세포치료제”는 “줄기세포 치료제”를 의미한다. In the present invention, the 'cell therapeutic agent' refers to the biological characteristics of cells by proliferating, selecting, or otherwise releasing autologous, allogenic, and xenogenic cells in vitro to restore the function of cells and tissues. Drugs used for the purpose of treatment, diagnosis and prevention through a series of actions such as changing. These cell therapies can be broadly classified into two categories. The first is a "stem cell therapy" for tissue regeneration, long-term function recovery or immune cell function control. It is an “immune cell therapy” for the regulation of immune response such as hyperactivity. Since the composition of the present invention and its components promote the production of stem cell-derived exosomes, thereby having an effect of enhancing the therapeutic efficacy of stem cells, within the present specification, "cell therapeutic agent" is "stem cell therapeutic agent" Means.
상기 줄기세포 치료제는 심근경색, 심부전 등의 심혈관 질환, 간암, 위암, 대장암, 전립선암, 방광암, 폐암 등의 암 질환, 아토피성 피부염, 관절염, 자가면역성 뇌척수염, 전신성 홍반성 루푸스, 대장염 및 다발성 경화증과 같은 염증성 질환, 또는 자가면역성 질환 등의 치료대상 질병을 치료하는 용도로 사용될 수 있으나, 이에 한정되지 않는다. The stem cell therapeutic agents include cardiovascular diseases such as myocardial infarction and heart failure, liver cancer, gastric cancer, colon cancer, prostate cancer, bladder cancer, lung cancer and other cancer diseases, atopic dermatitis, arthritis, autoimmune encephalomyelitis, systemic lupus erythematosus, colitis and multiple It may be used for the treatment of inflammatory diseases such as sclerosis, or diseases to be treated such as autoimmune diseases, but is not limited thereto.
상기 줄기세포 치료제는 상술한 본 발명의 줄기세포 유래 엑소좀 생성 촉진용 조성물과 구성성분을 공통으로 하기 때문에, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다. Since the stem cell therapeutic agent has the composition and components for promoting stem cell-derived exosomes of the present invention described above in common, the common content between the two is omitted in order to avoid excessive complexity of the present specification.
또한, 본 발명의 다른 일 양태에 따르면, 본 발명은 상술한 피오글리타존(pioglitazone), 메트포르민(metformin) 및 AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)로 이루어진 군으로부터 선택된 1 이상의 물질(또는 줄기세포 유래 엑소좀 생성 촉진용 조성물)로 전처리된 줄기세포로부터 유래한 엑소좀을 유효성분으로 포함하는 줄기세포 치료제를 제공한다. In addition, according to another aspect of the present invention, the present invention is one or more substances selected from the group consisting of pioglitazone (pioglitazone), metformin (metformin) and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) (or stem cell derived) The present invention provides a stem cell therapeutic agent comprising an exosome derived from stem cells pretreated with an exosome generation promoting composition) as an active ingredient.
상기 줄기세포 치료제는 상술한 피오글리타존(pioglitazone), 메트포르민(metformin) 및 AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)로 이루어진 군으로부터 선택된 1 이상의 물질로 전처리된 줄기세포에서 유래한 엑소좀과 구성성분을 공통으로 하기 때문에, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다. The stem cell therapeutic agent comprises exosomes and components derived from stem cells pretreated with one or more substances selected from the group consisting of pioglitazone, metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide). Because they are common, the content in common between the two is omitted in order to avoid excessive complexity of the present specification.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 피오글리타존(pioglitazone), 메트포르민(metformin) 및 AICAR(5-Aminoimidazole-4-carboxamide ribonucleotide)로 이루어진 군으로부터 선택된 1 이상의 물질을 포함하는 줄기세포 유래 엑소좀 생성 촉진용 조성물 및 이를 이용한 중간엽줄기세포 유래 엑소좀의 생성 촉진 방법에 관한 것이다.(A) The present invention is a composition for promoting stem cell-derived exosomes production comprising at least one substance selected from the group consisting of pioglitazone (pioglitazone), metformin and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) and using the same It relates to a method for promoting the production of mesenchymal stem cell-derived exosomes.
(b) 본 발명에 따른 줄기세포 유래 엑소좀 생성촉진용 조성물을 줄기세포 배양에 이용하는 경우, 줄기세포 유래 엑소좀의 생산량 및 엑소좀에서 분리되는 단백질과 RNA의 함량이 증가되므로 종래 알려진 방법에 비하여 보다 효율적으로 양질의 엑소좀을 대량 생산할 수 있으며, 이를 관련 연구개발 및 제품화에 유용하게 사용할 수 있다. (b) When the composition for promoting stem cell-derived exosomes production according to the present invention is used for stem cell culture, the production of stem cell-derived exosomes and the content of protein and RNA isolated from exosomes is increased compared to the conventionally known methods More efficient mass production of high quality exosomes, which can be useful for the relevant research and development and commercialization.
도 1은 본 발명의 구성성분인 피오글리타존(Pioglitazone), AICAR, 메트포르민(Metformin)을 처리한 중간엽 줄기세포 유래 엑소좀(미세소포체)의 전자현미경 이미지를 나타낸 도이다.
도 2는 본 발명의 구성성분인 피오글리타존(Pioglitazone), AICAR, 메트포르민(Metformin)을 처리한 중간엽 줄기세포 유래 엑소좀(미세소포체)의 크기 및 분포를 나타낸 도이다.
도 3은 본 발명의 구성성분인 피오글리타존(Pioglitazone), AICAR, 메트포르민(Metformin)을 처리한 중간엽 줄기세포 유래 엑소좀(미세소포체)의 단백질 마커를 확인한 도이다.
도 4는 본 발명의 구성성분인 피오글리타존(Pioglitazone), AICAR, 메트포르민(Metformin)을 처리한 중간엽 줄기세포 유래 엑소좀(미세소포체)의 수를 상기 물질을 미처리한 중간엽 줄기세포 유래 엑소좀의 수와 비교하여 나타낸 도이다.
도 5는 본 발명의 구성성분인 피오글리타존(Pioglitazone), AICAR, 메트포르민(Metformin)을 처리한 중간엽 줄기세포 유래 엑소좀(미세소포체) 내에서 분리되는 단백질의 양을 상기 물질을 미처리한 중간엽 줄기세포 유래 엑소좀 내 단백질의 양과 비교하여 나타낸 도이다.
도 6은 본 발명의 구성성분인 피오글리타존(Pioglitazone), AICAR, 메트포르민(Metformin)을 처리한 중간엽 줄기세포 유래 엑소좀(미세소포체) 내에서 분리되는 RNA의 함량을 상기 물질을 미처리한 중간엽 줄기세포 유래 엑소좀 내 RNA의 함량과 비교하여 나타낸 도이다.1 is a diagram showing an electron microscope image of mesenchymal stem cell-derived exosomes (microvesicles) treated with pioglitazone, AICAR, and metformin, which are components of the present invention.
2 is a diagram showing the size and distribution of mesenchymal stem cell-derived exosomes (microvesicles) treated with Pioglitazone, AICAR, and metformin, which are components of the present invention.
3 is a diagram confirming protein markers of mesenchymal stem cell-derived exosomes (microvesicles) treated with Pioglitazone, AICAR, and metformin (Metformin), which are components of the present invention.
Figure 4 shows the number of mesenchymal stem cell-derived exosomes (microvesicles) treated with pioglitazone, AICAR, and metformin, which are the constituents of the present invention, of the mesenchymal stem cell-derived exosomes without treatment of the material. It is a figure compared with the number.
Figure 5 is a mesenchymal stem untreated with the amount of protein isolated in the mesenchymal stem cell-derived exosomes (microvesicles) treated with Pioglitazone, AICAR, metformin (Metformin) as a component of the present invention Figure shows the amount of protein in cell-derived exosomes.
Figure 6 is a mesenchymal stem untreated with the substance content of RNA isolated in mesenchymal stem cell-derived exosomes (microvesicles) treated with Pioglitazone, AICAR, metformin (Metformin) as a component of the present invention Figure shows the content of RNA in the cell-derived exosomes.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
실시예Example
실시예Example 1. One. 피오글리타존Pioglitazone (( pioglitazonepioglitazone ), 메트포르민(), Metformin ( metforminmetformin ), ), AICARAICAR (5-(5- AminoimidazoleAminoimidazole -4--4- carboxamidecarboxamide ribonucleotide) 처리에 따른 줄기세포 유래 stem cell derivation following ribonucleotide treatment 엑소좀Exosomes 분리 detach
피오글리타존(pioglitazone) 4 μM, 메트포르민(metformin) 4 mM, 또는 AICAR 100 μM이 포함된 배양배지[high glucose DMEM (Gibco, Cat no.11995-065), 10% Fetal bovine Serum (HyClone), 1% MEM Non-Essential Amino Acids Solution (100X) (Gibco, Cat no.11140-050)]에서 제대조직 유래 중간엽 줄기세포를 1주일간 배양하였다. 제대조직 유래 중간엽줄기세포는 서울아산병원 줄기세포센터에서 태아의 제대조직을 자체적으로 분리하여 수립한 제대 조직 유래 중간엽줄기세포이다. 배양을 완료한 후 상기 각각의 전처리 물질[피오글리타존(pioglitazone) 4 μM, 메트포르민(metformin) 4 mM, AICAR 100 μM]이 전처리 된 중간엽줄기세포를 세척하고 엑소좀이 제거된 우태아혈청(Fetal Bovine Serum, FBS)를 10% 첨가한 배양배지에서 추가적으로 72시간 배양하였다. 엑소좀이 제거된 FBS를 사용하는 이유는 일반적으로 사용하는 FBS에는 소 혈청유래의 엑소좀이 매우 많이 포함되어 있기 때문에, 세포가 분비하는 엑소좀 외에 FBS 유래 엑소좀이 혼입되는 것을 방지하기 위함이다. 72시간 배양 후 각각의 전처리 물질이 처리된 중간엽줄기세포 배양배지를 수거하여 300 x g에서 10분간 원심분리하여 남아있는 세포와 세포잔여물을 제거하였다. 상층액을 취하여 0.22 μm 필터를 이용하여 여과한 다음, 고속원심분리기(high speed centrifuge)를 이용하여 10,000 x g, 4 ℃에서 70분간 원심분리하였다. 원심분리 된 상층액을 다시 취하여 초원심분리기 (ultracentrifuge)를 이용하여 100,000 x g, 4 ℃에서 90분간 원심분리하여 상층액을 제거하였다. 하층에 남아있는 엑소좀을 PBS(phosphate bufferd salin)에 희석하여 이하의 실험에 사용하였다.Culture medium containing 4 μM pioglitazone, 4 mM metformin, or 100 μM AICAR [high glucose DMEM (Gibco, Cat no.11995-065), 10% Fetal bovine Serum (HyClone), 1% MEM Umbilical cord tissue-derived mesenchymal stem cells were cultured for 1 week in Non-Essential Amino Acids Solution (100X) (Gibco, Cat no. 11140-050). Umbilical cord stem-derived mesenchymal stem cells are umbilical cord stem-derived mesenchymal stem cells established by separating the fetal umbilical cord tissues from the Asan Hospital Stem Cell Center. After the incubation was completed, the mesenchymal stem cells pretreated with the respective pretreatment materials (pioglitazone 4 μM, metformin 4 mM,
실시예Example 2. 2. 피오글리타존Pioglitazone (( pioglitazonepioglitazone ), 메트포르민(), Metformin ( metforminmetformin ), ), AICARAICAR (5-(5- AminoimidazoleAminoimidazole -4--4- carboxamidecarboxamide ribonucleotide) 처리에 따른 줄기세포 유래 stem cell derivation following ribonucleotide treatment 엑소좀Exosomes 확인 Confirm
상기 실시예 1에서와 같이 피오글리타존(pioglitazone) 4 μM, 메트포르민(metformin) 4 mM 또는 AICAR 100 μM를 처리한 중간엽줄기세포 배양액에서 엑소좀을 분리하고 nanoparticle tracking assay (NanoSight NS300, Malvern)를 통해 각각의 엑소좀의 크기를 확인하였다(도 2). 전자 현미경을 이용하여 엑소좀의 형태를 확인하였으며(도 1), 웨스턴 블랏으로 엑소좀 특이적 항원인 CD9과 CD63에 대한 항체를 이용하여 CD9과 CD63의 발현을 확인하였다(도 3).Exosomes were isolated from mesenchymal stem cell cultures treated with pioglitazone 4 μM, metformin 4 mM or
실시예Example 3. 3. 피오글리타존Pioglitazone (( pioglitazonepioglitazone ), 메트포르민(), Metformin ( metforminmetformin ), ), AICARAICAR (5-(5- AminoimidazoleAminoimidazole -4--4- carboxamidecarboxamide ribonucleotide) 처리에 따른 ribonucleotide treatment 엑소좀Exosomes 생산 production 효율 증가Increase efficiency
3-1. 3-1. 피오글리타존Pioglitazone (( pioglitazonepioglitazone ), 메트포르민(), Metformin ( metforminmetformin ), ), AICARAICAR (5-(5- AminoimidazoleAminoimidazole -4--4- carboxamidecarboxamide ribonucleotide) 처리에 따른 ribonucleotide treatment 엑소좀Exosomes 수 증가 비교 Number increase comparison
줄기세포 전처리 물질[피오글리타존(pioglitazone) 4 μM, 메트포르민(metformin) 4 mM, AICAR 100 μM]을 처리한 중간엽줄기세포 배양액에서 엑소좀을 분리하여 각각 nanoparticle tracking assay (NanoSight NS300, Malvern)를 통해 동일 중간엽줄기세포 유래 엑소좀 수를 비교하였다.Exosomes were isolated from the mesenchymal stem cell cultures treated with stem cell pretreatment materials (pioglitazone 4 μM, metformin 4 mM,
결과는 도 4에 나타내었다.The results are shown in FIG.
도 4에 나타낸 바와 같이, 아무 물질도 처리하지 않은 중간엽 줄기세포에 비하여, 줄기세포 전처리 물질[피오글리타존(pioglitazone) 4 μM, 메트포르민(metformin) 4 mM, AICAR 100 μM]을 전처리한 중간엽 줄기세포에서 적게는 약 22%에서 많게는 약 61% 많은 수의 엑소좀을 생산하였음을 확인하였다. 상기 결과로부터 본 발명에 따른 상기 줄기세포 전처리 물질을 중간엽 줄기세포에 처리시, 중간엽 줄기세포가 생산하는 엑소좀의 수가 증가함을 알 수 있다. As shown in Figure 4, compared with mesenchymal stem cells not treated with any substance, mesenchymal stem cells pretreated with stem cell pretreatment materials (pioglitazone 4 μM, metformin 4 mM,
3-2. 3-2. 피오글리타존Pioglitazone (( pioglitazonepioglitazone ), 메트포르민(), Metformin ( metforminmetformin ), ), AICARAICAR (5-(5- AminoimidazoleAminoimidazole -4--4- carboxamidecarboxamide ribonucleotide) 처리에 따른 ribonucleotide treatment 엑소좀Exosomes 유래 단백질 함량 비교 Derived Protein Content Comparison
줄기세포 전처리 물질[피오글리타존(pioglitazone) 4 μM, 메트포르민(metformin) 4 mM, AICAR 100 μM]을 처리한 중간엽 줄기세포 배양액에서 분리한 엑소좀에서 엑소좀 단백질 분리 킷트 (Total exosome RNA and protein isolation kit, Invitrogen)를 이용하여 단백질을 분리하고 bradford analysis를 통해 595 nm에서 흡광도를 측정하여 단백질을 정량하여 비교하였다. Total exosome RNA and protein isolation kit from exosomes isolated from mesenchymal stem cell culture treated with stem cell pretreatment materials (pioglitazone 4 μM, metformin 4 mM,
결과는 도 5에 나타내었다.The results are shown in FIG.
도 5에 나타낸 바와 같이, 아무 물질도 처리하지 않은 중간엽 줄기세포에 비하여, 줄기세포 전처리 물질[피오글리타존(pioglitazone) 4 μM, 메트포르민(metformin) 4 mM, AICAR 100 μM]을 전처리한 중간엽 줄기세포에서 약 3.4 배에서 약 4.6 배 많은 량의 엑소좀 유래 단백질을 생산하였음을 확인하였다. 이로부터 상기 줄기세포 전처리 물질을 중간엽 줄기세포에 처리시 엑소좀의 수 뿐만 아니라 엑소좀 유래 단백질(exosomal protein)의 함량 또한 증가함을 알 수 있다.5, mesenchymal stem cells pretreated with stem cell pretreatment materials (pioglitazone 4 μM, metformin 4 mM,
3-3. 3-3. 피오글리타존Pioglitazone (( pioglitazonepioglitazone ), 메트포르민(), Metformin ( metforminmetformin ), ), AICARAICAR (5-(5- AminoimidazoleAminoimidazole -4--4- carboxamidecarboxamide ribonucleotide) ribonucleotide) 처리에 따른 According to treatment 엑소좀Exosomes 유래 RNA 함량 비교 Derived RNA Content Comparison
줄기세포 전처리 물질[피오글리타존(pioglitazone) 4 μM, 메트포르민(metformin) 4 mM, AICAR 100 μM]를 처리한 중간엽줄기세포 배양액에서 분리한 엑소좀에서 RNA 분리용 킷트 (Total exosome RNA and protein isolation kit, Invitrogen)를 이용하여 전체 엑소좀 RNA를 분리하고 nanodrop을 이용하여 RNA의 농도를 측정하여 비교하였다. Kit for RNA separation from exosomes isolated from mesenchymal stem cell culture treated with stem cell pretreatment materials (pioglitazone 4 μM, metformin 4 mM,
결과는 도 6에 나타내었다.The results are shown in FIG.
도 6에 나타낸 바와 같이, 아무 물질도 처리하지 않은 중간엽 줄기세포에 비하여, 줄기세포 전처리 물질[피오글리타존(pioglitazone) 4 μM, 메트포르민(metformin) 4 mM, AICAR 100 μM]을 전처리한 중간엽 줄기세포에서 약 3.2 배에서 약 4.3 배 많은 량의 엑소좀 유래 RNA가 생산되었음을 확인하였다. 6, mesenchymal stem cells pretreated with stem cell pretreatment materials (pioglitazone 4 μM, metformin 4 mM,
3-4. 소결3-4. Sintered
상기 실시예 3-1 내지 3-3의 결과를 하기 표 1에 나타내었다.The results of Examples 3-1 to 3-3 are shown in Table 1 below.
(MSC: 미처리 중간엽 줄기세포, Pio-MSC: 피오글리타존-처리 중간엽 줄기세포, AICAR-MSC: AICAR-처리 중간엽 줄기세포, Met-MSC: 메트포르민-처리 중간엽 줄기세포)(MSC: untreated mesenchymal stem cells, Pio-MSC: pioglitazone-treated mesenchymal stem cells, AICAR-MSC: AICAR-treated mesenchymal stem cells, Met-MSC: metformin-treated mesenchymal stem cells)
상기 표 1에 나타낸 바와 같이, 본 발명에 따른 줄기세포 전처리 물질(피오글리타존, 메트포르민, AICAR)을 중간엽 줄기세포에 처리시 엑소좀의 수 뿐만 아니라 엑소좀 유래 단백질(exosomal protein)의 생산량 또한 증가하며, 엑소좀 유래 RNA(exosomal RNA)의 함량도 증가함을 알 수 있다. 따라서, 본 발명의 줄기세포 유래 엑소좀 생성 촉진용 조성물은 추가적인 연구가 필요하겠지만 단순히 줄기세포가 생성하는 엑소좀의 숫자만을 증가시키는 것이 아니라, 엑소좀 내 존재하는 단백질과 RNA의 함량 또한 증가시키므로 우수한 기능성을 가진 엑소좀을 대량으로 생산할 수 있는 것으로 추정된다.As shown in Table 1 above, when the stem cell pretreatment material (pioglitazone, metformin, AICAR) according to the present invention is treated to mesenchymal stem cells, the number of exosomes as well as the production of exosomal protein are increased. It can be seen that the content of exosome-derived RNA (exosomal RNA) also increases. Therefore, the composition for promoting stem cell-derived exosomes production of the present invention may require additional research, but not simply increase the number of exosomes produced by stem cells, but also increase the content of proteins and RNA present in the exosomes. It is estimated that a large amount of exosomes can be produced.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
Claims (12)
Composition for promoting stem cell-derived exosomes production comprising pioglitazone (pioglitazone).
The composition of claim 1, wherein the stem cells are embryonic stem cells, adult stem cells, or induced pluripotent stem cells (iPS).
The method of claim 2, wherein the adult stem cells are adult stem cells of human or animal tissue origin, mesenchymal stromal cells derived from human or animal tissue, or derived from induced pluripotent stem cells of human or animal tissue origin The mesenchymal stem cells, characterized in that the composition.
The composition of claim 3, wherein the mesenchymal stem cells are derived from a tissue selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, skin, amniotic membrane, and placenta.
The method of claim 3, wherein the adult stem cells of human or animal tissue origin are selected from the group consisting of hematopoietic stem cells, mammary stem cells, intestinal stem cells, vascular endothelial stem cells, neural stem cells, olfactory nerve stem cells and testicular stem cells The composition characterized in that.
The composition of claim 1, wherein the composition increases the number of exosomes, exosome-derived protein, and exosome-derived RNA.
(a) 피오글리타존(pioglitazone)을 포함하는 세포배양 배지에서 줄기세포를 배양하는 단계.
Method for producing stem cell-derived exosomes comprising the following steps:
(a) culturing the stem cells in a cell culture medium containing pioglitazone.
The method of claim 10, further comprising: (b) further culturing the cultured stem cells in cell culture medium; And (c) separating the exosomes from the cell culture medium.
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