KR20230023997A - Compositions for predicting stem cell therapy reactivity using extracellular vesicles or by the same method - Google Patents
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Abstract
Description
본 발명은 세포외소포를 측정하는 단계; 를 포함하는, 줄기세포 치료제 투여 후 환자의 예후 예측에 대한 정보 제공 방법 및 세포외소포를 포함하는, 뇌 질환 환자의 줄기세포 치료제에 대한 예후 예측용 바이오마커 조성물에 관한 것이다. The present invention comprises the steps of measuring extracellular vesicles; It relates to a biomarker composition for predicting the prognosis of a patient with brain disease, including a method for providing information on prognosis prediction of a patient after administration of stem cell therapy, and an extracellular vesicle.
다양한 질환에서 줄기세포, 특히 중간엽 줄기세포(mesenchymal stem cells; MSC)를 이용한 치료법과 긍정적인 임상 결과들이 보고되어 왔다. 그러나 아직까지 3상 연구에서 유효성이 검증되어 시판되고 있는 줄기세포치료제의 종류는 극히 일부분이다. 따라서, 현재 다양한 질환에서 다양한 생산법 및 전처치 방법으로 효능 증진된 줄기세포치료제가 개발되고 임상시험이 준비 중이다. Therapies using stem cells, especially mesenchymal stem cells (MSC), for various diseases and positive clinical results have been reported. However, only a few types of stem cell therapy products that have been validated in
줄기세포, 특히 중간엽줄기세포의 치료 효능은 대부분 줄기세포의 근거리 분비(paracrine) 효과로 인한 주변 세포의 재생과 보호능력의 증진 및 항염증 반응의 감소에 기인한 것으로 보고되어 있다. 그러나 아직까지 환자에서 줄기세포치료제 적용에 있어서 치료효과를 예측하는 줄기세포치료제 혈중 바이오마커에 대한 연구는 제한적이다. 전신성 홍반성 낭창(systemic lupus erythematosus) 환자에게 MSC를 주입하고 그 치료효과가 혈중 interferon-γ 수준과 관련 있다는 보고(Stem Cell Transl Med 2017;6:1777-85)와 류마티스성 관절염(rheumatoid arthritis) 환자에게 MSC를 주입 후 혈중 마이크로알엔에이(microRNA, miRNA)의 수준, 특히 miRNA-26b-5p, miRNA-487b-3p, miRNA-495-3p가 상승되었다는 보고(Stem Cell Transl Med 2017;6:1202-6)만이 제한적으로 있을 뿐이며, 뇌졸중 환자에서는 이와 같은 줄기세포 치료제 혈중 바이오 마커에 관해 보고된 바 없다. Stem cells, particularly mesenchymal stem cells, have been reported to have therapeutic efficacy due to the enhancement of regeneration and protective ability of surrounding cells and reduction of anti-inflammatory response due to the paracrine effect of stem cells. However, studies on blood biomarkers of stem cell therapy products that predict the therapeutic effect in the application of stem cell therapy products to patients are limited. A report that MSC was injected into a patient with systemic lupus erythematosus and the therapeutic effect was related to the level of interferon-γ in the blood (Stem Cell Transl Med 2017;6:1777-85) and a patient with rheumatoid arthritis reported that the level of microRNA (miRNA) in the blood, especially miRNA-26b-5p, miRNA-487b-3p, and miRNA-495-3p, was elevated after MSC injection (Stem Cell Transl Med 2017;6:1202 -6) is only limited, and there has been no report on such blood biomarkers for stem cell therapy in stroke patients.
한편 세포외소포란 크기에 따라 엑소좀(exosome)과 미세소포(microvesicle)로 분류하는데 엑소좀은 직경 30~150nm이며, 미세소포는 100~1,000nm의 크기를 갖는다. 세포외소포는 세포막 일부가 혈중으로 떨어져 나온 것으로, 단백질과 핵 성분 모두를 함유하고 있어 세포간 커뮤니케이션을 매개하는 것으로 알려져 있다. 세포외소포는 혈액, 소변, 타액, 모유, 흉수, 복수, 뇌척수액 등 대부분의 우리 몸에 정상적으로 혹은 질병 발생 시 발생하는 체액에 존재한다고 밝혀져 있어서 그 임상적 유용성이 대단히 높으며, 실제로 우리 몸의 체액에서 세포외소포를 분리하여 분석함으로써 암은 물론 각종 질환을 진단하는 방법으로 개발하려는 노력들이 국제적으로 매우 활발히 진행되고 있는 실정이다.Meanwhile, extracellular vesicles are classified into exosomes and microvesicles according to their size. Exosomes have a diameter of 30 to 150 nm, and microvesicles have a size of 100 to 1,000 nm. Extracellular vesicles are part of the cell membrane that have been released into the blood, and are known to mediate cell-to-cell communication because they contain both proteins and nuclear components. Extracellular vesicles have been found to exist in body fluids that occur normally or during disease in most of our bodies, such as blood, urine, saliva, breast milk, pleural fluid, ascites, and cerebrospinal fluid, so their clinical usefulness is very high. Efforts to develop a method for diagnosing various diseases as well as cancer by isolating and analyzing extracellular vesicles are being actively conducted internationally.
이에 본 발명자들은 줄기세포 치료제 투여 후 환자의 예후를 예측할 수 있는 바이오 마커에 관하여 연구하던 중, 줄기세포 치료제를 투여한 뇌 질환 환자에서 세포외소포 수준을 측정하여 줄기세포 치료제에 대한 환자의 예후를 예측할 수 있음을 확인하고, 본 발명을 완성하였다. Accordingly, the present inventors, while studying biomarkers capable of predicting the prognosis of patients after administration of stem cell therapy, measured the level of extracellular vesicles in patients with brain diseases to whom stem cell therapy was administered to determine the patient's prognosis for stem cell therapy. It was confirmed that it could be predicted, and the present invention was completed.
따라서, 본 발명의 목적은 1) 줄기세포 치료제를 투여한 뇌 질환 환자에서 세포외소포를 측정하는 단계; 를 포함하는, 줄기세포 치료제 투여 후 환자의 예후 예측에 대한 정보 제공 방법 및 세포외소포를 포함하는, 뇌 질환 환자의 줄기세포 치료제에 대한 예후 예측용 바이오마커 조성물을 제공하는 것이다. Therefore, an object of the present invention is 1) measuring extracellular vesicles in brain disease patients administered stem cell therapy; It is to provide a biomarker composition for predicting the prognosis of a brain disease patient, including a method for providing information on prognosis prediction of a patient after administration of stem cell therapy and extracellular vesicles, including.
상기 목적을 달성하기 위하여, 본 발명은 1) 줄기세포 치료제를 투여한 뇌 질환 환자에서 세포외소포를 측정하는 단계; 를 포함하는, 줄기세포 치료제 투여 후 환자의 예후 예측에 대한 정보 제공 방법을 제공한다. In order to achieve the above object, the present invention is 1) measuring extracellular vesicles in brain disease patients administered stem cell therapy; Including, it provides a method for providing information on the prognosis prediction of patients after administration of stem cell therapy.
또한 본 발명은 세포외소포를 포함하는, 뇌 질환 환자의 줄기세포 치료제에 대한 예후 예측용 바이오마커 조성물을 제공한다. In addition, the present invention provides a biomarker composition for predicting the prognosis of stem cell therapy for brain disease patients, including extracellular vesicles.
또한 본 발명은 세포외소포를 검출하는 제제를 포함하는, 뇌 질환 환자의 줄기세포 치료제에 대한 예후 예측용 조성물을 제공한다. In addition, the present invention provides a composition for predicting the prognosis of a stem cell therapeutic agent for brain disease patients, including an agent for detecting extracellular vesicles.
본 발명에 따르면 줄기세포 치료제 투여 후, 혈중 세포외소포 분석을 통해 뇌 질환 환자의 줄기세포 치료제 투여에 따른 반응성 및 예후 예측이 가능한 바, 환자 맞춤형 치료를 보다 효과적으로 제공할 수 있다. According to the present invention, it is possible to predict the responsiveness and prognosis of patients with brain diseases according to the administration of stem cell therapeutics through the analysis of extracellular vesicles in blood after administration of stem cell therapeutics, so that customized treatment for patients can be provided more effectively.
도 1은 MSC 처리 후 환자에서 혈장 세포외소포의 형태 및 크기 분포를 확인한 결과를 나타낸 도이다.
도 2는 세포외소포 양성 마커 및 음성 마커를 웨스턴 블랏을 통해 확인한 결과를 나타낸 도이다.
도 3은 MSC를 주입하기 전에 MSC 처리 대상 환자군의 순환 세포외소포 수준을 MSC 주입 하루 전(Time point 1) 및 MSC 주입 직전(Time point 2)에 연속적으로 측정한 결과를 나타낸 도이다.
도 4는 MSC 치료 직후 순환 세포외소포의 혈장 수준 측정을 통해, 세포외소포와 MSC 치료효과의 관련성을 확인한 결과를 나타낸 도이다(MSC, mesenchymal stem cell; EV, extracellular vesicles; MCID, minimal clinically important difference; FMA-T, a total score of Fugl-Meyer Assessment; FMA-UL, upper limb score of Fugl-Meyer Assessment; and FMA-LL, lower limb score of Fugl-Meyer Assessment., Mean±SEM, * p=0.001, †p=0.039, ‡p=0.023).
도 5는 MSC 처리군에서 순환 세포외소포 수준 및 임상적 개선과의 관련성을 확인한 결과를 나타낸 도이다.
도 6은 MSC 처리 24시간 후 혈청 내 순환 EV 수준과 신경 가소성의 DTI 및 rs-fMRI 지수 간의 연관성을 90 일에 측정한 결과를 나타낸 도이다(CST, corticospinal tract; FA, fractional anisotropy; M1, primary motor cortex; PMd, dorsal premotor area; PMv, ventral premotor area; SMA, supplementary motor area, p<0.05 in all cases except contralesional CTS).
도 7은 MSC 처리에 따른 혈장 내 성장인자 수준의 변화를 MSC 처리군 (A) 및 대조군 (B) 에서 확인한 결과를 나타낸 도이다(VEGF, vascular endothelial growth factor; BDNF, brain-derived neurotrophic factor; SDF-1, stromal cell-derived factor-1; NSE, neuron-specific enolas, p>0.05 in all cases).
도 8은 줄기세포 투여 후, 신경가소성 관련 microRNA인 has-miR-18a-5p의 상대적인 발현 증감을 확인한 결과를 나타낸 결과(A) 및 해당 microRNA의 표적 유전자에서의 기능을 GO (gene ontology) 경로 분석(B) 및 KEGG (Kyoto Encyclopedia of Genes and Genomes) 분석(C)을 통해 확인하여, has-miR-18a-5p가 신경계 발달, 신경 성장인자 수용체 신호전달 경로 및 축삭 유도와 관련된 신호 경로 및 생물학적 과정과 관련 있음을 확인한 결과를 나타낸 도이다. 1 is a diagram showing the results of confirming the shape and size distribution of plasma extracellular vesicles in patients after MSC treatment.
Figure 2 is a diagram showing the results of confirming the extracellular vesicle positive markers and negative markers through Western blotting.
Figure 3 is a diagram showing the results of continuously measuring the level of circulating extracellular vesicles in the MSC-treated patient group before MSC injection one day before MSC injection (Time point 1) and immediately before MSC injection (Time point 2).
Figure 4 is a diagram showing the results of confirming the relationship between extracellular vesicles and MSC treatment effects through measurement of plasma levels of circulating extracellular vesicles immediately after MSC treatment (MSC, mesenchymal stem cell; EV, extracellular vesicles; MCID, minimal clinically important difference; FMA-T, a total score of Fugl-Meyer Assessment; FMA-UL, upper limb score of Fugl-Meyer Assessment; and FMA-LL, lower limb score of Fugl-Meyer Assessment., Mean±SEM, * p= 0.001, †p=0.039, ‡p=0.023).
Figure 5 is a diagram showing the results of confirming the relationship between the level of circulating extracellular vesicles and clinical improvement in the MSC-treated group.
6 is a diagram showing the results of measuring the correlation between circulating EV levels in serum and DTI and rs-fMRI indices of neuroplasticity at 90 days after MSC treatment 24 hours (CST, corticospinal tract; FA, fractional anisotropy; M1, primary motor cortex; PMd, dorsal premotor area; PMv, ventral premotor area; SMA, supplementary motor area, p<0.05 in all cases except contralesional CTS).
Figure 7 is a diagram showing the results of confirming the change in the level of growth factors in plasma according to MSC treatment in the MSC treatment group (A) and control group (B) (VEGF, vascular endothelial growth factor; BDNF, brain-derived neurotrophic factor; SDF -1, stromal cell-derived factor-1; NSE, neuron-specific enolas, p>0.05 in all cases).
Figure 8 shows the result of confirming the relative expression increase or decrease of has-miR-18a-5p, a neuroplasticity-related microRNA, after stem cell administration (A) and GO (gene ontology) pathway analysis of the function of the microRNA in the target gene (B) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis (C) confirm that has-miR-18a-5p is a signal pathway and biological process related to nervous system development, nerve growth factor receptor signaling pathway, and axon guidance. It is a diagram showing the result of confirming that it is related to .
본 발명은 1) 줄기세포 치료제를 투여한 뇌 질환 환자에서 세포외소포를 측정하는 단계; 를 포함하는, 줄기세포 치료제 투여 후 환자의 예후 예측에 대한 정보 제공 방법을 제공한다. The present invention comprises the steps of 1) measuring extracellular vesicles in a brain disease patient administered a stem cell therapy; Including, it provides a method for providing information on the prognosis prediction of patients after administration of stem cell therapy.
본 발명에서 용어 "예후 예측에 대한 정보를 제공"은 줄기세포 치료제 투여에 의한 환자의 반응성, 병리 상태의 개선, 호전에 대한 정보를 제공하는 것을 의미한다. 본 발명의 목적상, 뇌 질환의 임상 증상이나 병리적 현상이 개선되는지 예측하는 것을 의미하며, 이와 같은 예후 예측을 통해 줄기세포 치료에 적합한 환자인지 아닌지 판단하여 환자 맞춤형 치료를 제공할 수 있다. In the present invention, the term "providing information on prognosis prediction" means providing information on the patient's responsiveness, pathological state improvement, and improvement by the administration of stem cell therapy. For the purpose of the present invention, it means to predict whether clinical symptoms or pathological phenomena of brain diseases are improved, and through such prognosis prediction, it is possible to determine whether a patient is suitable for stem cell treatment or not to provide a patient-specific treatment.
본 발명에서, 상기 방법은 2) 상기 1) 단계에서 측정된 세포외소포 수준과 줄기세포 치료제 투여 전 측정된 세포외소포의 수준을 비교하는 단계; 를 더 포함하는, 줄기세포 치료제 투여 후 환자의 예후 예측에 대한 정보 제공 방법일 수 있다. In the present invention, the method comprises: 2) comparing the level of extracellular vesicles measured in step 1) with the level of extracellular vesicles measured before administration of a stem cell therapy; It may be a method of providing information about predicting the patient's prognosis after administration of stem cell therapy, further comprising.
상기 2) 단계에서는 줄기세포 치료제 투여 전 측정된 세포외소포의 수준과 줄기세포 투여 후 측정된 세포외소포 수준을 비교하여, 줄기세포 투여 전 대비 투여 후 세포외소포 수준이 증가된 경우, 해당 뇌 질환 환자가 줄기세포 치료제를 통해 뇌 질환이 치료, 개선, 완화되어 좋은 예후를 나타낼 수 있는 것으로 예측할 수 있다. 반면 줄기세포 투여 전 대비 투여 후 세포외소포가 증가되지 않고, 감소 또는 동등한 수준으로 유지되는 경우, 해당 뇌 질환 환자는 줄기세포 치료제 투여에도 불구하고, 뇌 질환의 치료, 개선, 완화 효과가 나타나지 않아, 좋은 예후를 기대할 수 없는 것으로 예측할 수 있다. In step 2), the level of extracellular vesicles measured before administration of stem cell therapy and the level of extracellular vesicles measured after administration of stem cells are compared, and if the level of extracellular vesicles is increased after administration compared to before administration of stem cells, the corresponding brain It can be predicted that disease patients can treat, improve, and alleviate brain diseases through stem cell therapy, resulting in a good prognosis. On the other hand, if the amount of extracellular vesicles is not increased, decreased, or maintained at an equal level after administration compared to before stem cell administration, the patient with the brain disease does not show the effect of treating, improving, or alleviating the brain disease despite the administration of stem cell therapy. , it can be predicted that a good prognosis cannot be expected.
본 발명에 있어, 상기 1) 단계의 세포외소포 측정은 혈중 세포외소포의 반감기를 고려하여, 줄기세포 치료제 투여 후 240 시간 이내 측정하는 것을 특징으로 할 수 있으며, 바람직하게는 줄기세포 치료제 투여 후 168시간 이내, 더욱 바람직하게는 투여 후 120 시간 이내, 가장 바람직하게는 72시간 이내 측정하는 것을 특징으로 할 수 있다. 본 발명의 바람직한 일 구현예에서는 혈액 내 세포외소포가 줄기세포 치료제 투여 후 24시간 이내에 가장 급격하게 증가하는 것을 확인하였다. In the present invention, the measurement of extracellular vesicles in step 1) may be characterized in that it is measured within 240 hours after administration of stem cell therapy, taking into account the half-life of extracellular vesicles in blood, and preferably after administration of stem cell therapy It can be characterized in that it is measured within 168 hours, more preferably within 120 hours after administration, and most preferably within 72 hours. In a preferred embodiment of the present invention, it was confirmed that extracellular vesicles in the blood most rapidly increased within 24 hours after administration of the stem cell therapy.
한편, 줄기세포 치료제에 대한 반응성을 나타내는 환자에서 세포외소포가 증가함에 따라, 세포외소포 내 포함되어 있는 뇌질환 예후 개선과 관련된 miRNA 의 수준도 함께 높게 측정되는 것을 확인하였다. 구체적으로 신경가소성, 신경 재생, 신경소세포형성(oligodendrogenesis), 혈관 신생, 세포 보호, 항염증, 뇌졸중 후 회복과 관련된 miRNA, 바람직하게는 miR-17-3p, miR18a-5p, miR-20a-5p, miR-92-1, miR-133b, miR-126-5p, miR-132-3p, miR-181b-5p, miR-494-3p, miR-19a-3p, miR-146a-5p, miR-210-3p, miR-223-3p, miR-21-5p 또는 miR-196a-5p, 더욱 바람직하게는 miR-17-92 클러스터인 miR-17-3p, 18a-5p, miR-20a-5p, miR-92-1 및/또는 miR-133b의 발현 수준이 대조군 대비 증가되어 있는 것을 확인하였다. On the other hand, as extracellular vesicles increased in patients showing responsiveness to stem cell therapy, it was confirmed that the levels of miRNAs included in extracellular vesicles related to improving prognosis of brain diseases were also measured high. Specifically, miRNAs related to neuroplasticity, nerve regeneration, oligodendrogenesis, angiogenesis, cell protection, anti-inflammation, and post-stroke recovery, preferably miR-17-3p, miR18a-5p , miR-20a-5p, miR-92-1, miR-133b, miR-126-5p, miR-132-3p, miR-181b-5p, miR-494-3p, miR-19a-3p, miR-146a-5p, miR-210- 3p, miR-223-3p, miR-21-5p or miR-196a-5p, more preferably miR-17-3p, 18a-5p , miR-20a-5p, miR-92 cluster of miR-17-92 It was confirmed that the expression level of -1 and/or miR-133b was increased compared to the control group.
따라서, 본 발명의 예후 예측에 대한 정보 제공 방법은, 상기 뇌 질환 환자에 줄기세포 치료제 투여 이후, miR-17-3p, miR18a-5p, miR-20a-5p, miR-92-1, miR-133b, miR-126-5p, miR-132-3p, miR-181b-5p, miR-494-3p, miR-19a-3p, miR-146a-5p, miR-210-3p, miR-223-3p, miR-21-5p 또는 miR-196a-5p 의 발현 수준 변화를 측정하는 단계를 추가로 포함하는 것을 특징으로 할 수 있고, 바람직하게는 miR-17-92 클러스터인 miR-17-3p, 18a-5p, miR-20a-5p, miR-92-1 및/또는 miR-133b의 발현 수준 변화를 측정하는 단계를 추가로 포함하는 것을 특징으로 할 수 있다. Therefore, the method for providing information on prognosis prediction of the present invention, after administration of stem cell therapy to the brain disease patient, miR-17-3p, miR18a-5p , miR-20a-5p, miR-92-1, miR-133b , miR-126-5p, miR-132-3p, miR-181b-5p, miR-494-3p, miR-19a-3p, miR-146a-5p, miR-210-3p, miR-223-3p, miR -21-5p or miR-196a-5p may be characterized by further comprising the step of measuring the change in expression level, preferably miR-17-92 cluster miR-17-3p, 18a-5p , It may be characterized by further comprising the step of measuring the change in the expression level of miR-20a-5p, miR-92-1 and/or miR-133b.
본 발명에 있어서, 상기 줄기세포 치료제는 유래에 제한은 없으나, 자가, 타가 또는 동종이계 유래 줄기세포 치료제일 수 있고, 바람직하게는 자가(autologous) 줄기세포 치료제일 수 있다. In the present invention, the stem cell therapeutic agent is not limited in origin, but may be an autologous, allogeneic or allogeneic stem cell therapeutic agent, preferably an autologous stem cell therapeutic agent.
또한 본 발명의 줄기세포 치료제는 뇌 질환의 치료에 사용될 수 있는 것이라면 그 종류를 제한없이 포함할 수 있으며, 예컨대 중간엽 줄기세포, 만능줄기세포, 유도만능줄기세포 및 배아줄기세포로 이루어진 군에서 선택된 어느 하나 이상일 수 있고, 제대혈, 탯줄, 골수, 지방, 태반, 와튼 젤리(Wharton jelly), 양수, 양막 및 편도로 이루어진 군에서 선택된 1종에서 유래된 줄기세포일 수 있다. 본 발명의 바람직한 일 구현예에서는 골수 유래 중간엽 줄기세포 치료제를 이용하였다. In addition, the stem cell therapeutic agent of the present invention may include any type without limitation as long as it can be used for the treatment of brain diseases, for example, mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, and embryonic stem cells selected from the group consisting of It may be any one or more, and may be stem cells derived from one selected from the group consisting of umbilical cord blood, umbilical cord, bone marrow, fat, placenta, Wharton jelly, amniotic fluid, amnion, and tonsil. In a preferred embodiment of the present invention, a bone marrow-derived mesenchymal stem cell treatment agent was used.
본 발명에 있어서, 뇌 질환은 뇌에 발생할 수 있는 다양한 질환을 제한없이 포함할 수 있으나, 바람직하게 허혈성 뇌질환, 뇌 신경 손상질환 및 퇴행성 뇌질환으로 이루어진 군에서 선택된 1종 이상일 수 있다. In the present invention, the brain disease may include various diseases that may occur in the brain without limitation, but may preferably be at least one selected from the group consisting of ischemic brain disease, cranial nerve injury disease, and degenerative brain disease.
본 발명의 뇌 질환이 허혈성 뇌질환인 경우, 이는 허혈성 뇌졸중, 혈전증, 색전증, 일과성 허혈발작, 백질이상증 및 소경색으로 이루어진 군에서 선택된 1종 이상일 수 있고, 뇌 신경 손상질환인 경우, 뇌내출혈에 의한 뇌신경 손상 질환 및 외상으로 인한 뇌신경 손상 질환으로 이루어진 군에서 선택된 1종 이상일 수 있으며, 퇴행성 뇌질환인 경우, 치매, 알츠하이머 및 파킨슨으로 이루어진 군에서 선택된 1종 이상일 수 있다. When the brain disease of the present invention is an ischemic brain disease, it may be at least one selected from the group consisting of ischemic stroke, thrombosis, embolism, transient ischemic attack, leukodystrophy, and small infarction, and in the case of a cranial nerve damage disease, intracerebral hemorrhage It may be at least one selected from the group consisting of cranial nerve injury diseases caused by trauma and cranial nerve injury diseases due to trauma, and in the case of degenerative brain diseases, it may be at least one selected from the group consisting of dementia, Alzheimer's disease, and Parkinson's disease.
본 발명에 따르면, 줄기세포 치료제 투여 이후 혈중 세포외소포의 증가는 줄기세포 치료제에 의한 다양한 임상 및 병리학적 개선과 유의미한 상관 관계가 있으며, 보다 구체적으로, 운동 기능의 회복과 같은 임상적 호전과, DTI 및 rs-fMRI 와 같은 영상적 평가에서 확인할 수 있는 신경 가소성 회복, 동면 피질 척수로(ipsilesional corticospinal tract) 및 반구 내 운동 네트워크(intrahemispheric motor network)의 완전성 개선, 해부학적인 회복 및 기능 회복, 병변측 운동 네트워크 (ipsilesional motor network)와 유의미한 상관 관계를 나타냄을 확인하였다. According to the present invention, the increase in extracellular vesicles in blood after administration of stem cell therapy has a significant correlation with various clinical and pathological improvements by stem cell therapy, and more specifically, clinical improvement such as recovery of motor function, Restoration of neuroplasticity, improvement of integrity of ipsilesional corticospinal tract and intrahemispheric motor network, anatomical recovery and functional recovery, lesion side, as confirmed by imaging evaluations such as DTI and rs-fMRI It was confirmed that it showed a significant correlation with the ipsilesional motor network.
따라서, 줄기세포 치료제 투여 후 혈액 내 순환하는 순환 세포외소포 수준의 변화를 통해 환자의 예후를 예측할 수 있으며, 여기에서 환자의 예후란 줄기세포 치료제 투여로 인하여 개선될 수 있는 뇌 질환의 다양한 병리학적, 임상학적 상태를 의미하고, 예컨대 뇌 질환에 따른 운동 기능 손실 회복, 뇌 병변의 감소, 피질 척수로 (corticospinal tract) 손상의 개선 및 반구 내 운동 네트워크(intrahemispheric motor network)의 개선으로 이루어진 군에서 선택된 1종 이상을 의미할 수 있다. 상기 뇌병변의 감소는 뇌 영상 데이터를 통해 확인할 수 있고, 상기 뇌 영상 데이터는 뇌 질환의 병리학적 상태를 평가할 수 있는 당 분야에 공지된 다양한 기법을 통해 확인할 수 있으나, 예컨대 확산텐서영상(diffusion tensor imaging, DTI) 및 휴식상태 기능적 자기공명영상(resting state-functional MRI, rs-fMRI) 에 의해 얻어질 수 있다. Therefore, the patient's prognosis can be predicted through changes in the level of circulating extracellular vesicles circulating in the blood after administration of stem cell therapy. , Refers to a clinical condition, e.g., recovery from loss of motorfunction due to brain disease, reduction of brain lesions, improvement of corticospinal tract damage and improvement of intrahemispheric motor network selected from the group consisting of It may mean one or more types. The reduction of the brain lesion can be confirmed through brain image data, and the brain image data can be confirmed through various techniques known in the art that can evaluate the pathological state of brain disease, but, for example, diffusion tensor imaging imaging, DTI) and resting state-functional MRI (rs-fMRI).
본 발명에 있어, 예후 예측 마커로 사용되는 세포외소포는 혈중에서 순환하는 혈중 세포외소포일 수 있으며, 혈청, 혈장 내 세포외소포를 모두 포함하는 것을 의미한다. 상기 세포외소포는 당 분야에 공지된 다양한 방법을 통해 제한없이 수득하고 혈중 농도를 측정할 수 있으며, 본 발명의 일 구현예에서는 원심분리법 및 Exoquick을 사용하여 혈청 내 세포외 소포체를 분리 수득하였다. 혈중 세포외소포 수준은 환자마다 각각 상이할 수 있으나, 줄기세포 치료제 투여와 같은 처리없이는 환자의 혈액 내 순환 세포외소포 수준은 일정하게 유지되는 특징을 나타내고, 줄기세포 투여 전 후의 혈중 세포외소포 변화에 의하여 개별적으로 예후 예측이 가능한 것을 특징으로 할 수 있다. In the present invention, the extracellular vesicles used as prognostic markers may be extracellular vesicles circulating in the blood, and include both serum and plasma extracellular vesicles. The extracellular vesicles can be obtained without limitation through various methods known in the art and the concentration in blood can be measured. In one embodiment of the present invention, extracellular vesicles in serum are separated and obtained using centrifugation and Exoquick. Although the level of extracellular vesicles in blood may vary from patient to patient, the level of circulating extracellular vesicles in the blood of a patient is maintained constant without treatment such as administration of stem cell therapy, and the change of extracellular vesicles in blood before and after stem cell administration It can be characterized in that individual prognosis can be predicted by.
또한 본 발명은 세포외소포를 포함하는, 뇌 질환 환자의 줄기세포 치료제에 대한 예후 예측용 바이오마커 조성물, 또는 세포외소포를 검출하는 제제를 포함하는, 뇌 질환 환자의 줄기세포 치료제에 대한 예후 예측용 조성물에 관한 것이다. In addition, the present invention is a biomarker composition for predicting the prognosis of stem cell therapeutics for brain disease patients, including extracellular vesicles, or a prognostic prediction for stem cell therapeutics for brain disease patients, including an agent for detecting extracellular vesicles. It is about a composition for
상기 세포외소포를 검출하는 제제는 Nanosight, Exoview, Zetaview, qNano, FACS, Cytoflex, Exoid 등의 instrument에서 사용되는 세포외소포 농도 및 크기 분석을 위한 제제일 수 있다. The agent for detecting the extracellular vesicles may be an agent for analyzing the concentration and size of extracellular vesicles used in instruments such as Nanosight, Exoview, Zetaview, qNano, FACS, Cytoflex, and Exoid.
본 발명의 바이오마커 조성물 또는 예후 예측용 조성물을 이용하면, 뇌 질환 환자의 혈중 세포외소포 수준의 변화를 통해 줄기세포 치료제에 대한 환자의 예후를 예측할 수 있어, 환자 맞춤형 치료를 제공할 수 있다. Using the biomarker composition or the composition for predicting prognosis of the present invention, it is possible to predict the prognosis of a patient for stem cell therapy through changes in the level of extracellular vesicles in the blood of a patient with brain disease, thereby providing a patient-specific treatment.
본 발명에 있어 환자의 예후는 줄기세포 치료제 투여로 인하여 개선될 수 있는 뇌 질환의 다양한 병리학적, 임상학적 상태를 의미하고, 예컨대 뇌 질환에 따른 운동 기능 손실 회복, 뇌 병변의 감소, 피질 척수로 (corticospinal tract) 손상의 개선 및 반구 내 운동 네트워크(intrahemispheric motor network)의 개선으로 이루어진 군에서 선택된 1종 이상을 의미할 수 있다. 상기 뇌병변의 감소는 뇌영상 데이터를 통해 확인할 수 있고, 상기 뇌 영상 데이터는 뇌 질환의 병리학적 상태를 평가할 수 있는 당 분야에 공지된 다양한 기법을 통해 확인할 수 있으나, 예컨대 확산텐서영상(diffusion tensor imaging, DTI) 및 휴식상태 기능적 자기공명영상(resting state-functional MRI, rs-fMRI) 에 의해 얻어질 수 있다.In the present invention, the patient's prognosis refers to various pathological and clinical conditions of brain diseases that can be improved by the administration of stem cell therapeutics, such as recovery of motor and functional loss caused by brain diseases, reduction of brain lesions, cortical spinal cord It may refer to at least one selected from the group consisting of improvement of (corticospinal tract) damage and improvement of intrahemispheric motor network. The reduction of the brain lesion can be confirmed through neuroimaging data, and the brain image data can be confirmed through various techniques known in the art that can evaluate the pathological state of brain disease, but, for example, diffusion tensor imaging imaging, DTI) and resting state-functional MRI (rs-fMRI).
본 바이오 마커 조성물, 예후 예측용 조성물에 관한 세부사항은 앞서 기재된 줄기세포 치료제 투여 후 환자의 예후 예측에 대한 정보 제공 방법에 관한 설명을 동일하게 적용할 수 있다. The details of the biomarker composition and the composition for predicting prognosis can be equally applied to the description of the method for providing information on the prognosis prediction of a patient after administering a stem cell therapy described above.
본 발명에서는 세포외소포가 줄기세포 치료제를 이용한 뇌 질환에 대한 반응성 및 예후 예측 마커로 사용할 수 있음을 확인하였고, 나아가 줄기세포 투여 후 세포외소포 내 줄기세포의 치료 효능 및 뇌졸중 후 회복과 관련된 miRNA 의 수준이 증가할 수 있음을 확인하였다. 보다 구체적으로 신경 가소성, 신경재생, 신경소세포형성(oligodendrogenesis) 과 관련이 있는 miRNA로 miR-17-92 (miR-17-3p, miR 18a-5p, miR-20a-5p, miR-92-1), miR-133b를 선정하였고, 혈관 신생과 관련이 있는 miRNA 로 miR-126-5p, miR-132-3p, miR-181b-5p, miR-494-3p를 선정하였으며, 세포 보호 및 항염 관련 miR-19a-3p, miR-146a-5p, miR-210-3p, miR-223-3p, 및 뇌졸중 후 회복과 관련된 miR-21-5p, miR-196a-5p 를 확인하였다. 그 결과, 줄기세포 치료제 투여에 의해 반응성을 나타내는 환자군에서의 세포외소포 증가와 더불어, 세포외소포 내에 포함되어 있는 뇌질환 예후 개선과 관련된 miRNA 도 증가되어 있음을 확인하였고, 특히 예컨대 miR-17-92 클러스터인 miR-17-3p, 18a-5p, miR-20a-5p, miR-92-1 및/또는 miR-133b의 발현 수준이 증가되어 있음을 확인하였다. 이러한 결과를 통해 세포외소포가 줄기세포 치료제에 대한 예후 예측 마커가 될 수 있음을 확인하였고, 이의 수준 분석과 더불어 세포외소포 내 포함되어 있는 miRNA 수준 분석을 통해 줄기세포 치료제에 의해 효과를 나타낼 수 있는 환자군을 선별할 수 있음을 확인할 수 있다. In the present invention, it was confirmed that extracellular vesicles can be used as markers for predicting responsiveness and prognosis for brain diseases using stem cell therapy, and furthermore, miRNAs related to the therapeutic efficacy of stem cells in extracellular vesicles and recovery after stroke after stem cell administration It was confirmed that the level of More specifically, miR-17-92 (miR-17-3p,
중복되는 내용은 본 명세서의 복잡성을 고려하여 생략하며, 본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다.Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined in the present specification have meanings commonly used in the technical field to which the present invention belongs.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.
실시예 1. MSC 치료 임상 시험군 설정 및 치료 효과 평가 Example 1. MSC treatment clinical trial group establishment and treatment effect evaluation
1.1. 임상 시험군의 설정 1.1. Establishment of clinical trial groups
STARTING-2 임상시험(Trials 2013; 14:317) 대상자인 총 54명의 환자에서 혈청 샘플을 수득하였다. 환자들의 평균 나이는 63.4세였으며 여성은 27명이였다. 시험 참가자들을 표준 치료 외에 자가 골수-유래 MSC를 정맥 내로 주입받는 MSC 투여군 (MSC 군) 39명과 표준 치료만을 단독으로 받는 대조군 15명으로 무작위 배정하였다. 표준 치료는 일반적으로 뇌졸중 환자에게 처치되는 치료법을 의미하며, 항혈전제, 항염증제를 환자의 상태에 따라 투여하는 것을 의미한다. 두 군간의 나이, 성별, 뇌졸중 중증도, 위험인자 등 임상적인 차이는 없었다. 시험 참가자의 정보를 표 1에 나타내었다. Serum samples were obtained from a total of 54 patients who were subjects of the STARTING-2 clinical trial (Trials 2013; 14:317). The average age of the patients was 63.4 years old and there were 27 females. The trial participants were randomly assigned to 39 MSC-administered groups (MSC group) receiving intravenous infusion of autologous bone marrow-derived MSCs in addition to standard treatment and 15 control subjects receiving standard treatment alone. Standard treatment generally refers to treatment for stroke patients, and means administration of antithrombotic and anti-inflammatory drugs according to the patient's condition. There were no clinical differences between the two groups, such as age, gender, stroke severity, and risk factors. The information of the test participants is shown in Table 1.
(AMI, acute myocardial infarction; mRS, modified Rankin scale; NIHSS, National Institutes of Health stroke scale; mBI, modified Barthel Index) (AMI, acute myocardial infarction; mRS, modified Rankin scale; NIHSS, National Institutes of Health stroke scale; mBI, modified Barthel Index)
모든 참가자들은 입원환자 재활기간 동안 필요한 재활 치료를 받았으며, MSC 군의 환자들에게는 뇌졸중이 발병하고 55.9 ± 19.1 일 내에 1X106 cells/mL (최대 1.2 × 108 MSCs/kg)로 확장된 자가 MSC를 10분에 걸쳐 전주와정맥(antecubital vein) 을 통해 생리 식염수와 함께 5 × 106 MSCs/ml 가 되도록 천천히 주입하였다.All participants received necessary rehabilitation treatment during the inpatient rehabilitation period. Patients in the MSC group received autologous MSCs that expanded to 1X10 6 cells/mL (up to 1.2 × 10 8 MSCs/kg) within 55.9 ± 19.1 days after onset of stroke. 5 × 10 6 MSCs / ml was slowly injected with physiological saline through the antecubital vein over 10 minutes.
1.2 MSC 처리에 따른 뇌졸중 반응성 확인 1.2 Confirmation of Stroke Responsiveness to MSC Treatment
MSC 치료에 의한 뇌졸중 개선 효과를 확인하기 위하여, MSC 처리 후 운동 기능 회복과 같은 임상적 호전 정도와 함께 영상을 통한 뇌졸중 개선 효과를 확인하였다. 구체적으로, 세부 운동 기능의 변화는 수정된 Rankin 점수와 Fugl-Meyer Assessment (FMA)로 평가하였다. 각 점수의 변화는 무작위화 후 90 일 동안 연속적으로 측정하였다. 임상적 호전을 평가하기 위한 점수 지표로 [Top Stroke Rehabil 2011;18 Suppl 1:599; Top Stroke Rehabil 2016;23:233-9] 문헌에 기재된 지표를 동일하게 적용하여 이용하였다. In order to confirm the stroke amelioration effect by MSC treatment, the degree of clinical improvement such as motor function recovery after MSC treatment and the stroke amelioration effect through imaging were confirmed. Specifically, changes in fine motor function were evaluated using the modified Rankin score and the Fugl-Meyer Assessment (FMA). Changes in each score were measured continuously for 90 days after randomization. As a score index to evaluate clinical improvement [Top Stroke Rehabil 2011;18 Suppl 1:599; Top Stroke Rehabil 2016;23:233-9] The index described in the literature was applied and used in the same way.
영상적 회복 정도는 DTI 및 rs-fMRI를 90일째 시행하여 평가하였다. 구체적인 분석방법은 기존의 방법을 따라 진행하였다(Neuroimage Clin 2013;2:521-33; Neuroimage 2012;59:2771-82). The degree of imaging recovery was evaluated by performing DTI and rs-fMRI on the 90th day. The detailed analysis method was carried out according to the existing method (Neuroimage Clin 2013; 2:521-33; Neuroimage 2012;59:2771-82).
상기와 같은 기준에 따라, 임상적 평가 및 영상적 평가에서 모두 뇌졸중 개선 효과를 나타내는 환자군을 반응성 환자군(good responder group) 으로 분류하였고, 유의적인 개선 효과를 나타내지 않는 환자군을 저반응성 환자군 (poor responder group) 으로 분류하였다. 분류에 따라 MSC 군 39 명 중 9 명의 환자, 그리고 대조군 15명 중 2명의 환자가 90일 이후 FMA-T 에 따른 반응성 환자군으로 분류되었다 (p>0.05).According to the above criteria, the patient group showing a stroke improvement effect in both clinical evaluation and imaging evaluation was classified as a good responder group, and the patient group showing no significant improvement effect was classified as a poor responder group. ) were classified as According to the classification, 9 out of 39 patients in the MSC group and 2 out of 15 patients in the control group were classified as responsive patients according to FMA-T after 90 days (p>0.05).
MSC 처리에 의하여 뇌졸중 치료 효과를 나타낸 반응성 환자군과 MSC 처리에도 불구하고 뇌졸중 치료 효과를 나타내지 않은 비 반응성 환자군을 구별할 수 있는 MSC 처리에 대한 반응성 평가 지표를 확인하기 위하여, MSC 투여 전후 세포외소포, 성장인자의 변화와 반응성과의 관련성을 이하 실시예를 통해 확인하였다. In order to identify a responsive evaluation index for MSC treatment that can distinguish between a responsive patient group showing a stroke treatment effect by MSC treatment and a non-responsive patient group showing no stroke treatment effect despite MSC treatment, MSC treatment before and after extracellular parcels, The relationship between changes in growth factors and reactivity was confirmed through the following examples.
실시예 2. MSC 처리에 대한 반응성 평가 지표 확인 - 세포외소포 Example 2. Confirmation of reactivity evaluation index for MSC treatment - extracellular vesicles
2.1 세포외소포 수득 2.1 Obtaining extracellular vesicles
참가자들의 혈청을 MSC 정맥 투여 전과 후에 순차적으로 수득하였다. 혈청 수집 시기는 MSC 주입군의 경우 MSC 주입 2일 전과 1일 전 및 주입 후 24시간내 (postTxD0), 주입 후 3일째 (postTxD3), 7일째 (postTxD7), 14일째 (postTxD14) 및 90일째 (postTxD90)로 하였으며, 대조군의 경우 연구개시 30일째 (preTx), 44일째 (postTx14) 및 120일째 (postTx90) 시행하였다. 시트레이트된 전혈로부터 혈장 시료를 제조하고, 이후 실험을 위하여 즉시 2000 g에서 15분간 원심분리 후 -80℃에 보관하였다. 세포외소포를 분리하고 측정하기 위하여, 시트레이트 혈장 (citrate plasma) 을 PBS와 1:1로 희석한 후 15분간 10,000 g로 4℃에서 원심분리하였다. 미리 제거된 상층액 유래의 세포외소포를 1시간 동안 100,000 g로 원심분리하여 펠렛화하였다. 원심분리 과정은 4℃에서 Optima TLX ultracentrifuge (Beckman Coulter, Brea, CA, USA)과 TLA120.2 rotor를 이용하여 수행하였다. 펠렛을 여과된 PBS에 재현탁하고 동일한 튜브를 사용하여 4℃에서 60 분 동안 100,000g로 재원심분리하였다. 세포외소포를 포함하는 최종 펠릿을 100 μL PBS에 재현탁하였다. 이후 원심분리법과 Exoquick을 사용하여 혈청내 세포외소포를 분리하였다. Serum from participants was obtained sequentially before and after intravenous administration of MSCs. Serum collection times were 2 days before and 1 day before MSC injection and within 24 hours after injection (postTxD0), 3 days after injection (postTxD3), 7 days (postTxD7), 14 days (postTxD14) and 90 days in the case of the MSC injection group. postTxD90), and in the case of the control group, it was conducted on the 30th day (preTx), 44th day (postTx14) and 120th day (postTx90). Plasma samples were prepared from citrated whole blood, and immediately centrifuged at 2000 g for 15 minutes and stored at -80 °C for later experiments. To isolate and measure extracellular vesicles, citrate plasma was diluted 1:1 with PBS and centrifuged at 10,000 g for 15 minutes at 4°C. Extracellular vesicles from the previously removed supernatant were pelleted by centrifugation at 100,000 g for 1 hour. Centrifugation was performed at 4°C using an Optima TLX ultracentrifuge (Beckman Coulter, Brea, CA, USA) and a TLA120.2 rotor. The pellet was resuspended in filtered PBS and re-centrifuged at 100,000g for 60 minutes at 4°C using the same tube. The final pellet containing extracellular vesicles was resuspended in 100 μL PBS. Thereafter, extracellular vesicles in serum were separated using centrifugation and Exoquick.
2.2 세포외소포 분석 2.2 Extracellular vesicle analysis
최적의 분석을 위해 세포외소포를 소포가 없는 물에 미리 희석하였으며, 세포외소포의 농도 및 크기 분포를 NanoSight NS300 instrument (Malvern, Worcestershire, UK)를 통해 측정하였다. 세포외소포의 평균 크기 및 농도 (particles/mL) 는 3가지 개별 측정 데이터를 통합하여 계산하였다. 세포외소포는 Cryo-transmission electron microscopy (TEM) 를 이용하여 직접 시각화하였다. 세포외소포 양성 및 음성 마커를 확인하기 위하여, 20㎍ 세포외소포 단백질을 SDS-PAGE 를 통해 분리하고 이를 니트로셀룰로오스 막으로 이동시켰다(Bio-Rad, Hercules, CA, USA). 막을 CD63, Flotillin-1, 및 HSP70 (1:1000, Cell Signaling Technology, Beverly, MA, USA), 또는 calnexin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) 에 대한 1차 항체와 함께 4℃에서 밤새 배양 후, horseradish peroxidase (HRP)-접합된 2차 항체(1:1000, anti-rabbit, Cell Signaling Technology)와 함께 2시간 동안 반응시켰다. 단백질을 ThermoFisher Scientific, Inc. (Waltham, MA, USA)의 chemiluminescence substrate를 사용하여 검출하였고, X-선 필름 (Agfa, Mortsel, Belgium)으로 시각화하였다. MSC 처리 후 환자에서 혈장 세포외소포의 수준을 측정하였으며 혈장 세포외소포의 형태 및 크기분포를 Cryo-TEM 를 통해 확인하고 그 결과를 도 1에 나타내었고, 세포외소포 양성 및 음성 마커 검출 결과를 도 2에 나타내었다. For optimal analysis, extracellular vesicles were pre-diluted in vesicle-free water, and the concentration and size distribution of extracellular vesicles were measured using a NanoSight NS300 instrument (Malvern, Worcestershire, UK). The average size and concentration (particles/mL) of extracellular vesicles were calculated by integrating the data of three separate measurements. Extracellular vesicles were directly visualized using cryo-transmission electron microscopy (TEM). To identify extracellular vesicle positive and negative markers, 20 μg of extracellular vesicle protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Membranes were incubated with primary antibodies to CD63, Flotillin-1, and HSP70 (1:1000, Cell Signaling Technology, Beverly, MA, USA), or calnexin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After overnight incubation at 4°C, reaction was performed for 2 hours with horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000, anti-rabbit, Cell Signaling Technology). Proteins were purchased from ThermoFisher Scientific, Inc. (Waltham, MA, USA) and visualized with X-ray film (Agfa, Mortsel, Belgium). After MSC treatment, the level of plasma extracellular vesicles was measured in the patient, and the shape and size distribution of plasma extracellular vesicles were confirmed by cryo-TEM, and the results are shown in FIG. shown in Figure 2.
도 1에 나타낸 바와 같이, 대부분의 세포외소포는 전자 밀도 구조의 둥근 모양을 가졌으며 평균(SD) 직경은 각각 156.98 (17.5) nm 인 것을 확인하였다. As shown in Figure 1, it was confirmed that most of the extracellular vesicles had a round shape of electron density structure, and the average (SD) diameter was 156.98 (17.5) nm, respectively.
또한 도 2에 나타낸 바와 같이, 순환 세포외소포는 CD63, Flotillin 1 및 HSP70를 포함하는 세포외소포 마커에 대해서 양성을 나타내었으며, 세포외소포 음성 마커인 Calnexin 은 검출되지 않았다.In addition, as shown in FIG. 2, circulating extracellular vesicles were positive for extracellular vesicle markers including CD63,
2.3 MSC 처리에 대한 반응성과 세포외소포 변화 확인2.3 Confirmation of responsiveness and changes in extracellular vesicles to MSC treatment
MSC 처리 전 후 혈중 순환 세포외소포 수준이 변화되는지 여부를 확인하기 위한 실험을 수행하였다. 먼저 MSC를 주입하기 전에 MSC 처리 대상 환자군의 순환 세포외소포 수준을 MSC 주입 하루 전 및 MSC 주입 직전에 연속적으로 측정하였고, 그 결과를 도 3에 나타내었다 (n = 21). Experiments were performed to determine whether the level of circulating extracellular vesicles in blood was changed before and after MSC treatment. First, before MSC injection, the level of circulating extracellular vesicles in the MSC-treated patient group was continuously measured one day before MSC injection and immediately before MSC injection, and the results are shown in FIG. 3 (n = 21).
도 3에 나타낸 바와 같이, 환자마다 세포외소포 의 수준은 모두 상이 (평균 ± SD, 4.5 × 109 ± 7.9 × 109 EVs/ml) 하나, 대조군과 MSC 그룹간 유의적인 차이는 없었고 (p> 0.05), 세포외소포 수준은 1차 측정 시점에 평균 ± SD, 4.9 × 109 ± 8.7 × 109 EVs / ml, 2차 측정시점에 5.3×109 ± 8.8×109 EVs/ml 로 시간에 따라 변하지 않고 일정한 것을 확인하였다. 즉 혈청 내 세포외소포 수준은 MSC 치료 전에 안정적인 수준으로 각 환자에서 유지되고 있음을 확인하였다. As shown in FIG. 3, the level of extracellular vesicles was different for each patient (mean ± SD, 4.5 × 10 9 ± 7.9 × 10 9 EVs/ml), but there was no significant difference between the control group and the MSC group (p> 0.05), the level of extracellular vesicles was mean ± SD, 4.9 × 10 9 ± 8.7 × 10 9 EVs / ml at the time of the first measurement, and 5.3 × 10 9 ± 8.8 × 10 9 EVs / ml at the time of the second measurement. It was confirmed that it was constant and did not change. That is, it was confirmed that the level of extracellular vesicles in serum was maintained in each patient at a stable level before MSC treatment.
이 후, 혈중 세포외소포의 수가 MSC 치료 후 변화되는지 여부를 확인하기 위하여, MSC 치료 직후 세포외소포 수를 측정하였고, 그 결과를 도 4에 나타내었다. Thereafter, in order to confirm whether the number of extracellular vesicles in blood is changed after MSC treatment, the number of extracellular vesicles was measured immediately after MSC treatment, and the results are shown in FIG. 4 .
도 4에 나타낸 바와 같이, 세포외소포의 수준은 MSC 주입 후 24 시간 이내에 약 5 배 (평균 ± SD, 2.7 × 109 ± 2.2 × 109 EVs / ml에서 1.3 × 1010 ± 1.7 × 1010 EVs / ml로) 크게 증가하였다 (p = 0.001) (A). 반면 대조군에서는 증가하지 않고 일정한 수준을 유지하였다 (B). 보다 구체적으로 MSC 처리 환자군 중에서 Fugl-Meyer Assessment의 MCID (minimal clinically important difference) 를 나타내는 환자군 (C) 들은 MCID 를 나타내지 않는 환자군(D) 과 비교하여 유의한 수준의 세포외소포 증가를 나타내는 것을 확인하였다(EV number Log, p=0.039 for FMA-total, p=0.023 for FMA-upper, p=0.291 for FMA-lower). As shown in Fig. 4, the level of extracellular vesicles increased approximately 5-fold (mean ± SD, from 2.7 × 10 ± 2.2 × 10 EVs/ml to 1.3 × 10 ± 1.7 × 10 EVs within 24 h after MSC injection). / ml) increased significantly (p = 0.001) (A). On the other hand, the control group did not increase and maintained a constant level (B). More specifically, among the MSC-treated patient groups, it was confirmed that the patient group (C) showing the minimal clinically important difference (MCID) of the Fugl-Meyer Assessment showed a significant increase in extracellular vesicles compared to the patient group (D) without MCID. (EV number Log, p=0.039 for FMA-total, p=0.023 for FMA-upper, p=0.291 for FMA-lower).
MSC 치료에 대하여 우수한 반응성을 나타내어 좋은 예후를 보이는 환자군에서 혈청 내 순환 세포외소포가 더 많이 증가되어 있음을 보여주는 상기와 같은 결과를 통해, 세포외소포가 MSC 치료에 대한 환자의 반응성 및 예후 예측과 관련성이 있음을 예상하였다. 한편 혈중 세포외소포는 줄기세포치료제 주입 후 24시간 이내가 가장 현저하게 증가하며, 수일에 걸쳐 치료 전 수준으로 감소되었다. 혈액 내에서 세포외소포의 반감기가 수십분 정도로 매우 짧기 때문에 혈중 세포외소포의 측정시기는 24시간 이내가 가장 이상적일 것으로 판단하여, 이후에서는 MSC 처리 24시간 후 혈청 내 순환 세포외소포의 수준을 이후 세포외소포와 임상 개선 효과와의 관련성을 평가하는데 이용하였다. Through the above results showing that circulating extracellular vesicles in serum were increased more in patients with good prognosis due to excellent responsiveness to MSC treatment, extracellular vesicles are associated with prediction of patient's responsiveness and prognosis to MSC treatment A relationship was expected. On the other hand, extracellular vesicles in blood increased most significantly within 24 hours after injection of stem cell therapy, and decreased to the pre-treatment level over several days. Since the half-life of extracellular vesicles in the blood is very short, about several tens of minutes, the ideal time to measure extracellular vesicles in the blood is within 24 hours. It was used to evaluate the relationship between extracellular vesicles and clinical improvement effects.
2.4 MSC 처리에 대한 반응성과 세포외소포 관련성 검증 2.4 Verification of responsiveness to MSC treatment and relevance to extracellular vesicles
실시예 1.2에 나타낸 임상적 평가 및 영상적 평가로 확인된 뇌졸중 치료, 개선 효과와 세포외소포 의 관련성을 확인하기 위하여 상관관계 분석을 수행하였으며, 그 결과를 도 5에 나타내었다. Correlation analysis was performed to confirm the relevance of extracellular vesicles to the stroke treatment and improvement effects confirmed by the clinical evaluation and imaging evaluation shown in Example 1.2, and the results are shown in FIG. 5.
도 5에 나타낸 바와 같이, MSC 처리 후 순환 세포외소포의 수는 임상적인 호전 정도와 직접적인 연관 관계를 가지고 있음을 확인하였다. 상관 관계 분석에 따르면 MSC 처리 후 순환 세포외소포 수는 운동 기능의 개선과 유의한 상관 관계가 있었으나 (r = 0.444, P = 0.001) MSC 를 처리하지 않은 대조군에서는 뚜렷한 상관관계가 없는 것을 확인 (r = 0.359, p = 0.207) 하였다. As shown in Figure 5, it was confirmed that the number of circulating extracellular vesicles after MSC treatment had a direct correlation with the degree of clinical improvement. According to the correlation analysis, the number of circulating extracellular vesicles after MSC treatment was significantly correlated with the improvement of motor function (r = 0.444, P = 0.001), but there was no clear correlation in the control group that was not treated with MSC (r = 0.444, P = 0.001). = 0.359, p = 0.207).
다변수검정결과 연령, 성별, 초기 뇌졸중 중증도(NIHSS 점수) 및 줄기세포 주입을 보정하였으며, 줄기세포를 주입 후 혈청 내 세포외소포의 수만이 독립적으로 임상개선과 관련된 요인임을 확인하였다 (odds ratio for good responder, 5.718 for EV number Log, 95% 신뢰구간 1.144-28.589, p=0.034). As a result of the multivariate test, age, gender, initial stroke severity (NIHSS score), and stem cell injection were corrected, and it was confirmed that only the number of extracellular vesicles in serum after stem cell injection was an independent factor related to clinical improvement (odds ratio for good responder, 5.718 for EV number Log, 95% confidence interval 1.144-28.589, p=0.034).
2.5 MSC 처리에 대한 신경가소성 호전과 세포외소포 관련성 검증 2.5 Verification of association between improvement of neuroplasticity and extracellular vesicles in response to MSC treatment
혈청내 세포외소포 수준과 줄기세포치료제 주입 후 신경 가소성 호전과의 관련성을 확인하기 위하여, MSC 처리 24시간 후 혈청 내 순환 세포외소포 수준과 신경 가소성의 DTI 및 rs-fMRI 지수 간의 연관성을 90 일에 측정(n=54, 대조군 및 MSC 처리군 포함) 하고, 그 결과를 도 6에 나타내었다. To confirm the relationship between the level of extracellular vesicles in serum and the improvement of neuroplasticity after stem cell therapy injection, the correlation between the level of circulating extracellular vesicles in serum and the DTI and rs-fMRI indices of neuroplasticity after 24 hours of MSC treatment was examined for 90 days. was measured (n = 54, including control and MSC treatment groups), and the results are shown in FIG. 6.
도 6에 나타낸 바와 같이, 동면 피질 척수로(ipsilesional corticospinal tract) 및 반구 내 운동 네트워크(intrahemispheric motor network)의 완전성은 순환 세포외소포 수준과 유의한 상관 관계(두 경우 모두 p <0.05)가 있었으며, 줄기세포치료제 투여 후 24시간내 측정한 혈청 내 세포외소포의 수준이 영상학적인 호전 정도(확산텐서영상[diffusion tensor imaging]상 해부학적 신경회로 회복 및 휴식상태 기능적 자기공명영상[resting state-functional MRI]상 기능적 신경회로 회복)와 직접적으로 연관됨을 확인하였다. 이는 MSC 처리 뇌졸중 환자에서 MSC 처리 24시간 이내 증가된 세포외소포 수준이 해부학적인 회복 및 기능 회복, 병변측 운동 네트워크 (ipsilesional motor network)와 관련성이 있음을 보여주는 결과이다. As shown in Figure 6, the integrity of the ipsilesional corticospinal tract and intrahemispheric motor network was significantly correlated with the level of circulating extracellular cells (p <0.05 in both cases), The level of extracellular vesicles in the serum, measured within 24 hours after administration of stem cell therapy, is the degree of imaging improvement (anatomical neural circuit recovery on diffusion tensor imaging and resting state-functional magnetic resonance imaging [resting state-functional MRI]). ] It was confirmed that it was directly related to functional neural circuit recovery). This is a result showing that the increased level of extracellular vesicles within 24 hours of MSC treatment in MSC-treated stroke patients is related to anatomical recovery and functional recovery, and the ipsilesional motor network.
상기와 같은 결과를 통해, MSC 투여 직후 24시간 이내 혈청 내 세포외소포의 수준이 급격하게 증가되며, 증가한 세포외소포 수준이 MSC 치료에 의한 질환의 임상적 지표 개선 유무, 즉, 환자의 예후와 관련성이 있음을 확인하였다. Through the above results, the level of extracellular vesicles in serum increased rapidly within 24 hours immediately after MSC administration, and the increased level of extracellular vesicles was associated with the improvement of clinical indicators of the disease by MSC treatment, that is, the patient's prognosis and It was confirmed that there is a relationship.
실시예 3. MSC 처리에 대한 반응성 평가 지표 확인 - 성장인자 분석 Example 3. Confirmation of reactivity evaluation index for MSC treatment - growth factor analysis
세포외소포 외 MSC 처리 후 다른 MSC 분비 인자도 치료 예후 및 반응성과 관련이 있는지 확인하기 위하여, MSC 처리 후 순환 성장인자 및 케모카인의 수준을 측정하여 이들의 관련성을 확인하였다. 성장인자 및 케모카인 수준 측정을 위한 ELISA는 개별 제조업체의 메뉴얼에 따라 상용 키트로 수행하였다. 다음과 같은 ELISA 키트를 사용하였다: In order to confirm whether other MSC-secreted factors are also related to treatment prognosis and responsiveness after extracellular MSC treatment, their relevance was confirmed by measuring the levels of circulating growth factors and chemokines after MSC treatment. ELISA for measuring growth factor and chemokine levels was performed with a commercially available kit according to the individual manufacturer's manual. The following ELISA kits were used:
human VEGF (R&D systems, Minneapolis, MN, USA), human CXCL12/SDF-1 (R&D systems), human BDNF (R&D systems), human neuron-specific enolase (NSE, R&D systems). 모든 키트에는 표준 단백질이 포함되어 있으며, 단백질의 양은 각 키트의 표준 곡선에 기초하여 결정하였다. MSC 처리에 따른 혈장 내 성장인자 수준의 변화를 도 7에 나타내었다. human VEGF (R&D systems, Minneapolis, MN, USA), human CXCL12/SDF-1 (R&D systems), human BDNF (R&D systems), human neuron-specific enolase (NSE, R&D systems). A standard protein was included in all kits, and the amount of protein was determined based on the standard curve of each kit. Changes in plasma growth factor levels according to MSC treatment are shown in FIG. 7 .
도 7에 나타낸 바와 같이, MSC 치료 후 혈장 성장인자의 수준은 변화되지 않았으며, 순환 사이토카인, 케모카인 및 성장인자는 대조군 및 MSC 치료군에서 차이가 없는 것을 확인하였다. 이러한 결과는 혈중 성장인자는 MSC 처리에 따른 반응성 평가 지표로 적절하지 않음을 보여주는 결과이다. As shown in Figure 7, after MSC treatment, the level of plasma growth factor was not changed, and circulating cytokines, chemokines, and growth factors were confirmed to have no difference between the control group and the MSC treatment group. These results show that blood growth factors are not appropriate as an evaluation index for reactivity according to MSC treatment.
실시예 4. 세포외소포 내 함유물질 분석 Example 4. Analysis of substances contained in extracellular vesicles
실시예 2 및 3을 통해 세포외소포가 MSC 뇌졸중 치료에 대한 반응성 및 예후 예측 마커로 사용할 수 있음을 확인하였으므로, 추가적으로 MSC 처리에 따른 세포외소포 내 함유 물질의 변화를 확인하는 실험을 수행하였다. Since it was confirmed through Examples 2 and 3 that extracellular vesicles can be used as responsiveness and prognostic markers for MSC stroke treatment, an experiment was performed to determine changes in substances contained in extracellular vesicles according to additional MSC treatment.
4.1 MSC 처리 후 순환 세포외소포 내 신경재생 관련 miRNA 증가 확인 4.1 Confirmation of increase in miRNA related to nerve regeneration in circulating extracellular vesicles after MSC treatment
줄기세포 투여 후, 혈중 순환 세포외소포 증가 외 세포외소포 내 miRNA 또한 증가되는지 여부를 확인하기 위하여, 대조군과 MSC군 모두에서 세포외소포 내 miRNA의 수준을 순차적으로 (치료 전, 치료 당일, 치료 14 일, 치료 90 일) 측정하였다. 세포외소포내의 줄기 세포의 치료 효능 및 뇌졸중 후 회복과 관련된 miRNA의 수준을 정량적 PCR 을 사용하여 개별 환자에서 측정하고 2-ΔCt 정량적 방법으로 분석하였다. miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany)를 사용하여 세포외소포의 총 RNA를 추출하였다. RNA의 농도는 NanoDrop 1000 (NanoDrop, Wilmington, Delaware, USA)을 이용해 정량화하였다. miRNA 용 프라이머는 TaqMan®MicroRNA Assays (Applied Biosystems, Foster City, CA, USA)을 이용하였다. 총 15개의 MicroRNA를 선정하였다. 먼저 신경 가소성, 신경재생, 신경소세포형성(oligodendrogenesis) 과 관련이 있는 miRNA로 miR-17-92 (miR-17-3p, 18a-5p, miR-20a-5p, miR-92-1), miR-133b를 선정하였고, 혈관 신생과 관련이 있는 miRNA 로 miR-126-5p, miR-132-3p, miR-181b-5p, miR-494-3p를 선정하였으며, 세포 보호 및 항염 관련 miR-19a-3p, miR-146a-5p, miR-210-3p, miR-223-3p, 및 뇌졸중 후 회복과 관련된 miR-21-5p, miR-196a-5p를 선정하여 이들의 수준을 측정하였다. 발현된 miRNA의 표적 유전자에서의 기능을 보기 위해 KEGG (Kyoto Encyclopedia of Genes and Genomes) 및 GO (gene ontology) 경로 분석을 실시하였다. GO 분석 및 KEGG 경로 농축 분석은 miRWalk2.0 웹 기반 도구 (http://zmf.umm.uni heidelberg.de/apps/zmf/mirwalk2/)에 의해 수행하였다. 혈청 세포외소포내 함유된 miRNA의 발현 정도를 분석한 결과 중 신경가소성과 관련된 miR-18a-5p 발현 비교 결과를 도 8에 나타내었다. After stem cell administration, in order to confirm whether miRNAs in extracellular vesicles are also increased in addition to the increase in blood circulating extracellular vesicles, the level of miRNAs in extracellular vesicles in both the control group and the MSC group was sequentially measured (before treatment, on the day of treatment, treatment 14 days, 90 days of treatment). Levels of miRNAs associated with the therapeutic efficacy of stem cells in extracellular vesicles and post-stroke recovery were measured in individual patients using quantitative PCR and analyzed by the 2-ΔCt quantitative method. Total RNA from extracellular vesicles was extracted using the miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany). RNA concentration was quantified using NanoDrop 1000 (NanoDrop, Wilmington, Delaware, USA). As primers for miRNA, TaqMan®MicroRNA Assays (Applied Biosystems, Foster City, CA, USA) were used. A total of 15 microRNAs were selected. First, miR-17-92 (miR-17-3p, 18a-5p , miR-20a-5p, miR-92-1), miR- 133b was selected, and miR-126-5p, miR-132-3p, miR-181b-5p, and miR-494-3p were selected as miRNAs related to angiogenesis, and miR-19a-3p related to cell protection and anti-inflammatory , miR-146a-5p, miR-210-3p, miR-223-3p, and miR-21-5p and miR-196a-5p related to post-stroke recovery were selected and their levels were measured. KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (gene ontology) pathway analyzes were performed to see the function of the expressed miRNA in the target gene. GO analysis and KEGG pathway enrichment analysis were performed by the miRWalk2.0 web-based tool (http://zmf.umm.uni heidelberg.de/apps/zmf/mirwalk2/). Figure 8 shows the comparison of miR-18a-5p expression associated with neuroplasticity among the results of analyzing the expression level of miRNA contained in serum extracellular vesicles.
세포외소포의 증가와 더불어, 신경 가소성과 관련된 세포외소포 내의 miRNA의 상대적 발현은 치료 직후 MSC 그룹 환자에서 증가하였고, 도 8의 (A)에 나타낸 바와 같이, 특히 miR-17-92 cluster의 하나인 miR-18a-5p 발현은 MSC 처리군에서 유의하게 증가하였다 (p = 0.0479). 도 8의 (B) 및 (C) 에 나타낸 바와 같이, GO 및 KEGG 분석 결과 세포외소포 내에서 증가된 miR-18a-5p는 신경계 발달, 신경 성장인자 수용체 신호전달 경로 및 축삭 유도와 관련된 신호 경로 및 생물학적 과정과 관련이 있는 것을 확인하였다. 이를 통해, MSC 반응성 환자군에서 확인되는 MSC 처리에 의한 치료 직후 세포외소포 수 증가와 더불어 세포외소포 내 포함되어 있는 신경 가소성과 관련있는 miR-18a-5p 역시 대조군 대비 증가하였음을 확인하였고, 이는 MSC 를 이용한 뇌 질환 치료에 있어서, 세포외소포가 치료 효과와 관련된 지표가 될 수 있음을 보여주는 결과이며, 세포외소포 측정과 더불어 세포외소포 내 포함된 miRNA 발현 수준의 분석을 통해 줄기세포 치료제의 예후를 예측할 수 있음을 보여주는 결과이다. In addition to the increase in extracellular vesicles, the relative expression of miRNAs in extracellular vesicles related to neuroplasticity increased in MSC group patients immediately after treatment, and as shown in FIG. 8(A), in particular, one of the miR-17-92 cluster Phosphorus miR-18a-5p expression was significantly increased in the MSC-treated group (p = 0.0479). As shown in (B) and (C) of FIG. 8, as a result of GO and KEGG analysis, increased miR-18a-5p in extracellular vesicles is a signal pathway related to nervous system development, nerve growth factor receptor signaling pathway, and axon guidance. And it was confirmed that it is related to biological processes. Through this, it was confirmed that along with the increase in the number of extracellular vesicles immediately after treatment by MSC treatment confirmed in the MSC-responsive patient group, miR-18a-5p related to neuroplasticity included in extracellular vesicles also increased compared to the control group. In the treatment of brain diseases using vesicles, this result shows that extracellular vesicles can be an indicator related to the therapeutic effect, and the prognosis of stem cell therapy through the analysis of the miRNA expression level included in extracellular vesicles along with the measurement of extracellular vesicles This result shows that it is possible to predict
상기와 같은 내용을 종합적으로 고려하여, MSC 치료에 의하여 효과를 나타내는 뇌질환 환자군에서 순환 세포외소포가 뚜렷하게 증가한다는 것을 확인하였고, 이와 같이 증가되는 순환 세포외소포가 운동 기능의 개선 및 MRI 가소성 지표와 유의한 상관관계가 있음을 확인함으로써, 세포외소포가 뇌 질환 환자에서 MSC 치료에 대한 반응성을 예측하는 마커로 사용될 수 있음을 확인하였다. Considering the above information comprehensively, it was confirmed that the circulating extracellular vesicles increased significantly in the brain disease patient group that was effective by MSC treatment, and the increased circulating extracellular vesicles improved motor function and MRI plasticity index By confirming that there is a significant correlation with, it was confirmed that extracellular vesicles can be used as markers predicting responsiveness to MSC treatment in brain disease patients.
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다.In the above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (15)
1) measuring extracellular vesicles in brain disease patients administered stem cell therapy; A method for providing information on prognosis prediction of a patient after administration of stem cell therapy comprising a.
2) 상기 1) 단계에서 측정된 세포외소포 수준과 줄기세포 치료제 투여 전 측정된 세포외소포의 수준을 비교하는 단계; 를 더 포함하는, 줄기세포 치료제 투여 후 환자의 예후 예측에 대한 정보 제공 방법.
According to claim 1,
2) comparing the level of extracellular vesicles measured in step 1) with the level of extracellular vesicles measured before administering the stem cell therapy; Further comprising, providing information about the prognosis prediction of the patient after administration of stem cell therapy.
The method of claim 1, wherein the measurement of extracellular vesicles in step 1) is performed within 240 hours after administration of the stem cell therapy.
The method of claim 1, after administration of stem cell therapy to the brain disease patient, miR-17-3p, miR18a-5p , miR-20a-5p, miR-92-1, miR-133b, miR-126-5p, miR -132-3p, miR-181b-5p, miR-494-3p, miR-19a-3p, miR-146a-5p, miR-210-3p, miR-223-3p, miR-21-5p or miR-196a - A method for providing information about predicting a patient's prognosis after administration of stem cell therapy, further comprising the step of measuring a change in the expression level of 5p.
The method of claim 1, wherein the stem cell therapy is an autologous, allogeneic or allogeneic stem cell therapy, providing information about prediction of a patient's prognosis after administration of a stem cell therapy.
The method of claim 1, wherein the stem cells are at least one selected from the group consisting of mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, and embryonic stem cells. .
The method of claim 1, wherein the stem cells are stem cells derived from one species selected from the group consisting of umbilical cord blood, umbilical cord, bone marrow, fat, placenta, Wharton jelly, amniotic fluid, amnion, and tonsil. A method for providing information on predicting the prognosis of post-patients.
The method of claim 1, wherein the brain disease is at least one selected from the group consisting of ischemic brain disease, cranial nerve injury disease, and degenerative brain disease.
The method of claim 8, wherein the ischemic cerebrovascular disease is at least one selected from the group consisting of ischemic stroke, thrombosis, embolism, transient ischemic attack, leukodystrophy, and small infarction, providing information on prognosis prediction of patients after administration of stem cell therapy method
The method of claim 8, wherein the brain nerve injury disease is one or more selected from the group consisting of brain nerve injury disease due to intracerebral hemorrhage and brain nerve injury disease due to trauma, after administration of stem cell therapy.
The method of claim 8, wherein the degenerative brain disease is at least one selected from the group consisting of dementia, Alzheimer's, and Parkinson's.
The method of claim 1, wherein the extracellular vesicles are blood extracellular vesicles, and provide information about prediction of a patient's prognosis after administration of a stem cell therapy.
A biomarker composition for predicting the prognosis of stem cell therapy for brain disease patients, including extracellular vesicles.
[Claim 14] The biomarker composition according to claim 13, wherein the extracellular vesicles are extracellular vesicles in the blood, and predict the prognosis of stem cell therapy for brain disease patients.
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