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Mesenchymal stem cell-derived extracellular vesicles for immunomodulation and regeneration: a next generation therapeutic tool?

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• Review Article
• Open access
• Published: 04 July 2022

Mesenchymal stem cell-derived extracellular vesicles for immunomodulation and regeneration: a next generation therapeutic tool?

• Meng Kou, 
• Li Huang, 
• Jinjuan Yang, 
• Zhixin Chiang, 
• Shaoxiang Chen, 
• Jie Liu, 
• Liyan Guo, 
• Xiaoxian Zhang, 
• Xiaoya Zhou, 
• Xiang Xu, 
• Xiaomei Yan, 
• Yan Wang, 
• Jinqiu Zhang, 
• Aimin Xu, 
• Hung-fat Tse & 
• Qizhou Lian 

Cell Death & Disease volume 13, Article number: 580 (2022) Cite this article

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Abstract

Mesenchymal stem cells (MSCs) can be widely isolated from various tissues including bone marrow, umbilical cord, and adipose tissue, with the potential for self-renewal and multipotent differentiation. There is compelling evidence that the therapeutic effect of MSCs mainly depends on their paracrine action. Extracellular vesicles (EVs) are fundamental paracrine effectors of MSCs and play a crucial role in intercellular communication, existing in various body fluids and cell supernatants. Since MSC-derived EVs retain the function of protocells and have lower immunogenicity, they have a wide range of prospective therapeutic applications with advantages over cell therapy. We describe some characteristics of MSC-EVs, and discuss their role in immune regulation and regeneration, with emphasis on the molecular mechanism and application of MSC-EVs in the treatment of fibrosis and support tissue repair. We also highlight current challenges in the clinical application of MSC-EVs and potential ways to overcome the problem of quality heterogeneity.

초록
중간엽 줄기세포(MSCs)는 

골수, 탯줄, 지방 조직 등 다양한 조직에서 널리 분리될 수 있으며, 

자기 재생 능력과 다분화 잠재력을 갖추고 있습니다. 

MSC의 치료 효과는

 주로 그들의 파라크린 작용에 의존한다는 강력한 증거가 있습니다. 

세포외 소포(EVs)는 

MSC의 주요 파라크라인 효과자로, 

세포 간 통신에 중요한 역할을 하며 다양한 체액과 세포 상청액에 존재합니다. 

MSC 유래 EVs는 

원시 세포의 기능을 유지하며 

면역원성이 낮아 세포 치료법보다 우월한 장점을 가진 광범위한 치료적 응용 가능성을 가지고 있습니다. 

본 논문에서는 MSC-EV의 일부 특성을 설명하고, 

면역 조절 및 재생에서의 역할을 분자 메커니즘과 섬유화 치료 및 조직 복구 지원에서의 응용에 초점을 맞춰 논의합니다. 

또한 MSC-EV의 임상 적용에서의 현재 도전 과제와 품질 이질성 문제를 극복하기 위한 잠재적 방법을 강조합니다.

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Facts

• MSC-derived EVs have low-immunogenicity and strong potential for therapeutic applications.

• MSC-derived EVs were used to treat tissue fibrosis and promote tissue regeneration.

• MSC-derived EVs are proposed as a novel therapeutic agent to mediate immunomodulation and promote regeneration.

Open questions

• How can MSC-derived EVs mediate immunomodulation and regeneration?

• How can MSC-derived EVs be used to aid regeneration of fibrotic tissue?

• How can mass manufacturing of MSC-derived EVs be achieved and the problem of quality heterogeneity overcome?

• What are the challenges of MSC-derived EV-based immunomodulation and regeneration in clinical practice?

사실

• MSC 유래 엑소좀(EVs)은 낮은 면역원성과 치료적 응용에 대한 강력한 잠재력을 가지고 있습니다.
• MSC 유래 엑소좀은 조직 섬유화를 치료하고 조직 재생을 촉진하기 위해 사용되었습니다.
• MSC 유래 엑소좀은 면역 조절을 매개하고 재생을 촉진하는 새로운 치료제로 제안되었습니다.

개방된 질문

• MSC 유래 엑소좀은 어떻게 면역 조절과 재생을 매개할 수 있을까요?
• MSC 유래 엑소좀은 섬유화 조직의 재생을 돕기 위해 어떻게 사용될 수 있을까요?
• MSC 유래 EV의 대량 생산을 어떻게 달성하고 품질 이질성 문제를 극복할 수 있을까요?
• 임상 실무에서 MSC 유래 EV 기반 면역 조절 및 재생의 도전 과제는 무엇인가요?

Introduction

Mesenchymal stem cells (MSCs) exist in various tissues such as bone marrow (BMSCs), umbilical cord blood (UC-MSCs) and umbilical cord tissue, placental tissue (hPMSCs), adipose tissue (ADSCs), and menstrual blood (MenSCs). These cells have multidirectional differentiation potential [1] to become osteoblasts, chondrocytes or adipocytes in vitro [2], and have a unique function of cytokine secretion [3]. Cell models have been applied in proliferation, transplantation, and differentiation studies, and in identification of immune responses in vitro [4]. Numerous studies have shown that MSCs have great potential in immune regulation and regeneration [5]. The U.S. FDA has approved nearly 60 clinical trials [6], mainly focused on Hematopoietic Stem Cell Transplantation (HSCT) [7], tissue healing, Autoimmune Disease (AID), and genetic therapy vectors [8]. Recently, MSCs have been widely used in clinical studies as a regenerative agent and to treat a variety of conditions including osteoarthritis [9], pulmonary fibrosis, spinal cord injury, myocardial damage, knee cartilage injury, dental pulp regeneration, and organ transplantation [10]. An increasing number of studies has revealed that the powerful therapeutic effects of MSCs are due to paracrine-like secretion of cytokines (growth factors and chemokines) [11, 12] and extracellular vesicles (EVs) as well as their involvement in cellular communication [13,14,15,16].

Application of MSCs as cell therapy is based on regulating the inflammatory response and participating in tissue repair and regeneration [17]. The therapeutic effect of MSCs is mainly attributed to their immunomodulatory function regulated by the inflammatory environment [18]. When stimulated by inflammatory factors, MSCs produce a large number of immunomodulatory factors, cell chemokines, and growth factors, thereby regulating the tissue immune microenvironment and promoting tissue regeneration [19]. There is accumulating evidence that EVs derived from MSCs preserve the therapeutic action of the parent MSCs and their use avoids the safety concerns associated with live cell therapy [20, 21]. Therefore, use of MSC-EVs to replace MSCs as cell-free therapy may be the focus of future clinical treatments [20]. We review recent studies of the role of MSC-EVs in immunomodulation and regeneration, focusing on their molecular mechanisms in the treatment of osteoarthritis, spinal cord injury, skin injury, and liver, kidney, and lung fibrosis.

소개

중간엽 줄기세포(MSCs)는

골수(BMSCs), 탯줄 혈액(UC-MSCs) 및 탯줄 조직, 태반 조직(hPMSCs), 지방 조직(ADSCs), 월경 혈액(MenSCs) 등

다양한 조직에 존재합니다.

이 세포들은

다방향 분화 잠재력[1]을 가지고 있어

체외에서 골세포, 연골세포 또는 지방세포로 분화될 수 있으며[2],

사이토킨 분비라는 독특한 기능을 가지고 있습니다[3].

세포 모델은

증식, 이식, 분화 연구 및 체외에서 면역 반응 식별에 적용되었습니다 [4].

수많은 연구에서 MSC가

면역 조절 및 재생에 큰 잠재력을 가지고 있음을 보여주었습니다 [5].

미국 식품의약국(FDA)은

약 60건의 임상 시험을 승인했으며 [6],

주로 혈액 줄기세포 이식(HSCT) [7],

조직 치유, 자가면역 질환(AID), 유전자 치료 벡터 [8]에 초점을 맞추고 있습니다.

최근 MSC는

골관절염 [9], 폐 섬유화, 척수 손상, 심근 손상, 무릎 연골 손상, 치수 재생, 장기 이식 [10] 등

다양한 질환의 치료를 위한 재생 치료제로 임상 연구에서 널리 사용되고 있습니다.

최근 연구들은

MSC의 강력한 치료 효과가

사이토킨(성장 인자 및 케모카인)의 파라크린 유사 분비[11, 12] 및

세포외 소포(EVs)와 세포 간 통신에의 참여[13,14,15,16]에

기인함을 밝혀냈습니다.

MSCs를 세포 치료제로 적용하는 것은

염증 반응 조절과 조직 수리 및 재생에 참여하는 데 기반을 두고 있습니다 [17].

MSCs의 치료 효과는

주로 염증 환경에 의해 조절되는 면역 조절 기능에 기인합니다 [18].

염증 인자에 자극을 받으면

MSCs는 면역 조절 인자, 세포 케모카인, 성장 인자를 대량으로 생성하여

조직 면역 미세 환경을 조절하고 조직 재생을 촉진합니다 [19].

MSCs에서 유래한 엑소좀(EVs)이 모세포의 치료 효과를 유지하며,

살아있는 세포 치료와 관련된 안전성 문제를 회피할 수 있다는 증거가

누적되고 있습니다 [20, 21].

따라서

MSC-EVs를 세포 없는 치료법으로

MSC를 대체하는 것이 미래 임상 치료의 초점이 될 수 있습니다 [20].

우리는 골관절염, 척수 손상, 피부 손상, 간, 신장, 폐 섬유화 치료에서

MSC-EV의 면역 조절 및 재생 역할에 대한 최근 연구를 검토하며,

특히 분자 메커니즘에 초점을 맞췄습니다.

Extracellular vesicles composition

Extracellular vesicles (EVs) exist in body fluids, are released by cells, and have a membrane structure [22]. They can be divided into four subgroups according to their diameter: exosomes (30–150 nm), microvesicles (100–1000 nm), apoptotic bodies (50–5000 nm, generated during cell apoptosis) [23, 24], and oncosomes (1–10 μm), newly discovered and observed in cancer cells [25]. EVs encapsulate many bioactive molecules (proteins, lipids, nucleic acids, and organelles) [26,27,28] that can be delivered to target cells. Large amounts of data suggest that exosomes and microvesicles are vital mediators of EVs in numerous physiological (pathological) processes [29] (Fig. 1).

세포외 소포체 구성

세포외 소포체(EVs)는

체액에 존재하며 세포에 의해 방출되며 막 구조를 가지고 있습니다 [22].

그들은 직경에 따라 네 가지 하위 그룹으로 나눌 수 있습니다:

엑소좀(30–150 nm),

마이크로베시클(100–1000 nm),

아포토틱 바디(50–5000 nm, 세포 아포토시스 과정에서 생성됨) [23, 24],

및 온코소메(1–10 μm),

암 세포에서 새롭게 발견되고 관찰된 [25].

EV는

많은 생물활성 분자(단백질, 지질, 핵산, 세포 소기관)를 포함하며 [26,27,28],

표적 세포로 전달될 수 있습니다.

많은 데이터는

엑소좀과 미세소체가 다양한 생리적(병리적) 과정에서의

EV의 중요한 매개체임을 시사합니다 [29] (그림 1).

Fig. 1: The development and main types of extracellular vesicles.

A Exosomes are derived from the endosomal pathway. B Composition of exosomes.

Full size image

Exosomes

Exosomes are microscopic vesicles with a density of 1.11–1.19 g/mL. They have a typical “disk-like” structure and flat spherical shape when seen under an electron microscope [24]. Many kinds of cellsin various body fluids and cell supernatants can secrete exosomes under normal and pathological conditions. Exosomes were first discovered in 1983 in sheep reticulocytes and were named “Exosomes” by Johnstone in 1987 [30]. These tiny vesicles contain specific proteins, lipids, and nucleic acids that can be transmitted and serve as signaling molecules to alter the function of other cells [31, 32].

During the formation of exosomes, the extracellular components and cell membrane proteins are wrapped by the invaginated plasma membrane to form early endosomes. These can exchange materials with intracellular organelles and develop into late endosomes, eventually forming intracellular multivesicular bodies (MVBs) [33, 34]. MVBs contain many intraluminal vesicles (ILVs) [35]. They may be degraded and released into the cytoplasm by fusion with autophagosomes or lysosomes, or released into extracellular vesicles by fusion with plasma membrane, including ILVs, resulting in exosome formation [34]. Exosome-mediated intercellular communication is achieved by direct membrane fusion, receptor-mediated endocytosis, phagocytosis, caveolae, and micropinocytosis [36,37,38].

Proteins involved in exosome biogenesis (such as transport and fusion) include Rab GTPases [39,40,41], ESCRT (endosomal sorting complex required for transport) [42], annexin, lipid raft proteins, and four transmembrane proteins (CD63, CD81, and CD9) [43, 44]. In addition, they also contain biosynthetic antibodies (Alix and TSG101) involved in MVBs [45, 46], cholesterol, ceramide, phosphoglyceride that provides structural stability, and immune-related molecule MHC-II that is involved in antigen binding and presentation. Exosomes also carry functional mRNAs and miRNAs that can be transferred between cells [47]. Exosomes released by tumors contain single-stranded DNA, genomic DNA, cDNA, and a transposable element [48, 49]. It is clear that exosomes have many functions as biomarkers of disease.

엑소좀

엑소좀은

밀도 1.11–1.19 g/mL의 미세 소포체입니다.

전자 현미경으로 관찰할 때 전형적인 '원반형' 구조와 평평한 구형 모양을 보입니다 [24].

다양한 신체 액체와 세포 상청액에 존재하는 많은 종류의 세포는

정상적 및 병리적 조건에서

엑소좀을 분비합니다.

엑소좀은 1983년 양의 적혈구 전구세포에서 처음 발견되었으며,

1987년 존스턴에 의해 '엑소좀'이라는 이름이 부여되었습니다 [30].

이 작은 소포체는

특정 단백질, 지질, 핵산 등을 포함하며,

다른 세포의 기능을 변화시키기 위해 신호 분자로 작용하여 전달될 수 있습니다 [31, 32].

엑소좀 형성 과정에서 세포 외 성분과 세포막 단백질은

내향된 세포막에 의해 감싸져

초기 엔도좀을 형성합니다.

이 구조물은

세포 내 소기관과 물질 교환을 통해 후기 엔도좀으로 발달하며,

최종적으로 세포 내 다중 소포체(MVBs)를 형성합니다 [33, 34].

MVBs는

많은 내강 소포체(ILVs)를 포함합니다 [35].

이들은 오토파고소체나 리소좀과의 융합을 통해

세포질로 분해되어 방출되거나,

세포막과의 융합을 통해 세포외 소포체(ILVs 포함)로 방출되어

엑소좀 형성이 이루어집니다 [34].

엑소좀 매개 세포 간 통신은

직접적인 막 융합, 수용체 매개 내포작용, 식작용, 카베올라, 미세 내포작용을 통해

이루어집니다 [36,37,38].

엑소좀 생성에 관여하는 단백질(운반 및 융합 관련)에는 Rab GTPases [39,40,41], ESCRT(운반에 필요한 엔도소체 분류 복합체) [42], 안네신, 지질 래프트 단백질, 및 네 개의 트랜스막 단백질(CD63, CD81, 및 CD9) [43, 44]이 포함됩니다. 또한 MVBs와 관련된 생합성 항체(Alix 및 TSG101) [45, 46], 구조적 안정성을 제공하는 콜레스테롤, 세라마이드, 인산글리세리드, 항원 결합 및 제시와 관련된 면역 관련 분자 MHC-II를 포함합니다.

엑소좀은 세포 간 전달이 가능한

기능성 mRNA와 miRNA를 운반합니다 [47].

종양에서 방출된 엑소좀에는 단일 가닥 DNA, 게놈 DNA, cDNA, 및 전이 요소 [48, 49]가 포함되어 있습니다.

엑소좀이

질병의 바이오마커로서 다양한 기능을 갖는다는 것은

명확합니다.

Microvesicles

Microvesicles are also known as microparticles. Biogenesis of MVs differs to that of exosomes since they are released from outward budding and fission of plasma membrane when the cell is stimulated or apoptotic [50]. Nonetheless, they share characteristics of high biocompatibility, and low immunogenicity and targeting and can be used as drug carriers [51]. Studies have shown that the use of tumor cell-derived MVs to deliver chemotherapy drugs produces in better cancer treatment results with few side effects or adverse reactions [52, 53].

미세소포체

미세소포체는 미세입자라고도 불립니다. 미세소포체(MVs)의 생성은 엑소좀과 다릅니다. 이는 세포가 자극을 받거나 아포토시스 상태에 있을 때 세포막의 외부로 돌출되어 분열되는 과정에서 방출되기 때문입니다 [50]. 그러나 미세소포는 높은 생체적합성, 낮은 면역원성, 표적화 특성을 공유하며 약물 전달체로 활용될 수 있습니다 [51]. 연구 결과, 종양 세포 유래 미세소포를 화학요법 약물 전달에 활용하면 부작용이나 부작용이 적으면서 더 우수한 암 치료 효과를 나타내는 것으로 확인되었습니다 [52, 53].

MSC-derived extracellular vesicles

Although MSCs derive from a variety of sources, they can all be adherent in culture and differentiated into a variety of cell types with specific surface markers [54]. With the need for clinical treatment with MSCs, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) has proposed minimum criteria for identification of human MSCs: (1) Cultured under standard conditions they must adhere to plastic substrates; (2) On flow cytometry, the positive rate of CD105, CD73 and CD90 expression‎ in MSC surface markers should reach 95%, and negative expression‎ rate CD45, CD34, CD14 or CD11b, CD79a or CD19 or HLA-DR (human leukocyte antigen -DR) (≤2% positive); (3) After induction by standard methods in vitro, MSCs must be able to induce differentiation into osteoblasts, chondrocytes and adipocytes [55]. Nonetheless, further research has revealed that these standards do not fully define MSCs [56]. There is accumulating evidence that heterogeneous MSCs have multiple cell subpopulations with characteristic surface markers [57, 58], but the definition of surface markers and biological functions of these subpopulations requires ongoing exploration.

MSCs are easy to resuscitate and proliferate in vitro, enabling them to be mass-produced for clinical application [18]. In recent years, they have been the most studied stem cell type for clinical application, and have played an effective therapeutic role in graft-versus-host disease (GVHD) [7], kidney injury [59], tissue and organ transplantation, immune tolerance [60], nerve injury, rheumatic disease, and liver disease. At present, MSCs have attracted much attention in the context of the COVID-19 pandemic [61]. Leng et al. demonstrated that in an MSC treatment group, patients with COVID-19 infection were cured or their condition significantly improved as a result of regulation of increased interleukin 10 (IL 10) expression‎, inhibition of overactivated immune T cells and NK cells, and a significantly reduced TNF-α level [62].

Despite their advantages, there are aspects of MSC therapy that warrant consideration. First, the proliferation ability of MSCs is gradually weakened and accompanied by a certain degree of differentiation and even aging with increasing passages during in vitro culture. This impacts their regulatory and therapeutic ability [56, 63]. Second, in the in vivo environment, heredity factors and the self-renewal ability of MSCs cannot be controlled with consequent potential for tumorigenicity [64]. In addition, although MSCs have a strong regenerative regulatory potential, it is uncertain whether they can target or remain at the damaged site following intravenous injection [65]. There is some evidence that only a small number of MSCs reach the target site due to the host body’s scavenging capacity [66, 67]. Although in-situ injection can partially solve these problems, there remain problems with cell differentiation and aging, and the clinical effects are not optimistic [68]. MSCs have also been found to cause and promote the growth of various types of cancer [69]. In addition, there are the usual associated risks of cell therapy such as viral infection and immune rejection as well as problems with storage and transportation [70].

The discovery that most therapeutic effects of MSCs depend on their paracrine action and that EVs can replace their parent cells offers exciting prospects for researchers [21]. EVs offer great advantages [71]: they are not self-replicating and largely avoid the risk of tumorigenicity [72]; compared with cell therapy, EVs are safer; as nanoparticles they have both biocompatibility and low immunogenicity, enabling them to cross-protective barriers such as the blood-brain barrier [73]; they can be continuously secreted by immortalized cells to obtain a sufficient number [74]; EVs protect their internal biomolecular activity via their lipid membrane structure, can be preserved for a prolonged period at -80°C, and are not subject to deactivation, even after repeated freezing and thawing [75, 76]; and they have an encapsulation capability, can load specific drugs and transport them to target cells [77].

Notably, MSC-EVs express EV surface markers CD63, CD9 and CD81, as well as mesenchymal stem cell surface markers CD44, CD73, and CD90 [78]. In addition, proteins contained in the extracellular vesicles secreted by MSCs are a specific protein subclass that determines their unique biological functions [36]. At the same time, the encapsulated mRNA and miRNA in MSC-EVs form the molecular basis for their function [79]. Accordingly, MSC-EVs transmit information and communicate with target cells through internal substances, thus changing the activity and function of target cells [80].

With their unique advantages, MSC-EVs play an important role in immune regulation and regeneration. Studies of the promotion of regeneration through immune regulation are described in detail below. Meanwhile, in the treatment of autoimmune diseases, Wu et al. found that BM-MSC-derived EVs targeted inhibition of the cyclin I-activated ATM/ATR/p53 signaling pathway by upregulation of miR- 34a, thereby inhibiting RA fibroblast-like synoviocytes (RA-FLSs) and significantly ameliorating RA inflammation in vivo [81]. Another study on the regulation of type-I autoimmune diabetes mellitus (T1DM) showed that AD-MSC-derived exosomes ameliorated T1DM symptoms by upregulating the expression‎ of regulatory T cells, interleukin 4 (IL 4), IL 10 and transforming growth factor-beta (TGF-β) and down-regulating IL 17 and interferon-gamma (IFN-γ) [82]. Additional studies of autoimmune disease regulation have been summarized elsewhere [83]. Recently MSC-EVs have also been applied in clinical practice. Nassar et al. are in the process of eval‎uating the effect of human UC-MSC-derived EVs on islet β cells in patients with T1DM (trial NCT02138331). Recent clinical trials have been conducted to eval‎uate the safety and efficacy of MSC-EVs in patients with a variety of diseases based on their potential for immune regulation and regeneration (Table 1).

MSC 유래 세포외 소체

MSC는 다양한 출처에서 유래하지만, 모두 배양에서 부착 가능하며 특정 표면 마커를 가진 다양한 세포 유형으로 분화될 수 있습니다 [54].

MSC를 이용한 임상 치료의 필요성에 따라 국제 세포 치료학회(ISCT)의 중간엽 및 조직 줄기세포 위원회는 인간 MSC의 식별을 위한 최소 기준을 제안했습니다:

(1) 표준 조건 하에서 배양 시 플라스틱 기질에 부착되어야 합니다;

(2) 유동 세포 측정법에서 MSC 표면 마커인 CD105, CD73 및 CD90의 양성 발현율이 95% 이상이어야 하며, CD45, CD34, CD14 또는 CD11b, CD79a 또는 CD19 또는 HLA-DR(인간 백혈구 항원 -DR)의 음성 발현율이 2% 이하이어야 합니다;

(3) 표준 방법에 따라 체외에서 유도된 후, MSC는 골세포, 연골세포 및 지방세포로 분화할 수 있어야 합니다 [55].

그러나 추가 연구 결과, 이러한 기준이 MSC를 완전히 정의하지 못한다는 것이 밝혀졌습니다 [56]. 이질적인 MSC는 특이적인 표면 마커를 가진 다중 세포 하위 집단을 포함한다는 증거가 누적되고 있습니다 [57, 58], 그러나 이러한 하위 집단의 표면 마커 정의와 생물학적 기능은 지속적인 연구가 필요합니다.

MSCs는

체외에서 쉽게 회복되고 증식할 수 있어

임상 적용을 위해 대량 생산이 가능합니다 [18].

최근 몇 년간 임상 적용을 위한 가장 많이 연구된 줄기세포 유형으로,

이식 대 호스트 질환 (GVHD) [7],

신장 손상 [59], 조직 및 장기 이식, 면역 관용 [60], 신경 손상, 류마티스 질환, 간 질환 등에서

효과적인 치료 역할을 수행해 왔습니다.

현재 MSC는 코로나19 팬데믹 맥락에서 많은 관심을 받고 있습니다[61].

Leng 등[62]은 MSC 치료 그룹에서 코로나19 감염 환자의 증상이 완화되거나 크게 개선되었으며, 이는 인터루킨 10(IL-10) 발현 조절, 과활성화된 면역 T 세포 및 NK 세포 억제, TNF-α 수준 감소와 관련이 있음을 보여주었습니다.

그러나 MSC 치료에는 고려해야 할 측면이 있습니다.

첫째,

체외 배양 과정에서 세대 수가 증가함에 따라 MSC의 증식 능력이 점차 약화되며,

일정 수준의 분화 및 노화가 동반됩니다.

이는 그들의 조절 및 치료 능력을 저해합니다 [56, 63].

둘째,

체내 환경에서 MSC의 유전적 요인과 자기 재생 능력이 통제되지 않아

종양 형성 가능성이 있습니다 [64].

또한 MSC는 강한 재생 조절 잠재력을 가지고 있지만,

정맥 주사 후 손상 부위에 표적화되거나 남아 있을 수 있는지 불확실합니다 [65].

호스트 신체 의 청소 능력으로 인해 목표 부위에 도달하는

MSC의 수가 적다는 증거가 있습니다 [66, 67].

현장 주사는 이러한 문제를 부분적으로 해결할 수 있지만,

세포 분화 및 노화 문제와 임상 효과가 낙관적이지 않습니다 [68].

MSCs는 다양한 유형의 암의 발생과 증식을 유발하고 촉진한다는 것이 밝혀졌습니다 [69].

또한 세포 치료의 일반적인 위험인 바이러스 감염과 면역 거부 반응, 보관 및 운송 문제도 존재합니다 [70].

MSCs의 치료 효과 대부분이 파라크린 작용에 의존하며,

EVs가 부모 세포를 대체할 수 있다는 발견은 연구자들에게 흥미로운 전망을 제시합니다 [21].

EVs는 다음과 같은 큰 장점을 제공합니다 [71]:

자체 복제 능력이 없으며 종양 유발 위험을 크게 피합니다 [72];

세포 치료에 비해 안전합니다;

나노입자로서 생체 적합성과 낮은 면역 원성을 갖추어 혈액-뇌 장벽과 같은 보호 장벽을 통과할 수 있습니다 [73];

불멸화된 세포에서 지속적으로 분비되어 충분한 양을 얻을 수 있습니다 [74];

EV는 지질 막 구조를 통해 내부 생물분자 활성을 보호하며,

-80°C에서 장기간 보존 가능하며, 반복적인 동결 및 해동 후에도 비활성화되지 않습니다 [75, 76];

또한 캡슐화 능력을 갖추어 특정 약물을 탑재하고 표적 세포로 운반할 수 있습니다 [77].

특히, MSC-EV는 EV 표면 마커인 CD63, CD9, CD81 및 중간엽 줄기세포 표면 마커인 CD44, CD73, CD90을 발현합니다 [78]. 또한 MSC가 분비하는 세포외 소포에 포함된 단백질은 그들의 독특한 생물학적 기능을 결정하는 특정 단백질 하위 클래스입니다 [36]. 동시에 MSC-EV에 캡슐화된 mRNA와 miRNA는 그 기능의 분자적 기반을 형성합니다 [79]. 이에 따라 MSC-EV는 내부 물질을 통해 표적 세포와 정보를 전달하고 소통함으로써 표적 세포의 활동과 기능을 변화시킵니다 [80].

이러한 독특한 장점을 바탕으로 MSC-EV는 면역 조절과 재생에 중요한 역할을 합니다. 면역 조절을 통한 재생 촉진에 대한 연구는 아래에서 자세히 설명됩니다. 한편, 자가면역 질환 치료에서 Wu 등[81]은 BM-MSC 유래 엑소좀이 miR-34a 발현을 증가시켜 cyclin I 활성화 ATM/ATR/p53 신호전달 경로를 표적 억제함으로써 RA 섬유모세포 유사 활막세포(RA-FLS)를 억제하고 체내 RA 염증을 유의미하게 완화시켰습니다. 제1형 자가면역 당뇨병(T1DM) 조절에 대한 또 다른 연구에서는 AD-MSC 유래 엑소좀이 조절 T 세포, 인터루킨 4(IL-4), IL-10 및 변형 성장 인자-베타(TGF-β)의 발현을 증가시키고 IL-17 및 인터페론-감마(IFN-γ)를 억제함으로써 T1DM 증상을 완화시켰습니다[82]. 자가면역 질환 조절에 대한 추가 연구는 다른 곳에서 요약되어 있습니다 [83]. 최근 MSC-EV는 임상 실무에도 적용되었습니다. Nassar 등 연구진은 T1DM 환자의 췌장 베타 세포에 대한 인간 UC-MSC 유래 엑소좀의 효과를 평가하는 연구를 진행 중입니다(임상 시험 NCT02138331). 최근 면역 조절 및 재생 잠재력을 기반으로 다양한 질환 환자를 대상으로 MSC-EV의 안전성과 유효성을 평가하는 임상 시험이 진행되었습니다(표 1).

Table 1 Summary of registered clinical trials based on MSC-EVs with potential for immune regulation and regeneration.

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Application of MSC-EVs in immune regulation and regeneration

The therapeutic potential of MSC-EVs has been reported in immune regulation and tissue regeneration based on EV-mediated cellular communication between MSCs and several target cells, including macrophages, microglia, chondrocytes, articular chondrocytes, endothelial cells, fibroblasts, pericytes, neural stem cells (NSC), neurons, hepatic stellate cells, and podocytes. In this paper, we discuss the molecular mechanisms of MSC-EVs in tissue repair and anti-fibrosis, in which several clusters of miRNA and their downstream pathways have been revealed to play important roles in osteoarthritis, spinal cord injury, skin injury, liver fibrosis, kidney fibrosis, and lung fibrosis (Tables 2–7).

MSC-EV의 면역 조절 및 재생에 대한 적용

MSC-EV의 치료적 잠재력은

MSC와 대식세포, 미세아교세포, 연골세포, 관절 연골세포, 내피세포,

섬유아세포, 페리사이트, 신경줄기세포(NSC), 신경세포, 간성별세포, 포도세포 등

여러 표적 세포 간의 EV 매개 세포 간 통신을 기반으로

면역 조절 및 조직 재생 분야에서 보고되었습니다.

본 논문에서는 MSC-EVs의 조직 복구 및 항섬유화 메커니즘을 논의하며,

골관절염, 척수 손상, 피부 손상, 간 섬유화, 신장 섬유화, 폐 섬유화 등에서

여러 클러스터의 마이크로RNA(miRNA)와

그 하류 경로가 중요한 역할을 한다는 것이 밝혀졌습니다(표 2–7).

Table 2 Summary of studies on the role of extracellular vesicles in osteoarthritis.

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Table 3 Summary of studies on the role of extracellular vesicles in spinal cord injury.

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Table 4 Summary of studies on the role of extracellular vesicles in skin injury.

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Table 5 Summary of studies on the role of extracellular vesicles in liver fibrosis.

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Table 6 Summary of studies on the role of extracellular vesicles in kidney fibrosis.

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Table 7 Summary of studies on the role of extracellular vesicles in lung fibrosis.

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Support tissue repairOsteoarthritis

Osteoarthritis (OA) is the principal form of joint disease with unclear pathogenesis, presenting with pain and stiffness, and in some cases, disability [84]. Recently, MSC-EVs have been proven to have both regenerative and immunoregulatory benefits in OA (Table 2).

Several studies have reported that hBMSC-EVs play a significant role in the treatment of OA by inhibiting some pro-inflammatory pathways and factors, and enhancing the proliferation and migration of chondrocytes. Vonk et al. determined that MSC-EVs blocked NFκB signaling by inhibiting phosphorylation of IκBα, thereby down-regulating TNF-α-induced COX2 expression‎, and interleukins and collagenase activity. Additionally, MSC-EVs up-regulated the expression‎ of SOX9 and WNT7A, and promoted the production of proteoglycan and type II collagen in in vitro studies [85]. Li et al. concluded that hBMSC-EVs promoted OA-chondrocyte (OA-CH) proliferation and migration and reduced apoptosis via downregulation of MMP13, ALPL, IL-1β-activated pro-inflammatory Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling pathways and increased gene expression‎ of PRG4, BCL2, and ACAN (aggrecan) [86]. In addition, in OA-like chondrocytes, MSC-EVs induced the expression‎ of type II collagen and aggrecan (chondrocyte markers), while inhibiting MMP-13 and ADAMTS5 (catabolic) and iNOS (inflammatory markers). In a CIOA model, treated mice also exhibited reduced cartilage and bone degeneration [87]. In an OA model, Ruiz showed that the effect of MSC-EVs was due to the presence of TGFBI mRNA and protein [88]. Analogously, in the same model, BMSC-EVs promoted the conversion of RAW264.7 from M1 to M2, reduced the expression‎ of proinflammatory cytokines IL-1β, TNF-α, and IL-6, and enhanced the expression‎ of IL-10, chondrogenic genes, collagen II and SOX9 [89]. Interestingly, Woo et al. revealed in their monosodium iodoacetate (MIA) rat and the surgical destabilization of the medial meniscus (DMM) mouse model that MSC-EVs could ameliorate cartilage degeneration by increasing type II collagen synthesis and decreasing MMP-1, MMP-3, MMP-13 and ADAMTS-5 expression‎ in the presence of IL-1β [90].

Recent studies have also examined the effect of miRNAs in MSC-EVs. In synovial-derived MSC-EVs (SMSC-EVs), Tao et al. overexpressed miR-140-5p to block Wnt5a and Wnt5b to activate YAP via the Wnt signaling pathway and significantly reduce extracellular matrix (ECM) secretion [91]. Wang et al. found that exosomes derived from miR 155-5p–overexpressing SMSCs (SMSC-155-5p-Exos) promoted ECM secretion by targeting Runx2, which enhanced cartilage regeneration and ameliorated OA [92]. Likewise, SMSC-EVs highly expressed miR-31 and relieved OA via the KDM2A/E2F1/PTTG1 axis [93]. Of interest, hypoxia increased the expression‎ of miR-216a-3p in HIF-1α-induced BMSC-EVs and promoted down-regulation of JAK2, promoting proliferation, migration, and reduced apoptosis of chondrocytes via inhibition of the JAK2/STAT3 signaling pathway [94]. A combination of these miRNAs and MSC-EVs may serve as a potential therapy for OA. In contrast, several studies have shown that miRNAs cause side effects in OA. Intra-articular injection of antagomir-miR-100-5p dramatically attenuated the infrapatellar fat pad (IPFP) MSC-EV (MSCIPFP-EVs)-mediated protective effect on articular cartilage in vivo [95]. MiR-29b-3p targets FoxO3 gene and enhances chondrocyte destruction. lncRNA H19 from umbilical cord MSC-EVs could competitively bind to miR-29b-3p to attenuate its inhibition of the target gene FoxO3 [96].

조직 재생 지원

골관절염

골관절염(OA)은 병인이 명확하지 않은 관절 질환의 주요 형태로, 통증과 경직을 동반하며 일부 경우 장애를 유발합니다 [84]. 최근 MSC-EVs가 OA에서 재생 및 면역 조절 효과를 모두 갖는 것으로 입증되었습니다(표 2).

여러 연구에서 hBMSC-EVs가 일부 염증성 경로와 인자를 억제하고 연골세포의 증식 및 이동을 촉진함으로써 OA 치료에 중요한 역할을 한다는 것이 보고되었습니다. Vonk 등[84]은 MSC-EVs가 IκBα의 인산화를 억제함으로써 NFκB 신호전달을 차단하여 TNF-α에 의해 유도된 COX2 발현을 억제하고 인터루킨 및 콜라게나제 활성을 감소시킨다는 것을 확인했습니다. 또한 MSC-EVs는 체외 연구에서 SOX9 및 WNT7A의 발현을 증가시키고 프로테오글리칸 및 제2형 콜라겐의 생성을 촉진했습니다 [85]. Li 등[86]은 hBMSC-EVs가 OA-연골세포(OA-CH)의 증식과 이동을 촉진하고, MMP13, ALPL, IL-1β 활성화 프로염증성 Erk1/2, PI3K/Akt, p38, TAK1, 및 NF-κB 신호전달 경로를 억제함으로써 세포 사멸을 감소시키며, PRG4, BCL2, 및 ACAN(aggrecan) 유전자 발현을 증가시킨다고 결론지었습니다. 또한 OA 유사 연골세포에서 MSC-EV는 제2형 콜라겐과 아그레칸(연골세포 표지자)의 발현을 유도하며, MMP-13과 ADAMTS5(분해성 표지자) 및 iNOS(염증 표지자)를 억제했습니다. CIOA 모델에서 치료된 쥐는 연골 및 골 퇴행이 감소했습니다 [87]. OA 모델에서 Ruiz는 MSC-EV의 효과가 TGFBI mRNA 및 단백질의 존재 때문임을 보여주었습니다 [88]. 유사하게, 동일한 모델에서 BMSC-EVs는 RAW264.7 세포의 M1에서 M2로의 전환을 촉진했으며, 염증성 사이토킨 IL-1β, TNF-α, 및 IL-6의 발현을 감소시키고, IL-10, 연골 생성 유전자, 콜라겐 II 및 SOX9의 발현을 증가시켰습니다 [89]. 흥미롭게도 Woo 등[90]은 단일나트륨 요오도아세테이트(MIA) 쥐 모델과 내측 반월판 수술적 불안정화(DMM) 마우스 모델에서 MSC-EV가 IL-1β 존재 하에서 II형 콜라겐 합성을 증가시키고 MMP-1, MMP-3, MMP-13 및 ADAMTS-5 발현을 감소시켜 연골 퇴화를 완화한다는 것을 밝혀냈습니다.

최근 연구에서는 MSC-EV 내 마이크로RNA(miRNA)의 효과도 조사되었습니다. 관절막 유래 MSC-EV(SMSC-EV)에서 Tao 등[91]은 miR-140-5p를 과발현시켜 Wnt5a와 Wnt5b를 차단하고 Wnt 신호 경로를 통해 YAP를 활성화시켜 세포외 기질(ECM) 분비를 유의미하게 감소시켰습니다. Wang 등[92]은 miR-155-5p 과발현 SMSC(SMSC-155-5p-Exos)에서 유래한 엑소좀이 Runx2를 표적화하여 ECM 분비를 촉진함으로써 연골 재생과 OA를 개선했습니다. 마찬가지로, SMSC-EVs는 miR-31을 고발현하여 KDM2A/E2F1/PTTG1 축을 통해 OA를 완화했습니다[93]. 흥미롭게도 저산소 환경은 HIF-1α에 의해 유도된 BMSC-EVs에서 miR-216a-3p의 발현을 증가시켜 JAK2의 발현을 억제함으로써 JAK2/STAT3 신호전달 경로를 차단하여 연골세포의 증식, 이동성을 촉진하고 세포 사멸을 감소시켰습니다 [94]. 이러한 miRNA와 MSC-EVs의 조합은 OA 치료를 위한 잠재적 치료법으로 작용할 수 있습니다. 반면, 여러 연구에서 miRNA가 OA에서 부작용을 유발한다는 것이 밝혀졌습니다. 관절 내 주사된 antagomir-miR-100-5p는 체내에서 관절 연골에 대한 infrapatellar fat pad (IPFP) MSC-EV (MSCIPFP-EVs)의 보호 효과를 현저히 감소시켰습니다 [95]. MiR-29b-3p는 FoxO3 유전자를 표적화하여 연골세포 파괴를 촉진합니다. 제대혈 MSC-EV에서 유래한 lncRNA H19는 miR-29b-3p와 경쟁적으로 결합하여 표적 유전자 FoxO3에 대한 억제 효과를 감소시킵니다 [96].

Spinal cord injury

Spinal cord injury (SCI) arises following damage to its structure and function by various pathogenic factors, with consequent spinal cord dysfunction including that of movement, sensation, and reflexes [97]. Due to the limited regenerative ability of nerve components, MSC-EVs have been recently viewed as a promising clinical treatment for SCI (Table 3).

A rat model of SCI has commonly been applied to eval‎uate treatment with MSC-EVs. They have been found to be able to regulate immunity and restore function through a variety of pathways. First, Huang et al. studied the administration of hBMSC-Exos in an animal model, and demonstrated that inhibition of apoptosis protein (Bax) and pro-inflammatory factors (TNFα and IL 1β), and promotion of anti-apoptotic protein (Bcl-2), anti-inflammatory protein (IL 10) and angiogenesis, could improve motor function [98]. Interestingly, the reduced pericyte migration mediated by BMSC-EVs correlated with inhibition of the NF-KB P65 signaling pathway with consequent weakening of the blood-spinal cord barrier (BSCB) [99]. In addition, Zhou et al. showed that treatment with BMSC-Exos suppressed the expression‎ of caspase 1 and IL 1β by reducing pyroptosis, and enhanced neuronal regeneration to ameliorate motor ability in rats with spinal cord injury [100]. Han et al. found that TGF-β in BMSC-EVs enhanced the expression‎ of Smad6, inhibited the excessive differentiation of neural stem cells (NSCs) into astrocytes, and promoted regeneration of neurons [101]. Consecutively, Nakazaki et al. proposed that BMSC-EVs should be administered over 3 days to up-regulate transforming growth factor -β (TGF-β), TGF-β receptor, and relative proteins of tight junction [102]. More intriguingly, Zhou et al. provided evidence that exosomes secreted by hPMSCs increased the activation of proliferating endogenous nerve stem/progenitor cells in vivo, while promoting NSC proliferation and upregulating MEK, ERK, and CREB phosphorylation levels in vitro, resulting in functional recovery [103].

MiRNAs have always been potent biological effectors of MSC-EVs, and without exception, they play a strong role in immune regulation and regeneration in spinal cord injury. Jia et al. confirmed that overexpression‎ of miR-381 in MSC-EVs could promote SCI repair by up-regulating Ras homologous A (RhoA)/ RHO kinase activity and down-regulating BRD4 expression‎ and DRG cell apoptosis by WNT5A [104]. Li et al. observed that miR-133 carried by MSC-Exos could directly target and down-regulate the expression‎ of RhoA, and also promote expression‎ of ERK1/2 STAT3 and CREB signaling pathway proteins related to neuronal survival and axon regeneration, thus rescuing neuron apoptosis and promoting axon regeneration [105]. Of interest, when miR-17-92, miR-26a, and miR-216a-5p were enriched in BMSC-Exos, they respectively induced activation of mTOR/PI3K/Akt, PTEN/ Akt /mTOR, and the TLR4/NF-κB/PI3K/ Akt signaling pathway cascade, with consequent promotion of axonal regeneration and nerve function repair after SCI [106,107,108]. In addition, miRNA-22 encapsulated in BMSC-EVs promotes neurogenesis and inflammation suppression by downregulating the expression‎ of inflammatory cytokines and GSDMD, and blocking the pyroptosis of microglia after SCI [109]. Overexpression‎ of miR-199a-3p/145-5p in exosomes secreted by human umbilical cord-derived MSCs has been shown to activate the NGF/TrkA signaling pathway affecting TrkA ubiquitination, and improve locomotor function in rats with SCI [110].

Skin injury

Skin injury is quite common. Skin regeneration is typically accompanied by four overlapping processes: inflammation, angiogenesis, new tissue formation, and remodeling [111,112,113] (Table 4).

There is recent evidence that human-derived MSC-Exos effectively benefit skin damage and accelerate wound healing by modulating related signaling pathways. Intriguingly, Zhou et al. adopted a combination therapy, applying hADSC-Exos both locally and intravenously to accelerate skin wound healing. Mechanistically, hADSC-Exos achieved this effect by down-regulating TNF-α, IL-6, CD14, CD19, CD68, and C-caspase 3, and up-regulating VEGF, CD31, Ki67, PCNA, filaggrin, loricrin and AQP3 [114]. Jiang et al. demonstrated that hBMSC-Exos suppressed TGF-β1, Smad2, Smad3, and Smad4 by targeting the TGF-β/Smad signaling pathway, but increased the expression‎ of TGF-β3 and Smad7, thus improving scar formation and promoting wound healing [115]. Remarkably, fetal dermal mesenchymal stem cell-derived exosomes (FDMSC-Exos) have been shown to activate adult dermal fibroblast (ADFs) to promote cell proliferation, migration and secretion by targeting Jagged 1 ligand in the Notch signaling pathway, and ultimately accelerate wound healing [116].

Similar effects have also been observed for human-derived MSC-Exos carrying miRNAs. Of interest, He et al. showed that hBMMSCs and jaw bone marrow MSCs (JMMSCs) could induce macrophages toward M2 polarization and promote wound healing. The mechanism suggested that exosomes secreted by donors may regulate the polarization of macrophages by carrying miR-223 targeting Pknox1. Nonetheless, researchers cannot confirm‎ whether other miRNAs or factors carried by these exosomes are involved in the induction of M2 polarization, and further studies are needed [117]. Likewise, Wu et al. utilized BMSC-Exos treated with 50 µg/mL Fe3O4 nanoparticles and 100 mT SMF to form a functional exosome (mag-BMSC-Exos). Notably, miR-21-5p was overexpressed in mag-BMSC-Exos and promoted angiogenesis in vivo and in vitro to accelerate skin wound healing by targeting SPRY2 to activate the PI3K/AKT and ERK1/2 signaling pathways [118]. Additionally, Cheng et al. found that hUCMSCs-EVs are highly enriched with miR-27b and promote the expression‎ of JUNB and IRE1α by targeting the Itchy E3 ubiquitin-protein ligase (ITCH), thereby accelerating cutaneous wound healing [119]. In addition, hUMSC-Exos can be enriched with a set of microRNAs (miR-21, -23A, -125b, and -145) to attenuate excess myofibroblast formation and scarring via repression of the TGF-β2 /SMAD2 pathways [120]. Another study showed that hADSC-Exos derived miR-19b regulate the TGF-β pathway by targeting CCL1 [121]. Li et al. verified that hADSC-Exos down-regulated the expression‎ of Col1, Col3, α-SMA, IL-17RA, and P-SMad2/P-SMad3, and up-regulated the level of SIP1 by suppressing multiplication and migration of hypertrophic scar-derived fibroblasts (HSFs). In addition, miR-192-5p was highly enriched in ADSC-EXO and reduced the level of pro-fibrosis protein, improved hypertrophic scar fibrosis, and accelerated wound healing via targeted inhibition of IL-17RA expression‎ [122]. Alongside this, overexpression‎ of miR-486-5P in hADSC-EVs enhanced the migration of human skin fibroblasts (HSFs) and the angiogenic activity of human microvascular endothelial cells (HMECs) by targeting Sp5 and motivating CCND2 expression‎, thereby promoting wound healing [123]. Interestingly, Gao et al. found that overexpression‎ of Mir-135a in hAMSC-Exos significantly down-regulated LATS2, thereby increasing cell migration and promoting wound healing [124].

Anti-fibrosis

Liver fibrosis

Liver fibrosis is a pathophysiological process and refers to the abnormal proliferation of intrahepatic connective tissue due to various pathogenic factors [125]. Recently, use of MSC-EVs has been considered a new therapeutic approach to repair liver fibrosis (Table 5). Rong et al. showed that human bone MSC-EVs inhibited expression‎ of Wnt/β-catenin pathway components, α-SMA, and type I collagen, thereby preventing stellate cell activation and increasing hepatocyte regeneration. In vivo injection of hBMSC-Exos has been shown to effectively alleviate CCL4-induced liver fibrosis in rats and restore liver function [126]. Likewise, using a CCL4-induced liver fibrosis animal model, Ohara et al. proved that EVs from amnion-derived MSCs (AMSC-EVs) could significantly reduce the number of Kupffer cells (KCs), mRNA expression‎ of inflammatory factors, activation of hepatic stellate cells (HSC), and the lipopolysaccharide (LPS)/toll-like receptor 4 (TLR4) signaling pathway, thereby reducing inflammation and fibrosis [127].

The anti-fibrotic effect of miRNAs in MSC-EVs has become a focus of research into CCL4-induced liver fibrosis in rats. MiRNA-181-5p overexpression‎ in ADSC-EVs has been shown to down-regulate transcription 3 (STAT3) and Bcl-2 and activated autophagy in HST-T6 cells, alongside a significant decrease in collagen I, vimentin, a-SMA, and fibronectin in liver [128]. Similarly, high expression‎ of miR-122 in ADSC-EVs modulated the expression‎ of target genes such as insulin-like growth factor receptor 1 (IGF1R) cyclin G(CCNG1), and proline-4-hydroxylase A1(P4HA1), thereby more effectively blocking the proliferation of HSCs and collagen maturation [129]. Interestingly, Kim et al. reported that miR-486-5p was highly expressed in T-MSC-EVs that could target the hedgehog receptor, smoothened (Smo), and inhibit hedgehog signaling, thereby attenuate the activation of HSCs and liver fibrosis [130].

Kidney fibrosis

Renal fibrosis is a gradual pathophysiological process during which kidney function progresses from healthy to injured, then to damage with an ultimate loss of function [131]. Increasingly, MSC-EVs have been studied in the treatment of renal fibrosis using various models (Table 6).

Ji et al. determined that hUC-MSC-Exos repressed Yes-associated protein (YAP) through casein kinase 1δ (CK1δ) and E3 ubiquitin ligase β-TRCP in a rat model of unilateral ureteral obstruction (UUO), thus ameliorating renal fibrosis [132]. Similar effects in a UUO model were confirmed in Liu’s study. They revealed that hUC-MSC-Exos attenuated renal fibrosis by inhibiting the ROS-mediated p38MAPK/ERK signaling pathway [133]. Likewise, Shi et al. showed that milk fat globule–epidermal growth factor–factor 8 (MFG-E8) was included in BMSC-EVs, and ameliorated renal fibrosis by blocking the RhoA/ROCK pathway in a UUO model [134]. Of interest, in a UUO mouse model, BMSC-Exos loaded miR-34c-5p inhibited core fucosylation (CF) by cd81-EGFR complex, thereby improving renal interstitial fibrosis (RIF) [135]. Correspondingly, recent studies also suggest that exosomes from ADSCs ameliorate the development of DN via miRNAs. Jin et al. used miRNA-215-5p to inhibit ZEB2 and improved diabetic nephropathy (DN) symptoms. They also revealed that upregulated expression‎ of miR-486 could suppress the Smad1/mTOR signaling pathway in podocytes [136, 137]. MV-miR-451a from hUMSCs repressed cell cycle inhibitor P15 and P19 expression‎ by targeting their 3′-UTR sites, thereby decreasing α-SMA and increasing e-cadherin expression‎. This resulted in epithelial-mesenchymal transformation (EMT) reversal and improved DN symptoms [138]. In another study of amelioration of DN, BMSC-Exos significantly enhanced the expression‎ of LC3 and Beclin-1, and decreased the level of mTOR and fibrotic markers in a streptozotocin-induced rat model of diabetes mellitus [139]. Interestingly, Grange et al. reported that renal fibrosis and the expression‎ of collagen I were significantly ameliorated via multiple injections of HLSCs (human liver stem-like cells) and MSC-EVs in NOD/SCID/IL2Rγ KO (NSG) mice. Additionally, related genes (Serpina1a, FAS ligand, CCL3, TIMP1, MMP3, collagen I, and SNAI1) were significantly downregulated, thereby attenuating DN symptoms [140].

Lung fibrosis

Pulmonary fibrosis is a terminal change in lung disease characterized by fibroblast proliferation and accumulation of a large amount of extracellular matrix accompanied by inflammatory injury and destruction of tissue. Normal alveolar tissue is damaged and abnormal repair leads to structural abnormalities [141, 142]. The etiology in the vast majority of patients with pulmonary fibrosis is unknown [143]. Idiopathic pulmonary fibrosis (IPF) manifests mainly with pulmonary fibrotic lesions and is a serious interstitial lung disease that can lead to progressive loss of lung function. IPF has a higher mortality than most tumors and is considered a tumor-like disease [142]. Recently, MSC-EVs have become an effective treatment for pulmonary fibrosis (Table 7).

BMSC-Exos exert their therapeutic effect through immunomodulation. In a mouse model, BMSC-Exos have been shown to significantly ameliorate hyperoxia (HYRX)-induced bronchopulmonary dysplasia (BPD), alveolar fibrosis, and pulmonary vascular remodeling by suppressing M1 macrophage production and enhancing M2 macrophage generation [144]. Likewise, BMSC-Exos have been shown to significantly reverse fibrosis in a bleomycin-induced pulmonary fibrosis model by regulating total lung imbalance of MΦ phenotype [145]. In addition, the Wnt5a/BMP signaling pathway regulated by UC-MSC-Exos can enhance Wnt5a, Wnt11, BMPR2, BMP4, and BMP9 expression‎, and down-regulate that of β-catenin, Cyclin D1 and TGF-β1. In a monocrotaline (MCT)-induced rat model of pulmonary hypertension (PH), MSC-Exos were shown to significantly ameliorate pulmonary vascular remodeling and pulmonary fibrosis [146]. Of interest, Chaubey et al. showed that UC-MSC-Exos played a therapeutic role in improving pulmonary inflammation, pulmonary simplification, pulmonary hypertension, and right ventricular hypertrophy through immunomodulatory glycoprotein TSG-6 in a neonatal BPD mouse model [147].

Additionally, MSC-EVs can reverse lung injury and pulmonary fibrosis by expressing influential miRNAs. Wan et al. determined that high expression‎ of miR-29b-3p by BMSC-EVs ameliorated IPF by FZD6 [148]. Zhou et al. found that miR-186 enriched by BMSC-EVs repressed the expression‎ of SOX4 and Dickkopf-1 (Dkk1), thereby effectively inhibiting fibroblast development and attenuating IPF [149]. In addition, Lei’s study revealed that hPMSC -EVs could carry miR-214-3p and downregulate ATM/P53/P21 signaling, thus relieving radiation-induced lung inflammation and fibrosis [150]. In BLM-induced lung fibrosis and a mouse model of alveolar epithelial cell damage, exosomes secreted from MenSCs (MenSCs-Exos) have been shown to ameliorate pulmonary fibrosis by transferring miRNA Let-7 to suppress reactive oxygen species (ROS), mitochondrial DNA (mtDNA) damage, and activation of NLRP3 inflammasome [151]. Similarly, Xiao et al. used another LPS-induced Acute Lung Injury (ALI) mouse model and demonstrated that MSC-Exos repressed NF-κB and hedgehog pathways by transporting miR-23a-3p and miR-182-5p, thereby improving lung injury and fibrosis [152].

Challenges and application of MSC-EVS as an advanced therapy

Although MSC-EV-based therapy holds great promise as a novel “cell-free” therapeutic product, there remain many challenges to overcome prior to their clinical application. At present, several limitations restrict the clinical translation of MSC-EVs including the discrepancies in the components of EVs from various sources and the lack of standard operation processes for largescale production, both of which largely depend on quality control of the sources of EVs. It is plausible to overcome these hurdles by introducing a strategy to control the quality of MSCs from the original source of EVs.

The quality of MSC-derived EVs from different groups and batches is heterogeneous

MSCs are most commonly derived from bone marrow, fat, umbilical cord and other tissues, but maintaining consistent quality of MSCs and their EVs from different sources and across batches is difficult. This severely restricts the quality control and management of MSCs and their EVs as drugs, and increases the problem of drug resistance [153]. This results in limited reproducibility of functional measurements in vitro and in vivo [154].

In the angiogenesis study, BMSC-, ADSC-, and UCBMSC-derived EVs were compared and found to reduce myocardial apoptosis, facilitate angiogenesis, and improve cardiovascular function. Notably, EVs from ADSCs stimulated cardioprotection factors VEGF, bFGF, and HGF [155]. In addition, BMSC-derived EVs appeared to have a greater angiogenic potential than ADSC-derived EVs when compared in two independent ischemic model studies, with an approximately 4-fold increase in endothelial cell numbers compared with controls, and a 1.5-fold change in the latter [156, 157]. Nonetheless, another study showed that EVs from endometrial mesenchymal stem cells resulted in a greater level of angiogenesis than EVs from BMSCS or ADMSCs [158].

In studies of osteogenesis studies, in two separate rat skull defect studies, BMSC-EV treatment increased bone volume four-fold relative to the control group [159], while ADSC-EV increased bone volume by about 1.33 times [160]. In other studies, BMSC- and ADSC-derived EVs accelerated chondrocyte proliferation, migration, and osteogenic differentiation [161, 162].

Comparison of the immunomodulatory differences of MSC-derived EVs from different sources revealed that BMSC-EVs and ADSC-EVs could induce M2 polarization of macrophages in vivo and in vitro [163, 164]. Interestingly, in a separate experiment, Wang et al. showed that BMSC-EVs prompted a significant (3.2-fold) increase in the expression‎ of CD206 of M2-polarization marker in an acute lung injury mouse model [163]. Nonetheless Liu et al. reported that the M2 polarization ability of ADSC-EVs increased only by a factor of 1.5 in a mouse model [165].

The proliferation capacity of MSCs extracted from adult tissues was limited, and affected the largescale production of EVs

To develop MSC-EVs into commercially advanced therapeutic products (ATPs), quality assurance (QA) is required of the original material, including parental groups or cells used in the manufacture of MSCs. There remain many difficulties in mass production of EVs from adult tissues for clinical trials since proprietary MSCs have a limited number of passage times, age easily, and come at a high financial cost. In addition, their heterogenicity makes traditional cell culture inefficient in terms of time and cost.

MSCs derived from pluripotent stem cells overcome the problems of mass production of MSC-EVs and quality heterogeneity

The original source MSCs requires good, consistent, and controllable quality, with a strong ability to proliferate and to secrete large numbers of EVs. To achieve this, we established an induction system of MSCs using pluripotent stem cells to overcome the problems of mass production of MSC-EVs and variation in quality. We successfully induced MSCs from pluripotent stem cells (PSC) [166,167,168,169,170]. Compared with MSCs extracted from traditional sources, our MSCs were derived from the same parent PSCs, consequently overcoming the problem of EV heterogeneity when MSCs from a variety of sources are used. Recently, GMP-grade MSCs derived from human PSCs (hPSC) have been used in clinical trials for refractory graft-versus-host disease (GVHD) [171]. The therapeutic potential of MSC-EVs has been shown in preclinical studies of both acute GVHD (aGVHD)[172,173,174] and chronic GVHD (cGVHD) [175] models. The preliminary benefits of hPMSC-EVs have been reported in a patient with cutaneous cGVHD. The stiffening and dryness of skin were improved significantly after intravenous injection of hPMSC-EVs [176]. Based on the preliminary efficacy and safety profiles, a phase 1 study has been launched to eval‎uate the safety and efficacy of BM-MSC-derived EVs in patients with acute or chronic rejection following abdominal solid organ transplantation (NCT05215288, Table 1). It is plausible that hPSC-MSC-derived EVs will promote the clinical translation of MSC-EVs owing to the quality control and largescale productive advantages of hPSC-MSCs compared with traditional MSC. hPSC-MSCs have more passages (more than 30 generations), strong amplification ability, can withstand senescence [166, 167, 170], and have strong secretion ability (including cytokines and exosomes) [168] compared with the traditional MSCs. Nonetheless, the passage times of traditional MSCs are generally less than 10 generations, and the proliferation and differentiation abilities of MSCs are reduced after numerous passages in culture, and affects the secretion of extracellular vesicles. Therefore, our hPSC-MSCs have great advantages for large-scale production and cost control of EVs. Mass production of MSCs and their EVs is now possible using bioreactors and microcarriers to maximize MSC growth and EV release per unit surface area. We eval‎uated mesenchymal stem cells from different sources and found that PSC-MSCs had the highest EV production. To optimize EV production, we acquired hPSC-MSCs in a scalable cell factory-based culture and were able to overcome the major obstacles during transformation of MSC-EVs into ATPs.

Conclusions and future perspective

Extracellular vesicles derived from mesenchymal stem cells play a critical role in the development of immune regulation and regeneration. These EVs mimic the effects of stem cells and perform powerful functions by modulating immune pathways, promoting effector cell migration and proliferation, and reducing apoptosis. To date, 15 clinical trials have been registered in ClinicalTrial.gov, but none has been completed. Although EVs compared with MSC cell therapy incite a lower immune response and have a higher safety profile, there remain challenges to their clinical application [56]. In addition, the successful application of EVs depends on low cost for mass production, as well as improved separation efficiency and more accurate characterization methods. This review has discussed the therapeutic effects of EVs based on the function of MSCs or the introduction of specific molecules (such as miRNAs and lncRNAs). As work continues, researchers are actively developing engineered EVs that are more effective and capable of targeting, through loading of bioactive molecules and surface modification. Of interest, Feng et al. developed ε-polylysine-polyethylene-distearyl phosphatidylethanolamine (PPD) to modify MSC-EVs and invert their surface charge. As a result, the steric and electrostatic hindrance of cartilage matrix were alleviated, and the efficiency of MSC-EVs in the treatment of OA was improved [177]. These treatment strategies have achieved promising results at the initial stage and provide exciting new avenues for regenerative medicine therapy.

Data availability

All relevant data are included in this manuscript.

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