KR101193541B1 - Method of inducing differentiation of human embryonic stem cells into mesenchymal stem cells - Google Patents

Method of inducing differentiation of human embryonic stem cells into mesenchymal stem cells Download PDF

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KR101193541B1
KR101193541B1 KR1020090078088A KR20090078088A KR101193541B1 KR 101193541 B1 KR101193541 B1 KR 101193541B1 KR 1020090078088 A KR1020090078088 A KR 1020090078088A KR 20090078088 A KR20090078088 A KR 20090078088A KR 101193541 B1 KR101193541 B1 KR 101193541B1
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stem cells
mesenchymal stem
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hescs
mesoderm
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한용만
전납둥
박상욱
장미진
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한국과학기술원
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Abstract

본 발명은 조성물이 최적화된 배지에서 배양함으로써 인간 배아줄기세포를 중간엽 줄기세포로 분화시키는 방법에 관한 것으로, 구체적으로 TGF-β(Transforming growth factor-β), WNT 및 BMP(Bone morphogenic protein) 신호전달 경로를 활성화함으로써 중배엽을 유도한 후, bFGF 및/또는 PDGF-BB(Platelet-derived growth factor beta polypeptide)의 처리에 의해 중간엽 줄기세포를 제조하는 방법에 관한 것이다. 본 발명의 방법은 세포기반 치료에 유용한 중간엽 줄기세포를 대량으로 제공하는 데에 유용하게 이용될 수 있다.The present invention relates to a method for differentiating human embryonic stem cells into mesenchymal stem cells by culturing the composition in an optimized medium, specifically TGF-β (Transforming growth factor-β), WNT and BMP (Bone morphogenic protein) signal The present invention relates to a method for producing mesenchymal stem cells by treatment of bFGF and / or platelet-derived growth factor beta polypeptide (PDGF-BB) after inducing mesoderm by activating a delivery pathway. The method of the present invention can be usefully used to provide a large amount of mesenchymal stem cells useful for cell-based therapy.

중간엽 줄기세포, 중배엽, 인간 배아줄기세포, TGF-β(Transforming growth factor-β), WNT, BMP(Bone morphogenic protein), bFGF, PDGF-BB(Platelet-derived growth factor beta polypeptide)Mesenchymal stem cells, mesoderm, human embryonic stem cells, transforming growth factor-β (TGF-β), WNT, bone morphogenic protein (BMP), bFGF, and platelet-derived growth factor beta polypeptide (PDGF-BB)

Description

인간 배아줄기세포에서 중간엽 줄기세포로 분화시키는 방법{Method of inducing differentiation of human embryonic stem cells into mesenchymal stem cells}Method of inducing differentiation of human embryonic stem cells into mesenchymal stem cells}

본 발명은 조성물이 최적화된 배지에서 배양함으로써 인간 배아줄기세포를 중간엽 줄기세포로 분화시키는 방법에 관한 것이다.The present invention relates to a method for differentiating human embryonic stem cells into mesenchymal stem cells by culturing the composition in an optimized medium.

Friendenstein 연구단(Exp Hematol, 4(5):267-274, 1976)에 의해 골수에서 처음 확인된 중간엽줄기세포(Mesenchymal stem cells; MSCs)는 전능성 세포로, 재생 의학에서 큰 잠재성을 가진다. 상기 MSCs는 생체내 여러 유형의 중간엽 계통, 예를 들어 골세포(Osteocytes), 연골세포(Chondrocytes), 힘줄세포(tendinocytes), 지방세포(Adipocytes), 근육세포(Myocytes), 및 섬유아세포(Fibroblasts) 등으로 분화될 수 있다. 또한, 적절한 배지 조건에서는 신경 세포(Hung SC et al ., Stem Cells, 20(6):522-529, 2002; Sanchez-Ramos J et al ., Exp Neurol, 164(2):247-256, 2000), 심근세포(Kadivar M et al ., Biochem Biophys Res Commun, 340(2):639-647, 2006), 내피세포(Reyes M et al ., Blood, 98(9):2615-2625, 2001), 및 간세포(Kang XQ et al ., World J Gastroenterol, 11(47):7461-7465, 2005)로 교차 분화될 수 있다. 게다가 골수 MSCs는 클래스 II가 아닌 클래스 I MHC 항원을 발현하거나, MSCs가 면역원성 활성이 없는 것을 나타내는 상호 자극 분자들을 발현한다(Klyushnenkova E et al ., J Biomed Sci, 12(1):47-57, 2005). MSCs는 면역억제 활성을 나타내므로, 이식 촉진제 및 태아 이식(fatal graft) 및 숙주 질환의 억제제로 사용될 수 있다(Le Blanc K et al ., Lancet, 363(9419):1439-1441, 2004; El-Badri N.S et al ., Exp Hematol, 26(2):110-116, 1998).Friendenstein Research (Exp Mesenchymal stem cells (MSCs) first identified in bone marrow by Hematol , 4 (5): 267-274, 1976) are omnipotent and have great potential in regenerative medicine. The MSCs can be derived from several types of mesenchymal lineages in vivo, such as osteoblasts, chondrocytes, tendon cells, adipocytes, myocytes, and fibroblasts. ) And the like. In addition, under appropriate media conditions, neurons (Hung SC et al . , Stem Cells , 20 (6): 522-529, 2002; Sanchez-Ramos J et al . , Exp Neurol , 164 (2): 247-256, 2000), cardiomyocytes (Kadivar M et. al . , Biochem Biophys Res Commun , 340 (2): 639-647, 2006), endothelial cells (Reyes M et. al . , Blood , 98 (9): 2615-2625, 2001), and liver cells (Kang XQ et al . , World J Gastroenterol , 11 (47): 7461-7465, 2005). In addition, bone marrow MSCs express class I MHC antigens, but not class II, or express stimulatory molecules that indicate that MSCs lack immunogenic activity (Klyushnenkova E et. al . , J Biomed Sci , 12 (1): 47-57, 2005). Because MSCs exhibit immunosuppressive activity, they can be used as transplant promoters and inhibitors of fetal grafts and host diseases (Le Blanc K et. al . Lancet , 363 (9419): 1439-1441, 2004; El-Badri NS et al . , Exp Hematol , 26 (2): 110-116, 1998).

MSCs는 골수, 지방 조직, 제대혈(cord blood), 말초혈, 신생아 조직(neonatal tissues), 인간 태반 등의 다양한 성인 조직으로부터 분리될 수 있다(Kassis I et al ., Bone Marrow Transplant, 37(10):967-976, 2006; Wang HS et al ., Stem Cells, 22(7):1330-1337, 2004; Fukuchi Y et al ., Stem Cells, 22(5):649-658, 2004). 그러나 성인 조직에서 수득한 MSCs의 개수에는 한계가 있고, 공여자로부터 MSCs를 분리하기 위해서는 침습적인 절차가 요구되어, 공여자에게 예상하지 못한 위험을 끼칠 수 있다. 따라서 배아줄기세포(Embryonic Stem cells; ES 세포) 유래의 MSCs를 이용하는 것이 세포기반 치료시 세포를 제공하기 위한 하나의 대안이다. 게다가 인간 배아줄기세포(hESCs) 유래의 MSCs의 숙주 면역 거부반응은 hESC 라인의 뱅크가 충분히 클 경우, 핵전이, iPS(induced pluripotent stem) 세포주 또는 면역 호환성 동종이계 hESC의 이용에 의해서 극복 할 수도 있다. 현재까지 인간 ESC로부터 MSCs를 생성하기 위해 여러 가지 방법들이 독립적으로 개발되어 왔다. 예를 들면, Barberi T 연구단은 OP9 세포주를 영양 세포층으로 이용하여 hESC와 공동 배양함으로써 인간 MSCs를 제조하였으나, 시간이 상당히 소요되었고 효율도 낮았다(Barberi T et al ., PLoS Med, 2(6):e161, 2005). 다른 연구단은 10% FBS를 포함하는 DMEM 배지가 담긴 용기의 중앙 또는 모서리 부근의 hESC로부터 분화된 세포를 배양 및 수집함으로써 인간 MSCs를 개발하였다(Olivier EN et al ., Stem Cells, 24(8):1914-1922, 2006). 상기 방법은 영양세포층을 필요로 하지 않으나, 제조된 세포에서 여전히 ES 세포에서 발현하는 SSEA-4(Stage-specific embryonic antigen 4)를 발현함으로써, 상기 MSC가 여전히 비분화된 세포의 특성을 유지하는 것을 나타내었다. Pike & Shevde도 중간엽 특이적 배지에서 10 내지 12일 동안 배상체(embryoid body)를 배양함으로써 MSCs를 유도하고자 노력하였으나, 이전의 연구단과 동일한 문제에 직면하였다(Trivedi P & Hematti P, Exp Hematol, 36(3):350-359, 2008). 즉, 유도된 MSCs에서 여전히 전능성 마커인 Oct4 유전자가 발현되었다. Qizhou 연구단은 CD105 양성 및 CD24 음성으로 분화된 hESC로부터 임상적으로 호환성인 MSCs를 제조하였다(Lian Q et al ., Stem Cells, 25(2):425-436, 2007). 상기 연구에서 hESC를 젤라틴으로 코팅된 접시에서, bFGF(basic fibroblast growth factor) 및 PDGF-AB(Platelet Derived Growth Factor-AB)를 포함하는 배지에서 배양하였다. CD105 양성 및 CD24 음성 세포의 개체군은 FACS Aria에 의해 배열되었다. 상기 세포는 골수 유래 MSCs에서 발현하는 대부분의 유전자 마커를 발현하였고, 지방세포, 연골세포 및 골세포로 분화 될 수 있었다. 상기 연구는 이전의 한계점을 거의 해결하였으나, 전체 세포군으로부터 대략 5%의 CD105+, CD24- 세포만 형성되었다.MSCs can be isolated from various adult tissues such as bone marrow, adipose tissue, cord blood, peripheral blood, neonatal tissues, human placenta (Kassis I et al. al . , Bone Marrow Transplant , 37 (10): 967-976, 2006; Wang HS et al . , Stem Cells , 22 (7): 1330-1337, 2004; Fukuchi Y et al . , Stem Cells , 22 (5): 649-658, 2004). However, the number of MSCs obtained from adult tissues is limited, and invasive procedures are required to isolate MSCs from donors, which may pose unexpected risks to donors. Therefore, the use of MSCs derived from embryonic stem cells (ES cells) is one alternative to provide cells for cell-based treatment. In addition, host immune rejection of MSCs derived from human embryonic stem cells (hESCs) can be overcome by the use of nuclear transfer, induced pluripotent stem (iPS) cell lines or immune compatible allogeneic hESCs if the banks of the hESC lines are large enough. . To date, several methods have been developed independently to generate MSCs from human ESCs. For example, the Barberi T research group produced human MSCs by co-culturing with hESCs using OP9 cell lines as feeder cell layers, but it was very time consuming and inefficient (Barberi T et. al . , PLoS Med , 2 (6): e161, 2005). Other groups have developed human MSCs by culturing and collecting differentiated cells from hESCs near the center or corner of a vessel containing DMEM medium containing 10% FBS (Olivier EN et. al . , Stem Cells , 24 (8): 1914-1922, 2006). The method does not require a feeder cell layer, but by expressing the stage-specific embryonic antigen 4 (SSEA-4), which is still expressed in ES cells in the prepared cells, it is found that the MSC still retains the properties of undifferentiated cells. Indicated. Pike & Shevde also sought to induce MSCs by incubating embryoid bodies for 10-12 days in mesenchymal specific media, but faced the same problem as previous studies (Trivedi P & Hematti P, Exp Hematol , 36 (3): 350-359, 2008). That is, the Oct4 gene, which is still an omnipotent marker, was expressed in the induced MSCs. The Qizhou Research Group produced clinically compatible MSCs from hESCs differentiated into CD105 positive and CD24 negative (Lian Q et. al . , S tem Cells, 25 (2 ): 425-436, 2007). In this study hESCs were incubated in gelatin-coated dishes in a medium containing basic fibroblast growth factor (bFGF) and Platelet Derived Growth Factor-AB (PDGF-AB). Populations of CD105 positive and CD24 negative cells were arranged by FACS Aria. The cells expressed most of the genetic markers expressed in bone marrow-derived MSCs and were able to differentiate into adipocytes, chondrocytes and osteocytes. The study nearly solved the previous limitations, but only approximately 5% of CD105 +, CD24− cells were formed from the entire cell population.

이에, 본 발명자들은 이전의 한계점을 극복하며, 높은 비율로 인간 배아줄기세포로부터 중간엽 줄기세포를 짧은 기간에 효과적으로 생성할 수 있는 방법을 개발하기 위해, 몇 가지 신호전달 경로를 조절함으로써 hESCs로부터 중배엽 세포를 거쳐 중간엽 줄기세포로 유도시켰다. 상기 hESCs 유래 중간엽 줄기세포는 공지의 분화유도제에 의해 지방세포 또는 골수세포로 성공적으로 분화함으로써, 본 발명의 방법이 세포기반 치료에 유용한 중간엽 줄기세포를 대량으로 제공하는 데에 유용하게 이용될 수 있음을 확인함으로써 본 발명을 완성하였다.Accordingly, the present inventors overcome the previous limitations, and in order to develop a method capable of effectively producing mesenchymal stem cells from human embryonic stem cells at high rates in a short period of time, by regulating several signaling pathways, the mesoderm from hESCs The cells were induced through mesenchymal stem cells. The hESCs-derived mesenchymal stem cells are successfully differentiated into adipocytes or bone marrow cells by known differentiation inducing agents, so that the method of the present invention can be usefully used to provide a large amount of mesenchymal stem cells useful for cell-based therapy. The present invention has been completed by confirming that it can.

본 발명의 목적은 배아줄기세포(Embryonic Stem Cells; 이하, ESCs) 유래 중배엽 분화 유도용 조성물을 제공하는 것이다.An object of the present invention is to provide a composition for inducing mesoderm differentiation derived from embryonic stem cells (Embryonic Stem Cells; hereinafter, ESCs).

본 발명의 목적은 ESCs 유래 중배엽 분화 방법을 제공하는 것이다.It is an object of the present invention to provide a method for mesoderm differentiation derived from ESCs.

본 발명의 다른 목적은 ESCs 유래 중배엽으로부터 중간엽 줄기세포 분화 방법을 제공하는 것이다.Another object of the present invention is to provide a method for differentiating mesenchymal stem cells from ESCs-derived mesoderm.

본 발명의 다른 목적은 ESCs 유래 중배엽 유도 배지를 제공하는 것이다.Another object of the present invention is to provide a mesoderm derived medium derived from ESCs.

상기 목적을 달성하기 위하여, 본 발명은 Activin A, 6-브로모인디루빈-3'-옥심(6-bromoindirubin-3'-oxime; 이하, BIO) 및 BMP4를 포함하는 배아줄기세포(Embryonic Stem Cells; 이하, ESCs) 유래 중배엽 분화 유도용 조성물을 제공한다.In order to achieve the above object, the present invention is an embryonic stem cell containing Activin A, 6-bromoindirubin-3'-oxime (hereinafter BIO) and BMP4 (Embryonic Stem Cells It provides a composition for inducing mesoderm differentiation derived from ESCs).

또한, 본 발명은 In addition,

1) 영양 세포층이 있는 조건에서 배양된 ESCs를 마트리겔로 코팅된 배양 접시로 옮긴 후, 처리 배지(Conditioned Medium; CM)로 1 내지 3일 동안 배양하는 단계; 및,1) transferring ESCs cultured in the presence of a feeder cell layer to a culture dish coated with Matrigel, and then incubating for 1 to 3 days with Conditioned Medium (CM); And

2) 상기 영양 세포층이 없는 조건에서 배양된 ESCs를 Activin A, BIO 및 BMP4를 포함하는 중배엽 유도 배지로 3 내지 5일 동안 유도하는 단계를 포함하는 hESCs 유래 중배엽 분화 방법을 제공한다.2) It provides a hESCs-derived mesoderm differentiation method comprising the step of inducing ESCs cultured in the absence of the feeder cell layer with mesoderm induction medium containing Activin A, BIO and BMP4 for 3 to 5 days.

또한, 본 발명은 상기 중배엽 분화 방법에 이어서, 상기 방법으로 제조된 ESCs 유래 중배엽을 bFGF 및/또는 PDGF-BB(Platelet-derived growth factor beta polypeptide)를 포함하는 중간엽 유도 배지로 3 내지 6일 동안 유도하는 단계를 포함하는 ESCs 유래 중배엽으로부터 중간엽 줄기세포 분화 방법을 제공한다.In addition, the present invention, following the mesoderm differentiation method, the ESCs-derived mesoderm prepared by the method for 3 to 6 days in the mesenchymal induction medium containing bFGF and / or platelet-derived growth factor beta polypeptide (PDGF-BB) It provides a method for differentiating mesenchymal stem cells from ESCs-derived mesoderm comprising the step of inducing.

아울러, 본 발명은 Activin A, BIO 및 BMP4를 포함하는 ESCs 유래 중배엽 유도 배지를 제공한다.In addition, the present invention provides ESCs-derived mesoderm induction medium comprising Activin A, BIO and BMP4.

이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명자들은 인간 배아줄기세포(human Embryonic Stem Cells; hESCs)로부터 중배엽 세포를 거쳐 중간엽 줄기세포로 유도시킨 후, 지방세포 또는 골수세포로 분화하고자 하였다(도 1 참조). 본 발명의 구체적인 실시예에서, 3개의 세포내 신호전달 경로인 TGF-β(Transforming growth factor-β), WNT 및 BMP(Bone morphogenic protein) 신호전달 경로의 기능을 조절함으로써, hESCs를 중배엽으로 유도하였다. 즉, Activin A, BIO(6-bromoindirubin-3'-oxime) 및 BMP4 등의 TGF-β, WNT 및 BMP의 신호전달 활성제로 조절한 결과, hESCs를 영양세포층이 없는 조건에서, 2-5 ng/㎖ Activin A, 2-5 mM BIO, 및 10-20 ng/㎖ BMP4를 포함하는 비처리 배지(Unconditioned Medium; UM)를 이용하여 3 내지 5일간 배양함으로써 중배엽으로 성공적으로 분화하였다. 상기 신호전달 중 하나의 활성유도에 의해서는 중배엽로 유도하는 것이 충분하지 않았으나, 이것들을 조합함으로써 시너지 효과에 의해 중배엽에서만 특이적으로 중배엽 특이적 유전자의 발현을 증가시켰다(도 2 및 도 4 참조). 또한, DKK1, Noggin 및 SB431542 등의 신호전달 억제제에 의해서는 상기 중배엽 특이적 유전자의 발현을 유의하게 감소시켰다(도 3 참조).The present inventors attempt to induce into mesenchymal stem cells from human Embryonic Stem Cells (hESCs) via mesodermal cells, and then differentiate into adipocytes or bone marrow cells (see FIG. 1). In a specific embodiment of the present invention, hESCs were induced into mesoderm by regulating the functions of three intracellular signaling pathways, TGF-β (TGF-β), WNT and BMP (Bone morphogenic protein) signaling pathways. . In other words, TGF-β, WNT and BMP such as Activin A, BIO (6-bromoindirubin-3'-oxime) and BMP4 were regulated by signaling activators, resulting in hESCs 2-5 ng / Differentiation was successfully differentiated into mesoderm by incubation for 3 to 5 days using unconditioned medium (UM) containing ml Activin A, 2-5 mM BIO, and 10-20 ng / ml BMP4. Induction of mesoderm was not sufficient due to the activation of one of the signaling, but by combining them, the mesenchymal-specific gene expression was specifically increased only in the mesoderm by synergistic effects (see FIGS. 2 and 4). . In addition, signaling inhibitors such as DKK1, Noggin and SB431542 significantly reduced the expression of the mesoderm specific genes (see FIG. 3).

또한, 본 발명의 구체적인 실시예에서는, 상기 hESCs 유래 중배엽의 bFGF 및 PDGF 신호전달 경로의 조절에 의해 CD105 양성 세포로 연속적으로 분화되었다. 즉, bFGF 및/또는 PDGF-BB(Platelet-derived growth factor beta polypeptide)의 처리에 의해 중간엽 줄기세포 특이적 유전자의 발현을 확인하였다(도 5 참조). 이때, 20 ng/㎖ bFGF 및 20 ng/㎖ PDGF-BB의 조합이 CD105 양성 세포의 유도에 가장 최적인 조건이었다. 상기 hESCs 유래 중배엽은 상기 조건에서 배양함으로써, 시간이 지남에 따라 CD105 양성 세포로 분화되었고, 6일 배양 후에 CD105 양성 세포로의 분화가 최고지점(대략 36%)에 도달하였다. 상기 CD105 양성 세포를 자성활성 세포 분류기(magnetic-activated cell sorting, MACS)를 이용하여 분리한 후, 유전자 발현 양상을 확인한 결과 중간엽 특이적 유전자 등을 발현하였고, 음성 마커 유전자들은 발현하지 못하였다(도 7 참조).In a specific embodiment of the present invention, the hESCs-derived mesoderm were continuously differentiated into CD105 positive cells by regulation of the bFGF and PDGF signaling pathways. That is, the expression of mesenchymal stem cell-specific genes was confirmed by treatment of bFGF and / or platelet-derived growth factor beta polypeptide (PDGF-BB) (see FIG. 5). At this time, the combination of 20 ng / ml bFGF and 20 ng / ml PDGF-BB was the most optimal condition for induction of CD105 positive cells. The hESCs-derived mesoderm were differentiated into CD105 positive cells over time by culturing in the above conditions, and after 6 days of culture, the differentiation into CD105 positive cells reached the highest point (approximately 36%). The CD105 positive cells were isolated using magnetic-activated cell sorting (MACS), and then the expression patterns of the genes were expressed, and the mesenchymal-specific genes were expressed. See FIG. 7).

본 발명의 구체적인 실시예에서, 상기 분리된 CD105 양성 세포는 공지의 지방세포 및 골아세포 분화유도제를 처리함으로써, 지방세포 및 골아세포로 성공적으로 분화되었다. 즉, 상기 방법으로 제조된 지방세포에서 2주 후에는 지질 액포가 고농도로 거의 모든 유도된 세포에서 나타났고(도 8a 참조), 지방세포 특이적인 FABP-4(fatty acid binding protein-4) 단백질의 존재를 확인하였으며(도 8b 참조), FABP-4 및 PPARg2(peroxisome proliferator-activated receptor-g2)의 지방세포 특이적 유전자의 발현이 유도된 것으로 나타났다(도 8c 참조).In a specific embodiment of the present invention, the isolated CD105 positive cells were successfully differentiated into adipocytes and osteoblasts by treatment with known adipocytes and osteoblast differentiation agents. That is, after two weeks in the adipocytes prepared by the above method, lipid vacuoles appeared in almost all induced cells at high concentration (see FIG. 8A), and the fat cell-specific FABP-4 (fatty acid binding protein-4) protein The presence was confirmed (see FIG. 8B) and the expression of adipocyte specific genes of FABP-4 and peroxisome proliferator-activated receptor-g2 (PPARg2) was induced (see FIG. 8C).

본 발명은 Activin A, 6-브로모인디루빈-3'-옥심(6-bromoindirubin-3'-oxime; 이하, BIO) 및 BMP4를 포함하는 배아줄기세포(Embryonic Stem Cells; 이하, ESCs) 유래 중배엽 분화 유도용 조성물을 제공한다.The present invention relates to mesoderm derived from Embryonic Stem Cells (ESCs), including Activin A, 6-bromoindirubin-3'-oxime (hereinafter referred to as BIO) and BMP4. Provided is a composition for inducing differentiation.

Activin A, BIO 및 BMP4는 최종배지 내에 각각 2-5 ng/㎖, 2-5 mM 및 10-20 ng/㎖의 농도로 존재할 수 있도록 조성되고, 바람직하게는 5 ng/㎖, 2 mM 및 20 ng/㎖의 농도로 존재할 수 있도록 조성된다.Activin A, BIO and BMP4 are formulated to be present in final media at concentrations of 2-5 ng / ml, 2-5 mM and 10-20 ng / ml, respectively, preferably 5 ng / ml, 2 mM and 20 It is formulated to be present at a concentration of ng / ml.

상기 조성물은 중배엽을 분화를 위해 ESCs를 배양할 때 배양 배지에 혼합하여 사용할 수 있으며, 상기 Activin A, BIO 및 BMP4는 각각 상기 농도의 1 내지 100배로 농축된 스톡 용액(stock solution)의 상태로 제공되어, 상기 배양 배지에 희석하여 사용될 수 있다.The composition may be used by mixing the mesoderm in the culture medium when culturing the ESCs for differentiation, the Activin A, BIO and BMP4 is provided in the state of the stock solution concentrated to 1 to 100 times the concentration, respectively Can be diluted in the culture medium and used.

또한, 본 발명은 In addition,

1) 영양 세포층이 있는 조건에서 배양된 ESCs를 마트리겔로 코팅된 배양 접시로 옮긴 후, 처리 배지(Conditioned Medium; CM)로 1 내지 3일 동안 배양하는 단계; 및,1) transferring ESCs cultured in the presence of a feeder cell layer to a culture dish coated with Matrigel, and then incubating for 1 to 3 days with Conditioned Medium (CM); And

2) 상기 영양 세포층이 없는 조건에서 배양된 ESCs를 Activin A, BIO 및 BMP4를 포함하는 중배엽 유도 배지로 3 내지 5일 동안 유도하는 단계를 포함하는 hESCs 유래 중배엽 분화 방법을 제공한다.2) It provides a hESCs-derived mesoderm differentiation method comprising the step of inducing ESCs cultured in the absence of the feeder cell layer with mesoderm induction medium containing Activin A, BIO and BMP4 for 3 to 5 days.

배아줄기세포는 영양 세포층(feeder layer)을 기반으로 하여 체외에서 장기간 미분화 상태로 배양될 수 있으며, 영양 세포층 없이 처리 배지(영양 세포의 배양액)만을 이용한 배양도 연구되고 있다. 본 발명의 구체적인 실시예에서는 STO 세포를 배아줄기세포 배양을 위한 영양 세포층으로 사용하였고, STO 세포를 배양한 배양액을 단계 1)의 처리 배지로 사용하였다.Embryonic stem cells can be cultured in vitro for a long time in undifferentiated state on the basis of feeder layer, and culture using only the treatment medium (nutrient solution of nutrient cell) without the feeder cell layer is also being studied. In a specific embodiment of the present invention, STO cells were used as a feeder cell layer for culturing embryonic stem cells, and culture medium in which STO cells were cultured was used as a treatment medium of step 1).

단계 2)의 Activin A, BIO 및 BMP4는 각각 TGF-β(Transforming growth factor-β), WNT 및 BMP(Bone morphogenic protein) 신호전달 경로의 활성제로 사용하였다. 상기 Activin A, BIO 및 BMP4의 처리농도는 각각 2-5 ng/㎖, 2-5 mM 및 10-20 ng/㎖이고, 바람직하게는 5 ng/㎖, 2 mM 및 20 ng/㎖ BMP4이다.Activin A, BIO and BMP4 of step 2) were used as activators of the transforming growth factor-β (TGF-β), WNT and bone morphogenic protein (BMP) signaling pathways, respectively. The treatment concentrations of Activin A, BIO and BMP4 are 2-5 ng / ml, 2-5 mM and 10-20 ng / ml, respectively, preferably 5 ng / ml, 2 mM and 20 ng / ml BMP4.

또한, 본 발명은 In addition,

1) 영양 세포층이 있는 조건에서 배양된 ESCs를 마트리겔로 코팅된 배양 접시로 옮긴 후, 처리 배지(Conditioned Medium; CM)로 1 내지 3일 동안 배양하는 단계;1) transferring ESCs cultured in the presence of a feeder cell layer to a culture dish coated with Matrigel, and then incubating for 1 to 3 days with Conditioned Medium (CM);

2) 상기 영양 세포층이 없는 조건에서 배양된 ESCs를 Activin A, BIO 및 BMP4를 포함하는 중배엽 유도 배지로 3 내지 5일 동안 ESCs 유래 중배엽 분화를 유도하는 단계; 및,2) inducing ESCs-derived mesoderm differentiation for 3 to 5 days with ESCs cultured in the absence of the feeder cell layer with mesoderm induction medium comprising Activin A, BIO and BMP4; And

3) 상기 ESCs 유래 중배엽을 bFGF 및/또는 PDGF-BB(Platelet-derived growth factor beta polypeptide)를 포함하는 중간엽 유도 배지로 3 내지 6일 동안 유도하는 단계를 포함하는 ESCs 유래 중배엽으로부터 중간엽 줄기세포 분화 방법을 제공한다.3) mesenchymal stem cells from ESCs-derived mesoderm comprising inducing the ESCs-derived mesoderm into mesenchymal induction medium containing bFGF and / or platelet-derived growth factor beta polypeptide (PDGF-BB) for 3 to 6 days Provide differentiation methods.

상기 단계 3)의 bFGF 및/또는 PDGF-BB의 처리농도는 각각 5 내지 20 ng/㎖ 및/또는 5 내지 20 ng/㎖이고, 바람직하게는 bFGF 및 PDGF-BB가 20 ng/㎖ 및 20 ng/㎖이다.The treatment concentration of bFGF and / or PDGF-BB in step 3) is 5-20 ng / ml and / or 5-20 ng / ml, respectively, preferably bFGF and PDGF-BB are 20 ng / ml and 20 ng, respectively. / Ml.

아울러, 본 발명은 Activin A, BIO 및 BMP4를 포함하는 ESCs 유래 중배엽 유도 배지를 제공한다.In addition, the present invention provides ESCs-derived mesoderm induction medium comprising Activin A, BIO and BMP4.

Activin A, BIO 및 BMP4는 최종배지 내에 각각 2-5 ng/㎖, 2-5 mM 및 10-20 ng/㎖의 농도로 존재할 수 있도록 조성되고, 바람직하게는 5 ng/㎖, 2 mM 및 20 ng/㎖의 농도로 존재할 수 있도록 조성된다.Activin A, BIO and BMP4 are formulated to be present in final media at concentrations of 2-5 ng / ml, 2-5 mM and 10-20 ng / ml, respectively, preferably 5 ng / ml, 2 mM and 20 It is formulated to be present at a concentration of ng / ml.

본 발명의 인간 배아줄기세포로부터 유래된 중간엽 줄기세포를 제조하는 방법은 세포기반 치료에 유용한 중간엽 줄기세포를 대량으로 제공하는 데에 유용하게 이용될 수 있다.The method for producing mesenchymal stem cells derived from human embryonic stem cells of the present invention can be usefully used to provide a large amount of mesenchymal stem cells useful for cell-based therapy.

이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.

단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

<< 실시예Example 1> 중배엽 유도 1> mesoderm induction

<1-1> 인간 배아줄기세포의 영양 세포층 유무에 따른 배양<1-1> Culture of Human Embryonic Stem Cells with or without Feeder Cell Layer

본 발명에서는 두 개의 hESCs(human Embryonic Stem Cells; 인간 배아줄기세포) 세포주(SNUhES3[서울대병원] 및 CHA4-hES[차병원])가 사용되었다. 상기 hESCs는 Mitomycin C(Sigma, USA)가 처리된 STO(ATCC CRL-1503) 영양 세포층에서, 4 ng/㎖ bFGF(Basic Fibroblast Growth Factor; Invitrogen, USA)를 포함하는 비처리 배지(Unconditioned Medium; UM)에서, 37℃ 및 5% CO2의 조건으로 배양되었다. 20% 녹아웃 혈청 대체물, 1% 비필수 아미노산 및 0.1 mM 머캅토에탄올을 포함하는 DMEM/F12 배지(Invitrogen, USA)로 구성된 UM 배지를 이용하여 매일 교환해주었다.In the present invention, two hESCs (human Embryonic Stem Cells) cell lines (SNUhES3 [Seoul National University Hospital] and CHA4-hES [Phase Hospital]) were used. The hESCs were unconditioned medium (UM) containing 4 ng / ml bFGF (Introtrogen, USA) in a STO (ATCC CRL-1503) feeder cell layer treated with Mitomycin C (Sigma, USA). ) Were incubated at 37 ° C. and 5% CO 2 . Daily exchange was performed using UM medium consisting of DMEM / F12 medium (Invitrogen, USA) containing 20% knockout serum replacement, 1% non-essential amino acids and 0.1 mM mercaptoethanol.

영양 세포층을 사용하지 않는 배양시에는 공지 기술대로 배양하였다(Xu C et al., Nat Biotechnol, 19(10):971-974, 2001). 즉, 마트리겔(BD Biosciences, USA)로 코팅된 배양 접시에서 영양 세포층이 없이 hESCs를 배양하면서, 8 ng/㎖ bFGF가 보충된 STO 처리 배지(Conditioned Medium; CM)(STO를 배양한 후 회수한 배양액)에서 2일 동안 안정화하였다.In culture without the feeder cell layer, the cells were cultured according to a known technique (Xu C et. al ., Nat Biotechnol , 19 (10): 971-974, 2001). That is, while culturing hESCs without a feeder cell layer in a culture dish coated with Matrigel (BD Biosciences, USA), STO-treated medium (CM) supplemented with 8 ng / ml bFGF (CM) (STO) was recovered after culturing In the medium) for 2 days.

<1-2> 중배엽 유도<1-2> Mesoderm Induction

hESCs를 중배엽으로 분화하기 위해, 본 발명자들은 3개의 세포내 신호전달 경로인 TGF-β(Transforming growth factor-β), WNT 및 BMP(Bone morphogenic protein) 신호전달 경로의 기능을 대상으로 하였다.To differentiate hESCs into mesoderm, we targeted the function of three intracellular signaling pathways, transforming growth factor-β (TGF-β), WNT, and Bone morphogenic protein (BMP) signaling pathways.

구체적으로, Activin A(Ac), BIO(6-bromoindirubin-3'-oxime) 및 BMP4(B4)를 각각 TGF-β, WNT 및 BMP 신호전달 경로의 활성제로 사용하여, 실시예 1-2에서 준비한 마트리겔로 코팅된 접시에 안정화된 hESCs의 배양액을 2-5 ng/㎖ Activin A(Peprotech, USA), 2-5 mM BIO(Sigma, USA), 및 10-20 ng/㎖ BMP4(Peprotech, USA)을 포함하는 UM 배지로 바꿔준 후, 3 내지 5일간 배양하였다.Specifically, using Activin A (Ac), BIO (6-bromoindirubin-3'-oxime) and BMP4 (B4) as an activator of TGF-β, WNT and BMP signaling pathway, respectively, prepared in Example 1-2 Cultures of stabilized hESCs in plates coated with Matrigel were prepared using 2-5 ng / ml Activin A (Peprotech, USA), 2-5 mM BIO (Sigma, USA), and 10-20 ng / ml BMP4 (Peprotech, USA). ), And then cultured for 3 to 5 days.

또는 상기 활성제 대신에 100 ng/㎖ DKK1, 100 ng/㎖ Noggin 및 50 μM SB431542 등의 신호전달 억제제를 사용하여 배양하였다.Or in place of the active agent was cultured using a signaling inhibitor such as 100 ng / ml DKK1, 100 ng / ml Noggin and 50 μM SB431542.

이때, 상기 활성제를 단독으로, 또는 세 개의 활성제를 동시에(ABB) 또는 활성제와 억제제를 조합하여(ABD: Activin A, BMP4 및 DKK1; ABN: Activin A, BIO 및 Noggin; BBS: BIO, BMP4 및 SB431542; ABB: Acitivin A, BIO 및 BMP4) 사용하였다.At this time, the active agent alone, or three active agents simultaneously (ABB) or a combination of the active agent and the inhibitor (ABD: Activin A, BMP4 and DKK1; ABN: Activin A, BIO and Noggin; BBS: BIO, BMP4 and SB431542 ABB: Acitivin A, BIO and BMP4).

이때, 상기 UM 배지에 함유된 활성제의 농도에 따라 분화를 조사한 결과, 5 ng/㎖ Activin A, 2 mM BIO 및 20 ng/㎖ BMP4를 포함하는 UM 배지에서 최적이었다.At this time, the differentiation was investigated according to the concentration of the activator contained in the UM medium, it was optimal in UM medium containing 5 ng / ㎖ Activin A, 2 mM BIO and 20 ng / ㎖ BMP4.

<< 실시예Example 2> 중배엽 특이적 유전자의 발현량 확인 2> Confirmation of expression level of mesoderm specific genes

분화된 세포를 회수하여 실시간 RT-PCR, 웨스턴 블랏팅 및 면역 염색법을 수행하여 중배엽 특이적 유전자의 발현량을 분석하였다.Differentiated cells were harvested and subjected to real-time RT-PCR, western blotting and immunostaining to analyze the expression level of mesoderm specific genes.

<2-1><2-1> 전체 all RNARNA 추출 및 실시간  Extract and Real Time RTRT -- PCRPCR

TRIzol(Invitrogen)을 사용하여 제조사의 프로토콜에 따라 세포로부터 전체 RNA를 추출하였다. 대략 1-2 g의 전체 RNA로부터 Superscript II 역전사효소(Invitrogen)를 이용하여 cDNA를 합성하였고, 상기 cDNA는 Prime Q-Master mix(SYBR Green I; GENETBIO)를 이용한 실시간 PCR에 사용되었다. 증폭반응은 iCycler iQ5 Real-Time detection system(Bio-Rad Laboratories, USA)에서 수행되었고, 실시간 RT-PCR의 반응 조건은 95℃ 10분 후, 95℃ 30초, 60℃ 30초 및 72℃ 30초를 40회 반복 및 최종 신장 72℃ 5분이었다. 비특이적 피크가 없는 것을 확인하기 위해 녹는점 곡선 분석을 수행하였고, 모든 반응은 세 번 반복되었다. 실시간 PCR에서 사용된 유전자 특이적 프라이머는 표 1에 기재된 바와 같다.Total RNA was extracted from cells using TRIzol (Invitrogen) according to the manufacturer's protocol. CDNA was synthesized using Superscript II reverse transcriptase (Invitrogen) from approximately 1-2 g of total RNA, which was used for real-time PCR using Prime Q-Master mix (SYBR Green I; GENETBIO). The amplification reaction was carried out in the iCycler iQ5 Real-Time detection system (Bio-Rad Laboratories, USA), and the reaction conditions of real-time RT-PCR were 95 ° C 30 seconds, 95 ° C 30 seconds, 60 ° C 30 seconds, and 72 ° C 30 seconds after 95 ° C 10 minutes. Was repeated 40 times and the final elongation was 72 ° C. for 5 minutes. Melting point curve analysis was performed to confirm that there were no nonspecific peaks, and all reactions were repeated three times. Gene specific primers used in real time PCR are as described in Table 1.

그 결과, 도 2a에 나타난 바와 같이 BIO, BMP4 또는 Activin A는 단독으로는 T, MIXL1 , GSC ( Goosecoid ), MEOX1 및 WNT3 등의 중배엽 특이적 유전자의 발현량을 거의 유도하지 못하였다. 그러나 상기 활성제들을 함께 사용할 경우, 중배엽 특이적 유전자의 발현량이 강하게 유도되는 시너지 효과를 나타내었다. 또한, 흥미롭게는 Activin A, BIO 및 BMP4의 혼합 처리에 의해서는 외배엽(도 2b) 또는 내배엽(도 2c) 특이적 유전자는 유도되지 않았고, 오직 중배엽 특이적 유전자만 유도되었다.As a result, as shown in Figure 2a, BIO, BMP4 or Activin A alone, T, MIXL1 , GSC ( Goosecoid ), MEOX1 And little expression of mesodermal specific genes such as WNT3 . However, when used together with the active agent, the expression level of mesoderm specific genes showed a strong synergistic effect. Interestingly, the mixed treatment of Activin A, BIO and BMP4 did not induce ectoderm (FIG. 2B) or endoderm (FIG. 2C) specific genes, but only mesodermal specific genes were induced.

그러나 도 3에 나타난 바와 같이 DKK1, Noggin 및 SB431542과 같은 신호전달 억제제는 유도된 hESCs에서 중배엽 마커 유전자의 발현량을 유의하게 억제하였다.However, as shown in FIG. 3, signaling inhibitors such as DKK1, Noggin and SB431542 significantly inhibited the expression of mesodermal marker genes in induced hESCs.

유전자명Gene name 서열번호SEQ ID NO: 서열order TT 서열번호 1SEQ ID NO: 1 ATCACAAAGAGATGATGGAGGAAATCACAAAGAGATGATGGAGGAA 서열번호 2SEQ ID NO: 2 GGTGAGTTGTCAGAATAGGTTGGGGTGAGTTGTCAGAATAGGTTGG MIXL1MIXL1 서열번호 3SEQ ID NO: 3 GCAGATATGAGATTGCGGTAAACGCAGATATGAGATTGCGGTAAAC 서열번호 4SEQ ID NO: 4 TTGGGTAGGGGGCTCAGTCCTTAGTTTGGGTAGGGGGCTCAGTCCTTAGT GSCGSC 서열번호 5SEQ ID NO: 5 cggtcctcatcagaggagtccggtcctcatcagaggagtc 서열번호 6SEQ ID NO: 6 tcgtctgcttgtgcaagtcctcgtctgcttgtgcaagtcc MEOX1MEOX1 서열번호 7SEQ ID NO: 7 CCGCTGTTCTCCTAGGTCAGCCGCTGTTCTCCTAGGTCAG 서열번호 8SEQ ID NO: 8 CCCCAAAGCCTCAGATGTGCAGCCCCCAAAGCCTCAGATGTGCAGC WNT3WNT3 서열번호 9SEQ ID NO: 9 GGCTGTGACTCGCATCATAAGGCTGTGACTCGCATCATAA 서열번호 10SEQ ID NO: 10 CTTGCATTTGAGGTGCATGTCTTGCATTTGAGGTGCATGT ZIC1ZIC1 서열번호 11SEQ ID NO: 11 GCGCTCCGAGAATTTAAAGAGCGCTCCGAGAATTTAAAGA 서열번호 12SEQ ID NO: 12 CGTGGACCTTCATGTGTTTGCGTGGACCTTCATGTGTTTG PAX6PAX6 서열번호 13SEQ ID NO: 13 GTGTCCAACGGATGTGTGAGGTGTCCAACGGATGTGTGAG 서열번호 14SEQ ID NO: 14 CTAGCCAGGTTGCGAAGAACCTAGCCAGGTTGCGAAGAAC FOXA2FOXA2 서열번호 15SEQ ID NO: 15 ATGAACGGCATGAACACGTAATGAACGGCATGAACACGTA 서열번호 16SEQ ID NO: 16 TGGAGTTCATGTTGGCGTAGTGGAGTTCATGTTGGCGTAG CXCR4CXCR4 서열번호 17SEQ ID NO: 17 GGTGGTCTATGTTGGCGTCTGGTGGTCTATGTTGGCGTCT 서열번호 18SEQ ID NO: 18 TGCAATAGCAGGACAGGATGTGCAATAGCAGGACAGGATG a-FDa-FD 서열번호 19SEQ ID NO: 19 CAAACGATGAAGCAAGAGTTTCT CAAACGATGAAGCAAGAGTTTCT 서열번호 20SEQ ID NO: 20 AAAAGTTCACACCGAATGAAAGAAAAAGTTCACACCGAATGAAAGA

<2-2> 면역 염색 및 <2-2> immunostaining and 웨스턴Western 블랏팅Blotting

<2-2-1> 면역염색<2-2-1> Immunostaining

세포를 Ca2 +/Mg2 +-free PBS(Invitrogen)에 세척한 후, 4% 포름알데히드를 이용하여 실온에서 20분 동안 고정하였다. 핵단백질의 검출을 위해, 상기 세포를 0.1% Triton X-100 in PBS에서 30분 동안 투과시킨 후, 4% 정상 염소 혈청 또는 우태아혈청을 이용하여 실온에서 1시간 동안 차단하였다. 이후, Oct3/4(Santa Cruz Biotechnology, USA, 1:1000) 및 T(R&D, USA, 1:500)에 특이적인 항체를 차단용액으로 적절히 희석한 후, 4℃에서 밤새도록 배양하였다. 최종적으로, 상기 세포를 PBST(0.1% Tween-20 in PBS)로 수차례 세척한 후, Alexa-488- 또는 -594-결합- 2차 항체(Invitrogen)와 인큐베이션 하였다. 면역염색된 세포를 형광현미경(Olympus, Japan)으로 관찰하였다.After washing the cells, the Ca 2 + / Mg 2 + -free PBS (Invitrogen), using a 4% formaldehyde were fixed for 20 minutes at room temperature. For detection of nucleoproteins, the cells were permeated in 0.1% Triton X-100 in PBS for 30 minutes and then blocked for 1 hour at room temperature using 4% normal goat serum or fetal bovine serum. Subsequently, the antibodies specific for Oct3 / 4 (Santa Cruz Biotechnology, USA, 1: 1000) and T (R & D, USA, 1: 500) were properly diluted with a blocking solution and then incubated overnight at 4 ° C. Finally, the cells were washed several times with PBST (0.1% Tween-20 in PBS) and then incubated with Alexa-488- or -594-binding secondary antibody (Invitrogen). Immunostained cells were observed by fluorescence microscope (Olympus, Japan).

<2-2-2> <2-2-2> 웨스턴Western 블랏팅Blotting

전체단백질 추출을 위해, 세포를 PRO-PREPTM 단백질 추출 용액(Intronbio, Korea)에서 균일하게 용해한 후, 추출된 단백질을 브래드포드 검사법으로 정량하였다. 전체 단백질(20-30 g)을 10% SDS-PAGE 방법으로 분리한 후, 니트로셀룰로오스 멤브레인(Bio-Rad)으로 옮겼다. 상기 블랏팅된 멤브레인을 TBST(10 mM Tris-HCl, pH 7.5, 150 nM NaCl 및 0.1% Tween-20; Sigma)에 녹인 3-5% 스킴 밀크로 1시간 동안 실온에서 차단하였다. 이후 상기 멤브레인을 Oct3/4 및 T에 특이적인 1차 항체와 함께 밤새도록 4℃에서 인큐베이션 하였다. TBST로 세척한 후, 상기 시료를 4℃에서 1시간 동안 차단 용액에서 적절한 2차 항체로 처리하였다. 이후 상기 멤브레인을 세척한 후, ECL 시스템(Pierce, USA)을 이용하여 제조사의 권고 프로토콜에 따라 신호를 검출하였다.For whole protein extraction, the cells were treated with PRO-PREP ™ After homogeneously dissolving in the protein extraction solution (Intronbio, Korea), the extracted protein was quantified by the Bradford test. Total protein (20-30 g) was isolated by 10% SDS-PAGE method and then transferred to nitrocellulose membrane (Bio-Rad). The blotted membrane was blocked for 1 hour at room temperature with 3-5% scheme milk dissolved in TBST (10 mM Tris-HCl, pH 7.5, 150 nM NaCl and 0.1% Tween-20; Sigma). The membrane was then incubated at 4 ° C. overnight with primary antibodies specific for Oct3 / 4 and T. After washing with TBST, the samples were treated with the appropriate secondary antibody in blocking solution at 4 ° C. for 1 hour. After washing the membrane, the signal was detected using the ECL system (Pierce, USA) according to the manufacturer's recommended protocol.

그 결과, 도 4에 나타난 바와 같이 T의 단백질 발현량도 면역염색 및 웨스턴 블랏팅에 의해 확인되었다. 또한, 웨스턴 블랏팅 결과를 통해서, 전능성 마커인 Oct4가 유도된 중배엽 세포에서 억제된 것을 확인하였다. 상기 결과로부터 BIO, BMP4 및 Activin A의 시너지 효과에 의해 hESCs를 중배엽 전구세포로 효율적으로 분화된 것을 확인하였다.As a result, as shown in Figure 4, the protein expression amount of T was also confirmed by immunostaining and Western blotting. In addition, it was confirmed through Western blotting results that the omnipotent marker Oct4 was inhibited in induced mesoderm cells. From the above results, it was confirmed that hESCs were efficiently differentiated into mesodermal progenitor cells by synergistic effects of BIO, BMP4 and Activin A.

<< 실시예Example 3>  3> 중간엽Intermediate lobe 줄기세포 유도 Stem cell induction

<3-1> <3-1> 중간엽Intermediate lobe 줄기세포 유도 Stem cell induction

실시예 2의 hESCs 유래 중배엽으로부터 CD105 양성 세포를 유도하기 위해, 본 발명자들은 bFGF 및 PDGF 신호전달 경로를 조절하였다.To induce CD105 positive cells from the hESCs derived mesoderm of Example 2, we modulated the bFGF and PDGF signaling pathways.

구체적으로, 중배엽으로 분화된 세포를 bFGF 및/또는 PDGF-BB(Platelet-derived growth factor beta polypeptide)를 포함하는 UM 배지에서 추가로 3 내지 6일 동안 배양하였다. 다양한 농도의 bFGF 및 PDGF-BB를 이용하여 CD105 양성 세포의 유도에 최적인 조건을 확인한 결과, 20 ng/㎖ bFGF 및 20 ng/㎖ PDGF-BB의 조합이 CD105 양성 세포의 유도에 가장 최적인 조건이었다.Specifically, the mesoderm differentiated cells were cultured for additional 3 to 6 days in UM medium containing bFGF and / or platelet-derived growth factor beta polypeptide (PDGF-BB). Optimum conditions for induction of CD105 positive cells using various concentrations of bFGF and PDGF-BB were determined. The combination of 20 ng / ml bFGF and 20 ng / ml PDGF-BB was most optimal for induction of CD105 positive cells. It was.

<3-2> <3-2> 중간엽Intermediate lobe 줄기세포 특이적 유전자의 발현량 확인 Expression level of stem cell specific gene

실시예 2-1의 방법으로 상기 중간엽 줄기세포로부터 cDNA를 제조한 후, PCR PreMix(GENETBIO, Korea)를 이용하여 중간엽 줄기세포 특이적 유전자를 증폭하였다. RT-PCR 반응시 사용된 유전자 특이적 프라이머는 표 2에 기재된 바와 같다. RT-PCR 조건은 하기와 같다: 초기 변성 95℃에서 5분, 94℃에서 30초, 60℃에서 30초 및 72℃에서 30를 25 내지 35회 반복, 및 최종 신장 72℃에서 5분. 상기 PCR 산물은 1.5% 아가로스 겔에서 전기영동에 의해 분석되었다.After cDNA was prepared from the mesenchymal stem cells by the method of Example 2-1, mesenchymal stem cell specific genes were amplified using PCR PreMix (GENETBIO, Korea). Gene specific primers used in the RT-PCR reaction are as described in Table 2. RT-PCR conditions are as follows: initial denaturation 5 min at 95 ° C., 30 sec at 94 ° C., 30 sec at 60 ° C., and 30 to 30 cycles at 72 ° C., and 5 min at 72 ° C. final elongation. The PCR product was analyzed by electrophoresis on 1.5% agarose gel.

그 결과, 생성된 세포군은 도 5에 나타난 바와 같이 CD44 , CD49a , CD73 , CD105 및 CD166 등의 중간엽 줄기세포 특이적 유전자를 발현하였다.As a result, the resulting cell population expressed mesenchymal stem cell specific genes such as CD44 , CD49a , CD73 , CD105, and CD166 as shown in FIG.

유전자명Gene name 서열번호SEQ ID NO: 서열order CD105CD105 서열번호 21SEQ ID NO: 21 GCCAGCATTGTCTCACTTCAGCCAGCATTGTCTCACTTCA 서열번호 22SEQ ID NO: 22 ATGCGCAACAAGCTCTTTCTATGCGCAACAAGCTCTTTCT CD166CD166 서열번호 23SEQ ID NO: 23 CTTGCACAGCAGAAAACCAACTTGCACAGCAGAAAACCAA 서열번호 24SEQ ID NO: 24 CCAGTAGACGACACCAGCAACCAGTAGACGACACCAGCAA CD73CD73 서열번호 25SEQ ID NO: 25 CGCAACAATGGCACAATTACCGCAACAATGGCACAATTAC 서열번호 26SEQ ID NO: 26 ACTCGACACTTGGTGCAAAGACTCGACACTTGGTGCAAAG CD49aCD49a 서열번호 27SEQ ID NO: 27 TGGGTGCTTATTGGTTCTCCTGGGTGCTTATTGGTTCTCC 서열번호 28SEQ ID NO: 28 CTCCATTTGGGTTGGTGACTCTCCATTTGGGTTGGTGACT CD44CD44 서열번호 29SEQ ID NO: 29 TGGCACCCGCTATGTCCAGTGGCACCCGCTATGTCCAG 서열번호 30SEQ ID NO: 30 GTAGCAGGGATTCTGTCTGGTAGCAGGGATTCTGTCTG GAPDHGAPDH 서열번호 31SEQ ID NO: 31 TGGTATCGTGGAAGGACTCATGGTATCGTGGAAGGACTCA 서열번호 32SEQ ID NO: 32 CCTGCTTCACCACCTTCTTGCCTGCTTCACCACCTTCTTG

<< 실시예Example 4>  4> CD105CD105 양성 세포의 분리 Isolation of Positive Cells

<4-1> <4-1> MACSMACS 를 이용한 분리Separation

CD105 양성(CD105+) 세포를 분류하기 위해, 실시예 3의 이질성 세포군에 Accutase(Innovative Cell Technnologies, USA)로 5 내지 10분 동안 37℃에서 처리하여 단일 세포 현탁액을 제조하였다. 상기 처리된 세포에 부드럽게 파이펫팅하여 세포를 떼어놓고, 40-m 세포 여과기(BD Biosciences, USA)에 통과시켰다. 이후 CD105+ 세포를 자성활성 세포 분류기(magnetic-activated cell sorting, MACS) MicroBead 컬럼(Miltenyi Biotech, USA)을 이용하여 제조업자의 프로토콜에 따라 분화된 hESCs 유래 중배엽으로부터 분리하였다.To classify CD105 positive (CD105 + ) cells, the heterogeneous cell population of Example 3 was treated with Accutase (Innovative Cell Technnologies, USA) for 5-10 minutes at 37 ° C. to prepare a single cell suspension. The cells were detached by gently pipetting the treated cells and passed through a 40-m cell filter (BD Biosciences, USA). CD105 + cells were then isolated from hESCs derived mesoderm differentiated according to the manufacturer's protocol using a magnetic-activated cell sorting (MACS) MicroBead column (Miltenyi Biotech, USA).

<4-2> <4-2> 유세포Flow cell 분석 analysis

상기 분리된 세포를 2% FBS를 포함하는 PBS에 현탁한 후, CD105-APC(R&D Systems, USA) 또는 Isotope-APC(R&D Systems, USA)에 특이적인 항체를 이용하여 4℃에서 30 내지 45분 동안 표지하였다. 2% FBS를 포함하는 PBS로 세차례 세척한 후, 상기 항체 표지된 세포를 LSRII 유세포 분석기(Becton Dickson, USA)를 이용하여 제조업자의 프로토콜에 따라 분석하였다. FlowJo 소프트웨어(Tree Star, Inc., USA)를 이용하여 수득한 데이터를 분석하였다.The isolated cells were suspended in PBS containing 2% FBS, followed by 30 to 45 minutes at 4 ° C. using antibodies specific for CD105-APC (R & D Systems, USA) or Isotope-APC (R & D Systems, USA). Was labeled. After washing three times with PBS containing 2% FBS, the antibody labeled cells were analyzed using the LSRII flow cytometer (Becton Dickson, USA) according to the manufacturer's protocol. The data obtained was analyzed using FlowJo software (Tree Star, Inc., USA).

그 결과, 도 6에 나타난 바와 같이 이질성 세포군 유래의 CD105 양성 세포의 비율은 bFGF 및 PDGF-BB을 포함하는 UM 배지에서 시간이 지남에 따라 증가하였고, 6일 배양 후에 최고지점(대략 36%)에 도달하였다.As a result, as shown in FIG. 6, the proportion of CD105 positive cells from heterogeneous cell populations increased over time in UM medium containing bFGF and PDGF-BB, and reached the highest point (approximately 36%) after 6 days of culture. Reached.

<< 실시예Example 5>  5> CD105CD105 양성 세포의 유전자 발현 Gene Expression in Positive Cells

실시예 2-1의 방법으로 상기 CD105 양성 세포로부터 cDNA를 제조한 후, 실시예 3-2의 방법으로 표3의 프라이머 쌍을 이용하여 중간엽 특이적 유전자를 증폭하였다.After cDNA was prepared from the CD105 positive cells by the method of Example 2-1, the mesenchymal specific gene was amplified using the primer pairs of Table 3 by the method of Example 3-2.

그 결과, 도 7에 나타난 바와 같이 상기 세포는 중간엽 특이적 유전자인 CD29, CD44 , CD49a , CD73 , CD105 및 CD166 등을 발현하였으나, 중간엽의 음성 마커 유전자인 CD34 , CD45 및 HLA - DR 등은 발현하지 못하였다. 또한, OCT4는 CD105 양성 세포에서는 발현하지 않았다[데이타 기재하지 않음].As a result, as shown in Figure 7, the cells are mesenchymal-specific genes CD29, CD44 , CD49a , CD73 , CD105 And CD166 , but the negative marker gene of mesenchymal CD34 , CD45 And HLA - DR did not express. In addition, OCT4 was not expressed in CD105 positive cells [data not shown].

유전자명Gene name 서열번호SEQ ID NO: 서열order PDGF-b ReceptorPDGF-b Receptor 서열번호 33SEQ ID NO: 33 CAACTACATGGCCCCTTACGCAACTACATGGCCCCTTACG 서열번호 34SEQ ID NO: 34 TTCACAGATGAGCACGTTCCTTCACAGATGAGCACGTTCC CD29CD29 서열번호 35SEQ ID NO: 35 CAAAGGAACAGCAGAGAAGCCAAAGGAACAGCAGAGAAGC 서열번호 36SEQ ID NO: 36 ATTGAGTAAGACAGGTCCATAAGGATTGAGTAAGACAGGTCCATAAGG CD45CD45 서열번호 37SEQ ID NO: 37 TTCAACTTATACCCTTCGTGTCTTCAACTTATACCCTTCGTGTC 서열번호 38SEQ ID NO: 38 CCTGCTTTACTTTGTCCACTTCCCTGCTTTACTTTGTCCACTTC CD34CD34 서열번호 39SEQ ID NO: 39 TGAAGCCTAGCCTGTCACCTTGAAGCCTAGCCTGTCACCT 서열번호 40SEQ ID NO: 40 CGCACAGCTGGAGGTCTTATCGCACAGCTGGAGGTCTTAT HLA-DRHLA-DR 서열번호 41SEQ ID NO: 41 caaagaaggagacggtctggcaaagaaggagacggtctgg 서열번호 42SEQ ID NO: 42 gggctctctcagttccacaggggctctctcagttccacag OCT4OCT4 서열번호 43SEQ ID NO: 43 CGTGAAGCTGGAGAAGGAGAAGCTGCGTGAAGCTGGAGAAGGAGAAGCTG 서열번호 44SEQ ID NO: 44 CAAGGGCCGCAGCTTACACATGTTCCAAGGGCCGCAGCTTACACATGTTC

<< 실시예Example 6> 지방세포 및  6> fat cells and 골아세포로Into osteoblasts 분화 differentiation

본 발명자들은 Human Mesenchymal Stem Cell Functional Identification Kit(R&D system)를 이용하여 CD105 양성 세포를 지방세포 및 골아세포로 분화하였다.We differentiated CD105 positive cells into adipocytes and osteoblasts using the Human Mesenchymal Stem Cell Functional Identification Kit (R & D system).

<6-1> 지방세포 분화<6-1> Adipocyte Differentiation

CD105 양성 세포를 2.1×104 cells/㎝2의 밀도로 4웰 플레이트의 α-MEM 기본 배지에 접종한 후 밤새도록 배양하였다. 상기 세포가 80-100%까지 자라면, 기본배지를 지방세포 분화 유도제(R&D Systems, USA)가 포함된 배지로 바꿔주었다. 배지를 3 내지 4일 마다 교환하면서 1 내지 3주간 배양하였고, 5 내지 7일 후에 유도된 세포로부터 지질 액포가 나타나기 시작했다. 1 내지 3주 후에, 지방세포를 고정하여 Oil Red S 염색 및 면역염색에 사용하였다. 이때, 대조군은 지방세포 분화 유도제가 포함되지 않은 기본 배지에 세포를 접종하여 수행하였다. 그 결과, 도 8a에 나타난 바와 같이 2주 후에는 상기 지질 액포가 대조군과 비교하여 고농도로 거의 모든 유도된 세포에서 나타났다.CD105 positive cells were seeded in 4-well plate α-MEM basal medium at a density of 2.1 × 10 4 cells / cm 2 and then incubated overnight. When the cells grew to 80-100%, the basal medium was changed to a medium containing adipocyte differentiation inducing agent (R & D Systems, USA). The medium was incubated for 1 to 3 weeks with changing every 3 to 4 days, and after 5 to 7 days lipid vesicles started to appear from the induced cells. After 1-3 weeks, the adipocytes were fixed and used for Oil Red S staining and immunostaining. At this time, the control group was inoculated with the cells in a basal medium that does not contain adipocyte differentiation inducing agent. As a result, as shown in FIG. 8A, the lipid vacuoles appeared in almost all induced cells at a high concentration as compared with the control group.

또한, 지방세포 특이적인 FABP-4(fatty acid binding protein-4)에 특이적인 항체(R&D System, USA)를 이용하여 실시예 2-2-1의 방법으로 면역염색을 수행함으로써, 도 8b에 나타난 바와 같이 분화된 세포에서 FABP-4의 존재를 확인하였다.In addition, by immunostaining by the method of Example 2-2-1 using an antibody specific to fat cell-specific FABP-4 (fatty acid binding protein-4) (R & D System, USA), it is shown in Figure 8b The presence of FABP-4 was confirmed in differentiated cells as shown.

아울러, 표 4의 프라이머쌍을 이용하여 실시예 3-2의 방법으로 RT-PCR을 수행한 결과, 도 8c에 나타난 바와 같이 hESCs와 비교하여 FABP-4 및 PPARg2(peroxisome proliferator-activated receptor-g2)의 지방세포 특이적 유전자의 발현이 유도된 것으로 나타났다.In addition, as a result of performing RT-PCR by the method of Example 3-2 using the primer pairs of Table 4, FABP-4 and PPARg2 (peroxisome proliferator-activated receptor-g2) compared to hESCs as shown in Figure 8c The expression of adipocyte specific genes of was shown to be induced.

즉, CD105 양성 세포로부터 지방세포가 성공적으로 유도된 것을 확인하였다.That is, it was confirmed that adipocytes were successfully induced from CD105 positive cells.

유전자명Gene name 서열번호SEQ ID NO: 서열order FABP-4FABP-4 서열번호 45SEQ ID NO: 45 TGCAGCTTCCTTCTCACCTTTGCAGCTTCCTTCTCACCTT 서열번호 46SEQ ID NO: 46 TCACATCCCCATTCACACTGTCACATCCCCATTCACACTG PPARg2PPARg2 서열번호 47SEQ ID NO: 47 CTCCGTGGATCTCTCCGTAACTCCGTGGATCTCTCCGTAA 서열번호 48SEQ ID NO: 48 GATGCAGGCTCCACTTTGATGATGCAGGCTCCACTTTGAT

<6-2> <6-2> 골아세포Osteoblast 분화 differentiation

CD105 양성 세포를 4.2×103 cells/㎝2의 밀도로 4웰 플레이트의 α-MEM 기본 배지에 접종한 후 밤새도록 배양하였다. 상기 세포가 50-70%까지 자라면, 기본배지를 골아세포 분화 유도제(R&D Systems, USA)가 포함된 배지로 바꿔주었다. 배지를 3 내지 4일 마다 교환하면서 2 내지 3주간 배양한 후, 골아세포를 고정하여 Alizarin Red S 염색 및 면역염색에 사용하였다. 이때, 대조군은 골아세포 분화 유도제가 포함되지 않은 기본 배지에 세포를 접종하여 수행하였다. 그 결과, 도 9a에 나타난 바와 같이 3주 후에는 대조군과 비교하여 유도된 세포에는 칼슘 침착(Calcium deposit)이 매우 많이 나타났다.CD105 positive cells were seeded in α-MEM basal medium in 4-well plates at a density of 4.2 × 10 3 cells / cm 2 and incubated overnight. When the cells grew to 50-70%, the basal medium was changed to a medium containing osteoblast differentiation inducing agent (R & D Systems, USA). After culturing for 2-3 weeks with changing medium every 3-4 days, osteoblasts were fixed and used for Alizarin Red S staining and immunostaining. At this time, the control group was inoculated with the cells in a basal medium that does not contain osteoblast differentiation inducing agent. As a result, as shown in Figure 9a after 3 weeks the calcium deposits (Calcium deposit) was very high in the induced cells compared to the control group.

또한, 도 9b에 나타난 바와 같이 항인간 오스테오칼신(Osteocalcin) 항체(R&D System)를 이용하여 전형적인 골아세포 단백질인 오스테오칼신을 분명하게 검출하였다.In addition, as shown in Figure 9b using the anti-human osteocalcin antibody (R & D System) was clearly detected osteocalcin, a typical osteoblastic protein.

또한, Alkaline phosphatase kit(Sigma-Aldrich,USA)를 이용하여 AP 염색을 수행한 결과, 도 9c에 나타난 바와 같이 대조군과 비교하여 유도된 세포에는 알칼리성 인산분해효소(Alkaline phosphatase)가 매우 풍부하였다.In addition, as a result of AP staining using an Alkaline phosphatase kit (Sigma-Aldrich, USA), as shown in FIG. 9C, cells induced compared to the control group were very rich in alkaline phosphatase.

아울러, 표 5의 프라이머쌍을 이용하여 실시예 3-2의 방법으로 RT-PCR을 수행한 결과, 도 9d에 나타난 바와 같이 hESCs와 비교하여 RUNX2 및 오스테오칼신의 골아세포 특이적 유전자의 발현이 유도된 것으로 나타났다.In addition, as a result of performing RT-PCR by the method of Example 3-2 using the primer pairs of Table 5, the expression of osteoblast-specific genes of RUNX2 and osteocalcin was induced as compared to hESCs as shown in FIG. 9D. Appeared.

즉, CD105 양성 세포로부터 골아세포가 성공적으로 유도된 것을 확인하였다.That is, it was confirmed that osteoblasts were successfully induced from CD105 positive cells.

유전자명Gene name 서열번호SEQ ID NO: 서열order OsteocalcinOsteocalcin 서열번호 49SEQ ID NO: 49 GGCAGCGAGGTAGTGAAGAGGGCAGCGAGGTAGTGAAGAG 서열번호 50SEQ ID NO: 50 AGCAGAGCGACACCCTAGACAGCAGAGCGACACCCTAGAC Runx2Runx2 서열번호 51SEQ ID NO: 51 TAGGCGCATTTCAGATGATGTAGGCGCATTTCAGATGATG 서열번호 52SEQ ID NO: 52 GACTGGCGGGGTGTAAGTAAGACTGGCGGGGTGTAAGTAA

도 1은 인간 배아줄기세포로부터 중배엽 세포를 거쳐 중간엽 줄기세포로 유도시킨 후, 지방세포 또는 골수세포로 분화되는 전략을 나타낸 도이다.1 is a diagram showing the strategy of differentiation into adipocytes or bone marrow cells after induction into mesenchymal stem cells from human embryonic stem cells through mesodermal cells.

도 2는 본 발명에서 유도된 중배엽 세포에서 중배엽 특이적 유전자의 발현량을 확인한 도이다:Figure 2 is a diagram confirming the expression level of mesoderm specific genes in mesoderm cells induced in the present invention:

a: 중배엽, b) 외배엽 및 c) 내배엽.a: mesoderm, b) ectoderm and c) endoderm.

도 3은 본 발명에서 중배엽 세포 유도시 신호전달 억제제의 영향을 확인한 도이다:Figure 3 is a diagram confirming the effect of the signaling inhibitors in mesodermal cell induction in the present invention:

a: WNT 신호억제제, b) BMP 신호억제제 및 c) TGF-β 신호억제제.a: WNT signal inhibitor, b) BMP signal inhibitor and c) TGF-β signal inhibitor.

도 4는 본 발명에서 유도된 중배엽 세포에서 중배엽 특이적 유전자의 단백질 발현량을 확인한 도이다.Figure 4 is a diagram confirming the protein expression level of mesoderm specific genes in mesoderm cells induced in the present invention.

도 5는 본 발명에서 유도된 hESCs 유래 중간엽 줄기세포에서 중간엽 줄기세포 특이적 유전자의 발현량을 확인한 도이다.Figure 5 is a diagram confirming the expression level of mesenchymal stem cell-specific genes in hESCs-derived mesenchymal stem cells induced in the present invention.

도 6은 본 발명에서 유도된 hESCs 유래 중간엽 줄기세포를 FACS로 확인한 도이다.Figure 6 is a diagram confirming the hESCs derived mesenchymal stem cells derived from the present invention by FACS.

도 7은 본 발명에서 분리된 CD105 양성 세포의 중간엽 줄기세포 특이적 유전자의 발현량을 확인한 도이다.Figure 7 is a diagram confirming the expression level of mesenchymal stem cell specific genes of CD105 positive cells isolated in the present invention.

도 8은 본 발명에서 유도된 지방세포의 분화를 확인한 도이다:8 is a diagram confirming the differentiation of adipocytes induced in the present invention:

a: Oil Red S 염색;a: Oil Red S stain;

b: 면역염색; 및,b: immunostaining; And

c: RT-PCR.c: RT-PCR.

도 9는 본 발명에서 유도된 골아세포의 분화를 확인한 도이다:9 is a diagram confirming the differentiation of osteoblasts induced in the present invention:

a: Alizarin Red S 염색;a: Alizarin Red S stained;

b: 면역염색;b: immunostaining;

c: AP 염색; 및,c: AP staining; And

d: RT-PCR.d: RT-PCR.

<110> Korea Advanced Instititue of Science and Technology <120> Method of inducing differentiation of human embryonic stem cells into mesenchymal stem cells <130> 9P-07-15 <160> 52 <170> KopatentIn 1.71 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> T Forward primer <400> 1 atcacaaaga gatgatggag gaa 23 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> T Reverse primer <400> 2 ggtgagttgt cagaataggt tgg 23 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MIXL1 Forward primer <400> 3 gcagatatga gattgcggta aac 23 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> MIXL1 Reverse primer <400> 4 ttgggtaggg ggctcagtcc ttagt 25 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GSC Forward primer <400> 5 cggtcctcat cagaggagtc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GSC Reverse primer <400> 6 tcgtctgctt gtgcaagtcc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MEOX1 Forward primer <400> 7 ccgctgttct cctaggtcag 20 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MEOX1 Reverse primer <400> 8 ccccaaagcc tcagatgtgc agc 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WNT3 Forward primer <400> 9 ggctgtgact cgcatcataa 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WNT3 Reverse primer <400> 10 cttgcatttg aggtgcatgt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ZIC1 Forward primer <400> 11 gcgctccgag aatttaaaga 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ZIC1 Reverse primer <400> 12 cgtggacctt catgtgtttg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX6 Forward primer <400> 13 gtgtccaacg gatgtgtgag 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX6 Reverse primer <400> 14 ctagccaggt tgcgaagaac 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FOXA2 Forward primer <400> 15 atgaacggca tgaacacgta 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FOXA2 Reverse primer <400> 16 tggagttcat gttggcgtag 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCR4 Forward primer <400> 17 ggtggtctat gttggcgtct 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCR4 Reverse primer <400> 18 tgcaatagca ggacaggatg 20 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> a-FD Forward primer <400> 19 caaacgatga agcaagagtt tct 23 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> a-FD Reverse primer <400> 20 aaaagttcac accgaatgaa aga 23 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD105 Forward primer <400> 21 gccagcattg tctcacttca 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD105 Reverse primer <400> 22 atgcgcaaca agctctttct 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD166 Forward primer <400> 23 cttgcacagc agaaaaccaa 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD166 Reverse primer <400> 24 ccagtagacg acaccagcaa 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD73 Forward primer <400> 25 cgcaacaatg gcacaattac 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD73 Reverse primer <400> 26 actcgacact tggtgcaaag 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD49a Forward primer <400> 27 tgggtgctta ttggttctcc 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD49a Reverse primer <400> 28 ctccatttgg gttggtgact 20 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CD44 Forward primer <400> 29 tggcacccgc tatgtccag 19 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CD44 Reverse primer <400> 30 gtagcaggga ttctgtctg 19 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Forward primer <400> 31 tggtatcgtg gaaggactca 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Reverse primer <400> 32 cctgcttcac caccttcttg 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PDGF-b Receptor Forward primer <400> 33 caactacatg gccccttacg 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PDGF-b Receptor Reverse primer <400> 34 ttcacagatg agcacgttcc 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD29 Forward primer <400> 35 caaaggaaca gcagagaagc 20 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CD29 Reverse primer <400> 36 attgagtaag acaggtccat aagg 24 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CD45 Forward primer <400> 37 ttcaacttat acccttcgtg tc 22 <210> 38 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CD45 Reverse primer <400> 38 cctgctttac tttgtccact tc 22 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD34 Forward primer <400> 39 tgaagcctag cctgtcacct 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD34 Reverse primer <400> 40 cgcacagctg gaggtcttat 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HLA-DR Forward primer <400> 41 caaagaagga gacggtctgg 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HLA-DR Reverse primer <400> 42 gggctctctc agttccacag 20 <210> 43 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 04-Oct Forward primer <400> 43 cgtgaagctg gagaaggaga agctg 25 <210> 44 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 04-Oct Reverse primer <400> 44 caagggccgc agcttacaca tgttc 25 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FABP-4 Forward primer <400> 45 tgcagcttcc ttctcacctt 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FABP-4 Reverse primer <400> 46 tcacatcccc attcacactg 20 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARg2 Forward primer <400> 47 ctccgtggat ctctccgtaa 20 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARg2 Reverse primer <400> 48 gatgcaggct ccactttgat 20 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Osteocalcin Forward primer <400> 49 ggcagcgagg tagtgaagag 20 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Osteocalcin Reverse primer <400> 50 agcagagcga caccctagac 20 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Runx2 Forward primer <400> 51 taggcgcatt tcagatgatg 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Runx2 Reverse primer <400> 52 gactggcggg gtgtaagtaa 20 <110> Korea Advanced Instititue of Science and Technology <120> Method of inducing differentiation of human embryonic stem cells          into mesenchymal stem cells <130> 9P-07-15 <160> 52 <170> Kopatentin 1.71 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> T Forward primer <400> 1 atcacaaaga gatgatggag gaa 23 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> T223 primer <400> 2 ggtgagttgt cagaataggt tgg 23 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MIXL1 Forward primer <400> 3 gcagatatga gattgcggta aac 23 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> MIXL1 Reverse primer <400> 4 ttgggtaggg ggctcagtcc ttagt 25 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GSC Forward primer <400> 5 cggtcctcat cagaggagtc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GSC Reverse primer <400> 6 tcgtctgctt gtgcaagtcc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MEOX1 Forward primer <400> 7 ccgctgttct cctaggtcag 20 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MEOX1 Reverse primer <400> 8 ccccaaagcc tcagatgtgc agc 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WNT3 Forward primer <400> 9 ggctgtgact cgcatcataa 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WNT3 Reverse primer <400> 10 cttgcatttg aggtgcatgt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ZIC1 Forward primer <400> 11 gcgctccgag aatttaaaga 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ZIC1 Reverse primer <400> 12 cgtggacctt catgtgtttg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX6 Forward primer <400> 13 gtgtccaacg gatgtgtgag 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX6 Reverse primer <400> 14 ctagccaggt tgcgaagaac 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FOXA2 Forward primer <400> 15 atgaacggca tgaacacgta 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FOXA2 Reverse primer <400> 16 tggagttcat gttggcgtag 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCR4 Forward primer <400> 17 ggtggtctat gttggcgtct 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CXCR4 Reverse primer <400> 18 tgcaatagca ggacaggatg 20 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> a-FD Forward primer <400> 19 caaacgatga agcaagagtt tct 23 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> a-FD Reverse primer <400> 20 aaaagttcac accgaatgaa aga 23 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD105 Forward primer <400> 21 gccagcattg tctcacttca 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD105 Reverse primer <400> 22 atgcgcaaca agctctttct 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD166 Forward primer <400> 23 cttgcacagc agaaaaccaa 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <166> CD166 Reverse primer <400> 24 ccagtagacg acaccagcaa 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD73 Forward primer <400> 25 cgcaacaatg gcacaattac 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD73 Reverse primer <400> 26 actcgacact tggtgcaaag 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD49a Forward primer <400> 27 tgggtgctta ttggttctcc 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD49a Reverse primer <400> 28 ctccatttgg gttggtgact 20 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CD44 Forward primer <400> 29 tggcacccgc tatgtccag 19 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CD44 Reverse primer <400> 30 gtagcaggga ttctgtctg 19 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Forward primer <400> 31 tggtatcgtg gaaggactca 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Reverse primer <400> 32 cctgcttcac caccttcttg 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PDGF-b Receptor Forward primer <400> 33 caactacatg gccccttacg 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PDGF-b Receptor Reverse primer <400> 34 ttcacagatg agcacgttcc 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD29 Forward primer <400> 35 caaaggaaca gcagagaagc 20 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CD29 Reverse primer <400> 36 attgagtaag acaggtccat aagg 24 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CD45 Forward primer <400> 37 ttcaacttat acccttcgtg tc 22 <210> 38 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CD45 Reverse primer <400> 38 cctgctttac tttgtccact tc 22 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD34 Forward primer <400> 39 tgaagcctag cctgtcacct 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD34 Reverse primer <400> 40 cgcacagctg gaggtcttat 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HLA-DR Forward primer <400> 41 caaagaagga gacggtctgg 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HLA-DR Reverse primer <400> 42 gggctctctc agttccacag 20 <210> 43 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 04-Oct Forward primer <400> 43 cgtgaagctg gagaaggaga agctg 25 <210> 44 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 04-Oct Reverse primer <400> 44 caagggccgc agcttacaca tgttc 25 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FABP-4 Forward primer <400> 45 tgcagcttcc ttctcacctt 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FABP-4 Reverse primer <400> 46 tcacatcccc attcacactg 20 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARg2 Forward primer <400> 47 ctccgtggat ctctccgtaa 20 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARg2 Reverse primer <400> 48 gatgcaggct ccactttgat 20 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Osteocalcin Forward primer <400> 49 ggcagcgagg tagtgaagag 20 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Osteocalcin Reverse primer <400> 50 agcagagcga caccctagac 20 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Runx2 Forward primer <400> 51 taggcgcatt tcagatgatg 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Runx2 Reverse primer <400> 52 gactggcggg gtgtaagtaa 20  

Claims (16)

삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 1) 영양 세포층이 있는 조건에서 배양된 인간 배아줄기세포(human Embryonic Stem Cells; 이하 hESCs)를 배양 접시로 옮긴 후, 영양 세포층이 없는 조건에서 처리 배지(Conditioned Medium; CM)로 1 내지 3일 동안 배양하는 단계;1) Human Embryonic Stem Cells (hESCs) cultured in the presence of a feeder cell layer were transferred to a culture dish, and then treated with Conditioned Medium (CM) for 1 to 3 days in the absence of the feeder cell layer. Culturing; 2) 상기 영양 세포층이 없는 조건에서 배양된 hESCs를 2-5 ng/㎖ Activin A, 2-5 mM 6-브로모인디루빈-3'-옥심(6-bromoindirubin-3'-oxime; 이하, BIO) 및 10-20 ng/㎖ BMP4(Bone morphogenic protein 4)를 포함하는 중배엽 유도 배지로 3 내지 5일 동안 hESCs로부터 중배엽을 유도하는 단계; 및,2) 2-5 ng / ml Activin A, 2-5 mM 6-bromoindirubin-3'-oxime (6-bromoindirubin-3'-oxime; hereinafter referred to as BIO) cultured in the absence of the feeder cell layer Inducing mesoderm from hESCs for 3-5 days with mesoderm induction medium comprising 10-20 ng / ml BMP morphogenic protein 4); And 3) 상기 hESCs 유래 중배엽을 5-20 ng/㎖ bFGF(basic fibroblast growth factor) 및 5-20 ng/㎖ PDGF-BB(Platelet-derived growth factor beta polypeptide)를 포함하는 중간엽 유도 배지로 3 내지 6일 동안 유도하는 단계를 포함하는 것을 특징으로 하는 hESCs로부터 중간엽 줄기세포(Mesenchymal Stem Cells; MSCs)를 제조하는 방법.3) The hESCs-derived mesoderm is a mesenchymal induction medium containing 5-20 ng / ml basic fibroblast growth factor (bFGF) and 5-20 ng / ml platelet-derived growth factor beta polypeptide (PDGF-BB). Method for producing mesenchymal stem cells (MSCs) from hESCs comprising the step of inducing for one day. 제 9항에 있어서, Activin A, BIO 및 BMP4의 처리농도는 5 ng/㎖ Activin A, 2 mM BIO 및 20 ng/㎖ BMP4인 것을 특징으로 하는 hESCs로부터 중간엽 줄기세포를 제조하는 방법.10. The method of claim 9, wherein the treatment concentrations of Activin A, BIO and BMP4 are 5 ng / ml Activin A, 2 mM BIO and 20 ng / ml BMP4. 제 9항에 있어서, bFGF 및 PDGF-BB의 처리농도는 20 ng/㎖ bFGF이고 20 ng/㎖ PDGF-BB인 것을 특징으로 하는 hESCs로부터 중간엽 줄기세포를 제조하는 방법.The method of claim 9, wherein the treatment concentration of bFGF and PDGF-BB is 20 ng / ml bFGF and 20 ng / ml PDGF-BB method for producing mesenchymal stem cells from hESCs. 제 9항에 있어서, 상기 영양 세포층은 STO 배양 세포층인 것을 특징으로 하는 hESCs로부터 중간엽 줄기세포를 제조하는 방법.10. The method of claim 9, wherein said feeder cell layer is an STO culture cell layer. 제 9항에 있어서, 상기 처리 배지는 STO 배양액인 것을 특징으로 하는 hESCs로부터 중간엽 줄기세포를 제조하는 방법.10. The method of claim 9, wherein the treatment medium is STO culture. 제 9항에 있어서, 상기 배양 접시는 마트리겔로 코팅된 것을 특징으로 하는 hESCs로부터 중간엽 줄기세포를 제조하는 방법.The method of claim 9, wherein the culture dish is coated with Matrigel method for producing mesenchymal stem cells from hESCs. 제 9항에 있어서, 중간엽 줄기세포는 지방세포로 분화가 가능한 중간엽 줄기세포인 것을 특징으로 하는 hESCs로부터 중간엽 줄기세포를 제조하는 방법.10. The method of claim 9, wherein the mesenchymal stem cells are mesenchymal stem cells capable of differentiating into adipocytes. 제 9항에 있어서, 중간엽 줄기세포는 골아세포로 분화가 가능한 중간엽 줄기세포인 것을 특징으로 하는 hESCs로부터 중간엽 줄기세포를 제조하는 방법.10. The method of claim 9, wherein the mesenchymal stem cells are mesenchymal stem cells capable of differentiation into osteoblasts.
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