Mesenchymal Stem Cells for Regenerative Medicine

Abstract

In recent decades, the biomedical applications of mesenchymal stem cells (MSCs) have attracted increasing attention. MSCs are easily extracted from the bone marrow, fat, and synovium, and differentiate into various cell lineages according to the requirements of specific biomedical applications. As MSCs do not express significant histocompatibility complexes and immune stimulating molecules, they are not detected by immune surveillance and do not lead to graft rejection after transplantation. These properties make them competent biomedical candidates, especially in tissue engineering. We present a brief overview of MSC extraction methods and subsequent potential for differentiation, and a comprehensive overview of their preclinical and clinical applications in regenerative medicine, and discuss future challenges.

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Review
Mesenchymal Stem Cells for Regenerative Medicine
Yu Han 1,2,†, Xuezhou Li 1 ,2 , †, Yanbo Zhang 3,* , Yuping Han 4,*, Fei Chang 1,* and
Jianxun Ding 2
1Department of Orthopedics, The Second Hospital of Jilin University, 218 Ziqiang Street,
Changchun 130041, China
2Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry,
Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, China
3Department of Orthopedics, China-Japan Union Hospital of Jilin University, 126 Xiantai Street,
Changchun 130033, China
4Department of Urology, China-Japan Union Hospital of Jilin University, 126 Xiantai Street,
Changchun 130033, China
*Correspondence: zhangyb2012@jlu.edu.cn (Y.Z.); hyp181818@126.com (Y.H.); ccfei_cn@hotmail.com (F.C.)
†These authors contributed equally to this work.
Received: 12 July 2019; Accepted: 6 August 2019; Published: 13 August 2019


Abstract:
In recent decades, the biomedical applications of mesenchymal stem cells (MSCs) have
attracted increasing attention. MSCs are easily extracted from the bone marrow, fat, and synovium,
and differentiate into various cell lineages according to the requirements of specific biomedical
applications. As MSCs do not express significant histocompatibility complexes and immune
stimulating molecules, they are not detected by immune surveillance and do not lead to graft rejection
after transplantation. These properties make them competent biomedical candidates, especially in
tissue engineering. We present a brief overview of MSC extraction methods and subsequent potential
for differentiation, and a comprehensive overview of their preclinical and clinical applications in
regenerative medicine, and discuss future challenges.
Keywords: mesenchymal stem cell; extraction; cell differentiation; biomedical application
1. Introduction
Since the discovery of spindle-shaped, bone marrow-derived plastic-adherent cells in the
mid-1970s [
1
], science has come a long way, and studies have found that these cells could
differentiate into osteoblasts and chondrocytes [
2
,
3
]. Techniques for extraction, culture, and induction
of mesenchymal stem cells (MSCs) have improved, with almost all MSC types derived from
various tissues now capable of differentiation into osteocytes and end-stage lineages [
4
]. The rapid
development of molecular biology and transplantation techniques has benefitted MSC applications in
regenerative medicine.
MSCs are an ideal cell source for tissue regeneration, owing to the excellent properties as follows.
MSCs exist in almost all tissues, including bone marrow, adipose, and synovium [
5
], and are easily
extracted. MSCs can differentiate into almost any end-stage lineage cells to enable their seeding
in specific scaffolds (Figure 1) [
6
]. Their immunological properties, including anti-inflammatory,
immunoregulatory, and immunosuppressive capacities, contribute to their potential role as immune
tolerant agents [7,8].
Numerous studies have explored MSCs for tissue regeneration in several animal models
in vitro
;
trials have not been limited to preclinical validation. Several clinical reports verify the potential efficacy
of MSC-based cell therapy; although its effectiveness remains limited, the outcomes are inspiring.
We present a brief overview of MSC extraction methods and subsequent potential for differentiation and
Cells 2019,8, 886; doi:10.3390/cells8080886 www.mdpi.com/journal/cells

Cells 2019,8, 886 2 of 32
provide a comprehensive overview of future applications of various MSCs in regenerative medicine,
as well as the challenges.
Cells 2019, 8, x 2 of 36
outcomes are inspiring. We present a brief overview of MSC extraction methods and subsequent
potential for differentiation and provide a comprehensive overview of future applications of
various MSCs in regenerative medicine, as well as the challenges.
Figure 1. Schematic diagram of regenerative medicine based on mesenchymal stem cells (MSCs).
The MSCs can be easily extracted from varies tissues, and the multilineage differentiation and
immunoregulatory properties of MSCs make them an ideal cell therapeutic candidate.
2. Discovery and Extraction of MSCs from Different Sources
The rich source of MSCs is the critical basis for their extensive researches and applications. It is
known that MSCs can be isolated from various tissues, such as bone marrow, adipose, and
synovium, and human umbilical cord blood, and bone marrow is one of the essential sources of
MSCs.
Figure 2. Typical extraction process of adipose-derived mesenchymal stem cells from adipose tissue
of mouse.
MSCs exist in various tissues and organs apart from bone marrow, with multilineage cells
from human umbilical cord blood, first reported in early 2000 [9]. Adipose tissue was subsequently
demonstrated as a rich source of MSCs in 2001 [10], and synovium-derived MSCs (SMSCs) were
successfully isolated [11]. MSCs from other tissues or organs were detected, and protocols were
Figure 1.
Schematic diagram of regenerative medicine based on mesenchymal stem cells (MSCs).
The MSCs can be easily extracted from varies tissues, and the multilineage differentiation and
immunoregulatory properties of MSCs make them an ideal cell therapeutic candidate.
2. Discovery and Extraction of MSCs from Different Sources
The rich source of MSCs is the critical basis for their extensive researches and applications. It is
known that MSCs can be isolated from various tissues, such as bone marrow, adipose, and synovium,
and human umbilical cord blood, and bone marrow is one of the essential sources of MSCs.
MSCs exist in various tissues and organs apart from bone marrow, with multilineage cells
from human umbilical cord blood, first reported in early 2000 [
9
]. Adipose tissue was subsequently
demonstrated as a rich source of MSCs in 2001 [
10
], and synovium-derived MSCs (SMSCs) were
successfully isolated [
11
]. MSCs from other tissues or organs were detected, and protocols were
established for their extraction, identification, and culture (Figure 2and Table 1) [
12
–
30
]. Figure 2and
Table 1describe the general protocols used for MSC extraction. Briefly, the process involves isolation
of various tissues, digestion to obtain cells, and culture for three to five days, followed by discarding
non-adherent cells and continuous culture of adherent cells to the desired passage. The primary culture
medium for MSCs includes low-glucose Dulbecco’s modified Eagle medium (LG-DMEM) with 1%
(W/V) antibiotic/antimycotic and 10% (V/V) fetal bovine serum (FBS). Additionally, Table 1lists a
variety of markers expressed on the MSC surface. Notably, rabbit is the most frequently used animal
model for experiments, involving cartilage or bone tissue regeneration, and should receive increased
focus concerning MSC identification. Moreover, the surface markers of rabbit tissue-derived MSCs
require further verification.

Cells 2019,8, 886 3 of 32
Cells 2019, 8, x 2 of 36
outcomes are inspiring. We present a brief overview of MSC extraction methods and subsequent
potential for differentiation and provide a comprehensive overview of future applications of
various MSCs in regenerative medicine, as well as the challenges.
Figure 1. Schematic diagram of regenerative medicine based on mesenchymal stem cells (MSCs).
The MSCs can be easily extracted from varies tissues, and the multilineage differentiation and
immunoregulatory properties of MSCs make them an ideal cell therapeutic candidate.
2. Discovery and Extraction of MSCs from Different Sources
The rich source of MSCs is the critical basis for their extensive researches and applications. It is
known that MSCs can be isolated from various tissues, such as bone marrow, adipose, and
synovium, and human umbilical cord blood, and bone marrow is one of the essential sources of
MSCs.
Figure 2. Typical extraction process of adipose-derived mesenchymal stem cells from adipose tissue
of mouse.
MSCs exist in various tissues and organs apart from bone marrow, with multilineage cells
from human umbilical cord blood, first reported in early 2000 [9]. Adipose tissue was subsequently
demonstrated as a rich source of MSCs in 2001 [10], and synovium-derived MSCs (SMSCs) were
successfully isolated [11]. MSCs from other tissues or organs were detected, and protocols were
Figure 2.
Typical extraction process of adipose-derived mesenchymal stem cells from adipose tissue
of mouse.

Cells 2019,8, 886 4 of 32
Table 1. Extraction, discrimination, and culture of MSCs derived from various tissues.
MSC Type Source Extraction Approach Culture Medium Marker Reference
BMSCs
Human: tubular
bones and iliac crest
bone marrow
1. Aspirate 1 mL of bone marrow for bone canal;
2. Extraction is diluted in PBS (1:1) and
centrifuged for 30 min at 3000 rpm;
3. The obtained buffy coat is isolated, washed,
and plated on culture flasks for incubation
LG-DMEM with 1% (W/V)
antibiotic/antimycotic,
10% (V/V) FBS
CD29+, CD44+, CD73+, CD90+, CD105+,
Sca-1+, CD14−, CD34−, CD45−, CD19−,
CD11b−, CD31−, CD86−, Ia−,
and HLA-DR−
[13–15]
Mouse, rat, and rabbit:
tubular bones, e.g.,
femurs and tibias
1. Collect femurs and tibias, cleanse the tissue
with scissors, and wash the bones with 70%
(V/V) ethanol and then PBS;
2. Cut offthe proximal and distal parts of bones,
and flush out bone marrow from bone canal by a
spring to culture flasks for incubation;
3. At days 3–5, non-adherent cells are removed
Mouse: CD29+, CD44+, CD73+, CD90+,
CD105+, Sca-1+, CD14−, CD34−, CD45−,
CD11b−, CD31−, Vcam-1−, C-Kit−,
CD135−, CD11b−, Ia−, and CD86−
[12–14,16]
Rat: CD29+, CD44+, CD54+, CD73+,
CD90+, CD105+, CD106+, Sca-1+, CD14−,
CD34−, CD45−, and CD11b−
[17,31]
Rabbit: CD29+, CD44+, CD73+, CD81+,
CD90+, CD166+, CD14−, CD34−, CD45−,
CD117−, and HLD-DR−
[15]
ADSCs
Human:
subcutaneous adipose
in abdomen, buttocks,
and abdominal zone
1. Separate adipose from host body, and mince it
with scissors or scalpel;
2. Digested by collagenase type I for 1 h at 37
◦
C
gently shaking in a water bath;
3. Centrifuge the sample and discard the
superior lipid layer;
4. Filtered through 100 and 40, or 70 µm filters;
5. Washed by 10 mL PBS and centrifuged again;
6. Discard the supernatant, resuspend the cells,
and transfer them to culture flask for incubation
DMEM with 1% (W/V)
P/S, 10% (V/V) FBS
Human: CD29+, CD44+, CD73+, CD90+,
CD105+, CD146+, CD166+, MHC-I+,
CD31−, CD45−, and HLA-DR−
[18–21]
Mouse, rat, and rabbit:
subcutaneous adipose
Mouse: CD34+, CD44+, CD45+, CD90+,
MHC-I+, MHC-II+, and CD117−.[30]
Rat: CD44+, CD73+, CD90+, MHC-I+,
CD31−, and CD45−
Rabbit: CD44+, CD105+, NG2+, CD34−,
and CD45−[22,23]

Cells 2019,8, 886 5 of 32
Table 1. Cont.
MSC Type Source Extraction Approach Culture Medium Marker Reference
SMSCs
Synovium, especially
in knee joints, of
human, mouse, rat,
rabbit, pig, etc.
1. Separate synovium from host knee joint,
and mince it with scissors or scalpel;
2. Digested by collagenase type II, D or P at 37
◦C, and filtered through a 70 µm nylon filter;
3. The released cells are washed and
resuspended in a culture medium for incubation
DMEM or
α
MEM with 1%
(W/V) P/S, 250 ng mL−1
amphotericin B, and 10%
(V/V) FBS
Human: CD10+, CD13+, CD49+, CD44+,
CD73+, CD90+, CD105+, CD147+,
CD166+, CD14−, CD20−, CD31−, CD34−,
CD45
−
, CD62
−
, CD68
−
, CD113
−
, CD117
−
,
HLA-DR−, and ALP−
[24]
Mouse: CD29+, CD44+, CD90+, CD34−,
CD45−, and CD107−[25]
Rat: CD90+, CD11b−, and CD45−[31]
Rabbit: CD44+, CD90+, and CD105+[26,27]
UCB-MSCs Umbilical cord blood
of human
1. Harvest of human umbilical cord blood;
2. Mononuclear cells (MNC) are isolated from
the buffy coat layer;
3. Seed into 25 cm
2
flask, and non-adherent cells
are removed after 48 h
LG-DMEM, 1% P/S, 250
ng mL−1amphotericin B,
and 10% (V/V) FBS
CD29+, CD44+, CD73+, CD90+, CD105+,
CD166+, CD14−, CD31−, CD34−, CD45−,
CD106−, and HLA-DR−
[28]

Cells 2019,8, 886 6 of 32
3. Differentiation Potentials of MSC Types
The multi-directional differentiation potential is one of the most critical characteristics of MSCs.
In addition, different tissue sources affect the differentiation tendency and proliferation capability
of MSCs.
There is an increasing number of publications addressing the heterogeneity of MSCs [
47
].
The transcriptome, proteome, immunophenotype, and immunomodulatory activities of various MSC
types differ, implying that MSCs exhibit unique differentiation potentials. As a critical MSC-specific
property, differentiation potential affects MSC fate; different tissue-derived MSCs display distinct
tendencies to differentiate into different end-stage lineage cells, such as osteoblasts and chondrocytes.
As a critical source of MSCs for tissue engineering, bone marrow-derived MSCs (BMSCs) exhibit
superior capacities for osteogenesis and chondrogenesis under standard differentiation protocols [
48
],
and SMSCs show more significant proliferation and chondrogenic potential than adipose-derived
MSCs (ADSCs) [
49
]. Umbilical cord blood-derived MSCs (UCB-MSCs) exhibit biological advantages
relative to other adult sources, including their capability for longer culture times, larger-scale expansion,
more significant retardation of senescence, and higher anti-inflammatory effects [
50
]. Researchers must
choose the desired MSC type according to the specific purpose. Table 2summarizes fundamental and
in vivo experiments and identifies differentiation conditions based on previous studies.

Cells 2019,8, 886 7 of 32
Table 2. Summary of differentiation researches and application potential of MSCs.
Differentiation
Direction *
Preferred
MSC Type
Basic Induction Medium Identify Methods Application Field Reference
Basic Medium Induce Agents Staining IHC RT-PCR Others
Osteoblast BMSCs
LG-DMEM, 10% (V/V)
FBS, 1% (W/V)
antibiotic/antimycotic
(In some studies,
the osteogenic medium
used HG-DMEM
solution)
10.0 mM β-glycerophosphate,
50.0 µg mL−1ascorbic acid,
and 100 nM dexamethasone
Alizarin red
staining, Von
Kossa
Staining
Col I, OCN,
OPN
Col I, OCN, OPN, ALP,
BSP, Osterix, RUNX2
ALP activity, Calcium
assay kit
Bone regeneration
[32]
Chondrocyte
50.0 µM ascorbic acid, 100 nM
dexamethasone, 10.0 ng mL−1
TGF-β1/TGF-β3
Alcian blue
staining,
Toluidine
blue staining
Col II
Col II, SOX-9, Aggrecan,
SOX-5, SOX-6, NOX 4, Col
X, Chondroitin
4-sulfotransferase
GAG assay kit Cartilage
regeneration [33,34]
Neurocyte BMSCs,
ADSCs
10.0 ng mL−1EGF, 20 ng mL−1
HGF, 20 ng mL−1VEGF; 8 days
later, 200 µM BHA, 5.0 mM KCl,
2.0 µM valproic acid, 10 µM
forskolin, 1.0
µ
M hydrocortisone,
and 5.0 µM insulin are added to
the medium
—
Enolase,
Tubulin-βIII,
GFAP, S100,
MBP, MAP2,
NF
Tubulin-βIII,GFAP,
Enolase, NeuN, NCAM,
Glial cell marker, NANOG,
OCT4 and SOX-2, MAP2,
NF-M, GAP 43
—Nerve
regeneration [35–37]
Cardiomyocyte
ADSCs
10.0 µg L−1bFGF, 10.0 µM
5-azacytidine;
one day later, the medium
maintained in the same
conditions without 5-azacytidine
for four weeks
—
Desmin,
M-cadherin,
MHC,
α-cardiac
actin, cTnI
Desmin, MYOD1, MYOG,
MHC, α-cardiac actin,
cTnT, MYF5/6, MEF2C,
TNNI1/2, CKM, Myosin2,
HCN2, HCN4
Heterotypic Cell Fusion
Assay
Myocardial
regeneration [38–40]
Hepatocyte PDSCs
1X ITS, 10−8M dexamethasone,
20.0 ng mL−1EGF, 20.0 ng mL−1
FGF, 40.0 ng mL−1OsM, 40 ng
mL−1HGF;
After two weeks, the medium is
replaced with hepatic
differentiation medium with an
increased concentration of
dexamethasone at 10
−5
M and/or
1.0 µM TSA
PAS staining
ALB, AFP,
CK-18,
PanCK, CK 19,
Transthyretin
ALB, AFP,β-actin, CK-18,
HNF-4α, Transthyretin,
TDO2, and CYP7A1
LDL/CM-Dil uptake
assay; Cell morphology;
Ammonia clearance;
Albumin production;
ELISA assay
Liver
regeneration [41–44]
Keratocyte
No
comparative
studies
LG-DMEM: F-12 3:1,
5% FBS, 1% (W/V)
antibiotic/antimycotic
Induction medium: without
pyruvate, 25.0 ng mL−1BMP-4,
1.0 mM all-trans retinoic acid,
and 10.0 ng mL−1EGF;
Differentiation medium: 5.0 µg
mL−1insulin, 2.0 nM
tri-iodothyronine, 2.0 nM
adenine, and 10.0 ng mL−1EGF
H&E staining
CK3,
β1-integrin,
and E-cadherin,
p63, CK12,
CK8, CK14,
CK15
ABCG2, β1-integrin,
CEBP
δ
, CK3, and p63, Oct4,
Sox2, Nanog, Rex1, DSC1,
and DSG1
Transepithelial
electrical resistance
Corneal
regeneration [45,46]
* The culture media for another end-stage lineage cells have not been standardized except osteoblasts and chondrocytes. The bold words in identified methods mean the main identified
staining, proteins of immunohistochemistry, genes of real-time reverse transcription polymerase chain reaction (RT-PCR), and other methods.

Cells 2019,8, 886 8 of 32
4. MSC-Based Regenerative Medicine
So far, MSCs have been widely studied and applied in regenerative medicine. In this section,
we summarize reports concerning the latest preclinical and clinical trials of various MSC types for
tissue engineering. The topics mainly focus on the reconstruction of fragile tissues, including those
associated with the musculoskeletal system, nervous system, myocardium, liver, cornea, trachea,
and skin, as shown in Figure 3.
Cells 2019, 8, x 9 of 36
Figure 3. Applications of mesenchymal stem cells with multiple differentiation potential for repair
of various tissues.
3. Differentiation Potentials of MSC Types
The multi-directional differentiation potential is one of the most critical characteristics of
MSCs. In addition, different tissue sources affect the differentiation tendency and proliferation
capability of MSCs.
There is an increasing number of publications addressing the heterogeneity of MSCs [47]. The
transcriptome, proteome, immunophenotype, and immunomodulatory activities of various MSC
types differ, implying that MSCs exhibit unique differentiation potentials. As a critical MSC-specific
property, differentiation potential affects MSC fate; different tissue-derived MSCs display distinct
tendencies to differentiate into different end-stage lineage cells, such as osteoblasts and
chondrocytes. As a critical source of MSCs for tissue engineering, bone marrow-derived MSCs
(BMSCs) exhibit superior capacities for osteogenesis and chondrogenesis under standard
differentiation protocols [48], and SMSCs show more significant proliferation and chondrogenic
potential than adipose-derived MSCs (ADSCs) [49]. Umbilical cord blood-derived MSCs (UCB-
MSCs) exhibit biological advantages relative to other adult sources, including their capability for
longer culture times, larger-scale expansion, more significant retardation of senescence, and higher
anti-inflammatory effects [50]. Researchers must choose the desired MSC type according to the
Figure 3. Applications of mesenchymal stem cells with multiple differentiation potential for repair of
various tissues.
4.1. Bone Regeneration
Bone defects frequently accompany recovery from trauma, revision arthroplasty, or tumor resection
surgeries. Autologous bone grafting represents the gold standard therapeutic strategy, despite its many
drawbacks, including (1) the limited supply of autologous bone, (2) increased operation time and blood
loss, (3) temporary disruption of bone structure in the donor site, and (4) donor site morbidity [
51
].
Allografting carries a risk of disease and/or infection [
52
]. Therefore, MSC-based bone regeneration is
considered an optimal approach [53].

Cells 2019,8, 886 9 of 32
The MSC osteoblast-differentiation capacity has been identified [
2
,
3
], with BMSCs representing
the most frequently applied cells for osteoblast differentiation [
2
]. Comparative studies evaluating the
osteogenic ability of other MSC types yielded no definitive conclusions. By contrast, UCB-MSCs show
better angiogenic capacity, supporting more abundant blood supply during bone regeneration [
54
],
which promotes rapid tissue reconstruction. In addition to BMSCs, human dental pulp stem cells
(hDPSCs) show excellent vascular differentiation potential while differentiating into osteoblasts,
which subsequently support bone regeneration [
55
]. However, these hDPSCs are screened from a
stromal vascular dental pulp fraction; therefore, this represents a limited source for further research
and application. Since ADSCs can be routinely isolated from lipoaspirate with a high degree of purity
with minimal donor site morbidity or patient discomfort, ADSCs are considered to have the most
significant potential as a primary source for clinical bone tissue engineering [
48
,
56
,
57
]. Additional
comparative and screening studies are necessary to identify other cell sources with applications in
bone reconstruction.
Stimulating factors play an important role in directing MSC differentiation into target cells
in vitro
.
The most commonly used inducing factor for osteogenesis is the bone morphogenetic protein-2 (BMP-2),
which is usually immobilized on scaffolds to promote osteoblast differentiation. BMP-2 exhibits a
strong osteogenic ability, which can be tested by the osteoblast activity and/or expression of bone
markers, such as alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) [
58
–
60
].
BMP-7 activates the transforming growth factor-
β
(TGF-
β
)/SMAD signaling in CD105
+
MSCs to
enhance the expression of osteogenesis-related genes [
61
]; Wnt11 enhances the osteogenic potential of
BMP-9 [
62
]. Nano-hydroxyapatite [
58
] and strontium [
63
] are used as osteogenic regulators in tissue
engineering to promote osteogenic differentiation of MSCs while changing the physical properties of
the scaffolds.
Studies of MSC-based cell therapy for bone defects and the use of novel scaffolds describe
inspiring advances
in vitro
and
in vivo
[
64
,
65
]. Clinical applications of MSCs in bone reconstruction
have been described, including those involving implantation of scaffolds seeded with MSCs into bone
defect sites. Specifically, dentists have used this technique to address alveolar cleft defects, jaw defect
reconstruction, and maxillary sinus augmentation, with excellent outcomes [
66
–
68
]. Defects in or
non-union of human tubular bone have been tentatively treated via local implantation of MSCs with or
without scaffolds [69,70].
4.2. Cartilage Repair
Cartilage defect repair is one of the significant challenges faced by orthopedic surgeons. Due to
the inherent avascular nature of cartilage and the proliferation of mature chondrocytes, cartilage
is greatly limited in its ability to repair itself. Currently, the clinically applied cartilage repair
techniques, such as bone marrow stimulation and osteochondral transplantation, have their limitations.
Fibrocartilage produced by bone marrow stimulation is not strong enough, and grafts for osteochondral
transplantation are challenging to integrate.
MSCs offer a new strategy for the repair of damaged cartilage, as they can differentiate
into chondrocytes [
2
,
3
]. An integrated cartilage reconstruction unit comprises cells, scaffolds,
and stimulatory factors, with BMSCs [
71
], ADSCs [
56
], and SMSCs [
72
], used as the primary cell
sources. Among these, BMSCs displayed better chondrogenic capacity
in vitro
and
in vivo
[
33
,
34
,
71
],
although SMSCs show better proliferation and differentiation potential and less hypertrophy than
BMSCs and ADSCs [
72
]. Cartilage reconstruction requires a combination of multiple stimulating
factors; co-culture of chondrocytes and MSCs would achieve better results than the application of
MSCs alone [73].
Novel bioactive three-dimensional (3D) scaffolds, such as hydrogels [
55
] and electrospun
scaffolds [
74
], have undergone constant improvement. These scaffolds provide an optimal 3D
microenvironment for cartilage regeneration. Moreover, hydrogels can regulate MSC proliferation
and differentiation due to their high water content and biocompatibility and similar properties to the

Cells 2019,8, 886 10 of 32
extracellular matrix (ECM) [
55
]. Injectable hydrogels enable minimally invasive treatment of large
areas of cartilage defects [
75
]; thus, hydrogels loaded with MSCs and stimulating factors are highly
efficacious for the repair of cartilage damage. The development of electrospun scaffolds suggests that
the arrangement of nanofibers also affects cell differentiation and provides a different approach to
cartilage repair [
74
]. Stimulating factors are necessary for cartilage engineering and responsible for
inducing, accelerating, and/or enhancing cartilage formation. Common stimulating factors include
BMP-2/-4, insulin-like growth factor (IGF)-1, and TGF-
β
1/-
β
3 [
76
–
78
]. Moreover, physical stimuli,
such as hydrostatic pressure and dynamic compression, have been explored to induce MSC-mediated
cartilage formation [79].
The first preclinical trial of MSC application for cartilage repair occurred in 1994 [
80
]. MSCs were
seeded into a collagen (Col) gel to treat a full-thickness defect in rabbit femoral cartilage, resulting in
better outcomes than those observed in a control group. This defect model was subsequently used
as a classical cartilage defect model for cartilage regeneration; subsequently, numerous trials have
been conducted in both animal models and humans to evaluate MSC-based therapy for cartilage
damage [81].
Despite clinical trials being conducted, there are no commercially available products for MSC-based
cartilage reconstruction [
82
,
83
]. Several studies investigated the effects of expanded MSCs
in vivo
on damage to human articular cartilage. Transplantation of expanded autologous BMSCs improved
cartilage quality in patients with chronic knee osteoarthritis [
83
], although the clinical improvement was
not significant [
82
]. Other studies reported the injection of allogeneic MSCs into joints in the presence
or absence of pre-mixing with autologous chondrocytes [
81
]. All the clinical outcomes indicated the
safety of these therapeutic approaches, and their ability to relieve some symptoms, although their
ability to repair the effects of cartilage damage was not always apparent. MSC transplantation showed
better results for early lesions [81].
There remain many challenges for MSC-based cartilage regeneration, including the identification
of optimal cell sources. Additional studies are needed to enable the use of MSC-based materials as
commercial products for implantation to promote cartilage regeneration.
4.3. Regeneration of Other Musculoskeletal Tissues
Recent studies investigated the MSC-mediated regeneration of musculoskeletal tissues outside
the bone and cartilage, including the meniscus, tendons and ligaments, and intervertebral discs (IVD).
Meniscus regeneration has received increasing attention. Intra-articular administration of MSCs
to promote meniscal regeneration was first performed with favorable outcomes [
84
]. Similar to its
use for cartilage regeneration, hydrogels [
85
] and electrospun scaffolds [
86
] loaded with MSCs were
used to reconstruct the meniscus. Moreover, the meniscus-derived decellularized matrix shows better
histocompatibility and is more capable of inducing MSC differentiation as compared with natural
or synthetic polymer materials [
87
]. Scaffold-free tissue-engineered constructs show promise as an
MSC-based implantation technique to repair meniscal lesions [
88
]. Tarafder et al. [
89
] proposed the
recruitment of synovial MSCs through connective tissue TGF and TGF-
β
3 to repair meniscus injury,
thereby avoiding the disadvantages of cell-based techniques. Mechanical stimulation is crucial for
meniscus growth and maintenance, with mechanical stimuli, such as dynamic compression and tensile
loads applied for meniscus repair [
90
]. Although satisfactory results were obtained in animal models,
there remains a lack of evidence in humans regarding the capability of MSCs for forming durable
tissues similar to the meniscus [91].
Tendon injury is a common problem associated with sports [
92
]. BMP-14 induces myogenic
differentiation of BMSCs via the sirtuin-1
−
Janus N-terminal kinase (JNK)/SMAD1-peroxisome
proliferator-activated receptor-
γ
signaling pathway [
93
]. Studies describing tendogenic differentiation
of MSCs were not limited to stimulating factors [
94
] and scaffolds [
95
] but also referred to mechanical
stimuli that play essential roles in MSC differentiation into tendon lineages. Uniaxial cyclic stretching

Cells 2019,8, 886 11 of 32
promoted tendogenic differentiation of MSCs
in vitro
and
in vivo
[
96
]; however, MSCs did not repair
tendon injury but only delayed lesion progression [97].
With the increasing age of populations, IVD degeneration has become prevalent. MSCs represent
promising candidates for disc regeneration; scaffolds made of Col provide readily available support for
chondrogenic differentiation of MSCs
in vitro
, although the phenotype of the differentiated MSCs is not
yet equivalent to that of nucleus pulposus (NP) cells [
98
]. The acellular matrices derived from NP cells
stimulated by TGF-
β
3 also enhance MSC differentiation [
99
]. Transfection of adenoviral expression of
SOX-9 and BMP-2 in BMSCs increased Col II and aggrecan expression, and promoted IVD repair [
100
].
Varma et al. [
101
] used a hydrogel loaded with two different concentrations of MSCs to repair NP,
and showed that MSC inoculation at a lower density resulted in a better NP-specific matrix phenotype.
A systematic review of MSC-based cell therapy for IVD indicated the safety and effectiveness of
short-term MSC transplantation, as well as the necessity for human-based clinical trials [
102
]. In 2011,
expanded autologous BMSCs injected into patients with lumbar disc degeneration revealed several
advantages and better prognosis relative to the current gold standard treatments [
103
]. Clinical
percutaneous injection of autologous bone marrow concentrate cells into a patient with degenerative
IVD resulted in decreased lumbar discogenic pain within 13 years [
104
]. Clinical studies [
105
] indicated
that MSC transplantation represents a safe treatment option for degenerative IVD; the specific effects
need verification by additional clinical trials.
4.4. Central Nervous System Rebuilding
The adult central nervous system (CNS) lacks the ability to repair damaged neurons, so the
damage of CNS is irreversible, and there is currently no effective repair method for CNS injury in
clinical practice repair. In the area of CNS regeneration, MSC-based therapy mainly focuses on two
areas: Damage or injury of the CNS caused by severe trauma and continuous ischemia and CNS
dysfunction caused by neurologic diseases. To date, BMSCs and ADSCs are the most extensively
studied cell sources for CNS repair, with each showing similar neuronal differentiation potential [
35
,
36
].
BMSCs can reduce scar formation around spinal cord injury (SCI) lesions and promoted axonal
regeneration [
106
]; however, ADSCs might represent a more suitable cell source owing to their easy
extraction and abundant sources. ADSCs inhibit inflammation of the nervous system and improve
the recovery of function from traumatic brain injury via neural stem cells [
107
]. UCB-MSCs can be
induced to differentiate into neuron-like cells
in vitro
[
37
]; DPSCs can differentiate into neurons and
express multiple factors that promote neuronal and axonal regeneration [108].
MSC expression of neuronal or astrocytic markers has been observed
in vitro
[
109
] and
in vivo
[
110
].
To promote MSC-based CNS restoration, gene-modified MSCs, such as neurotrophin-3-transferred
BMSCs, showed improved neuronal differentiation
in vivo
[
111
]. Persistent release of specific
cytokines and growth factors, which can facilitate neurogenesis, angiogenesis, and synaptogenesis,
creates a favorable microenvironment for angiogenesis or remyelination during reconstruction [
112
].
IGF-1-transfected spinal cord-derived neural stem cells displayed higher viability and the ability to
differentiate into oligodendrocytes [
113
]. Moreover, MSCs can induce T cell tolerance and release of
paracrine anti-inflammatory factors, such as TGF-β, that promote neuroprotective effects [114].
Animal models of traumatic and ischemic brain injury or SCI have been used to evaluate
MSC-based therapy [
115
,
116
]. A meta-analysis of 1568 rats with traumatic SCI showed that MSC
therapy provided a substantial beneficial effect on locomotor recovery [
116
]. Clinical studies indicated
MSC-based therapy as a safe and feasible technique for patients with SCI and/or traumatic brain
injury [
117
,
118
]. Migration of MSC pretreated under hypoxic conditions to the peri-cerebral injury
area of cerebral hemorrhagic stroke victimized rats resulted in the release of various growth factors to
promote neurogenesis and neurological recovery [
119
]. For neurological diseases, non-expanded or
expanded MSCs have been widely used for the treatment of multiple sclerosis [
120
], amyotrophic lateral
sclerosis [
121
], ischemic stroke [
122
], and Parkinson’s disease [
123
]. Most of the beneficial effects of MSCs
on neurological diseases are associated with their immunomodulatory and neuroprotective properties

Cells 2019,8, 886 12 of 32
exerted following local injection of non-expanded or expanded autologous MSCs, with clinical trials
assessing their ability to achieve promising outcomes and different degrees of remission.
4.5. Peripheral Nervous System Rebuilding
Peripheral nervous system (PNS) injury is mainly caused by severe trauma, usually accompanied
by bone fracture and vascular damage. Autologous nerve grafting (autologous nerve bridging) is
the gold standard for peripheral nerve repair; however, limited donor nerve resources and other
issues preclude the search for new therapeutic strategies. Schwann cells, neurotrophic factors,
and anti-inflammatory cells work together to promote peripheral nerve regeneration, with this process
involving axonal sprouting and fiber myelination.
There are few comparative studies concerning the effects of different MSC types on animal
models of peripheral nerve injury. ADSCs are more suitable cell sources for neural regeneration
in vitro
[
35
], and a nerve growth factor transcript has been identified in ADSC-secreted nanovesicles
that promotes synaptic growth
in vitro
and repair of sciatic nerve damage
in vivo
[
124
].
Sun et al.
[
125
]
proposed a new protocol called intermittent induction that alternates complete and incomplete
induction media to induce ADSC differentiation into SLCs. Compared with traditional protocols,
SLCs obtained by intermittent induction secrete neurotrophic factors and promote axonal growth
in vitro
and more effectively repair rat sciatic nerve injury
in vivo
. Notably, SLCs seeded in acellular
nerve grafts show better functional recovery as compared with MSCs [
126
]. BMSCs [
111
], ADSCs [
127
],
and UCB-MSCs [
128
] have also been seeded onto a variety of biodegradable scaffolds, with almost
all resulting in better recovery relative to controls. MSCs form a neuroblast-like sheath following
transplantation at the site of nerve injury and secrete neurotrophic factors that provide physical and
chemical barriers for the inner nerve fibers [
110
]. Compared with polymers, acellular neural matrix
hydrogels show better biocompatibility and tissue specificity and support Schwann cell proliferation
in vitro
and repair rat sciatic nerve defects
in vivo
[
129
]. Furthermore, 3D-bioprinting technology has
enabled the development of 3D scaffolds with complex structures to address the challenges of nerve
tissue regeneration [130].
For nerve regeneration studies, sciatic nerve crush and nerve gap animal models were
established [
131
,
132
], and the effects of local implantation [
133
] or intravenous administration [
134
,
135
]
of neural stem cells or MSCs in peripheral nerve injury models have been investigated, resulting in
excellent outcomes relative to controls. ADSCs displayed relevant therapeutic potential not only via
their direct release of growth factors but also through the indirect modulation of neurocyte behavior in
an animal model of acute axonal injury [
134
]. Moreover, intravenously infused MSCs ameliorated
function recovery post-acute peripheral nerve injury in a sciatic nerve crush model [135].
Although preclinical studies show the feasibility of MSC-based therapy in animal models of
peripheral nerve injury, there are few reports of its clinical application [126].
4.6. Myocardium Restoration
Cardiac disease is characterized by substantial morbidity and mortality, and serious adverse
consequences. In addition to congenital heart disease, almost all cardiac diseases involve insufficient
blood supply to critical regions, resulting in myocardial damage and necrosis. Although myocardium
has limited regenerative capacity, restoration of severe damage to cardiomyocytes due to catastrophic
myocardial infarction or other myocardial diseases is inadequate.
A role for MSC in attenuating myocardium damage was first reported in 2002 [
136
]; purified BMSCs
engrafted in the murine myocardium appeared to differentiate into cardiomyocytes. Several subsequent
studies evaluated the potential of different MSC sources to differentiate into cardiomyocytes [
38
–
40
],
finding ADSCs as the most suitable. Spraying was found to be a more cost-effective and less
invasive method for transferring ADSCs into a pig heart infarction model to promote cardiac function
recovery [
137
]. MSC-specific mechanisms associated with the repair of damaged cardiomyocytes
involve three factors: (1) myogenic and angiogenic capacities; (2) the ability to supply massive

Cells 2019,8, 886 13 of 32
amounts of angiogenic, anti-apoptotic, and mitogenic factors; and (3) the inhibition of myocardial
fibrosis (Figure 4) [
138
,
139
]. Butler et al. [
140
] demonstrated the safety of MSC therapy, and that it
improved the left ventricular ejection fraction in patients with non-ischemic cardiomyopathy via its
immunomodulatory effects. Co-culture of MSCs with cardiomyocytes promotes resistance to high
oxidative stress in heart tissue after myocardial infarction [
141
]. 5-Azacytidine is an effective factor
for inducing MSC differentiation into cardiomyocytes [
142
]. IGF-1-transfected MSCs protected the
myocardium from fibrosis and cardiomyocyte apoptosis and reduced infarct size after myocardial
infarction in rats [
143
]. Interleukin (IL)-7 enhances the fusion of MSCs with cardiomyocytes to improve
cardiac function, with this attributed to the ability of IL-7 to promote cell proliferation and support
damaged myocardial regeneration [
144
]. In addition to their ability to differentiate into cardiomyocytes,
MSCs promoted angiogenesis by secreting vascular endothelial growth factor (VEGF) in a critical limb
ischemia model [145], resulting in cardiac reconstruction.
MSC-based therapy is a feasible strategy to improve cardiac function, according to preclinical and
clinical findings [
146
]. Furthermore, MSC therapy has been intensively investigated as a treatment for
myocardial infarction [
147
], peripheral ischemic vascular diseases [
148
], dilated cardiomyopathy [
138
],
and pulmonary hypertension [149].
Cells 2019, 8, x 15 of 36
Figure 4. Schematic mechanisms of mesenchymal stem cells (MSCs) for cardiac regeneration.
Angiogenesis, vasculogenesis, and cardiomyocytes differentiation capacities of MSCs make them
possible for cardiac repair. Moreover, the paracrine effects of MSCs provide different kinds of
growth and anti-inflammatory factors for the immunoregulation after ischemia of heart [139].
4.7. Liver Regeneration
The liver is an essential human organ, failure of which causes fatal illnesses. Until recently, the
only effective therapy for liver failure was organ transplantation [150]; however, the availability of
transplantable livers is scarce, and many patients do not survive the wait time for transplantation.
Cell therapy provides a possible solution by either building a partial or full liver for transplantation
or addressing the damage to the liver. Although hepatocytes isolated from the livers of donors have
been studied, their therapeutic efficacy is questionable owing to their immunogenicity. Recently,
MSCs have become a therapeutic option owing to their ability to exert potent immunosuppressive
and anti-inflammatory effects [8]. Importantly, the multilineage potential of MSCs to differentiate
into different types of end-stage cells, including hepatocyte, makes them an attractive candidate for
liver-specific therapeutics [151].
The clinical use of MSCs to treat liver failure is mainly based on their immunomodulatory
capacity [152]. Preclinical and clinical studies revealed the mechanisms associated with
immunoregulation following MSC-based treatment, including transdifferentiation, fusion,
inhibition of Col deposition, paracrine effects, modulation of matrix metalloproteinase expression
and activity, neoangiogenesis, and vascular support [153–155]. A clinical phase II trial showed that
transplantation of autologous BMSCs inhibited tissue fibrosis and improved liver function in
alcoholic cirrhosis [156]; intravenous injection of allogeneic MSCs positively affected the treatment
of autoimmune cirrhosis [157]. Administration of UCB-MSCs reduces the liver inflammatory
response and hepatocyte injury, as well as the possibility of liver failure, by inhibiting T cell and B
cell proliferation and upregulating the levels of regulatory T cells [158].
Figure 4.
Schematic mechanisms of mesenchymal stem cells (MSCs) for cardiac regeneration.
Angiogenesis, vasculogenesis, and cardiomyocytes differentiation capacities of MSCs make them
possible for cardiac repair. Moreover, the paracrine effects of MSCs provide different kinds of growth
and anti-inflammatory factors for the immunoregulation after ischemia of heart [139].
4.7. Liver Regeneration
The liver is an essential human organ, failure of which causes fatal illnesses. Until recently,
the only effective therapy for liver failure was organ transplantation [
150
]; however, the availability
of transplantable livers is scarce, and many patients do not survive the wait time for transplantation.

Cells 2019,8, 886 14 of 32
Cell therapy provides a possible solution by either building a partial or full liver for transplantation
or addressing the damage to the liver. Although hepatocytes isolated from the livers of donors have
been studied, their therapeutic efficacy is questionable owing to their immunogenicity. Recently,
MSCs have become a therapeutic option owing to their ability to exert potent immunosuppressive
and anti-inflammatory effects [
8
]. Importantly, the multilineage potential of MSCs to differentiate
into different types of end-stage cells, including hepatocyte, makes them an attractive candidate for
liver-specific therapeutics [151].
The clinical use of MSCs to treat liver failure is mainly based on their immunomodulatory
capacity [
152
]. Preclinical and clinical studies revealed the mechanisms associated with immunoregulation
following MSC-based treatment, including transdifferentiation, fusion, inhibition of Col deposition,
paracrine effects, modulation of matrix metalloproteinase expression and activity, neoangiogenesis,
and vascular support [
153
–
155
]. A clinical phase II trial showed that transplantation of autologous BMSCs
inhibited tissue fibrosis and improved liver function in alcoholic cirrhosis [
156
]; intravenous injection
of allogeneic MSCs positively affected the treatment of autoimmune cirrhosis [
157
]. Administration of
UCB-MSCs reduces the liver inflammatory response and hepatocyte injury, as well as the possibility
of liver failure, by inhibiting T cell and B cell proliferation and upregulating the levels of regulatory T
cells [158].
In addition to their immunomodulatory effects, MSCs can differentiate into hepatocytes to
promote liver regeneration. MSCs can differentiate into several types of liver cells under certain
physiopathological conditions [
151
]. Functional hepatocyte-like cells were obtained from multipotent
adult progenitor cells [
159
]. Hepatocyte growth factor (HGF) and oncostatin M have been used
to successfully induce human BMSCs and human UCB-MSCs to differentiate into hepatocyte-like
cells [
160
]. Hepatocyte-like cells have been derived from BMSCs [
161
], ADSCs [
41
,
42
], UCB-MSCs [
43
],
and placenta-derived MSCs (PDSCs) [
44
] via appropriate culture conditions, including co-culture
with hepatocytes on 2D or 3D scaffolds [
142
], floating culture, or treatment with serum collected from
rats after partial hepatectomy [
162
]. Liver reconstruction using differentiated MSCs has only been
demonstrated in the animal models, although recently, a phase II trial showed that transplantation of
differentiated autologous MSCs could be used as a potential clinical treatment for liver cirrhosis [14].
There remain two obstacles to MSC-based liver regeneration: transplanted MSCs cannot efficiently
differentiate into hepatocyte-like cells, and transplanted MSCs might promote fibrogenesis [
163
].
Moreover, a previous study suggested that transplanted MSCs might contribute to the myofibroblast
pool to enhance fibrotic processes within the liver [
164
]. However, a clinical study [
165
] reported
that MSC injections were sufficiently safe, but their immunosuppressive effects following liver
transplantation were insufficient to replace immunosuppressants, despite the severe side effects of
immunosuppressants commonly used in clinical practice.
4.8. Corneal Reconstruction
The cornea represents a transparent avascular connective tissue that provides most of the refractive
ability of the eye and acts as the primary barrier against infection and mechanical damage to internal
structures. Because the cornea is fragile and directly exposed to the external environment, a variety
of clinical disorders, such as aniridia and Stevens-Johnson syndrome, or chemical, mechanical,
and thermal injury potentially causes corneal injury. Human corneal epithelial cells can be renewed by
stem cells located in the peripheral region of the cornea, containing limbal epithelial stem cells (LESCs).
However, if the entire corneal layer is damaged, vascularization, conjunctivalization, keratinization,
corneal scarring, and opacification occur and lead to impaired vision and even blindness. Currently,
keratoplasty is the primary method used for corneal restoration, because sources of donor tissue are
restricted, and post-operation immune rejection compromises transplantation efficacy.
Transplantation of corneal epithelial stem cells by limbal allograft restored useful vision to some
patients with severe ocular surface disorders [
166
]. LESCs have been investigated as the main cell
therapeutic candidates for corneal disorders, with clinical and/or preclinical studies demonstrating

Cells 2019,8, 886 15 of 32
that LESCs cultured on an amniotic membrane effectively inhibited inflammation and reconstruct the
injured corneal surface [
167
]. However, although LESCs have been widely used in clinical practice,
visual recovery after successful transplantation remains sub-optimal [
168
]. MSCs were used for corneal
repair include BMSCs [
45
], ADSCs [
169
], and UCB-MSCs [
170
]. Allogeneic MSCs exhibited good
immunosuppressive effects in a pre-sensitized rat model of corneal transplantation [
171
]; however,
no comparative studies are evaluating the effects of different MSC types on cornea reconstruction.
MSC transplantation successfully reconstructed the damaged corneal surface in a rat model,
and the main therapeutic effect of MSCs was not epithelial differentiation but instead an inhibition of
inflammation and angiogenesis following transplantation [
172
]. Transplanted amniotic fluid-derived
MSCs reduce neovascularization and promote an anti-inflammatory and anti-fibrotic environment [
167
].
Several studies have investigated the differentiation processes associated with corneal reconstruction.
Rabbit BMSCs promoted the healing of injured corneal epithelium and could be induced to differentiate
into corneal epithelial-like cells expressing CK3, a corneal epithelium-specific marker,
in vivo
and
ex vivo [
173
], and differentiated MSCs cultured on an amniotic membrane expressed the CK3
marker [
46
]. MSCs cultured in the keratocyte-conditioned medium differentiated into keratocyte-like
cells [
169
], and Yamashita et al. [
170
] induced UCB-MSC differentiation into tissue-engineered corneal
endothelial cells
in vitro
, which subsequently displayed improved preservation of corneal thickness
and transparency. Further investigation is necessary to reveal whether MSCs play a role in corneal
reconstruction through their transdifferentiation effects.
The immunoregulatory, anti-inflammatory, and immunosuppressive capacities ofMSCs play essential
roles in their therapeutic effects and have been demonstrated in studies of their transdifferentiation
effects [
172
,
174
]. The associated mechanisms include downregulation of CD45 and IL-2 expression [
172
],
downregulation of macrophage inflammatory protein-1
α
and VEGF [
175
], secretion of soluble
factors [
176
], suppression of T cell proliferation [
177
], reduction of activation and migration of chemokine
receptor-7-antigen-presenting cells [
174
], and abortion of early inflammatory responses by secretion of
tumor necrosis factor-
α
-stimulated gene/protein-6 (TSG-6) [
178
]. MSCs employed as therapeutic agents
for reconstructing corneas in animal models have resulted in mostly good prognoses [46].
Most available studies describe the strategies of MSC-based corneal reconstruction as involving
their anti-inflammatory effects associated with corneal angiogenesis and their ability to differentiate
into keratocyte-like cells; however, their differentiation efficiency and purity need to be improved.
4.9. Tracheal Reconstruction
Tracheal stenosis and other severe diseases, such as tracheobronchial cancer, require partial
tracheal resection and reconstruction. Recently, clinical trials have been undertaken to promote tracheal
reconstruction using tracheal substitutes, including autologous grafts, homografts, and prostheses [
179
].
Based on the progress in MSC-based tissue regeneration, investigations have been conducted concerning
their efficacy for tracheal reconstruction.
For regeneration of airway epithelium, different culture methods, such as co-culture with epithelial
cells or in combination with inducing factors, such as VEGF, brain-derived neurotrophic factor, TGF-
β
1,
and activin A, have been explored. These four factors play a vital role in MSC differentiation into
epithelial cells by triggering the appropriate signaling pathways [
180
]. Other induction factors also
promote epithelial-lineage differentiation, including epidermal growth factor (EGF), keratinocyte
growth factor, HGF, and IGF-2 [
181
]. Physical culture conditions are also involved in regulating
epithelial-lineage differentiation, with studies revealing that compartmentalized or polarized culture
conditions provide a suitable environment for MSC differentiation into epithelial progenitor cells with
tight junction formation [
182
]. Similarly, BMSCs and porous tracheal scaffolds implanted
in vivo
after
co-culture
in vitro
maintain their structural integrity and significantly reduce immune rejection [
183
].
For whole trachea regeneration, preclinical trials using animal models verified the efficacy and
safety of MSC-based tracheal reconstruction. Seeding MSCs onto decellularized trachea scaffolds
represents a promising means of trachea engineering in rats [
184
]; an acellular tracheal matrix inoculated

Cells 2019,8, 886 16 of 32
with BMSCs showed excellent biocompatibility and immunogenicity [
185
]. A 3D-bioprinted artificial
scaffold coated with MSCs seeded in fibrin was constructed to repair partial tracheal defects, resulting in
successful restoration in the absence of graft rejection in four rabbits [
186
]. An aortic allograft for trachea
replacement with MSC transplantation was also demonstrated as a feasible reconstruction method for
tracheal defects [
187
]; Jorge et al. [
188
] used an acellular amniotic membrane for tracheal reconstruction,
revealing that the membrane promoted cartilage regeneration, neovascularization, and epithelialization
and reduced the risk of postoperative complications, such as tracheal stenosis. However, low porosity of
the acellular tracheal matrix might lead to incomplete cartilage regeneration [
189
]; therefore, they used
laser micropore technology to alter scaffold porosity, with
in vivo
results confirming that the technique
improved the cartilage matrix and the mechanical strength of the scaffolds. Furthermore, artificial
biomaterial-based scaffolds seeded with MSCs, such as Col-based electrospun scaffolds [
190
] and
core
−
shell nanofibrous scaffolds [
191
], represent viable options for replacing a damaged trachea,
although scaffolds loaded with MSCs promote epithelium formation and angiogenesis
in vivo
but in
the absence of cartilage formation [192].
A tissue regeneration trial involving tracheal reconstruction supported the use of MSCs for
clinical tracheal tissue engineering based on their contribution to grafted tissue integration and
angiogenesis [
193
]. In 2008, the first transplantation of a tissue-engineered trachea was performed on a
30-year-old woman with end-stage left-main bronchus malacia and involved a trachea comprising
decellularized donor trachea, epithelial cells, and BMSC-derived chondrocytes [
194
]. At a five-year
follow-up, the patient exhibited normal lung function, ciliary function, cough reflex, and mucus
clearance in the absence of stem cell-related teratoma and anti-donor antibodies [
195
]. This result
demonstrated that the cell-seeded scaffold was clinically safe and feasible, and was followed by another
BMSC-based tissue-engineered tracheal replacement in a 12-year-old boy, which also showed good
results at a two-year follow-up [196].
To reconstruct a functional trachea, collaborative techniques, including those associated with stem
cell biology, biomaterials, and tissue engineering, are essential, with the trachea required to assemble
around the differentiating MSCs and matrix components.
4.10. Skin Regeneration
Skin is the first line of defense against microorganisms or physical damage, with infections
and/or trauma potentially leading to skin defects. Treatment of large-scale skin damage is sometimes
inadequate because autologous transplantation of skin is limited by skin tissue availability. Additionally,
allogeneic skin grafts always cause immunological rejection or communicable illnesses.
Wound healing is a complex process involving interactions between soluble mediators, the ECM,
and infiltrating blood cells [
197
]. MSC-based therapy combined with artificial scaffolds offers a
promising strategy to promote wound healing or complete reconstruction of full-thickness skin
(Figure 5) [
198
]. Feldman et al. [
199
] used TGF-
β
3, albumin-based scaffolds, and MSCs to treat pressure
ulcers, with good therapeutic results. Recent progress has also been reported in skin regeneration via
MSC-based therapy; MSCs significantly improve wound condition and angiogenesis. MSC-secreted
TSG-6 improves wound healing by limiting macrophage activation, inflammation, and fibrosis [
200
];
VEGF secreted by MSCs promotes keratinocyte-mediated wound healing [
201
]. Angiotensin II promotes
BMSC differentiation into keratinocytes through the mitogen-activated protein kinase/JNK/Janus
kinase 2 signaling pathways [
202
]. Furthermore, gene-modified MSCs provide another possible
way to promote skin regeneration. EGF-transfected MSCs [
203
], adenovirus-transfected C-X-C motif
chemokine receptor-4-overexpressing BMSCs [
204
], VEGF-modified human UCB-MSCs [
205
], stromal
cell-derived factor-1-transfected BMSCs [
206
], and ectodysplasin-modified MSCs [
207
] promote
MSC-mediated wound healing activity in skin defects, as does physical stimuli, such as laser
therapy [
208
]. A large number of scaffolds, such as those involving fibrin hydrogels [
209
], 3D-hybridized
chitosan (CS) and poly(
ε
-caprolactone) (PCL) [
210
], Col
−
CS [
211
], sodium carboxymethylcellulose [
212
],
and electrospun nanofibrous silk fibroin [
213
], have been developed to support MSC-based regeneration

Cells 2019,8, 886 17 of 32
of defective skin. Qi et al. [
214
] developed a photo-cross-linkable sericin hydrogel to repair full-thickness
skin damage; this hydrogen inhibited inflammation, stimulate angiogenesis, and recruit MSCs to the
site of injury to regenerate skin appendages.
Recently autologous and allogeneic MSCs were both transplanted into humans to promote the
regeneration of skin defects, and several clinical trials have been conducted involving the efficacy of MSC
transplantation alone or combined with grafts for treating severe skin burns [
215
], perianal fistula [
216
],
non-healing ulcers due to diabetes [
217
], dystrophic epidermolysis bullosa [
218
], and radiation-related
skin lesions [
219
]. Estrogen treatment repairs diabetic wounds by significantly increasing MSC
viability and proliferation and promoting neovascularization [
220
]. For appendage regeneration,
Sheng et al.
[
221
] reported the successful transplantation of MSCs to regenerate functional sweat glands
in five patients in 2009, followed by subsequent reports of this procedure was successfully performed,
with ectodysplasin-modified MSCs [
207
] and BMSCs on EGF-loaded scaffolds [
222
] demonstrated to
differentiate into the functional sweat-gland cells.
Cells 2019, 8, x 19 of 36
was successfully performed, with ectodysplasin-modified MSCs [207] and BMSCs on EGF-loaded
scaffolds [222] demonstrated to differentiate into the functional sweat-gland cells.
Figure 5. Strategies for wound healing: various mesenchymal stem cells (MSCs) are isolated and
identified, and then the cells are augmented and differentiated in a specific culture condition. To
realize the skin regeneration, MSCs secrete numerous factors to modulate inflammation and induce
angiogenesis.
4.11. Other Examples of MSC-Based Therapeutics
Systemic administration of MSCs alters the course of kidney injury via paracrine and/or
endocrine mechanisms [223]. Intratracheal injection of BMSCs was demonstrated as a feasible
strategy for managing lung diseases [30]; another study described the promotion of bladder
regeneration by MSCs seeded onto PCL/CS scaffolds [224]. MSCs loaded into bioprinted
vascularized tissue resulted in thicker tissue than that produced using current bioprinting methods
and capable of surviving for only short periods [225]. Current evidence supports the use of MSCs
for tissue regeneration in a variety of scenarios.
5. Potential Risk of Implanting MSCs
Although a large number of preclinical and clinical studies have been reported, the safety of
MSC-related therapies remains the biggest problem for clinical applications. The principal risks of
MSCs are tumorigenicity, proinflammation, and fibrosis.
Tumorigenicity is one of the most severe risk factors. On the one hand, MSCs have the ability
to develop into tumors, and some studies have shown that Ewing’s sarcoma cells are derived from
MSCs [226]. Additionally, a case of gliomas has occurred four years after stem cell transplantation
for ataxia-telangiectasia, and the tumor cells were shown to be derived from grafts [227]. On the
other hand, MSCs promote the development of tumors. Excess cytokines produced by MSCs, such
Figure 5.
Strategies for wound healing: various mesenchymal stem cells (MSCs) are isolated and
identified, and then the cells are augmented and differentiated in a specific culture condition.
To realize the skin regeneration, MSCs secrete numerous factors to modulate inflammation and
induce angiogenesis.
4.11. Other Examples of MSC-Based Therapeutics
Systemic administration of MSCs alters the course of kidney injury via paracrine and/or endocrine
mechanisms [
223
]. Intratracheal injection of BMSCs was demonstrated as a feasible strategy for
managing lung diseases [
30
]; another study described the promotion of bladder regeneration by MSCs
seeded onto PCL/CS scaffolds [
224
]. MSCs loaded into bioprinted vascularized tissue resulted in

Cells 2019,8, 886 18 of 32
thicker tissue than that produced using current bioprinting methods and capable of surviving for only
short periods [
225
]. Current evidence supports the use of MSCs for tissue regeneration in a variety
of scenarios.
5. Potential Risk of Implanting MSCs
Although a large number of preclinical and clinical studies have been reported, the safety of
MSC-related therapies remains the biggest problem for clinical applications. The principal risks of
MSCs are tumorigenicity, proinflammation, and fibrosis.
Tumorigenicity is one of the most severe risk factors. On the one hand, MSCs have the ability
to develop into tumors, and some studies have shown that Ewing’s sarcoma cells are derived from
MSCs [
226
]. Additionally, a case of gliomas has occurred four years after stem cell transplantation
for ataxia-telangiectasia, and the tumor cells were shown to be derived from grafts [
227
]. On the
other hand, MSCs promote the development of tumors. Excess cytokines produced by MSCs, such as
chemokines and growth factors, directly act on receptors on the surface of cancer cells, thereby
regulating tumor growth. The immunosuppression ability of MSCs contributes to tumor growth and
tumor cell metastasis [
228
,
229
]. Additionally, MSCs have pro-angiogenic functions in the context of
tumor development [230].
MSCs show immunosuppressive effects when exposed to sufficiently high levels of pro-
inflammatory cytokines. However, they promote inflammatory responses in the presence of low levels
of TNF-
α
and IFN-
γ
[
231
]. It demonstrates that MSCs need to be triggered by inflammatory cytokines
to become immunosuppressants, and the inflammatory environment is a crucial factor affecting the
immune regulation of MSCs.
In addition to tissue repair, MSCs also produce fibrotic reactions. For instance, MSCs can
differentiate into myofibroblasts [
232
]. Additionally, the balance between repair and fibrosis of MSCs
is broken in the process of injury repair, which leads to fibrotic lung disease [233].
In order to improve the therapeutic effect of MSCs and reduce the potential risks, some measures
should be implemented, such as reducing excessive cytokines, further exploring the immunomodulatory
effects of MSCs, and establishing strict preclinical biosafety testing rules. Fortunately, major adverse
events were rare according to clinical trials evaluation of MSC therapy [
234
]. However, this study only
proves that MSCs are well tolerated and safe in the short term. As the development of cancer is a
continuous process, further longer and larger controlled clinical trials are still necessary to determine
the safety of MSCs.
6. Conclusions and Perspectives
The magic capability of regeneration of damaged parts of the body to regain lost function has long
been a dream of humanity. It has been 50 years since MSCs were first identified, and advancements in
the MSC-based tissue engineering have followed. In recent years, optimizations of extraction, culture,
and differentiation methods have allowed MSCs to progress closer to clinical applications for disease
therapy and tissue reconstruction. Three MSC properties make them optimal for tissue regeneration:
(1) Immunoregulatory capacity beneficial to alleviate abnormal immune responses, (2) paracrine or
autocrine functions that generate growth factors, and (3) the ability to differentiate into target cells.
Previous studies of MSC-based regenerative medicine mainly focused on musculoskeletal tissues;
however, recent progress has expanded their applications into other tissues, including the CNS, heart,
liver, cornea, and trachea. Induction factors are one of the most critical factors affecting the outcome of
MSC therapy, which sharply accelerate the repair process of MSCs on tissues. Scaffolds provide the
environment for proliferation and differentiation of MSCs, and produce some mechanical stimulation to
MSCs, which is beneficial for further applications of MSCs. Moreover, scaffolds loaded with induction
factors enhance the therapeutic effects of MSCs, which is also worthy of further study. Scaffolds and
induction factors remain indispensable agents in these processes; therefore, future investigation of

Cells 2019,8, 886 19 of 32
advanced materials and efficient inducing factors will promote the further applications of MSCs in
regenerative medicine.
Although MSCs have several advantages, there are still many challenges to overcome. The unique
immunomodulatory properties of MSCs are essential for their functions, but the mechanisms of MSC
immune regulation have not been elucidated. Different researchers have their distinct methods of
isolating and culturing MSC, although the primary medium is similar. However, different culture
conditions, such as FBS, supplements, cell seeding density, and oxygen, may affect cell proliferation
and differentiation potential [
235
–
237
]. Therefore, a standard protocol needs to be formulated for
the
in vitro
culture of MSCs. Additionally, cryopreserved MSCs have low viability, which will affect
the further applications of cells [
238
]. Moreover, the age of the donors also affects the proliferation
and differentiation potential of MSCs, and MSCs from young donors show lower damage and better
proliferation [
239
]. We conclude that many factors influence the therapeutic potential of MSCs, such as
induction factors, oxygen concentrations, and mechanical stimuli. Therefore, optimizing the culture
conditions of MSCs may be an effective means to improve the therapeutic potential of MSCs to achieve
tissue repair successfully.
For clinical applications, autologous and allogeneic MSCs have both been reported as sources
for tissue regeneration. Specifically, autologous MSCs represent the primary sources considered
safe for transplantation and minimization of immunological risk, despite the lack of documented
complaints regarding allogeneic MSC-based therapy. The effect of MSCs on human clinical outcomes
has not readily achieved the predominantly positive outcomes in murine. Furthermore, the oncogenic
potential of uncontrolled MSC differentiation needs to be further investigated. The differentiation
potential, surface markers, and transcription of various tissue-derived MSCs are challenging to be
unified, which undoubtedly become a hindrance to the clinical transformation of MSCs.
In the clinic, the effect of MSCs on human clinical outcomes has not readily achieved
the predominantly positive outcome in murine. The different results mainly attribute to the
immunocompatibility and fitness of MSCs. Expanding the indications of diseases and reducing
the differences among different individuals are challenges in the future research of mesenchymal stem
cells. Further research is required on cell physiology about how MSCs function
in vivo
and how to
achieve accurate administration.
Despite the current challenges, MSC-based tissue engineering represents a promising clinical
strategy in the field of regenerative medicine. Moreover, improving the cultural environment of MSCs
and selecting appropriate scaffolds and induction factors are essential for MSC therapy.
Author Contributions:
Conceptualization, Y.Z., F.C., and J.D.; Writing—original draft preparation, Y.H. and X.L.;
Writing—review and editing, Y.Z., Y.P.H., and J.D.; Funding acquisition, F.C. and J.D.
Funding:
This work was finically supported by the National Natural Science Foundation of China (Grant Nos.
51873207, 51803006, 81701811, 51673190, and 81671804), the Development and Reform Commission of Jilin
Province (Grant No. 2018C052-4), the Health and Family Planning Commission of Jilin Province (Grant No.
20165061), the Science and Technology Development Project of Jilin Province (Grant No. 20190201068JC), the Youth
Talents Promotion Project of Jilin Province (Grant No. 181909), the Youth Innovation Promotion Association of
Chinese Academy of Sciences (Grant No. 2019005), and the Natural Science Foundation of Shandong Province
(Grant No. ZR2018BH008).
Conflicts of Interest: The authors declare no conflict of interest.
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