KR101686315B1 - A method for differentiation of tonsil-derived mesenchymal stem cell into schwann cells - Google Patents
A method for differentiation of tonsil-derived mesenchymal stem cell into schwann cells Download PDFInfo
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- KR101686315B1 KR101686315B1 KR1020150003572A KR20150003572A KR101686315B1 KR 101686315 B1 KR101686315 B1 KR 101686315B1 KR 1020150003572 A KR1020150003572 A KR 1020150003572A KR 20150003572 A KR20150003572 A KR 20150003572A KR 101686315 B1 KR101686315 B1 KR 101686315B1
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Abstract
본 발명은 편도 유래 줄기세포로부터 슈반 세포의 분화방법 및 이를 이용한 세포 치료제에 관한 것이다.
본 발명의 분화 방법은 슈반 세포로의 높은 분화능을 보여 다량의 슈반 세포의 확보가 가능하며, 본 발명에 따라 분화된 슈반 세포는 버려지는 자가 조직을 사용함으로써 세포 공여부의 신경 손상 없이 제조될 수 있으며, 신경 세포를 지지하고 신경을 재생하여 신경 관련 질환의 세포 치료제로써 우수한 이용가능성을 보인다. The present invention relates to a method of differentiating Schwann cells from one-way stem cells and a cell therapy agent using the same.
The Schwann cells differentiated according to the present invention can be produced without the nerve damage of the cell donor part by using the abandoned autologous tissue. , Supporting neurons and regenerating neurons, thus showing excellent utility as a cell therapy for neuronal diseases.
Description
본 발명은 편도 유래 중간엽 줄기세포로부터 슈반 세포의 분화방법 및 이를 이용한 세포 치료제에 관한 것이다. The present invention relates to a method of differentiating Schwann cells from one-way derived mesenchymal stem cells and a cell therapy agent using the same.
줄기세포(stem cell)는 생물 조직을 구성하는 다양한 세포들로 분화할 수 있는 세포로서 배아, 태아 및 성체의 각 조직에서 얻을 수 있는 분화되기 전 단계의 미분화 세포들을 총칭한다. 줄기세포는 분화 자극(환경)에 의하여 특정 세포로 분화가 진행되고, 세포분열에 의해 자신과 동일한 세포를 생산(self-renewal)할 수 있는 특성이 있으며, 분화 자극에 따라 상이한 세포로도 분화될 수 있는 유연성(plasticity)을 가지고 있는 것이 특징이다. Stem cells are the cells that can differentiate into various cells constituting biological tissue and collectively referred to as the undifferentiated cells in the pre-differentiation stage which can be obtained from each tissue of embryo, fetus and adult body. Stem cells are characterized by the ability to differentiate into specific cells by differentiation stimuli (environment), self-renewal by the division of cells by self-renewal, and differentiated into different cells by differentiation stimuli It is characterized by having plasticity.
줄기세포는 그 분화능에 따라 만능(pluripotency), 다분화능(multipotency) 및 단분화능(unipotency) 줄기세포로 나눌 수 있다. 만능줄기세포(pluripotent stem cells)는 모든 세포로 분화될 수 있는 잠재력을 지닌 전분화능(pluripotency)의 세포이며, 일부 줄기세포는 다분화능 또는 단분화능의 잠재력을 지닌다. Stem cells can be divided into pluripotency, multipotency, and unipotency stem cells according to their differentiation potential. Pluripotent stem cells are pluripotent cells with the potential to differentiate into all cells, and some stem cells have the potential of multipotential or mono-differentiability.
상기 줄기세포들이 가지는 분화능을 기초로 세포 치료제로 이용 가능성이 있어 이와 관련된 연구 개발이 활발히 진행중이다. 하지만, 배아 줄기세포를 이용한 세포 치료제의 경우 윤리적 문제나 조직 적합성 불일치 문제가 제기되고 있으며, 역분화 줄기세포를 세포 치료제로 사용할 경우 종양 발생 가능성의 문제가 있다. The stem cells can be used as a cell therapy agent on the basis of the differentiation ability of the stem cells, and research and development related thereto are actively underway. However, in the case of cell therapy using embryonic stem cells, problems of ethical problems and inconsistency of tissue compatibility are raised, and there is a problem of possibility of tumor development when using the degenerated stem cell as a cell therapy agent.
이에 따라, 분화능이 낮은 것으로 알려져 있지만 상대적으로 안전한 중간엽줄기세포를 이용한 연구가 많이 진행되고 있다. 중간엽 줄기세포(mesenchymal stem cell, MSCs)는 성체 골수 등에 있는 multi-potential non-hematopoietic progenitor cell로서 지방, 연골, 뼈, 근육, 피부 등 다양한 종류의 세포로 분화할 수 있는 세포를 말한다. 이러한 중간엽 줄기세포를 이용하여 다양한 조직 재생을 위한 임상 연구가 진행되고 있으며, 장기이식 분야에도 적용가능성을 보이고 있다. Thus, studies using mesenchymal stem cells, which are known to have low ability to differentiate, are relatively safe. Mesenchymal stem cells (MSCs) are multi-potential non-hematopoietic progenitor cells in the adult bone marrow. They are cells capable of differentiating into various types of cells such as fat, cartilage, bone, muscle and skin. Clinical studies for regeneration of various tissues using such mesenchymal stem cells are under way, and they are applicable to organ transplantation.
그러나, 중간엽 줄기세포 중에서도 몇몇 줄기세포는 그 이용에 있어서 세포를 얻는데 큰 제한이 있기 때문에 그 이용이 어렵다. 예를 들어, 제대혈, 지방조직으로부터 유래한 중간엽 줄기세포는 침습적인 방법을 이용해 얻어야만 한다. 가장 비침습적인 방법을 이용해 얻을 수 있는 세포는 골수채취를 통한 중간엽 줄기세포이지만, 골수채취는 마취가 필요하고 고통을 유발하여, 그 이용에 제한이 있다. 이에 대한 대안으로 환자 맞춤형 줄기세포를 분리하기 위해 말초혈액을 이용한 세포획득법 등이 요구되고 있으나 말초혈액만으로는 성인에서 분리할 수 있는 중간엽 줄기세포의 수가 너무 적고 분리방법이 경제적이지 못하며, 분리해낸다고 해도 세포치료에 사용가능한 양만큼 증식이 원활하지 않은 경우가 대부분이기에 좀 더 실용성을 높일 수 있는 대체 방안이 필요하다. However, among mesenchymal stem cells, some stem cells are difficult to use because of their great limitations in obtaining cells for their use. For example, mesenchymal stem cells derived from umbilical cord blood and adipose tissue must be obtained using invasive methods. The most non-invasive cells are mesenchymal stem cells through bone marrow harvesting, but bone marrow harvesting requires anesthesia and causes pain, which limits its use. In order to isolate patient-tailored stem cells, a cell harvesting method using peripheral blood is required as an alternative. However, the number of mesenchymal stem cells that can be isolated from adults is too small, the separation method is not economical, However, since it is difficult to proliferate by an amount that can be used for cell therapy, an alternative method that can increase practicality is needed.
또한, 고령인 환자에서 얻는 성체 줄기세포는 낮은 연령에게서 얻는 세포들에 비해 증식능력이 현저히 떨어지며 각종 인자들의 분비 및 줄기세포의 병변으로 이동 능력 등이 떨어지기 때문에, 저연령의 환자로부터 자연스럽게 분리될 수 있거나 버려지는 조직으로부터 세포를 얻을 필요성이 있다. 또한, 이렇게 얻어진 세포는 실험에 용이한 양적 확보가 가능하고 세포의 계대 배양 시에 분화능이 잘 유지될 필요성이 있다. In addition, adult stem cells obtained from elderly patients have a significantly lower proliferative capacity than the cells obtained from lower ages, and secretion of various factors and migration ability to stem cell lesions are reduced. Therefore, adult stem cells are naturally separated from patients of lower ages There is a need to obtain cells from tissues that can or are discarded. In addition, the cells obtained in this manner can be easily quantitated in the experiment, and it is necessary that the differentiation ability is maintained well when the cells are subcultured.
한편, 신경병증과 관련된 다양한 질환에서 줄기세포로부터 분화된 세포 치료제와 관련된 연구 개발이 요구되고 있다. 예컨대, 유전성 신경병증으로 샤르코 마리 투스 병 (Charot-Marie-Tooth disease) 또는 유전성 신경 편향 압박마비증세 (Hereditary neuropathy with liability to pressure palsies); 말초신경 퇴화 (Peripheral nerve degeneration) 또는 말초신경 절단에 의한 외상성 신경 질환; 또는 대사성 신경병증으로 당뇨병의 합병증으로 발생한 신경병변, 예컨대 당뇨병성 말초 신경병증 (diabetes neuropathy) 등의 질환에서 손상 부위의 기능적 재생을 위한 세포 치료제에 대한 연구 개발이 지속적으로 요구되고 있다. 이를 위해서는 신경계를 구성하는 여러 세포들 중, 특히 신경의 축삭을 감싸고 있는 슈반세포가 중요한 역할을 담당하는바, 세포 치료제로써 슈반 세포에 대한 다량 확보가 필요하다. On the other hand, in various diseases related to neuropathy, research and development related to a cell therapy agent differentiated from stem cells is required. For example, hereditary neuropathy may be caused by Charot-Marie-Tooth disease or hereditary neuropathy with liability to pressure palsies; Traumatic neuropathy due to peripheral nerve degeneration or peripheral nerve shearing; Or metabolic neuropathy caused by complications of diabetes, such as diabetic neuropathy (diabetes neuropathy), such as diseases, such as the functional regeneration of the cell for therapeutic treatment for the development of research and development is required. To do this, Schwann cells, which encircle the axons of the nerves, play an important role among the various cells forming the nervous system, and it is necessary to secure a large amount of Schwann cells as a cell therapy agent.
그런데, 슈반 세포는 그 공급이 원활하지 않고 생체 외 배양이 쉽지 않은 문제점이 있다. 예를 들어, 슈반세포(Schwann cell)를 얻기 위해서는 세포 공여부의 신경 손상이 불가피하고 세포의 양적확보가 어렵다는 단점이 있기 때문에 관련 연구 개발이 쉽지 않다. 또한, 슈반 세포 분화용 배지를 이용하여 성체 줄기세포로부터 슈반 세포로 분화를 유도하는 연구들이 일부 있지만, 분화 효율 면에서 그 이용이 매우 제한적이다. 또한, 상이한 유전적 기원, 유래 및/또는 배경을 가지는 중간엽 줄기세포들의 경우 중간엽 줄기세포를 규정하는 일부 기준들에 대해서는 서로 유의적 차이를 나타내지 아니하지만 통상적으로 각각의 생체 내 활성에 있어서는 매우 큰 차이를 나타내는바, 특정 유래의 줄기세포로부터 분화 방법 및 분화된 세포 특성에 대한 확인 역시 필요하다. However, Schwann cells are not smoothly supplied and in vitro culturing is difficult. For example, in order to obtain schwann cells, nerve damage of the cell donor is inevitable, and it is difficult to obtain quantitative cells, so that research and development is difficult. In addition, there are some studies that induce differentiation from adult stem cells into Schwann cells using the Schwann cell differentiation medium, but its use in the efficiency of differentiation is very limited. In addition, in the case of mesenchymal stem cells having different genetic origins, origins and / or backgrounds, there is no significant difference in some criteria defining mesenchymal stem cells, And it is also necessary to confirm the differentiation method and the differentiated cell characteristics from the stem cells of a specific origin.
이에 따라, 슈반 세포로의 높은 분화능을 보일 수 있는 특정 줄기세포로부터 최적의 분화 방법을 확인하여 인체 적용에 적합한 슈반 세포를 확보하는 방안에 대한 연구 개발이 필요한 실정이다. Therefore, it is necessary to research and develop a method for securing Schwann cells suitable for human application by confirming the optimal differentiation method from specific stem cells which can show high differentiation ability to Schwann cells.
본 발명은 표피 성장 인자 (Epidermal growth factor), 섬유 아세포 성장 인자 (basic fibroblast growth factor) 및 B27 첨가제를 포함하는 배지에서 편도 유래 중간엽 줄기세포를 배양하는 단계를 포함하는 편도 유래 중간엽 줄기세포로부터 신경구 (Neurosphere) 분화 방법에 관한 것이다. The present invention relates to a method for producing a mesenchymal stem cell from a mesenchymal stem cell comprising a step of culturing a mesenchymal stem cell from a mononuclear cell in a culture medium containing an epidermal growth factor, a basic fibroblast growth factor and a B27 additive The present invention relates to a method of differentiating neurospheres.
본 발명은 또한 (a) 표피 성장 인자 (Epidermal growth factor), 섬유아세포 성장 인자 (basic fibroblast growth factor) 및 B27 첨가제를 포함하는 배지에서 편도 유래 중간엽 줄기세포를 배양하는 단계를 포함하는 편도 유래 중간엽 줄기세포로부터 신경구 (Neurosphere)를 유도하는 단계; 및The present invention also relates to a method for the treatment of one or more metastatic mesenchymal stem cells comprising the steps of: (a) culturing one-way derived mesenchymal stem cells in a medium comprising an epidermal growth factor, a fibroblast growth factor, Inducing a neurosphere from a leaf stem cell; And
(b) 상기 (a) 단계의 신경구(Neurosphere)를 혈소판 유래 성장인자 (Platelet-derived growth factor), 섬유아세포 성장 인자 (basic fibroblast growth factor), 헤레굴린-β(Heregulin-β) 및 포스콜린 (Forskolin) 하에서 배양하여 슈반세포를 유도하는 단계를 포함하는 편도 유래 중간엽 줄기세포로부터 슈반 세포 분화 방법에 관한 것이다. (b) comparing the Neurosphere of step (a) with Platelet-derived growth factor, basic fibroblast growth factor, Heregulin-beta and phoscholine Forskolin) to induce schwann cells. The present invention also relates to a method for differentiating schwann cells from one-way derived mesenchymal stem cells.
본 발명은 또한 (a) 표피 성장 인자 (Epidermal growth factor), 섬유아세포 성장 인자 (basic fibroblast growth factor) 및 B27 첨가제를 포함하는 배지에서 편도 유래 중간엽 줄기세포를 배양하는 단계를 포함하는 편도 유래 중간엽 줄기세포로부터 신경구 (Neurosphere)를 유도하는 단계;The present invention also relates to a method for the treatment of one or more metastatic mesenchymal stem cells comprising the steps of: (a) culturing one-way derived mesenchymal stem cells in a medium comprising an epidermal growth factor, a fibroblast growth factor, Inducing a neurosphere from a leaf stem cell;
(b) 상기 (a) 단계의 신경구(Neurosphere)를 혈소판 유래 성장인자 (Platelet-derived growth factor), 섬유아세포 성장 인자 (basic fibroblast growth factor), 헤레굴린-β(Heregulin-β) 및 포스콜린 (Forskolin) 하에서 배양하여 슈반 세포를 분화시키는 단계; 및(b) comparing the Neurosphere of step (a) with Platelet-derived growth factor, basic fibroblast growth factor, Heregulin-beta and phoscholine Forskolin) to differentiate Schwann cells; And
(c) 상기 (b) 단계에서 분화된 슈반 세포를 배양하여 슈반세포로부터 분비된 신경 영양인자를 포함하는 배양 배지를 얻는 단계를 포함하는, 신경 영양인자 조성물의 제조방법에 관한 것이다. (c) culturing Schwann cells differentiated in step (b) to obtain a culture medium containing a neurotrophic factor secreted from schwann cells.
본 발명은 또한 상기 편도 유래 중간엽 줄기세포로부터 슈반 세포 분화 방법에 따라 제조된 슈반 세포에 관한 것이다.The present invention also relates to Schwann cells produced by the Schwann cell differentiation method from said one-shot-derived mesenchymal stem cells.
본 발명은 또한 상기 신경 영양인자 조성물의 제조방법에 따라 제조된 신경 영양인자 조성물에 관한 것이다. The present invention also relates to a neurotrophic factor composition prepared according to the method for preparing said neurotrophic factor composition.
본 발명자들은 인체 적용에 적합한 슈반 세포의 대량 생산 방법을 연구하던 중, 편도 유래 중배엽 줄기 세포로부터 슈반 세포를 단기간에 대량 생산하는 방법을 발명하여 본 발명을 완성하였다. The inventors of the present invention completed the present invention by inventing a method for mass production of schwann cells from one-shot-derived mesenchymal stem cells in a short period of time while studying a method for mass production of schwann cells suitable for human application.
본 발명에 있어서, 편도 유래 중간엽 줄기세포란 목의 안쪽과 코의 뒷부분에 위치하여 외부에서 침입하는 세균 등의 물질로부터 일차적으로 우리 몸을 방어함과 동시에 림프상피 면역조직으로 작용을 수행하는 조직인 편도에서 유래된 자기 복제 능력을 가지면서 두 개 이상의 새로운 세포로 분화하는 능력을 가진 미분화된 줄기세포를 의미한다. In the present invention, a mesenchymal stem cell originating from one side is located in the back of the throat and the nose, and is a tissue that acts primarily as a lymphocyte immune tissue while defending the body from substances such as bacteria that enter from the outside Means an undifferentiated stem cell having the ability to replicate into two or more new cells while having self-replicating ability derived from one-way.
본 발명에 있어서, 신경구(Neurosphere)란 신경 줄기세포의 free-floating cluster로 태아나 성인의 중추신경계로부터 유래한 신경 줄기세포와 형태학적 측면및 분화 가능성의 측면에서 유사한 성격을 띠는 세포로, 슈반 세포, 신경세포 (neuron), 신경교세포, 성상교세포(astrocyte), 또는 희소돌기아교세포(oligodendrocyte)등의 신경 관련 세포로 분화가 가능한 세포를 의미한다. In the present invention, the neurosphere is a free-floating cluster of neural stem cells, which is similar in morphological aspect and differentiability to neural stem cells derived from the central nervous system of the fetus or adult. Refers to a cell capable of differentiating into neuron-related cells such as cells, neurons, glial cells, astrocyte, or oligodendrocytes.
본 발명에 있어서, 슈반 세포란 말초 신경계의 신경교세포로 신경의 발생, 분화에 중요한 역할을 할 뿐 아니라 신경이 손상된 경우 축삭의 재생과 재수초화(remyelination)에 필수적인 역할을 하는 세포를 의미한다. 특히, 축삭이 모여 다발을 이룬 것을 신경 섬유라고 부르는데, 슈반세포는 축삭을 둘러싸는 가장 바깥쪽의 막인 신경 섬유초를 형성하며, 슈반세포에 의해 수초화를 이룬 유수신경섬유는 수초의 절연체 역할에 의해 빠른 신경전도 속도를 가질 수 있는 반면 수초에 손상이 있을 경우 전도 속도가 느려지고 비수초화에 의한 축삭의 이차적 손상을 야기 시킬 수 있다. In the present invention, Schwann cells are peripheral nervous system glial cells, which play an important role not only in the development and differentiation of nerves, but also in cells that play an essential role in axon regeneration and remyelination when the nerve is damaged. In particular, the bundle of axons is called the nerve fiber. Schwann cells form the outermost layer of the nerve fiber, which surrounds the axon, and the anterior nerve fibers, which have been hydrated by Schwann cells, While it can have fast nerve conduction velocity, damage to the myelin can slow conduction velocity and cause secondary damage of axon by non-herbalization.
본 발명에 있어서, 신경 영양인자 조성물은 본 발명의 분화 방법에 따라 편도 유래 중간엽 줄기세포로부터 분화된 슈반 세포의 배양에 따라 제조되는 슈반세포로부터 분비된 신경 영양인자를 포함하는 조성물로 축삭, 신경극 등의 신경 세포의 성장 및 회복을 유도하는 신경 영양 인자를 포함하는 조성물을 의미한다. 상기 신경 영양인자 조성물은 Desert Hedgehog (DHH), Neurotrophin-3 (NT-3), Nerve growth factor (NGF), Glial cell line-derived neurotrophic factor (GDNF), F-Spondin, Brain-derived neurotrophic factor (BDNF)등의 신경 영양 인자를 포함하며, 슈반 세포로부터 분비 가능한 신경 영양 인자라면 이에 제한되지 않고 본 발명의 범주 내에 포함될 수 있다. In the present invention, the neurotrophic factor composition is a composition comprising a neurotrophic factor secreted from Schwann cells produced by culturing Schwann cells differentiated from a mesenchymal stem cell according to the present invention, Quot; means a composition comprising a neurotrophic factor that induces the growth and recovery of nerve cells such as nerve cells. The neurotrophic factor composition may be selected from the group consisting of Desert Hedgehog (DHH), Neurotrophin-3 (NT-3), Nerve growth factor (NGF), Glial cell line-derived neurotrophic factor (GDNF), F- ), And may be included in the scope of the present invention without being limited thereto, as long as it is a neurotrophic factor capable of releasing from Schwann cells.
본 발명은 올트랜스레티놀산 (all trans-retinoic acid, ATRA), B27 첨가제, N2 첨가제, 인슐린 유사 성장 인자 (Insulin-like growth factor, IGF), 신경 성장 인자 (Nerve growth factor, NGF), 헤파린 (heparin), 섬유 아세포 성장 인자 (basic fibroblast growth factor, bFGF) 또는 표피 성장 인자 (Epidermal growth factor, EGF)로부터 선택되는 어느 하나 이상을 포함하는 배지에서 편도 유래 중간엽 줄기세포를 배양하는 단계를 포함하는 편도 유래 중간엽 줄기세포로부터 신경구 (Neurosphere) 분화 방법을 제공한다. The present invention relates to a pharmaceutical composition comprising all trans-retinoic acid (ATRA), B27 additive, N2 additive, insulin-like growth factor (IGF), nerve growth factor (NGF) cultured mesenchymal stem cells in a culture medium containing at least one selected from heparin, basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) And provides a method of differentiating neurospheres from one-way derived mesenchymal stem cells.
바람직하게, 본 발명은 표피 성장 인자 (Epidermal growth factor), 섬유 아세포 성장 인자 (basic fibroblast growth factor) 및 B27 첨가제를 포함하는 배지에서 편도 유래 중간엽 줄기세포를 배양하는 단계를 포함하는 편도 유래 중간엽 줄기세포로부터 신경구 (Neurosphere) 분화 방법을 제공한다. Preferably, the present invention provides a method for the treatment of a mesenchymal stem cell comprising the step of culturing a mesenchymal stem cell from a mononuclear cell in a medium comprising an epidermal growth factor, a basic fibroblast growth factor and a B27 additive. Provides a method of differentiating neurospheres from stem cells.
본 발명에 있어서, 표피 성장 인자 (Epidermal growth factor)는 표피 증식, 케라틴화 및 사람 섬유아세포 증식 등을 촉진하는 폴리펩티드성 인자를 의미한다. In the present invention, epidermal growth factor refers to a polypeptide factor that promotes epidermal proliferation, keratinization, and human fibroblast proliferation.
본 발명에 있어서, 섬유 아세포 성장 인자 (basic fibroblast growth factor)는 섬유아세포를 자극하여 강한 증식성을 유도하는 성장 인자로 신경세포의 성장을 촉진하는 성장인자를 의미한다. In the present invention, a basic fibroblast growth factor refers to a growth factor that stimulates fibroblasts to induce strong proliferation and promotes growth of neurons.
본 발명에 있어서, B-27 첨가제는 줄기세포로부터 신경구 유도에 사용되는 첨가제로 상업적으로 구입가능(Gibco 등)하며, serum-free 첨가제로 해마와 중추신경계의 신경 세포를 키우는데 생존과 성장을 돕는 기능을 가진 첨가제를 의미한다. In the present invention, the B-27 additive is commercially available as an additive for inducing nerve root from stem cells (Gibco et al.), And is a serum-free additive to nerve cells of the hippocampus and central nervous system, ≪ / RTI >
본 발명에 있어서, 상기 배양은 6일 내지 15일 동안 수행되는 것이 바람직하다. 또한, 신경구 유도 배양액 5 ml 당 2.5×106 개~ 4×106 개의 세포를 부유 상태에서 배양하는 것이 바람직하다. 또한, 상기 배지는 alpha MEM(alpha Minimum Essential Medium Eagle), Neurobasal medium, 또는 DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12)으로부터 선택되는 어느 하나가 바람직하며, 보다 바람직하게 DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12)를 사용한다. 또한, 상기 표피 성장인자는 10 ng/ml 내지 50 ng/ml, 섬유 아세포 성장 인자는 5 ng/ml 내지 50 ng/ml 및 B27은 0.5% 내지 5%의 농도(상업적으로 이용가능한 B27 첨가제를 이용하였을 때, 배지 총 중량에 대한 중량 %)로 첨가되는 것이 바람직하다. In the present invention, the culturing is preferably carried out for 6 to 15 days. It is also preferable to cultivate 2.5 x 10 6 to 4 x 10 6 cells per 5 ml of the nerve root induction medium in a floating state. The medium is preferably selected from alpha MEM (alpha minimum essential medium medium), Neurobasal medium, or DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), more preferably DMEM / F12 Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12). In addition, the epidermal growth factor has a concentration of 10 ng / ml to 50 ng / ml, a fibroblast growth factor of 5 ng / ml to 50 ng / ml and B27 at a concentration of 0.5% to 5% (commercially available B27 additive By weight based on the total weight of the culture medium).
본 발명의 신경구 분화 방법은 편도 유래 중간엽 줄기세포를 사용하여, 타 성체 줄기세포를 사용한 방법과 대비하여 현저한 분화능을 보이며, 세포구의 형성 속도 및 부피 성장의 속도가 빠르다는 장점을 가진다. 또한, 신경구 역시 계대 배양이 가능한데, 10-40 gauge needle을 이용하여 분쇄함으로 쉽고 안정적으로 이루어진다는 장점을 가진다. 또한, 본 발명의 분화 방법에 따라 분화된 신경구는 슈반 세포, 신경세포, 성상교세포, 희소돌기아교세포 등의 신경 관련 세포로 분화 가능하여, 신경구로부터 분화된 이러한 세포는 여러 신경 손상 질병의 세포 치료제나 혹은 약물 약효 검사의 대상으로 이용 가능하다. 뿐만 아니라 신경세포의 전구체인 신경구를 분화 전 상태로 이식하여 신경구 자체를 세포치료제로서 이용도 가능하다.The neural differentiation method of the present invention has the advantage of remarkably differentiating ability compared with the method using mesenchymal stem cells using one-way derived mesenchymal stem cells, and has a rapid rate of cell sphere formation and volume growth. In addition, the nerve can also be subcultured, and it has an advantage that it is easy and stable by grinding using 10-40 gauge needle. In addition, according to the differentiation method of the present invention, the differentiated neural nerve can be differentiated into neural-related cells such as Schwann cells, neurons, astrocytes, rarely proliferating glial cells and the like, It can be used as a target of me or drug drug testing. In addition, the neurons, which are precursors of neurons, can be transplanted into the differentiated state, and the neurons themselves can be used as a cell therapy agent.
본 발명은 또한, 올트랜스레티놀산 (all trans-retinoic acid, ATRA), B27 첨가제, N2 첨가제, 인슐린 유사 성장 인자 (Insulin-like growth factor, IGF), 신경 성장 인자 (Nerve growth factor, NGF), 헤파린 (heparin), 섬유 아세포 성장 인자 (basic fibroblast growth factor, bFGF) 또는 표피 성장 인자 (Epidermal growth factor, EGF)로부터 선택되는 어느 하나 이상을 포함하는 배지에서 편도 유래 중간엽 줄기세포를 배양하는 단계를 포함하는 편도 유래 중간엽 줄기세포로부터 신경구 (Neurosphere)를 유도하는 단계; 및 상기 (a) 단계의 신경구를 헤파린(heparin), 라미닌 (laminin), Nerve growth factor (NGF), 아스콜빅 엑시드 (ascorbic acid), glial growth factor-2 (GGF-2), N2 supplement, 글루타민 glutamine, 헤레귤린(heregulin), 포스콜린(forskolin), 혈소판 유래 성장인자(PDGF) 또는 섬유 아세포 성장 인자 (basic fibroblast growth factor)으로부터 선택된 어느 하나 이상을 포함하는 배지 하에서 배양하여 슈반 세포를 유도하는 단계를 포함하는 편도 유래 중간엽 줄기세포로부터 슈반 세포 분화 방법을 제공한다. The present invention also relates to a pharmaceutical composition comprising all trans-retinoic acid (ATRA), B27 additive, N2 additive, insulin-like growth factor (IGF), nerve growth factor (NGF) Culturing the mesenchymal stem cells from a medium containing at least one selected from heparin, basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) Inducing neurospheres from the mesenchymal stem cells comprising the monocyte; Wherein the nerve of step (a) is selected from the group consisting of heparin, laminin, Nerve growth factor (NGF), ascorbic acid, glial growth factor-2 (GGF- inducing Schwann cells by culturing under an atmosphere containing at least one selected from the group consisting of glutamine, heregulin, forskolin, platelet-derived growth factor (PDGF) or basic fibroblast growth factor Derived mesenchymal stem cells containing Schwann cells.
바람직하게, 본 발명은 (a) 표피 성장 인자 (Epidermal growth factor), 섬유아세포 성장 인자 (basic fibroblast growth factor) 및 B27 첨가제를 포함하는 배지에서 편도 유래 중간엽 줄기세포를 배양하는 단계를 포함하는 편도 유래 중간엽 줄기세포로부터 신경구 (Neurosphere)를 유도하는 단계; 및 (b) 상기 (a) 단계의 신경구(Neurosphere)를 혈소판 유래 성장인자 (Platelet-derived growth factor), 섬유아세포 성장 인자 (basic fibroblast growth factor), 헤레굴린-β(Heregulin-β) 및 포스콜린 (Forskolin) 하에서 배양하여 슈반 세포를 유도하는 단계를 포함하는 편도 유래 중간엽 줄기세포로부터 슈반 세포 분화 방법을 제공한다. Preferably, the present invention relates to a method for the treatment of amygdala, comprising the steps of: (a) culturing one-way derived mesenchymal stem cells in a medium comprising Epidermal growth factor, basic fibroblast growth factor and B27 additive; Inducing a neurosphere from the derived mesenchymal stem cells; And (b) comparing the Neurosphere of step (a) with Platelet-derived growth factor, basic fibroblast growth factor, Heregulin-beta and Phoscholine And inducing Schwann cells by culturing under Forskolin. The present invention also provides Schwann cell differentiation from unilateral derived mesenchymal stem cells.
본 발명에 있어서, 상기 (a) 단계의 신경구 (Neurosphere)를 유도하는 단계는 상기 편도 유래 중간엽 줄기세포로부터 신경구 (Neurosphere) 분화 방법과 같이 적용될 수 있다. In the present invention, the step of inducing the neurosphere of step (a) may be applied as a neurosphere differentiation method from the mesenchymal stem cells derived from the unilateral.
상기 (b) 단계는 신경구 (Neurosphere)로부터 슈반세포를 유도함으로써 슈반 세포를 분화한다. The step (b) differentiates Schwann cells by inducing schwann cells from Neurosphere.
본 발명에 있어서, 혈소판 유래 성장인자 (Platelet-derived growth factor)는 혈소판 과립에 존재하는 당단백질로 간엽계 세포의 증식을 촉진하는 인자를 말한다. In the present invention, the platelet-derived growth factor is a glycoprotein present in platelet granules and refers to a factor promoting the proliferation of mesenchymal cells.
본 발명에 있어서, 섬유아세포 성장 인자 (basic fibroblast growth factor)는 앞서 정의한 바와 같다. In the present invention, the basic fibroblast growth factor is as defined above.
본 발명에 있어서, 헤레굴린-β(Heregulin-β)는 Neuregulin 1이 alternative splicing에 의해 생성되는 isotype 중 하나로 리셉터 타이로신 카이나제 (receptor tyrosine kinase)인 ERBB2와 반응한다. 다양한 신호 전달을 통해 신경세포의 성장과 분화를 촉진 시키는 역할을 하는데 특히 슈반 세포와 관련해서는 축삭으로부터 분비되는 헤레굴린-β가 슈반세포의 이동을 유도하여 수초화를 시키고, 또한 슈반세포가 자가 사멸에 들어가는 것을 억제하는 기능을 한다.In the present invention, heregulin-beta (Heregulin-beta) is one of the isotypes produced by alternative splicing of
본 발명에 있어서, 포스콜린 (Forskolin)은 하기 화학식 1의 구조를 가지는 화합물로, 아데닐릴 싸이클레이즈(adenylyl cyclase)의 직접적인 활성인자로서 헤레굴린-β와 함께 슈반세포의 증식을 높이는 시너지 효과를 보이는 단백질을 의미한다. In the present invention, Forskolin is a compound having a structure represented by the following formula (1), and has synergistic effect of increasing the proliferation of Schwann cells together with herlegulin-beta as a direct activator of adenylyl cyclase Protein.
[화학식 1][Chemical Formula 1]
본 발명에 있어서, 상기 (b) 단계의 배양은 바람직하게 8 내지 15일 동안 수행되어 신경구로부터 슈반 세포를 분화한다. 또한, 상기 (b) 단계의 배양 배지는 alpha MEM(alpha Minimum Essential Medium Eagle), Neurobasal medium, 또는 DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12)으로부터 선택되는 어느 하나이며, 바람직하게 DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) 배지이다. 또한, 상기 (b) 단계에서 바람직하게 상기 혈소판 유래 성장인자는 1 ng/ml 내지 10 ng/ml, 상기 섬유아세포 성장 인자는 1 ng/ml 내지 20 ng/ml, 상기 헤레굴린-β(Heregulin-β)는 10 ng/ml 내지 400 ng/ml, 및 상기 포스콜린은 2 uM 내지 25 uM의 농도로 첨가된다. 또한, 상기 신경구는 배양 전 분쇄되어 뭉쳐있는 세포가 분쇄될 수 있으며, 예컨대, 10-40 gauge needle을 이용하여 분쇄할 수 있다. 또한, 상기 배양은 poly-L-lysine, collagen 또는 laminin으로부터 선택된 물질로 코팅된 배양접시, 바람직하게 laminin이 1ug/ml 내지 10 ug/ml 코팅된 배양접시 내에서 배양된다. 또한, 상기 (b) 단계에서 배양 배지는 FBS(Fetal bovine serum)를 더 포함할 수 있으며, 바람직하게 5 내지 12%(상업적으로 이용가능한 FBS를 이용하였을 때, 배지 총 중량에 대한 중량 %)로 포함한다. In the present invention, the culture of step (b) is preferably performed for 8 to 15 days to differentiate Schwann cells from the nerve root. The culture medium of step (b) is any one selected from alpha MEM, Neurobasal medium, or DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) medium. Preferably, the platelet-derived growth factor is 1 ng / ml to 10 ng / ml, the fibroblast growth factor is 1 ng / ml to 20 ng / ml, the Heregulin- β) is added at a concentration of 10 ng / ml to 400 ng / ml, and the phosholin is added at a concentration of 2 uM to 25 uM. In addition, the nerve root can be pulverized before culturing, and the pulverized cells can be pulverized, for example, by using a 10-40 gauge needle. The culture is also cultivated in a culture dish coated with a material selected from poly-L-lysine, collagen or laminin, preferably in a culture dish coated with 1 ug / ml to 10 ug / ml of laminin. In addition, in the step (b), the culture medium may further contain FBS (Fetal bovine serum), preferably 5 to 12% (when using commercially available FBS, weight% based on the total weight of the culture medium) .
또한, 본 발명에 따른 분화 방법에 있어서, 추가로 glutamine, insuline 등을 첨가제로 추가할 수 있다. Further, in the differentiation method according to the present invention, glutamine, insulin, etc. may be added as an additive.
본 발명의 슈반 세포 분화 방법은 편도 유래 중간엽 줄기세포를 사용하여, 타 유래의 성체 줄기세포를 사용한 방법과 대비하여 현저한 분화능 및 증식능을 보이며, 특히 Schwann cell 분화에 전반적으로 발현되어지는 발현 인자들의 높은 발현효과를 볼 때, 타 유래 줄기세포로부터의 분화능 및 증식능과 대비하여 현저한 분화능 및 증식능을 가진다. The Schwann cell differentiation method of the present invention shows remarkable differentiation and proliferative ability compared to the method using adult stem cells derived from unilateral MSF using mesenchymal stem cells, and particularly the expression factors expressed in Schwann cell differentiation In view of its high expression effect, it has remarkable differentiation and proliferation ability as compared with the differentiation and proliferation ability from other stem cells.
본 발명은 또한, 상기 슈반 세포 분화 방법에 따라 제조된 슈반 세포를 제공한다. 본원 발명에 따라 제조된 슈반 세포는 편도 유래 줄기세포와 대비하여 증가된 GFAP(Glial fibrillary acidic protein), NGFR(nerve growth factor receptor)의 발현을 보인다. 또한, 편도 유래 줄기세포와 대비하여 CAD19(cadherin 19)의 감소를 보이며, KROX20(early growth response 2), S100B (S100 calcium binding protein B)의 증가를 보인다. 특히, Schwann cell 분화 전반적으로 발현되어지는 GFAP, NGFR, KROX20 또는 S100B가 타 유래 중간엽 줄기세포로부터 분화된 세포와 대비하여 전반적인 높은 발현을 보여, 슈반 세포로써 우수한 특성을 가짐을 알 수 있다. 본 발명에 따른 슈반 세포는 신경 영양인자 분비를 통한 축삭, 신경극 등의 신경 세포 재생 및 성장 유도 효과를 가지며, 후근 신경절과 공동 배양시 후근 신경절의 감각 신경 세포의 축삭의 성장을 유도하여 결합한 후 수초화 시키는 특성을 가진다. The present invention also provides schwann cells prepared according to the Schwann cell differentiation method. The Schwann cells produced according to the present invention exhibit increased expression of GFAP (glial fibrillary acidic protein) and NGFR (nerve growth factor receptor) in comparison with unilateral stem cells. In addition, CAD19 (cadherin 19) is decreased compared with one-way stem cells, and KROX20 (early growth response 2) and S100B (S100 calcium binding protein B) are increased. In particular, the expression of Schwann cell differentiation as a whole GFAP, NGFR, KROX20, or S100B show high overall expression as compared to cells differentiated from other originated mesenchymal stem cells, and have excellent characteristics as Schwann cells. The Schwann cells according to the present invention have neuronal regeneration and growth-inducing effects such as axons and nerve poles through the secretion of neurotrophic factors and induce axonal growth of the sensory neurons of the posterior ganglia during co-culture with the posterior ganglion, It has the property of weeding.
본 발명에 따른 슈반 세포 분화 방법에 따라 제조된 슈반 세포를 유효성분으로 포함하는 신경 손상 질환 예방 또는 치료용 조성물을 제공한다. 즉, 개체로부터 분리, 배양 및 특수한 조작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품으로 사용되어 신경 손상 질환에 사용될 수 있다. The present invention provides a composition for preventing or treating neuropathic diseases, which comprises Schwann cells according to the Schwann cell differentiation method according to the present invention as an active ingredient. That is, it can be used as a medicine used for the purpose of treatment, diagnosis and prevention by cells and tissues prepared by separation, culture and special manipulation from an individual, and can be used for nerve damage diseases.
신경 손상 질환은 유전적, 대사적 또는 외상에 의해 신경 관련 조직에 손상이 발생하여 유발되는 질환을 말한다. 예를 들어, 상기 신경 손상 질환은 유전성 신경병증으로 샤르코 마리 투스 병 (Charot-Marie-Tooth disease) 또는 유전성 신경 편향 압박마비증세 (Hereditary neuropathy with liability to pressure palsies)로부터 선택된 어느 하나일 수 있다. 또한, 상기 신경 손상 질환은 외상성 신경 질환으로 말초신경 퇴화 (Peripheral nerve degeneration) 또는 말초신경 절단에 의한 외상성 신경 질환일 수 있다. 또한, 상기 신경 손상 질환은 대사성 신경병증으로 당뇨병의 합병증으로 발생한 신경병변, 예컨대 당뇨병성 말초 신경병증 (diabetes neuropathy)일 수 있다. Neurological damage is a disease caused by genetic, metabolic, or traumatic damage to nerve tissue. For example, the nerve damage disorder can be any one selected from Charot-Marie-Tooth disease or Hereditary neuropathy with liability to pressure palsies as hereditary neuropathy. In addition, the nerve injury disease is a traumatic neurological disease, which may be a peripheral nerve degeneration or a traumatic neuropathy due to peripheral nerve shearing. In addition, the nerve damage disease may be a neurological lesion caused by diabetic complication due to metabolic neuropathy, such as diabetic neuropathy.
상기 신경 손상 질환 예방 또는 치료용 조성물은 약학적 분야의 통상의 방법에 따라 환자의 신체 내 투여에 적합한 단위투여형의 약학적 제제로 제형화시켜 투여할 수 있으며, 상기 제제는 1회 또는 수회 투여에 의해 효과적인 투여량을 포함한다. 이러한 목적에 적합한 제형으로는 비경구투여 제제로서 주사제, 주입제, 분무제 등이 바람직하다. 또한, 상기 신경 손상 질환 예방 또는 치료용 조성물은 약학적으로 허용가능한 통상의 불활성 담체를 포함할 수 있다. 또한, 당업계에서 통상적으로 사용하는 투여방법을 이용하여 이식 및 투여될 수 있으며, 바람직하게는 치료가 필요한 환자의 질환 부위에 직접 생착 또는 이식이 가능하나 이에 한정되지는 않는다. 또한, 상기 투여는 카테터를 이용한 비외과적 투여 및 질환부위 절개 후 주입 또는 이식 등 외과적 투여방법 모두 가능하다. 투여량은 1.0×104 내지 1.0×1010세포/kg 체중, 바람직하게는 1.0×105 내지 1.0×109세포/kg 체중을 1회 또는 수회로 나누어 투여할 수 있다. 그러나, 유효성분의 실제 투여량은 치료하고자 하는 질환, 질환의 중증도, 투여경로, 환자의 체중, 연령 및 성별 등의 여러 관련 인자에 비추어 결정되어야 하는 것으로 이해되어야 하며, 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The composition for preventing or treating neuropathic diseases may be formulated into a unit dosage form of a pharmaceutical preparation suitable for administration to a patient in accordance with a conventional method in the pharmaceutical field. The preparation may be administered once or several times ≪ / RTI > As formulations suitable for this purpose, injectable preparations, injecting agents and spraying agents are preferred as parenteral administration preparations. In addition, the composition for preventing or treating the nerve injury disease may comprise a conventional inert carrier which is pharmaceutically acceptable. Also, it can be transplanted and administered using a method of administration commonly used in the art, and preferably it can be transplanted or transplanted directly to a diseased part of a patient in need of treatment, but is not limited thereto. In addition, the above administration is possible both in a non-surgical administration using a catheter and in a surgical administration method such as implantation or transplantation after incision of a disease site. The dose may be administered once or several times in the range of 1.0 × 10 4 to 1.0 × 10 10 cells / kg body weight, preferably 1.0 × 10 5 to 1.0 × 10 9 cells / kg body weight. It should be understood, however, that the actual dosage of the active ingredient should be determined in light of various relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the body weight, age and sex of the patient, The scope of the present invention is not limited thereto.
본 발명은 또한, (a) 표피 성장 인자 (Epidermal growth factor), 섬유아세포 성장 인자 (basic fibroblast growth factor) 및 B27 첨가제를 포함하는 배지에서 편도 유래 중간엽 줄기세포를 배양하는 단계를 포함하는 편도 유래 중간엽 줄기세포로부터 신경구 (Neurosphere)를 유도하는 단계; (b) 상기 (a) 단계의 신경구(Neurosphere)를 혈소판 유래 성장인자 (Platelet-derived growth factor), 섬유아세포 성장 인자 (basic fibroblast growth factor), 헤레굴린-β(Heregulin-β) 및 포스콜린 (Forskolin) 하에서 배양하여 슈반 세포를 분화시키는 단계; 및 (c) 상기 (b) 단계에서 분화된 슈반 세포를 배양하여 슈반세포로부터 분비된 신경 영양인자를 포함하는 배양 배지를 얻는 단계를 포함하는, 신경 영양인자 조성물의 제조방법을 제공한다. The present invention also relates to a method for the treatment of osteoporosis, comprising the steps of: (a) culturing one-way derived mesenchymal stem cells in a medium comprising Epidermal growth factor, basic fibroblast growth factor and B27 additive Inducing a neurosphere from mesenchymal stem cells; (b) comparing the Neurosphere of step (a) with Platelet-derived growth factor, basic fibroblast growth factor, Heregulin-beta and phoscholine Forskolin) to differentiate Schwann cells; And (c) culturing the Schwann cells differentiated in step (b) to obtain a culture medium containing a neurotrophic factor secreted from Schwann cells.
본 발명의 상기 (a) 편도 유래 중간엽 줄기세포로부터 신경구 (Neurosphere)를 유도하는 단계 및 (b) 슈반 세포를 분화시키는 단계는 앞서 살핀 바와 같다. The step of inducing neurosphere from the (a) one-way derived mesenchymal stem cells of the present invention and the step of (b) differentiating Schwann cells are as described above.
본 발명에 따라 상기 (c) 단계에서 분화된 슈반 세포를 배양하는 배지는 alpha MEM(alpha Minimum Essential Medium Eagle), Neurobasal medium, 또는 DMEM (Dulbecco's Modified Eagle Medium)으로부터 선택되는 어느 하나의 배지, 바람직하게 DMEM (Dulbecco's Modified Eagle Medium) 하에서 배양 할 수 있다. 배양은 1 내지 7일간 진행될 수 있으며, 배양액은 당업계의 통상의 방법에 따라 분리하여 신경 영양인자 조성물의 제조한다. 예컨대, 원심분리방법을 통해 신경 영양인자 조성물만을 따로 분리할 수 있다. 본 발명의 제조 방법에 따라 제조된 상기 신경 영양인자 조성물은 Desert Hedgehog (DHH), Neurotrophin-3 (NT-3), Nerve growth factor (NGF), Glial cell line-derived neurotrophic factor (GDNF), F-Spondin, Brain-derived neurotrophic factor (BDNF)등의 신경 영양 인자를 포함하며, 슈반 세포로부터 분비 가능한 신경 영양 인자라면 이에 제한되지 않고 본 발명의 범주 내에 포함될 수 있다. 본 발명의 제조 방법에 따라 제조된 신경 영양인자 조성물은 축삭, 신경극 등의 성장을 유도하고 신경 재생 회복 등에 효과를 보인다. The medium for culturing the Schwann cells differentiated in step (c) according to the present invention may be any one medium selected from alpha MEM (alpha minimum essential medium), Neurobasal medium, or DMEM (Dulbecco's modified Eagle medium) And cultured under DMEM (Dulbecco's Modified Eagle Medium). The culturing can be carried out for 1 to 7 days, and the culture liquid is separated according to a conventional method in the art to prepare a neurotrophic factor composition. For example, only the neurotrophic factor composition can be separated by centrifugation. The neurotrophic factor composition prepared according to the method of the present invention can be used for the treatment of various diseases such as desert hedgehog (DHH), neurotrophin-3 (NT-3), nerve growth factor (NGF), glial cell line-derived neurotrophic factor Spondin, and Brain-derived neurotrophic factor (BDNF), and may be included within the scope of the present invention without being limited thereto, as long as it is a neurotrophic factor that can secrete from Schwann cells. The neurotrophic factor composition prepared according to the production method of the present invention induces growth of the axon, nerve pole and the like and has an effect on nerve regeneration and the like.
본 발명은 또한 상기 신경 영양인자 조성물 제조방법에 따라 제조된 신경 영양인자 조성물을 제공하며, 신경 영양인자 조성물은 신경 영양 인자로 Desert Hedgehog (DHH), Neurotrophin-3 (NT-3), Nerve growth factor (NGF), Glial cell line-derived neurotrophic factor (GDNF), F-Spondin, Brain-derived neurotrophic factor (BDNF)등을 포함 할 수 있으나, 이에 제한되지 않는다. The present invention also provides a neurotrophic factor composition prepared according to the method for producing a neurotrophic factor composition, wherein the neurotrophic factor composition is selected from the group consisting of Desert Hedgehog (DHH), Neurotrophin-3 (NT-3) But are not limited to, NGF, Glial cell line-derived neurotrophic factor (GDNF), F-Spondin, Brain-derived neurotrophic factor (BDNF)
본 발명의 분화 방법은 슈반 세포로의 높은 분화능을 보여 다량의 슈반 세포의 확보가 가능하며, 본 발명에 따라 분화된 슈반 세포는 버려지는 자가 조직을 사용함으로써 세포 공여부의 신경 손상 없이 제조될 수 있으며, 신경 세포를 지지하고 신경을 재생하여 신경 관련 질환의 세포 치료제로써 우수한 이용가능성을 보인다. The Schwann cells differentiated according to the present invention can be produced without the nerve damage of the cell donor part by using the abandoned autologous tissue. , Supporting neurons and regenerating neurons, thus showing excellent utility as a cell therapy for neuronal diseases.
도 1은 편도 유래 중간엽 줄기세포로부터 슈반세포를 분화시키기 위한 분화 단계별 방법 및 시기별 세포 형태를 나타낸 도이다.
도 2는 편도 유래 중간엽 줄기세포로부터 슈반세포를 분화시킨 후 계대한 샘플이 슈반 세포로의 특성을 가지는 것을 웨스턴 블랏팅을 통하여 확인한 결과를 나타낸다.
도 3은 편도 유래 중간엽 줄기세포로부터 슈반세포를 분화시킨 후 세포가 슈반세포로의 특성을 가지는 것을 면역형광 염색을 통하여 확인한 결과를 나타낸다.
도 4는 편도 유래 중간엽 줄기세포로부터 슈반세포를 분화시킨 조건 배양액(conditioned media)을 NSC34 세포에 처리하여 신경극 발아 유도를 확인한 결과를 나타낸다.
도 5는 편도 유래 중간엽 줄기세포로부터 분화된 슈반세포와 후근신경절을 공동배양에 따른 수초화(myelination) 현상을 면역형광염색을 통해 확인한 결과를 나타낸다.
도 6은 편도 유래 중간엽 줄기세포 또는 지방 조직 유래 줄기세포를 슈반세포로 분화시켜 CAD19, GFAP, NGFR, KROX20, 및 S100B의 발현 변화를 RT-PCR을 통해 확인한 결과를 나타낸다. Brief Description of the Drawings Fig. 1 is a diagram showing a method for differentiating Schwann cells from a mesenchymal-derived mesenchymal stem cell and a morphology of a cell according to a period.
FIG. 2 shows Western blotting results of differentiation of Schwann cells from one-way derived mesenchymal stem cells to show that the sample has Schwann cell characteristics.
FIG. 3 shows the results of immunofluorescence staining of Schwann cells differentiated from one-way derived mesenchymal stem cells and showing that the cells have Schwann cells.
FIG. 4 shows the results of confirming induction of neuronal germination by treating conditioned medium prepared by differentiating Schwann cells from one-way derived mesenchymal stem cells to NSC34 cells.
FIG. 5 shows the result of immunofluorescence staining of myelination caused by co-culture of Schwann cells and the posterior ganglia differentiated from one-way derived mesenchymal stem cells.
FIG. 6 shows the results of RT-PCR analysis of changes in expression of CAD19, GFAP, NGFR, KROX20, and S100B by differentiating mesenchymal stem cells or adipose tissue-derived stem cells into Schwann cells.
본 발명의 이해를 돕기 위하여 실시예, 제조예를 제시한다. 하기의 실시예, 제조예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예, 제조예에 의해 본 발명의 내용이 한정되는 것은 아니다.Examples and production examples are provided to facilitate understanding of the present invention. The following examples and preparation examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples and the production examples.
<< 실시예Example 1> 편도 유래 1> One way origination 중간엽Intermediate lobe 줄기세포 및 지방 조직 유래 줄기세포의 Stem cells and adipose tissue-derived stem cells 배양culture
편도 유래 중간엽 줄기세포(Tonsil-derived mesenchymal stem cell, TMSC) 는 이대목동병원 이비인후-두경부외과에서 편도적출술을 시행하는 환자로부터 적출된 편도조직(4-20세의 저연령층 조직, 임상윤리위원회 심의 통과: ECT 11-53-02)으로부터 얻어, 10% FBS (Gibco), 1% 페니실린/스트렙토마이신 (Gibco)이 보충된 low glucose DMEM (Dulbecco's modified Eagle's medium, hyclone)하에 배양되었다. Tonsil-derived mesenchymal stem cells (TMSCs) were isolated from patients who underwent tonsillectomy at post-nasopharyngeal surgery in Ewha Womans University Hospital (4-20-year-old younger group, (Dulbecco's modified Eagle's medium, hyclone) supplemented with 10% FBS (Gibco), 1% penicillin / streptomycin (Gibco).
한편, 지방 조직 유래 줄기세포(adipose-derived stem cell, ASC)는 ATCC로부터 구입하여, Mesenchymal Stem Cell Growth Kit (ATCC)가 보충된 Mesenchymal Stem Cell Basal Medium (ATCC)에서 배양하였다. Adipose-derived stem cells (ASC) were purchased from ATCC and cultured in Mesenchymal Stem Cell Basal Medium (ATCC) supplemented with Mesenchymal Stem Cell Growth Kit (ATCC).
<< 실시예Example 2> 슈반세포로의 분화 유도 2> induction of differentiation into schwann cells
편도 유래 중간엽 줄기세포 및 지방 조직 유래 줄기세포로부터 슈반 세포의 분화를 유도하기 위한 첫 번째 단계로 신경구(Neurosphere)를 형성하였다. 신경구는 60 mm 페트리접시(petri dish)에 20ng/ml EGF (PeproTech), 20ng/ml basic FGF (PeproTech) 및 2% B27 supplement (Gibco)가 첨가된 DMEM/F12 신경구 유도 배양액 5 ml당 2,500,000개~ 4,000,000개의 세포를 부유하여 7일 동안 세포 응집을 유도하여 제조하였으며, 신경구 유도 배양액은 3일마다 갈아주었다. Neurosphere was formed as the first step to induce differentiation of schwann cells from mesenchymal stem cells derived from tonsil and adipose tissue derived stem cells. Neural nerves were cultured in a 60-mm Petri dish at 2,500,000 cells per 5 ml DMEM / F12 nerve root induction medium supplemented with 20 ng / ml EGF (PeproTech), 20 ng / ml basic FGF (PeproTech) and 2% B27 supplement (Gibco) 4,000,000 cells were suspended and induced to induce cell aggregation for 7 days. The nerve root induction medium was changed every 3 days.
형성된 신경구를 슈반 세포로 분화시키기 위해 27 gauge needle로 분쇄시켜 2ug/ml laminin(Sigma)이 코팅된 배양접시 내 커버 슬립(cover slip) 위에 재부착(replating)을 시키고, 5ng/ml human Recombinant PDGF-AA(PeproTech), 10ng/ml basic FGF(PeproTech), 200ng/ml human Recombinant Heregulinβ-1(PeproTech), 14μM Forskolin (Sigma) 및 10% FBS(Gibco)가 첨가된 DMEM/F12배양액 내에서 10일 동안 배양하였다. 슈반 세포로의 분화 과정 중, 배양액은 매 3일마다 갈아주었다.In order to differentiate the formed neurons into Schwann cells, they were pulverized with a 27 gauge needle and replated on a cover slip in a culture dish coated with 2 ug / ml laminin (Sigma). 5 ng / ml human Recombinant PDGF- The cells were cultured in DMEM / F12 medium supplemented with AA (PeproTech), 10 ng / ml basic FGF (PeproTech), 200 ng / ml human Recombinant Heregulinβ-1 (PeproTech), 14 μM Forskolin (Sigma) and 10% FBS Lt; / RTI > During the differentiation into Schwann cells, the culture medium was changed every 3 days.
상기 편도 유래 중간엽 줄기세포로부터 슈반 세포 분화 유도의 프로토콜 과정 중 일부를 도 1에 나타내었다. 도 1에 나타낸 바와 같이, 세포 형태학적으로 편도 유래 중간엽 줄기세포(TMC)로부터 신경구(Neurosphere)가 유도되고 슈반 세포(Schwann cell)로 분화가 이루어졌음을 확인하였다. FIG. 1 shows a part of the protocol process for inducing Schwann cell differentiation from the one-way-derived mesenchymal stem cells. As shown in FIG. 1, it was confirmed that neurospheres were induced from schistosome-derived mesenchymal stem cells (TMC), and schwann cells were differentiated from cytoskeletons.
<< 실시예Example 3> 분화된 슈반세포와 3> differentiated Schwann cells and 후근신경절(Dorsal root ganglia)의Of the dorsal root ganglia 공동배양 Co-culture
분화된 슈반세포와 후근신경절 (Dorsal root ganglia, DRG)의 공동 배양을 통한 수초화를 확인하기 위하여, 분화된 슈반 세포와 후근신경절을 공동 배양하였다. Differentiated schwann cells and dorsal root ganglia (DRG) were co-cultured with differentiated Schwann cells and the posterior ganglia in order to confirm herniation through co-cultivation of differentiated Schwann cells and DRGs.
임신한 암컷 ICR 마우스로부터 13일째의 배아를 적출하여 배아의 후근신경절을 핀셋을 이용하여 분리하였다. 분리된 후근신경절을 분화된 슈반 세포 위에 올려주어 공동배양 시켜주었다. 공동배양을 위해 처음 4일간은 ITS supplement (Gibco), 0.2% BSA (Bovogen), 4mg/ml D-글루코즈 (Sigma), 2mM L-글루타민 (Sigma), 50ng/ml human Recombinant β-NGF (PeproTech) 및 1% 페니실린/스트렙토마이신 (GIBCO)이 첨가된 MEM (Gibco) 배지에서 배양해 주었고, 이후의 14일은 동일한 배양액에 15%의 FBS (Hyclone)와 10μM Ascorbic acid (Sigma)를 첨가해 주었다. 배양액은 매 3일 마다 갈아주었다.Embryos were removed from pregnant female ICR mice on day 13, and the posterior ganglia of the embryos were separated using tweezers. The separated posterior ganglia were placed on differentiated Schwann cells and co-cultured. For co-cultivation, ITS supplement (Gibco), 0.2% BSA (Bovogen), 4 mg / ml D-glucose (Sigma), 2 mM L-glutamine (Sigma), 50 ng / ml human Recombinant β-NGF (PeproTech) And 15% FBS (Hyclone) and 10 μM Ascorbic acid (Sigma) were added to the same culture medium on the 14th day after incubation. The cells were cultured in MEM (Gibco) medium supplemented with 1% penicillin / streptomycin (GIBCO) The culture medium was changed every 3 days.
상기 슈반 세포와 후근신경절 공동 배양 과정 프로토콜의 일부를 도 1에 나타내었다. 도 1에 나타낸 바와 같이, 세포 형태학적으로 슈반세포와 후근신경절 공동 배양에 따라 수초화가 이루어졌음을 확인하였다. Some of the Schwann cells and the posterior ganglia co-culturing protocol are shown in FIG. As shown in Fig. 1, it was confirmed that the herbalization was carried out according to cytopathologic co-culture of Schwann cells and the posterior ganglion.
<< 실시예Example 4> 4> 웨스턴Western 블랏을Blat 통한 편도 유래 One way through 중간엽Intermediate lobe 줄기세포의 슈반세포로의 분화 확인 Identification of stem cells into Schwann cells
상기 미분화 편도 유래 중간엽 줄기세포와 분화 단계별 세포를 취하여 분화에 따른 슈반 세포 분화 표지인자들의 발현 변화를 확인하였다. The mesenchymal stem cells derived from undifferentiated monocyte and the differentiated cells were differentiated to confirm the expression of Schwann cell differentiation markers.
이를 위하여, 분화 단계별로 취한 세포를 단백질 분해효소 억제제(Roche)가 포함된 Lysis 완충액에 넣고 파쇄하였다. 전체 단백질(10-30μg)을 확인하고자하는 1차 항체로 면역블롯팅하였고, GAPDH (Abcam)는 내부 대조군으로 사용하였다. 슈반 세포 분화 표지인자로써 GFAP 및 NGFR가 사용되었다. GFAP은 슈반세포의 발달 과정 중 상대적으로 후기 단계에 발현하며 슈반세포가 수초화(myelination)를 이루기 전까지 슈반세포의 세포 골격을 형성하고 유지시켜주는 중간섬유로써 역할을 하는 단백질이며, NGFR은 리셉터 타이로신 카이네이즈(receptor tyrosine kinase)의 한 종류로 세포 내로의 신호 전달을 포함한 다양한 기능을 가지고 있고 신경 세포의 생존과 축삭(axon)의 성장, 신경극 발아(neurite outgrowth), 및 반세포의 이동(migration)에 중요한 역할을 하는 단백질이다. LAS-3000 (Fuji film)를 이용하여 밴드의 세기를 정량화하고 GAPDH의 세기로 표준화하였으며, 그 결과를 도 2에 나타내었다. For this purpose, the cells taken at different stages of differentiation were placed in Lysis buffer containing protease inhibitor (Roche) and disrupted. The whole protein (10-30 μg) was immunoblotted with the primary antibody to be identified, and GAPDH (Abcam) was used as an internal control. GFAP and NGFR were used as Schwann cell differentiation markers. GFAP is a protein that acts as an intermediate fiber that forms and maintains the cytoskeleton of Schwann cells until Schwann cells develop myelination. Expression of GFAP is relatively late in the development of Schwann cells, and NGFR is a receptor tyrosine kinase is a type of receptor tyrosine kinase that has various functions including signaling into the cell and is important for neuronal survival and axon growth, neurite outgrowth, and migration of the half cells It is a protein that plays a role. The intensity of the band was quantified using LAS-3000 (Fuji film) and standardized to the intensity of GAPDH. The results are shown in FIG.
도 2의 1에 나타낸 바와 같이, GFAP의 경우 미분화된 편도 유래 중간엽 줄기세포 (TMSC)에 비해 편도 유래 중간엽 줄기세포로부터 분화된 슈반 세포 (TMSC-SC)에서 발현량이 증가하는 것을 확인하였다. 또한, 도 2의 2에 나타낸 바와 같이, NGFR 단백질의 발현은 미분화된 편도 유래 중간엽 줄기세포 (TMSC)에서는 발현되지 않다가 편도 유래 중간엽 줄기세포로부터 분화된 슈반 세포 (TMSC-SC)에서 발현량이 크게 증가하는 것을 확인하였다. 또한, 도 2의 3에 나타낸 바와 같이, 편도 유래 중간엽 줄기세포로부터 분화된 슈반 세포 (TMSC-SC)의 계대 증가에 따라 웨스턴블랏팅을 하였을 때, GFAP, NGFR 단백질들의 발현을 유지되는 것을 확인하였다. 상기 결과들을 종합하여 볼 때, 편도 유래 중간엽 줄기세포 (TMSC)로부터 분화된 슈반세포가 분화후에 계대가 증가함에도 슈반세포로서 특성을 유지함을 확인하였다. As shown in Fig. 2, the expression level of GFAP was increased in Schwann cells (TMSC-SC) differentiated from unilocated mesenchymal stem cells compared to undifferentiated one-way mesenchymal stem cells (TMSC). 2, expression of NGFR protein was not expressed in undifferentiated one-way derived mesenchymal stem cells (TMSC) but expressed in Schwann cells (TMSC-SC) differentiated from unilateral MSCs And the amount of the reaction increased. Further, as shown in FIG. 2, when Western blotting was performed according to an increase in the number of Schwann cells (TMSC-SC) differentiated from the mesenchymal stem cells derived from unilateral MSCs, the expression of GFAP and NGFR proteins was confirmed to be maintained Respectively. Taken together, these results indicate that Schwann cells differentiated from TMSCs retain their characteristics as Schwann cells even after passage through differentiation.
<< 실시예Example 5> 면역 형광 염색을 통한 편도 유래 5> Origin of one-way through immunofluorescence staining 중간엽Intermediate lobe 줄기세포의 슈반세포로의 분화 확인 Identification of stem cells into Schwann cells
상기 미분화 편도 유래 중간엽 줄기세포(TMSC)와 분화 유도된 슈반 세포 (TMSC-SC)의 면역형광염색을 실시하여 분화를 확인하였다. The differentiation was confirmed by immunofluorescence staining of the undifferentiated monocyte-derived mesenchymal stem cell (TMSC) and differentiated Schwann cells (TMSC-SC).
상기 미분화 편도 유래 중간엽 줄기세포와 분화 유도된 슈반 세포 분화(TMSC-SC)는 커버 슬립(cover slip) 위에서 각 조건 하에 배양하였으며, 4% 파라포름알데히드 (paraformaldehyde) 용액으로 15분간 실온에서 고정한 후 PBS로 세척하였다. 세척된 세포는 0.1% Tween-20 및 2% 소혈청알부민(Bovine serum albumin)을 첨가한 PBS 용액에서 1시간 처리하고, 발현을 확인하고자 하는 항체는 제조사의 매뉴얼에 따라 비율대로 희석하고 PBS에 첨가한 후 실온에서 1시간 또는 냉장 상태에서 밤새 반응시켰다. 뒤이어 다시 PBS 용액으로 세척한 후, 실온 또는 냉장 상태에서 TRITC (tetrarhodamine isothiocyanate) 또는 FITC (fluorescein isothiocyanate) 접합체의 2차 항체를 1차 항체와 동일 방법으로 처리하였다. 대조 염색으로 세포핵을 염색하기 위해 DAPI가 첨가된 마운팅 용액(Vectashield)을 사용하여 마운팅한 후 형광현미경으로 관찰하였다.The differentiation-induced mesenchymal stem cells and differentiated Schwann cell differentiation (TMSC-SC) were cultured on cover slips under various conditions, fixed in 4% paraformaldehyde solution for 15 minutes at room temperature And washed with PBS. The washed cells were treated for 1 hour in PBS solution supplemented with 0.1% Tween-20 and 2% bovine serum albumin. The antibody to be expressed was diluted in proportions according to the manufacturer's manual and added to PBS And then reacted overnight at room temperature or in a refrigerated state. After washing with PBS solution, the secondary antibody of TRITC (tetrarhodamine isothiocyanate) or FITC (fluorescein isothiocyanate) conjugate was treated at the room temperature or in the cold state in the same manner as the primary antibody. In order to stain the nuclei with contrast dye, mounting was performed using mounting solution (DACI) with Vectashield and observed with fluorescence microscope.
그 결과를 도 3에 나타내었다. 도 3에서 확인되는 바와 같이, 미분화된 편도 유래 중간엽 줄기세포(TMSC)에서는 GFAP의 발현을 관찰하기 힘든데 반하여, 분화 유도된 슈반 세포 (TMSC-SC)에서는 GFAP의 발현이 강하게 나타나는 것을 확인하였다. 또한, NGFR도 GFAP와 마찬가지로 미분화 상태의 편도 유래 중간엽 줄기세포(TMSC)에서는 발현되지 않지만 분화 유도된 슈반 세포 (TMSC-SC)에서는 발현이 강한 것을 확인하였다. The results are shown in Fig. As shown in FIG. 3, it was difficult to observe GFAP expression in undifferentiated one-way derived mesenchymal stem cells (TMSC), whereas GFAP expression was strongly observed in differentiated Schwann cells (TMSC-SC). In addition, NGFR was not expressed in undifferentiated one-way-derived mesenchymal stem cells (TMSC) as in GFAP, but it was confirmed that expression was strong in differentiated Schwann cells (TMSC-SC).
<< 실시예Example 6> 조건 배양액 ( 6> conditioned medium ( ConditionedConditioned MediaMedia )을 이용한 ) NSC34NSC34 세포 배양을 통한 분화된 슈반세포의 특성 및 Characterization of differentiated Schwann cells by cell culture and 분화능Ability to distinguish 확인 Confirm
슈반세포는 구조적, 기능적으로 말초 신경의 재생(peripheral nerve regeneration)에 중요한 역할을 하는 세포로 다양한 신경 영양 인자(neurotrophic factors)를 세포 외부로 분비하여 축삭(axon) 혹은 신경극(neurite)의 성장을 유도하고 안내하는 역할을 하는 것이 알려져 있다. 이에 따라 편도 유래 중간엽 줄기세포로부터 분화된 슈반 세포가 신경극 발아(neurite outgrowth)를 유도하는 영양 인자를 분비하는지 확인하기 위하여, 분화된 슈반세포로부터 조건 배양액 (conditioned media, CM)을 모아 마우스 운동 신경 세포인 NSC34 배양에 사용하였다. Schwann cells are structurally and functionally responsible for peripheral nerve regeneration. They secrete various neurotrophic factors into the extracellular space to produce axons or neurites. It is known that it plays a role of inducing and guiding. In order to determine whether Schwann cells differentiated from one-way derived mesenchymal stem cells secrete nutrients that induce neurite outgrowth, conditioned medium (CM) was collected from differentiated schwann cells and transferred to mouse movement NSC34, a neuronal cell, was used for culture.
배양 접시 내 80% 정도의 밀도로 분화된 슈반세포로부터 조건 배양액 (conditioned media)을 모으기 위해 PBS 워싱을 2회 해주었다. 2% FBS (Gibco)가 들어간 DMEM을 넣고 2일간 배양한 후 사용된 배양액을 모아 원심분리기로 1000g, 5분 처리하고 상층액을 분리하여 조건 배양액 (conditioned media, CM)을 준비하였다. 10% FBS (Gibco), 1% Penicillin/Streptomycin (Gibco)이 첨가된 DMEM 일반 배양액과 조건 배양액 (conditioned media)을 각각 NSC34 운동 신경 세포에 처리하여 세포가 자라나는 morphology 변화를 확인하였다. PBS washings were performed twice to collect conditioned media from differentiated Schwann cells at a density of about 80% in the culture dish. DMEM containing 2% FBS (Gibco) was added and cultured for 2 days. The culture medium was collected, treated with 1000 g for 5 minutes using a centrifuge, and conditioned medium (CM) was prepared by separating the supernatant. DMEM medium supplemented with 10% FBS (Gibco), 1% Penicillin / Streptomycin (Gibco) and conditioned medium were each treated with NSC34 motor neurons to confirm morphology changes.
그 결과를 도 4에 나타내었다. 도 4에 나타낸 바와 같이, 일반 배양액에 키운 대조군이 구형의 모양을 띄는 것에 비해 조건 배양액 (conditioned media)을 이용해 키운 경우 이틀째부터 세포의 신경극이 돌출이 일어나고, 시간 경과에 따라 신경극의 길이 역시 점점 길어지는 것을 확인하였다. 이를 통해 편도 유래 줄기세포로부터 분화된 슈반세포가 신경극 성장을 자극하는 영양 물질들을 세포 외부로 분비하는 것을 확인하였다. The results are shown in Fig. As shown in FIG. 4, when the conditioned medium was used to grow the control group, the nerve pole protrudes from the second day of the incubation period, and the length of the nerve pole And it was confirmed that it became longer. These results suggest that Schwann cells differentiated from one - way stem cells secrete nutrients that stimulate nerve pole growth into the extracellular space.
<< 실시예Example 7> 7> 후근신경절(Dorsal root ganglia)과Dorsal root ganglia and 공동배양을 통한 분화된 슈반 세포의 특성과 Characterization of differentiated Schwann cells through co-culture 분화능Ability to distinguish
편도 유래 중간엽 줄기세포로부터 분화된 슈반 세포가 축삭을 수초화시키는 기능을 수행하는지 확인하기 위해, 13일된 마우스 배아로부터 후근신경절(Dorsal root ganglia, DRG)을 분리하여 슈반세포와 상기 실시예 3의 방법과 동일하게 공동배양하였다. 총 18일의 공동배양 후 실시예 5의 방법과 동일하게 면역형광염색을 하여 수초화를 확인하였다. 면역형광염색에서 표지인자로 사용된 MBP는 슈반세포가 신경수초화(neural myelination)를 형성할 때에 발현되는 단백질로 슈반세포가 다중막(multi-layered sheath)을 형성할 때에 수초막에 있는 지질과 반응하여 수초막이 바른 구조를 형성할 수 있도록 하는 단백질이다. 상기 면역형광염색 분석 결과를 도 5에 나타내었다. 도 5에 나타낸 바와 같이, 인간 미토콘드리아(human mitochondria, Human Mito)와 MBP가 이중염색(double stainning)된 것이 확인되었고, 후근신경절에서 뻗어져 나온 신경세포를 편도 유래 중간엽 줄기세포로부터 분화된 슈반 세포가 감싸는 모양을 확인하였다. In order to confirm whether Schwann cells differentiated from one-way derived mesenchymal stem cells perform the function of planting axons, a dorsal root ganglia (DRG) was isolated from a 13-day-old mouse embryo and the Schwann cells and the method of Example 3 . ≪ / RTI > After a total of 18 days of co-cultivation, immunofluorescence staining was performed in the same manner as in Example 5 to confirm the herbalization. MBP, used as a marker in immunofluorescent staining, is a protein that is expressed when Schwann cells form neural myelination. When Schwann cells form a multi-layered sheath, MBP reacts with lipid in the canal Is a protein that allows the weed membrane to form the right structure. The results of the immunofluorescence staining analysis are shown in FIG. As shown in FIG. 5, it was confirmed that human mitochondria (Human Mito) and MBP were double-stained, and neurons extended from the posterior ganglion were stimulated with Schwann cells differentiated from unilateral mesenchymal stem cells .
<< 실시예Example 8> 편도 유래 8> One way origination 중간엽Intermediate lobe 줄기세포의 슈반 세포로의 높은 분화 효과 확인 Identification of the high differentiation effect of stem cells into schwann cells
Real-time PCR을 통해 본 발명 방법에 따른 편도 유래 중간엽 줄기 세포로부터의 슈반세포의 분화능 측면에 있어서 타 유래 줄기세포를 사용하는 것보다 높은 효과를 가짐을 확인하였다. It was confirmed through real-time PCR that the effect of the present invention on the differentiation ability of Schwann cells from one-way derived mesenchymal stem cells was higher than that of using other stem cells.
Real-time PCR을 위하여 미분화 상태의 편도 유래 중간엽 줄기세포(TMSC) 및 지방 조직 유래 줄기세포(ASC)와 슈반세포로 분화된 상태의 세포에서 총 RNA 추출을 위해 RNeasy mini kit (Qiagen Inc.)를 이용하여 제조자의 지시에 따라 총 RNA를 추출하였다. Superscript II (Invitrogen)과 oligo-d(T)20 프라이머를 이용하여 42℃에서 1시간, 80℃에서 10분 반응하여 cDNA를 합성하였다. cDNA로부터의 표적 서열(primer)은 7500 Fast Real time PCR system(Applied Biosystem)를 이용하여 실시간 중합효소연쇄반응을 시키되 그 과정은 SYBR Premix Ex Taq (Takara)를 사용하여, 95℃에서 30초 동안 최초 변성(denaturation) 시키고, 95℃에서 3초간 변성시키고, 60℃에서 30초간 표적 유전자 증폭과정을 총 40회 반복 시킨 후, 멜팅 커브(Melting curve)를 그리기 위해 95℃에서 15초, 60℃에서 1분, 95℃에서 15초 과정을 진행하였다. GAPDH 발현량을 내부 대조군으로 사용하여 표준화시켰다. For real-time PCR, RNeasy mini kit (Qiagen Inc.) was used for total RNA extraction from undifferentiated one-way derived mesenchymal stem cell (TMSC) and adipose tissue derived stem cell (ASC) And the total RNA was extracted according to the manufacturer's instructions. CDNA was synthesized by using Superscript II (Invitrogen) and oligo-d (T) 20 primers at 42 ° C for 1 hour and 80 ° C for 10 minutes. The target sequence from the cDNA was subjected to real-time PCR using 7500 Fast Real-time PCR system (Applied Biosystem). The procedure was performed using SYBR Premix Ex Taq (Takara) Denaturation was performed at 95 ° C. for 3 seconds and the target gene amplification process was repeated 40 times at 60 ° C. for 30 seconds. After that, 15 cycles of 95 ° C. for 15 seconds, 60 ° C. for 1 minute to draw a melting curve, Min and 95 ° C for 15 seconds. GAPDH expression levels were normalized using an internal control.
표지인자로, Neural precursor 단계에만 제한적으로 발현되는 CAD19, 수초형성(myelination)이 일어나지 않은 immature Schwann cell 단계를 비롯하여 Schwann cell 분화에 전반적으로 발현되어지는 GFAP, NGFR, KROX20, S100B 및 수초형성(myelination)이 일어났을 때만 특이적으로 발현이 증가하는 MBP를 사용하였다. As a marker, CAD19, which is expressed only in the stage of neural precursor, immature Schwann cell stage in which myelination does not occur, GFAP, NGFR, KROX20, S100B and myelination, which are expressed in Schwann cell differentiation, MBP, which is specifically expressed only when it occurs, was used.
그 결과를 도 6에 나타내었다. The results are shown in Fig.
Neural precursor marker로 슈반세포 분화 후에 발현량이 감소될 것으로 예상되었던 CAD19의 발현이 TMSC-SC와 ASC-SC (ASC-derived Schwann cell) 두 경우 모두 미분화단계에 비해 분화 후에 감소하는 패턴을 보임을 확인하였다. The expression of CAD19, which was expected to decrease after Schwann cell differentiation as a neural precursor marker, was found to decrease after differentiation compared to the undifferentiated stage in both TMSC-SC and ASC-SC (ASC-derived Schwann cell) .
반면, 수초 형성이 일어나지 않은 단계에서 GFAP, NGFR, KROX20 및 S100B의 발현은 미분화 단계의 편도 유래 중간엽 줄기세포에 비해서 편도 유래 중간엽 줄기세포로부터 분화된 슈반 세포에서 모두 증가한 양상을 보임을 확인하였다. 그러나, 지방 조직 유래 줄기세포로부터 유래된 슈반 세포의 경우, GFAP, NGFR 및 S100B의 발현이 미분화 상태의 지방 조직 유래 줄기세포에 비해 오히려 줄어드는 양상을 보였다. On the other hand, GFAP, NGFR, KROX20 And S100B were increased in Schwann cells differentiated from unilateral MSFs compared with unilateral MSFs in undifferentiated stage. However, in the case of Schwann cells derived from adipose tissue-derived stem cells, the expression of GFAP, NGFR and S100B decreased rather than that of undifferentiated adipose tissue-derived stem cells.
즉, 본원 발명의 방법에 따라 편도 유래 줄기세포로부터 슈반 세포로의 분화 유도시에 타 유래 세포와 대비하여 슈반 세포로의 분화능 측면에서 현저한 효과가 있음이 확인되었다. 상기 실험 결과들을 토대로, 본원 발명의 방법에 따른 슈반 세포로의 분화 방법은 사용되는 줄기세포와 분화 방법을 최적화하여 높은 효율로 슈반 세포를 다량 생산할 수 있음을 확인하였다. That is, according to the method of the present invention, it was confirmed that when inducing differentiation from Schistosoma cells derived from one-shot-derived stem cells, Schwann cells were remarkably effective in differentiating into Schwann cells compared to other cells. Based on the above experimental results, it was confirmed that the method of differentiating Schwann cells according to the method of the present invention can produce a large amount of Schwann cells at a high efficiency by optimizing the stem cells and the differentiation method used.
Claims (16)
(b) 상기 (a) 단계의 신경구(Neurosphere)를 혈소판 유래 성장인자 (Platelet-derived growth factor), 섬유아세포 성장 인자 (basic fibroblast growth factor), 헤레굴린-β(Heregulin-β) 및 포스콜린 (Forskolin) 하에서 배양하여 슈반 세포를 유도하는 단계를 포함하는 편도 유래 중간엽 줄기세포로부터 슈반 세포 분화 방법. (a) culturing a mesenchymal stem cell (MSC) derived from one end in an medium containing epidermal growth factor, basic fibroblast growth factor and B27 additive, Inducing a neurosphere; And
(b) comparing the Neurosphere of step (a) with Platelet-derived growth factor, basic fibroblast growth factor, Heregulin-beta and phoscholine Forskolin) to induce Schwann cells. The method for differentiating Schwann cells from monolayer-derived mesenchymal stem cells comprises the steps of:
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