KR20230138786A - Composition for Facilitating the Differentiation of Stem Cell Organoids Comprising Transforming Growth Factor-β1 as an Effective Ingredient - Google Patents
Composition for Facilitating the Differentiation of Stem Cell Organoids Comprising Transforming Growth Factor-β1 as an Effective Ingredient Download PDFInfo
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- KR20230138786A KR20230138786A KR1020220036842A KR20220036842A KR20230138786A KR 20230138786 A KR20230138786 A KR 20230138786A KR 1020220036842 A KR1020220036842 A KR 1020220036842A KR 20220036842 A KR20220036842 A KR 20220036842A KR 20230138786 A KR20230138786 A KR 20230138786A
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- stem cell
- stem cells
- growth factor
- transforming growth
- osteogenic differentiation
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Abstract
본 발명은 줄기세포 오르가노이드의 골분화를 촉진시키기 위한 조성물 및 이를 이용한 골분화 촉진 방법에 관한 것이다. 본 발명에 따르면, 전환성장인자 베타1(Transforming Growth Factor-β1)를 유효성분으로 포함하는 줄기세포 오르가노이드의 골분화 촉진용 조성물은 줄기세포 오르가노이드의 형태를 유지하면서 줄기세포 오르가노이드의 골분화를 촉진시키는 효과가 있으므로, 줄기세포 오르가노이드의 골분화를 촉진시키고자 할 때 본 발명의 줄기세포 오르가노이드의 골분화 촉진용 조성물이 유용하게 이용될 수 있다.The present invention relates to a composition for promoting osteogenic differentiation of stem cell organoids and a method for promoting osteogenic differentiation using the same. According to the present invention, a composition for promoting osteogenic differentiation of stem cell organoids containing Transforming Growth Factor-β1 as an active ingredient promotes osteogenic differentiation of stem cell organoids while maintaining the shape of the stem cell organoids. Since it has the effect of promoting , the composition for promoting osteogenic differentiation of stem cell organoids of the present invention can be usefully used when trying to promote osteogenic differentiation of stem cell organoids.
Description
본 발명은 전환성장인자 베타1를 유효성분으로 포함하는 줄기세포 오르가노이드의 골분화 촉진용 조성물 및 이를 이용한 골분화 촉진 방법에 관한 것이다.The present invention relates to a composition for promoting osteogenic differentiation of stem cell organoids containing transforming growth factor beta 1 as an active ingredient and a method for promoting osteogenic differentiation using the same.
3차원 오르가노이드, 생명공학, 장기 칩 기술과 같은 최근 개발된 기술은 다세포 조직을 구성하고 기관을 생성할 수 있는 큰 잠재력을 가지고 있다(Matsumoto R., et al., 2021, J Mol Sci 22:). 일반적으로 사용되는 2차원 배양 방법과 비교하여 세포를 스페로이드로 배양하면 세포 간 교류를 자극하고 생체 내 환경을 보다 정확하게 모방하여 재생 잠재력이 향상된다 (Findeisen L., et al., 2021, BMC Musculoskelet Disord 22: 401). 기존 연구에 따르면 하이브리드 신경 혈관 스페로이드는 인간 유도 만능 줄기 세포 유래 피질 신경 전구 세포 스페로이드, 내피 세포 스페로이드 및 지지하는 인간 중간엽 줄기 세포의 융합에 의해 구성되었다(Song L., et al., 2019, Sci Rep 9: 5977). 최근 몇 년 동안 세포 자가 조립은 인 비트로(in vitro)의 일정 조건에서 공동 배양된 필수 세포 유형이 오르가노이드를 생성하는 것으로 나타난 경우 뇌, 소장, 간 및 신장과 같은 장기 구조의 복잡성을 달성하기 위한 방법으로 부상하였다(Wan ACA, 2016, Trends Biotechnol 34: 711-721). 또한, 인 비트로(in vitro)에서 건 형성 및 인대 형성을 위한 스캐폴드가 없는 3차원 오르가노이드 모델이 설명되었다(Chu J., et al., 2021, Eur Cell Mater 41: 20-33).Recently developed technologies such as 3D organoids, biotechnology, and organ-on-a-chip technology have great potential to construct multicellular tissues and generate organs (Matsumoto R., et al., 2021, J Mol Sci 22: ). Compared to commonly used two-dimensional culture methods, culturing cells as spheroids improves regenerative potential by stimulating cell-cell exchange and more accurately mimicking the in vivo environment (Findeisen L., et al., 2021, BMC Musculoskelet Disord 22: 401). According to previous studies, hybrid neurovascular spheroids were constructed by the fusion of human induced pluripotent stem cell-derived cortical neural progenitor cell spheroids, endothelial cell spheroids, and supporting human mesenchymal stem cells (Song L., et al., 2019, Sci Rep 9: 5977). In recent years, cell self-assembly has been shown to generate organoids when essential cell types co-cultured under certain conditions in vitro have been shown to achieve complexity in the structure of organs such as the brain, small intestine, liver, and kidney. emerged as a method (Wan ACA, 2016, Trends Biotechnol 34: 711-721). Additionally, a scaffold-free three-dimensional organoid model for tendon formation and ligament formation in vitro has been described (Chu J., et al., 2021, Eur Cell Mater 41: 20-33).
줄기세포는 특히 다양한 질병의 치료에 오랫동안 큰 관심을 받아왔다. 줄기세포는 다양한 기능을 가지는 것으로 알려져 있다(Tae, J.Y., et al., 2019. Exp Ther Med 18: 326-331). 줄기세포는 다양한 조직으로 분화할 수 있을 뿐만 아니라 다양한 인자를 분비하며, 파라크라인 효과(paracrine effect)라고 불리우는 이 기능이 주변 조직에 영향을 미치는 것으로 보고되어 있다(Lee, H., et al., 2018. Adv Clin Exp Med 27: 971-977). 최근에는 줄기세포가 조직재생에 적용되고 있다 (Kang, S.H., et al., 2019. J Periodontal Implant Sci 49: 258-267).Stem cells have long been of great interest, especially in the treatment of various diseases. Stem cells are known to have various functions (Tae, J.Y., et al., 2019. Exp Ther Med 18: 326-331). Stem cells can not only differentiate into various tissues but also secrete various factors, and this function, called the paracrine effect, has been reported to affect surrounding tissues (Lee, H., et al. , 2018. Adv Clin Exp Med 27: 971-977). Recently, stem cells have been applied to tissue regeneration (Kang, S.H., et al., 2019. J Periodontal Implant Sci 49: 258-267).
연골 분화에 대한 중간엽 줄기세포의 조절은 관절 연골 결함 및 골관절염에 대한 줄기세포 치료에서 중요한 문제다. 한편, 에탄올, 아스코르브산, 덱사메사손, 프로스타글란딘 E2, 스타우로스포린, 카토제닌, 그리고 옥소피페라진 유도체를 포함한 저분자 화합물들이 중간엽 줄기세포의 연골 분화를 효과적으로 유도하는 것으로 보고되고 있다. 그러나, 더욱 뛰어난 연골 분화를 위한 효과적인 단분자 화합물은 지속적으로 연구되고 있다.Regulation of mesenchymal stem cells on chondrogenic differentiation is an important issue in stem cell therapy for articular cartilage defects and osteoarthritis. Meanwhile, low-molecular-weight compounds including ethanol, ascorbic acid, dexamethasone, prostaglandin E2, staurosporine, katogenin, and oxopiperazine derivatives have been reported to effectively induce chondrogenic differentiation of mesenchymal stem cells. However, effective single-molecule compounds for better cartilage differentiation are continuously being researched.
특히, 중간엽 줄기세포(mesenchymal stem cell)로부터 특정 단백질 또는 유전자를 이용하여 조골세포로 분화시키는 기술들이 보고되고 있다. 중간엽 줄기세포(mesenchymal stem cells, MSCs)는 중간엽 계통으로 분화할 수 있는 다능성 세포이며 다양한 조직에서 분리할 수 있으며 인 비트로(in vitro)에서 쉽게 배양할 수 있다(Castro-Manrreza, M.E., 2015, Journal of immunology research 394917-394917). In particular, technologies for differentiating mesenchymal stem cells into osteoblasts using specific proteins or genes have been reported. Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into the mesenchymal lineage and can be isolated from various tissues and easily cultured in vitro (Castro-Manrreza, M.E., 2015, Journal of immunology research 394917-394917).
종래의 연구에 따르면 치은(gingiva) 조직에서 유래된 중간엽 줄기세포는 임상적으로 뼈 재건과 같은 골재생에 있어 줄기세포 기반 치료에 새로운 공급원이 될 수 있다. 또한, 인간 치은(gingiva) 유래 중간엽 줄기세포는 재생 의학에서 세포 치료를 위한 골수(bone marrow) 유래 중간엽 줄기세포보다 우수함이 밝혀졌다(Tomar GB, Srivastava RK, Gupta N, Barhanpurkar AP, Pote ST, Jhaveri HM, et al. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine. Biochem Biophys Res Commun 2010;393:377-383.). 줄기세포의 다른 공급원과 마찬가지로 치은 유래 중간엽 줄기세포에서 면역 조절 성질이 발견되었다. 치은 조직으로부터 유래된 줄기세포는 격리의 용이성, 접근 가능한 조직 공급원 및 빠른 생체 외 팽창을 포함한 몇 가지 장점을 가지고 있다.According to previous studies, mesenchymal stem cells derived from gingiva tissue can be a new source for stem cell-based treatment in bone regeneration such as clinical bone reconstruction. Additionally, human gingiva-derived mesenchymal stem cells have been shown to be superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine (Tomar GB, Srivastava RK, Gupta N, Barhanpurkar AP, Pote ST , Jhaveri HM, et al. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine. Biochem Biophys Res Commun 2010;393:377-383.). As with other sources of stem cells, immunomodulatory properties have been discovered in gingival-derived mesenchymal stem cells. Stem cells derived from gingival tissue have several advantages, including ease of isolation, accessible tissue source, and rapid in vitro expansion.
많은 연구에서 치은 유래 중간엽 줄기세포(gingiva-derived mesenchymal stem cell, GMSC)에 대한 일부 성장인자의 효과와 GMSC의 골분화를 유도시키는 능력을 시험했다(Lee, H., et al., 2019, Exp Ther Med 18: 2867-2876; Lee, H., et al., 2018, Adv Clin Exp Med 27: 971-977; Lee, H., et al., 2017, Biomed Rep 7: 291-296). 이전 보고서에 따르면, 골형성 단백질-4는 콜라겐 I 및 Sp7 발현의 상향 조절을 통해 골분화를 향상시켰다(Lee, H., et al., 2019, Exp Ther Med 18: 2867-2876). 섬유아세포 성장인자는 세포 생존능을 유지하면서 줄기세포 스페로이드의 골분화에 관여하는 것으로 나타났으며, 또한 성장인자의 농도와 증식 또는 분화 활동 간의 연관성도 연구되었다(Son, J., et al., 2020, Exp Ther Med 20: 2013-2020).Many studies have tested the effects of some growth factors on gingiva-derived mesenchymal stem cells (GMSCs) and their ability to induce osteogenic differentiation of GMSCs (Lee, H., et al., 2019, Exp Ther Med 18: 2867-2876; Lee, H., et al., 2018, Adv Clin Exp Med 27: 971-977; Lee, H., et al., 2017, Biomed Rep 7: 291-296). According to a previous report, osteogenic protein-4 enhanced osteogenic differentiation through upregulation of collagen I and Sp7 expression (Lee, H., et al., 2019, Exp Ther Med 18: 2867-2876). Fibroblast growth factors have been shown to be involved in osteogenic differentiation of stem cell spheroids while maintaining cell viability, and the correlation between growth factor concentration and proliferation or differentiation activity has also been studied (Son, J., et al., 2020, Exp Ther Med 20: 2013-2020).
본 명세서에서 언급된 특허문헌 및 참고문헌은 각각의 문헌이 참조에 의해 개별적이고 명확하게 특정된 것과 동일한 정도로 본 명세서에 참조로 삽입된다.Patent documents and references mentioned herein are herein incorporated by reference to the same extent as if each individual document was individually and specifically identified by reference.
본 발명자들은 치은(gingiva) 유래 중간엽 줄기세포 오르가노이드의 골분화에 전환성장인자 베타1를 사용할 수 있는지 연구하였고, 전환성장인자 베타1를 골분화 유도 배지에 첨가할 경우에 치은(gingiva) 유래 중간엽 줄기세포 오르가노이드의 골분화를 촉진하는 것으로 확인함으로써 본 발명을 완성하였다. The present inventors studied whether transforming growth factor beta 1 could be used for osteogenic differentiation of gingiva-derived mesenchymal stem cell organoids, and when transforming growth factor beta 1 was added to the osteogenic differentiation induction medium, gingiva-derived mesenchymal stem cell organoids were used for osteogenic differentiation. The present invention was completed by confirming that it promotes osteogenic differentiation of mesenchymal stem cell organoids.
따라서, 본 발명의 목적은 전환성장인자 베타1(Transforming Growth Factor-β1; TGF-β1)를 유효성분으로 포함하는 줄기세포 오르가노이드(organoid)의 골분화 촉진용 조성물을 제공하는 것에 있다.Therefore, the purpose of the present invention is to provide a composition for promoting osteogenic differentiation of stem cell organoids containing Transforming Growth Factor-β1 (TGF-β1) as an active ingredient.
본 발명의 다른 목적은 전환성장인자 베타1(Transforming Growth Factor-β1)를 유효성분으로 포함하는 줄기세포 배양용 배지를 제공하는 것에 있다.Another object of the present invention is to provide a medium for stem cell culture containing Transforming Growth Factor-β1 as an active ingredient.
본 발명의 또 다른 목적은 분리된 줄기세포에 전환성장인자 베타1(Transforming Growth Factor-β1)를 처리하는 단계를 포함하는 줄기세포 오르가노이드의 골분화 촉진 방법을 제공하는 것에 있다.Another object of the present invention is to provide a method for promoting osteogenic differentiation of stem cell organoids, which includes treating isolated stem cells with Transforming Growth Factor-β1.
본 발명의 또 다른 목적은 본 발명의 골분화 촉진용 조성물로 분화된 조골세포를 유효성분으로 포함하는 골결손증 예방 또는 치료용 조성물을 제공하는 것에 있다.Another object of the present invention is to provide a composition for preventing or treating bone defects containing osteoblasts differentiated with the composition for promoting osteogenic differentiation of the present invention as an active ingredient.
본 발명의 다른 목적 및 기술적 특징은 이하의 발명의 상세한 설명, 청구의 범위 및 도면에 의해 보다 구체적으로 제시된다.Other objects and technical features of the present invention are presented in more detail by the following detailed description, claims, and drawings.
본 발명의 일 양태에 따르면, 본 발명은 전환성장인자 베타1(Transforming Growth Factor-β1)를 유효성분으로 포함하는 줄기세포 오르가노이드(organoid)의 골분화 촉진용 조성물을 제공한다.According to one aspect of the present invention, the present invention provides a composition for promoting osteogenic differentiation of stem cell organoids containing Transforming Growth Factor-β1 as an active ingredient.
본 발명의 일 구현예에 따르면, 상기 줄기세포는 성체줄기세포 및 각종 분화 세포를 초기화시킨 유도 다능성 줄기세포(ips 세포)를 포함할 수 있으나, 이에 한정되지 않는다. 바람직하게는 성체줄기세포일 수 있고, 더욱 바람직하게는 치은(gingiva) 유래 중간엽 줄기세포일 수 있다. According to one embodiment of the present invention, the stem cells may include adult stem cells and induced pluripotent stem cells (ips cells) that initialize various differentiated cells, but are not limited thereto. Preferably, it may be an adult stem cell, and more preferably, it may be a gingiva-derived mesenchymal stem cell.
본 발명에서 “줄기세포”는 개체의 모든 조직의 세포로 분화할 수 있는 다능성(多能性, pluripotent)이거나 전능성(全能性, totipotent)이 있는 자가-재생산능(self-renewal)을 갖는 세포를 의미하며, 유도 다능성 줄기세포 및 성체줄기세포를 포함한다.In the present invention, “stem cells” are pluripotent or totipotent cells that can differentiate into cells of all tissues of an organism. It means induced pluripotent stem cells and adult stem cells.
본 발명에서 “유도 다능성 줄기세포”는 분화된 세포들이 인위적인 역분화 과정을 통해 다능성 분화능을 가지도록 유도된 세포들을 일컫는 말로서, 역분화 줄기세포(iPSC: induced pluripotent stem cells)이라고도 한다.In the present invention, “induced pluripotent stem cells” refers to cells induced to have pluripotent differentiation ability through an artificial dedifferentiation process, and are also called induced pluripotent stem cells (iPSC).
본 발명에서 “성체줄기세포”는 인간을 포함한 포유동물의 조직에서 분리해 낸, 분화되기 직전의 원시세포로서, 무한정으로 증식할 수 있는 능력 및 여러 가지 형태의 세포(예를 들면, 지방세포, 연골세포, 근육세포, 뼈세포등)로 분화할 수 있는 줄기세포이다. 성체줄기세포는 조혈 줄기세포(hematopoietic stem cell), 재생의학의 재료로 각광 받고 있는 중간엽 줄기세포(mesenchymal stem cell) 및 신경 줄기세포(neural stem cell) 등이 있다.In the present invention, “adult stem cells” are primitive cells isolated from the tissues of mammals, including humans, just before differentiation, and have the ability to proliferate indefinitely and form various types of cells (e.g., adipocytes, It is a stem cell that can differentiate into cartilage cells, muscle cells, bone cells, etc.). Adult stem cells include hematopoietic stem cells, mesenchymal stem cells, and neural stem cells, which are in the spotlight as materials for regenerative medicine.
본 발명에서 “분화”는 덜 특화된 세포가 특정한 세포로 발달하여 세포의 크기나 모양, 막전위, 대사활성, 신호에 대한 반응이 특정한 유형의 세포로 변화하는 현상을 의미한다. 상기 분화는 주로 다세포 생물이 단일 접합자(zygote)에서 복잡한 조직을 형성하는 과정 중에 일어나거나, 성인이 되었을 때 손상된 조직을 복구하는 경우 성체줄기세포가 특정한 세포로 분화하게 된다.In the present invention, “differentiation” refers to a phenomenon in which less specialized cells develop into specific cells and the size, shape, membrane potential, metabolic activity, and response to signals change to a specific type of cell. The differentiation mainly occurs during the process of multicellular organisms forming complex tissues from a single zygote, or when repairing damaged tissues in adulthood, adult stem cells differentiate into specific cells.
본 발명의 일 구현예에 따르면, 상기 골분화는 줄기세포가 조골세포로 분화하는 것일 수 있다.According to one embodiment of the present invention, the osteogenic differentiation may be the differentiation of stem cells into osteoblasts.
본 발명의 일 구현예에 따르면, 상기 줄기세포 오르가노이드의 골분화 촉진용 조성물에는 상기 전환성장인자 베타1이 0.0001 내지 1000 ng/ml의 함량으로 포함될 수 있고, 바람직하게는 0.0005 내지 500 ng/ml의 함량으로 포함될 수 있으며, 더욱 바람직하게는 0.001 내지 200 ng/ml의 함량으로 포함될 수 있으나, 이에 한정하지 아니한다.According to one embodiment of the present invention, the composition for promoting osteogenic differentiation of stem cell organoids may contain the transforming growth factor beta 1 in an amount of 0.0001 to 1000 ng/ml, preferably 0.0005 to 500 ng/ml. It may be included in an amount of 0.001 to 200 ng/ml, but is not limited thereto.
본 발명의 다른 양태에 따르면, 상기 전환성장인자 베타1(Transforming Growth Factor-β1)를 유효성분으로 포함하는 줄기세포 배양용 배지를 제공한다.According to another aspect of the present invention, a medium for stem cell culture containing the Transforming Growth Factor-β1 as an active ingredient is provided.
상기 전환성장인자 베타1는 줄기세포 배양용 배지에 일 성분으로 포함될 수 있다.The transforming growth factor beta 1 may be included as a component in the medium for stem cell culture.
상기 줄기세포 배양용 배지는 줄기세포의 배양에 통상적으로 이용되는 모든 배지를 포함하고, 예를 들어 DMEM(Dulbeco's Modified Eagle's Medium), MEM(Minimal essential Medium), BME(Basal Medium Eagle), RPMI1640, F-10, F-12, MEM(Minimal essential Medium), GMEM(Glasgow's Minimal essential Medium), IMDM(Iscove's Modified Dulbecco's Medium)등과 같은 기본 배지, 또는 골분화를 유도할 수 있는 성분이 함유된 배지일 수 있으나, 이에 한정되지 아니한다.The medium for stem cell culture includes all media commonly used for culturing stem cells, for example, DMEM (Dulbeco's Modified Eagle's Medium), MEM (Minimal essential medium), BME (Basal Medium Eagle), RPMI1640, F It may be a basic medium such as -10, F-12, MEM (Minimal essential Medium), GMEM (Glasgow's Minimal essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), or a medium containing ingredients that can induce osteogenic differentiation. , but is not limited to this.
본 발명의 일 구현예에 따르면, 상기 줄기세포 배양용 배지로 배양되는 줄기세포는 성체줄기세포 및 각종 분화 세포를 초기화시킨 유도 다능성 줄기세포(ips 세포)를 포함할 수 있으나, 이에 한정되지 않는다. 바람직하게는 성체줄기세포일 수 있고, 더욱 바람직하게는 치은(gingiva) 유래 중간엽 줄기세포일 수 있다. According to one embodiment of the present invention, the stem cells cultured in the stem cell culture medium may include, but are not limited to, adult stem cells and induced pluripotent stem cells (ips cells) that have initialized various differentiated cells. . Preferably, it may be an adult stem cell, and more preferably, it may be a gingiva-derived mesenchymal stem cell.
본 발명의 일 구현예에 따르면, 상기 줄기세포 배양용 배지에 포함되는 전환성장인자 베타1이 0.0001 내지 1000 ng/ml의 함량으로 포함될 수 있고, 바람직하게는 0.0005 내지 500 ng/ml의 함량으로 포함될 수 있으며, 더욱 바람직하게는 0.001 내지 200 ng/ml의 함량으로 포함될 수 있으나, 이에 한정하지 아니한다.According to one embodiment of the present invention, transforming growth factor beta 1 contained in the medium for stem cell culture may be included in an amount of 0.0001 to 1000 ng/ml, and preferably in an amount of 0.0005 to 500 ng/ml. It may be included, and more preferably, may be contained in an amount of 0.001 to 200 ng/ml, but is not limited thereto.
본 발명의 또 다른 양태에 따르면, 분리된 줄기세포에 전환성장인자 베타1(Transforming Growth Factor-β1)를 처리하는 단계를 포함하는 줄기세포 오르가노이드의 골분화 촉진 방법을 제공한다.According to another aspect of the present invention, a method for promoting osteogenic differentiation of stem cell organoids is provided, comprising treating isolated stem cells with Transforming Growth Factor-β1.
본 발명의 줄기세포 오르가노이드의 골분화 촉진 방법은 분리된 줄기세포에 전환성장인자 베타1를 처리하는 단계 및 상기 전환성장인자 베타1이 처리된, 분리된 줄기세포를 배양하는 단계를 포함한다.The method of promoting osteogenic differentiation of stem cell organoids of the present invention includes the steps of treating isolated stem cells with transforming growth factor beta 1 and culturing the isolated stem cells treated with transforming growth factor beta 1.
상기 골분화 촉진의 대상이 되는 줄기세포는 인 비트로(in vitro) 상태일 수 있고, 상기 전환성장인자 베타1는 상기 줄기세포 배양용 배지의 형태로 줄기세포에 처리될 수 있다. 또한, 골분화를 촉진하기 위하여 줄기세포는 3차원 지지체에서 배양될 수 있다. 상기 지지체를 구성하는 성분은 생체적합한 물질을 사용할 수 있고, 지지체에서 배양되어 분화된 조골세포는 지지체로부터 별도로 분리하지 않고 골질환 치료를 위하여 지지체에 부착된 상태로 생체에 이식될 수 있다.Stem cells subject to the promotion of osteogenic differentiation may be in an in vitro state, and the transforming growth factor beta 1 may be treated to the stem cells in the form of a medium for culturing the stem cells. Additionally, stem cells can be cultured on a three-dimensional scaffold to promote osteogenic differentiation. Components constituting the scaffold may be made of biocompatible materials, and osteoblasts cultured and differentiated on the scaffold may be transplanted into a living body while attached to the scaffold for the treatment of bone diseases without being separated from the scaffold.
본 발명의 일 구현예에 따르면, 상기 줄기세포 오르가노이드의 골분화 촉진 방법으로 배양되는 줄기세포는 성체줄기세포 및 각종 분화 세포를 초기화시킨 유도 다능성 줄기세포(ips 세포)를 포함할 수 있으나, 이에 한정되지 않는다. 바람직하게는 성체줄기세포일 수 있고, 더욱 바람직하게는 치은(gingiva) 유래 중간엽 줄기세포일 수 있다.According to one embodiment of the present invention, the stem cells cultured by the method of promoting osteogenic differentiation of the stem cell organoid may include adult stem cells and induced pluripotent stem cells (ips cells) that have initialized various differentiated cells. It is not limited to this. Preferably, it may be an adult stem cell, and more preferably, it may be a gingiva-derived mesenchymal stem cell.
본 발명의 일 구현예에 따르면, 상기 줄기세포 오르가노이드의 골분화 촉진 방법에 포함되는 전환성장인자 베타1이 0.0001 내지 1000 ng/ml의 함량으로 포함될 수 있고, 바람직하게는 0.0005 내지 500 ng/ml의 함량으로 포함될 수 있으며, 더욱 바람직하게는 0.001 내지 200 ng/ml의 함량으로 포함될 수 있으나, 이에 한정하지 아니한다.According to one embodiment of the present invention, transforming growth factor beta 1 included in the method for promoting osteogenic differentiation of stem cell organoids may be included in an amount of 0.0001 to 1000 ng/ml, preferably 0.0005 to 500 ng/ml. It may be included in an amount of 0.001 to 200 ng/ml, but is not limited thereto.
본 발명의 또 다른 양태에 따르면, 본 발명의 줄기세포 오르가노이드의 골분화 촉진용 조성물로 분화된 조골세포를 유효성분으로 포함하는 골결손증 예방 또는 치료용 조성물을 제공한다.According to another aspect of the present invention, there is provided a composition for preventing or treating bone defects containing osteoblasts differentiated with the composition for promoting osteogenic differentiation of stem cell organoids of the present invention as an active ingredient.
본 발명에서 “골결손증”은 골분화 및 재생을 필요로 하는 골에 관련된 질환을 의미하며, 골절 등 외상성 골손상 및 수술 후 발생한 골결손을 포함한다.In the present invention, “bone defect” refers to a bone-related disease that requires bone differentiation and regeneration, and includes traumatic bone damage such as fractures and bone defects that occur after surgery.
본 발명의 분화된 연골세포는 각종 세포 치료에 이용될 수 있다. 분화된 조골세포는 콜라겐이나 히드록시아파타이트 스캐폴드 등 다양한 생체보조제와 혼합하여 외과수술로 골조직에 이식할 수 있다.The differentiated chondrocytes of the present invention can be used in various cell treatments. Differentiated osteoblasts can be mixed with various bioauxiliaries such as collagen or hydroxyapatite scaffolds and transplanted into bone tissue through surgery.
본 발명에 따른 골결손증 예방 또는 치료용 조성물은 상기 분화된 조골세포 외에 보조성분으로서 콜라겐이나 골지지체 등을 더 함유할 수 있다.The composition for preventing or treating bone defects according to the present invention may further contain collagen or bone support as auxiliary ingredients in addition to the differentiated osteoblasts.
본 발명에 따른 골결손증 예방 또는 치료용 조성물 중 분화된 연골세포는 질병의 유형, 투여경로, 환자의 나이 및 성, 및 질병의 정도에 따라 수를 조절할 수 있고, 바람직하게는 성인의 경우 1~5×107 cell/cm3 스캐폴드로, 1회 또는 수회로 나누어 투여할 수 있다.The number of differentiated chondrocytes in the composition for preventing or treating bone defects according to the present invention can be adjusted according to the type of disease, route of administration, age and sex of the patient, and severity of the disease, and is preferably 1 to 1 for adults. It is a 5×10 7 cell/cm 3 scaffold and can be administered once or in multiple doses.
본 발명의 조성물에 있어서, 상기 치료용 조성물은 골질환 치료를 위해 사용되는 것을 특징으로 한다.In the composition of the present invention, the therapeutic composition is used for treating bone diseases.
본 발명에서 상기 용어 “골질환”이란 골분화 및 골재생을 필요로 하는 골에 관련된 질환을 의미하며, 골절등 외상성 골손상 및 수술 후 발생한 골결손을 포함한다.In the present invention, the term “bone disease” refers to a bone-related disease that requires bone differentiation and bone regeneration, and includes traumatic bone damage such as fractures and bone defects occurring after surgery.
본 발명에 따른 전환성장인자 베타1을 유효성분으로 포함하는 줄기세포 오르가노이드의 골분화 촉진용 조성물은 줄기세포를 연골세포로 촉진을 유도시키는 효과가 현저하여, 줄기세포를 조골세포로 유도하고자 할 때 유용하며, 유도된 조골세포를 치과 영역을 비롯한 조골세포가 필요한 다양한 재생의료시장에 적용이 가능하므로 그 활용도가 높은 이점이 있다.The composition for promoting osteogenic differentiation of stem cell organoids containing transforming growth factor beta 1 as an active ingredient according to the present invention has a remarkable effect of inducing stem cells to become chondrocytes, and thus can be used to induce stem cells into osteoblasts. It is useful for this purpose, and the induced osteoblasts can be applied to various regenerative medicine markets that require osteoblasts, including the dental field, so it has the advantage of being highly usable.
또한, 본 발명에 따른 전환성장인자 베타1을 유효성분으로 포함하는 줄기세포 오르가노이드의 골분화 촉진용 조성물을 이용하여 줄기세포의 조골세포로의 분화를 유도하여 골결손증의 예방 및 치료제로서 사용하게 할 수 있는 이점이 있다.In addition, the composition for promoting osteogenic differentiation of stem cell organoids containing transforming growth factor beta 1 according to the present invention as an active ingredient can be used to induce differentiation of stem cells into osteoblasts and to use it as a preventive and therapeutic agent for bone defects. There are benefits to doing this.
도 1은 골형성 배지에서 전환성장인자 베타1을 다양한 농도(1, 10 및 20ng/ml)로 처리한 후 배양 7일, 9일, 11일 및 14일자의 줄기세포 형태 변화를 도립 현미경을 사용하여 관찰한 이미지이다. 스케일바는 100 μm을 나타낸다(원래 배율 x 200).
도 2는 배양 7일, 9일, 11일 및 14일자의 줄기세포 오르가노이드의 직경을 나타낸 이미지이다. 전환성장인자 베타 1 처리 후 배양 7일차에 통계적으로 유의한 차이는 확인되지 않았다(P>0.05). 또한, 전환성장인자 베타 1 처리 후 배양 9일 및 11일 그룹에서 더 긴 배양한 그룹과 비교하여 통계적 유의차를 나태내지 않았다(P>0.05).
도 3은 배양 14일자의 CCK-8 분석에 의한 세포 생존도를 나타내는 그래프이다. 전환성장인자 베타1 처리는 세포 생존도에 유의한 변화를 일으키지 않았다(P>0.05).
도 4는 배양 7, 9, 11, 14일자의 알칼리성 포스파타제(alkaline phosphatase) 활성을 나타내는 그래프이다. 배양 14 일자의 전환성장인자 베타1을 처리한 그룹에서 통계적 유의차를 나타냈다(P<0.05).
도 5a는 배양 14 일자의 실시간 중합효소 연쇄반응에 의한 TGF-β mRNA 발현에 대한 정량분석을 나타내는 그래프이다. 전환성장인자 베타1을 0 ng/ml 처리한 그룹과 비교하여 통계적 유의차를 나타내지 않았다(P>0.05).
도 5b는 배양 14 일자의 실시간 중합효소 연쇄반응에 의한 RUNX2 mRNA 발현에 대한 정량분석을 나타내는 그래프이다. 전환성장인자 베타1을 0 ng/ml 처리한 그룹과 비교하여 통계적 유의차를 나타내지 않았다(P>0.05).
도 5c는 배양 14 일자의 실시간 중합효소 연쇄반응에 의한 OCN mRNA 발현에 대한 정량분석을 나타내는 그래프이다. 전환성장인자 베타1을 0 ng/ml 처리한 그룹과 비교하여 통계적 유의차를 나타내지 않았다(P>0.05).
도 5d는 배양 14 일자의 실시간 중합효소 연쇄반응에 의한 SOX9 mRNA 발현에 대한 정량분석을 나타내는 그래프이다. 전환성장인자 베타1을 0 ng/ml 처리한 그룹과 비교하여 통계적 유의차를 나타내지 않았다(P>0.05).
도 5e는 배양 14 일자의 실시간 중합효소 연쇄반응에 의한 COL1A1 mRNA 발현에 대한 정량분석을 나타내는 그래프이다. 전환성장인자 베타1을 0 ng/ml 처리한 그룹과 비교하여 통계적 유의차를 나타내지 않았다(P>0.05).Figure 1 shows changes in stem cell morphology on days 7, 9, 11, and 14 of culture after treatment with various concentrations (1, 10, and 20 ng/ml) of transforming growth factor beta 1 in osteogenic medium using an inverted microscope. This is the image observed. Scale bar represents 100 μm (original magnification × 200).
Figure 2 is an image showing the diameter of stem cell organoids on days 7, 9, 11, and 14 of culture. No statistically significant difference was identified on the 7th day of culture after treatment with transforming growth factor beta 1 (P>0.05). In addition, there was no statistically significant difference in the 9 and 11 day culture groups after treatment with transforming growth factor beta 1 compared to the longer culture group (P>0.05).
Figure 3 is a graph showing cell viability by CCK-8 analysis on day 14 of culture. Transforming growth factor beta1 treatment did not cause significant changes in cell viability (P>0.05).
Figure 4 is a graph showing alkaline phosphatase activity on days 7, 9, 11, and 14 of culture. There was a statistically significant difference in the group treated with transforming growth factor beta 1 on day 14 of culture (P<0.05).
Figure 5a is a graph showing quantitative analysis of TGF-β mRNA expression by real-time polymerase chain reaction on day 14 of culture. There was no statistically significant difference compared to the group treated with 0 ng/ml transforming growth factor beta 1 (P>0.05).
Figure 5b is a graph showing quantitative analysis of RUNX2 mRNA expression by real-time polymerase chain reaction on day 14 of culture. There was no statistically significant difference compared to the group treated with 0 ng/ml transforming growth factor beta 1 (P>0.05).
Figure 5c is a graph showing quantitative analysis of OCN mRNA expression by real-time polymerase chain reaction on day 14 of culture. There was no statistically significant difference compared to the group treated with 0 ng/ml transforming growth factor beta 1 (P>0.05).
Figure 5d is a graph showing quantitative analysis of SOX9 mRNA expression by real-time polymerase chain reaction on day 14 of culture. There was no statistically significant difference compared to the group treated with 0 ng/ml transforming growth factor beta 1 (P>0.05).
Figure 5e is a graph showing quantitative analysis of COL1A1 mRNA expression by real-time polymerase chain reaction on day 14 of culture. There was no statistically significant difference compared to the group treated with 0 ng/ml transforming growth factor beta 1 (P>0.05).
본 명세서에서 설명된 구체적인 실시예는 본 발명의 바람직한 구현예 또는 예시를 대표하는 의미이며, 이에 의해 본 발명의 범위가 한정되지는 않는다. 본 발명의 변형과 다른 용도가 본 명세서 특허청구범위에 기재된 발명의 범위로부터 벗어나지 않는다는 것은 당업자에게 명백하다.The specific embodiments described in this specification are meant to represent preferred embodiments or examples of the present invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that modifications and other uses of the present invention do not depart from the scope of the invention as set forth in the claims herein.
실시예 Example
실험 재료 및 방법Experimental materials and methods
1. 치은(gingiva) 유래 중간엽 줄기세포의 분리 및 배양1. Isolation and culture of gingiva-derived mesenchymal stem cells
가톨릭대학교 의과대학 서울성모병원의 임상시험연구심사위원회에서 승인받아, 치주치료 진행 중인 건강한 환자로부터 건강한 치은(gingiva) 조직을 얻었다. 상기 치은 조직은 즉시 100 U/㎖ 페니실린(시그마 알드리치, 미국) 및 100 ㎍/㎖ 스트렙토마이신(시그마 알드리치, 미국)이 포함된 살균된 인산완충식염수(PBS, Welgene)에 넣어 4℃에 두었다. 상기 치은 조직은 탈-상피화(de-epithelialize)하여 분쇄하였고, 콜라게나아제 Ⅳ(시그마 알드리치)로 소화시킨 후, 5% CO2의 습윤 배양기에서 37℃로 배양하였다. 배양 24시간 후, 부착되지 않은 세포는 씻어내고, α-MEM 배지를 기본으로 하는 배지로 교체하였다. 상기 배지는 15% 소태아혈청(fetal bovine serum, Gibco), 100 U/㎖ 페니실린, 100 ㎍/㎖ 스트렙토마이신, 200 mM L-글루타민(시그마 알드리치, 미국) 및 10 mM 아스코르브산 2-인산염(시그마 알드리치, 미국)를 포함한다. 그 이후, 2-3일 마다 배지를 갈아줌으로써, 인간 치은(gingiva) 유래 중간엽 줄기세포(성체줄기세포)를 확립하였다.With approval from the Clinical Trial Review Board of Seoul St. Mary's Hospital, Catholic University College of Medicine, healthy gingiva tissue was obtained from a healthy patient undergoing periodontal treatment. The gingival tissue was immediately placed in sterile phosphate buffered saline (PBS, Welgene) containing 100 U/ml penicillin (Sigma Aldrich, USA) and 100 μg/ml streptomycin (Sigma Aldrich, USA) and placed at 4°C. The gingival tissue was de-epithelialized, pulverized, digested with collagenase IV (Sigma Aldrich), and then cultured at 37°C in a humidified incubator with 5% CO 2 . After 24 hours of culture, non-attached cells were washed away and replaced with medium based on α-MEM medium. The medium contains 15% fetal bovine serum (Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin, 200 mM L-glutamine (Sigma Aldrich, USA), and 10 mM ascorbic acid 2-phosphate (Sigma Aldrich, USA). After that, by changing the medium every 2-3 days, mesenchymal stem cells (adult stem cells) derived from human gingiva were established.
상기 세포는 콜로니-형성 능력, 부착능(plastic adherence) 및 다양한 계통으로 분화능(골형성, 지방형성, 연골형성)과 같은 줄기세포 특성을 나타내었고, 또한 상기세포는 유동 세포 분석법에 의해 CD44, CD73, CD90 및 CD105는 발현하지만, CD14, CD45, CD34 및 CD19는 발현하지 않는 것이 확인되었다.The cells showed stem cell characteristics such as colony-forming ability, adhesive ability (plastic adherence), and differentiation ability into various lineages (osteogenesis, adipogenesis, chondrogenesis), and the cells also showed CD44, CD73, and CD44 cells by flow cytometry. , it was confirmed that CD90 and CD105 were expressed, but CD14, CD45, CD34, and CD19 were not expressed.
2. 치은 유래 중간엽 줄기세포를 이용한 세포 오르가노이드의 형성2. Formation of cell organoids using gingiva-derived mesenchymal stem cells
배양 7일, 9일, 11일 및 14일차에 가능한 한 많은 세포 배양 배지를 배양물에서 제거하였다. 이 후, 기저막 매트릭스(basement membrane matrix, Matrigel®) 부피의 2배 이상이 되는 미리 냉각된 세포 회수 용액(cell recovery solution, 제품 번호 354253, Corning)을 추가하였다. 다음으로 넓은 orifice 피펫 팁을 사용하여 부드럽게 위아래로 피펫팅하고 3차원 배양물을 손상시키지 않고 기저막 매트릭스(Matrigel®)를 부수었다. 4°C에서 약 20분 동안 세포 회수 용액(cell recovery solution, Corning)과 함께 배양물을 배양하였다. 현미경으로 배양물을 시각화하여 기저막 매트릭스(basement membrane matrix, Matrigel®)가 완전히 분해되고 3차원 배양물이 기저막 매트릭스(basement membrane matrix, Matrigel®)에서 떠 있는지 확인하였다. 세포회수액을 제거하고 다시 세포회수액을 도포하는 과정을 반복하였다. 3차원 배양에서 기저막 매트릭스(basement membrane matrix, Matrigel®)를 제거한 후 배양액을 간단히 원심분리하여 용액을 분리하였다. 그런 다음 세포 회수 용액(cell recovery solution, Corning)을 제거하고 차가운 인산완충식염수(LB 004-02, Welgene, 경상북도 경산시)로 배양액을 여러 번 세척하였다. 인산완충식염수(Welgene)를 제거한 후, 4℃에 보관되어 있던 Trizol 시약(TR 118, TRI Reagent®, Molecular Research Center, Inc., Cincinnati, OH, USA) 1ml를 즉시 첨가하고 약 2분 후, 피펫으로 시료를 채취하여 1.5ml 튜브에 담아 -80℃에서 보관하였다.On days 7, 9, 11, and 14 of culture, as much cell culture medium as possible was removed from the cultures. Afterwards, a pre-cooled cell recovery solution (product number 354253, Corning) in an amount more than twice the volume of the basement membrane matrix (Matrigel®) was added. Next, using a wide orifice pipette tip, gently pipet up and down to break the basement membrane matrix (Matrigel®) without damaging the 3D culture. Cultures were incubated with cell recovery solution (Corning) for approximately 20 minutes at 4°C. The culture was visualized under a microscope to confirm that the basement membrane matrix (Matrigel®) was completely decomposed and that the three-dimensional culture was floating on the basement membrane matrix (Matrigel®). The process of removing the cell recovery solution and applying the cell recovery solution again was repeated. After removing the basement membrane matrix (Matrigel®) from the 3D culture, the culture medium was briefly centrifuged to separate the solution. Then, the cell recovery solution (Corning) was removed, and the culture was washed several times with cold phosphate-buffered saline (LB 004-02, Welgene, Gyeongsan-si, Gyeongsangbuk-do). After removing the phosphate-buffered saline solution (Welgene), 1 ml of Trizol reagent (TR 118, TRI Reagent®, Molecular Research Center, Inc., Cincinnati, OH, USA) stored at 4°C was immediately added, and after about 2 minutes, pipetted. Samples were collected, placed in 1.5ml tubes, and stored at -80°C.
3. 세포 생존도의 정량적 측정3. Quantitative measurement of cell viability
배양 1, 4 및 7 일자에 줄기세포의 세포 생존도에 대한 정량분석을 위해, 수용성 테트라졸륨염(tetrazolium salt)을 기반으로 하는, Cell Counting Kit-8 (CK04-11, Dojindo, Tokyo, 일본) 분석 키트을 사용하여 생존도 평가를 수행했다. 테트라졸륨, 일나트륨염을 첨가한 후 줄기세포 오르가노이드를 37℃에서 60분 동안 배양하였다. 흡광도를 450 nm에서 측정되었다.For quantitative analysis of cell viability of stem cells on days 1, 4 and 7 of culture, Cell Counting Kit-8 (CK04-11, Dojindo, Tokyo, Japan), based on water-soluble tetrazolium salt. Survival assessment was performed using an assay kit. After adding tetrazolium and monosodium salt, the stem cell organoids were cultured at 37°C for 60 minutes. Absorbance was measured at 450 nm.
4. 알칼리 포스파타제 활성 수준(골분화능)의 평가4. Evaluation of alkaline phosphatase activity level (osteogenic ability)
줄기세포 오르가노이드는 골 형성 배지와 함께 5% CO2를 포함하는 37°C 가습 인큐베이터에서 배양하였다. 알칼리 포스파타제 활성 수준의 평가를 위해 시판되는 키트(AS-72146, SensoLyte® pNPP Alkaline Phosphatase Assay Kit, AnaSpec Inc., Freemont, CA, 미국)를 사용하였다. 파라-니트로페닐포스페이트(para-nitrophenylphosphate)를 기반으로 하는 비색 분석을 사용하여 알칼리 포스파타제의 활성을 평가하였다. 상등액을 p-니트로페닐포스페이트 기질(p-nitrophenylphosphate substrate)과 혼합하고 실온에서 30분 동안 배양하였다. 생성된 p-니트로페놀의 흡광도를 405 nm에서 분광광도법으로 측정하였다.Stem cell organoids were cultured in a humidified incubator at 37°C containing 5% CO 2 with osteogenic medium. A commercially available kit (AS-72146, SensoLyte® pNPP Alkaline Phosphatase Assay Kit, AnaSpec Inc., Freemont, CA, USA) was used to evaluate the level of alkaline phosphatase activity. The activity of alkaline phosphatase was assessed using a colorimetric assay based on para-nitrophenylphosphate. The supernatant was mixed with p-nitrophenylphosphate substrate and incubated at room temperature for 30 minutes. The absorbance of the produced p-nitrophenol was measured spectrophotometrically at 405 nm.
5. 정량적 실시간 중합효소 연쇄반응(qPCR)에 의한 TGF-β1, RUNX2, OCN, SOX9 및 COL1A1 mRNA의 total RNA 추출 및 정량화 평가5. Total RNA extraction and quantification evaluation of TGF-β1, RUNX2, OCN, SOX9, and COL1A1 mRNA by quantitative real-time polymerase chain reaction (qPCR)
total RNA 추출은 배양 14일차에 제조업체 지침에 따라 상업적으로 이용 가능한 키트(Thermo Fisher Scientific, Inc., Waltham, MA, 미국)를 사용하여 수행되었다.Total RNA extraction was performed on day 14 of culture using a commercially available kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions.
RNA 품질은 키트(RNA 6000 Nano Chip; Agilent Technologies)를 사용하여 bioanalyzer(Agilent 2100)로 평가하였고, RNA 양은 분광광도계(ND-2000, Thermo Fisher Scientific, Inc.)를 사용하여 260 nm 및 280 nm에서의 흡광도의 비율로 평가하였다.RNA quality was assessed by a bioanalyzer (Agilent 2100) using a kit (RNA 6000 Nano Chip; Agilent Technologies), and RNA quantity was assessed using a spectrophotometer (ND-2000, Thermo Fisher Scientific, Inc.) at 260 nm and 280 nm. It was evaluated by the ratio of absorbance.
RNA는 역전사효소(SuperScript II; Invitrogen, Carlsbad, CA, 미국)를 적용한 역전사 주형으로 사용되었다. mRNA 발현은 배양 14일차에 qPCR에 의해 검출되었다. GenBank를 사용하여 PCR용 센스(sense) 및 안티센스(antisense) 프라이머를 설계하였다. 프라이머의 염기서열은 하기 표 1에 기재하였다.RNA was used as a reverse transcription template applying reverse transcriptase (SuperScript II; Invitrogen, Carlsbad, CA, USA). mRNA expression was detected by qPCR on day 14 of culture. Sense and antisense primers for PCR were designed using GenBank. The base sequences of the primers are listed in Table 1 below.
명칭primer
designation
(수탁번호: NM_000660.7)TGF-β1
(Accession number: NM_000660.7)
(수탁번호: NM_001015051.3)RUNX2
(Accession number: NM_001015051.3)
(수탁번호: NM_199173.6)OCN
(Accession number: NM_199173.6)
(수탁번호: NM_000346.4)SOX9
(Accession number: NM_000346.4)
(수탁번호: NM_000088.4)COL1A1
(Accession number: NM_000088.4)
실험결과Experiment result
1. 줄기세포 오르가노이드 형태 평가1. Stem cell organoid morphology evaluation
줄기세포 오르가노이드에 전환성장인자 베타1을 1, 10 및 20ng/ml로 처리한 후, 배양 7일차에 배양된 오르가노이드의 형태를 도 1에 나타내었다. 줄기세포 오르가노이드는 배양 시간이 길수록 더 커졌으나 손상되지 않았다.After treating stem cell organoids with transforming growth factor beta 1 at 1, 10, and 20 ng/ml, the morphology of the cultured organoids on the 7th day of culture is shown in Figure 1. The longer the culture time, the larger the stem cell organoids became, but they remained intact.
배양 7일째에 전환성장인자 베타1의 최종농도 0, 1, 10 및 20ng/ml 처리에 따른 줄기세포 오르가노이드의 직경은 각각 113.2 ± 24.7, 129.7 ± 10.4, 142.2 ± 21.5 및 166.2 ± 42.3μm 이였다(P>0.05).On the 7th day of culture, the diameters of stem cell organoids treated with final concentrations of 0, 1, 10, and 20 ng/ml of transforming growth factor beta 1 were 113.2 ± 24.7, 129.7 ± 10.4, 142.2 ± 21.5, and 166.2 ± 42.3 μm, respectively ( P>0.05).
배양 14일째에 전환성장인자 베타1의 최종농도 0, 1, 10 및 20ng/ml 처리에 따른 줄기세포 오르가노이드의 직경은 각각 222.2 ± 9.6, 186.1 ± 4.8, 197.2 ± 9.6 및 211.1 ± 19.2μm 이였다(P<0.05). 이를 도 2에 나타내었다.On day 14 of culture, the diameters of stem cell organoids treated with final concentrations of transforming growth factor beta 1 at 0, 1, 10, and 20 ng/ml were 222.2 ± 9.6, 186.1 ± 4.8, 197.2 ± 9.6, and 211.1 ± 19.2 μm, respectively ( P<0.05). This is shown in Figure 2.
2. 세포 생존도 평가2. Cell viability assessment
배양 14일째의 세포 생존도에 대한 정량값을 도 3에 나타내었다. 전환성장인자 베타1의 최종농도 1, 10 및 20ng/ml 처리에 따른 상대값은 대조군을 100%로 간주할 때(100.0 % ± 9.6%), 각각 98.7% ± 5.6%, 101.5% ± 9.4% 및 105.5% ± 8.3% 이였다(P>0.05).Quantitative values for cell viability on day 14 of culture are shown in Figure 3. The relative values of transforming growth factor beta 1 at final concentrations of 1, 10, and 20 ng/ml were 98.7% ± 5.6%, 101.5% ± 9.4%, and 100.0% ± 9.6%, respectively, when considering the control group as 100% (100.0% ± 9.6%). It was 105.5% ± 8.3% (P>0.05).
3. 알칼리 포스파타제(Alkaline phosphatase, ALP) 활성(골분화능) 분석 평가3. Alkaline phosphatase (ALP) activity (osteogenic differentiation potential) analysis evaluation
배양 7일, 9일, 11일, 14일차에 전환성장인자 베타1로 처리된 알칼리 포스파타제(Alkaline phosphatase, ALP) 활성분석 결과를 도 4에 나타내었다.The results of alkaline phosphatase (ALP) activity analysis of cells treated with transforming growth factor beta 1 on days 7, 9, 11, and 14 of culture are shown in Figure 4.
배양 7일째에 전환성장인자 베타1의 처리농도 1, 10 및 20ng/ml 처리에 따른 상대값은 대조군을 100%로 간주할 때(100.0 % ± 26.7%), 각각 84.3% ± 5.7%, 132.2% ±17.3% 및 110.6% ± 3.3% 이였다(P<0.05).On the 7th day of culture, the relative values of transforming growth factor beta 1 at concentrations of 1, 10, and 20 ng/ml were 84.3% ± 5.7% and 132.2%, respectively, when considering the control group as 100% (100.0% ± 26.7%). ±17.3% and 110.6% ± 3.3% (P<0.05).
배양 14일째에 전환성장인자 베타1의 처리농도 1, 10 및 20ng/ml 처리에 따른 상대값은 대조군을 100%로 간주할 때(440.7% ± 10.9%), 각각 511.5% ± 9.0%, 489.4% ± 17.6% 및 495.7% ± 23.0% 이였다(P<0.05).On the 14th day of culture, the relative values of transforming growth factor beta 1 at concentrations of 1, 10, and 20 ng/ml were 511.5% ± 9.0% and 489.4%, respectively, when considering the control group as 100% (440.7% ± 10.9%). ± 17.6% and 495.7% ± 23.0% (P<0.05).
4. 정량적 실시간 중합효소 연쇄반응(qPCR)에 의한 TGF-β1, RUNX2, OCN, SOX9 및 COL1A1 mRNA의 total RNA 추출 및 정량화 평가4. Total RNA extraction and quantification evaluation of TGF-β1, RUNX2, OCN, SOX9, and COL1A1 mRNA by quantitative real-time polymerase chain reaction (qPCR)
배양 14일차에 전환성장인자 베타1로 처리된 TGF-β1, RUNX2, OCN, SOX9 및 COL1A1 mRNA의 total RNA 추출 및 정량화를 도 5a 내지 도 5e에 나타내었다.Total RNA extraction and quantification of TGF-β1, RUNX2, OCN, SOX9, and COL1A1 mRNA treated with transforming growth factor beta 1 on day 14 of culture are shown in Figures 5A to 5E.
배양 14일차에 정량적 실시간 중합 효소 연쇄 반응에 의하여 전환성장인자 베타 1의 처리농도 0, 1, 10 및 20ng/ml에 따라 각각 TGF-β1의 mRNA 수준이 1.001 ± 0.058, 1.384 ± 0.864, 1.761 ± 0.527 및 2.030 ± 0.382인 것으로 확인되었다(P>0.05).On the 14th day of culture, the mRNA levels of TGF-β1 were 1.001 ± 0.058, 1.384 ± 0.864, and 1.761 ± 0.527 depending on the treatment concentration of transforming growth factor beta 1 at 0, 1, 10, and 20 ng/ml, respectively, by quantitative real-time polymerase chain reaction. and 2.030 ± 0.382 (P>0.05).
배양 14일차에 정량적 실시간 중합 효소 연쇄 반응에 의하여 전환성장인자 베타 1의 처리농도 0, 1, 10 및 20ng/ml에 따라 각각 RUNX2의 mRNA 수준이 1.044 ± 0.394, 1.504 ± 0.520, 1.294 ± 0.527 및 0.755 ± 0.212인 것으로 확인되었다(P>0.05).On the 14th day of culture, the mRNA levels of RUNX2 were 1.044 ± 0.394, 1.504 ± 0.520, 1.294 ± 0.527, and 0.755 depending on the treatment concentration of transforming growth factor beta 1 at 0, 1, 10, and 20 ng/ml, respectively, by quantitative real-time polymerase chain reaction. It was confirmed to be ± 0.212 (P>0.05).
배양 14일차에 정량적 실시간 중합 효소 연쇄 반응에 의하여 전환성장인자 베타 1의 처리농도 0, 1, 10 및 20ng/ml에 따라 각각 OCN의 mRNA 수준이 1.004 ± 0.118, 2.502 ± 1.191, 1.286 ± 0.475 및 0.974 ± 0.132인 것으로 확인되었다(P>0.05).On the 14th day of culture, the mRNA levels of OCN were 1.004 ± 0.118, 2.502 ± 1.191, 1.286 ± 0.475, and 0.974 depending on the treatment concentration of transforming growth factor beta 1 at 0, 1, 10, and 20 ng/ml, respectively, by quantitative real-time polymerase chain reaction. It was confirmed to be ± 0.132 (P>0.05).
배양 14일차에 정량적 실시간 중합 효소 연쇄 반응에 의하여 전환성장인자 베타 1의 처리농도 0, 1, 10 및 20ng/ml에 따라 각각 SOX9의 mRNA 수준이 1.053 ± 0.387, 1.666 ± 0.470, 1.228 ± 0.603 및 1.220 ± 0.297인 것으로 확인되었다(P>0.05).By quantitative real-time polymerase chain reaction on the 14th day of culture, the mRNA levels of SOX9 were 1.053 ± 0.387, 1.666 ± 0.470, 1.228 ± 0.603, and 1.220 at treatment concentrations of transforming growth factor beta 1 of 0, 1, 10, and 20 ng/ml, respectively. It was confirmed to be ± 0.297 (P>0.05).
배양 14일차에 정량적 실시간 중합 효소 연쇄 반응에 의하여 전환성장인자 베타 1의 처리농도 0, 1, 10 및 20ng/ml에 따라 각각 COL1A1의 mRNA 수준이 1.001 ± 0.050, 1.656 ± 0.218, 1.509 ± 0.043 및 1.167 ± 0.177인 것으로 확인되었다(P>0.05).On the 14th day of culture, the mRNA levels of COL1A1 were 1.001 ± 0.050, 1.656 ± 0.218, 1.509 ± 0.043, and 1.167 depending on the treatment concentration of transforming growth factor beta 1 at 0, 1, 10, and 20 ng/ml, respectively, by quantitative real-time polymerase chain reaction. It was confirmed to be ± 0.177 (P>0.05).
<110> The Catholic University of Korea Industry-Academic Cooperation Foundation <120> Composition for Facilitating the Differentiation of Stem Cell Organoids Comprising Transforming Growth Factor-b1 as an Effective Ingredient <130> DPC220232 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward PCR primer for TGF-b1 <400> 1 gagcctgagg ccgactacta 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse PCR primer for TGF-b1 <400> 2 agatttcgtt gtgggtttcc 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward PCR primer for RUNX2 <400> 3 cagttcccaa gcatttcatc c 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse PCR primer for RUNX2 <400> 4 aggtggctgg atagtgcatt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward PCR primer for OCN <400> 5 ggtgcagagt ccagcaaagg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse PCR primer for OCN <400> 6 gcgcctgggt ctcttcacta 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward PCR primer for SOX9 <400> 7 ctgggaacaa cccgtctaca 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse PCR primer for SOX9 <400> 8 ggatcatctc ggccatcttc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward PCR primer for COL1A1 <400> 9 taccccactc agcccagtgt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse PCR primer for COL1A1 <400> 10 ccgaaccaga catgcctctt 20 <110> The Catholic University of Korea Industry-Academic Cooperation Foundation <120> Composition for Facilitating the Differentiation of Stem Cell Organoids Comprising Transforming Growth Factor-b1 as an Effective Ingredient <130>DPC220232 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward PCR primer for TGF-b1 <400> 1 gagcctgagg ccgactacta 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse PCR primer for TGF-b1 <400> 2 agatttcgtt gtgggtttcc 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward PCR primer for RUNX2 <400> 3 cagttcccaa gcatttcatc c 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse PCR primer for RUNX2 <400> 4 aggtggctgg atagtgcatt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward PCR primer for OCN <400> 5 ggtgcagagt ccagcaaagg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse PCR primer for OCN <400> 6 gcgcctgggt ctcttcacta 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward PCR primer for SOX9 <400> 7 ctgggaacaa cccgtctaca 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse PCR primer for SOX9 <400> 8 ggatcatctc ggccatcttc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward PCR primer for COL1A1 <400> 9 taccccactc agcccagtgt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse PCR primer for COL1A1 <400> 10 ccgaaccaga catgcctctt 20
Claims (15)
상기 줄기세포는 성체줄기세포인 것을 특징으로 하는 줄기세포 오르가노이드의 골분화 촉진용 조성물.According to paragraph 1,
A composition for promoting osteogenic differentiation of stem cell organoids, wherein the stem cells are adult stem cells.
상기 성체줄기세포는 치은(gingiva) 유래 중간엽 줄기세포인 것을 특징으로 하는 줄기세포 오르가노이드의 골분화 촉진용 조성물.According to paragraph 2,
A composition for promoting osteogenic differentiation of stem cell organoids, wherein the adult stem cells are gingiva-derived mesenchymal stem cells.
상기 골분화는 줄기세포가 조골세포로 분화하는 것을 특징으로 하는 줄기세포 오르가노이드의 골분화 촉진용 조성물.According to paragraph 1,
A composition for promoting osteogenic differentiation of stem cell organoids, wherein the osteogenic differentiation occurs when stem cells differentiate into osteoblasts.
상기 전환성장인자 베타1는 0.0001 내지 1000 ng/ml의 함량으로 포함되는 것을 특징으로 하는 줄기세포 오르가노이드의 골분화 촉진용 조성물.According to paragraph 1,
A composition for promoting osteogenic differentiation of stem cell organoids, wherein the transforming growth factor beta 1 is contained in an amount of 0.0001 to 1000 ng/ml.
상기 줄기세포는 성체줄기세포인 것을 특징으로 하는 줄기세포 배양용 배지 According to clause 6,
A medium for stem cell culture, wherein the stem cells are adult stem cells.
상기 성체줄기세포는 치은(gingiva) 유래 중간엽 줄기세포인 것을 특징으로 하는 줄기세포 배양용 배지.In clause 7,
A medium for stem cell culture, wherein the adult stem cells are gingiva-derived mesenchymal stem cells.
상기 전환성장인자 베타1는 0.0001 내지 1000 ng/ml의 함량으로 포함되는 것을 특징으로 하는 줄기세포 배양용 배지.According to clause 6,
A medium for stem cell culture, characterized in that the transforming growth factor beta 1 is contained in an amount of 0.0001 to 1000 ng/ml.
상기 줄기세포는 성체줄기세포인 것을 특징으로 하는 줄기세포 오르가노이드의 골분화 촉진 방법.According to clause 10,
A method of promoting osteogenic differentiation of stem cell organoids, wherein the stem cells are adult stem cells.
상기 성체줄기세포는 치은(gingiva) 유래 중간엽 줄기세포인 것을 특징으로 하는 줄기세포 오르가노이드의 골분화 촉진 방법.According to clause 11,
A method of promoting osteogenic differentiation of stem cell organoids, wherein the adult stem cells are gingiva-derived mesenchymal stem cells.
상기 전환성장인자 베타1는 0.0001 내지 1000 ng/ml의 함량으로 포함되는 것을 특징으로 하는 줄기세포 오르가노이드의 골분화 촉진 방법.According to clause 10,
A method for promoting osteogenic differentiation of stem cell organoids, wherein the transforming growth factor beta 1 is included in an amount of 0.0001 to 1000 ng/ml.
상기 줄기세포 오르가노이드는 오목한 표면에서 배양하는 것을 특징으로 하는 줄기세포 오르가노이드의 골분화 촉진 방법.According to clause 10,
A method of promoting osteogenic differentiation of stem cell organoids, characterized in that the stem cell organoids are cultured on a concave surface.
A composition for preventing or treating bone defects comprising osteoblasts differentiated according to any one of claims 10 to 14 as an active ingredient.
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