Preclinical Experimental Applications of miRNA Loaded BMSC Extracellular Vesicles

Abstract

Bone marrow mesenchymal stem cells have been investigated for many years, especially for tissue regeneration, and have inherent limitations. One of the rapidly developing fields in the scientific world in recent years is extracellular vesicles. Especially, bone marrow mesenchymal stem cell originated extracellular vesicles are known to have positive contributions in tissue regeneration, and these extracellular vesicles have also been used as gene transfer systems for cellular therapy. Through gene expression analysis and bioinformatics tools, it is possible to determine which genes have changed in the targeted tissue or cell and which miRNAs that can correct this gene expression disorder. This approach connecting the stem cell, extracellular vesicles, epigenetics regulation and bioinformatics fields is one of the promising areas for the treatment of diseases in the future. With this review, it is aimed to present the studies carried out for the use of bone marrow stem cell-derived extracellular vesicles loaded with targeted miRNAs in different in vivo and in vitro human disease models and to discuss recent developments in this field.

Full text

Preclinical Experimental Applications of miRNA Loaded BMSC
Extracellular Vesicles
Zafer Cetin
1,2
&Eyup I. Saygili
3,4
&Gokhan Görgisen
5
&Emel Sokullu
6,7
Accepted: 8 November 2020
#Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
Bone marrow mesenchymal stem cells have been investigated for many years, especially for tissue regeneration, and have
inherent limitations. One of the rapidly developing fields in the scientific world in recent years is extracellular vesicles.
Especially, bone marrow mesenchymal stem cell originated extracellular vesicles are known to have positive contributions in
tissue regeneration, and these extracellular vesicles have also been used as gene transfer systems for cellular therapy. Through
gene expression analysis and bioinformatics tools, it is possible to determine which genes have changed in the targeted tissue or
cell and which miRNAs that can correct this gene expression disorder. This approach connecting the stem cell, extracellular
vesicles, epigenetics regulation and bioinformatics fields is one of the promising areas for the treatment of diseases in the future.
With this review, it is aimed to present the studies carried out for the use of bone marrow stem cell-derived extracellular vesicles
loaded with targeted miRNAs in different in vivo and in vitro human disease models and to discuss recent developments in this
field.
Keywords Bone marrow Mesenchymal stem cells .miRNA modified BMSCs .Extracellular vesicles .miRNA .Experimental
animal models .Cancer .Spinal cord injury .Fibrosis .Rheumatoid Arthiritis .Myocardial infarction .Cerebral ischemia
Abbreviations
3’UTR 3′-Untranslated Region
ADAM9 ADAM Metallopeptidase Domain 9
ADAMTS A Disintegrin And Metalloproteinase
With Thrombospondin Motifs
AGAP2 ArfGAP With GTPase Domain, Ankyrin
Repeat And PH Domain 2
AGEs Advanced Glycation end Products
AGO2 Argonaute-2
Akt Protein Kinase B
ALI Acute Lung Injury
alpha-SMA α-Smooth Muscle Actin
AMH Anti Müllerain Hormon
AMI Acute Myocardial Infarction
AML12 Alpha Mouse Liver 12
ASK1 Signal-Regulating Kinase 1
Bad Bcl-2-Antagonist Of Cell Death Protein
BALF Bronchoalveolar Lavage Fluid
Bax Bcl-2 Associated X Protein
BBB Basso Beattie Bresnahan
This article belongs to the Topical Collection: Special Issue on Exosomes
and Microvesicles: from Stem Cell Biology to Translation in Human
Diseases
Guest Editor: Giovanni Camussi
*Zafer Cetin
zcetin@sanko.edu.tr
1
Department of Medical Biology, School of Medicine, SANKO
University, Gaziantep, Turkey
2
Department of Biological and Biomedical Sciences, Graduate
Education Institute, SANKO University, Gaziantep, Turkey
3
Department of Medical Biochemistry, School of Medicine, SANKO
University, Gaziantep, Turkey
4
Department of Molecular Medicine, Graduate Education Institute,
SANKO University, Gaziantep, Turkey
5
Department of Medical Biology, School of Medicine, Van Yüzüncü
Yil University, Van, Turkey
6
Department of Biophysics, School of Medicine, KOÇ University,
Istanbul, Turkey
7
Research Center for Translational Medicine, KOÇ University,
Istanbul, Turkey
Stem Cell Reviews and Reports
https://doi.org/10.1007/s12015-020-10082-x

Bcl-2 B-Cell CLL/Lymphoma 2
BIP Binding-Immunoglobulin Protein
BMD Bone Mineral Density
BMECs Brain Microvascular Endothelial Cells
BMP-2 Bone Morphogenetic Protein 2
BMS Basso Mouse Scale
BMSCs Bone Marrow Mesenchymal Stem Cells
CaMKII Calcium/Calmodulin-Dependent
Protein Kinase II
CCNE1 Cyclin E1
CD Clusters of Differentiations
CDK-4 Cyclin Dependent Kinase 4
CFA Complete Freund’s Adjuvant
COL10A1 Collagen type X alpha 1 chain
COL2A1 Collagen Type II Alpha 1 Chain
COL4A1 Collagen Type IV Alpha 1 Chain
COL9A1 Collagen Type IX Alpha 1 Chain
COMP Cartilage Oligomeric Matrix Protein
COX2 Cyclooxygenase-2
CREB CAMP Responsive Element
Binding Protein
CTGF Connective Tissue Growth Factor
CTX Cyclophosphamide
CYP2J2 Cytochrome P450 2J2
DCX Doublecortin
DDIT4 DNA Damage Inducible Transcript 4
DFO Deferoxamine
DGCR8 DiGeorge Syndrome Critical
Region gene 8
DMBA 7,12-Dimetilbenz [a] antrasen
DMOG Dimethyl Oxaloylglycine
E2 Estradiol
EBV Epstein-Barr Virus
ECM Extracellular Matrix
EGFR Epidermal Growth Factor Receptor
EGM-2 Endothelial Cell Growth Medium
EMT Epithelial Mesenchymal Transition
ERK Extracellular Signal-Regulated Kinase
ESCRT Endosomal Sorting Complex Required
for Transport”
ESM1 Endothelial Cell-Specific Molecule-1
EZH2 Enhancer of Zeste Homolog 2
FasL Fas Ligand
FFT Foot False Test
FLS Fibroblast-Like Synoviocytes
FOXA2 Forkhead Box protein A2
FSH Follicle Stimulating Hormone
FSP1 Ferroptosis Suppressor Protein 1
GAP43 Growth Associated Protein 43
GFAP Glial Fibrillary Acidic Protein
Gli3 GLI Family Zinc Finger 3
GSK-3 beta Glycogen Synthase Kinase 3 beta
GVHD Graft Versus Host Disease
H/R Hypoxia/Reoxygenation
HDAC3 Histone Deacetylase 3
HGF Hepatocyte Growth Factor
HHS Histological Hepatitis Score
HIF1-αHypoxia-Inducible Factor 1-Alpha
HSP Heat Shock Protein
HUVECs Human Umbilical Vein Endothelial Cells
I/R Ischemia/Reperfusion
ICHÇ Intracerebral Hemorrhage
IkB-alpha IkappaBalpha
INPP4B Inositol Polyphosphate-4-Phosphatase
Type II B
IRF2 Interferon Regulatory Factor 2
ITGA2 Integrin Subunit Alpha 2b
IVD Intravertebral Disc
iNOS Inducible Nitric Oxide Synthase
JNK JUN N-Terminal Kinase
KDR Kinase Insert Domain Receptor
KGF Keratinocyte Growth Factor
KIM1 Kidney Injury Molecule 1
LAD Left Anterior Descending Coronary
Artery Ligation
LCN2 Lipocalin2
LDH Lactate Dehydrogenase
LH luteinizing Hormone
LMP1 Latent Membrane Protein 1
LPS Lipopolysaccharide
LVEDD Left Ventricular End Diastolic Dimension
LVEDP Left Ventricular End Diastolic Pressure
LVEF Left Ventricular Ejection Fraction
LVESD Left Ventricular End Systolic Dimension
LVFS Left Ventricular Fractional Shortening
LVSP Left Ventricular Systolic Pressure
MAPK Mitogen-Activated Protein Kinase
MCAO Middle Cerebral Artery Occlusion
MCP-1 Monocyte Chemotactic Protein 1
MDA Malondialdehyde
MDI Motor Deficit Index
MI Myocardial Infarction
MLS Macrophage-Like Synoviocytes
MMP14 Matrix Metallopeptidase 14
mNSS Modified Neurologic Severity Score
MPO Myeloperoxidase
MSCs Mesenchymal Stem Cells
mTOR Mammalian Target of Rapamycin
MVBs Multi-Vesicular Bodies
ncRNA noncoding RNAs
NeuN Neuronal Nuclei
NF Neurofibromin
NF-200 Neurofilament 200
NFH Neurofilament Heavy
NF-kB Nuclear Factor-kappa B
NLRP3 NLR Family Pyrin Domain Containing 3
Stem Cell Rev and Rep

NOX4 NADPH Oksidaz 4
NPC Neuroprogenic Cells
NSCLC Non-Small Cell Lung Cancer
OA Osteoarthiritis
OGD Oxygene Glucose Deprivation
OPMD Oral Potentially Malignant Disorders
P38MAPK P38 MAP Kinase
PACT Protein Activator of
Interferon-Induced Protein Kinase
PARP Poly (ADP-ribose) Polymerase
PCNA Proliferating Cell Nuclear Antigen
PDCD4 Programmed Cell Death 4
Peli1 Pellino-1
PGE2 Prostaglandin E2
PI3K Phosphoinositide 3-Kinase
POF Premature Ovarian Failure
pre-miRNA Precursor miRNA
pri-miRNAs Primary miRNAs
PTEN Phosphatase And Tensin Homolog
PTGS2 Prostaglandin-Endoperoxide Synthase 2
RA Rheumatoid Arthritis
RABEPK Rab Effector Protein with Kelch Motifs
RAC2 Rac Family Small GTPase 2
RASA1 RAS P21 Protein Activator 1
RhoA Ras Homolog Family Member A
RHPN2 Rhophilin Rho GTPase Binding Protein 2
RISC RNA Induced Silencing Complex
ROS Reactive Oxygen Species
RUNX2 RUNX Family Transcription Factor 2
RVG+Lamp2b) Rabies Virus Glycoprotein +
Lysosome-Associated
Membrane Glycoprotein 2b
SAA3 Serum Amyloid A3
SAH Subarachnoid Hemorrhage
SCI Spinal Cord Injury
SCID Severe Combined Immunodeficiency
SEMA3A Semaphorin 3A
SIRT7 Sirtun 7
SMSCs Synovial Mesenchymal Stem Cells
SNAIL1 Snail Family Transcriptional Repressor 1
SOD Superoxide Dismutase
SOX2 SRY-Box Transcription Factor 2
SOX9 SRY-Box Transcription Factor 9
SP1 Specificity Protein 1
STAT3 Signal Transducer And Activator Of
Transcription 3
TFF3 Trefoil Factor-3
TGF-beta 1 Transforming Growth Factor Beta 1
TGF-beta Transforming Growth Factor Beta
TGF-BR1 Transforming Growth Factor Beta (TGF-
beta) Receptor Type 1
TNBS 2,4,6-Trinitrobenzene Sulfonic Acid
TNF-alpha Tumor Necrosis Factor Alpha
TRBP Transactivation Response Element RNA-
Binding Protein
Trx Thioreductin
TXNIP Thioreduxin- Interacting Protein
UUO Unilateral Ureteral Obstruction
VEGFA Vascular Endothelial Growth Factor A
VEGFR Vascular Endothelial Growth Factor
Receptor
VPA Valproic Acid
VSMC Vascular Smooth Muscle Cells
WNT5A Wnt Family Member 5A
XPO5 Exportin-5
ZEB Zinc Finger E-Box Binding Homeobox
Introduction
Mesenchymal Stem Cells (MSCs) are multipotent stem cells
that remain in adult tissues, have immunomodulatory effects,
homing properties and capacity of MSC to differentiate into
cells from all three germ layers significantly augment the re-
generative capacity of many tissues [1,2]. MSCs can be iso-
lated from almost all tissues including adipose tissue, umbil-
ical cord, dental pulp etc. [3]. Several preclinical studies dem-
onstrated that implanted MSCs can home to the injured tissue
and differentiate to functional cells to replace damaged cells.
However, only a low percentage of implanted MSCs survive
in vivo and engraftments in injured tissues. This strongly sug-
gests that therapeutic effects of MSCs might be attributed to
their secretory activity rather than their tissue-homing and
differentiation capacity [4,5].
MSCs are commonly used in clinical trials for hematolog-
ical pathologies, graft versus host disease (GVHD), cardiovas-
cular, neurological, bone and cartilage, chronic inflammatory
and autoimmune diseases, liver, lung, and kidney injury, and
organ transplantation [6–10].
Although MSCs exhibits great potential in the treatment of
various diseases, MSC transplantation based therapy is limited
by cell-related disadvantages including: phenotype drifting,
infusional toxicity caused by the large cells physically trapped
in the lung or kidney microvasculature, immunological rejec-
tion, genetic variation, carcinogenic risk, complicated opera-
tion steps, uncontrolled action of implanted cells, operation
costs and cell storage difficulties [11–13]. Also it has been
proposed that another limitation factor for MSC transplanta-
tion based therapies is low survival rate of MSCs in the host as
a result of hypoxia, inflammatory cytokines, and proapoptotic
factors [14,15].
Researchers are focused on different strategies to enhance
the MSC functions and to minimalize the limitations. First
approach for enhancement of MSC functions for tissue engi-
neering and regenerative medicine was preconditioning of
MSCs. The conditions that have shown improvements in
Stem Cell Rev and Rep

MSC transplantation success include: cytokines, hypoxia, tro-
phic factors and physical factors. Preconditioning with other
chemical and biological factors mimic the effect of the hyp-
oxia include: Valproic Acid (VPA), CoCl2, Deferoxamine
(DFO), and Dimethyl Oxaloylglycine (DMOG) [16,17].
Second appoach was co-administration of MSCs with adju-
vant agents such as Melatonine which has anti-inflammatory
and antioxidant properties [14]. More recently, in vivo studies
demonstrated that the therapeutic benefit of MSCs is princi-
pally orchestrated by extracellular vesicles (EVs) secreted by
MSCs. Therefore researchers are focused on third approach
which is based on transplantation of extracellular vesicles in-
cluding secreted by MSCs instead of direct MSCs injection
[18,19]. For example, Lui et al., showed that MSC-derived
(EVs) provide protection similar to that of MSCs against in-
testinal ischemia-reperfusion-mediated acute lung injury [20].
Bone Marrow Mesenchymal Stem Cells (BMSCs) are
multipotent cells that can differentiate into various cell types,
such as chondrocytes, osteoblasts, and adipocytes [21]. They
are characterized based on their plastic adherence properties:
tri-lineage differentiation potential including osteogenic,
adipogenic, and chondrogenic lineages and expression of spe-
cific Clusters of Differentiations (CD) including CD90 and
CD105, besides CD17, CD29, CD44, and CD106, while lack-
ing in the expression of hematopoietic stem cell markers in-
cluding CD14, CD19, CD31, CD34, CD133 and Kinase
Insert Domain Receptor (KDR) [3]. Relatively simple cultiva-
tion, rapid proliferation, high genetic stability, multidirection-
al differentiation capacity and low immunogenicity properties
makes BMSCs preferred in terms of their usability in the treat-
ment of various diseases [22].
Extracellular Vesicles
The term exosomes is commonly utilized for Multi-Vesicular
Bodies (MVBs)-derived extracellular vesicles [23]. Based on
the ISEV consensus conference’s suggestion to use the term
EVs for all vesicles released from the cell, this publication
uses the termextracellular vesicle instead of the term exosome
[24]. EVs could be isolated from almost all biological fluids,
such as blood, sperm, milk and urine. As a consequence of
their endosomal origin, nearly all EVs contain proteins in-
volved in membrane transport and fusion (Rab GTPases,
Annexins, Flotillin), MVB biogenesis (Alix and TSG101),
Heat Shock Proteins (HSP70 and HSP90), integrins and
tetraspanins (CD63, CD9, CD81 and CD82) [25]. Although
the exact mechanism underlying their biogenesis remains an
area of intense investigation, it is generally considered that
they can originate from an “Endosomal Sorting Complex
Required for Transport”(ESCRT)-dependent or ESCRT-
independent mechanisms [3]. EVs are small nanosized mem-
brane vesicles with diameter of 30–100 nm, released by all
kinds of cells into the extracellular environment. EVs are
thought to function as intercellular communication vehicles
to transfer biomolecules to the target cells including receptors,
ligands, cytokines, lipids, mRNAs, miRNAs, noncoding
RNAs (ncRNA), ribosomal RNAs and genomic DNA be-
tween cells to elicit biological responses in recipient cells [1,
26]. EVs have been demonstrated to be an important mode of
cellular communication as they are involved in multiple phys-
iologic and pathologic functions, including proliferation, ap-
optosis, inflammation and tissue regeneration [27]. Stem cell-
derived EVs have similar effects as the stem cells from which
they originate in relation to the issues outlined below. 1) stem
cell-derived EVs do not cause overt immune reactions, as they
have a suppressive effects on T lymphocytes, natural killer
cells, dendritic cells and B lymphocytes, like the cells from
which they originate when they administered to xenogenic
animals [28], 2) intrinsic homing capabilities similar to their
parental cell type related to membrane bound integrin and
connexins [29,30]. In addition to these, stem cell derived
exosomes have some superior aspects compared to stem cells
from which they originate; 1) Exosomes have no risk of an-
euploidy because they dont contain chromosomes [31], 2)
when stored in proper conditions and thawed, their efficiency
is higher compared to the cell from which they originated,
because they are not living structures [32], 3) They have a
lower immune rejection potential compared to the cells from
which they originated due to the smaller amount of protein
attached to the extracellular vesicle membrane [33], 4) Stem
cells cannot cross the blood brain barrier while extracellular
vesicles can cross the blood brain barrier freely [34]5) EVs
that are very small in size are more stable in circulation as they
are saved from being broken down by mononuclear phago-
cytes [35]. EVs may show immunogenicity depending on the
surface antigens of the cells from which they originate [36].
MSC-derived EVs are normally hypo-immunogenic due to
the lack of MHC-II and low expression of MHC-I similar to
their parental cells [37].
Efficiency of BMSC Derived Extracellular
Vesicles (EVs) in Animal Disease Models
Recent studies suggest that MSCs and MSC derived EVs
yield similar therapeutic benefits in various conditions includ-
ing repairing damaged tissues, gene theraphy, inhibiting in-
flammatory reaction and regulating immune responses [27,
38]. MSCs produce EVs, and many regenerative properties
previously credited to stem cells may actually be attributed
to secreted EVs [17]. A growing number of researchs regard
transplantation of EVs as a potential alternative to stem cell
transplantation [11]. Specific surface ligands on EVs can bind
to target cells, allowing EVs to deliver RNAs, proteins or
cytokines into the target cells to stimulate particular biological
Stem Cell Rev and Rep

functions [39]. Due to their many excellent attributes includ-
ing wide distribution in biological fluids, intrinsic homing
capability and penetrable to blood brain barrier, EVs undoubt-
edly could be an ideal drug delivery vehicles [40].
Recently, MSCs-derived EVs have been identified as a
new therapeutic strategy for immunomodulation in autoim-
mune related disorders, regenerative therapies, anti-tumor
therapy, and drug delivery [41]. EVs derived from different
cells contain different contents and influence the response of
the other cells and play important role in cell-to-cell commu-
nication [42,43]. Meckes et al.,demonstrated the presence of
various growth factor signaling pathway components and on-
cogenic protein and miRNAs encoded by the virus in extra-
cellular vesicles obtained from nasopharyngeal carcinoma
cells infected with Epstein-Barr Virus (EBV) or transfected
with the Latent Membrane Protein 1 (LMP1) gene responsible
for the oncogenic characteristic of EBV. In addition, they
determined that these EVs have the ability to modulate the
tumor microenvironmentby fusing with endothelial and fibro-
blast cells and transferring these components to the host cells
[44]. Recent studies indicated that BMSC-derived EVs are
efficacious in animal models of erectile dysfunction, spinal
cord injury, hypoxic-ischemia, cerebral ischemia, cutaneous
wound healing, kidney diseases, liver injury, myocardial
ischemia/reperfusion (I/R) injury, healing of diabetic skin de-
fects and osteochondral injury [5,13,14,27,45–52]. In this
regard, EVs are considered as suitable carriers for RNA inter-
ference techniques applied to achieve gene modulation in tar-
get cells [38,53].
miRNAs
MicroRNAs are endogenous small non-coding RNA mole-
cules with 18–25 nucleotides in length that regulate gene ex-
pression based on complementarity between the 5′region of
the microRNA and the 3′-Untranslated Region (3’UTR) of
the target mRNA [54]. They are transcribed as primary
miRNAs (pri-miRNAs) by RNA Pol II and Pol III in the
nucleus and contain an m7G Cap at the 5′end, and a poly
(A) tail at the 3’UTR region [55]. These pri-miRNA tran-
scripts are processed in the nucleus by the nuclear RNase III
enzyme (DROSHA) and its essential cofactor DiGeorge
Syndrome Critical Region gene 8 (DGCR8/Pasha) [56].
Within the pri-miRNA, DGCR8 binds at the N6-
methyladenylated GGAC motifs and DROSHA cleaves the
pri-miRNA duplex to produce a ~ 70 nucleotides in lenght
hair-pin shaped structure called precursor miRNA (pre-
miRNA). A transporter complex consists of Exportin-5
(XPO5) and Ran-GTPase carries pre-miRNAs to the cyto-
plasm [57]. In the cytoplasm, a micro-scissor called Dicer
coupled with the Transactivation Response Element RNA-
Binding Protein (TRBP) and Protein Activator of Interferon-
Induced Protein Kinase (PACT) proteins removes the terminal
loop to produce mature miRNA: miRNA duplex [56].
Helicase unwind miRNA::miRNA complex and develops ma-
ture miRNA molecule which participating in formation of
RNA Induced Silencing Complex (RISC) comprising of
Argonaute-2 (AGO2) protein. The mature miRNA name is
determined by the directionality of the miRNA strand. For
instance, the 5p form of a mature miRNA originates from
the 5′endwhilethe3pstrandarisesfrom3′end of the pre-
miRNA. The critical region in a miRNA comprises nucleo-
tides between 2 and 7 from the 5′end so-called ‘seed’region.
This region make Watson–Crick base pairing with 3’UTR
region of target mRNA. Upon binding of mature miRNA to
3’UTR region of target mRNA molecule RISC complex can
induce the suppression of mRNA translation or the mRNA
degradation [54,58]. miRNAs have regulatory functions in
many biological and pathological processes including devel-
opment, differentiation, angiogenesis, proliferation, apoptosis,
and immune responses etc. The biological and physiological
effects of miRNAs through epigenetic mechanisms suggest
that they can be used in the treatment of various pathological
conditions [59].
Artificial miRNA Expression Systems
The first step of artificial miRNA synthesis for a target gene is
the prediction of the gene targeted by the miRNA. There are
different target prediction tools which use different data min-
ing algorithms. These databases often use algorithms trained
by high throughput microarray data or crosslinking and im-
munoprecipitation (CLIP) sequencing data [60,61]. During
artificial miRNA synthesis, the mature miRNA sequence de-
signed for the target gene is placed in a ready-made stem loop
back bone [62]. There are different expression cassette sys-
tems for increasing the expression of miRNAs in transferred
cells, and these are artificial miRNA’s (in the form of precur-
sor miRNA or primary miRNA forms) and short hairpin-like
miRNA expression cassettes [63].ThedeliveryofmiRNAsin
the form of shRNA has an off target effect, disrupts endoge-
nous miRNA machinery and activate interferon responce in
the host cell while the delivery of miRNAs in the form of
artificial miRNA results in high target gene silencing efficien-
cy compared to short hairpin form [64,65]. Although different
backbone structures can be used to produce artificial miRNA,
validated human miRNA libraries have been prepared using
miR-30 backbone. The first generation miRNA backbones
have pre-miRNA structure while the second generation
miRNA backbones have pri-miRNA structure whose three-
dimensional structures are more similar to natural miRNAs
[65]. Both short hairpin and artificial miRNAs can be synthe-
sized from RNA Polymerase II and RNA polymerase III pro-
moters. However, RNA Polymerase II promoters are preferred
Stem Cell Rev and Rep

promoters because they are inducible and tissue-specific pro-
moters [66].
MiRNA transfection to cells is not a standardized method
and different tools and methods can be used for this purpose.
Viral and non-viral methods can be applied to transfer miRNA
to cells. Adenovirus, adeno-associated virus, lentivirus and
retroviruses can be used as vectors for transferring miRNAs
into cells. Among these vectors, differences are observed in
terms of factors such as vector titers, efficiency, insert capac-
ity, toxicity, immunogenicity, genome integration, insertional
mutagenesis, expression time and tropism [67]. It is important
to select the appropriate vector to be used for miRNA transfer,
considering factors such as whether the expression of miRNA
to be transferred is long-term, whether non-dividing cells are
desired to be infected, and whether the innate immune system
is activated or not [68]. Of these viral vectors, Retroviruses
can only transfer miRNA to cells that can divide, while
Adenoviruses, AAVs, and Lentiviruses can deliver miRNA
to both dividing and non-dividing cells. Adenoviruses are
double stranded DNA viruses and cause transient gene expres-
sion since they are not capable of integrating transferred gene
fragments into the host’sgenome[69]. Another disadvantage
of adenoviral vectors is that they are highly immunogenic.
The most important disadvantage of adeno-associated virus
vectors is the need for a helper virus to reproduce [70].
Among them, lentivirus-based vectors came to the fore be-
cause it is known that its integration into the genome and its
long-term expression are safe when administered in humans
[71]. Apart from these, there are various non-viral approaches
that can be used to transfer miRNA to cells including; lipid-
based nanocarriers, polymeric vectors/dendrimer-based
vectors, cell-derived membrane vesicles, inorganic
material-based and 3D scaffold-based delivery systems
[72]. Although liposomes are the most commonly used
non-viral artificial miRNA transfer tools in in vitro
studies, they have disadvantages such as unwanted im-
mune responses and toxic effects [73]. Depending on
which approaches given above are used, the level of
miRNA expression to be obtained in target cells and
the effect of gene suppression may differs.
In the literature, there are also publications examining the
effects of miRNA loaded adipose tissue, Wharton’sJelly,cho-
rionic plate, endometrium, umbilical cord mesenchymal stem
cell-derived in vitro and in vivo disease models on gene ex-
pression [74–78]. However, most of the studies in this area
have been carried out using BMSC EVs, since BMSCs
are the most well-enlightened mesenchymal stem cells.
Therefore, in this review, it is aimed to present the
studies that have been performed using miRNA loaded
BMSC EVs so far and to provide implications for the
studies planned to be carried out in the future. Recent
developments in this field will be discussed in subse-
quent sections (Table 1).
Experimental Applications of miRNA
Modified BMSC Derived EVs in Diseases
miRNA Modified BMSC Derived EVs in Cancer Therapy
miRNA loaded BMSC EV treatment approach is evaluated
for glioblastomas, multiple myeloma, acute myeloid leukemia
(AML), head and neck cancers, pancreatic, colorectal prostate,
bladder and lung cancers. These studies will be discussed
under the following subheadings.
miRNA Modified BMSC Derived EVs in Glioblastoma
miRNA loaded BMSC EV treatment approach is evaluated
for glioblastomas in different studies. Epidermal Growth
Factor Receptor (EGFR) gene amplification is a highly fre-
quent genetic change in glioblastomas (approximately 40%)
and upregulated EGFR expression is associated with the in-
vasiveness of cancer cells. One of the miRNAs targeting
EGFR mRNA, miR-146b is down-regulated in gliomas and
its transfection reduces glioma cell invasion, migration, via-
bility and EGFR expression. [79]. Intratumor administration
of miR-146b modified rat BMSC EVs in Fischer rats with
human primary brain tumor xenograft model resulted in a
decrease in tumor volume and cell growth. Treatment of rat
glioma cell line 9 L with these EVs resulted in a decrease both
in EGFR and Nuclear Factor-kappa B (NF-kB) expression
[80]. Another important miRNA in glioma is miR-124a which
suppresses aberrant glioma cell proliferation [81]. Based on
this knowledge, the researchers decided to examine the effi-
ciency of transferring miR-124a to glioblastoma cells via
BMSC EVs, which they determined to have a binding site in
the 3’UTR region of Forkhead Box protein A2 (FOXA2)
mRNA by bioinformatics analysis. Intraarterial administration
of miR-124a modified BMSC EVs in athymic nude micewith
intracranial glioblastoma stem cell line xenograft model re-
sulted in a decrease in FOXA2 expression. Treatment of glio-
blastoma stem cell lines GSC276 and GSC6–27 with these
EVs resulted in a reduction in FOXA2 expression,
clonogenity, cell viability and proliferation while an increase
in apoptotic marker cleaved Poly (ADP-ribose) Polymerase
(PARP) expression. These results demonstrated the anti-
proliferative role of miR-124a through the targeted degrada-
tion of FOXA2 followed by modulation of lipid metabolism
and activation of PARP [82]. miR-584 is a major tumor sup-
pressor miRNA in various cancers and inhibits tumor cell
growth, invasion and metastasis. As a result of their bioinfor-
matic analysis, the researchers determined that miR-584 has a
binding site in the 3’UTR region of Cytochrome P450 2 J2
(CYP2J2) mRNA and investigated the effects of transferring
this miRNA via BMSC-derived EVs on glioblastoma.
Administration of miR-584-5p modified commercial BMSC
EVs in athymic nude mice with subcutaneous human primary
Stem Cell Rev and Rep

Table 1 Results of miRNA loaded BMSC extracellular vesicles for therapoutic purposes in in vitro and in vivo disease models
Model
organism
BMSC
Source
Loaded
miRNA
Administration
Method
Animal Disease
Model
In vivo Gene
Expression
Analysis
Administration
Results
Clinical
Evaluation
In vitro Model In vitro Gene
Expression
Analysis
Reference
Fischer rats Rat Bone
marrow
miR-146b Intratumor Primary brain
tumor xenograft
model
NA Tumor volume↓
Cell growth↓
NA 9 L cells EGFR↓
NF-kB↓
(80)
Atymic
nude
mice
Commercial miRNA-124a Intra-arterial Intracranial GSC
xenograft
FOXA2↓Viability↓
Proliferation↓
Clonogeneity↓
Longevity↑GSC276
GSC6–27
FOXA2↓
Cleaved
PARP↑
(82)
Atymic
nude
mice
Commercial miRNA-584-5p NA Subcutaneous
xenograft
NA Tm growth↓
Tm weight↓
Proliferation↓
Migration↓
Apoptosis↑
NA U87 CYP2J2↓
Bcl-2↓
Bax↑
P-Akt↓
P-MAPK↓
(83)
Nude mice C57BL/6
Femur and
Tibia
miRNA-133b Intraperitoteal (Subcutaneous U87)
xenograft tumor
model
EZH2↓
Wnt↓
P-GSK-3B↓
B-Catenin↓
GSK-3B↑
Tumor volume↓
Tumor weight↓
Tumorigenesis↓
NA U87 EZH2↓
Wnt↓
P-GSK-3B↓
B-Catenin↓
GSK-3B↑
(86)
NA Human
primary
BMSC
miR-199a NA NA NA Proliferation↓
Invasion↓
Migration↓
Apoptosis↑
NA U251 AGAP2↓(87)
NA Human
BMSC
miR-222-3p NA NA NA Proliferation↓
Apoptosis↑
NA THP-1 IRF2↓
INPP4B↓
(88)
BALB/c
nude
mice
Old Human
BMSC
miR-340 matrigel plug assay
administration
BALB/c nude mice
Subcutaneous
matrigel plug
assay RPMI8226
cell line
NA Angiogenesis↓
Proliferation↓
Tube formation↓
NA HUVECs
RPMNI8226-HR
c-MET↓
HGF↓
(89)
Syrian
golden
hamsters
C57BL/6 miR-185 Buccal mucosa DMBA induced
OPMD
IL1B↓
IL-16↓
IL-6↓
IL10↑
P-Akt↓
P-NF-kB↓
PCNA↓
VEGFA↓
Caspase3↑
Caspase9↑
Inflammatory
exudation↓
White patch size↓
Inflammatory cell
Infiltration↓
Hyperplasia↓
Proliferation↓
Microvascularisation↓
Apoptosis↑
NA NA NA (91)
BALB/c
nude
mice
Human
primary
BMSC
miRNA-101-3p Intratumor (Subcutaneous
TCA8113)
xenograft tumor
model
COL10A1↓Invasion↓
Migration↓
Colony forming
ability↓
Tumor volume↓
Tumor weight↓
NA TCA8113 COL10A1↓(92)
Athymic
BALB/c
Human
BMSC
miR-1231 Tail IV NA Proliferation↓
Cell cycle arrest↑
Tumor size↓
Tumor weight↓
BxPC3
PANC-1
EGFR↓
Cyclin E↓
(94)
Stem Cell Rev and Rep

Table 1 (continued)
Model
organism
BMSC
Source
Loaded
miRNA
Administration
Method
Animal Disease
Model
In vivo Gene
Expression
Analysis
Administration
Results
Clinical
Evaluation
In vitro Model In vitro Gene
Expression
Analysis
Reference
Nude mice
(SJA)
(Subcutaneous
BxPC3) xenograft
tumor model
Migration↓
Adhesion↓
Tumor volume↓
BALB/c mice Human
primary
FSF cells
miRNA-126-3p NA (Subcutaneous
PANC-1)
xenograft tumor
model
COX2↓
ADAM9↓
MMP14↓
Metastasis↓
Proliferation↓
Migration↓
Invasion↓
Apoptosis↑
Tumor size↓
Growth rate↓
PANC-1 ADAM9
Ki67
VEGF
COX2
MMP14
(95)
BALB/c nude
mice
Commercial miRNA-16-5p Ex vivo (Subcutaneous
Caco2)
xenograft tumor
model
ITGA2↓Proliferation↓
Invasion↓
Migration↓
Apoptosis↑
Tumor growth↓
Tumor volume↓
CaCo2 ITGA2↓
MMP2↓
MMP9↓
(96)
BALB/c nude
mice
Femur and
tibia
miR-9-3p Tail vein (Subcutaneous
UMUC-3)
xenograft
tumor model
ESM1↓
MMP2↓
MMP9↓
Viability
Migration
Invasion
Apoptosis
Tm weight ↓
Tm volume↓
Liver metastasis↓
Lymph node
meastasis↓
UMUC-3 ESM1↓
Ki-67↓
PCNA↓
MMP2↓
MMP9↓
(98)
Nude Mice Human
primary
bone
marrow
miRNA-205 Tail vein (Subcutaneous
LNCaP)
xenograft tumor
model
RHPN2↓
MMP2↓
MMP9↓
Tumor volume↓
Tumor weight↓
Proliferation↓
Invasion↓
Migration↓
Apoptosis↑
NA LNCaP Ki67↓
PCNA↓
Bcl-2↓
MMP-2↓
MMP-9↓
Bax↑
(100)
BALB/c nude
mice
Commercial miR-143 Before the xenograft (Subcutaneous
PC-3)
xenograft tumor
model
TFF3↓
MMP2↓
Tumor volume↓
Growth ability↓
Invasion↓
Proliferation↓
PC-3 Ki-67↓
PCNA↓
MMP-2↓
MMP9↓
(101)
BALB/c nude
mice
Primary
human
miR144-3p Tail vein IV BALB/c nude
mice
Subcutaneous
xenograft
A549
CCNE1↓
CCNE2↓
Ki67↓
PCNA↓
Tm volume↓
Tm weight↓
Go/G1 arrest↑
S phase arrest↓
Colony formation↓
Proliferation↓
NA A549 CCNE1↓
CCNE2↓
Ki67↓
PCNA↓
(102)
NA Human
commer-
cial
miRNA-143 NA NA NA Migration↓NA 143B ostesarcoma
cel line
NA (103)
BALB/c mice Commercial miRNA-199a-5p Tail vein Renal Ischemia
Reperfusion
model
Caspase3 Apoptosis↓
E.R. stress↓
Urea secretion↓
CREA secretion↓
NRK-52E Caspase3↓
BIP↓
(106)
SCID Commercial miRNA-10
miRNA-486
IV Gliserol induced
AKI Model
BUN↓
Creatin↓
Hyalin cast↓
Renal functions↑
mTEC proliferation↑
Tubular necrosis ↓
NA Hypoxi treated
murine tubular
epithelial cells
NA (107)
BALB/c Human
BMSC
miR-199a-3p Tail IV BALB/c
Bilateral kidney
I/R model
Caspase3↓
SEMA3A↓
Apoptosis↓
Tubular dilation↓
Brush border loss↓
BUN↓
Creatinine↓
Hypoxia
Reoxygenation
induced HK-2
Caspase3↓
Bax↓
Bcl-2 ↑
(108)
Stem Cell Rev and Rep

Table 1 (continued)
Model
organism
BMSC
Source
Loaded
miRNA
Administration
Method
Animal Disease
Model
In vivo Gene
Expression
Analysis
Administration
Results
Clinical
Evaluation
In vitro Model In vitro Gene
Expression
Analysis
Reference
Nuclear loss↓
Cast formation↓
ASK1↓
DDIT4↓
SP1↓
SEMA3A↓
P-Akt↑
P-ERK↑
C57BL/6 mice C57BL/6
BMSC
commer-
cial
miR223-3p IV S100/CFA induced
experimental
Autoimmun
Hepatitis model
STAT3↓
P-STAT3↓
Mononuclear cell
Infiltration↓
Liver injuri↓
ALT↓
AST↓
HSS score↓
IL-1B↓
IL-6↓
IL-17↓
Th17↓
IL10↑
Tregs↑
RAW264.7 IL-1B↓
IL-6↓
STAT3↓
P-STAT3↓
(109)
C57BL/6 Femur and
tibia
miRNA-223 Intraperitoneal S100 induced
autoinflammatory
hepatitis model
NLRP3↓
Caspase1↓
Inflammation↓ALT↓
AST↓
TNF-a↓
IL-1B↓
IL-17a↓
AML12 IL-1B↓
NLRP3↓
Caspase1↓
LDH release↓
(110)
C57BL/6 Commercial
human
BMSC
miR-Let7c IV UOO surgery
model
Kim1↓
COL4A1↓
a-SMA↓
TGF-BR1↓
TGF-B1↓
Tubular daliation↓
Inflammatoty cell ↓
Fibosis markers ↓
Collagen
accumulation↓
NA TGF-B treated
NRK52E cells
COL4A1↓
a-SMA↓
TGF-BR1↓
(111)
SD rats Femur miR-200b Caudal IV TNBS induced
colonic fibrosis
Fibronectin↓
Collagen I↓
Collagen III↓
a-SMA↓
Vimentin↓
E-Cadherin↑
FSP1↓
ZEB1↓
ZEB2↓
Fibrosis↓
Collagen formation↓
EMT↓
Body weight↑
Colon lenght↑
Colon weight↓
IEC-6 E-Cadherin↑
Vimentin↓
ZEB1↓
ZEB2↓
(113)
C57BL/6 Femur and
tibia
miR-30b-3p Jugular vein LPS induced
ALI model
SAA3↓
IL-1B↓
TNF-a↓
IL-6↓
IL-10↑
KGF↑
Ordered alveolar cell
s↑
Alveolar septum
Edema ↓
Alveolar septum
tickening ↓
Hemorrhage ↓
Infiltration ↓
MPO activity ↓
Cell proliferation↑
Apoptosis ↓
NA MLE-12 SAA3↓
P-NF-kB↓
IkB-a↓
ERK↓
MEK1/2↓
P38MARK↓
JNK↓
(114)
Stem Cell Rev and Rep

Table 1 (continued)
Model
organism
BMSC
Source
Loaded
miRNA
Administration
Method
Animal Disease
Model
In vivo Gene
Expression
Analysis
Administration
Results
Clinical
Evaluation
In vitro Model In vitro Gene
Expression
Analysis
Reference
W/D ratio↓
BALF neutrophiles↓
NA Rat femur
and tibia
miR-21 NA NA NA Apoptosis↓NA H2O2 induced Rat
C-kit + cardiac
stem cells
PTEN↓
Caspase-3↓
P-Akt↑
(116)
C57BL/6 C57BL/6
femur and
tibia
miR-132 Ex vivo
intramyocardial
injection
HUVEC+ matrigel
plug assay
subcutaneous
inguinal injection
C57BL/6 model
NA Tube lenght↑
Number of meshes↑
Angiogenesis↑
Blood perfused
vessel↑
Vessel density↑
LVEF↑
FS↑
HUVECs RASA1↓(117)
mice B6129 tibia
and femur
miRNA-21a-5p Pericardial salc LAD MI mice
model
PDCD4↓
PTEN↓
Peli1↓
FasL↓
Caspase3↓
Infarct size↓NA H9c2 PCDC4↓
FasL↓
PTEN↓
(118)
S.D Rat Femur and
tibia
miR-125b Left ventricular ligation
area
Rat LAD I/R
model
SIRT7↓Apoptosis↓
Infarct size↓
Viability↑
Inflammation↓
LVEF↑
LVFS↑
LVSP↑
LVESD↓
LVEDD↓
LVEDP↓
Primary
cardiomyocytes
SIRT7↓
Bax↓
Caspase3↓
Bcl-2↑
IL-1 beta↓
IL-6↓
TNF-alpha↓
(119)
NA S.D. rat
primary
miRNA-214 NA NA NA Apoptosis↓NA C-kit+ primary
cardiac stem
cells
ROS↓
Caspase3↓
Bax↓
Bcl-2↑
MDA↓
SOD↑
CaMKII↓
(120)
S.D Rat Femur and
tibia
miR-301 Periferal area of
myocardial
infarction
Rat LAD
AMI model
LC3-II/LC3-I ↓
P62↑
Autophagy↓LVEF↑
LVFS↑
LVESD↓
LVEDD↓
NA NA (121)
S.D Rat S.D. femur miRNA-133a Myocardial injection Rat LAD AMI
model
SNAIL-1↓
Collagen-1↓
a-SMA↓
Apoptosis↓
Infiltration↓
Fibrosis↓
LVEF↑
SF↑
LV mass↑
LV volume↑
Primary rat
cardiomyocyte
SNAIL-1↓(123)
C57B/L10 Human iliac
crest
primary
miRNA-92a-3p Injected Collagenase
VII induced
O.A model
COL2A1↑
Aggrecan↑
WNT5A↓
MMP13↓
Progression↓
Articular cartilage
damage↓
NA Human OA
primary knee
joint cartilage
chondrocyte
Aggrecan↑
COL2A1↑
SOX9↑
COL9A1↑
COMP↑
COL10A1↓
RUNX2↓
(127)
Stem Cell Rev and Rep

Table 1 (continued)
Model
organism
BMSC
Source
Loaded
miRNA
Administration
Method
Animal Disease
Model
In vivo Gene
Expression
Analysis
Administration
Results
Clinical
Evaluation
In vitro Model In vitro Gene
Expression
Analysis
Reference
MMP13↓
WNT5A↓
Wistar rats Human
ilium
primary
miRNA-26a-5p Intrajoint injection Rat surgical
O.A. model
MMP-13↓
MMP-3↓
PTGS2↓
Proliferation↓
Inflammation↓
Apoptosis↑
IL-1B↓Human primary
synovial
fibroblast
PTGS2↓
Bax↑
Caspase-3↑
Bcl-2↓
IL-6↓
IL-8↓
TNF-a↓
(129)
Wistar rats Wistar rat
femur and
tibia
miRNA-192-5p Articular cavity Collagen II induced
arthritis model
RAC2↓Histopathological
score↓
Clinical score↓
Synovial
Hyperplasia↓
BMD↑
Thickness↑
TRAP↓activity↓
PGE2↓
IL-1B↓
TNE-a↓
NO↓
iNOS↓
Commercial
HFLS-RA
HFLS
RAC2↓(133)
DBA/1 J DBA/1 J
mice
femur and
tibia
miR-150-5p I.P. Collagen Induced
Arthiritis model
CD31↓
MMP14↓
VEGF↓
Angiogenesis↓
Lining thickness↓
Migration↓
Invasion↓
Tube formation↓
Arthritis score↓
Hind paw
thickness↓
Human primary
R.A synovial
fluid FLS
HUVECs
MMP14↓
VEGF↓
(134)
NA Rat BMSC miR-134 NA NA NA Apoptosis↓NA Rat OGD
induced
oligodendrocyte
progenitor cells
Caspase 8↓(136)
S.D. Rats S.D. rat
femur
miR-29b-3p Intracerebroventricular
stereotactic injection
S.D. MCAO
model
PTEN
P_Akt
VEGFA
VEGFR2
CD31
Apoptosis↓
Tube formation↑
Angiogenesis↑
Injured brain
volume ↓
BMECs
Primary cortical
neurons
Bax↓
Caspase-3↓
Bcl-2 ↑
VEGFA↑
VEGFR2↑
(46)
Wistar rats Primary rat
bone
marrow
miRNA-17-92 Intravenous MCAO rat
model
P-NF-H↑
PTEN↓
P-Akt↑
P-mTOR↑
P-GSK-3B↑
Synaptophysin↑
Axon myelin bundle
Density↑
Neurite brunching↑
Dendritic plasticity↑
Oligodendrocyte
number↑
FFT↑
mMSS↑
NA NA (137)
C56BL/6 mice Primary
mice
femur
miRNA-124 Tail I.V. Photothrombosis
ischemia
model
Sox2↓
Nestin↓
Gli3↓
Stat3↓
DCX↑
Neuronal progenitors NA NA NA (139)
Stem Cell Rev and Rep

Table 1 (continued)
Model
organism
BMSC
Source
Loaded
miRNA
Administration
Method
Animal Disease
Model
In vivo Gene
Expression
Analysis
Administration
Results
Clinical
Evaluation
In vitro Model In vitro Gene
Expression
Analysis
Reference
Wistar rat Rat primary miRNA-133b Tail I.V. MCAO Rat
model
intraluminal
vascular
occlusion
NF-200↑
Synaptophysin↑
CTGF↓
RhoA↓
Neurite remodeling↑
Cortical axon
density↑
Adhesive removal
↑
FFT↑
NA NA (140)
C57BL/6 C57BL/6
femur
primary
miRNA-138-5p NA C57BL/6
MCAO model
IL-6↓
IL-1B↓
TNF-a↓
Caspase-3↓
Bax↓
Bcl-2↑
Apoptosis↓
Cerebral infarction↓
LDH content↓
Neuron number↑
Proliferation↑
Migration↑
NA C57BL/6 primary
astrocyte
CDK-4↑
CyclinD1↑
CyclinE↑
Caspase-3↓
Bax↓
Bcl-2↑
(141)
Wistar rat NA miRNA-133b IA Intraluminal
Vascular
Occlusion
MCAO model
P-NFH↑
RABEPK↓
Functional recovery↑
Axonal density↑
Synaptogenesis↑
Synaptic plasticity↑
Exosome release↑
FTT↑
mNSS↑
Primary rat
cortical
astrocyte
RABEPK↓(143)
C57BL/6 mice Mice femur
primary
miR-193-3p Suprachiasmatic
cistern
Subarachnoid
hemorrhage
C57BL/6 mice
HDAC3↓
ac-p65↑
Caspase3↓
Bcl-2↑
IL1B↓
IL6↓
TNF alpha↓
Brain edema↓
FJC + cells↓
BBB permeability ↓
Neuronal scrore↑NA NA (144)
SD rats Femur and
tibia
miR-133b Tail IV Autologous
arterial blood
ICH model
Rho-A↓
P-ERK↑
P-CREB↑
Neuronal apoptosis↓
neurodegeneration↓
NA NA NA (146)
SD rats Femur and
tibia
miR-133b Tail IV Aneurysm clip
SCI model
NeuN↑
GAP43↑
NF↑
RhoA↓
P-ERK1/2↑
P-STAT↑
P-CREB↑
Mature neuron
number↑
Axonal outgrowth↑
Neuronal apoptosis↓
Neurite outgrowth↑
Hindlimb
locomotor
function↑
Lesion area↓
NA NA (147)
SD rats Femur and
tibia
miR-29b Tail IV Striking device
SCI model
NF200↑
GAP43↑
GFAP↓
Neuronal
regeneration↑
BBB score↑
Contarctile nerve
cell ↓
NA NA (148)
SD Rats Human
primary
miRNA-21 Intradiscal IVD degeneration
SD rat model
Caspase-3↓Apoptosis↓
Proliferation↑
IVD degeneration
score↓
TNF-a induced
Human primary
NPC cells
Caspase3↓
Bad↓
Bax↓
PTEN↓
P-Akt↑
Bcl-2↑
(149)
SD Rats Obese rat
primary
miRNA-21 Tail vein SCI SD rat model PDCD↓Apoptosis↓
Lesion cavity↓
BMS score↑NA NA (150)
S.D rats miRNA-25 Intratechal NOX4↓Intact motor neuron↑MDI score↓NA NA (152)
Stem Cell Rev and Rep

Table 1 (continued)
Model
organism
BMSC
Source
Loaded
miRNA
Administration
Method
Animal Disease
Model
In vivo Gene
Expression
Analysis
Administration
Results
Clinical
Evaluation
In vitro Model In vitro Gene
Expression
Analysis
Reference
S.D. rat
commer-
cial
Ischemia
reperfusion
SCI rat model
Il-1B↓
TNF-a↓
MDA↓
SOD↑
S.D. rats Femur and
tibia
miR-544 Tail vein Extradural
compression
SCI rat model
IL-1 alpha↓
TNF- alpha↓
IL-17 beta↓
IL-36 beta↓
BBB score ↑
Spinal cord neuron
survival↑
NA NA NA (153)
C57BL/6 NA miR-644-5p Tail I.V. Cisplatin induced
C57BL/6 POF
model
Caspase3↓
P53↓
Follicle atresia↓
Apoptosis ↓
Corpus luteum↑
E2↑Cisplatin induced
primary mice
granulosa cells
Caspase3↓
P53↓
Bcl2↑
(154)
S.D. Rats S.D. Rat
femur and
tibia
miR-144-5p Intrapertoneally
injected
Cyclophosphamide
induced S.D.
POF model
Caspase 3 ↓
Caspae 9↓
PTEN↓
Attretic follicle↓
Apoptotic cells↓
FSH↓
LH↓
E2↑
AMH↑
CTX induced
primary mice
granulosa cells
PTEN↓
p-Akt↑
(155)
S.D. rats S.D. rat
whole
bone
miR-146a NA NA NA Calsification ↓
Osteogenetic
differentiation↓
NA AGEs and High
Glucose
induced Rat
aortic VSMCs
Runx↓
BMP-2↓
ALP activity↓
TXNIP↓
ROS ↓
MDA↓
SOD↑
(158)
Spraque
Dawley Rat
Human
BMSC
miR26a Caudal vein Corticosterone
induced
depression
Bcl-2↑
Bax↓
SOD↑
MDA↓
LDH↓
TNF-alpha↓
IL1-B↓
Cellular vaculoes↓
Edema↓
Apoptotic neurons ↓
Nissle bodies ↑
Neuron proliferation↑
Apoptosis↓
Rat weight↑
Sucrose preference
index↑
Horizontal
movement↑
Rat primary
hippocampal
neuron
Bcl-2↑
Bax↓
SOD↑
MDA↓
LDH↓
TNF-alpha↓
IL1-B↓
(160)
AKI; Acute Kidney Injury, BBB; Basso Beattie Bresnahan BMEC; Brain Microvascular Endothelial Cells, BMSC; Bone Marrow Mesenchymal Stem cell, CTX; Cyclophosphamide, DMBA; 7,12-
Dimethylbenz[a]anthracene, FFT; Foot False Test HFLS; Human Fibroblast-Like Synoviocytes, HUVECs; Human Umbilical Vein Endothelial Cells, ICH; intracranial hemorrhage, I.V.; Intravenous
IVD; Intravertebral Disc Degeneration, LPS; Lipopolysaccaride, OPMD; Oral Potentially Malignant Disorder, TNBS; Trinitrobenzene sulfonic acid, LAD/AMI; Left Anterior Descending /Acute
Myocardial Infarction, MCAO; Middle Cerebral Artery Occlusion, mNSS; modified Neurologic Severity Score) NPC; Neural Progenitor Cell, O.A.; Osteoarthritis, OGD; Oxygen-Glucose Deprivation,
SCI; Spinal Cord Injury SD; Spraque Dawley rat, UUO; Unilateral Ureteral Obstruction VSMC; Vascular Smooth Muscle Cells
Stem Cell Rev and Rep

glioblastoma cell line U87 xenograft model resulted in a de-
crease in tumor growth and weight. Treatment of U87 with
these EVs resulted in a decrease in antiapoptotic protein B
Cell CLL/Lymphoma 2 (Bcl-2) expression, survival and pro-
liferation pathway proteins CYP2J2, phosphorylated Protein
Kinase B (Akt) and phosphorylated Mitogen-Activated
Protein Kinase (MAPK), proliferation and migration abilities
of the cells while an increase in proapoptotic protein Bcl-2
Associated X Protein (Bax) expression and apoptosis. These
results showed that miR-584-5p transferred via BMSC-
derived EVs has a tumor suppressor role by inducing the deg-
radation of CYP2J2 mRNA in glioma cells [83]. Another
protein that is upregulated in gliomas and associated with
the invasiveness and metastatic properties of the disease is
Enhancer of Zeste Homolog 2 (EZH2), which activates the
Wnt/Beta-Catenin pathway [84]. MiR-133b, which sup-
presses EZH2, has tumor suppressor activity and is down-
regulated in glioblastomas [85]. Intraperitoneal administration
of miR-133b modified tibiafemoral BMSC EVs in C57BL/6
nude mice with subcutaneous human primary glioblastoma
cell line U87 xenograft tumor model and treatment of U87
cell line with these EVs resulted in a decrease in EZH2,
Wnt, phosphorylated Glycogen Synthase Kinase 3 beta
(GSK-3 beta), Beta-Catenin expression, tumor volume and
weight, tumorigenesis while in an increase in GSK-3 Beta
expression. These results demonstrate that miR-133b trans-
ferred via BMSC-derived EVs functions as an antitumorigenic
factor for glioblastoma by inducing degradation of EZH2 and
subsequently inactivating Wnt/Beta-Catenin signaling [86].
Studies in glioma tissue samples and U251, LN229, T98G,
LN-18, SF-539 and A172 glioma cell lines have shown that
ArfGAP With GTPase Domain, Ankyrin Repeat And PH
Domain 2 (AGAP2), which plays a role in the endosomal
pathway, is upregulated, and miRNA-199a is downregulated.
Bioinformatics analysis has shown that AGAP2 mRNA is one
of the targets of miRNA-199a. Treatment of glioblastoma cell
line U251 with miRNA-199a loaded human primary BMSC
EVs resulted in a decrease in AGAP2 expression, prolifera-
tion, invasion, migration while an increase in apoptosis [87].
Although these findings are not supported by in vivo studies
these findings shows that miRNA loaded BMSC EV treat-
ment succesfully suppress brain tumors through targeting
PI3K/Akt, Ras/Raf/MAPK/NFkB, Wnt/Beta-Catenin path-
ways which are activated during glioblastoma development.
miRNA Modified BMSC Derived EVs in Hematological Cancers
This approach also evaluated in multiple myeloma and Acute
Myeloid Leukamia (AML) models. Interferon regulatory fac-
tor family of proteins consists of transcription factors involved
in proliferation, apoptosis, lymphocyte differentiation and he-
matopoietic stem cell development. Interferon Regulatory
Factor 2 (IRF2) binds promotor of Inositol Polyphosphate-4-
Phosphatase Type II B (INPP4B) and enhance its expression.
Activation of IRF2/INPPB4 signaling promotes AML cell
proliferation and survival while inhibits apoptosis.
Bioinformatic analysis revealed that miR222-3p have a bind-
ing site at 3’UTR region of IRF2 mRNA. Treatment of acute
monocytic leukemia cell line THP-1 with miR-222-3p modi-
fied human BMSC EVs resulted in a decrease in IRF2 and
INPP4B expression and proliferation while an increase in ap-
optosis. However, in vivo studies that need to be performed to
support these findings have not been performed by the authors
[88]. Old human BMSCs results in increased angiogenesis in
multiple myeloma extracellular matrix while young human
BMScs are less effective in improvement of angiogenesis.
miR-340 have been differentially expressed between young
and older BMSC EVs. Matrigel plug administration of miR-
340 loaded elder human BMSC EVs in BALB/C nude mice
with hypoxia-resistant RPMI8226-HR multiple myeloma cell
line seeded subcutaneous matrigel plug assay resulted in a
decrease in angiogenesis. Co-culturing of Human Umbilical
Vein Endothelial Cells (HUVECs) and RPMI8226-HR cell
lines with miR-340 loaded old human BMSC EVs resulted
in a decreased expression of c-MET receptor tyrosine kinase
in HUVECs and Hepatocyte Growth Factor (HGF) expression
from RPMI8226-HR cells proliferation, tube formation [89].
miRNA Modified BMSC Derived EVs in Head and Neck Cancers
miRNA modified BMSC EV treatment approach in head and
neck squamous cell carcinoma is applied in two different stud-
ies. The first of these studies was based on the finding that
miR-185 is downregulated in oral squamous cell carcinoma
tissue samples [90]. PI3K/Akt/NF-kB signaling is important
in secretion of proinflammatory cytokines IL-1B and IL-6 and
miR-185 have a binding motif at 3’UTR region of Akt
mRNA and target it for degradation. Administration of miR-
185 modified C57BL/6 BMSC EVs in Syrian golden hamster
with 7,12-Dimetilbenz [a] antrasen (DMBA) induced Oral
Potentially Malignant Disorders (OPMD) resulted in a de-
crease in proinflammatory cytokines IL-1B, IL-16, IL-6, pro-
liferation pathway proteins phospho-Akt, phospho-NF-kB,
proliferating cell marker Proliferating Cell Nuclear Antigen
(PCNA), angiogenesis marker Vascular Endothelial Growth
Factor A (VEGFA), inflammatory exudation, white patch
size, inflammatory cell infiltration, hyperplasia, proliferation
and microvascularisation while an increase in anti inflamma-
tory cytokine IL-10, apoptosis associated proteins Caspase-3
and Caspase-9 and apoptosis. These findings suggest that
miR-185 acts as an anti-inflammatory molecule in OPMD
by stimulating the destruction of Akt [91]. Second study in
oral cancers was based on the finding that miR-101-3p is
downregulated while Collagen type X alpha 1 chain
(COL10A1) is upregulated in primary oral cancer tissues
and in CAL27, TCA8113, SCC9, SCC25 and HN4 oral cavity
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cancer cell lines. As a result of bioinformatics analysis, re-
searchers who found that miR-101-3p has a binding site in
the 3’UTR region of the COL10A1 mRNA examined the
effect of transferring these miRNAs via BMSC-derived EVs
on subcutaneous tongue squamous cell carcinomas.
Intratumor administration of miR-101-3p modified human
primary BMSC EVs in BALB/c nude mice with subcutaneous
tongue squamous cell carcinoma TCA8113 cell line xenograft
tumor model and treatment of TCA8113 cell line with
these EVs resulted in a decrease in COL10A1 expres-
sion with tumor volume and weight, invasion, migration
and colony forming ability of the cells. miR-101-3p act
its antitumorigenic effect throughtargeteddegradation
of COL10A1 mRNA [92]. These two studies show that
miRNA loaded BMSC EV treatment suppress head and
neck cancers through PI3K/Akt signaling pathway which is
frequently activated in oral cancers and COL10A1 which is
activated in hypoxic contitions.
miRNA Modified BMSC Derived EVs in Gastrointestinal
Cancers
The efficiency of miRNA transfer via BMSC-derived EVs on
pancreatic cancers was investigated in two different studies.
The first of these studies is based on the knowledge that miR-
1231 has inhibitory effects on the proliferation, migration,
invasion and adhesion ability of pancreatic cancer cells [93].
Bioinformatics analyses have shown that miR-1231 has bind-
ing sides in the 3’UTR regions of EGFR and Cyclin E1
(CCNE1) mRNAs. Tail vein administration of miR-1231
loaded human BMSC EVs in athymic BALB/c nude mice
with subcutaneous BxPC3 cell line xenograft tumor model
resulted in a decrease in tumor weight and volume.
Treatment of BxPC3 and PANC-1 cell lines with these EVs
resulted in a decrease in EGFR, CCNE1 expressions, prolif-
eration, migration, adhesion while in an increase in cell cycle
arrest. These findings suggest that miR-1231 has
antitumorigenic activity for pancreatic cancer by inducing
the degradation of EGFR and CCNE1 mRNA molecules
[94]. miR-126 is another poorly expressed miRNA in pancre-
atic cancer patient tissue samples and pancreatic cell lines
including: PANC1, SW1990, Capan-1, asPC-1, PC-3 and
MIAPaCa. miR-126-3p negatively regulates ADAM
Metallopeptidase Domain 9 (ADAM9) expression and in-
hibits invasion and metastasis of pancreatic cancers.
ADAM9 is a matrix metalloproteinase which activates
proligands of EGFR through cutting them. miR-126-3p have
a binding site at 3’UTR region of ADAM9 mRNA.
Administration of miRNA-126-3p modified human primary
BMSC EVs in BALB/c mice with subcutaneous pancreatic
ductal carcinoma cell line PANC-1 xenograft tumor model
resulted in a decrease in Cyclooxygenase-2 (COX2),
Extracellular Matrix (ECM) degradation markers ADAM9
and Matrix Metallopeptidase 14 (MMP14) expression, metas-
tasis, tumor size and growth rate. Treatment of PANC-1
cell line with these EVs resulted in a decrease in pro-
liferation marker Ki67, angiogenesis marker VEGF and
COX2, ADAM9, MMP14, proliferation, migration, inva-
sion while an increase in apoptosis. miR126-3p act its
antitumorigenic activity through targeted degradation of
ADAM9 [95]. These results show that destruction of EGFR
signaling pathway components by miRNA transfer via
BMSC-derived EVs may be effective in the treatment of pan-
creatic cancer.
There is only one study to investigate the effectiveness of
miRNA transfer via BMSC-derived EVs on colorectal can-
cers. This study is based on the knowledge that Integrin
Subunit Alpha 2b (ITGA2) is upregulated while miR-16-5p
is down-regulated in primary colorectal cancer tissue samples
and Caco2 and LoVo colorectal cancer cell lines.
Bioinformatics analysis showed that miRNA-16-5p has a
binding site in the 3’UTR region of the ITGA2 mRNA. Ex
vivo administration of miR-16-5p modified BMSC EVs in
BALB/c nude mice with subcutaneous Caco2 human epithe-
lial colorectal adenocarcinoma cell line xenograft tumor mod-
el resulted in a decrease in ITGA2 expression, tumor growth
and volume. Treatment of CaCo2 cell line with these EVs
resulted in a decrease in ITGA2, extracellular matrix degrada-
tion related proteins MMP2 and MMP9 expression, prolifer-
ation, invasion and migration while an increase in apoptosis.
miRNA-16-5p exert its effects through targeted degradation
of ITGA2 mRNA [96]. These findings showed that targeted
degradation of ITGA2 which is an important mediator in cell-
extracellular matrix interaction, cell proliferation and migra-
tion through miRNA loaded BMSC EV treatment suppress
invasiveness and migration capability properties of colorectal
cancers.
miRNA Modified BMSC Derived EVs in Genitourinary Cancers
There is only one study to investigate the effectiveness of
miRNA transfer via BMSC-derived EVs on bladder cancer.
This study is based on the knowledge of overexpression of
Endothelial Cell-Specific Molecule-1 (ESM1), which has an-
giogenic property through the VEGFA pathway, also known
as Endocan, in blood vessels, urine and serum samples of
invasive bladder cancer patients [97]. MiR-9-3p, which has
a binding motif in the 3’UTR region of the ESM1 mRNA, is
downregulated in UM-UC-3, BIU-87, EJ, T24, and 5637
bladder cancer cell lines. Tail vein injection of miR-9-3p mod-
ified tibiafemoral BMSC EVs in BALB/c nude mice with
subcutaneous UMUC-3 cell line xenograft tumor model re-
sulted in a decrease in ESM1, invasion markers MMP2 and
MMP9 expression, cell viability, migration, invasion, tumor
weight and volume, lymph node and liver metastasis while an
increase in apoptosis. Treatment of human bladder transitional
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cell carcinoma cell line UM-UC-3 with these EVs resulted in a
decrease in ESM1, proliferation markers Ki-67 and PCNA,
invasion markers MMP2 and MMP9 expression. miR-9-3p
exert its antitumorigenic effect through targeted degradation
of ESM1 mRNA [98]. These studies indicate that targeted
degradation of Endocan which is a signaling component of
Vascular Endothelial Growth Factor Receptor (VEGFR)
pathway through miRNA loaded BMSC EV treatment
suppress agresiveness and metastatic properties of blad-
der cancers.
There is two studies to investigate the effectiveness of
miRNA transfer via BMSC-derived EVs on prostate cancer.
The first study is based on the knowledge that Rhophilin Rho
GTPase Binding Protein 2 (RHPN2) is overexpressed in pros-
tate cancer specimens while miR-205 is downregulated [99].
Bioinformatics analysis showed that miR-205 have a binding
site at 3’UTR region of RHPN2 mRNA. Tail vein adminis-
tration of miR-205 loaded human primary BMSC EVs in nude
mice with subcutaneous LNCaP androgen-sensitive human
prostate adenocarcinoma cell line xenograft model resulted
in a decrease in RHPN2, MMP2 and MMP9 expression, tu-
mor weight and volume. Treatment of LNCaP cell line with
these EVs resulted in a decrease in proliferation markers Ki67
and PCNA, antiapoptotic protein Bcl-2, ECM degredation
markers MMP-2 and MMP-9 expression, proliferation, inva-
sion and migration, and in increase in apoptosis and
proapoptotic protein Bax expression.miR-205 exert its
proapoptotic role through targeted degradation of its target
RHPN2 mRNA.These findings showed that targeted degra-
dation of RHPN2 which thought to be involved in actin skel-
eton turnover and Rho signaling through miRNA loaded
BMSC EV treatment suppress proliferation and migration ca-
pability properties of prostate cancer cells [100]. In another
study, bioinformatics analysis showed that Trefoil Factor-3
(TFF3) mRNA was highly expressed in prostate cancer sam-
ples compared with normal tissue samples and miR-143 have
a negative effect on its expression level. The expression level
of miR-143 was found to be decreased in primary prostate
cancer tissue samples and also 22Rv1, VCaP, LNCaP,
Du145, and PC-3 prostate cancer cell lines compared to nor-
mal tissues and cell lines. Inversely, TFF3 mRNA expression
level was found to be increased in prostate cancer cell lines
compared to normal prostatic cell lines. BMSCs transfected
with miR-143 and extracellular vesicles of these cells were
collected. Treatment of PC3 cells with miR-143 treated
BMSC EVs suppressed the expression of TFF3 in PC-3 cells.
Also, this treatment decreased the expression of proliferation
related markers (Ki-67 and PCNA), invasion related markers
(MMP-2 and MMP4) and migration ability while increased
the the apoptosis rate. Treatment of BALB/c nude mice PC-3
xenograft model with these EVs resulted in decreased tumor
growth ability and tumor volume, decreased invasion marker
MMP-2 and TFF-3 expression [101].
miRNA Modified BMSC Derived EVs in Miscellaneous Cancers
There is only one study to investigate the effectiveness of
miRNA transfer via BMSC-derived EVs on lung cancers.
CCNE1 is an important factor for G1/S entry in the cell cycle,
and small molecule inhibitors of CCNE1 block cell cycle pro-
gression in lung cancers. CCNE1 and CCNE2 are highly
expressed proteins in Non-Small Cell Lung Cancer
(NSCLC) tissue samples and A549, NCI-H1975, NCI-
H1299, SPC-A1 cell lines. miR-144-3p is found to be down-
regulated in NSCLC patient tissue samples and lung cancer
cell lines. Bioinformatics analysis showed that miR-144-3p
has a binding site in the 3’UTR region of CCNE1 and
CCNE2 mRNAs. Tail vein intravenous injection of miR-
144-3p modified primary human BMSC EVs in BALB/c nude
mice with subcutaneous A549 xenograft model and treatment
of human alveolar basal epithelial adenocarcinoma A549 cell
line with these EVs resulted in a decrease in G1/S-specific
cyclin molecules CCNE1 and CCNE2, proliferation markers
Ki67 and PCNA expressions, tumor weight and volume, S-
phase arrest, colony formation ability and proliferation while
an increase in Go/G1 arrest. miR-144-3p exerts its antiprolif-
erative and antimitotic effect throught targeted degradation of
CCNE1 and CCNE2 mRNAs [102]. These findings showed
that degradation of CCNE1 and CCNE2 mRNAs which are
important in cell cycle progression through miR-144-3p load-
ed BMSC EV treatment suppress proliferation capability of
lung cancer cells.
There is only one study to investigate the effectiveness of
miRNA transfer via BMSC-derived EVs on osteosarcoma.
This study is based on the knowledge that miR-143 is down-
regulated in the 143B human osteosarcoma cell line, which
has high metastatic ability to the lung. Treatment of 143B
ostesarcoma cell line with miR-143 modified human BMSC
EVs inhibited its migration capacity. However, the studies
required to show that these findings are the same in in vivo
animal models have not been carried out by the researchers
[103]. All studies discussed above show that miRNA loaded
BMSC EV targeted mRNA modulation approach could be
succesfully applied in various types of cancers.
miRNA Modified BMSC Derived EVs in Renal I/R Injury
miRNA loaded BMSC EV treatment strategy is evaluated in
in vitro studies and in vivo renal I/R injury models in three
studies. Renal I/R injury leads to renal failure and impaired
endoplasmic reticulum homeostasis is usually involved in this
process [104]. BMSC EVs suppress renal I/R injury in rats
[105]. Bioinformatics analysis in the first study showed that
miR-199a5p has a binding site in the 3’UTR region of the
Binding-Immunoglobulin Protein (BIP) mRNA. Tail vein ad-
ministration of miR-199a-5p modified BMSC EVs in BALB/
c mice with renal I/R model resulted in a decrease in
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proapoptotic Caspase-3 expression, apoptosis, endoplasmic
reticulum stress, renal function markers urea and creatin se-
cretion. Treatment of NRK-52E cells with these EVs under
the hypoxia/reoxygenation (H/R) conditions resulted in a de-
crease in Caspase-3 and endoplasmic reticulum stress related
protein BIP expression. These findings suggest that miR-
199a-5p, transferred via BMSC-derived EVs, is effective in
inhibiting endoplasmic reticulum pathway stress caused by
renal I/R by inducing degradation of BIP mRNA [106]. In
the second study, intravenous administration of miR-486
andmiR-10modifiedBMSCEVsinSevereCombined
Immunodeficiency (SCID) mice with gliserol induced acute
kidney injury model resulted in a decrease in renal function
markers BUN and Creatin, tubular cell damage marker Hyalin
cast and tubular necrosis while resulted in an increase in renal
functions. miR-486 and miR-10 modified BMSC EV admin-
istration on H/R treated murine tubular epithelial cells resulted
in increased medullary thymic epithelial cell proliferation
[107]. The third study is based on the knowledge that H/R
administration increases the expression of the apoptotic pro-
teins Caspase-3 and Bax protein, while decreasing the expres-
sion of the antiapoptotic protein Bcl-2 and that miR-199a-3p
inhibits I/R-induced renal cell apoptosis. miR-199a-3p have a
binding motif at 3’UTR region of Semaphorin 3A
(SEMA3A) mRNA. Tail vein administration of miR-199a-
3p modified human BMSC EVs in BALB/c mice with bilat-
eral kidney I/R model resulted in a decrease in proapoptotic
Caspase-3, SEMA3A expression, apoptosis, tubular dilata-
tion, brush border loss, nuclear loss, cast formation, serum
BUN and Creatin expression. Treatment of H/R induced prox-
imal tubular epithelial cell line HK-2 with these EVs resulted
in a decrease in proapoptotic Caspase-3 and Bax expression, I/
R induced renal injury related proteins Apoptosis Signal-
Regulating Kinase 1 (ASK1), DNA Damage Inducible
Transcript 4 (DDIT4), Specificity Protein 1 (SP1) and
SEMA3A expression and an increase in phosphorylated forms
of survival related proteins Akt and Extracellular Signal-
Regulated Kinase (ERK), antiapoptotic Bcl-2 expression.
These findings show that miR-199a-3p, transferred via
BMSC-derived EVs, induces the destruction of the
SEMA3A mRNA that activates Caspase-3 associated apopto-
sis and activates antiapoptotic and survival activities [108].
These results showed that miRNA targeted degradation of
endoplasmic reticulum stress proteins and apoptotic signaling
proteins through miRNA loaded BMSC EV treatment regress
I/R injury.
miRNA Modified BMSC Derived EVs in Autoimmune
and Autoinflammatory Hepatitis Models
The efficiency of miRNA transfer via BMSC-derived EVs has
also been investigated in autoimmune hepatitis and
autoinflammatory hepatitis models. The first study is based
on the knowledge that cytokines released from leaky mono-
nuclear cells promote the differentiation of pure T-
lymphocytes into Th17 cells and miR-223-3p negatively reg-
ulates proinflammatory cytokines. Intravenous administration
of miR-223-3p modified C57BL/6 BMSC EVs in C57BL/6
mice with S100 emulsified with Complete Freund’sAdjuvant
(CFA) induced experimental autoinflammatory hepatitis mod-
el resulted in a decrease in proinflammatory signaling path-
way related protein Signal Transducer And Activator Of
Transcription 3 (STAT3), phospho-STAT3 expression,
mononuclear cell infiltration, liver injury markers ALT and
AST, Histological Hepatitis Score (HHS), proinflammatory
cytokines IL-1B, IL-6, IL-17 expression and Th17 cell num-
ber while an increase in anti-inflammatory cytokine IL-10
expression and regulatory T cell number. Treatment of
Lipopolysaccharide (LPS) induced monocyte/macrophage-
like cell line RAW264.7 with these EVs resulted in a decrease
in IL-1B, IL-6, STAT3, phospho-STAT3 expressions [109].
The second study is based on the information that IL-17, IL-6
and IL-1 Beta are upregulated in the liver tissues of patients
with autoinflammatory hepatitis, and miR-223 negatively reg-
ulates the expression of inflammatory genes, including IL-6
and NLR Family Pyrin Domain Containing 3 (NLRP3).
Intraperitoneal administration of miR-223 modified
tibiafemoral BMSC EVs in C57BL/6 mouse with S100 in-
duced autoinflammatory hepatitis model resulted in a decrease
in inflammasome related proteins NLRP3 and Caspase-1 ex-
pression, liver damage markers ALT and AST, proin-
flammatory cytokines Tumor Necrosis Factor alpha
(TNF-alpha), IL-1B and IL-17a. Treatment of LPS/
ATP-treated Alpha Mouse Liver 12 (AML12) cell line
with these EVs resulted in a decrease in IL-1B, NLRP3,
Caspase-1 expressions and cytotosicity marker Lactate
Dehydrogenase (LDH) release [110]. These results
showed that miRNA targeted degradation of signaling
components in inflammatory pathways through miRNA
loaded BMSC EV treatment regress acute inflammatory
and autoimmune hepatitis models.
miRNA Modified BMSC Derived EVs in Fibrosis
Therapy
miRNA loaded BMSC EV treatment strategy is evaluated in
in vitro studies and in vivo models for kidney, colonic and
lung fibrosis. The study applied by Wang B, et al.,(2016) is
based on the knowledge that miR-Let7c is down-regulated
under fibrotic conditions in the renal tubular cell line HK2
and treatment of NRK52E rat kidney epithelial cell line with
Transforming Growth Factor Beta (TGF-beta) results in over-
expression of Collagen Type IV Alpha 1 Chain (COL4A1).
Intravenous administration of miR-Let7c modified human
BMSC EVs in C57BL/6 mouse with Unilateral Ureteral
Obstruction (UUO) model resulted in a decrease in fibrosis
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related proteins Kidney Injury Molecule 1 (KIM1), COL4A1,
α-Smooth Muscle Actin (alpha-SMA), Transforming Growth
Factor-beta (TGF-beta) Receptor Type 1 (TGF-BR1) and
Transforming Growth Factor Beta 1 (TGF-beta 1) ex-
pression, inflammatory cell number and Collagen accu-
mulation. miR-Let7c modified human BMSC EV ad-
ministration on TGF-beta treated NRK52E resulted in
a decreased expression of fibrosis markers COL4A1,
alpha-SMA and TGF-BR1 [111].
The efficiency of miRNA transfer via BMSC-derived EVs
in colonic fibrosis has been investigated only in one study. In a
previous study, it was stated that miR-200b suppresses
Epithelial Mesenchymal Transition (EMT) and protects intes-
tinal epithelial cells from in vitro fibrogenesis [112]. In TGF-
beta induced rat small intestinal epithelial cell line IEC-6 cells
E-Cadherin expression was found to be downregulated while
EMT is propagated. Caudal intravenous administration of
miR-200b modified femoral BMSC EVs in Sprague Dawley
rats with 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS) in-
duced colonic fibrosis model resulted in a decrease in fibrosis
markers Fibronectin, Collagen I, Collagen III, alpha-SMA and
Ferroptosis Suppressor Protein 1 (FSP1), EMT markers
Vimentin, Zinc Finger E-Box Binding Homeobox 1
and 2 (ZEB1 and ZEB2) expression, fibrosis, Collagen
formation, EMT, colon weight while an increase in ep-
ithelial marker E-Cadherin expression, body weight and
colon lenght. Treatment of TGF-beta induced IEC-6
cells with these EVs resulted in an increase in E-
Cadherin, while a decrease in Vimentin, ZEB1, and
ZEB2 expressions [113].
The efficiency of miRNA transfer via BMSC-derived EVs
in lung fibrosis has been investigated only in one study.
Jugular vein administration of miR-30b-3p modified
tibiafemoral BMSC EVs in C57BL/6 mice with LPS induced
acute lung injury (ALI) model resulted in a decrease in proin-
flammatory cytokines IL-1B, TNF-alpha and IL-6, Serum
Amyloid A3 (SAA3), septum edema, alveolar septum
tickening, hemorrhage, inflammatory cell infiltration, inflam-
matory and oxidative stress marker Myeloperoxidase (MPO)
activity, apoptosis, lung wet/dry ratio and Bronchoalveolar
Lavage Fluid (BALF) neutrophiles and an increase in anti-
inflammatory cytokine IL-10, epithelial cell specific mitogen
Keratinocyte Growth Factor (KGF), ordered alveolar cells and
alveolar cell proliferation. Treatment of LPS induced murine
alveolar epithelial cell line MLE-12 with these EVs resulted in
a decrease in SAA3, phospho-NF-kB, IkappaBalpha (IkB-al-
pha), ERK, MEK1/2, P38 MAP Kinase (P38MAPK)
and JUN N-Terminal Kinase (JNK) expressions [114].
All studies discussed above show that miRNA loaded
BMSC EV targeted mRNA modulation approach could
be succesfully applied in repair of various types of tis-
sue injuries or fibrosis including kidney, intestines and
lung.
miRNA Modified BMSC Derived EVs in Myocardial
Infarction Therapy
Myocardial infarction (MI) which generally caused by throm-
botic occlusion of a coronary artery is characterized by
ischemia-induced disruption of aerobic metabolism in
cardiomyocytes followed by cardiomyocyte death and inflam-
matory processes involved in reparative scar formation [115].
The efficiency of miRNA transfer via BMSC-derived EVs in
MI has been investigated in seven studies. The first study is
based on the knowledge that inactivation of Phosphatase And
Tensin Homolog (PTEN) reduces apoptosis by activating the
Akt signaling pathway, miR-21 has a binding site on PTEN
mRNA, and miR-21 protects C-kit + cardiac stem cells from
apoptosis.Administration of miR-21 modified rat tibiafemoral
BMSC EVs on H2O2 induced rat C-kit + cardiac stem cells
resulted in a decrease in proapoptotic proteins PTEN and
Caspase-3 expression and apoptosis while an increase in sur-
vival related protein phospho-Akt expression. miR-21 act as a
prosurvival factor through degradation of its target PTEN
mRNA. No in vivo studies have been conducted to confirm
these findings by the authors [116]. The second study is based
on the knowledge that pericyte progenitor cells constitutively
secrete miR-132, stimulating the destruction of RAS P21
Protein Activator 1 (RASA1), followed by activation of the
MAPK pathway, promoting endothelial angiogenesis in the
mouse hind limb ischemia model. Ex vivo intramyocardial
injection of miR-132 modified mice tibiafemoral BMSC
EVs in C57BL/6 mice with Left Anterior Descending
Coronary Artery Ligation (LAD) Acute Myocardial
Infarction (AMI) model and subcutaneous inguinally injected
HUVEC plus matrigel plug assay resulted in an increase in
tube lenght, number of meshes, angiogenesis, blood perfused
vessels, vessel density, Left Ventricular Ejection Fraction
(LVEF) and shortening fraction. Treatment of HUVECs cul-
tivated in Endothelial Cell Growth Medium EGM-2 with
miR-132 loaded BMSC EVs resulted in a decrease in
RASA1 expression in vitro [117]. The third study is based
on the knowledge that BMSC-derived EVs have
cardioprotective effects and that down-regulation of miR-
21a-5p reduces the cardioprotective effects of BMSC-
derived EVs. miR-21a-5p modified mice tibiafemoral
BMSC EVs in B6129 with pericardial salc LAD-AMI mice
model resulted in a decrease in proapoptotic proteins Pellino-1
(Peli1), Programmed Cell Death 4 (PDCD4), PTEN, Fas
Ligand (FasL), Caspase-3 and infarct size. Treatment of
H9c2 cell line with these EVs under the Oxygene Glucose
Deprivation (OGD) conditions resulted in a decrease in
miR-21a-5p targets PCDC4, FasL and PTEN expres-
sions [118].
The fourth study is based on the knowledge that overex-
pression of miR-125b inhibits I/R-induced apoptosis of myo-
cardial cells by inhibiting p53-mediated apoptotic signaling
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and miR-125b has a binding site in the 3’UTR region of
Sirtuin 7 (SIRT7) mRNA. Left ventricular ligation area injec-
tion of miR-125b modified Sprague Dawley rat tibiafemoral
BMSC EVs in rat with LAD I/R model resulted in a decrease
in SIRT7 expression, infarct size, inflammation, Left
Ventricular End-Systolic Dimension (LVESD), Left
Ventricular End Diastolic Dimension (LVEDD) and Left
Ventricular End-Diastolic Pressure (LVEDP) while in an
increse in LVEF, Left Ventricular Fractional Shortening
(LVFS) and Left Ventricular Systolic Pressure (LVSP).
Treatment of primary rat cardiomyocytes with these EVs re-
sulted in a decrease in SIRT7, proapoptotic Bax and Caspase-
3, proinflammatory cytokines IL-1 Beta, IL-6, TNF-alpha ex-
pression and apoptosis while an increase in cell viability and
antiapoptotic protein Bcl-2 expression. These findings show
that miR-125b exerts its anti-inflammatory and anti-apoptotic
effects by inducing the destruction of the SIRT7 mRNA [119].
The fifth study is based on the knowledge that miR-214 pro-
tects cardiac myocytes from H2O2-induced injury and miR-
214 expression suppresses proapoptotic effectors of calcium
signaling molecules, including Calcium/Calmodulin-
Dependent Protein Kinase II (CaMKII). Treatment of C-kit+
primary cardiac stem cells with miR-214 modified Sprague
Dawley rat primary BMSC EVs resulted in an increase in
antiapoptotic Bcl-2 and antioxidant Superoxide Dismutase
(SOD) protein expressions while a decrease in proapoptotic
Caspase-3 and Bax expression, apoptotic cell number,
Reactive Oxygen Species (ROS) level, oxidative stress marker
Malondialdehyde (MDA) level, cardiac myocyte apoptosis
related protein CaMKII. Although these findings are not sup-
ported by in vivo studies it might be suggested that miR-214
plays a cardioprotective role by inducing the destruction of
CaMKII mRNA [120]. In the sixth study, MI periferal area
injection of miR-301 modified rat tibiafemoral BMSC EVs in
Sprague Dawley rat LAD AMI model resulted in a decrease in
LC3-II/LC3-I ratio, autophagy, LVESD and LVEDD while
an increase in P62 expression, LVEF and LVFS [121]. In a
study conducted, it was reported that cardiac expression of
miR-133 was significantly decreased in MI patients. [122].
The seventh study is based on the knowledge that the eleva-
tion of miR-133 reduces hypoxia, oxidative stress, endoplas-
mic reticulum-induced cardiac cell apoptosis in vitro,and that
Snail Family Transcriptional Repressor 1 (SNAIL1), one of
the main regulators of EMT is an inducer of fibrogenesis in
disease conditions. Bioinformatics analysis has shown that
miR-133a has a binding site in the 3’UTR region of the
SNAIL1 mRNA. Myocardial injection of miR-133a modified
rat femur BMSC EVs in Sprague Dawley rat with LAD AMI
model resulted in a decrease in fibrogenesis marker SNAIL-1,
ECM deposition marker Collagen-1, myofibroblast marker
alpha-SMA, apoptosis, infiltration and fibrosis while an in-
crease in LVEF, Shortening Fraction, Left Ventricular Mass
and Volume. Treatment of primary rat cardiomyocytes with
these EVs under hypoxic conditions resulted in a decrease in
SNAIL-1 expression [123]. All studies discussed above show
that miRNA loaded BMSC EV targeted mRNA modulation
approach could be applied in repair of MI through, inhibiting
p53, CAMKII and Fas/Fas-Ligand mediated apoptosis,
fibrogenesis and autophagy pathways and activation of
PI3K/Akt, Ras/Raf/MAPK survival pathways.
miRNA Modified BMSC Derived EVs in Arthritis
Therapy
The efficiency of miRNA transfer via BMSC-derived EVs in
arthritis has been investigated in four studies. Osteoarthiritis
(OA) is characterized by activation of inflammatory pathways
at joints and upregulation of cartilage extracellular matrix
degrading proteases including: matrix metalloproteases
MMP-1, MMP-3, MMP-13, and A Disintegrin And
Metalloproteinase With Thrombospondin Motifs
(ADAMTS), activation of MAC, MAP kinases, and NF-κB
pathways in articular chondrocytes, collagen type II downreg-
ulation, impaired cartilage repair [124]. miR-92a-3p expres-
sion have been found to be elevated in chondrogenic and
hypertrophic human MSC, while reduced in OA cartilage
compared with normal cartilage [125]. Development of artic-
ular joints is highly dependent on Wnt signaling pathway and
Wnt Family Member 5A (WNT5A) increase the expression of
inflammatory cytokines in Synovial Mesenchymal Stem Cells
(SMSCs) from OA patients [126]. The first study is based on
the knowledge that WNT5A activates chondrocyte prolifera-
tion in the early stages of cartilage formation while reducing
cartilage formation in the late stages, and miR-92a-3p has a
binding site in the 3’UTR region of the WNT5A mRNA.
Injection of miR-92a-3p modified human iliac crest primary
BMSC EVs in C57B/L10 with Collagenase VII induced OA
mice model resulted in an increase in chondrogenesis markers
Collagen Type II Alpha 1 Chain (COL2A1) and Aggrecan
expression, while a decrease in WNT5A and MMP13 expres-
sion, disease progression and articular cartilage damage.
Treatment of human OA primary knee joint cartilage
chondrocytes with these EVs resulted in an increase in carti-
lage matrix genes Aggrecan, COL2A1, SRY-Box
Transcription Factor 9 (SOX9), Collagen Type IX Alpha 1
Chain (COL9A1), Cartilage Oligomeric Matrix Protein
(COMP) while a decrease in cartilage degradation markers
COL10A1, RUNX Family Transcription Factor 2 (RUNX2),
MMP13 and WNT5A expression. miR-92a-3p targets
WNT5A for degredation [127]. The COX-2/prostaglandin
E2 (PGE2) pathway is one of the common pathways to induce
inflammation. When the tissue is hurt or becomes infected, the
pathway will be activated throught inducing COX-2 [128].
Second study is based on the knowledge that Prostaglandin-
Endoperoxide Synthase 2 (PTGS2) is overexpressed in pa-
tients with OA and inhibition of PTGS2 provides a protective
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effect on OA. Intrajoint injection of miR-26a-5p modified
human ilium primary BMSC EVs in Wistar rats with surgical
OA model resulted in a decrease in MMP-3 and MMP-13
expression, PTGS2, IL-1 Beta expression, proliferation and
inflammation, while an increase in apoptosis. Treatment of
human primary synovial fibroblasts with these EVs resulted
in an increase in proapoptotic molecules Bax and Caspase-3
while a decrease in PTGS2, antiapoptotic protein Bcl-2, pro-
inflammatory cytokines IL-6, IL-8 and TNF-alpha [129]. Rac
Family Small GTPase 2 (RAC2), an upstream molecule of
NF-κB pathway which is in a pivotal regulator of inflamma-
tion in Rheumatoid Arthritis (RA) [130]. RA is a chronic,
inflammatory syndrome characterized by presence of autoan-
tibodies, joint inflammation, Macrophage-Like Synoviocytes
(MLS) and Fibroblast-Like Synoviocytes (FLS) proliferation,
destruction of articular joints, ECM component release, acti-
vation of bone-resorbing osteoclasts, functional loss of joints
[131]. miR-192-5p have been found to be downregulated in
RA patients [132]. The third study is based on the knowledge
that RAC2 is overexpressed in the RA synovium and that the
miR-192-5p has a binding site in the 3’UTR region of RAC2
mRNA. Articular cavity administration of miR-192-5p mod-
ified rat tibiafemoral BMSC EVs in Wistar rat Collagen II
induced arthritis model and treatment of commercial RA hu-
man FLS cells with these EVs resulted in an increase in Bone
Mineral Density (BMD) and thickness while a decrease in
histopathological score, clinical score, synovial hyperplasia,
tartrate-resistant acid phosphatase activity, RAC2 expression,
proinflammatory cytokines PGE2, IL-1 beta, TNF-alpha,
Inducible Nitric Oxide Synthase (iNOS) expression and nitric
oxide level. miR-192-5p act as an immune regulator molecule
through targeted downregulation of RAC2 mRNA [133].
The fourth study is based on the knowledge that VEGF and
MMP14 are upregulated in the serum and synovial tissues of
RA patients, whereas miR-150-5p is downregulated.
Bioinformatics analysis has shown thatmiR-150-5p has bind-
ing sites in the 3’UTR region of VEGF and MMP14 mRNAs.
Intraperitoneal administration of miR-150-5p modified mice
tibiafemoral BMSC EVs in collagen induced DBA/1 J mice
arthritis model resulted in a decrease in angiogenesis marker
CD31, transmembrane proteinase MMP14, angiogenic factor
VEGF, angiogenesis, lining thickness, arthritis score, hind
paw thickness in in vivo studies. Treatment of human primary
RA synovial fluid FLS cells with these EVs resulted in a
decrease in MMP14 and VEGF expressions, migration, inva-
sion and tube formation abilities in vitro. miR-150-5p inhibits
synoviocyte hyperplasia and angiogenesis through targeted
degradation of VEGF and MMP14 mRNAs [134]. All studies
discussed above show that miRNA loaded BMSC EV
targeted gene expression modulation approach could be ap-
plied in arthritis therapy through, inhibiting Wnt5a/RAC2/
NFkB mediated inflammation pathways and VEGF related
angiogenesis pathways and antiapoptotic signaling molecules.
miRNA Modified BMSC Derived EVs in Stroke
There are two main types of stroke including ischemic and
hemorrhagic stroke. Both types of stroke results in oxygen and
energy defficiency for neurons, increased neuronal apoptosis
and necrosis caused by excitotoxicity, mitochondrial dysfunc-
tion, inflammation, and oxidative/nitritative stress and activat-
ed proinflammatory pathways [135]. The efficiency of
miRNA transfer via BMSC-derived EVs in stroke has been
investigated extensively. In OGD induced oligodendrocyte
progenitor cells, miR-134 expression was found to be down-
regulated while Caspase-8 was upregulated. Treatment of rat
OGD induced oligodendrocyte progenitor cells with miR-134
modified rat BMSC EVs resulted in a decrease in apoptosis by
targeted degradation of Caspase-8 mRNA. In vivo studies are
required to determine whether these findings are the same
under in vivo conditions [136].
miR-29b-3p have been found to be downregulated and
PTEN was upregulated in the brain of Middle Cerebral
Artery Occlusion (MCAO) rats and in OGD-treated cultured
neurons while ectopic expression of miR-29b-3p reverse these
alterations through targeting PTEN expression and activating
the Akt signaling pathway. Bioinformatics analysis has shown
that miR-29b-3p has a binding site in the 3’UTR region of the
PTEN mRNA. Intracerebroventricular stereotactic injection
of miR-29b-3p modified rat femoral BMSC EVs in Sqrague
Dawley rats with MCAO model resulted in a decraese in
PTEN expression and injured brain volume while an increase
in survival pathway protein phospho-Akt, angiogenesis relat-
ed VEGFA and VEGFR2 and endothelial cell marker CD31
expressions. Treatment of Brain Microvascular Endothelial
Cells (BMECs) and primary cortical neurons with these EVs
under OGD conditions resulted in a decrease in proapoptotic
Bax and Caspase-3 expression and apoptosis while an in-
crease in antiapoptotic Bcl-2, angiogenic proteions VEGFA
and VEGFR2 expression, tube formation and angiogenesis
[46]. miR-17-92 cluster plays an important role in neuronal
progenitor cell function by increasing cell proliferation and
inhibiting cell death. Bioinformatics analyzes have shown that
miRNA-17-92 has a binding site in the 3’UTR region of the
PTEN mRNA. Intravenous administration of miRNA-17-92
modified primary rat BMSC EVs in Wistar rats with MCAO
model resulted in an increase in filament stability marker
phospho-NF-H, phosphorylated forms of survival proteins
Akt, Mammalian Target of Rapamycin (mTOR) and GSK-3
Beta, synapsis marker Synaptophysin, axon myelin bundle
density, neurite brunching, dendritic plasticity, oligodendro-
cyte number, Foot False Test (FFT) and Modified Neurologic
Severity Score (mNSS) while a decrease in proapoptotic
PTEN expression [137].
Another miRNA that has been found to have neuroprotec-
tive and anti-inflammatory effects in stroke is miRNA-124
[138]. In stroke SRY-Box Transcription Factor 2 (Sox2) and
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Nestin expression have been found to be upregulated in in-
jured area. Tail vein injection of primary mice femur Rabies
Virus Glycoprotein plus Lysosome-Associated Membrane
Glycoprotein 2b (RVG + Lamp2b) and miR-124 modified
BMSC EVs in C56BL/6 mice with photothrombosis ischemia
model resulted in an increase in immature neuronal marker
Doublecortin (DCX) and neuronal progenitors while a de-
crease in adult neural progenitor markers Sox2, Nestin,
miRNA124 target genes GLI Family Zinc Finger 3 (Gli3)
and STAT3. miR-124 act its role as promoting adult
neurogenesis after the stroke [139]. In another study, tail vein
injection of miR-133b modified rat primary BMSC EVs in
Wistar rat with MCAO model resulted in an increase in
Neurofilament 200 (NF-200) (as a marker of large cortical
pyramidal neurons apical dendrites) and synapsis marker
Synaptophysin expression, neurite remodeling, cortical axon
density, adhesive removal while a decrease in Connective
Tissue Growth Factor (CTGF) and Ras Homolog Family
Member A (RhoA) expression and FFT [140]. Lipocalin2
(LCN2) have been overexpressing in ischemic stroke and it
can be secreted from by activated astrocytes to promote neu-
ron death and enhance inflammatory response. Bioinformatics
analysis showed that miR-138-5p has a binding site in the 3’
UTR region of the LCN2 mRNA. Administration of miR-
138-5p modified femoral primary BMSC EVs in C57BL/6
MCAO model resulted in an increase in antiapoptotic Bcl-2
expression and neuron number while a decrease in proinflam-
matory cytokines IL-6, IL-1 beta, TNF-alpha, apoptotic pro-
teins Caspase-3 and Bax expression, cerebral infarction and
LDH content. Treatment of C57BL/6 primary astrocyte
cells with these EVs under OGD conditions resulted in
an increase in proliferation, migration, cell cycle related
proteins Cyclin Dependent Kinase 4 (CDK-4), CyclinD1,
CyclinE and antiapoptotic protein Bcl-2 expression while a
decrease in LCN2, apoptotic proteins Caspase-3 and Bax ex-
pression [141].
In a previous study, it has been noted that treatment of rats
exposed to MCAO with BMSCs overexpressing miR-133b
resulted in neurite remodeling and brain plasticity [142].
Bioinformatics analysis has shown that the Rab Effector
Protein with Kelch Motifs (RABEPK) mRNA of miR-133b
has a binding site in the 3’UTR region. Intra-arterial admin-
istration of miR-133b modified BMSC EVs in Wistar rats
with MCAO model results in an increase in filament stability
marker phosphorylated Neurofilament Heavy (NFH), synap-
togenesis marker Synaptophysin, functional recovery, axonal
density, synaptic plasticity, EV release, FFT and mNSS and a
decrease in RABEPK expression. Treatment of primary rat
cortical astrocytes with these EVs under OGD conditions re-
sulted in an decrease in RABEPK expression in in vitro stud-
ies [143]. All studies discussed above clearly show that
miRNA loaded BMSC EV targeted mRNA modulation ap-
proach could be applied in stroke through, inhibiting LCN2
induced apoptotic pathways and CTGF related fibrosis path-
ways and activating Phosphoinositide 3-Kinase (PI3K)/Akt
survival pathways.
Another study showed that miR-193-3p was down-
regulated in serum EVs of Subarachnoid Hemorrhage
(SAH) patients, and Bcl-2 expression was downregulated
while expression of the proinflammatory cytokines IL-1
Beta, IL-6 and TNF-alpha and Caspase-3 increased in the
animal SAH model. Suprachiasmatic cistern application of
Lamp2b-RVG and miR-193-3p modified mice femoral pri-
mary BMSC EVs in C57BL/6 mice with SAH resulted in an
increase in NfKB pathway related molecule ac-p65,
antiapoptotic molecule Bcl-2, neuronal score and in a decrease
in epigenetic modulator Histone Deacetylase 3 (HDAC3),
proapoptotic Caspase-3, proinflammatory cytokines IL1B,
IL6 and TNF-alpha, brain edema, floro jade C positive cell
number and blood brain barrier permeability [144]. The last
study is based on the knowledge that ERK1/2 and CAMP
Responsive Element Binding Protein (CREB) play an impor-
tant role in neuroprotection after brain ischemia and reperfu-
sion injury, and miR-133b has protective effects in the MCAO
model. Bioinformatics analysis showed that the Rho-A
mRNA of miR-133b has a binding site in the 3’UTR region.
Tail vein injection of miR-133b modified rat tibiafemoral
BMSC EVs in Sprague Dawley rats with autologous arterial
blood Intracerebral Hemorrhage (ICH) model resulted in an
increase in phosphorylated forms of ERK and CREB and a
decrease in Rho-A, neuronal apoptosis, and neurodegenera-
tion [145]. All of these studies suggest that miRNA loaded
BMSC EV targeted mRNA modulation approach could be
applied in both ischemic and hemorrhagic stroke.
miRNA Modified BMSC Derived EVs in Spinal Cord
Injury Therapy
The efficiency of miRNA transfer via BMSC-derived EVs in
Spinal Cord Injury (SCI) has been investigated in six studies.
A previous study indicated that RhoA expression is upregu-
lated following SCI in rats and ectopic expression of miR-
133b improves functional recovery after SCI in mice and
zebrafish [146]. The first study is based on the knowledge that
miR-133b expression level is decreased in injured spinal cords
and miR-133b promotes neurite outgrowth in vitro.
Bioinformatics analysis showed that miR-133b has a binding
site in the 3’UTR region of the RhoA mRNA. Tail vein
injection of EVs obtained from miR-133b rat tibiafemoral
BMSC in Sprague Dawley rats with aneurysm clip SCI model
resulted in a decrease in RhoA expression, neuronal apoptosis
and lesion area, while an increase in neuronal regeneration
markers Growth Associated Protein 43 (GAP43),
Neurofibromin (NF), Neuronal Nuclei (NeuN), cell survival
protein phospho-ERK1/2, neurite outgrowth related phospho-
STAT, phospho-CREB expression, mature neuron number,
Stem Cell Rev and Rep

axonal and neurite outgrowth and hindlimb locomotor func-
tion [147]. The second study is based on the knowledge that
miRNA-29 is associated with tissue repair in myocardial
ischemia-reperfusion injury, skeletal muscle injury, and liver
injury. Tail vein injection of EVs obtained from miR-29b
modified rat tibiafemoral BMSC in Sprague Dawley rats with
striking device SCI model resulted in a decrease in Glial
Fibrillary Acidic Protein (GFAP), contractile nerve cell num-
ber and an increase in neuronal regeneration markers NF200
and GAP43 expression, neuronal regeneration rate and Basso
Beattie Bresnahan (BBB) score and motor function [148]. The
third study is based on the knowledge that downregulation of
miR-21 in TNF-alpha-treated Neuroprogenic Cells (NPC) re-
sults in cell death. Intradiscal injection of miR-21 modified
BMSC EVs in Sprague Dawley rats with Intravertebral Disc
(IVD) degeneration model results in a decrease in Caspase-3
expression, and IVD degeneration score. Treatment of TNF-
alpha induced human primary NPCs with these EVs resulted
in a decrease in proapoptotic proteins Caspase-3, Bcl-2-
Antagonist Of Cell Death Protein (Bad), Bax and PTEN and
apoptotic cell number while an increase in antiapoptotic pro-
teins phospho-Akt and Bcl-2 expression levels and neural
progenitor cell proliferation [149]. The fourth study is based
on the knowledge that obese rat BMSC EVs have impaired
protective effects on SCI which is related to downregulated
expression of miR-21 and low miR-21 levels results in in-
creased proapoptotic protein PDCD4 levels and apoptosis.
Injection of EVs derived from miR-21 modified obese rat
primary BMSC through tail vein in Sprague Dawley rats with
SCI model results in a decrease in PDCD4 expression, apo-
ptotic cell number and lesion cavity values whereas an in-
crease in Basso Mouse Scale (BMS) score and locomotor
activity [150].
miR-25 have been found to be downregulated in injured
spinal cords and ectopic expression of miR-25 protects rat
adrenal medulla cell line PC-12 cells against H
2
O
2
-induced
oxidative damage, suppress cell apoptosis, increase cell via-
bility, decrease the level of ROS, Hypoxia-Inducible Factor 1-
Alpha (HIF-α), γH2A, inflammatory mediators IL-1β,TNF-
alpha, IL-6, and Monocyte Chemotactic Protein 1 (MCP-1)
[151]. Bioinformatics analysis showed that miR-25 has a
binding site in the 3’UTR region of NADPH Oxidase 4
(NOX4) mRNA. In the fifth study, intratechal application of
EVs obtained from miR-25 modified rat BMSC in I/R induced
SCI Sprague Dawley rat model resulted in a decrease of
NOX4, inflammatory cytokines IL-1B, TNF-alpha, oxidative
stress marker MDA levels and Motor Deficit Index (MDI)
score while an increase in antioxidant enzyme SOD activity
and intact motor neuron numbers [152]. The sixth study is
based on the knowledge that miR-544 is downregulated in
SCI and ectopic administration of miR-544 attenuates inflam-
mation. Tail vein administration of miR-544 modified
tibiafemoral BMSC EVs in Sprague Dawley rats with
extradural compression SCI model resulted in a decrease in
proinflammatory molecules IL-1 Alpha, TNF- alpha, IL-17
Beta and IL-36 Beta expression while an increase in BBB
score and spinal cord neuron survival rate [153].
All studies discussed above show that miRNA loaded
BMSC EV targeted mRNA modulation approach could be
applied in SCI through, activation of survival pathways asso-
ciated with PI3K/Akt and ERK/CREB axis, NF200 and
GAP43 related neuron and axonal growth pathways and
inhibitingNOX4 related ROS production, and PDCD4 related
apoptosis.
miRNA Modified BMSC Derived EVs in Premature
Ovarian Failure Therapy
Premature Ovarian Failure (POF) is caused by follicular dys-
function. Granulosa cells are a layer of cells wrapped around
the surface of follicules. Apoptosis of granulosa cells leads to
follicular atresia and improve POF phenotype. The efficiency
of miRNA transfer via BMSC-derived EVs in POF has been
investigated in two studies. The first study is based on the
information that p53 and Caspase-3 expressions are increased
and Bcl-2 expression is decreased in ovarian tissues of mice
with the POF model. Tail vein administration of miR-644-5p
modified BMSC EVs in C57BL/6 mice with Cisplatin in-
duced C57BL/6 POF model resulted in a decrease in
Caspase-3 and p53 expressions, follicle atresia and apoptosis
and an increase in corpus luteum and serum Estradiol (E2)
level. Treatment of Cisplatin induced primary mice granulosa
cells with these EVs resulted in a decrease in proapoptotic
Caspase-3 and p53 expressions and an increase in
antiapoptotic Bcl-2 expression [154]. The second study is
based on the knowledge that miRNA-144-5p is downregulat-
ed in ovarian tissues of animal POF models. Bioinformatics
analysis showed that miR-144-5p have a binding site at 3’
UTR region of PTEN mRNA. Intraperitoneal injection of
miR-144-5p modified rat tibiafemoral BMSC EVs in
Sprague Dawley rats with Cyclophosphamide (CTX) induced
POF model resulted in a decrease in proapoptotic proteins
PTEN, Caspase-3 and Caspase-9 expression in ovarian tis-
sues, athretic follicle, apoptotic cells, serum Follicle
Stimulating Hormone (FSH) and Luteinizing Hormone (LH)
levels while an increase in serum E2 and anti-Müllerian hor-
mone (AMH) levels. Treatment of CTX induced primary rat
granulosa cells with these EVs resulted in a decrease in PTEN
expression while an increase in phospho-Akt expression
[155].
miRNA Modified BMSC Derived EVs in Miscellaneous
Diseases
miRNA loaded BMSC EVs therapeutic applications also eval-
uated in depression and Vascular Smooth Muscle Cells
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(VSMC) calcification models. Vascular calcification is char-
acterized bythe deposition of calcium phosphate in cardiovas-
cular structures, transition of VSMC into osteoblast like cells
and upregulation of osteogenesis related genes. Increased
smooth muscle cell endoplasmic reticulum stress, DNA dam-
age response signaling, apoptosis, and disorders of calcium-
phosphate homeostasis are involved in this process [156,157].
Advanced Glycation end Products (AGEs) induce VSMCs
calcification and ROS production and miR-146a decreases
high glucose induced ROS production in endothelial cells.
Thioreduxin Interacting Protein (TXNIP) is a target of
miRNA-146a. TXNIP inactivates Thioreductin (Trx) leading
to an increase in ROS level. TXNIP overexpression leads to
increased ROS production and AGE treatment decrease the
expression of miR146a in VSMCs. Administration of miR-
146a modified AGE pretreated rat BMSC EVs on AGEs/high
glucose induced Sprague Dawley rat aortic VSMCs resulted
in a decrease in calcification, osteogenic differentiation, oste-
ogenic differentiation markers Runx and Bone
Morphogenetic Protein 2 (BMP-2) expression, alkaline
A Simple Guide for miRNA Loaded BMSC Extracellular Vesicle Preclinical Research Anlaysis
In Vitro Analysis
Step 10. Select Cells (Cell Lines or Primary Cell Cultures)
Step 11. Generate in vitro Disease Model Through Treatment the
Cells With Disease Related Conditions or Compounds
Step 12. Treat the Cells with Extracellular Vesicles
-Naive extracellular vesicles, miRNA mimic (+)
Extracellular vesicles and miRNA negative control (+)
extracellular vesicles
Step 13. Analyse the Gene Expression Changes in Target Cells
-miRNA expression by Quantitative RT-PCR
-Expression analysis of Target mRNA/Protein
-Expression Analysis of Target Cellular Pathways
Step 14. Analyse the Cellular Phenotypes
In Vivo Analysis
Step 10. Select the Animal
Step 11. Generate in vivo Disease Model Through Treatment the
Animals With Disease Related Conditions/Compounds/
Surgical intervention
Step 12. Treat the Animals with Extracellular Vesicles
-Shem, Naive extracellular vesicles (+), miRNA mimic (+)
extracellular vesicles and miRNA negative control (+)
extracellular vesicles
Step 13. Analyse the Clinical Findings (Biochemical/Physical)
Step 14. Analyse the Gene Expression Changes in Tissue Cells
-miRNA expression by Quantitative RT-PCR
-Expression analysis of Target mRNA/Protein
-Expression Analysis of Target Cellular Pathways
Step 15. Analyse the Histological Alterations
Step 1. Identify the disease You Want to Study
Step 2. Identify the mRNA You Want to Target
-Bioinformatics analysis of GEO Datasets
-Gene expression analysis of cell lines or patient tissue samples
Step 3. Select the miRNA’s that Targets Your Selected mR NA
-Bioinformatics analysis in miRNA Target Detection Databases
Step 4. Determine the type of stem cell from which you will collect extracellular vesicles
-BMSC, AMSC, EMSC, Umbilical Cord, Wartons Jel etc. (Primary/ Commercial)
Step 5. For Primary BMSCs Verify Trilineage Differentiation
-Positivity for CD90, CD105, CD17, CD29, CD44, CD106 markers
-Negativity for CD14, CD19, CD31, CD34, CD133 and KDR
Step 6. Transfect Your BMSCs with Selected miRNA Mimic Vectors and Proliferate
-Unthreated / miRNA mimic / negative control miRNA mimic
Step 7. Collect the Extracellular Vesicles and Characterise Them
-Nanoparticle Tracking Analysis, Dynamic Light Scattering, Resistive Pulse Sensing Atomic Force
Microscopy, Transmission Electron Microscopy
-Flow Cytometry Alix, TSG101, HSP70, HSP90, CD63, CD9, CD81, CD82 Positivity
Step 8. Verify The Extracellular Vesicles Contain Your miRNA (Real-Time PCR)
Step 9. You Can Tag Your Extracellular Vesicles with Fluorescent Dyes to Keep Track of Their in vivo Distribution
Fig. 1 A Simple Flowchart for miRNA Loaded BMSC Exosome Preclinical Research Anlaysis
Stem Cell Rev and Rep

phosphatase activity, oxidative stress associated enzymes
TXNIP and MDA expression and ROS level, while an in-
crease in antioxidant SOD activity [158].
miRNA loaded BMSC EV treatment approch is also eval-
uated in depression model. In a previous study, miR-26a has
been shown to be important in neuronal plasticity [159].
miR26a induces angiogenesis in rat models of cerebral infarc-
tion through PI3K/Akt and MAPK/ERK signaling pathways.
Rat caudal vein administration of miR26a modified human
BMSC EVs in Sprague Dawley with Corticosterone induced
depression model and treatment of rat primary hippocampal
neurons with these EVs resulted in a decrease in apoptotic
protein Bax, inflammation markers MDA, LDH release,
TNF-alpha, IL1-B, cellular vaculoes, edema and apoptotic
neurons while an increase in nissle bodies, rat weight, sucrose
preference index, horizontal movement, antiapoptotic protein
Bcl-2 expression, antioxydant enzyme SOD activity and neu-
ronal proliferation [160]. This results indicate that miRNA
loaded BMSC EV treatment approach could be applied
succesfully in depression and VSMC calcification models.
Conclusion Remarks
When presented articles are examined, it is seen that there are a
wide range of studies including cancer, chronic disease and
degeneration models. Among the cancer studies most of the
studies have been performed on glioblastoma. These studies
have shown that miR-146b, miR-124a, miR-584, miR-133b,
miR-199a loaded BMSC EVs act on different cellular path-
ways, inhibiting the survival and proliferation of glioblastoma
cells and activating apoptosis pathways. In addition, studies
have also been conducted in OPMD, subcutaneous tongue
squamous cell carcinoma, pancratic cancer, colorectal cancer,
bladder cancer, prostate cancer, lung cancer, osteosarcoma,
multiple myeloma and AML, and the number of these studies
is less than the studies performed in glioblastoma. Among the
studies other than cancer, studies conducted on MI, renal I/R
Injury, fibrosis, stroke and SCI models stand out. One of the
most researched disease models is MI. These studies showed
that BMSC EVs loaded with miR-21, miR-132, miR-21a-5p,
miR-125b, miR-214, miR-301 and miR-133 have a
cardioprotective effects. As a result of studies conducted on
renal I/R injury it has been showed that BMSC EVs loaded
with miR-199a-5p, miR-486, miR-10 and miR-199a-3p inhibit
endoplasmic reticulum stress proteins and apoptotic proteins,
and reduce I/R induced renal cell death. In addition, miRNA
loaded BMSC EVs have been shown to reduce fibrosis in lung,
colon, and kidney fibrosis models. BMSC EVs loaded with
miR-134 miR-29b-3p, miR-17-92, miR-124, miR-133b, miR-
138-5p and miR-193-3p have been reported to have neuropro-
tective effects in ischemic and hemorrhagic stroke models.
Also, BMSC EVs loaded with miR-133b, miR-29b, miR-21,
miR-25 and miR-544 have been shown to contribute to neuro-
nal regeneration in SCI models. There are some obstacles to
compare these studies with each other. These factors can be
explained as follows: in some studies only in vitro studies or
in vivo studies were conducted, different cell lines or different
in vivo disease models were used in the studies, there were not
enough studies in some disease models, different miRNAs de-
termined by bioinformatics methods were used in each study,
the difference in methods used in miRNA transfer and EV
isolation. BMSC derived EVs loaded with selected miRNAs
are promising cell-free drug transport systems for delivery of
therapeutic agents to target tissue on different disease models.
In this approach detection of miRNAs, through bioinformatics
analysis, which is downregulated in a disease model and/or of
miRNAs which targets an upregulated proteins in a specific
signaling pathway in a disease model, allows the specific and
individualized therapy model. The work flow chart applied in
the studies performed to determine the efficiency of miRNA
modified stem cell EVs in in vitro and in vivo animal models is
presented in Fig. 1. EVs have advantages over the cell based
transplantation strategies and non-biological drug transporta-
tion approaches. There is no clinical trial study to examine
the applicability of this approach, which has been demonstrated
in different disease models created in vivo and in vitro. The lack
of immunogenicity and toxicity studies in most of these studies
may be considered an obstacle for this approach to move into
the clinical trial phase studies. This may be also due to a lack of
tissue or organ-specific targeting of EVs. Several approaches
have been developing to purpose of EVs membrane protein
modifications and generate decorated EVs to enhance the cell
specific targeting. These are approaches include; transfection-
based ligand overexpression, chemical assembling of ligands
on exosomal surface, pH gradient/surface charge-driven
targeting and magnetism-guided targeted delivery. RVG/
Lamp2b decorated EVs for nerve cell targeting, could be men-
tioned among these approaches. In adition to these approaches
superparamagnetic iron oxide nanoparticle modification of EVs
surfaces, chemical assembling of ligands on exosomal surface,
pH gradient/surface charge-driven targeting may be usefull to
concentrate and to target the EVs in a particular tissue/organs
might be usefull to enhance the effectivenes of miRNA loaded
EV applications.
Acknowledgements All of the persons contributed to the manuscript was
placed as an Author in the manuscript.
Availability of Data and Materials In this ‘Review’no publicly available
datasets were used. All of the articles discussed were cited.
Authors’Contributions Prof. Dr. Zafer Cetin conducted literature surveys
and prepared the main text, Fig. 1and Table 1, Assist Prof. Gökhan
Görgişen contributed the section 6, Assist. Prof. Emel Sokullu contribut-
ed to sections 2/3 and edited the manuscript, and Prof. Dr. Eyup Ilker
Saygili contributed to the configuration of the manuscript.
Stem Cell Rev and Rep

Funding ‘This work is not supported by any institution’.
Compliance with Ethical Standards
Competing Interests This manusript is only prepared for scientific pur-
poses and ‘There is no conflict of interests’.
Consent for Publication All authors of the manuscript; have read and
agreed the Journal BioMed Central‘Copyright and License Policy’,have
read and agreed to its content and are accountable for all aspects of the
accuracy and integrity of the manuscript in accordance with ICMJE
criteria and decleare that ‘The Article’is original, has not already been
published in a journal, and is not currently under consideration by another
journal.
Ethics Approval and Consent to Participate Not applicable.
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