Re: 중간엽 줄기세포 Extracellular vesicles(엑소좀) 치료법 2015년 ..

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ReviewVolume 23, Issue 5p812-823May 2015Open Archive

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Mesenchymal Stem Cell-derived Extracellular Vesicles: Toward Cell-free Therapeutic Applications

Sweta Rani1 sweta.rani@nuigalway.ie ∙ Aideen E Ryan2 ∙ Matthew D Griffin1 ∙ Thomas Ritter1

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Abstract

Mesenchymal stem (stromal) cells (MSCs) are multipotent cells with the ability to differentiate into several cell types, thus serving as a cell reservoir for regenerative medicine. Much of the current interest in therapeutic application of MSCs to various disease settings can be linked to their immunosuppressive and anti-inflammatory properties. One of the key mechanisms of MSC anti-inflammatory effects is the secretion of soluble factors with paracrine actions. Recently it has emerged that the paracrine functions of MSCs could, at least in part, be mediated by extracellular vesicles (EVs). EVs are predominantly released from the endosomal compartment and contain a cargo that includes miRNA, mRNA, and proteins from their cells of origin. Recent animal model-based studies suggest that EVs have significant potential as a novel alternative to whole cell therapies. Compared to their parent cells, EVs may have a superior safety profile and can be safely stored without losing function. In this article, we review current knowledge related to the potential use of MSC-derived EVs in various diseases and discuss the promising future for EVs as an alternative, cell-free therapy.

초록

중간엽 줄기세포(MSCs)는 

다양한 세포 유형으로 분화할 수 있는 다능성 세포로, 

재생 의학의 세포 저장고 역할을 합니다. 

MSCs의 다양한 질환 모델에 대한 치료적 적용에 대한 현재의 관심은 

주로 그들의 면역억제 및 항염증 성질과 연관되어 있습니다. 

MSCs의 항염증 효과의 주요 메커니즘 중 하나는 

파라크린 작용을 하는 

용해성 인자의 분비입니다. 

최근 연구에서 

MSC의 파라크라인 기능이 적어도 일부는 

세포외 소포(EVs)를 통해 매개될 수 있다는 것이 밝혀졌습니다. 

EVs는 주로 엔도소체 부위에서 분비되며, 

기원 세포의 miRNA, mRNA, 단백질 등을 포함한 화물을 포함합니다. 

최근 동물 모델 기반 연구는 

EVs가 전체 세포 치료법의 새로운 대안으로 상당한 잠재력을 가지고 있음을 시사합니다. 

모세포에 비해 EVs는 우수한 안전성 프로파일을 갖추고 있으며, 

기능 손실 없이 안전하게 보관될 수 있습니다. 

본 논문에서는 

MSC 유래 EVs의 다양한 질환에서의 잠재적 활용과 관련한 최신 지식을 검토하며, 

EVs가 세포 없는 치료법으로서의 유망한 미래를 논의합니다.

Introduction

Regenerative medicine focuses on the restoration of lost, damaged, or aging cells and tissues in the human body. Ferrari et al.1 demonstrated the value of a stem cell-based regenerative treatment for muscular dystrophies using bone marrow (BM)-derived myogenic progenitor cells. Since then numerous stem cell types have been investigated for use in tissue regeneration in both animal models and human clinical studies, with varying degrees of success.

Mesenchymal stem (or stromal) cells (MSCs) have emerged as a potential solution for tissue repair and wound healing.2 MSCs are multipotent, nonhematopoietic adult stem cells, which can be isolated from BM, umbilical cord,3,4 placental or adipose tissue. MSCs have the potential to differentiate into osteoblasts, chondrocytes, and adipocytes5 as well as endothelial, cardiovascular, and neurogenic cell types and are gaining credibility as a therapeutic agent because of their ex vivo expansion capacity and ethical acceptability.6 More recently, it has been discovered that, in addition to their direct role in tissue regeneration, MSCs have potent anti-inflammatory and/or immunosuppressive properties.7 Extensive research and clinical trials are currently underway for the use of MSCs as regenerative agents in many diseases including spinal cord injury, multiple sclerosis, Alzheimer's disease, liver cirrhosis and hepatitis, osteoarthritis, myocardial infarction, kidney disease, inflammatory bowel disease, diabetes mellitus, knee cartilage injuries, organ transplantation, and graft-versus-host disease (http://www.clinicaltrials.gov; accessed November 2014).

소개

재생의학은

인간 몸에서 손실되거나 손상된 세포 및 조직을 복원하는 데 초점을 맞춥니다.

Ferrari et al.1은

골수(BM) 유래 근원성 전구세포를 활용한 줄기세포 기반 재생 치료법의 가치를

근육 위축증 치료에 적용하여 입증했습니다.

이후 동물 모델과 인간 임상 연구에서

조직 재생에 활용하기 위해 다양한 줄기세포 유형이 연구되었으며,

성공 정도는 다양했습니다.

중간엽 줄기세포(또는 간질 줄기세포)(MSCs)는

조직 복구 및 상처 치유를 위한 잠재적 해결책으로 부상했습니다.2

MSCs는

골수, 탯줄,3,4 태반 또는 지방 조직에서 분리될 수 있는 다능성, 비혈액 생성성 성인 줄기세포입니다.

MSCs는

골세포, 연골세포, 지방세포5로 분화할 수 있으며,

내피세포, 심혈관 세포, 신경 발생 세포 유형으로도 분화 가능하며,

ex vivo 확장 능력과 윤리적 수용성으로 인해 치료제로서의 신뢰성을 얻고 있습니다.6

최근에는 조직 재생의 직접적인 역할 외에도 MSCs가

강력한 항염증 및/또는 면역 억제 특성을 갖는다는 것이 발견되었습니다. 7

현재 척수 손상, 다발성 경화증, 알츠하이머 병, 간경변 및 간염, 골관절염, 심근경색, 신장 질환, 염증성 장 질환, 당뇨병, 무릎 연골 손상, 장기 이식, 이식편 대 숙주 질환(http://www.clinicaltrials.gov; 2014년 11월 접근) 등 다양한 질환에서 MSC를 재생 치료제로 활용하기 위한 광범위한 연구와 임상 시험이 진행 중입니다.

Paracrine Actions of MSCs

González et al.8 studied the contact-dependent mechanism of human adipose-derived MSCs in regulating inflammatory cytokines. In their study, they determined that human adipose-derived MSCs and macrophages both produce high levels of interleukin-10 (IL-10) only after cell-to-cell contact is maintained.8

Although potentially triggered by cell-to-cell contact events, the regenerative potential of MSC therapies has been found—at least in part—to be mediated via paracrine actions.9 For example, the paracrine effect of MSC-conditioned medium (CM) was observed to protect cardiomyocytes by interfering with the mitochondria-mediated apoptotic pathway. In this study, application of MSC-CM to cardiomyocytes exposed to hypoxia/reoxygenation reduced apoptosis through inhibition of the release of cytochrome C from mitochondria and reduction of caspase-3 activation.10 Similarly, renoprotective effects of human umbilical cord blood-derived MSCs (hUCB-MSCs) in streptozotocin-induced diabetic rats was reportedly mediated through paracrine action.11 In this case, the authors studied the effects of hUCB-MSC-CM on transforming growth factor (TGF)-β1-activated rat renal proximal tubular epithelia (NRK-52E) cells and observed attenuated expression‎ of TGF-β1, α-smooth muscle actin, collagen I, and heat shock protein-47 mRNA and increased expression‎ of E-cadherin and bone morphogenic protein-7 mRNA, thereby preventing diabetes kidney disease.11

Although it was initially believed that the potential of MSCs to differentiate into various cell types plays a crucial role in their therapeutic effects, the mechanism of action of transplanted MSCs does not predominantly include differentiating into a specific cell type for promoting or repairing the tissue damage in most disease settings.12,13,14 Several studies have demonstrated the predominance of short-lived paracrine mechanisms among the therapeutic actions of MSCs. In one such study, Toma et al.15 injected human MSCs (hMSCs) tagged with β-galactosidase into the left ventricle of immunodeficient mice. The majority of hMSCs were found in the spleen, lung, and liver, 4 days after injection. They also reported that only 0.44% of the injected hMSCs survived and, with time, they were morphologically indistinguishable from the surrounding cardiomyocytes. Other studies on systemically administered MSCs have also reported that <1% of the administered cells survive for more than 1 week and that the benefits of MSC therapy could be attributed to their secreted factors.16,17,18

In acute kidney injury (AKI), the protective effect of MSC administration was not attributed to MSCs differentiating into a tubular or endothelial cell phenotype, but to enhanced regulation of anti-inflammatory and organ-protective mediators such as IL-10, basic fibroblast growth factor, TGF-α, and B-cell lymphoma 2 (Bcl-2), reflecting primarily the paracrine function of MSCs.19 Tögel et al.20 reported the paracrine nature of cytoprotection in the immediate vicinity of administered MSCs in AKI. The authors demonstrated the production of renotropic factors—hepatocyte growth factor, and insulin-like growth factor 1 —that are known to decrease apoptosis and stimulate proliferation of renal epithelial cells.

Although these studies, and many others, provide strong evidence for the potency of MSC-secreted factors in mediating tissue repair and regeneration, the precise mechanisms by which MSCs act in a paracrine fashion are not fully understood. In addition to secreting an array of soluble factors, it has also been recognized that MSCs release large numbers of extracellular vesicles (EVs). Thus, it is of interest to consider the possibilities that the complex paracrine regenerative actions of exogenously administered MSCs and other stem cells communicate by transferring information and regulatory genes mediated, to some degree, by released EVs9,21,22 and that EVs derived from cultured MSCs have the potential to constitute a safe, effective cell-free therapy.

MSC의 파라크라인 작용

González et al.8은

인간 지방 유래 MSC가

염증성 사이토카인의 조절에 미치는 접촉 의존적 메커니즘을 연구했습니다.

그들의 연구에서,

인간 지방 유래 MSC와 대식세포는

세포 간 접촉이 유지될 때만 인터루킨-10(IL-10)을 높은 수준으로 생성한다는 것을 확인했습니다.8

세포 간 접촉 사건에 의해 유발될 수 있지만,

MSC 치료의 재생 잠재력은 적어도 일부는 파라크라인 작용을 통해 매개된다는 것이 밝혀졌습니다.9

예를 들어, MSC 조건화 매체(CM)의 파라크라인 효과는

미토콘드리아 매개 세포 사멸 경로를 방해함으로써

심근 세포를 보호하는 것으로 관찰되었습니다.

이 연구에서

저산소/재산소화 조건에 노출된 심근 세포에 MSC-CM을 적용했을 때,

미토콘드리아에서 사이토크롬 C의 방출을 억제하고

카스파제-3 활성화를 감소시켜 세포 사멸이 감소되었습니다.10

유사하게,

스트렙토조토신으로 유도된 당뇨병 쥐에서 인간 제대혈 유래 MSC(hUCB-MSC)의 신장 보호 효과는

파라크린 작용을 통해 매개되었다고 보고되었습니다.11

이 경우, 저자들은 hUCB-MSC-CM이 변형 성장 인자(TGF)-β1로 활성화된 쥐 신장 근위 세뇨관 상피세포(NRK-52E)에 미치는 영향을 연구했으며, TGF-β1, α-평활근 액틴, 콜라겐 I, 열 충격 단백질-47 mRNA의 발현이 감소하고 E-카데린과 뼈 형성 단백질-7 mRNA의 발현이 증가하여 당뇨병 신장 질환을 예방하는 것을 관찰했습니다.11

초기에는

MSC의 다양한 세포 유형으로 분화하는 잠재력이 치료 효과에 중요한 역할을 한다고 믿어졌지만,

이식된 MSC의 작용 메커니즘은

대부분의 질병 환경에서 조직 손상을 촉진하거나 복구하기 위해

특정 세포 유형으로 분화하는 것을 주된 메커니즘으로 포함하지 않습니다.12,13,14

여러 연구에서

MSC의 치료 작용에서 단기적인 파라크라인 메커니즘이 우세함을 보여주었습니다.

한 연구에서 Toma et al.15은

면역결핍 마우스의 좌심실에 β-갈락토시다아제 표지된 인간 MSC(hMSC)를 주입했습니다.

주입 후 4일 후, 대부분의 hMSC는 비장, 폐, 간에서 발견되었습니다.

또한 주입된 hMSCs의 0.44%만 생존했으며, 시간이 지나면서 주변 심근 세포와 형태학적으로 구분되지 않았다고 보고했습니다.

전신 투여된 MSC에 대한 다른 연구에서도 투여된 세포의 1% 미만이 1주 이상 생존했으며,

MSC 치료의 효과는 분비된 인자에 기인할 수 있다고 보고되었습니다.16,17,18

급성 신장 손상(AKI)에서 MSC 투여의 보호 효과는

MSC가 관상 세포나 내피 세포로 분화하는 것보다는

IL-10, 기본 섬유아세포 성장 인자, TGF-α, B-세포 림프종 2(Bcl-2)와 같은

항염증 및 장기 보호 매개체의 조절 강화에 기인하며,

이는 주로 MSC의 파라크린 기능을 반영합니다. 19

Tögel et al.20은 AKI에서 투여된 MSC의 즉시 주변에서 세포 보호의 파라크라인 성질을 보고했습니다.

저자들은 신장 상피 세포의 사멸을 감소시키고 증식을 자극하는 것으로 알려진

신장 특이적 인자—간세포 성장 인자 및 인슐린 유사 성장 인자 1—의 생산을

입증했습니다.

이러한 연구들과 많은 다른 연구들은

MSC가 분비하는 인자가 조직 수복 및 재생에 미치는 강력한 효과를 입증하지만,

MSC가 파라크린 방식으로 작용하는 정확한 메커니즘은 완전히 이해되지 않았습니다.

MSC는

다양한 용해성 인자를 분비하는 것 외에도

대량의 세포외 소포(EVs)를 방출한다는 점이 인정되고 있습니다.

따라서,

외부에서 투여된 MSC 및 기타 줄기세포의 복잡한 파라크라인 재생 작용이

분비된 EVs를 통해 정보와 조절 유전자를 전달함으로써

일부 정도 매개되는 가능성을 고려하는 것이 흥미롭습니다.9,21,22

또한 배양된 MSC에서 유래한 EVs는

안전하고 효과적인 세포 무함유 치료법으로 활용될 잠재력을 가지고 있습니다.

Extracellular Vesicles

EVs were first clearly described by Pan and Johnstone in 1983.23 Initially, the release of EVs was thought to represent a disposal mechanism by which cells eliminate unwanted proteins and other molecules. After years of subsequent research, however, EV release has emerged as an important mediator of cell-to-cell communication that is not only involved in normal physiological process but also plays a role in the development and progression of diseases. Among the subtypes of EV, the most numerous, referred to as exosomes, have a diameter of 40–100 nm, can be isolated by centrifugation at 100,000 ×g and can be concentrated at the interface of 0.8 and 2.7M sucrose layers. Preparations of EVs, typically a mixture of exosomes and other subtypes, can be isolated from all types of body fluids including blood, urine, bronchoalveolar lavage fluid, breast milk, amniotic fluid, synovial fluid, pleural effusions, and ascites.24 EVs can also be isolated from culture supernatants of many cell types, including T-cells, B-cells, dendritic cells, platelets, mast cells, epithelial cells, endothelial cells, neuronal cells, cancerous cells, and, as we describe in detail later, MSCs.25,26,27,28,29,30,31,32,33,34,35,36,37

Biogenesis of EVs

The modes of biogenesis for exosomes and microvesicles (MVs) are completely distinct and are described in this section.

Exosome biogenesis

Although the term “exosome” has been frequently used to describe all vesicles released by cells into the extracellular milieu, it is now known that there are multiple different types of EV. The major EV subtypes that are currently recognized are listed along with their basic characteristics in Table 1. Because of lack of specific markers it is very difficult to distinguish between different subtypes of vesicles within mixed preparations as they have overlapping composition, density, and size. Therefore, the International Society for Extracellular Vesicles suggested that the term EVs be used preferentially to describe preparations of vesicles from body fluids and cell cultures.38

세포외 소포

EV는 1983년 Pan과 Johnstone에 의해 처음으로 명확히 기술되었습니다.23

초기에는 EV의 방출이

세포가 불필요한 단백질 및 기타 분자를 제거하는 배출 메커니즘으로 여겨졌습니다.

그러나 후속 연구를 통해 EV 방출은

정상적인 생리적 과정에 관여할 뿐만 아니라

질병의 발생과 진행에도 역할을 하는 세포 간 통신을 매개하는 중요한 요소로 부상했습니다.

EV의 하위 유형 중 가장 많은 수를 차지하는 엑소좀은

지름 40–100nm를 가지며,

100,000 ×g 속도로 원심분리하여 분리할 수 있으며,

0.8M과 2.7M 설탕층의 경계면에서 농축될 수 있습니다.

EV의 준비물은 일반적으로 엑소좀과 다른 하위 유형의 혼합물로,

혈액, 소변, 기관지 알베올라 세척액, 모유, 양수, 관절액, 흉막 삼출액, 복수 등

모든 유형의 체액에서 분리될 수 있습니다. 24

엑소좀은

T세포, B세포, ден드리틱 세포, 혈소판, 마스트 세포, 상피 세포, 내피 세포, 신경 세포, 암세포, 그리고

후술할 MSCs를 포함한 다양한 세포 유형의 배양 상청액에서도

분리될 수 있습니다.25,26,27,28,29,30,31,32,33,34,35,36,37

EV의 생합성

엑소좀과 미세소체(MV)의 생합성 메커니즘은 완전히 다르고, 이 섹션에서 설명됩니다.

엑소좀 생합성

세포가

세포외 환경으로 방출하는 모든 소체를 지칭하는 용어로 “엑소좀”이 자주 사용되어 왔지만,

현재는 다양한 유형의 EV가 존재한다는 것이 알려져 있습니다.

현재 인정되는 주요 EV 하위 유형은

기본 특성과 함께 표 1에 정리되어 있습니다.

특이적인 표지자가 부족하기 때문에 혼합된 시료 내에서

다양한 소포의 하위 유형을 구분하는 것은 매우 어렵습니다.

이는 구성, 밀도, 크기가 중첩되기 때문입니다.

따라서

세포외 소포 국제 학회는 체액 및 세포 배양에서 유래한 소포 시료를 설명할 때

“EVs”라는 용어를 우선적으로 사용하도록 제안했습니다.38

VesiclesSize (diameter)Sucrose gradientOrigin

Exosomes	40-100 nm	1.13-1.19g/ml	Luminal budding into MVBs; release by fusion of MVB with cell membrane
Microvesicles	50-1,000 nm	1.04-1.07 g/ml	Outward budding of cell membrane
Apoptotic bodies	1-5,000 nm	1.16 and 1.28 g/ml	Outward blebbing of apoptotic cell membrane

Table 1

Different types of vesicles derived from various fluids and CM

MVB, multivesicular body.

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Exosomes are EVs of endosomal origin. The endosomal sorting complex required for transport and its associated proteins are involved in the formation of multivesicular bodies (MVBs) and intraluminal vesicles (ILV).39 Exosome membranes are enriched in lipids such as cholesterol, ceramide, and sphingolipids that are involved in the budding of ILVs into MVBs.40,41 As was first described during reticulocyte differentiation, ILVs are released from cells as a consequence of MVB fusion with the plasma membrane and, once released, are then termed as exosomes.23,42 Tan et al.41 further confirmed the endosomal origin of MSC-derived exosomes by detecting the components of lipid rafts. Table 2 provides additional details about proteins involved in MVB and exosome biogenesis. Exosomes may subsequently be internalized by other cells via direct membrane fusion, endocytosis or cell-type specific phagocytosis.43,44,45 Figure 1 illustrates the intracellular sources, release and uptake mechanisms associated with exosomes and other major subtypes of EV.

Figure viewer

Figure 1 EVs origin and internalization. Origin of EVs are generally via (a) endocytosis or inward budding of plasma membrane that consist of lipid rafts and is mediated by clathrin-dependent or caveolae-dependent pathway, This gives rise to (b) early endosomes leading to the formation of numerous ILVs within a membrane maturing to MVBs. Finally MVBs fuse with plasma membrane releasing ILVs as exosomes. (c) Ectosomes are vesicles shed from the cell surface and (d) apoptotic bodies are also known as apobodies and are released by cells undergoing apoptosis. EVs are internalized by the target cells through several pathways including (e) endocytosis, (f) fusion, and (g) phagocytosis.

그림 1 EVs의 기원 및 내화 과정. EVs의 기원은 일반적으로 (a) 지질 랠프를 포함하는 세포막의 내막화 또는 내향성 돌출을 통해 발생하며, 이는 클라트린 의존적 또는 카베올라 의존적 경로를 통해 매개됩니다. 이 과정은 (b) 초기 엔도좀을 형성하며, 이는 막 성숙을 통해 다수의 ILVs로 발전하여 MVBs를 형성합니다. 최종적으로 MVBs는 세포막과 융합되어 ILVs를 엑소좀으로 방출합니다. (c) 엑소좀은 세포 표면에서 분비되는 소포이며, (d) 아포토시스 바디는 아포토시스 과정을 겪는 세포에서 방출되는 구조물로 아포바디라고도 불립니다. EVs는 표적 세포에 의해 내재화되며, 이 과정은 (e) 내포작용, (f) 융합, 및 (g) 식작용을 포함한 여러 경로를 통해 이루어집니다.

FunctionProteinsReferences

MVB biogenesis	ESCRT-0, -I, -II, and -III; Vps4, VTA1, ALIX, Tsg101, CHMP4, ARF6, clathrin, and PLD2	127–137
Exosome Cargo	Vps4, Vps27, Tsg101, ALIX, HRS, Hsc70, Hsp90, 14-3-3 epsilon, and PKM2	39,138–141
MVE docking	RAB27a, RAB35	142,143
Exosome trafficking	RAB2B, RAB9A, RAB5A, RAB27B, syndecan, syntenin, ALIX, RAP1B, RHO	58,144–146
Exosome Release	Slp4, Slac2b, DGKα kinase, TfR, VAMP7, VAMP3, PLD2	144,147–151
Fusion of MVBs	SNAP receptors (SNAREs; v-SNAREs, t-SNAREs)	152–154

Table 2

Proteins associated with exosome biogenesis

ALIX, ALG-2-interacting protein X; ARF6, ADP-ribosylation factor 6;

CHMP4: charged multivesicular body protein 4; DGKα, diacylglycerol kinase a; ESCRT, endosomal sorting complex required for transport; HRS, hepatocyte growth factor-regulated tyrosine kinase substrate; Hsc70, heat shock cognate 70 kDa protein; Hsp90, heat-shock proteins; MVB, multivesicular body; MVE, multivesicular endosomes; PLD2, phospholipase D2; PKM2, pyruvate kinase M2; RAB27a, ras-related protein Rab-27A; RAP1B, Ras-related protein Rap-1B; RHO, rhodopsin; SNAREs, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; Slac2b, synaptotagmin-like homolog lacking C2 domains b; Slp4, synaptotagmin-like protein 4; t-SNAREs, target SNAREs; TfR, transferrin receptor; Tsg101, tumor susceptibility gene 101; Vps4, vacuolar protein sorting 4; VTA1, vesicle (multivesicular body) trafficking 1; VAMP7, vesicle-associated membrane protein 7; v-SNAREs, vesicular SNAREs.

Microvesicle biogenesis

MVs result from outward budding and fission of plasma membrane. Membrane budding initiated by the activity of aminophospholipid translocases to translocate phosphatidylserine to the outer membrane.46,47,48 ADP-ribosylation factor 6 plays an important role in enabling MV budding by stimulating phospholipase D activity, which in turn facilitates extracellular signal-regulated kinase activation.49,50 Contractile protein myosin light chain kinase 2 (which contracts cytoskeleton) is phosphorylated by extracellular signal-regulated kinase, which in turn stimulates serine phosphorylation of myosin II that ultimately triggers the release of MVs.46,50,51,52

Regulation of EV Biogenesis

Earlier literature has shown that MSCs release EVs differently depending on external stimulation suggesting that this process is likely to be regulated by cross-talk between MSCs and their surrounding microenvironment.53,54 For example, hypoxia or inflammatory conditioning of MSCs has been shown to regulate protein packaging into EVs and to affect their functional properties.53,54 Several pathways, which may be relevant to the microenvironment in which MSCs reside, have been reported to regulate biogenesis and secretion of EVs. Tumor suppressor-activated pathway 6 is found to regulate EV formation55 and is transcriptionally regulated by p53 thereby enhancing EV production.56,57 An alternative cross-talk pathway was suggested by Baietti et al.58 who described that syndecans interact with syntenin to regulate intraluminal budding of endosomal membrane domains containing CD63 and ALIX.

미세소포 생합성

미세소포(MVs)는

세포막의 외부로 돌출된 부위에서 분열을 통해 형성됩니다.

세포막 돌출은

아미노인산지질 트랜스포터의 활성에 의해

인산세린을 외막으로 이동시키는 과정에서 시작됩니다.46,47,48

ADP-리보실화 인자 6은 인산리파아제 D 활성을 자극하여

MV 돌출을 가능하게 하며,

이는 다시 세포외 신호 조절 키나아제 활성화를 촉진합니다. 49,50

수축 단백질 마이오신 경쇄 키나제 2(세포 골격을 수축시키는 단백질)는

세포외 신호 조절 키나제에 의해 인산화되며,

이는 다시 마이오신 II의 세린 인산화를 자극하여 최종적으로 MV의 방출을 유발합니다.46,50,51,52

EV 생합성의 조절

이전 연구들은 MSC가

외부 자극에 따라 EV를 다르게 분비한다는 것을 보여주었으며,

이는 이 과정이 MSC와 주변 미세환경 간의 교신에 의해 조절될 가능성이 높음을 시사합니다. 53,54

예를 들어,

MSC의 저산소 또는 염증 조건은

EV로의 단백질 포장 및 기능적 특성에 영향을 미치는 것으로 보고되었습니다.53,54

MSC가 존재하는 미세환경과 관련될 수 있는 여러 경로가 EV의 생성과 분비를 조절하는 것으로 보고되었습니다.

종양 억제제 활성화 경로 6은

EV 형성을 조절하는 것으로 밝혀졌으며,55

p53에 의해 전사적으로 조절되어 EV 생산을 증가시킵니다.56,57

Baietti et al.58은 syndecans가 syntenin과 상호작용하여

CD63과 ALIX를 포함하는 내막막 도메인의 내막 분열을 조절한다고 제안했습니다.

Therapeutic Effects of MSC-Derived EVs (MSC-EVs)

As described earlier, EVs facilitate cell-to-cell communication via the transfer of functionally relevant biomolecules59,60 (see Table 3) and thus, may be harnessed for therapeutic purposes in a similar fashion to their parent cells. From a translational perspective, EVs derived from MSCs have shown encouraging therapeutic effects in various animal models (see Figure 2), and their isolation from MSCs is potentially sustainable and reproducible. Furthermore, in comparison to whole cell-based therapies, MSC-EVs may offer specific advantages for patient safety such as lower propensity to trigger innate and adaptive immune responses61 and inability to directly form tumors. For example, it has been shown that MSC-derived EVs induced anti-inflammatory cytokines as well as triggering apoptosis in activated T-cells.62 MSC-EVs also carry mRNAs encoding immunoregulatory mediators including cytokine receptor-like factor 1, interleukin 1 receptor, and metallothionein 1X.63

MSC 유래 엑소좀(MSC-EVs)의 치료 효과

앞서 설명된 바와 같이,

엑소좀은

기능적으로 중요한 생체 분자의 전달을 통해 세포 간 통신을 촉진합니다59,60 (표 3 참조)이며,

따라서

모세포와 유사한 방식으로 치료 목적으로 활용될 수 있습니다.

전임상 연구 측면에서 MSC에서 유래한 EVs는

다양한 동물 모델에서 유망한 치료 효과를 보여주었습니다(그림 2 참조),

그리고

MSC로부터의 분리 과정은

잠재적으로 지속 가능하고 재현 가능합니다.

또한 전체 세포 기반 치료법과 비교할 때 MSC-EVs는

선천적 및 적응성 면역 반응을 유발할 가능성이 낮고 종양을 직접 형성하지 않는 등

환자 안전성에 대한 특정 이점을 제공할 수 있습니다.

예를 들어,

MSC에서 유래한 EVs는

활성화된 T 세포에서 항염증성 사이토카인을 유도하고

아포토시를 유발하는 것으로 나타났습니다.62

MSC-EVs는 또한

사이토카인 수용체 유사 인자 1, 인터루킨 1 수용체, 메탈로티오닌 1X와 같은

면역 조절 매개체를编码하는 mRNA를 운반합니다.63

Figure viewer

Figure 2 Potential clinical applications of EVs. Therapeutic benefits and mechanisms of action of MSC-derived EVs in: (a) various heart conditions, (b) kidney injury, (c) liver injury, (d) lung injury, and (e) wound healing.

Source of exosomesProtein content

Endosome-associated proteins	Rab GTPase, SNAREs, Annexins, flotillin, ALIX, Tsg101
Membrane proteins	CD63, CD81, CD82, CD53, and CD37
Lipid raft protein	Glycosylphosphatidylinositol-anchored proteins and flotillin
RNA	Structural RNAs, tRNA fragments, vault RNA, Y RNA, and small interfering RNAs

Table 3

Molecular composition of EVs

EV, extracellular vesicle; SNAREs, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; Tsg101, tumor susceptibility gene 101.

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In the remaining sections and in Table 4, we examine the evidence to-date for beneficial effects of MSC-EVs in several important disease areas and discuss some of the future needs and challenges that may be of critical importance to their successful clinical translation.

ConditionsModel/cause of injuryOrigin of EVs/mode of administrationAmount (volume)Therapeutic capacityReference

Myocardial Infarction	Mice/heart excision and aortic root canulation	MSC/intravenously	20 μl unfractionated MSC-CM(10-220 nm), <1,000 kDa fraction(10-100 nm), >1,000-kDa fraction, or saline	>1,000 kDa fraction1. Confer cardio-protection 2. ↓Infarct size	64
MyocardialIR injury	Mouse Langendorff heart model/heart excision, aortic root canulation, and perfusion	Human ESC-derivedMSC/intravenously	0.4 μg of F1 fraction protein; 3 μgCM protein	F1 fraction + CM protein1. ↓Infarct size	32
MyocardialInfarction	C57Bl6/J mice/ temporary left coronary artery ligation	MSC/intravenously via the tail vein	0.4 μg/ml	MSC exosomes1. ↓Infarct size by 45% 2. Prevents left ventricular dilatation 3. Improves cardiac performance 4. ↓Inflammation	65
Acute myocardial infarction	Wistar rats/permanent ligation of the left anterior descending coronary artery	Human BM MSCs / intramyocardial injection	MSCs (2 × 106 cells); MSC-EVs (80 μg)	MSC-Evs1. ↑ Proliferation, migration, and tube formation of HUVECs 2. ↓Infarct size 3. Improved cardiac function 4. Angiogenesis	67
AKI	Sprague-Dawley rats/ bilateral renal ischemia	hUCB-MSC/left carotid artery	MVs dissolved in 0.5 ml PBS; control MV; IFNγtreated MV	MSC-MVs1. ↑ Formation of T-cells with Treg phenotype 2. Ameliorated kidney dysfunction and acute tubular necrosis	53
Renal injury	C57BL6/J mice/5/6 subtotal Nx	Mouse MSC/injected through caudal veins	Nx + MSC group, 1 × 106/mouse, second day of surgery; Nx + MV group, 30 μg MV/mouse, day 2, 3, 5 after surgery	Nx + MSC and Nx + MV1. Ameliorated renal injury 2. Prevent renal fibrosis 3. Preserved the remnant renal function	76
Chronic kidney disease	Sprague-Dawley rats/ IR injury	BM-MSCs; human fibroblasts/ intravenously	30 μg	MSC-MVs1. ↓Apoptosis tubular cells 2. ↑ Tubular cell proliferation 3. Protect against chronic kidney disease 4. ↓Accumulation of matrix in the glomeruli	77
AKI	SCID mice/rhabdomyolisis- induced AKI	Human BM-MSCs / intravenous injection into the tail vein	15 μg of MSC-MVs; 15 μg human fibroblasts-MVs; 75,000 BM-MSCs in 150 μl saline	MSC-MV1. ↑In vitro proliferation 2. ↑In vitro apoptosis resistance 3. ↑ Morphologic recovery of AKI in vivo 4. MVs accumulated within the lumen of injured tubules	63
AKI	SCID mice/cisplatin	BM-MSCs/tail vein	Single injection—100 μg; Multiple injection—50 μg (days 2, 6, 10, 14, and 18)	MSC-MVs1. ↓Mortality induced by cisplatin 2. Improved renal function 3. Inhibited apoptosis induced by cisplatin in vitro	78
AKI	CD1 nude mice/intramuscular injection of glycerol	BM-MSCs/intravenously	200 μg	MSC-EVs accumulate specifically in kidneys	80
Liver injury	C57BL/six mice/carbon tetrachloride (CCl4)	MSCs/intrasplenic injection	0.4 μg (100 μl PBS)	MSC-EV1. Reverse CCl4-induced injury 2. ↑Proliferation of hepatocytes 3. Up-regulated cell-proliferation markers 4. Induced hepatocyte-regenerative genes expression‎ in liver tissue after CCl4-induced injury	85
Liver injury	Mice/CCl4	hucMSCs/injected into livers	250 μg (330 μl PBS)	hucMSC-Ex1. CCl4 -induced liver fibrosissignificantly alleviated 2. Inhibit epithelial-to- mesenchymal transition 3. Ameliorate CCl -induced liver4 fibrosis	84
ALI	C57BL/six mice/endotoxin from E. coli	hMSCs/intravenously, external jugular vein or intratracheal	30 μl of MVs released by 1.5 × 106 serum starved MSCs; 750,000 MSCs	MSC-MVs1. ↓Influx of inflammatory cells 2. ↓Edema 3. Transfer of KGF mRNA	86
ALI	HPH mouse/HPH	hWJMSC/jugular vein, tail vein	0.1 and 10 μg	Exosome treatment1. Suppress hypoxic inflammation 2. Inhibits lung vascular remodeling 3. Prevents hypoxic pulmonary hypertension	90
Skin deep second- degree burn wound	Sprague-Dawley rats/injured with 80oC water for 8 seconds to create 16 mm diameter wound	hucMSC/subcutaneous	200 μg exosome (200 μl PBS); 1 × 106 cells (hucMSC and HFL1)	Exosome treatment1. Cell proliferation 2. ↑Re-epithelialization 3. Inhibits heat stress-induced apoptosis in vitro 4. Prompt wound healing	91
Multiple myeloma (MM)	SCID mice/N/A	BM-MSCs (healthy subjects, relapsed/refractory MM patients/implanted subcutaneously)	3 × 106 cells/tissue-engineered bones; 1 μg exosomes	MM BM-MSC-derived exosomes1. ↑MM cell growth in vitro 2. ↑Tumor growth in vivo 3. ↑BM homing	109
Angiogenesis, tumor growth	BALB/c nu/nu mice /N/A	Human BM-MSC, human lung fibroblast/ subcutaneous injections	SGC-7901 cells alone (1 × 106); SGC-7901 cells (1 × 106) mixed with MSCs (1 × 106); SGC-7901 cells (1 × 106) mixed with MSC exosomes (200 μg/ ml)	SGC-7901 cells mixed with exosomes1. ↑Tumor growth 2. ↑Proliferation of tumor cells invivo 3. ↑Tumor angiogenesis	110
Angiogenesis	BALB/c mice	Mouse BM-derived MSCs/subcutaneous injections	100 μg (100 μl PBS); 2 × 105 4T1 cells mixed with 100 μg of MSC- derived exosomes or 2 × 105 4T1 cellsmixed with 200 μg of MSC-derived exosomes	MSC-derived EVs1. ↓VEGF expression‎ in 4T1 cells 2. ↓Angiogenesis in vitro and in vivo 3. ↓Tumor growth in vivo	111
Bladder tumor growth	BALB/c nu/nu mice	hWJMSC/subcutaneous injection	1 × 107 T24 cells; 1 × 107 T24 cells mixed with 1 × 107 hWJMSCs; 1 × 107 T24 cells mixed with 200 μg protein hWJMSC-MVs; 200 μg protein hWJMSC-MVs.	hWJMSC-EVs + hWJMSCs1. ↓ Significantly tumor size 2. ↑ Apoptosis	92
Hepatoma growth	SCID mice	HLSCs/intratumor injection	100 μg of EVs (20 μl)	HLSC-derived EVs1. ↓Significantly tumor size 2. ↑Apoptosis	94
Breast cancer	CB-17/Icr-scid/scidJc1 mice	BM MSC	BM2 cells (20,000) treated with 3 μg of BM-MSC-derived EVs were then injected in mammary fat pad (100 μl injections of PBS containing 1 × 105 BM2 cells)	BM-MSC-derived EV-treated cells1. ↓Proliferation 2. ↓Tumor formation	96

Table 4

Information of MSC-derived EVs in different studies

Up arrow (↑) indicates increased and down arrow (↓) indicates decreased activity.

AKI, acute kidney injury; ALI, acute lung injury; BM, bone marrow; CM, conditioned medium; EV, extracellular vesicle; HLSCs, human adult live stem cells; HLSC, human adult liver stem cell; HPH, hypoxia-induced pulmonary hypertension; HUVEC, human umbilical vein endothelial cells; hWJMSC, human umbilical cord Wharton's jelly MSC; IR injury, ischemia/reperfusion injury; KGF, keratinocyte growth factor; MSC, mesenchymal stem (stromal) cell; MV, Microvesicle; Nx, nephrectomy; PBS, phosphate-buffered saline; SCID, severe combined immunodeficient.

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MSC-EVs in cardiovascular disease

The CM obtained from hMSCs was shown by Timmers et al.64 to have the potential to reduce myocardial infarct size by 60% in a porcine model of cardiac ischemia/reperfusion (IR) injury. In this same study, fractionation of the CM revealed that the cardio-protective effect was confined to the fraction containing products >1,000 kDa (100–220 nm). In a mouse model of myocardial infarction, Lai et al.32 then directly demonstrated that the active, cardio-protective component of MSC-derived CM is, in fact, the EVs. In this study, administration of purified MSC-EVs reduced infarct size by ~40%.

Subsequently, Arslan et al.65 reported reduced infarct size following a single intravenous injection of MSC-EVs which could be attributed to the fact that EVs are internalized by target cells at the infarct site via endocytosis or phagocytosis. To further prove that intact MSC-EVs were required for therapeutic benefit, these authors demonstrated that homogenized EVs failed to reduce infarct size.65

Other studies have explored mechanisms by which the number and proangiogenic effects of EVs released by MSCs can be enhanced.66 For example, in a study of placental MSCs, under hypoxic conditions, Salomon et al.54 observed 3.3- and 6.7-fold increases in EV release in the presence of 1% and 3% O2 when compared with placental MSCs maintained at 8% O2. The resulting placental MSCs-derived EVs induced a significant, dose-dependent increase in tube formation by placental microvascular endothelial cells when compared with vehicle-treated cells.54 It was speculated that the increased proangiogenic effect of MSC-EVs derived under hypoxic conditions may be conferred by transcriptional activities of the hypoxia inducible factor family of proteins.54

Following on from the above result, Bian et al.67 isolated EVs from MSCs cultured under hypoxic conditions. In an in vitro angiogenesis assay, MSC-EVs at a concentration of 80 μg/ml, promoted human umbilical vein endothelial cell migration and tube formation that was comparable to that induced by vascular endothelial growth factor (VEGF). In vivo studies confirmed that intramyocardial injection of hypoxia-conditioned MSC-EVs significantly improved cardiac function and reduced myocardial infarct size with similar potency to that observed in a whole-cell MSC-treated group.67

Micro-RNAs associated with MSC-EVs also play an important role in cardio-protection. For instance, it was found that cardiac remodeling following myocardial infarction is regulated by miR-22-loaded EVs via targeting of methyl CpG binding protein 2.68 Similarly, the level of miR-221 is significantly higher in MSC-EVs when compared with their parent MSCs, and this miRNA was shown to enhance cardio-protection by reducing the expression‎ of p53 upregulated modulator of apoptosis.69

MSC-유래 엑소좀(MSC-EVs)과 심혈관 질환

Timmers et al.64는

인간 MSC에서 추출한 세포외기질(CM)이

심근 허혈/재관류(IR) 손상 모델을 가진 돼지 모델에서 심근 경색 크기를 60% 감소시킬 수 있음을 보여주었습니다.

이 연구에서 CM의 분획화는 심장 보호 효과가 1,000 kDa(100–220 nm) 이상의 분획에 국한됨을 밝혔습니다. 마우스 심근경색 모델에서 Lai et al.32는 MSC 유래 CM의 활성 심장 보호 성분이 실제로 EVs임을 직접 입증했습니다. 이 연구에서 정제된 MSC-EVs 투여는 심근경색 크기를 약 40% 감소시켰습니다.

이후 Arslan et al.65는 MSC-EV의 단일 정맥 주사 후 심근 경색 크기가 감소했으며, 이는 EVs가 심근 경색 부위의 표적 세포에 내포작용 또는 식작용을 통해 내재화되기 때문일 수 있다고 보고했습니다.

치료 효과를 위해 완전한 MSC-EV가 필요함을 추가로 입증하기 위해,

이 연구자들은 균질화된 EVs가 심근 경색 크기를 감소시키지 못함을 보여주었습니다.65

다른 연구에서는 MSC에서 방출되는 EV의 수와 혈관新生 효과를 증강시키는 메커니즘을 탐구했습니다.66 예를 들어, 태반 MSC를 대상으로 한 연구에서 저산소 조건 하에서 Salomon et al.54는 1% 및 3% O2 조건에서 태반 MSC를 8% O2 조건에서 유지한 경우와 비교해 EV 방출량이 각각 3.3배 및 6.7배 증가함을 관찰했습니다.

결과적으로 태반 MSC에서 유래한 EVs는 대조군 세포와 비교해 태반 미세혈관 내피 세포의 관 형성 증가를 유의미하고 용량 의존적으로 유도했습니다.54 저자들은 저산소 조건 하에서 유도된 MSC-EVs의 증가된 혈관新生 촉진 효과가 저산소 유도 인자 단백질 가족의 전사 활성에 의해 매개될 수 있다고 추측했습니다.54

위 결과에 이어 Bian et al.67은 저산소 조건에서 배양된 MSC에서 EVs를 분리했습니다. in vitro 혈관新生 실험에서 80 μg/ml 농도의 MSC-EVs는 인간 태반 정맥 내피 세포의 이동 및 관 형성을 촉진했으며, 이는 혈관 내피 성장 인자(VEGF)에 의해 유도된 것과 유사한 수준이었습니다. In vivo 연구에서 저산소 조건에서 처리된 MSC-EV의 심근 내 주사는 심장 기능 개선과 심근 경색 크기 감소 효과가 전체 세포 MSC 처리 그룹과 유사한 효능을 보였습니다.67

MSC-EV와 관련된 마이크로RNA도 심장 보호에 중요한 역할을 합니다. 예를 들어, 심근경색 후 심장 구조 재편은 miR-22를 함유한 엑소좀이 메틸 CpG 결합 단백질 2를 표적화함으로써 조절된다는 것이 밝혀졌습니다.68 마찬가지로, MSC-EVs의 miR-221 수준은 모세포 MSC에 비해 유의미하게 높았으며, 이 마이크로RNA는 p53에 의해 조절되는 세포 사멸 조절 인자의 발현을 감소시켜 심장 보호 효과를 강화하는 것으로 나타났습니다.69

MSC-EVs in AKI

AKI is a major cause of morbidity and mortality among hospitalized patients and is most commonly caused by IR injury, exposure to nephrotoxic compounds, and severe volume loss or obstruction to urine flow.70 It has been well established in animal models of renal IR and other forms of kidney injury that systemic or localized administration of MSCs results in amelioration of AKI.71,72,73 MSCs downregulate proinflammatory cytokines in T-cells and consequently induce regulatory T-cells (T-regs) in the spleen.71 Anti-inflammatory and immunoregulatory properties of MSCs have become one of the important mechanistic approaches to the treatment of AKI. A broad range of growth factors, cytokines, and chemokines secreted from MSCs have been identified including hepatocyte growth factor, insulin-like growth factor 1, VEGF, IL-1, IL-4, IL-5, IL-6, keratinocyte-derived chemokine, chemokine (C-X-C motif) ligand 16 (CXCL16), chemokine (C–C motif) ligand 2 (CCL2), CCL3, chemokine (C-X3-C motif) ligand 1 (CX3CL1), and CCL5.20,74 In experimental models, mediators such as these have been associated with enhanced cell proliferation and reduced cell apoptosis, identifying MSCs as uniquely providing multimodal therapeutic effects in AKI.75

Similar to MSCs, MSC-EVs are capable of modulating T-cell as well as innate immune cell functions.53 To date, there are few reported studies that directly compare the effect of MSCs and MSC-EVs in the setting of AKI. However, in a study involving mouse 5/6 subtotal nephrectomy (Nx)—a model of chronic kidney disease–He et al.76 reported that both MSC- and MSC-EV-treated mice showed strikingly similar benefits including reduced fibrosis and interstitial lymphocyte infiltration and reduced or absent tubular atrophy when compared with the untreated control group.

In the rat model of renal IR, Gatti et al.77 found that intravenous injection of 30 μg of MSC-EVs prevented AKI. The administered EVs were shown to transiently accumulate within glomeruli and injured tubules in association with increased proliferation and reduced apoptosis of tubular epithelial cells.77 This study also reported that the protective effect was specific to MSC-EVs as fibroblast-EVs were ineffective. Similarly, Bruno et al.63 also reported that human BM-derived MSC-EVs accelerated renal morphologic and functional recovery in glycerol-induced AKI in immunodeficient mice by inducing proliferation of tubular cells. In this study, they also reported that the effect of MSC-EVs on the recovery of AKI was similar to that of hMSCs.63

The effects of human MSC-EVs were also studied in severe combined immunodeficient (SCID) mice with AKI induced by the chemotherapeutic agent cisplatin.78 In this study, MSC-EVs significantly improved the survival (40% at day 21) by improving renal function and morphology, but were unable to prevent chronic tubular injury (see Table 4). Multiple injections of MSC-EVs, however, further decreased mortality in association with normal histology and renal function.78 MSC-EVs were found to upregulate antiapoptotic genes, including B-cell lymphoma-extra large, Bcl-2 and baculoviral IAP repeat containing 8, and downregulating cell apoptosis genes including, Caspase-1 (Casp1), Caspase-8 (Casp8) and lymphotoxin α in cisplatin-treated human tubular epithelial cells.78 Renoprotection was also conferred by horizontal transfer of insulin-like growth factor-1 receptor via BM–MSC–EV.79

Grange et al.80 studied the biodistribution of intravenously injected MSC-EVs in an AKI mouse model. They observed the specific accumulation of EVs at the site of injury as compared to healthy mice receiving the same quantity of MSC-EVs.80 Overall, of the disease areas studied, AKI, caused by a variety of clinically relevant insults, represents one of the most convincing examples of a distinct therapeutic benefit of systemic MSC-EV injection.78,80,81

MSC-EVs와 급성 신부전 (AKI)

급성 신부전 (AKI)은 입원 환자에서 주요 사망 및 장애 원인 중 하나로, 주로 신장 허혈 손상, 신독성 물질 노출, 심각한 체액 손실 또는 요류 장애에 의해 발생합니다.70 신장 허혈 및 기타 신장 손상 동물 모델에서 MSC의 전신 또는 국소 투여가 AKI의 증상 완화에 효과적이라는 것이 잘 확립되어 있습니다. 71,72,73 MSC는 T세포에서 염증성 사이토카인을 억제하고 결과적으로 비장에서 조절 T세포(T-regs)를 유도합니다.71 MSC의 항염증 및 면역조절 특성은 AKI 치료의 중요한 기전적 접근법 중 하나로 부상했습니다. MSCs에서 분비되는 다양한 성장 인자, 사이토킨, 케모카인에는 간세포 성장 인자, 인슐린 유사 성장 인자 1, VEGF, IL-1, IL-4, IL-5, IL-6, 케라티노사이트 유래 케모카인, 케모카인(C-X-C 모티프) 리간드 16(CXCL16), 케모카인(C–C 모티프) 리간드 2(CCL2), CCL3, 케모카인(C-X3-C 모티프) 리간드 1(CX3CL1), 및 CCL5가 포함됩니다.20,74 실험 모델에서 이러한 매개체는 세포 증식 증가와 세포 사멸 감소와 연관되어, MSC가 AKI에서 다모달 치료 효과를 제공하는 독특한 역할을 한다는 것이 밝혀졌습니다.75

MSCs와 유사하게, MSC-EVs는 T세포 및 선천성 면역 세포의 기능을 조절할 수 있습니다.53 현재까지 AKI 환경에서 MSCs와 MSC-EVs의 효과를 직접 비교한 연구는 거의 없습니다. 그러나 쥐의 5/6 부분 신장 절제술(Nx) 모델—만성 신장 질환 모델—에서 He et al.76은 MSC 및 MSC-EV로 치료받은 쥐가 대조군과 비교해 섬유화 감소, 간질 림프구 침윤 감소, 관상 위축 감소 또는 소실 등 유사한 혜택을 보였다고 보고했습니다.

신장 허혈 재관류(IR) 쥐 모델에서 Gatti et al.77은 정맥 내 30 μg의 MSC-EV 투여가 AKI를 예방했다고 보고했습니다. 투여된 EVs는 사구체와 손상된 관상 세포 내에서 일시적으로 축적되며, 관상 상피 세포의 증식 증가와 사멸 감소와 연관되었습니다.77 이 연구는 보호 효과가 MSC-EV에 특이적임을 보여주었으며, 섬유모세포-EV는 효과적이지 않았습니다. Bruno et al.63도 면역결핍 마우스에서 글리세롤로 유발된 AKI에서 인간 골수 유래 MSC-EVs가 관상 세포 증식을 유도하여 신장 형태학적 및 기능적 회복을 가속화했다고 보고했습니다. 이 연구에서는 MSC-EVs의 AKI 회복 효과는 hMSCs와 유사하다고도 보고되었습니다.63

인간 MSC-EV의 효과는 화학요법제 시스플라틴으로 유발된 AKI를 가진 중증 복합 면역결핍(SCID) 마우스에서도 연구되었습니다.78 이 연구에서 MSC-EV는 신장 기능과 형태를 개선하여 생존율을 유의미하게 향상시켰지만(21일차에 40%), 만성 관상 손상을 예방하지는 못했습니다(표 4 참조). 그러나 MSC-EVs의 다중 투여는 정상적인 조직학 및 신장 기능과 연관되어 사망률을 추가로 감소시켰습니다. 78 MSC-EV는 시스플라틴 처리된 인간 관상 상피 세포에서 항아포토시스 유전자(B-세포 림프종-엑스트라 라지, Bcl-2 및 바쿨로바이러스 IAP 반복 포함 8)의 발현을 증가시키고, 아포토시스 유전자(카스파제-1(Casp1), 카스파제-8(Casp8) 및 림프톡신 α)의 발현을 감소시키는 것으로 확인되었습니다. 78 인슐린 유사 성장 인자-1 수용체의 수평 전달을 통해 신장 보호 효과가 BM–MSC–EV를 통해 부여되었습니다.79

Grange et al.80은 AKI 마우스 모델에서 정맥 내 주사된 MSC-EV의 생체 분포를 연구했습니다. 건강한 쥐와 동일한 양의 MSC-EV를 투여받은 경우와 비교해 손상 부위에서 EV의 특이적 축적이 관찰되었습니다.80 전체적으로, 연구된 질환 영역 중 다양한 임상적으로 관련 있는 손상으로 인한 AKI는 전신 MSC-EV 주사의 명확한 치료적 이점을 보여주는 가장 설득력 있는 사례 중 하나입니다.78,80,81

MSC-EVs in liver disease

MSCs have been shown to be of benefit in a range of acute and chronic liver disease models and clinical translation of this work is currently underway in a number of centers.82 For example, injection of MSCs into the portal vein has been reported to protect the liver in a rat model of hepatic IR injury after partial hepatectomy. In this study, MSC administration was shown to reduce hepatocyte apoptosis and enhance liver regeneration.83

Fewer studies have addressed the potential benefits of MSC-EVs in chronic liver disease models. In one such study, human umbilical cord-MSC (hucMSC)-EVs were shown to specifically localize to the liver and to alleviate liver fibrosis in carbon tetrachloride (CCl4)-induced injury by reducing hepatocyte apoptosis and hepatic lobule destruction.84 MSC-EV administration suppressed epithelial to mesenchymal transdifferentiation via reduced TGF-β1 expression‎ and Smad2 phosphorylation.84 Other in vivo studies have shown that MSC-EVs promote hepatocyte regeneration after CCl4-induced injury by inducing the IL-6/STAT3 pathway and cell cycle progression.85 In this case, the authors validated the direct hepatoprotective effects of MSC-EVs using the cell lines TAMH (an immortalized mouse hepatocyte line derived from transgenic MT42 male mice overexpressing TGF-α), THLE-2 (an immortalized primary human hepatocyte) and HuH-7 (a human hepatocarcinoma cell line) exposed in vitro to acetaminophen and hydrogen peroxide.85 Increased cytoprotection compared to control-treated cells was observed following treatment with 0.1 μg/ml MSC-EVs. Thus, both in vivo and in vitro studies have confirmed that MSC-EV therapy has the potential to promote liver regeneration following acute injury by directly enhancing hepatocyte survival and proliferation85 (see Table 4).

MSC-EVs와 간 질환

MSCs는 급성 및 만성 간 질환 모델에서 유익한 효과를 보였으며, 이 연구의 임상 적용은 현재 여러 기관에서 진행 중입니다.82 예를 들어, 부분적 간 절제술 후 간 허혈 손상 쥐 모델에서 문맥 정맥에 MSC를 주입한 것이 간 보호 효과를 보였다는 보고가 있습니다. 이 연구에서 MSC 투여는 간세포 사멸을 감소시키고 간 재생 능력을 향상시켰습니다.83

만성 간 질환 모델에서 MSC-EV의 잠재적 이점을 탐구한 연구는 상대적으로 적습니다. 한 연구에서 인간 제대혈 MSC (hucMSC)-EV는 간에 특이적으로 침투하여 탄소 테트라클로라이드 (CCl4)로 유발된 간 손상에서 간 섬유화를 완화시켰으며, 이는 간세포 사멸과 간 소엽 파괴를 감소시킴으로써 이루어졌습니다. 84 MSC-EV 투여는 TGF-β1 발현 감소와 Smad2 인산화 억제를 통해 상피-중간엽 전환을 억제했습니다.84 다른 in vivo 연구에서는 MSC-EV가 CCl4로 유발된 손상 후 간세포 재생을 촉진하기 위해 IL-6/STAT3 경로와 세포 주기 진행을 유도한다는 것이 밝혀졌습니다. 85 이 연구에서 저자들은 TGF-α를 과발현하는 전유전자 MT42 수컷 마우스에서 유래한 불멸화 마우스 간세포 라인 TAMH, 불멸화 인간 간세포 라인 THLE-2, 인간 간암 세포 라인 HuH-7을 아세트아미노펜과 과산화수소에 in vitro 노출시켜 MSC-EV의 직접적인 간 보호 효과를 검증했습니다. 85 MSC-EVs 0.1 μg/ml 처리 후 대조군 처리 세포에 비해 세포 보호 효과가 증가했습니다. 따라서 in vivo 및 in vitro 연구 모두 급성 손상 후 간 재생 촉진 잠재력을 확인했으며, 이는 간세포 생존 및 증식을 직접적으로 향상시킴으로써 달성되었습니다85 (표 4 참조).

MSC-EVs in lung diseases

Endotoxin-induced acute lung injury (ALI) in mice results in increased lung protein permeability causing an inflammatory response in the alveoli that is commonly used as a model of human ALI associated with severe pneumonia or sepsis. In this model, it has recently been shown by Zhu et al.86 that administration of MSC-EVs decreased the influx of total inflammatory cells into the lung by 36% and influx of neutrophils by 73%. The suppression of lung inflammation was accompanied by reduced protein permeability, thereby preventing the formation of pulmonary edema. From a mechanistic perspective, keratinocyte growth factor (KGF) has been shown to reduce lung edema and inflammation in various ALI models.87,88 Lee et al. 89 reported that hMSCs produced KGF and that its secretion as a paracrine soluble factor mediated the restoration of alveolar fluid clearance in vivo. Thus, Zhu et al. 86 hypothesized that MSC-EVs transfer KGF mRNA to the injured alveolar epithelium and to verify this, they transfected the MSCs with KGF-specific small interfering RNA before isolating EVs. In keeping with this mechanism, the therapeutic effect of EVs from KGF-depleted MSCs was reduced compared to that of control MSC-EVs.

In a mouse model of hypoxia-induced pulmonary hypertension, the injection of MSC-EVs resulted in a delayed pulmonary influx of macrophages and reduced production of proinflammatory mediators compared to injection of EVs-derived from mouse lung fibroblasts.90 MSC-EVs, upon low dose multiple administration, also ameliorated pulmonary hypertension via increasing the levels of miR-204,90 ventricular hypertrophy, and lung vascular remodelling.90 The authors further tested the efficacy of two sequential injections of a higher dose of MSC-EVs and observed similar beneficial effects on early and later outcomes.90 Finally, MSC-EVs have been found to suppress hypoxic activation of signal transducer and activator of transcription 3 (STAT3) by up-regulating miR-17.90

MSC-유래 엑소좀(MSC-EVs)과 폐 질환

엔도톡신에 의한 급성 폐 손상(ALI)은 쥐에서 폐 단백질 투과성을 증가시켜 폐포에서 염증 반응을 유발하며, 이는 중증 폐렴이나 패혈증과 관련된 인간 ALI의 모델로 널리 사용됩니다. 이 모델에서 Zhu et al.86은 MSC-EVs 투여가 폐로 유입되는 총 염증 세포 수를 36%, 중성구 유입을 73% 감소시켰다는 것을 최근에 보여주었습니다. 폐 염증 억제는 단백질 투과성 감소와 동반되어 폐부종 형성을 방지했습니다. 기전적 관점에서, 케라티노사이트 성장 인자(KGF)는 다양한 ALI 모델에서 폐 부종과 염증을 감소시키는 것으로 알려져 있습니다.87,88 Lee et al. 89는 hMSCs가 KGF를 생성하며, 이 인자가 파라크린 용해성 인자로 분비되어 in vivo에서 폐포 액체 제거 회복을 매개한다는 것을 보고했습니다. 따라서 Zhu et al. 86은 MSC-EV가 손상된 폐포 상피에 KGF mRNA를 전달한다고 가설을 세우고, 이를 검증하기 위해 MSC에 KGF 특이적 소형 간섭 RNA를 전사한 후 EV를 분리했습니다. 이 메커니즘과 일치하게, KGF가 제거된 MSC에서 유래한 EV의 치료 효과는 대조군 MSC-EV에 비해 감소했습니다.

저산소증으로 유발된 폐 고혈압 마우스 모델에서 MSC-EVs 주사는 마우스 폐 섬유아세포에서 유래한 EVs 주사에 비해 폐로의 대식세포 유입이 지연되고 염증 매개체 생산이 감소했습니다.90 MSC-EVs는 저용량 다회 투여 시 miR-204 수준 증가,90 심실 비대, 및 폐 혈관 재형성을 통해 폐 고혈압을 완화했습니다. 90 저자들은 MSC-EV의 고용량 두 차례 연속 투여의 효능을 추가로 테스트했으며, 초기 및 후기 결과에 대한 유사한 유익한 효과를 관찰했습니다.90 마지막으로, MSC-EV는 miR-17 발현을 증가시켜 저산소성 신호 전달 및 전사 활성화 인자 3(STAT3)의 활성화를 억제하는 것으로 확인되었습니다.90

MSC-EVs in cutaneous wound healing

In a recently reported study by Zhang et al. 91, the effects of locally injected hucMSC and hucMSC-EVs were studied in a rat deep second degree burn injury model. Using a range of histological and molecular indexes of healing, the authors found that injection of hucMSCs and hucMSC-EVs resulted in comparable and significant increase in re-epithelialization when compared with burn wounds that were treated with saline, human lung fibroblasts (HFL1) or HFL1-EVs. The epithelial healing effects were replicated in vitro in keratinocyte and dermal fibroblast cell lines in the form of increased cell proliferation and reduced apoptosis and were shown to be mediated by MSC-EV-delivered Wnt4 resulting in activation of β-catenin signaling and by activation of the AKT signaling pathway.91 Although additional studies are needed to confirm‎ these striking observations in other preclinical models, the results suggest that cutaneous injury and ulceration represent one of the most promising clinical translational avenues for MSC-EV preparations.

Antitumor Activity of MSC-EVs

MSCs have also been shown to have anticancer activities. Wu et al.92 demonstrated that human umbilical cord Wharton's jelly MSC (hWJMSC)-derived EVs reduce the growth of T24 bladder carcinoma cells in vitro and in vivo. The authors reported that incubation of T24 cells with various concentration of hWJMSC-EVs (0, 50, 100, 200 μg/ml protein) resulted in cell-cycle arrest and tumor cell apoptosis.92 Similarly, Bruno et al.93 reported inhibited cell-cycle progression and induced apoptosis in HepG2 (liver) and Kaposi's cells, and necrosis in Skov-3 (ovarian cell line) when treated with MSC-EVs.

In a study carried out using human adult liver stem cell (HLSC)-EVs, Fonsato et al.94 reported induction of apoptosis in HepG2 hepatoma and primary hepatocellular carcinoma cells. Significant reduction in tumor growth was also observed in the presence of MV-HLSC in SCID mice inoculated with primary hepatocellular carcinoma cells.94 The authors concluded that the antitumor effects of HLSC-EVs could be because of selective delivery of miRNAs—a mechanism that may also explain the potential antitumor effects of MSC-EVs in some settings.

MicroRNA-9 has been associated with drug resistance via increasing the expression‎ of P-glycoprotein.95 Munoz et al.95 reported that anti-miR-9-Cy5 was transferred from MSCs to glioblastoma multiforme cells via EVs, blocking the increase of P-glycoprotein and reversing the chemoresistance. Ono et al.96 reported that BM-MSC-EVs contributed to the dormant state of BM2 cells through EV-mediated transfer of miRNA.

MSC-EVs for Drug Delivery

EVs are natural transporters that may potentially reach a wide range of tissues following systemic administration, including the central nervous system as they have been reported to cross the blood–brain barrier.97 As EVs consist of a bilayered lipid membrane with an aqueous core they may potentially be loaded with both hydrophilic and lipophilic drugs.98 Furthermore, drugs could be either loaded into purified preparations of EVs99 or applied to parent cells and incorporated during EV biogenesis.100 Small molecules including siRNAs can also be loaded into the EVs either by electoporation or by chemical disruption.97,101 Although little explored to date, MSC-EVs may constitute a particularly promising vehicle for drug delivery given their inherent ability to exert disease-modulatory effects and the extensive literature documenting in vitro modification of MSCs using genetic and nongenetic approaches. As an example, Pascucci et al.102 observed that paclitaxel-treated MSCs mediated strong antitumorigenic effects because of their capacity to take up the drug and later release it in EVs. In this study, paclitaxel -treated MSC-EVs induced a dose-dependent inhibition of CFPAC-1 (human pancreatic adenocarcinoma) cell proliferation as well as 50% inhibition of tumor growth.

MSC-EVs를 활용한 약물 전달

EVs는

전신 투여 후 중추 신경계를 포함한 다양한 조직에 도달할 수 있는 자연적인 운반체로,

혈액-뇌 장벽을 통과할 수 있다는 보고가 있습니다.97

EVs는

이중층 지질 막과 수성 핵으로 구성되어 있어

친수성 및 친유성 약물을 모두 탑재할 수 있습니다.98

또한 약물은 정제된 EVs 준비물에 탑재되거나99

모세포에 적용되어 EVs 생합성 과정에서 통합될 수 있습니다. 100

siRNA를 포함한 소분자는

전기천공법이나 화학적 파괴를 통해 EV에 탑재될 수 있습니다.97,101

현재까지 연구가 부족하지만, MSC-EV는 질병 조절 효과를 발휘하는 내재적 능력과 유전적 및 비유전적 접근법을 통해 MSC를 in vitro에서 수정하는 광범위한 문헌 기록으로 인해 약물 전달을 위한 특히 유망한 운반체로 작용할 수 있습니다. 예를 들어, Pascucci et al.102는 파클리탁셀로 처리된 MSC가 약물을 흡수하고 나중에 EV를 통해 방출하는 능력으로 인해 강력한 항종양 효과를 나타냈다고 보고했습니다. 이 연구에서 파클리탁셀 처리된 MSC-EV는 CFPAC-1(인간 췌장 선암종) 세포 증식을 용량 의존적으로 억제했으며 종양 성장의 50% 억제를 유도했습니다.

Clinical Translation of MSC-EVs: Unresolved Issues and Future Priorities

Tumorigenesis and other potential adverse effects of MSC-EVs

Despite reported antitumor effects in some settings, there is also theoretical potential for whole cell MSC therapy to directly or indirectly induce cancerous tumors or to accelerate the progression of pre-existing cancers. Although this concern has not, thus far, been borne out in human clinical trials, subpopulations of MSC-like cells have been found in the tumor microenvironment of several human cancers including gastric adenocarcinoma103 and osteosarcoma.104 Furthermore, some animal model studies have demonstrated preferential migration of intravenously administered MSCs to tumors.105,106 Although EVs clearly lack the potential to directly form tumors following in vivo administration, this does not imply that MSC-EV administration to human subjects is without any risk of promoting neoplasia. For instance, multiple myeloma (MM) cell proliferation has been shown to be increased in the presence of either autocrine or paracrine secretory factors of BM-MSCs.107,108 Roccaro et al.109 isolated EVs from BM-MSCs derived from both MM patients and healthy controls. In this study, the MM BM-MSC-derived EVs were found to promote MM tumor/cell growth, whereas normal BM-MSC-derived EVs inhibited the growth of MM tumor/cells both in vitro and in vivo. The MM BM-MSC-derived EVs were also found to induce cell dissemination and metastasis to distant BM niches.109

MSC-EVs have been found to modulate the tumor microenvironment, creating a niche for cancer cell metastasis and have been proven to mimic the effects of MSCs to promote tumor growth. Zhu et al.110 showed that MSC-EVs co-implanted with SGC-7901 (human gastric cancer) cells increased tumor growth and angiogenesis when compared with SGC-7901 cells alone. However, Lee et al.111 reported contradictory results suggesting that MSC-EVs suppress angiogenesis in vitro by downregulating the mRNA and protein levels of VEGF in tumor cells in a concentration-dependent manner. They speculated that this inconsistency could be because of different tumor types or MSC heterogeneity.111

Intravascular infusion of MSCs has been documented to cause embolism and death in experimental animals,112 whereas MSCs inoculated into infarcted myocardium were reported to induce adverse cellular growth such as cardiac sympathetic nerve sprouting.113 For adverse effects such as these, it appears likely that the risk associated with MSC-EV administration will be significantly lower or perhaps absent. However, as evidence of striking efficacy in a variety of disease settings now exists, it is incumbent on the research community to carefully eval‎uate the short- and long-term safety of biologically active EVs. Based on this limited information, it is clear that successful translation of MSC-EVs as a clinical therapy will require a significant amount of additional preclinical investigation of the interaction between MSC-EVs and tumor cells.

MSC-EV의 임상적 적용: 미해결 문제와 미래 과제

MSC-EV의 종양 발생 및 기타 잠재적 부작용

일부 연구에서 항종양 효과가 보고되었음에도 불구하고, 전체 세포 MSC 치료가 직접적으로 또는 간접적으로 암 종양을 유발하거나 기존 암의 진행을 가속화할 수 있는 이론적 가능성이 존재합니다. 이 우려는 현재까지 인간 임상 시험에서 확인되지 않았지만, 위선암103 및 골육종 등 여러 인간 암의 종양 미세환경에서 MSC 유사 세포의 하위 집단이 발견되었습니다. 104 또한 일부 동물 모델 연구에서는 정맥 내 투여된 MSC가 종양으로의 선택적 이동을 보여주었습니다.105,106 그러나 EVs는 in vivo 투여 후 종양을 직접 형성할 잠재력이 없지만, 이는 인간 대상에 MSC-EV를 투여하는 것이 신생물 촉진 위험이 전혀 없다는 것을 의미하지 않습니다. 예를 들어, 골수성 다발성 골수종(MM) 세포의 증식이 골수 유래 MSC의 자가분비 또는旁분비 분비 인자의 존재 하에서 증가한다는 것이 밝혀졌습니다.107,108 Roccaro et al.109는 MM 환자 및 건강한 대조군에서 유래한 골수 유래 MSC로부터 EVs를 분리했습니다. 이 연구에서 MM BM-MSC 유래 EV는 MM 종양/세포의 성장을 촉진한 반면, 정상 BM-MSC 유래 EV는 in vitro 및 in vivo에서 MM 종양/세포의 성장을 억제했습니다. MM BM-MSC 유래 EV는 또한 세포 확산과 원격 골수 미세환경으로의 전이를 유도하는 것으로 확인되었습니다.109

MSC-EV는 종양 미세환경을 조절하여 암 세포 전이를 위한 미세환경을 형성하며, MSC의 효과를 모방하여 종양 성장을 촉진한다는 것이 입증되었습니다. Zhu et al.110은 SGC-7901(인간 위암) 세포와 함께 이식된 MSC-EV가 SGC-7901 세포 단독 대비 종양 성장과 혈관新生을 증가시켰다고 보고했습니다. 그러나 Lee et al.111은 MSC-EVs가 종양 세포의 VEGF mRNA 및 단백질 수준을 농도 의존적으로 감소시켜 in vitro에서 혈관新生을 억제한다는 상반된 결과를 보고했습니다. 그들은 이 불일치가 종양 유형이나 MSC 이질성 때문일 수 있다고 추측했습니다.111

MSC의 혈관 내 투여는 실험 동물에서 색전증과 사망을 유발한 것으로 보고되었습니다.112 반면, 심근 경색 부위에 이식된 MSC는 심장 교감 신경 분지 등 유해한 세포 성장 효과를 유발한 것으로 보고되었습니다.113 이러한 부작용과 관련하여 MSC-EV 투여와 관련된 위험은 현저히 낮거나 전혀 없을 가능성이 높습니다. 그러나 다양한 질환 환경에서 놀라운 효능의 증거가 존재함에 따라, 연구 커뮤니티는 생물학적 활성을 가진 EV의 단기 및 장기 안전성을 신중히 평가해야 합니다. 이 제한된 정보에 따르면, MSC-EV를 임상 치료제로 성공적으로 전환하려면 MSC-EV와 종양 세포 간의 상호작용에 대한 추가적인 전임상 연구가 필요할 것입니다.

Large-scale EV production for clinical use

Although MSCs are relatively easy to expand using conventional tissue flasks and bioreactors, their growth in culture is finite and their biological properties may become altered with repeated passage. In order to facilitate large-scale MSC-EV production, new batches of MSCs will have to be periodically derived with significant impact on the costs of derivation, testing, and validation.114 Strategies such as MSC immortalization by natural selection or by genetic modification or clonal isolation could be used to overcome this limitation although this would also raise specific safety issues.115,116 Chen et al.117 proposed a robust scalable manufacturing process for therapeutic EVs through oncogenic immortalization of human embryonic stem cell (ESC)-derived MSCs. As EVs are isolated from media conditioned by cells, MSC culture in serum-free media would be of specific value to limit extraneous biological activity within the final therapeutic product. Other approaches to enhancing the purity of MSC-EVs preparations could include sequential centrifugation, filtration, and ultracentrifugation followed by sucrose density gradient to remove contaminating protein aggregates, cell debris, and genetic material.118,119,120 To scale up the amount of EVs isolated, bioreactors could be used to culture the MSCs.121 In this regard, a small number of studies have documented significant increases in EV yield from cells cultured in bioreactor systems when compared with conventional tissue culture flasks.122 It will be important, however, to also determine whether bioreactor culture conditions result in alterations to EV protein and RNA content that may impact on therapeutic efficacy.123,124 There are many challenges related to bioreactor culture including adequacy of oxygen supply, hydrodynamic shear stress, metabolic byproducts build-up, and pH balance.125,126 One should also be mindful that the impacts of such parameters are likely to differ for different cell types.

임상용 대규모 EV 생산

MSC는

전통적인 조직 배양 용기와 생물반응기를 사용하여 비교적 쉽게 증식시킬 수 있지만,

배양에서의 성장에는 한계가 있으며

반복적인 배양으로 생물학적 특성이 변할 수 있습니다.

대규모 MSC-EV 생산을 위해 새로운 MSC 배치를 주기적으로 생산해야 하며,

이는 생산, 검사, 검증 비용에 큰 영향을 미칠 것입니다. 114 

자연 선택, 유전적 변형, 또는 클론 분리 등을 통한 MSC 불멸화 전략이

이 한계를 극복하는 데 사용될 수 있지만,

이는 특정 안전성 문제를 유발할 수 있습니다.115,116 

Chen et al.117은 인간 배아 줄기 세포(ESC) 유래 MSC의 종양 유발성 불멸화를 통해 치료용 EV의 견고하고 확장 가능한 제조 공정을 제안했습니다. EV는 세포가 배양된 배지에서 분리되기 때문에, 최종 치료 제품 내 외부 생물학적 활성을 제한하기 위해 MSC를 혈청 없는 배지에서 배양하는 것이 특정 가치를 가질 수 있습니다. MSC-EV 제제의 순도를 향상시키기 위한 다른 접근 방식에는 순차적 원심분리, 필터링, 초고속 원심분리 후 설탕 밀도 그라디언트를 통해 오염 단백질 집합체, 세포 잔여물, 유전 물질을 제거하는 방법이 포함될 수 있습니다.118,119,120 EV의 양을 대량화하기 위해 MSC 배양에 바이오리액터를 사용할 수 있습니다. 121 이 점에서, 생물반응기 시스템에서 배양된 세포에서 전통적인 조직 배양 플라스크와 비교해 EV 수율이 유의미하게 증가했다는 소수의 연구 결과가 있습니다.122 그러나 생물반응기 배양 조건이 EV 단백질 및 RNA 함량에 변화를 초래해 치료 효능에 영향을 미칠 수 있는지 확인하는 것이 중요할 것입니다. 123,124 바이오리액터 배양에는 산소 공급의 적절성, 수력학적 전단 응력, 대사 부산물 축적, pH 균형 등 많은 도전 과제가 있습니다.125,126 또한 이러한 파라미터의 영향은 세포 유형에 따라 다를 수 있다는 점을 유의해야 합니다.

Conclusion

As we have summarized in this article, EVs can be readily isolated from MSCs of various origin and MSC-EVs are now known to have striking therapeutic benefits in a range of animal disease models. In some cases, these effects have been clearly shown to be of equal potency to those observed with whole cell MSC administration. The mechanisms underlying the anti-inflammatory and proregenerative effects of MSC-EVs have not yet been fully elucidated and are likely to vary from one disease target to another. Nonetheless, the fundamental basis for MSC-EV therapeutic effects lies in their ability to transmit biological information—in the form of proteins, glycoproteins, lipids, and ribonucleic acids—from stem cells to injured cells.

MSC-EVs have theoretical advantages over intact MSCs as a medicinal product and may, in the future, gain preference over whole cells in the discipline of regenerative medicine. However, in order for the field to advance to widespread clinical use of MSC-EVs for common human diseases, a range of important questions regarding their definition, standardization, cost-effective production, optimal dosing, and, most importantly, safety must be methodically addressed and answered.

결론
이 기사에서 요약한 바와 같이, 다양한 출처의 MSC에서 EV를 쉽게 분리할 수 있으며, 

MSC-EV는 다양한 동물 질환 모델에서 놀라운 치료 효과를 나타내는 것으로 알려져 있습니다. 

일부 경우 이러한 효과는 전체 세포 MSC 투여 시 관찰된 효과와 동일한 효능을 보인 것으로 명확히 입증되었습니다. 

MSC-EV의 항염증 및 재생 촉진 효과의 기전은 아직 완전히 규명되지 않았으며, 질병 목표에 따라 다를 가능성이 높습니다. 

그럼에도 불구하고, MSC-EV의 치료 효과의 근본적인 기반은 

줄기세포에서 손상된 세포로 생물학적 정보(단백질, 글리코프로틴, 지질, 리보핵산 등)를 

전달하는 능력에 있습니다.

MSC-EV는 

의약품으로서 전체 MSC에 비해 이론적 우위를 가지고 있으며, 

미래에는 재생 의학 분야에서 전체 세포보다 선호될 수 있습니다. 

그러나 

MSC-EV를 일반적인 인간 질환의 임상적 적용으로 확대하기 위해서는

 정의, 표준화, 비용 효율적인 생산, 최적의 투여량, 그리고 

무엇보다도 안전성에 대한 중요한 질문들이 체계적으로 해결되어야 합니다.

Acknowledgments

The authors are supported by grants from the Health Research Board of Ireland (grant numbers HRA_POR/2013/341 (S.R., M.D.G., and T.R.) and HRA_HSR/2010/63 (M.D.G.)); the Irish Cancer Society (grant number CRF12RYA (A.E.R.)); Science Foundation Ireland [grant numbers 09/SRC/B1794 (M.D.G. and T.R.) and 12/IA/1624 (T.R.)); the European Union Framework 7 program (Health Collaborative Project VISICORT, grant number 602470 (M.D.G. and T.R.)); and the European Regional Development Fund.

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ReviewVolume 23, Issue 5p812-823May 2015Open Archive

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Mesenchymal Stem Cell-derived Extracellular Vesicles: Toward Cell-free Therapeutic Applications

Sweta Rani1 sweta.rani@nuigalway.ie ∙ Aideen E Ryan2 ∙ Matthew D Griffin1 ∙ Thomas Ritter1

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Abstract

Mesenchymal stem (stromal) cells (MSCs) are multipotent cells with the ability to differentiate into several cell types, thus serving as a cell reservoir for regenerative medicine. Much of the current interest in therapeutic application of MSCs to various disease settings can be linked to their immunosuppressive and anti-inflammatory properties. One of the key mechanisms of MSC anti-inflammatory effects is the secretion of soluble factors with paracrine actions. Recently it has emerged that the paracrine functions of MSCs could, at least in part, be mediated by extracellular vesicles (EVs). EVs are predominantly released from the endosomal compartment and contain a cargo that includes miRNA, mRNA, and proteins from their cells of origin. Recent animal model-based studies suggest that EVs have significant potential as a novel alternative to whole cell therapies. Compared to their parent cells, EVs may have a superior safety profile and can be safely stored without losing function. In this article, we review current knowledge related to the potential use of MSC-derived EVs in various diseases and discuss the promising future for EVs as an alternative, cell-free therapy.

Introduction

Regenerative medicine focuses on the restoration of lost, damaged, or aging cells and tissues in the human body. Ferrari et al.1 demonstrated the value of a stem cell-based regenerative treatment for muscular dystrophies using bone marrow (BM)-derived myogenic progenitor cells. Since then numerous stem cell types have been investigated for use in tissue regeneration in both animal models and human clinical studies, with varying degrees of success.

Mesenchymal stem (or stromal) cells (MSCs) have emerged as a potential solution for tissue repair and wound healing.2 MSCs are multipotent, nonhematopoietic adult stem cells, which can be isolated from BM, umbilical cord,3,4 placental or adipose tissue. MSCs have the potential to differentiate into osteoblasts, chondrocytes, and adipocytes5 as well as endothelial, cardiovascular, and neurogenic cell types and are gaining credibility as a therapeutic agent because of their ex vivo expansion capacity and ethical acceptability.6 More recently, it has been discovered that, in addition to their direct role in tissue regeneration, MSCs have potent anti-inflammatory and/or immunosuppressive properties.7 Extensive research and clinical trials are currently underway for the use of MSCs as regenerative agents in many diseases including spinal cord injury, multiple sclerosis, Alzheimer's disease, liver cirrhosis and hepatitis, osteoarthritis, myocardial infarction, kidney disease, inflammatory bowel disease, diabetes mellitus, knee cartilage injuries, organ transplantation, and graft-versus-host disease (http://www.clinicaltrials.gov; accessed November 2014).

Paracrine Actions of MSCs

González et al.8 studied the contact-dependent mechanism of human adipose-derived MSCs in regulating inflammatory cytokines. In their study, they determined that human adipose-derived MSCs and macrophages both produce high levels of interleukin-10 (IL-10) only after cell-to-cell contact is maintained.8

Although potentially triggered by cell-to-cell contact events, the regenerative potential of MSC therapies has been found—at least in part—to be mediated via paracrine actions.9 For example, the paracrine effect of MSC-conditioned medium (CM) was observed to protect cardiomyocytes by interfering with the mitochondria-mediated apoptotic pathway. In this study, application of MSC-CM to cardiomyocytes exposed to hypoxia/reoxygenation reduced apoptosis through inhibition of the release of cytochrome C from mitochondria and reduction of caspase-3 activation.10 Similarly, renoprotective effects of human umbilical cord blood-derived MSCs (hUCB-MSCs) in streptozotocin-induced diabetic rats was reportedly mediated through paracrine action.11 In this case, the authors studied the effects of hUCB-MSC-CM on transforming growth factor (TGF)-β1-activated rat renal proximal tubular epithelia (NRK-52E) cells and observed attenuated expression‎ of TGF-β1, α-smooth muscle actin, collagen I, and heat shock protein-47 mRNA and increased expression‎ of E-cadherin and bone morphogenic protein-7 mRNA, thereby preventing diabetes kidney disease.11

Although it was initially believed that the potential of MSCs to differentiate into various cell types plays a crucial role in their therapeutic effects, the mechanism of action of transplanted MSCs does not predominantly include differentiating into a specific cell type for promoting or repairing the tissue damage in most disease settings.12,13,14 Several studies have demonstrated the predominance of short-lived paracrine mechanisms among the therapeutic actions of MSCs. In one such study, Toma et al.15 injected human MSCs (hMSCs) tagged with β-galactosidase into the left ventricle of immunodeficient mice. The majority of hMSCs were found in the spleen, lung, and liver, 4 days after injection. They also reported that only 0.44% of the injected hMSCs survived and, with time, they were morphologically indistinguishable from the surrounding cardiomyocytes. Other studies on systemically administered MSCs have also reported that <1% of the administered cells survive for more than 1 week and that the benefits of MSC therapy could be attributed to their secreted factors.16,17,18

In acute kidney injury (AKI), the protective effect of MSC administration was not attributed to MSCs differentiating into a tubular or endothelial cell phenotype, but to enhanced regulation of anti-inflammatory and organ-protective mediators such as IL-10, basic fibroblast growth factor, TGF-α, and B-cell lymphoma 2 (Bcl-2), reflecting primarily the paracrine function of MSCs.19 Tögel et al.20 reported the paracrine nature of cytoprotection in the immediate vicinity of administered MSCs in AKI. The authors demonstrated the production of renotropic factors—hepatocyte growth factor, and insulin-like growth factor 1 —that are known to decrease apoptosis and stimulate proliferation of renal epithelial cells.

Although these studies, and many others, provide strong evidence for the potency of MSC-secreted factors in mediating tissue repair and regeneration, the precise mechanisms by which MSCs act in a paracrine fashion are not fully understood. In addition to secreting an array of soluble factors, it has also been recognized that MSCs release large numbers of extracellular vesicles (EVs). Thus, it is of interest to consider the possibilities that the complex paracrine regenerative actions of exogenously administered MSCs and other stem cells communicate by transferring information and regulatory genes mediated, to some degree, by released EVs9,21,22 and that EVs derived from cultured MSCs have the potential to constitute a safe, effective cell-free therapy.

Extracellular Vesicles

EVs were first clearly described by Pan and Johnstone in 1983.23 Initially, the release of EVs was thought to represent a disposal mechanism by which cells eliminate unwanted proteins and other molecules. After years of subsequent research, however, EV release has emerged as an important mediator of cell-to-cell communication that is not only involved in normal physiological process but also plays a role in the development and progression of diseases. Among the subtypes of EV, the most numerous, referred to as exosomes, have a diameter of 40–100 nm, can be isolated by centrifugation at 100,000 ×g and can be concentrated at the interface of 0.8 and 2.7M sucrose layers. Preparations of EVs, typically a mixture of exosomes and other subtypes, can be isolated from all types of body fluids including blood, urine, bronchoalveolar lavage fluid, breast milk, amniotic fluid, synovial fluid, pleural effusions, and ascites.24 EVs can also be isolated from culture supernatants of many cell types, including T-cells, B-cells, dendritic cells, platelets, mast cells, epithelial cells, endothelial cells, neuronal cells, cancerous cells, and, as we describe in detail later, MSCs.25,26,27,28,29,30,31,32,33,34,35,36,37

Biogenesis of EVs

The modes of biogenesis for exosomes and microvesicles (MVs) are completely distinct and are described in this section.

Exosome biogenesis

Although the term “exosome” has been frequently used to describe all vesicles released by cells into the extracellular milieu, it is now known that there are multiple different types of EV. The major EV subtypes that are currently recognized are listed along with their basic characteristics in Table 1. Because of lack of specific markers it is very difficult to distinguish between different subtypes of vesicles within mixed preparations as they have overlapping composition, density, and size. Therefore, the International Society for Extracellular Vesicles suggested that the term EVs be used preferentially to describe preparations of vesicles from body fluids and cell cultures.38

VesiclesSize (diameter)Sucrose gradientOrigin

Exosomes	40-100 nm	1.13-1.19g/ml	Luminal budding into MVBs; release by fusion of MVB with cell membrane
Microvesicles	50-1,000 nm	1.04-1.07 g/ml	Outward budding of cell membrane
Apoptotic bodies	1-5,000 nm	1.16 and 1.28 g/ml	Outward blebbing of apoptotic cell membrane

Table 1

Different types of vesicles derived from various fluids and CM

MVB, multivesicular body.

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Exosomes are EVs of endosomal origin. The endosomal sorting complex required for transport and its associated proteins are involved in the formation of multivesicular bodies (MVBs) and intraluminal vesicles (ILV).39 Exosome membranes are enriched in lipids such as cholesterol, ceramide, and sphingolipids that are involved in the budding of ILVs into MVBs.40,41 As was first described during reticulocyte differentiation, ILVs are released from cells as a consequence of MVB fusion with the plasma membrane and, once released, are then termed as exosomes.23,42 Tan et al.41 further confirmed the endosomal origin of MSC-derived exosomes by detecting the components of lipid rafts. Table 2 provides additional details about proteins involved in MVB and exosome biogenesis. Exosomes may subsequently be internalized by other cells via direct membrane fusion, endocytosis or cell-type specific phagocytosis.43,44,45 Figure 1 illustrates the intracellular sources, release and uptake mechanisms associated with exosomes and other major subtypes of EV.

Figure viewer

Figure 1 EVs origin and internalization. Origin of EVs are generally via (a) endocytosis or inward budding of plasma membrane that consist of lipid rafts and is mediated by clathrin-dependent or caveolae-dependent pathway, This gives rise to (b) early endosomes leading to the formation of numerous ILVs within a membrane maturing to MVBs. Finally MVBs fuse with plasma membrane releasing ILVs as exosomes. (c) Ectosomes are vesicles shed from the cell surface and (d) apoptotic bodies are also known as apobodies and are released by cells undergoing apoptosis. EVs are internalized by the target cells through several pathways including (e) endocytosis, (f) fusion, and (g) phagocytosis.

FunctionProteinsReferences

MVB biogenesis	ESCRT-0, -I, -II, and -III; Vps4, VTA1, ALIX, Tsg101, CHMP4, ARF6, clathrin, and PLD2	127–137
Exosome Cargo	Vps4, Vps27, Tsg101, ALIX, HRS, Hsc70, Hsp90, 14-3-3 epsilon, and PKM2	39,138–141
MVE docking	RAB27a, RAB35	142,143
Exosome trafficking	RAB2B, RAB9A, RAB5A, RAB27B, syndecan, syntenin, ALIX, RAP1B, RHO	58,144–146
Exosome Release	Slp4, Slac2b, DGKα kinase, TfR, VAMP7, VAMP3, PLD2	144,147–151
Fusion of MVBs	SNAP receptors (SNAREs; v-SNAREs, t-SNAREs)	152–154

Table 2

Proteins associated with exosome biogenesis

ALIX, ALG-2-interacting protein X; ARF6, ADP-ribosylation factor 6;

CHMP4: charged multivesicular body protein 4; DGKα, diacylglycerol kinase a; ESCRT, endosomal sorting complex required for transport; HRS, hepatocyte growth factor-regulated tyrosine kinase substrate; Hsc70, heat shock cognate 70 kDa protein; Hsp90, heat-shock proteins; MVB, multivesicular body; MVE, multivesicular endosomes; PLD2, phospholipase D2; PKM2, pyruvate kinase M2; RAB27a, ras-related protein Rab-27A; RAP1B, Ras-related protein Rap-1B; RHO, rhodopsin; SNAREs, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; Slac2b, synaptotagmin-like homolog lacking C2 domains b; Slp4, synaptotagmin-like protein 4; t-SNAREs, target SNAREs; TfR, transferrin receptor; Tsg101, tumor susceptibility gene 101; Vps4, vacuolar protein sorting 4; VTA1, vesicle (multivesicular body) trafficking 1; VAMP7, vesicle-associated membrane protein 7; v-SNAREs, vesicular SNAREs.

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Microvesicle biogenesis

MVs result from outward budding and fission of plasma membrane. Membrane budding initiated by the activity of aminophospholipid translocases to translocate phosphatidylserine to the outer membrane.46,47,48 ADP-ribosylation factor 6 plays an important role in enabling MV budding by stimulating phospholipase D activity, which in turn facilitates extracellular signal-regulated kinase activation.49,50 Contractile protein myosin light chain kinase 2 (which contracts cytoskeleton) is phosphorylated by extracellular signal-regulated kinase, which in turn stimulates serine phosphorylation of myosin II that ultimately triggers the release of MVs.46,50,51,52

Regulation of EV Biogenesis

Earlier literature has shown that MSCs release EVs differently depending on external stimulation suggesting that this process is likely to be regulated by cross-talk between MSCs and their surrounding microenvironment.53,54 For example, hypoxia or inflammatory conditioning of MSCs has been shown to regulate protein packaging into EVs and to affect their functional properties.53,54 Several pathways, which may be relevant to the microenvironment in which MSCs reside, have been reported to regulate biogenesis and secretion of EVs. Tumor suppressor-activated pathway 6 is found to regulate EV formation55 and is transcriptionally regulated by p53 thereby enhancing EV production.56,57 An alternative cross-talk pathway was suggested by Baietti et al.58 who described that syndecans interact with syntenin to regulate intraluminal budding of endosomal membrane domains containing CD63 and ALIX.

Therapeutic Effects of MSC-Derived EVs (MSC-EVs)

As described earlier, EVs facilitate cell-to-cell communication via the transfer of functionally relevant biomolecules59,60 (see Table 3) and thus, may be harnessed for therapeutic purposes in a similar fashion to their parent cells. From a translational perspective, EVs derived from MSCs have shown encouraging therapeutic effects in various animal models (see Figure 2), and their isolation from MSCs is potentially sustainable and reproducible. Furthermore, in comparison to whole cell-based therapies, MSC-EVs may offer specific advantages for patient safety such as lower propensity to trigger innate and adaptive immune responses61 and inability to directly form tumors. For example, it has been shown that MSC-derived EVs induced anti-inflammatory cytokines as well as triggering apoptosis in activated T-cells.62 MSC-EVs also carry mRNAs encoding immunoregulatory mediators including cytokine receptor-like factor 1, interleukin 1 receptor, and metallothionein 1X.63

Figure viewer

Figure 2 Potential clinical applications of EVs. Therapeutic benefits and mechanisms of action of MSC-derived EVs in: (a) various heart conditions, (b) kidney injury, (c) liver injury, (d) lung injury, and (e) wound healing.

Source of exosomesProtein content

Endosome-associated proteins	Rab GTPase, SNAREs, Annexins, flotillin, ALIX, Tsg101
Membrane proteins	CD63, CD81, CD82, CD53, and CD37
Lipid raft protein	Glycosylphosphatidylinositol-anchored proteins and flotillin
RNA	Structural RNAs, tRNA fragments, vault RNA, Y RNA, and small interfering RNAs

Table 3

Molecular composition of EVs

EV, extracellular vesicle; SNAREs, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; Tsg101, tumor susceptibility gene 101.

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In the remaining sections and in Table 4, we examine the evidence to-date for beneficial effects of MSC-EVs in several important disease areas and discuss some of the future needs and challenges that may be of critical importance to their successful clinical translation.

ConditionsModel/cause of injuryOrigin of EVs/mode of administrationAmount (volume)Therapeutic capacityReference

Myocardial Infarction	Mice/heart excision and aortic root canulation	MSC/intravenously	20 μl unfractionated MSC-CM(10-220 nm), <1,000 kDa fraction(10-100 nm), >1,000-kDa fraction, or saline	>1,000 kDa fraction1. Confer cardio-protection 2. ↓Infarct size	64
MyocardialIR injury	Mouse Langendorff heart model/heart excision, aortic root canulation, and perfusion	Human ESC-derivedMSC/intravenously	0.4 μg of F1 fraction protein; 3 μgCM protein	F1 fraction + CM protein1. ↓Infarct size	32
MyocardialInfarction	C57Bl6/J mice/ temporary left coronary artery ligation	MSC/intravenously via the tail vein	0.4 μg/ml	MSC exosomes1. ↓Infarct size by 45% 2. Prevents left ventricular dilatation 3. Improves cardiac performance 4. ↓Inflammation	65
Acute myocardial infarction	Wistar rats/permanent ligation of the left anterior descending coronary artery	Human BM MSCs / intramyocardial injection	MSCs (2 × 106 cells); MSC-EVs (80 μg)	MSC-Evs1. ↑ Proliferation, migration, and tube formation of HUVECs 2. ↓Infarct size 3. Improved cardiac function 4. Angiogenesis	67
AKI	Sprague-Dawley rats/ bilateral renal ischemia	hUCB-MSC/left carotid artery	MVs dissolved in 0.5 ml PBS; control MV; IFNγtreated MV	MSC-MVs1. ↑ Formation of T-cells with Treg phenotype 2. Ameliorated kidney dysfunction and acute tubular necrosis	53
Renal injury	C57BL6/J mice/5/6 subtotal Nx	Mouse MSC/injected through caudal veins	Nx + MSC group, 1 × 106/mouse, second day of surgery; Nx + MV group, 30 μg MV/mouse, day 2, 3, 5 after surgery	Nx + MSC and Nx + MV1. Ameliorated renal injury 2. Prevent renal fibrosis 3. Preserved the remnant renal function	76
Chronic kidney disease	Sprague-Dawley rats/ IR injury	BM-MSCs; human fibroblasts/ intravenously	30 μg	MSC-MVs1. ↓Apoptosis tubular cells 2. ↑ Tubular cell proliferation 3. Protect against chronic kidney disease 4. ↓Accumulation of matrix in the glomeruli	77
AKI	SCID mice/rhabdomyolisis- induced AKI	Human BM-MSCs / intravenous injection into the tail vein	15 μg of MSC-MVs; 15 μg human fibroblasts-MVs; 75,000 BM-MSCs in 150 μl saline	MSC-MV1. ↑In vitro proliferation 2. ↑In vitro apoptosis resistance 3. ↑ Morphologic recovery of AKI in vivo 4. MVs accumulated within the lumen of injured tubules	63
AKI	SCID mice/cisplatin	BM-MSCs/tail vein	Single injection—100 μg; Multiple injection—50 μg (days 2, 6, 10, 14, and 18)	MSC-MVs1. ↓Mortality induced by cisplatin 2. Improved renal function 3. Inhibited apoptosis induced by cisplatin in vitro	78
AKI	CD1 nude mice/intramuscular injection of glycerol	BM-MSCs/intravenously	200 μg	MSC-EVs accumulate specifically in kidneys	80
Liver injury	C57BL/six mice/carbon tetrachloride (CCl4)	MSCs/intrasplenic injection	0.4 μg (100 μl PBS)	MSC-EV1. Reverse CCl4-induced injury 2. ↑Proliferation of hepatocytes 3. Up-regulated cell-proliferation markers 4. Induced hepatocyte-regenerative genes expression‎ in liver tissue after CCl4-induced injury	85
Liver injury	Mice/CCl4	hucMSCs/injected into livers	250 μg (330 μl PBS)	hucMSC-Ex1. CCl4 -induced liver fibrosissignificantly alleviated 2. Inhibit epithelial-to- mesenchymal transition 3. Ameliorate CCl -induced liver4 fibrosis	84
ALI	C57BL/six mice/endotoxin from E. coli	hMSCs/intravenously, external jugular vein or intratracheal	30 μl of MVs released by 1.5 × 106 serum starved MSCs; 750,000 MSCs	MSC-MVs1. ↓Influx of inflammatory cells 2. ↓Edema 3. Transfer of KGF mRNA	86
ALI	HPH mouse/HPH	hWJMSC/jugular vein, tail vein	0.1 and 10 μg	Exosome treatment1. Suppress hypoxic inflammation 2. Inhibits lung vascular remodeling 3. Prevents hypoxic pulmonary hypertension	90
Skin deep second- degree burn wound	Sprague-Dawley rats/injured with 80oC water for 8 seconds to create 16 mm diameter wound	hucMSC/subcutaneous	200 μg exosome (200 μl PBS); 1 × 106 cells (hucMSC and HFL1)	Exosome treatment1. Cell proliferation 2. ↑Re-epithelialization 3. Inhibits heat stress-induced apoptosis in vitro 4. Prompt wound healing	91
Multiple myeloma (MM)	SCID mice/N/A	BM-MSCs (healthy subjects, relapsed/refractory MM patients/implanted subcutaneously)	3 × 106 cells/tissue-engineered bones; 1 μg exosomes	MM BM-MSC-derived exosomes1. ↑MM cell growth in vitro 2. ↑Tumor growth in vivo 3. ↑BM homing	109
Angiogenesis, tumor growth	BALB/c nu/nu mice /N/A	Human BM-MSC, human lung fibroblast/ subcutaneous injections	SGC-7901 cells alone (1 × 106); SGC-7901 cells (1 × 106) mixed with MSCs (1 × 106); SGC-7901 cells (1 × 106) mixed with MSC exosomes (200 μg/ ml)	SGC-7901 cells mixed with exosomes1. ↑Tumor growth 2. ↑Proliferation of tumor cells invivo 3. ↑Tumor angiogenesis	110
Angiogenesis	BALB/c mice	Mouse BM-derived MSCs/subcutaneous injections	100 μg (100 μl PBS); 2 × 105 4T1 cells mixed with 100 μg of MSC- derived exosomes or 2 × 105 4T1 cellsmixed with 200 μg of MSC-derived exosomes	MSC-derived EVs1. ↓VEGF expression‎ in 4T1 cells 2. ↓Angiogenesis in vitro and in vivo 3. ↓Tumor growth in vivo	111
Bladder tumor growth	BALB/c nu/nu mice	hWJMSC/subcutaneous injection	1 × 107 T24 cells; 1 × 107 T24 cells mixed with 1 × 107 hWJMSCs; 1 × 107 T24 cells mixed with 200 μg protein hWJMSC-MVs; 200 μg protein hWJMSC-MVs.	hWJMSC-EVs + hWJMSCs1. ↓ Significantly tumor size 2. ↑ Apoptosis	92
Hepatoma growth	SCID mice	HLSCs/intratumor injection	100 μg of EVs (20 μl)	HLSC-derived EVs1. ↓Significantly tumor size 2. ↑Apoptosis	94
Breast cancer	CB-17/Icr-scid/scidJc1 mice	BM MSC	BM2 cells (20,000) treated with 3 μg of BM-MSC-derived EVs were then injected in mammary fat pad (100 μl injections of PBS containing 1 × 105 BM2 cells)	BM-MSC-derived EV-treated cells1. ↓Proliferation 2. ↓Tumor formation	96

Table 4

Information of MSC-derived EVs in different studies

Up arrow (↑) indicates increased and down arrow (↓) indicates decreased activity.

AKI, acute kidney injury; ALI, acute lung injury; BM, bone marrow; CM, conditioned medium; EV, extracellular vesicle; HLSCs, human adult live stem cells; HLSC, human adult liver stem cell; HPH, hypoxia-induced pulmonary hypertension; HUVEC, human umbilical vein endothelial cells; hWJMSC, human umbilical cord Wharton's jelly MSC; IR injury, ischemia/reperfusion injury; KGF, keratinocyte growth factor; MSC, mesenchymal stem (stromal) cell; MV, Microvesicle; Nx, nephrectomy; PBS, phosphate-buffered saline; SCID, severe combined immunodeficient.

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MSC-EVs in cardiovascular disease

The CM obtained from hMSCs was shown by Timmers et al.64 to have the potential to reduce myocardial infarct size by 60% in a porcine model of cardiac ischemia/reperfusion (IR) injury. In this same study, fractionation of the CM revealed that the cardio-protective effect was confined to the fraction containing products >1,000 kDa (100–220 nm). In a mouse model of myocardial infarction, Lai et al.32 then directly demonstrated that the active, cardio-protective component of MSC-derived CM is, in fact, the EVs. In this study, administration of purified MSC-EVs reduced infarct size by ~40%.

Subsequently, Arslan et al.65 reported reduced infarct size following a single intravenous injection of MSC-EVs which could be attributed to the fact that EVs are internalized by target cells at the infarct site via endocytosis or phagocytosis. To further prove that intact MSC-EVs were required for therapeutic benefit, these authors demonstrated that homogenized EVs failed to reduce infarct size.65

Other studies have explored mechanisms by which the number and proangiogenic effects of EVs released by MSCs can be enhanced.66 For example, in a study of placental MSCs, under hypoxic conditions, Salomon et al.54 observed 3.3- and 6.7-fold increases in EV release in the presence of 1% and 3% O2 when compared with placental MSCs maintained at 8% O2. The resulting placental MSCs-derived EVs induced a significant, dose-dependent increase in tube formation by placental microvascular endothelial cells when compared with vehicle-treated cells.54 It was speculated that the increased proangiogenic effect of MSC-EVs derived under hypoxic conditions may be conferred by transcriptional activities of the hypoxia inducible factor family of proteins.54

Following on from the above result, Bian et al.67 isolated EVs from MSCs cultured under hypoxic conditions. In an in vitro angiogenesis assay, MSC-EVs at a concentration of 80 μg/ml, promoted human umbilical vein endothelial cell migration and tube formation that was comparable to that induced by vascular endothelial growth factor (VEGF). In vivo studies confirmed that intramyocardial injection of hypoxia-conditioned MSC-EVs significantly improved cardiac function and reduced myocardial infarct size with similar potency to that observed in a whole-cell MSC-treated group.67

Micro-RNAs associated with MSC-EVs also play an important role in cardio-protection. For instance, it was found that cardiac remodeling following myocardial infarction is regulated by miR-22-loaded EVs via targeting of methyl CpG binding protein 2.68 Similarly, the level of miR-221 is significantly higher in MSC-EVs when compared with their parent MSCs, and this miRNA was shown to enhance cardio-protection by reducing the expression‎ of p53 upregulated modulator of apoptosis.69

MSC-EVs in AKI

AKI is a major cause of morbidity and mortality among hospitalized patients and is most commonly caused by IR injury, exposure to nephrotoxic compounds, and severe volume loss or obstruction to urine flow.70 It has been well established in animal models of renal IR and other forms of kidney injury that systemic or localized administration of MSCs results in amelioration of AKI.71,72,73 MSCs downregulate proinflammatory cytokines in T-cells and consequently induce regulatory T-cells (T-regs) in the spleen.71 Anti-inflammatory and immunoregulatory properties of MSCs have become one of the important mechanistic approaches to the treatment of AKI. A broad range of growth factors, cytokines, and chemokines secreted from MSCs have been identified including hepatocyte growth factor, insulin-like growth factor 1, VEGF, IL-1, IL-4, IL-5, IL-6, keratinocyte-derived chemokine, chemokine (C-X-C motif) ligand 16 (CXCL16), chemokine (C–C motif) ligand 2 (CCL2), CCL3, chemokine (C-X3-C motif) ligand 1 (CX3CL1), and CCL5.20,74 In experimental models, mediators such as these have been associated with enhanced cell proliferation and reduced cell apoptosis, identifying MSCs as uniquely providing multimodal therapeutic effects in AKI.75

Similar to MSCs, MSC-EVs are capable of modulating T-cell as well as innate immune cell functions.53 To date, there are few reported studies that directly compare the effect of MSCs and MSC-EVs in the setting of AKI. However, in a study involving mouse 5/6 subtotal nephrectomy (Nx)—a model of chronic kidney disease–He et al.76 reported that both MSC- and MSC-EV-treated mice showed strikingly similar benefits including reduced fibrosis and interstitial lymphocyte infiltration and reduced or absent tubular atrophy when compared with the untreated control group.

In the rat model of renal IR, Gatti et al.77 found that intravenous injection of 30 μg of MSC-EVs prevented AKI. The administered EVs were shown to transiently accumulate within glomeruli and injured tubules in association with increased proliferation and reduced apoptosis of tubular epithelial cells.77 This study also reported that the protective effect was specific to MSC-EVs as fibroblast-EVs were ineffective. Similarly, Bruno et al.63 also reported that human BM-derived MSC-EVs accelerated renal morphologic and functional recovery in glycerol-induced AKI in immunodeficient mice by inducing proliferation of tubular cells. In this study, they also reported that the effect of MSC-EVs on the recovery of AKI was similar to that of hMSCs.63

The effects of human MSC-EVs were also studied in severe combined immunodeficient (SCID) mice with AKI induced by the chemotherapeutic agent cisplatin.78 In this study, MSC-EVs significantly improved the survival (40% at day 21) by improving renal function and morphology, but were unable to prevent chronic tubular injury (see Table 4). Multiple injections of MSC-EVs, however, further decreased mortality in association with normal histology and renal function.78 MSC-EVs were found to upregulate antiapoptotic genes, including B-cell lymphoma-extra large, Bcl-2 and baculoviral IAP repeat containing 8, and downregulating cell apoptosis genes including, Caspase-1 (Casp1), Caspase-8 (Casp8) and lymphotoxin α in cisplatin-treated human tubular epithelial cells.78 Renoprotection was also conferred by horizontal transfer of insulin-like growth factor-1 receptor via BM–MSC–EV.79

Grange et al.80 studied the biodistribution of intravenously injected MSC-EVs in an AKI mouse model. They observed the specific accumulation of EVs at the site of injury as compared to healthy mice receiving the same quantity of MSC-EVs.80 Overall, of the disease areas studied, AKI, caused by a variety of clinically relevant insults, represents one of the most convincing examples of a distinct therapeutic benefit of systemic MSC-EV injection.78,80,81

MSC-EVs in liver disease

MSCs have been shown to be of benefit in a range of acute and chronic liver disease models and clinical translation of this work is currently underway in a number of centers.82 For example, injection of MSCs into the portal vein has been reported to protect the liver in a rat model of hepatic IR injury after partial hepatectomy. In this study, MSC administration was shown to reduce hepatocyte apoptosis and enhance liver regeneration.83

Fewer studies have addressed the potential benefits of MSC-EVs in chronic liver disease models. In one such study, human umbilical cord-MSC (hucMSC)-EVs were shown to specifically localize to the liver and to alleviate liver fibrosis in carbon tetrachloride (CCl4)-induced injury by reducing hepatocyte apoptosis and hepatic lobule destruction.84 MSC-EV administration suppressed epithelial to mesenchymal transdifferentiation via reduced TGF-β1 expression‎ and Smad2 phosphorylation.84 Other in vivo studies have shown that MSC-EVs promote hepatocyte regeneration after CCl4-induced injury by inducing the IL-6/STAT3 pathway and cell cycle progression.85 In this case, the authors validated the direct hepatoprotective effects of MSC-EVs using the cell lines TAMH (an immortalized mouse hepatocyte line derived from transgenic MT42 male mice overexpressing TGF-α), THLE-2 (an immortalized primary human hepatocyte) and HuH-7 (a human hepatocarcinoma cell line) exposed in vitro to acetaminophen and hydrogen peroxide.85 Increased cytoprotection compared to control-treated cells was observed following treatment with 0.1 μg/ml MSC-EVs. Thus, both in vivo and in vitro studies have confirmed that MSC-EV therapy has the potential to promote liver regeneration following acute injury by directly enhancing hepatocyte survival and proliferation85 (see Table 4).

MSC-EVs in lung diseases

Endotoxin-induced acute lung injury (ALI) in mice results in increased lung protein permeability causing an inflammatory response in the alveoli that is commonly used as a model of human ALI associated with severe pneumonia or sepsis. In this model, it has recently been shown by Zhu et al.86 that administration of MSC-EVs decreased the influx of total inflammatory cells into the lung by 36% and influx of neutrophils by 73%. The suppression of lung inflammation was accompanied by reduced protein permeability, thereby preventing the formation of pulmonary edema. From a mechanistic perspective, keratinocyte growth factor (KGF) has been shown to reduce lung edema and inflammation in various ALI models.87,88 Lee et al. 89 reported that hMSCs produced KGF and that its secretion as a paracrine soluble factor mediated the restoration of alveolar fluid clearance in vivo. Thus, Zhu et al. 86 hypothesized that MSC-EVs transfer KGF mRNA to the injured alveolar epithelium and to verify this, they transfected the MSCs with KGF-specific small interfering RNA before isolating EVs. In keeping with this mechanism, the therapeutic effect of EVs from KGF-depleted MSCs was reduced compared to that of control MSC-EVs.

In a mouse model of hypoxia-induced pulmonary hypertension, the injection of MSC-EVs resulted in a delayed pulmonary influx of macrophages and reduced production of proinflammatory mediators compared to injection of EVs-derived from mouse lung fibroblasts.90 MSC-EVs, upon low dose multiple administration, also ameliorated pulmonary hypertension via increasing the levels of miR-204,90 ventricular hypertrophy, and lung vascular remodelling.90 The authors further tested the efficacy of two sequential injections of a higher dose of MSC-EVs and observed similar beneficial effects on early and later outcomes.90 Finally, MSC-EVs have been found to suppress hypoxic activation of signal transducer and activator of transcription 3 (STAT3) by up-regulating miR-17.90

MSC-EVs in cutaneous wound healing

In a recently reported study by Zhang et al. 91, the effects of locally injected hucMSC and hucMSC-EVs were studied in a rat deep second degree burn injury model. Using a range of histological and molecular indexes of healing, the authors found that injection of hucMSCs and hucMSC-EVs resulted in comparable and significant increase in re-epithelialization when compared with burn wounds that were treated with saline, human lung fibroblasts (HFL1) or HFL1-EVs. The epithelial healing effects were replicated in vitro in keratinocyte and dermal fibroblast cell lines in the form of increased cell proliferation and reduced apoptosis and were shown to be mediated by MSC-EV-delivered Wnt4 resulting in activation of β-catenin signaling and by activation of the AKT signaling pathway.91 Although additional studies are needed to confirm‎ these striking observations in other preclinical models, the results suggest that cutaneous injury and ulceration represent one of the most promising clinical translational avenues for MSC-EV preparations.

Antitumor Activity of MSC-EVs

MSCs have also been shown to have anticancer activities. Wu et al.92 demonstrated that human umbilical cord Wharton's jelly MSC (hWJMSC)-derived EVs reduce the growth of T24 bladder carcinoma cells in vitro and in vivo. The authors reported that incubation of T24 cells with various concentration of hWJMSC-EVs (0, 50, 100, 200 μg/ml protein) resulted in cell-cycle arrest and tumor cell apoptosis.92 Similarly, Bruno et al.93 reported inhibited cell-cycle progression and induced apoptosis in HepG2 (liver) and Kaposi's cells, and necrosis in Skov-3 (ovarian cell line) when treated with MSC-EVs.

In a study carried out using human adult liver stem cell (HLSC)-EVs, Fonsato et al.94 reported induction of apoptosis in HepG2 hepatoma and primary hepatocellular carcinoma cells. Significant reduction in tumor growth was also observed in the presence of MV-HLSC in SCID mice inoculated with primary hepatocellular carcinoma cells.94 The authors concluded that the antitumor effects of HLSC-EVs could be because of selective delivery of miRNAs—a mechanism that may also explain the potential antitumor effects of MSC-EVs in some settings.

MicroRNA-9 has been associated with drug resistance via increasing the expression‎ of P-glycoprotein.95 Munoz et al.95 reported that anti-miR-9-Cy5 was transferred from MSCs to glioblastoma multiforme cells via EVs, blocking the increase of P-glycoprotein and reversing the chemoresistance. Ono et al.96 reported that BM-MSC-EVs contributed to the dormant state of BM2 cells through EV-mediated transfer of miRNA.

MSC-EVs for Drug Delivery

EVs are natural transporters that may potentially reach a wide range of tissues following systemic administration, including the central nervous system as they have been reported to cross the blood–brain barrier.97 As EVs consist of a bilayered lipid membrane with an aqueous core they may potentially be loaded with both hydrophilic and lipophilic drugs.98 Furthermore, drugs could be either loaded into purified preparations of EVs99 or applied to parent cells and incorporated during EV biogenesis.100 Small molecules including siRNAs can also be loaded into the EVs either by electoporation or by chemical disruption.97,101 Although little explored to date, MSC-EVs may constitute a particularly promising vehicle for drug delivery given their inherent ability to exert disease-modulatory effects and the extensive literature documenting in vitro modification of MSCs using genetic and nongenetic approaches. As an example, Pascucci et al.102 observed that paclitaxel-treated MSCs mediated strong antitumorigenic effects because of their capacity to take up the drug and later release it in EVs. In this study, paclitaxel -treated MSC-EVs induced a dose-dependent inhibition of CFPAC-1 (human pancreatic adenocarcinoma) cell proliferation as well as 50% inhibition of tumor growth.

Clinical Translation of MSC-EVs: Unresolved Issues and Future PrioritiesTumorigenesis and other potential adverse effects of MSC-EVs

Despite reported antitumor effects in some settings, there is also theoretical potential for whole cell MSC therapy to directly or indirectly induce cancerous tumors or to accelerate the progression of pre-existing cancers. Although this concern has not, thus far, been borne out in human clinical trials, subpopulations of MSC-like cells have been found in the tumor microenvironment of several human cancers including gastric adenocarcinoma103 and osteosarcoma.104 Furthermore, some animal model studies have demonstrated preferential migration of intravenously administered MSCs to tumors.105,106 Although EVs clearly lack the potential to directly form tumors following in vivo administration, this does not imply that MSC-EV administration to human subjects is without any risk of promoting neoplasia. For instance, multiple myeloma (MM) cell proliferation has been shown to be increased in the presence of either autocrine or paracrine secretory factors of BM-MSCs.107,108 Roccaro et al.109 isolated EVs from BM-MSCs derived from both MM patients and healthy controls. In this study, the MM BM-MSC-derived EVs were found to promote MM tumor/cell growth, whereas normal BM-MSC-derived EVs inhibited the growth of MM tumor/cells both in vitro and in vivo. The MM BM-MSC-derived EVs were also found to induce cell dissemination and metastasis to distant BM niches.109

MSC-EVs have been found to modulate the tumor microenvironment, creating a niche for cancer cell metastasis and have been proven to mimic the effects of MSCs to promote tumor growth. Zhu et al.110 showed that MSC-EVs co-implanted with SGC-7901 (human gastric cancer) cells increased tumor growth and angiogenesis when compared with SGC-7901 cells alone. However, Lee et al.111 reported contradictory results suggesting that MSC-EVs suppress angiogenesis in vitro by downregulating the mRNA and protein levels of VEGF in tumor cells in a concentration-dependent manner. They speculated that this inconsistency could be because of different tumor types or MSC heterogeneity.111

Intravascular infusion of MSCs has been documented to cause embolism and death in experimental animals,112 whereas MSCs inoculated into infarcted myocardium were reported to induce adverse cellular growth such as cardiac sympathetic nerve sprouting.113 For adverse effects such as these, it appears likely that the risk associated with MSC-EV administration will be significantly lower or perhaps absent. However, as evidence of striking efficacy in a variety of disease settings now exists, it is incumbent on the research community to carefully eval‎uate the short- and long-term safety of biologically active EVs. Based on this limited information, it is clear that successful translation of MSC-EVs as a clinical therapy will require a significant amount of additional preclinical investigation of the interaction between MSC-EVs and tumor cells.

Large-scale EV production for clinical use

Although MSCs are relatively easy to expand using conventional tissue flasks and bioreactors, their growth in culture is finite and their biological properties may become altered with repeated passage. In order to facilitate large-scale MSC-EV production, new batches of MSCs will have to be periodically derived with significant impact on the costs of derivation, testing, and validation.114 Strategies such as MSC immortalization by natural selection or by genetic modification or clonal isolation could be used to overcome this limitation although this would also raise specific safety issues.115,116 Chen et al.117 proposed a robust scalable manufacturing process for therapeutic EVs through oncogenic immortalization of human embryonic stem cell (ESC)-derived MSCs. As EVs are isolated from media conditioned by cells, MSC culture in serum-free media would be of specific value to limit extraneous biological activity within the final therapeutic product. Other approaches to enhancing the purity of MSC-EVs preparations could include sequential centrifugation, filtration, and ultracentrifugation followed by sucrose density gradient to remove contaminating protein aggregates, cell debris, and genetic material.118,119,120 To scale up the amount of EVs isolated, bioreactors could be used to culture the MSCs.121 In this regard, a small number of studies have documented significant increases in EV yield from cells cultured in bioreactor systems when compared with conventional tissue culture flasks.122 It will be important, however, to also determine whether bioreactor culture conditions result in alterations to EV protein and RNA content that may impact on therapeutic efficacy.123,124 There are many challenges related to bioreactor culture including adequacy of oxygen supply, hydrodynamic shear stress, metabolic byproducts build-up, and pH balance.125,126 One should also be mindful that the impacts of such parameters are likely to differ for different cell types.

Conclusion

As we have summarized in this article, EVs can be readily isolated from MSCs of various origin and MSC-EVs are now known to have striking therapeutic benefits in a range of animal disease models. In some cases, these effects have been clearly shown to be of equal potency to those observed with whole cell MSC administration. The mechanisms underlying the anti-inflammatory and proregenerative effects of MSC-EVs have not yet been fully elucidated and are likely to vary from one disease target to another. Nonetheless, the fundamental basis for MSC-EV therapeutic effects lies in their ability to transmit biological information—in the form of proteins, glycoproteins, lipids, and ribonucleic acids—from stem cells to injured cells.

MSC-EVs have theoretical advantages over intact MSCs as a medicinal product and may, in the future, gain preference over whole cells in the discipline of regenerative medicine. However, in order for the field to advance to widespread clinical use of MSC-EVs for common human diseases, a range of important questions regarding their definition, standardization, cost-effective production, optimal dosing, and, most importantly, safety must be methodically addressed and answered.

Acknowledgments

The authors are supported by grants from the Health Research Board of Ireland (grant numbers HRA_POR/2013/341 (S.R., M.D.G., and T.R.) and HRA_HSR/2010/63 (M.D.G.)); the Irish Cancer Society (grant number CRF12RYA (A.E.R.)); Science Foundation Ireland [grant numbers 09/SRC/B1794 (M.D.G. and T.R.) and 12/IA/1624 (T.R.)); the European Union Framework 7 program (Health Collaborative Project VISICORT, grant number 602470 (M.D.G. and T.R.)); and the European Regional Development Fund.

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ReviewVolume 23, Issue 5p812-823May 2015Open Archive

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Mesenchymal Stem Cell-derived Extracellular Vesicles: Toward Cell-free Therapeutic Applications

Sweta Rani1 sweta.rani@nuigalway.ie ∙ Aideen E Ryan2 ∙ Matthew D Griffin1 ∙ Thomas Ritter1

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Abstract

Mesenchymal stem (stromal) cells (MSCs) are multipotent cells with the ability to differentiate into several cell types, thus serving as a cell reservoir for regenerative medicine. Much of the current interest in therapeutic application of MSCs to various disease settings can be linked to their immunosuppressive and anti-inflammatory properties. One of the key mechanisms of MSC anti-inflammatory effects is the secretion of soluble factors with paracrine actions. Recently it has emerged that the paracrine functions of MSCs could, at least in part, be mediated by extracellular vesicles (EVs). EVs are predominantly released from the endosomal compartment and contain a cargo that includes miRNA, mRNA, and proteins from their cells of origin. Recent animal model-based studies suggest that EVs have significant potential as a novel alternative to whole cell therapies. Compared to their parent cells, EVs may have a superior safety profile and can be safely stored without losing function. In this article, we review current knowledge related to the potential use of MSC-derived EVs in various diseases and discuss the promising future for EVs as an alternative, cell-free therapy.

Introduction

Regenerative medicine focuses on the restoration of lost, damaged, or aging cells and tissues in the human body. Ferrari et al.1 demonstrated the value of a stem cell-based regenerative treatment for muscular dystrophies using bone marrow (BM)-derived myogenic progenitor cells. Since then numerous stem cell types have been investigated for use in tissue regeneration in both animal models and human clinical studies, with varying degrees of success.

Mesenchymal stem (or stromal) cells (MSCs) have emerged as a potential solution for tissue repair and wound healing.2 MSCs are multipotent, nonhematopoietic adult stem cells, which can be isolated from BM, umbilical cord,3,4 placental or adipose tissue. MSCs have the potential to differentiate into osteoblasts, chondrocytes, and adipocytes5 as well as endothelial, cardiovascular, and neurogenic cell types and are gaining credibility as a therapeutic agent because of their ex vivo expansion capacity and ethical acceptability.6 More recently, it has been discovered that, in addition to their direct role in tissue regeneration, MSCs have potent anti-inflammatory and/or immunosuppressive properties.7 Extensive research and clinical trials are currently underway for the use of MSCs as regenerative agents in many diseases including spinal cord injury, multiple sclerosis, Alzheimer's disease, liver cirrhosis and hepatitis, osteoarthritis, myocardial infarction, kidney disease, inflammatory bowel disease, diabetes mellitus, knee cartilage injuries, organ transplantation, and graft-versus-host disease (http://www.clinicaltrials.gov; accessed November 2014).

Paracrine Actions of MSCs

González et al.8 studied the contact-dependent mechanism of human adipose-derived MSCs in regulating inflammatory cytokines. In their study, they determined that human adipose-derived MSCs and macrophages both produce high levels of interleukin-10 (IL-10) only after cell-to-cell contact is maintained.8

Although potentially triggered by cell-to-cell contact events, the regenerative potential of MSC therapies has been found—at least in part—to be mediated via paracrine actions.9 For example, the paracrine effect of MSC-conditioned medium (CM) was observed to protect cardiomyocytes by interfering with the mitochondria-mediated apoptotic pathway. In this study, application of MSC-CM to cardiomyocytes exposed to hypoxia/reoxygenation reduced apoptosis through inhibition of the release of cytochrome C from mitochondria and reduction of caspase-3 activation.10 Similarly, renoprotective effects of human umbilical cord blood-derived MSCs (hUCB-MSCs) in streptozotocin-induced diabetic rats was reportedly mediated through paracrine action.11 In this case, the authors studied the effects of hUCB-MSC-CM on transforming growth factor (TGF)-β1-activated rat renal proximal tubular epithelia (NRK-52E) cells and observed attenuated expression‎ of TGF-β1, α-smooth muscle actin, collagen I, and heat shock protein-47 mRNA and increased expression‎ of E-cadherin and bone morphogenic protein-7 mRNA, thereby preventing diabetes kidney disease.11

Although it was initially believed that the potential of MSCs to differentiate into various cell types plays a crucial role in their therapeutic effects, the mechanism of action of transplanted MSCs does not predominantly include differentiating into a specific cell type for promoting or repairing the tissue damage in most disease settings.12,13,14 Several studies have demonstrated the predominance of short-lived paracrine mechanisms among the therapeutic actions of MSCs. In one such study, Toma et al.15 injected human MSCs (hMSCs) tagged with β-galactosidase into the left ventricle of immunodeficient mice. The majority of hMSCs were found in the spleen, lung, and liver, 4 days after injection. They also reported that only 0.44% of the injected hMSCs survived and, with time, they were morphologically indistinguishable from the surrounding cardiomyocytes. Other studies on systemically administered MSCs have also reported that <1% of the administered cells survive for more than 1 week and that the benefits of MSC therapy could be attributed to their secreted factors.16,17,18

In acute kidney injury (AKI), the protective effect of MSC administration was not attributed to MSCs differentiating into a tubular or endothelial cell phenotype, but to enhanced regulation of anti-inflammatory and organ-protective mediators such as IL-10, basic fibroblast growth factor, TGF-α, and B-cell lymphoma 2 (Bcl-2), reflecting primarily the paracrine function of MSCs.19 Tögel et al.20 reported the paracrine nature of cytoprotection in the immediate vicinity of administered MSCs in AKI. The authors demonstrated the production of renotropic factors—hepatocyte growth factor, and insulin-like growth factor 1 —that are known to decrease apoptosis and stimulate proliferation of renal epithelial cells.

Although these studies, and many others, provide strong evidence for the potency of MSC-secreted factors in mediating tissue repair and regeneration, the precise mechanisms by which MSCs act in a paracrine fashion are not fully understood. In addition to secreting an array of soluble factors, it has also been recognized that MSCs release large numbers of extracellular vesicles (EVs). Thus, it is of interest to consider the possibilities that the complex paracrine regenerative actions of exogenously administered MSCs and other stem cells communicate by transferring information and regulatory genes mediated, to some degree, by released EVs9,21,22 and that EVs derived from cultured MSCs have the potential to constitute a safe, effective cell-free therapy.

Extracellular Vesicles

EVs were first clearly described by Pan and Johnstone in 1983.23 Initially, the release of EVs was thought to represent a disposal mechanism by which cells eliminate unwanted proteins and other molecules. After years of subsequent research, however, EV release has emerged as an important mediator of cell-to-cell communication that is not only involved in normal physiological process but also plays a role in the development and progression of diseases. Among the subtypes of EV, the most numerous, referred to as exosomes, have a diameter of 40–100 nm, can be isolated by centrifugation at 100,000 ×g and can be concentrated at the interface of 0.8 and 2.7M sucrose layers. Preparations of EVs, typically a mixture of exosomes and other subtypes, can be isolated from all types of body fluids including blood, urine, bronchoalveolar lavage fluid, breast milk, amniotic fluid, synovial fluid, pleural effusions, and ascites.24 EVs can also be isolated from culture supernatants of many cell types, including T-cells, B-cells, dendritic cells, platelets, mast cells, epithelial cells, endothelial cells, neuronal cells, cancerous cells, and, as we describe in detail later, MSCs.25,26,27,28,29,30,31,32,33,34,35,36,37

Biogenesis of EVs

The modes of biogenesis for exosomes and microvesicles (MVs) are completely distinct and are described in this section.

Exosome biogenesis

Although the term “exosome” has been frequently used to describe all vesicles released by cells into the extracellular milieu, it is now known that there are multiple different types of EV. The major EV subtypes that are currently recognized are listed along with their basic characteristics in Table 1. Because of lack of specific markers it is very difficult to distinguish between different subtypes of vesicles within mixed preparations as they have overlapping composition, density, and size. Therefore, the International Society for Extracellular Vesicles suggested that the term EVs be used preferentially to describe preparations of vesicles from body fluids and cell cultures.38

VesiclesSize (diameter)Sucrose gradientOrigin

Exosomes	40-100 nm	1.13-1.19g/ml	Luminal budding into MVBs; release by fusion of MVB with cell membrane
Microvesicles	50-1,000 nm	1.04-1.07 g/ml	Outward budding of cell membrane
Apoptotic bodies	1-5,000 nm	1.16 and 1.28 g/ml	Outward blebbing of apoptotic cell membrane

Table 1

Different types of vesicles derived from various fluids and CM

MVB, multivesicular body.

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Exosomes are EVs of endosomal origin. The endosomal sorting complex required for transport and its associated proteins are involved in the formation of multivesicular bodies (MVBs) and intraluminal vesicles (ILV).39 Exosome membranes are enriched in lipids such as cholesterol, ceramide, and sphingolipids that are involved in the budding of ILVs into MVBs.40,41 As was first described during reticulocyte differentiation, ILVs are released from cells as a consequence of MVB fusion with the plasma membrane and, once released, are then termed as exosomes.23,42 Tan et al.41 further confirmed the endosomal origin of MSC-derived exosomes by detecting the components of lipid rafts. Table 2 provides additional details about proteins involved in MVB and exosome biogenesis. Exosomes may subsequently be internalized by other cells via direct membrane fusion, endocytosis or cell-type specific phagocytosis.43,44,45 Figure 1 illustrates the intracellular sources, release and uptake mechanisms associated with exosomes and other major subtypes of EV.

Figure viewer

Figure 1 EVs origin and internalization. Origin of EVs are generally via (a) endocytosis or inward budding of plasma membrane that consist of lipid rafts and is mediated by clathrin-dependent or caveolae-dependent pathway, This gives rise to (b) early endosomes leading to the formation of numerous ILVs within a membrane maturing to MVBs. Finally MVBs fuse with plasma membrane releasing ILVs as exosomes. (c) Ectosomes are vesicles shed from the cell surface and (d) apoptotic bodies are also known as apobodies and are released by cells undergoing apoptosis. EVs are internalized by the target cells through several pathways including (e) endocytosis, (f) fusion, and (g) phagocytosis.

FunctionProteinsReferences

MVB biogenesis	ESCRT-0, -I, -II, and -III; Vps4, VTA1, ALIX, Tsg101, CHMP4, ARF6, clathrin, and PLD2	127–137
Exosome Cargo	Vps4, Vps27, Tsg101, ALIX, HRS, Hsc70, Hsp90, 14-3-3 epsilon, and PKM2	39,138–141
MVE docking	RAB27a, RAB35	142,143
Exosome trafficking	RAB2B, RAB9A, RAB5A, RAB27B, syndecan, syntenin, ALIX, RAP1B, RHO	58,144–146
Exosome Release	Slp4, Slac2b, DGKα kinase, TfR, VAMP7, VAMP3, PLD2	144,147–151
Fusion of MVBs	SNAP receptors (SNAREs; v-SNAREs, t-SNAREs)	152–154

Table 2

Proteins associated with exosome biogenesis

ALIX, ALG-2-interacting protein X; ARF6, ADP-ribosylation factor 6;

CHMP4: charged multivesicular body protein 4; DGKα, diacylglycerol kinase a; ESCRT, endosomal sorting complex required for transport; HRS, hepatocyte growth factor-regulated tyrosine kinase substrate; Hsc70, heat shock cognate 70 kDa protein; Hsp90, heat-shock proteins; MVB, multivesicular body; MVE, multivesicular endosomes; PLD2, phospholipase D2; PKM2, pyruvate kinase M2; RAB27a, ras-related protein Rab-27A; RAP1B, Ras-related protein Rap-1B; RHO, rhodopsin; SNAREs, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; Slac2b, synaptotagmin-like homolog lacking C2 domains b; Slp4, synaptotagmin-like protein 4; t-SNAREs, target SNAREs; TfR, transferrin receptor; Tsg101, tumor susceptibility gene 101; Vps4, vacuolar protein sorting 4; VTA1, vesicle (multivesicular body) trafficking 1; VAMP7, vesicle-associated membrane protein 7; v-SNAREs, vesicular SNAREs.

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Microvesicle biogenesis

MVs result from outward budding and fission of plasma membrane. Membrane budding initiated by the activity of aminophospholipid translocases to translocate phosphatidylserine to the outer membrane.46,47,48 ADP-ribosylation factor 6 plays an important role in enabling MV budding by stimulating phospholipase D activity, which in turn facilitates extracellular signal-regulated kinase activation.49,50 Contractile protein myosin light chain kinase 2 (which contracts cytoskeleton) is phosphorylated by extracellular signal-regulated kinase, which in turn stimulates serine phosphorylation of myosin II that ultimately triggers the release of MVs.46,50,51,52

Regulation of EV Biogenesis

Earlier literature has shown that MSCs release EVs differently depending on external stimulation suggesting that this process is likely to be regulated by cross-talk between MSCs and their surrounding microenvironment.53,54 For example, hypoxia or inflammatory conditioning of MSCs has been shown to regulate protein packaging into EVs and to affect their functional properties.53,54 Several pathways, which may be relevant to the microenvironment in which MSCs reside, have been reported to regulate biogenesis and secretion of EVs. Tumor suppressor-activated pathway 6 is found to regulate EV formation55 and is transcriptionally regulated by p53 thereby enhancing EV production.56,57 An alternative cross-talk pathway was suggested by Baietti et al.58 who described that syndecans interact with syntenin to regulate intraluminal budding of endosomal membrane domains containing CD63 and ALIX.

Therapeutic Effects of MSC-Derived EVs (MSC-EVs)

As described earlier, EVs facilitate cell-to-cell communication via the transfer of functionally relevant biomolecules59,60 (see Table 3) and thus, may be harnessed for therapeutic purposes in a similar fashion to their parent cells. From a translational perspective, EVs derived from MSCs have shown encouraging therapeutic effects in various animal models (see Figure 2), and their isolation from MSCs is potentially sustainable and reproducible. Furthermore, in comparison to whole cell-based therapies, MSC-EVs may offer specific advantages for patient safety such as lower propensity to trigger innate and adaptive immune responses61 and inability to directly form tumors. For example, it has been shown that MSC-derived EVs induced anti-inflammatory cytokines as well as triggering apoptosis in activated T-cells.62 MSC-EVs also carry mRNAs encoding immunoregulatory mediators including cytokine receptor-like factor 1, interleukin 1 receptor, and metallothionein 1X.63

Figure viewer

Figure 2 Potential clinical applications of EVs. Therapeutic benefits and mechanisms of action of MSC-derived EVs in: (a) various heart conditions, (b) kidney injury, (c) liver injury, (d) lung injury, and (e) wound healing.

Source of exosomesProtein content

Endosome-associated proteins	Rab GTPase, SNAREs, Annexins, flotillin, ALIX, Tsg101
Membrane proteins	CD63, CD81, CD82, CD53, and CD37
Lipid raft protein	Glycosylphosphatidylinositol-anchored proteins and flotillin
RNA	Structural RNAs, tRNA fragments, vault RNA, Y RNA, and small interfering RNAs

Table 3

Molecular composition of EVs

EV, extracellular vesicle; SNAREs, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; Tsg101, tumor susceptibility gene 101.

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In the remaining sections and in Table 4, we examine the evidence to-date for beneficial effects of MSC-EVs in several important disease areas and discuss some of the future needs and challenges that may be of critical importance to their successful clinical translation.

ConditionsModel/cause of injuryOrigin of EVs/mode of administrationAmount (volume)Therapeutic capacityReference

Myocardial Infarction	Mice/heart excision and aortic root canulation	MSC/intravenously	20 μl unfractionated MSC-CM(10-220 nm), <1,000 kDa fraction(10-100 nm), >1,000-kDa fraction, or saline	>1,000 kDa fraction1. Confer cardio-protection 2. ↓Infarct size	64
MyocardialIR injury	Mouse Langendorff heart model/heart excision, aortic root canulation, and perfusion	Human ESC-derivedMSC/intravenously	0.4 μg of F1 fraction protein; 3 μgCM protein	F1 fraction + CM protein1. ↓Infarct size	32
MyocardialInfarction	C57Bl6/J mice/ temporary left coronary artery ligation	MSC/intravenously via the tail vein	0.4 μg/ml	MSC exosomes1. ↓Infarct size by 45% 2. Prevents left ventricular dilatation 3. Improves cardiac performance 4. ↓Inflammation	65
Acute myocardial infarction	Wistar rats/permanent ligation of the left anterior descending coronary artery	Human BM MSCs / intramyocardial injection	MSCs (2 × 106 cells); MSC-EVs (80 μg)	MSC-Evs1. ↑ Proliferation, migration, and tube formation of HUVECs 2. ↓Infarct size 3. Improved cardiac function 4. Angiogenesis	67
AKI	Sprague-Dawley rats/ bilateral renal ischemia	hUCB-MSC/left carotid artery	MVs dissolved in 0.5 ml PBS; control MV; IFNγtreated MV	MSC-MVs1. ↑ Formation of T-cells with Treg phenotype 2. Ameliorated kidney dysfunction and acute tubular necrosis	53
Renal injury	C57BL6/J mice/5/6 subtotal Nx	Mouse MSC/injected through caudal veins	Nx + MSC group, 1 × 106/mouse, second day of surgery; Nx + MV group, 30 μg MV/mouse, day 2, 3, 5 after surgery	Nx + MSC and Nx + MV1. Ameliorated renal injury 2. Prevent renal fibrosis 3. Preserved the remnant renal function	76
Chronic kidney disease	Sprague-Dawley rats/ IR injury	BM-MSCs; human fibroblasts/ intravenously	30 μg	MSC-MVs1. ↓Apoptosis tubular cells 2. ↑ Tubular cell proliferation 3. Protect against chronic kidney disease 4. ↓Accumulation of matrix in the glomeruli	77
AKI	SCID mice/rhabdomyolisis- induced AKI	Human BM-MSCs / intravenous injection into the tail vein	15 μg of MSC-MVs; 15 μg human fibroblasts-MVs; 75,000 BM-MSCs in 150 μl saline	MSC-MV1. ↑In vitro proliferation 2. ↑In vitro apoptosis resistance 3. ↑ Morphologic recovery of AKI in vivo 4. MVs accumulated within the lumen of injured tubules	63
AKI	SCID mice/cisplatin	BM-MSCs/tail vein	Single injection—100 μg; Multiple injection—50 μg (days 2, 6, 10, 14, and 18)	MSC-MVs1. ↓Mortality induced by cisplatin 2. Improved renal function 3. Inhibited apoptosis induced by cisplatin in vitro	78
AKI	CD1 nude mice/intramuscular injection of glycerol	BM-MSCs/intravenously	200 μg	MSC-EVs accumulate specifically in kidneys	80
Liver injury	C57BL/six mice/carbon tetrachloride (CCl4)	MSCs/intrasplenic injection	0.4 μg (100 μl PBS)	MSC-EV1. Reverse CCl4-induced injury 2. ↑Proliferation of hepatocytes 3. Up-regulated cell-proliferation markers 4. Induced hepatocyte-regenerative genes expression‎ in liver tissue after CCl4-induced injury	85
Liver injury	Mice/CCl4	hucMSCs/injected into livers	250 μg (330 μl PBS)	hucMSC-Ex1. CCl4 -induced liver fibrosissignificantly alleviated 2. Inhibit epithelial-to- mesenchymal transition 3. Ameliorate CCl -induced liver4 fibrosis	84
ALI	C57BL/six mice/endotoxin from E. coli	hMSCs/intravenously, external jugular vein or intratracheal	30 μl of MVs released by 1.5 × 106 serum starved MSCs; 750,000 MSCs	MSC-MVs1. ↓Influx of inflammatory cells 2. ↓Edema 3. Transfer of KGF mRNA	86
ALI	HPH mouse/HPH	hWJMSC/jugular vein, tail vein	0.1 and 10 μg	Exosome treatment1. Suppress hypoxic inflammation 2. Inhibits lung vascular remodeling 3. Prevents hypoxic pulmonary hypertension	90
Skin deep second- degree burn wound	Sprague-Dawley rats/injured with 80oC water for 8 seconds to create 16 mm diameter wound	hucMSC/subcutaneous	200 μg exosome (200 μl PBS); 1 × 106 cells (hucMSC and HFL1)	Exosome treatment1. Cell proliferation 2. ↑Re-epithelialization 3. Inhibits heat stress-induced apoptosis in vitro 4. Prompt wound healing	91
Multiple myeloma (MM)	SCID mice/N/A	BM-MSCs (healthy subjects, relapsed/refractory MM patients/implanted subcutaneously)	3 × 106 cells/tissue-engineered bones; 1 μg exosomes	MM BM-MSC-derived exosomes1. ↑MM cell growth in vitro 2. ↑Tumor growth in vivo 3. ↑BM homing	109
Angiogenesis, tumor growth	BALB/c nu/nu mice /N/A	Human BM-MSC, human lung fibroblast/ subcutaneous injections	SGC-7901 cells alone (1 × 106); SGC-7901 cells (1 × 106) mixed with MSCs (1 × 106); SGC-7901 cells (1 × 106) mixed with MSC exosomes (200 μg/ ml)	SGC-7901 cells mixed with exosomes1. ↑Tumor growth 2. ↑Proliferation of tumor cells invivo 3. ↑Tumor angiogenesis	110
Angiogenesis	BALB/c mice	Mouse BM-derived MSCs/subcutaneous injections	100 μg (100 μl PBS); 2 × 105 4T1 cells mixed with 100 μg of MSC- derived exosomes or 2 × 105 4T1 cellsmixed with 200 μg of MSC-derived exosomes	MSC-derived EVs1. ↓VEGF expression‎ in 4T1 cells 2. ↓Angiogenesis in vitro and in vivo 3. ↓Tumor growth in vivo	111
Bladder tumor growth	BALB/c nu/nu mice	hWJMSC/subcutaneous injection	1 × 107 T24 cells; 1 × 107 T24 cells mixed with 1 × 107 hWJMSCs; 1 × 107 T24 cells mixed with 200 μg protein hWJMSC-MVs; 200 μg protein hWJMSC-MVs.	hWJMSC-EVs + hWJMSCs1. ↓ Significantly tumor size 2. ↑ Apoptosis	92
Hepatoma growth	SCID mice	HLSCs/intratumor injection	100 μg of EVs (20 μl)	HLSC-derived EVs1. ↓Significantly tumor size 2. ↑Apoptosis	94
Breast cancer	CB-17/Icr-scid/scidJc1 mice	BM MSC	BM2 cells (20,000) treated with 3 μg of BM-MSC-derived EVs were then injected in mammary fat pad (100 μl injections of PBS containing 1 × 105 BM2 cells)	BM-MSC-derived EV-treated cells1. ↓Proliferation 2. ↓Tumor formation	96

Table 4

Information of MSC-derived EVs in different studies

Up arrow (↑) indicates increased and down arrow (↓) indicates decreased activity.

AKI, acute kidney injury; ALI, acute lung injury; BM, bone marrow; CM, conditioned medium; EV, extracellular vesicle; HLSCs, human adult live stem cells; HLSC, human adult liver stem cell; HPH, hypoxia-induced pulmonary hypertension; HUVEC, human umbilical vein endothelial cells; hWJMSC, human umbilical cord Wharton's jelly MSC; IR injury, ischemia/reperfusion injury; KGF, keratinocyte growth factor; MSC, mesenchymal stem (stromal) cell; MV, Microvesicle; Nx, nephrectomy; PBS, phosphate-buffered saline; SCID, severe combined immunodeficient.

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MSC-EVs in cardiovascular disease

The CM obtained from hMSCs was shown by Timmers et al.64 to have the potential to reduce myocardial infarct size by 60% in a porcine model of cardiac ischemia/reperfusion (IR) injury. In this same study, fractionation of the CM revealed that the cardio-protective effect was confined to the fraction containing products >1,000 kDa (100–220 nm). In a mouse model of myocardial infarction, Lai et al.32 then directly demonstrated that the active, cardio-protective component of MSC-derived CM is, in fact, the EVs. In this study, administration of purified MSC-EVs reduced infarct size by ~40%.

Subsequently, Arslan et al.65 reported reduced infarct size following a single intravenous injection of MSC-EVs which could be attributed to the fact that EVs are internalized by target cells at the infarct site via endocytosis or phagocytosis. To further prove that intact MSC-EVs were required for therapeutic benefit, these authors demonstrated that homogenized EVs failed to reduce infarct size.65

Other studies have explored mechanisms by which the number and proangiogenic effects of EVs released by MSCs can be enhanced.66 For example, in a study of placental MSCs, under hypoxic conditions, Salomon et al.54 observed 3.3- and 6.7-fold increases in EV release in the presence of 1% and 3% O2 when compared with placental MSCs maintained at 8% O2. The resulting placental MSCs-derived EVs induced a significant, dose-dependent increase in tube formation by placental microvascular endothelial cells when compared with vehicle-treated cells.54 It was speculated that the increased proangiogenic effect of MSC-EVs derived under hypoxic conditions may be conferred by transcriptional activities of the hypoxia inducible factor family of proteins.54

Following on from the above result, Bian et al.67 isolated EVs from MSCs cultured under hypoxic conditions. In an in vitro angiogenesis assay, MSC-EVs at a concentration of 80 μg/ml, promoted human umbilical vein endothelial cell migration and tube formation that was comparable to that induced by vascular endothelial growth factor (VEGF). In vivo studies confirmed that intramyocardial injection of hypoxia-conditioned MSC-EVs significantly improved cardiac function and reduced myocardial infarct size with similar potency to that observed in a whole-cell MSC-treated group.67

Micro-RNAs associated with MSC-EVs also play an important role in cardio-protection. For instance, it was found that cardiac remodeling following myocardial infarction is regulated by miR-22-loaded EVs via targeting of methyl CpG binding protein 2.68 Similarly, the level of miR-221 is significantly higher in MSC-EVs when compared with their parent MSCs, and this miRNA was shown to enhance cardio-protection by reducing the expression‎ of p53 upregulated modulator of apoptosis.69

MSC-EVs in AKI

AKI is a major cause of morbidity and mortality among hospitalized patients and is most commonly caused by IR injury, exposure to nephrotoxic compounds, and severe volume loss or obstruction to urine flow.70 It has been well established in animal models of renal IR and other forms of kidney injury that systemic or localized administration of MSCs results in amelioration of AKI.71,72,73 MSCs downregulate proinflammatory cytokines in T-cells and consequently induce regulatory T-cells (T-regs) in the spleen.71 Anti-inflammatory and immunoregulatory properties of MSCs have become one of the important mechanistic approaches to the treatment of AKI. A broad range of growth factors, cytokines, and chemokines secreted from MSCs have been identified including hepatocyte growth factor, insulin-like growth factor 1, VEGF, IL-1, IL-4, IL-5, IL-6, keratinocyte-derived chemokine, chemokine (C-X-C motif) ligand 16 (CXCL16), chemokine (C–C motif) ligand 2 (CCL2), CCL3, chemokine (C-X3-C motif) ligand 1 (CX3CL1), and CCL5.20,74 In experimental models, mediators such as these have been associated with enhanced cell proliferation and reduced cell apoptosis, identifying MSCs as uniquely providing multimodal therapeutic effects in AKI.75

Similar to MSCs, MSC-EVs are capable of modulating T-cell as well as innate immune cell functions.53 To date, there are few reported studies that directly compare the effect of MSCs and MSC-EVs in the setting of AKI. However, in a study involving mouse 5/6 subtotal nephrectomy (Nx)—a model of chronic kidney disease–He et al.76 reported that both MSC- and MSC-EV-treated mice showed strikingly similar benefits including reduced fibrosis and interstitial lymphocyte infiltration and reduced or absent tubular atrophy when compared with the untreated control group.

In the rat model of renal IR, Gatti et al.77 found that intravenous injection of 30 μg of MSC-EVs prevented AKI. The administered EVs were shown to transiently accumulate within glomeruli and injured tubules in association with increased proliferation and reduced apoptosis of tubular epithelial cells.77 This study also reported that the protective effect was specific to MSC-EVs as fibroblast-EVs were ineffective. Similarly, Bruno et al.63 also reported that human BM-derived MSC-EVs accelerated renal morphologic and functional recovery in glycerol-induced AKI in immunodeficient mice by inducing proliferation of tubular cells. In this study, they also reported that the effect of MSC-EVs on the recovery of AKI was similar to that of hMSCs.63

The effects of human MSC-EVs were also studied in severe combined immunodeficient (SCID) mice with AKI induced by the chemotherapeutic agent cisplatin.78 In this study, MSC-EVs significantly improved the survival (40% at day 21) by improving renal function and morphology, but were unable to prevent chronic tubular injury (see Table 4). Multiple injections of MSC-EVs, however, further decreased mortality in association with normal histology and renal function.78 MSC-EVs were found to upregulate antiapoptotic genes, including B-cell lymphoma-extra large, Bcl-2 and baculoviral IAP repeat containing 8, and downregulating cell apoptosis genes including, Caspase-1 (Casp1), Caspase-8 (Casp8) and lymphotoxin α in cisplatin-treated human tubular epithelial cells.78 Renoprotection was also conferred by horizontal transfer of insulin-like growth factor-1 receptor via BM–MSC–EV.79

Grange et al.80 studied the biodistribution of intravenously injected MSC-EVs in an AKI mouse model. They observed the specific accumulation of EVs at the site of injury as compared to healthy mice receiving the same quantity of MSC-EVs.80 Overall, of the disease areas studied, AKI, caused by a variety of clinically relevant insults, represents one of the most convincing examples of a distinct therapeutic benefit of systemic MSC-EV injection.78,80,81

MSC-EVs in liver disease

MSCs have been shown to be of benefit in a range of acute and chronic liver disease models and clinical translation of this work is currently underway in a number of centers.82 For example, injection of MSCs into the portal vein has been reported to protect the liver in a rat model of hepatic IR injury after partial hepatectomy. In this study, MSC administration was shown to reduce hepatocyte apoptosis and enhance liver regeneration.83

Fewer studies have addressed the potential benefits of MSC-EVs in chronic liver disease models. In one such study, human umbilical cord-MSC (hucMSC)-EVs were shown to specifically localize to the liver and to alleviate liver fibrosis in carbon tetrachloride (CCl4)-induced injury by reducing hepatocyte apoptosis and hepatic lobule destruction.84 MSC-EV administration suppressed epithelial to mesenchymal transdifferentiation via reduced TGF-β1 expression‎ and Smad2 phosphorylation.84 Other in vivo studies have shown that MSC-EVs promote hepatocyte regeneration after CCl4-induced injury by inducing the IL-6/STAT3 pathway and cell cycle progression.85 In this case, the authors validated the direct hepatoprotective effects of MSC-EVs using the cell lines TAMH (an immortalized mouse hepatocyte line derived from transgenic MT42 male mice overexpressing TGF-α), THLE-2 (an immortalized primary human hepatocyte) and HuH-7 (a human hepatocarcinoma cell line) exposed in vitro to acetaminophen and hydrogen peroxide.85 Increased cytoprotection compared to control-treated cells was observed following treatment with 0.1 μg/ml MSC-EVs. Thus, both in vivo and in vitro studies have confirmed that MSC-EV therapy has the potential to promote liver regeneration following acute injury by directly enhancing hepatocyte survival and proliferation85 (see Table 4).

MSC-EVs in lung diseases

Endotoxin-induced acute lung injury (ALI) in mice results in increased lung protein permeability causing an inflammatory response in the alveoli that is commonly used as a model of human ALI associated with severe pneumonia or sepsis. In this model, it has recently been shown by Zhu et al.86 that administration of MSC-EVs decreased the influx of total inflammatory cells into the lung by 36% and influx of neutrophils by 73%. The suppression of lung inflammation was accompanied by reduced protein permeability, thereby preventing the formation of pulmonary edema. From a mechanistic perspective, keratinocyte growth factor (KGF) has been shown to reduce lung edema and inflammation in various ALI models.87,88 Lee et al. 89 reported that hMSCs produced KGF and that its secretion as a paracrine soluble factor mediated the restoration of alveolar fluid clearance in vivo. Thus, Zhu et al. 86 hypothesized that MSC-EVs transfer KGF mRNA to the injured alveolar epithelium and to verify this, they transfected the MSCs with KGF-specific small interfering RNA before isolating EVs. In keeping with this mechanism, the therapeutic effect of EVs from KGF-depleted MSCs was reduced compared to that of control MSC-EVs.

In a mouse model of hypoxia-induced pulmonary hypertension, the injection of MSC-EVs resulted in a delayed pulmonary influx of macrophages and reduced production of proinflammatory mediators compared to injection of EVs-derived from mouse lung fibroblasts.90 MSC-EVs, upon low dose multiple administration, also ameliorated pulmonary hypertension via increasing the levels of miR-204,90 ventricular hypertrophy, and lung vascular remodelling.90 The authors further tested the efficacy of two sequential injections of a higher dose of MSC-EVs and observed similar beneficial effects on early and later outcomes.90 Finally, MSC-EVs have been found to suppress hypoxic activation of signal transducer and activator of transcription 3 (STAT3) by up-regulating miR-17.90

MSC-EVs in cutaneous wound healing

In a recently reported study by Zhang et al. 91, the effects of locally injected hucMSC and hucMSC-EVs were studied in a rat deep second degree burn injury model. Using a range of histological and molecular indexes of healing, the authors found that injection of hucMSCs and hucMSC-EVs resulted in comparable and significant increase in re-epithelialization when compared with burn wounds that were treated with saline, human lung fibroblasts (HFL1) or HFL1-EVs. The epithelial healing effects were replicated in vitro in keratinocyte and dermal fibroblast cell lines in the form of increased cell proliferation and reduced apoptosis and were shown to be mediated by MSC-EV-delivered Wnt4 resulting in activation of β-catenin signaling and by activation of the AKT signaling pathway.91 Although additional studies are needed to confirm‎ these striking observations in other preclinical models, the results suggest that cutaneous injury and ulceration represent one of the most promising clinical translational avenues for MSC-EV preparations.

Antitumor Activity of MSC-EVs

MSCs have also been shown to have anticancer activities. Wu et al.92 demonstrated that human umbilical cord Wharton's jelly MSC (hWJMSC)-derived EVs reduce the growth of T24 bladder carcinoma cells in vitro and in vivo. The authors reported that incubation of T24 cells with various concentration of hWJMSC-EVs (0, 50, 100, 200 μg/ml protein) resulted in cell-cycle arrest and tumor cell apoptosis.92 Similarly, Bruno et al.93 reported inhibited cell-cycle progression and induced apoptosis in HepG2 (liver) and Kaposi's cells, and necrosis in Skov-3 (ovarian cell line) when treated with MSC-EVs.

In a study carried out using human adult liver stem cell (HLSC)-EVs, Fonsato et al.94 reported induction of apoptosis in HepG2 hepatoma and primary hepatocellular carcinoma cells. Significant reduction in tumor growth was also observed in the presence of MV-HLSC in SCID mice inoculated with primary hepatocellular carcinoma cells.94 The authors concluded that the antitumor effects of HLSC-EVs could be because of selective delivery of miRNAs—a mechanism that may also explain the potential antitumor effects of MSC-EVs in some settings.

MicroRNA-9 has been associated with drug resistance via increasing the expression‎ of P-glycoprotein.95 Munoz et al.95 reported that anti-miR-9-Cy5 was transferred from MSCs to glioblastoma multiforme cells via EVs, blocking the increase of P-glycoprotein and reversing the chemoresistance. Ono et al.96 reported that BM-MSC-EVs contributed to the dormant state of BM2 cells through EV-mediated transfer of miRNA.

MSC-EVs for Drug Delivery

EVs are natural transporters that may potentially reach a wide range of tissues following systemic administration, including the central nervous system as they have been reported to cross the blood–brain barrier.97 As EVs consist of a bilayered lipid membrane with an aqueous core they may potentially be loaded with both hydrophilic and lipophilic drugs.98 Furthermore, drugs could be either loaded into purified preparations of EVs99 or applied to parent cells and incorporated during EV biogenesis.100 Small molecules including siRNAs can also be loaded into the EVs either by electoporation or by chemical disruption.97,101 Although little explored to date, MSC-EVs may constitute a particularly promising vehicle for drug delivery given their inherent ability to exert disease-modulatory effects and the extensive literature documenting in vitro modification of MSCs using genetic and nongenetic approaches. As an example, Pascucci et al.102 observed that paclitaxel-treated MSCs mediated strong antitumorigenic effects because of their capacity to take up the drug and later release it in EVs. In this study, paclitaxel -treated MSC-EVs induced a dose-dependent inhibition of CFPAC-1 (human pancreatic adenocarcinoma) cell proliferation as well as 50% inhibition of tumor growth.

Clinical Translation of MSC-EVs: Unresolved Issues and Future PrioritiesTumorigenesis and other potential adverse effects of MSC-EVs

Despite reported antitumor effects in some settings, there is also theoretical potential for whole cell MSC therapy to directly or indirectly induce cancerous tumors or to accelerate the progression of pre-existing cancers. Although this concern has not, thus far, been borne out in human clinical trials, subpopulations of MSC-like cells have been found in the tumor microenvironment of several human cancers including gastric adenocarcinoma103 and osteosarcoma.104 Furthermore, some animal model studies have demonstrated preferential migration of intravenously administered MSCs to tumors.105,106 Although EVs clearly lack the potential to directly form tumors following in vivo administration, this does not imply that MSC-EV administration to human subjects is without any risk of promoting neoplasia. For instance, multiple myeloma (MM) cell proliferation has been shown to be increased in the presence of either autocrine or paracrine secretory factors of BM-MSCs.107,108 Roccaro et al.109 isolated EVs from BM-MSCs derived from both MM patients and healthy controls. In this study, the MM BM-MSC-derived EVs were found to promote MM tumor/cell growth, whereas normal BM-MSC-derived EVs inhibited the growth of MM tumor/cells both in vitro and in vivo. The MM BM-MSC-derived EVs were also found to induce cell dissemination and metastasis to distant BM niches.109

MSC-EVs have been found to modulate the tumor microenvironment, creating a niche for cancer cell metastasis and have been proven to mimic the effects of MSCs to promote tumor growth. Zhu et al.110 showed that MSC-EVs co-implanted with SGC-7901 (human gastric cancer) cells increased tumor growth and angiogenesis when compared with SGC-7901 cells alone. However, Lee et al.111 reported contradictory results suggesting that MSC-EVs suppress angiogenesis in vitro by downregulating the mRNA and protein levels of VEGF in tumor cells in a concentration-dependent manner. They speculated that this inconsistency could be because of different tumor types or MSC heterogeneity.111

Intravascular infusion of MSCs has been documented to cause embolism and death in experimental animals,112 whereas MSCs inoculated into infarcted myocardium were reported to induce adverse cellular growth such as cardiac sympathetic nerve sprouting.113 For adverse effects such as these, it appears likely that the risk associated with MSC-EV administration will be significantly lower or perhaps absent. However, as evidence of striking efficacy in a variety of disease settings now exists, it is incumbent on the research community to carefully eval‎uate the short- and long-term safety of biologically active EVs. Based on this limited information, it is clear that successful translation of MSC-EVs as a clinical therapy will require a significant amount of additional preclinical investigation of the interaction between MSC-EVs and tumor cells.

Large-scale EV production for clinical use

Although MSCs are relatively easy to expand using conventional tissue flasks and bioreactors, their growth in culture is finite and their biological properties may become altered with repeated passage. In order to facilitate large-scale MSC-EV production, new batches of MSCs will have to be periodically derived with significant impact on the costs of derivation, testing, and validation.114 Strategies such as MSC immortalization by natural selection or by genetic modification or clonal isolation could be used to overcome this limitation although this would also raise specific safety issues.115,116 Chen et al.117 proposed a robust scalable manufacturing process for therapeutic EVs through oncogenic immortalization of human embryonic stem cell (ESC)-derived MSCs. As EVs are isolated from media conditioned by cells, MSC culture in serum-free media would be of specific value to limit extraneous biological activity within the final therapeutic product. Other approaches to enhancing the purity of MSC-EVs preparations could include sequential centrifugation, filtration, and ultracentrifugation followed by sucrose density gradient to remove contaminating protein aggregates, cell debris, and genetic material.118,119,120 To scale up the amount of EVs isolated, bioreactors could be used to culture the MSCs.121 In this regard, a small number of studies have documented significant increases in EV yield from cells cultured in bioreactor systems when compared with conventional tissue culture flasks.122 It will be important, however, to also determine whether bioreactor culture conditions result in alterations to EV protein and RNA content that may impact on therapeutic efficacy.123,124 There are many challenges related to bioreactor culture including adequacy of oxygen supply, hydrodynamic shear stress, metabolic byproducts build-up, and pH balance.125,126 One should also be mindful that the impacts of such parameters are likely to differ for different cell types.

Conclusion

As we have summarized in this article, EVs can be readily isolated from MSCs of various origin and MSC-EVs are now known to have striking therapeutic benefits in a range of animal disease models. In some cases, these effects have been clearly shown to be of equal potency to those observed with whole cell MSC administration. The mechanisms underlying the anti-inflammatory and proregenerative effects of MSC-EVs have not yet been fully elucidated and are likely to vary from one disease target to another. Nonetheless, the fundamental basis for MSC-EV therapeutic effects lies in their ability to transmit biological information—in the form of proteins, glycoproteins, lipids, and ribonucleic acids—from stem cells to injured cells.

MSC-EVs have theoretical advantages over intact MSCs as a medicinal product and may, in the future, gain preference over whole cells in the discipline of regenerative medicine. However, in order for the field to advance to widespread clinical use of MSC-EVs for common human diseases, a range of important questions regarding their definition, standardization, cost-effective production, optimal dosing, and, most importantly, safety must be methodically addressed and answered.

Acknowledgments

The authors are supported by grants from the Health Research Board of Ireland (grant numbers HRA_POR/2013/341 (S.R., M.D.G., and T.R.) and HRA_HSR/2010/63 (M.D.G.)); the Irish Cancer Society (grant number CRF12RYA (A.E.R.)); Science Foundation Ireland [grant numbers 09/SRC/B1794 (M.D.G. and T.R.) and 12/IA/1624 (T.R.)); the European Union Framework 7 program (Health Collaborative Project VISICORT, grant number 602470 (M.D.G. and T.R.)); and the European Regional Development Fund.

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