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Review Article
Isolation, Cultivation, and Characterization of
Human Mesenchymal Stem Cells
Dolly Mushahary,1 Andreas Spittler,2 Cornelia Kasper,1 Viktoria Weber,3 Verena Charwat1*
1
Department of Biotechnology, University
of Natural Resources and Life Sciences,
1190 Vienna, Austria
2
Core Facility Flow Cytometry & Surgical
Research Laboratories, Medical
University of Vienna, 1090 Vienna,
Austria
3
Christian Doppler Laboratory for
Innovative Therapy Approaches in
Sepsis, Danube University Krems, 3500
Krems, Austria
Received 21 August 2017; Accepted 28
August 2017
*Correspondence to: Verena Charwat;
Department of Biotechnology, University
of Natural Resources and Life Sciences,
Vienna, Muthgasse 18, 1190 Vienna, Austria. E-mail: verena.charwat@boku.ac.at
Published online 00 Month 2017 in Wiley
Online Library (wileyonlinelibrary.com)
DOI: 10.1002/cyto.a.23242
Abstract
Mesenchymal stem cells (MSC) exhibit a high self-renewal capacity, multilineage differentiation potential and immunomodulatory properties. This set of exceptional features
makes them an attractive tool for research and clinical application. However, MSC are
far from being a uniform cell type, which makes standardization difficult. The exact
properties of human MSC (hMSC) can vary greatly depending on multiple parameters
including tissue source, isolation method and medium composition. In this review we
address the most important influence factors. We highlight variations in the differentiation potential of MSC from different tissue sources. Furthermore, we compare enzymatic isolation strategies with explants cultures focusing on adipose tissue and
umbilical cords as two relevant examples. Additionally, we address effects of medium
composition and serum supplementation on MSC expansion and differentiation. The
lack of standardized methods for hMSC isolation and cultivation mandates careful
evaluation of different protocols regarding efficiency and cell quality. MSC characterization based on a set of minimal criteria defined by the International Society for Cellular Therapy is a widely accepted practice, and additional testing for MSC functionality
can provide valuable supplementary information. The MSC secretome has been identified as an important signaling mechanism to affect other cells. In this context, extracellular vesicles (EVs) are attracting increasing interest. The thorough characterization
of MSC-derived EVs and their interaction with target cells is a crucial step toward a
more complete understanding of MSC-derived EV functionality. Here, we focus on
flow cytometric approaches to characterize free as well as cell bound EVs and address
potential differences in the bioactivity of EVs derived from stem cells from different
C 2017 International Society for Advancement of Cytometry
V
sources.
C 2017 International Society for
V
Advancement of Cytometry
Key terms
human mesenchymal stem cells; extracellular vesicles; cell isolation; explant culture;
enzymatic isolation
MESENCHYMAL STEM CELLS FROM DIFFERENT HUMAN TISSUES
Isolation of MSC From Different Tissue Sources
MSCS are adult multipotent progenitor cells with self-renewal potential and the
ability to differentiate into various specialized cell types. Since no single biomarker is
available for identification of human MSC (hMSC), a set of markers and cell characteristics has been proposed in 2006 by the International Society for Cellular Therapy
(1). These so-called minimal criteria of MSC are still widely accepted today and
include (i) the ability to self-renew, (ii) multipotency with osteogenic, chondrogenic,
and adipogenic potentials, and (iii) expression of a characteristic set of surface
markers, such as cluster of differentiation (CD)73, CD90, and CD105, while lacking
expression of CD14, CD34, CD45, and human leukocyte antigen-DR (HLA-DR).
Moreover, MSCs show plastic-adherent growth and are expandable in vitro over several passages (2,3). Traditionally, bone marrow (BM) has been the prevailing source
of MSC in humans (4). However, while BM is a rich source of hematopoietic stem
cells, it constitutes only a rare MSC population(5). Additionally, due to the painful
Cytometry Part A 00A: 00 00, 2017
Review Article
harvesting procedure, which requires general anesthesia, the
supply of BM-derived MSC (bmMSC) and their application
in research and in the clinical setting are limited. Over time, a
number of other tissues have been identified as alternative
sources for hMSC. Today, MSC can be isolated from multiple
tissues (6) including adipose tissue (7,8), dental tissues (9),
skin and foreskin, salivary gland (10), limb buds, menstrual
blood, and perinatal tissues (11–14). In the following we will
focus mainly on adipose tissue (AT) and umbilical cord (UC)
tissue as relevant and promising sources for hMSC. AT is a
rich source of MSC with ubiquitous availability. Since large
numbers of MSC can be obtained from AT by minimal invasive procedures, adMSC have become an important candidates for autologous and allogeneic stem cell based therapies
and tissue engineering (15).Conversely, perinatal tissues such
as the amnion, chorion and UC are promising MSC sources
due to the young donor age (16). Although MSCs have been
derived from multiple perinatal tissues, UC tissue (Wharton’s
jelly, WJ) and umbilical cord blood (UCB) (17–19) are the
most frequently used sources, with UC tissue being a richer
MSC source compared to UCB (18,20–22).
Differentiation Potential of MSC From Different
Tissue Sources
Since the first discovery of the multilineage differentiation potential of MSCs (23), a variety of human sources have
been used for producing terminally differentiated populations
of MSCs such as bone (24,25), cartilage (26), tendon (27,28),
muscle (29,30), adipose tissue (31,32), and hematopoieticsupporting stroma (32). Table 1 exemplifies some of the currently used tissue sources and the respective confirmed differentiation potentials. Since MSC quality is influenced by many
factors during isolation and cultivation, additional information on isolation procedures and media composition is provided. Several studies have demonstrated the potential of
MSCs to differentiate into multiple cell lines from sources
such as heart (32), peripheral blood (49), cord blood (50),
muscle (51), adipose tissue (4), lung (52) , trabecular bone
(53), intestine (54), kidney (55), liver (56), pancreas (57),
synovium (58), skin (59), and even in the brain (60). Some
studies examined the adipogenic and osteogenic potential
(61,62), while others merely focused on the adipogenic
(55,63–65), chondrogenic (66–68) or osteogenic (69–72) differentiation capacity. Although there are literature reporting
no significant morphological differences between osteogenic
and chondrogenic differentiation (4), many experiments also
suggest that there exist a varying degree of difference between
source material (69,73–75) and its age (69). For example,
mixed cord, WJ, and artery showed the most consistent differentiation for all the tested mesenchymal lineages, showing
consistently down adipogenic and osteogenic lineages. The
cells isolated from vein were the most variable with consistently poor osteogenesis but better adipogenesis in comparison (74). Also, juvenile adMSCs showed higher adipogenic
differentiation as compared to the adult adMSCs (69).
2
MSC ISOLATION PROCEDURES
Explant Culture Versus Enzymatic Isolation
The application of MSC in the clinical setting depends
on their quality either as progenitors for the regeneration of a
damaged tissue or as producers of secreted factors with pharmacological properties. However, the lack of standardized
procedures and the absence of a universal marker remain
major challenges for consistent MSC characterization. A crucial aspect that needs to be considered in any effort toward
standardization is the harvesting procedure. This initial step
of MSC retrieval from the donor tissue defines the cell population that will be propagated in vitro. MSCs are usually isolated as plastic-adherent cell population using simple
procedures involving tissue mincing, optional enzymatic
digestion and cell outgrowth on a plastic surface. The procedures can generally be divided into enzymatic and explant
protocols.
The explant culture (Fig. 1A) is one of the earliest techniques of cell isolation and in vitro cell cultivation. The source
tissue is rinsed to remove blood cells and then mechanically
split (e.g., cutting or chopping) into small pieces of no more
than a few millimeter in length. Reducing the size of tissue
pieces improves the diffusion of gases and nutrients toward
the cells (76). However, excessive mincing can lead to mechanical destruction of the cells. The tissue pieces are then placed
into plastic culture vessels with growth medium. The MSC
grow out from tissue pieces onto the culture dish surface.
After several days the tissue pieces can be removed. Enzymatic
protocol (Fig 1B) comprises additional steps where the
coarsely chopped tissue is incubated with an enzyme solution
that degrades the extracellular matrix (ECM). Single cells or
small cellular aggregates are released from the tissue and transferred to medium containing culture dishes.
Although no comprehensive study to compare the different isolation protocols is available to date, several authors
have compared specific aspects of explant procedures versus
enzymatic isolation methods. The explant method has been
reported to harvest less heterogeneous cell populations exhibiting higher proliferation rates and cell viability when compared to the enzymatic method (35,37,42). This could be due
to the presence of intact tissue pieces and undissociated ECM
during explant culture, which keep the cells protected from
proteolytic and mechanical stress and hence provide favorable
environment for the migrating cells (77,78). Further advantages of explant culture lies in the release of cytokines and
growth factors into the medium (78), higher yield of stromal
cells (79), shorter proliferation time (79,80), and simultaneous expression of surface markers CD73, CD90, and
CD105, and absence of CD14, CD31, CD34, and CD45 in all
MSC populations (81). The outcome of enzymatic digestion
in terms of yield, efficiency, and viability depends on the type
and concentration of the enzyme used for dissociation
(12,82,83). Enzymatic digestion leads to dissociation of the
ECM resulting in low yield (84,85) and double the time for
cell adherence (86). Comparison between enzymatic digestion
and enzymatic with mechanical dissociation (87) reported
hMSC
Cytometry Part A 00A: 00 00, 2017
5
4
3
2
1
SL.NO.
ISOLATION TECHNIQUE
MEDIA 1 SERUM
CELL SURFACE MARKERS
Table 1. Biological features of MSCs isolated from different sources using different isolation techniques
LINEAGE DIFFERENTIATION
Bone marrow
Ficoll density gradient
centrifugation
Knock out DMEM,
DMEM 1 10% FBS
Adipogenic, Chondrogenic,
Positive: SH2, SH3, CD29,
Osteogenic
CD44, CD49e, CD71, CD73,
CD90, CD105, CD106,
CD166, CD120a, CD124
Negative: CD34, CD45, CD19,
CD3, CD31, CD11b, HLA-DR
Adipogenic, Chondrogenic,
Positive: CK8, CK18, CK19,
Umbilical cord,
Ficoll-Hypaque density gradient DMEM 1 10% FBS, 10% FCS,
Osteogenic, Endothelial-like
MSCGM 1 10% FCS
CD10, CD13, CD29, CD44,
Umbilical cord blood centrifugation, Enzymatic
cells, Neuron-like cells
CD73, CD90, CD105, CD106,
digestion (0.25% trypsinHLA-I, HLA-II
EDTA), Explant culture
Negative: CD14, CD31, CD33,
CD34, CD45, CD38, CD79,
CD133, vWF, HLA-DR
Adipogenic, Osteogenic
DMEM/DMEM-F12 1 10% FBS Positive: CD13, CD29, CD44,
Wharton’s jelly
Explant culture, Enzymatic
CD73, CD90, CD105, HLA-I
digestion (0.1% collagenase II,
Negative: CD14, CD34, CD45,
1 mg/ml collagenase
CD31, HLA II
B1trypsin, collagenase1hyaluronidase1trypsin,
trypsin-EDTA, 0.26% collagenase I 1 0.07%
hyaluronidase 1 0.125%
trypsin)
Adipogenic, Chondrogenic,
Positive: CD13, CD29, CD44,
Adipose tissue
Enzymatic digestion (1.5 mg/ml DMEM-Low Glucose1
Osteogenic, Neurogenic,
CD73, CD90, CD105, CD166,
MCDB201 1 2% FCS,
collagenase I, 1 mg/ml
Muscular
HLA-I, HLA-ABC
DMEM 1 20% FBS,
collagenase-I in 0.1% BSA)
Negative: CD10, CD14, CD24,
MesenproRS
CD31, CD34, CD36, CD38,
CD45, CD49d, CD117,
CD133, SSEA4, CD106, HLAII, HLA-DR
Positive: SH2, SH3, SH4, CD29, Adipogenic, Osteogenic,
Amniotic fluid
Density gradient centrifugation, a-MEM 1 20% FBS, DMEMCD44, CD49, CD54, CD58,
F12 1 10% FBS, high glucose
Enzymatic digestion (0.25%
Neurogenic
CD71, CD73, CD90, CD105,
DMEM 1 20% hESC-defined
trypsin 1 1.2 units/ml of
CD123, CD166, HLA-ABC.
FBS, KSR-based media
dispase 1 2 mg/ml collagenase
Negative: CD10, CD11, CD14,
I
CD31, CD34, CD49, CD50,
CD117, HLA-DR, DP, DQ,
EMA.
SOURCE
(39–41)
(2,7,38)
(35–37)
(17–19)
(4,33,34)
REF
Review Article
3
4
Skin
Placenta
Salivary gland
Synovial fluid
7
8
9
10
SOURCE
Dental tissues
6
SL.NO.
TABLE 1. Continued
ISOLATION TECHNIQUE
MEDIA 1 SERUM
DMEM-F12 1 10/5/0.5% FBS,
DMEM 1 20% FBS
D-MEM-F12, a-MEM 1 2%
FCS
Ficoll gradient, Enzymatic diges- DMEM low glucose 1 20% FBS
tion (1 mg/ml Dispase, 300 U/
ml Collagenase, 100 U/ml
Hyalluronidase,
80 U/ml DNAse I, 0.25% trypsin-EDTA)
DMEM 1 10% FCS, DMEMEnzymatic digestion (1.67 mg/
F12 1 3% FBS
ml collagenase 1 1.33 mg/ml
hyaluronidase, 1.67 mg/ml
dispase; 5.01 mg/ml collagenase 2 1 3.99 mg/ml hyaluronidase, 33.4 mg/ml
dispase)
a-MEM 1 10% FBS, 20% FBS
Ficoll–Paque density gradient
centrifugation, Enzymatic
digestion (3mg/ml collagenase
V)
Enzymatic digestion (0.6 U/ml
dispase, 0.62 Wunsch U/ml
Liberase Blendzyme 1,
1.25 mg/ml collagenase I)
Enzymatic treatment (387.1 U/
mg collagenase 1 0.70 U/mg
dispase II)
CELL SURFACE MARKERS
LINEAGE DIFFERENTIATION
REF
(10,46)
(47,48)
Positive: CD10, CD166, CD44, Adipogenic, Chondrogenic,
Osteogenic
CD54, CD90, CD105, CD147,
D7-FIB, STRO-1
Negative: CD31, CD34, CD45,
CD106, CD117, CD166,
VEGFR2, Flk-1, CXCR4,
BMPR-1A, NGFR
(11,17)
(44,45)
(9,42,43)
Adipogenic, Chondrogenic,
Positive: CD13, CD29, CD44,
Osteogenic, Pancreatic
CD49f, Thy-1, CD90, CD104,
endocrine,
p75NGFR, b2-microglobulin,
CD130
Negative: CD34, CD38, CD45,
CD133
Positive: CD90, CD73, CD105,
SSEA4
Negative: CD14, CD45, CD34, ckit, CD133,
SSEA3, Oct-4, TRA 1–60, TRA
1–81, HLA-DR
Adipogenic, Endothelial-like
Positive: CD29, CD44, CD73,
cells, Neurogenic, Osteogenic
CD90, CD105
Negative: CD45, CD34, HLA-DR
Adipogenic, Chondrogenic,
Positive: CD29, CD44, CD90,
CD105, CD, SH2, SH3, HLA- Myogenic, Osteogenic
DR, CD117, CD146, DPSCEZ, DPSC-OG
Negative: CD10, CD14, CD34,
CD45, HLA-DR, Stro-1,
NGFR
Adipogenic, Myogenic,
Osteogenic
Review Article
hMSC
Review Article
Figure 1. Representative figure demonstrating MSC isolation steps by (A) explant culture method and (B) enzymatic method. [Color figure
can be viewed at wileyonlinelibrary.com]
70% increase in the yield using additional mechanical method,
suggesting the advantage of combining both these methods.
Overall, several researchers have undertaken different
strategies to establish reliable, robust, and standardized MSC
isolation procedures. In the following section of this review,
UC and AT are exemplified as highly relevant tissue sources
for hMSC and specific effects of different isolation approaches
are highlighted.
Isolation of MSC From Umbilical Cords
The human UC is formed during the fifth week of
embryogenesis and weighs about 40 g with a mean diameter
of 1.5 cm in normal full-term pregnancies. UcMSCs are considered adult stem cells but besides adult stem cell markers
CD73, CD90, and CD105, they also express embryonic stem
cell (ESC) markers, such as Tra-1–60, Tra-1–81, stage-specific
embryonic antigen-1 (SSEA-1), SSEA-4, and alkaline phosphatase (88). UcMSC also exhibit a higher degree of multipotency than bmMSCs or adMSCs (89). MSC have been isolated
from different parts of the UC, including blood, umbilical vein
sub-endothelium, and WJ (73,81). With 10–15 3 103 MSC/cm
(84), human WJ represents a valuable source for MSC isolation,
which usually starts by removing blood and vessels. The cord is
cut into pieces, which are then processed by different methods
including explant cultures (35,86,87,90–94), and enzymatic
digestion (36,73,86,95). Alternative methods without removal
of vessels have also been described (18,96).
Literature values of doubling times reported for ucMSC
isolated by enzymatic or explant culture are listed in Table 2.
Enzymatic isolation seems to yield slightly shorter doubling
times on average. However, the large variations within the
Table 2. Population doubling of ucMSCs derived by explant or enzymatic methods
SL. NO.
PART OF UC USED FOR MSC ISOLATION
1
Whole UC, Wharton’s jelly
2
3
4
5
6
7
Whole UC
Wharton’s jelly
Wharton’s jelly
UC
Perivascular cells
Wharton’s jelly
Explant
Explant
Explant
Explant
Enzymatic
Enzymatic
8
Wharton’s jelly
Enzymatic
9
Wharton’s jelly
Enzymatic
Cytometry Part A 00A: 00 00, 2017
ISOLATION METHOD
POPULATION DOUBLING
REFERENCES
Explant
23 h (P6) (UC)
24 h (P6) (WJ)
27 h (P4)
28.2 6 2.5 h (P5)
39.0 6 7.8 h (P1)
48 h (P1)
2.8 6 0.35 on day 20 (P3)
33.06 6 1.36 h in
simple medium (P6)
24.39 6 0.27 h in
complex medium
(P6)
69.5 6 8.5 h (P1)
41.3 6 7.5 h (P1)
31.9 6 6.8 h (P10)
(93)
(95)
(97)
(88)
(20)
(74)
(98)
(88)
(36)
5
Review Article
same category (enzymatic or explant) indicate that different
studies cannot be compared directly due to multiple biasing
factors including donor variation, medium composition, protocol variations. Only few studies have directly compared
explant UC cultures to enzymatic isolation strategies. For
example, Buyl et al. (98) and Iftimia-Mander et al. (99) performed an explant culture and enzymatic isolation using
0.05% (m/v) collagenase type II solution (98) or 0.075% Collagenase type I solution (99) respectively of uc-MSC. Both
reported similar cell morphology and comparable doubling
times for the different procedures. Overall, so far no significant differences in efficiency of the different isolation strategies for ucMSC have been confirmed. However, clearly more
detailed studies are needed to establish optimized and standardized procedures.
Isolation of MSC From Adipose Tissue
For isolation of adMSC, the white, subcutaneous AT is
used as it is a common surgical waste material in the form of
lipo-aspirates or larger tissue pieces. Since its first description
as a source of adult stem cells in 2001 (100), AT has been recognized as particularly rich source of MSC with upto 3% stem
and progenitor cells in its stroma-vascular fraction. For comparison, the frequency and yield of MSC in BM (6–60 3103
from 1 ml) is about 2500 times lower than from AT (5 3 104
to 2 3 105 from 1 g) (101,102). However, the numbers differ
depending on the donor characteristics, such as age, gender,
body mass index, location and type of fat tissue, tissue collection method, and culture conditions (102).
Several groups have isolated MSC from human AT using
the enzymatic method (103–105) according to a protocol
modified from Zuk et al. (100). The original protocol involved
washing of lipo-aspirates with PBS followed by digestion of
the ECM with 0.075% collagenase at 378C for 30 min, pelleting of the stromal vascular fraction (SVF) by centrifugation,
lysis of red blood cells and filtration through a 100-mm nylon
mesh to remove cellular debris. The cell fraction was then
incubated overnight in plastic culture dishes, followed by a
washing step to remove nonadherent cells. Later variations of
the protocol differ with respect to the type of enzyme or
enzyme cocktail used (38,103,105), enzyme concentration
ranging from 0.05 to 0.15% (71,103,106–108), digestion time
of 30–90 min (7,109,110), different buffers for inhibiting collagenase activity (7,103,106,107,110), centrifugation speed
(111), and different sized nylon mesh filters ranging from 70mm (112,113) to 100–250 mm to remove cell debris at the final
stage of isolation (38,112,113).
BLOOD-DERIVED MEDIA SUPPLEMENTS IN MSC
CULTURES
Role of Serum in MSC Expansion
The clinical applications of MSC require their ex vivo
expansion since the cell numbers needed for therapeutic
applications are much larger than what can be isolated from
the tissue itself. Therefore, identification of optimal culture
conditions is essential for MSC culture, which include
medium compositions, culture substrates, cell seeding density,
6
physico-chemical environment, such as dissolved O2 and CO2
concentrations, pH and temperature. Because of the high
complexity of media compositions, development of optimized
culture media remains an extremely complicated task due to
which blood-derived media supplements are still routinely
used in many laboratories. Here we survey the current transition from fetal bovine serum (FBS) to human-derived blood
products as media supplements and their effects on qualitative
and quantitative cell yield.
FBS-containing media have been commonly used for
hMSC expansion culture at a concentration of 10–20% (v/v)
(72,114,115). FBS contains high concentration of cell growth
and attachment factors along with nutritional and physicochemical compounds required for cell maintenance (116).
However, serum is a complex xenogenic compound with high
lot-to-lot variability, risk of pathogen contamination (e.g.,
virus, mycoplasma, and prions), and immunizing effects
(114–117). Additionally, a negative influence of FBS on the
therapeutic outcome has also been debated (115). These disadvantages raised safety and regulatory concerns regarding the
use of animal serum. Hence, there is a strong trend toward
autologous or allogeneic human blood-derived materials,
including human serum, platelet lysate (hPL) and cord blood
serum. Comparison of human serum to FBS at 10% v/v
showed higher proliferation capacity of MSC in human
serum, which was more prominent by 2.5-fold at 15% human
serum (72). hPL contains considerable growth promoting
properties and their use in hMSC expansion has been shown
to maintain the differentiation, therapeutic and immunomodulatory potential of hMSCs (118–121) along with a significant
3-fold increased proliferation rate (122) as compared with
FBS-cultured MSCs. Other reports also suggest significantly
higher MSC expansion in 7.5% hPL (P 5 0.01) (123) or 5%
hPL (P 5 0.02) (119) as compared to 10% FBS, with higher
cell recovery in hPL (54.8 3 106 to 365 3 106) compared to
FBS (2.7 3 106 to 31 3 106) (119). Some authors have also
replaced FBS with allogeneic human serum from UCB (124)
and placenta (125) as these tissues are rich source of growth
factors (123). Overall, existing data shows that human derived
supplements do not only overcome some of the general disadvantages associated with FBS but also outperform it with
regard to MSC expansion efficiency. However, no comprehensive studies including several human supplements applied to
MSC from different tissue sources under varying conditions
exist.
Role of Serum in MSC Differentiation
While human blood-derived media supplements show
clear advantages over FBS for MSC expansion, contradictory
results have been reported regarding MSC differentiation.
Gottipamula et al.(126) demonstrated comparatively more
osteogenic potential using 10% hPL in Dulbecco’s modified
Eagle medium (DMEM) containing low glucose (LG) compared with that having 10% FBS. Bm-MSCs cultured both in
FBS and HPL showed similar adipogenic differentiation
potential whereas those supplemented with hPL showed
higher potential of chondrogenic differentiation than FBS
hMSC
Review Article
supplemented medium. Tateishi et al.(72) reported no significant differences in the chondrogenic and osteogenic differentiation potential with 10% FBS, while Naaijkens et al.(122)
showed that MSCs in hPL group showed increased osteogenic
differentiation and decreased adipogenic differentiation. The
cord-blood serum (CBS)-expanded MSCs also exhibited a
unique differentiation potential characterized by a shift from
adipogenic to osteogenic differentiation (124). Some other
studies reported data showing reduced osteogenic or adipogenic differentiation potential when hMSCs were cultured in
media containing allogeneic human blood derivatives
(125,127,128). Moreover, there is also a report illustrating that
although cell proliferation was greatly enhanced, the use of
hPL altered the expression of some hMSC surface molecules
resulting in their decreased in vitro immunosuppressive
capacity (129). The use of autologous serum may also not be
applicable for elderly patients as its capacity to support cell
growth is likely to decrease with age. The allogeneic human
growth supplements might also present a risk of contamination with human pathogens which cannot be detected by routine screening of blood donors.
Serum Free MSC Media
The disadvantages related to the use of poorly defined
serum or human-sourced supplements made it necessary to
develop defined serum free media. This can be advantageous
in reducing the problems associated with undesirable downstream research and therapeutic applications due to illdefined nature of blood derivatives that provides inconsistent
performance and lot-to-lot variations, making data comparisons (i.e., cellular differentiation, genomics, and proteomics)
more difficult. In the development of a defined medium consisting of minimum essential components, growth factors
have been shown to be essential for the expansion of hMSCs
and has attracted significant attention. Chase et al. (130)
R
tested the ability of serum-free medium (SFM; StemProV
MSC SFM, Invitrogen) to support the expansion of hMSCs
and revealed a similar phenotype, expression profile and differentiation characteristics compared with cells expanded in
traditional serum-containing medium. Other studies have
also used growth factors, such as Transforming Growth Factor
beta 1 (TGF-b1) (131,132) Platelet Derived Growth Factor
(PDGF) (131,133), and basic Fibroblast growth Factor (bFGF)
(131), epidermal growth factor (134) that support the expansion of undifferentiated hMSCs either alone or together (131).
R
Likewise, other defined media such as MesencultV from Stem
Cell Technologies (Vancouver, Canada) and TheraPEAKTM
MSCGM-CDTM from Lonza (Basel, Switzerland) are also
available. However, the composition of the base medium, the
substrate and other supplementary factors used for cell expansion are confidential. This becomes a major drawback in using
the commercially available defined media as basal medium
components (salts, amino acids, vitamins, fatty acids, trace
elements, etc.) require strict quality control to ensure optimal
cell growth, and media formulations greatly affect the growth
frequency and morphology of primary and passaged hMSC
cultures. Hudson et al. (135) introduced mTeSR (Stem Cell
Cytometry Part A 00A: 00 00, 2017
Technologies) a defined medium designed to enhance hMSC
expansion containing additional factors (136,137), and
including LiCl (activator of canonical Wnt signaling
(138,139), and high insulin concentration (22.8 mg 5 ml) to
activate insulin-like growth factor-1 receptor (138,140) and
TGF-b1 (131,138). These systematically optimized defined formulations prevent overgrowth of undesired cells in primary
cultures and lead to production of more homogeneous
hMSCs. With the well-defined nature of the medium, the formulations can be modified to enhance the expression of specific genes to achieve an optimal profile (141), which is crucial
for enhancing the clinical efficacy of stem cells with desired
properties, and can facilitate cell bioprocessing protocols.
EXTRACELLULAR VESICLES
It is now generally conceived that MSC ameliorate disease
via the secretion of paracrine factors and stimulation of host
cells rather than via direct engraftment and cell replacement.
In this context, there is growing evidence for the significance
of stem cell derived extracellular vesicles (EVs), carrying and
transferring cargo such as regulatory miRNAs, cytokines and
growth factors, as well as signaling lipids. Research in the field
was stimulated by two landmark studies demonstrating that
fractions enriched from stem cell supernatants by centrifugation (142) or by size exclusion chromatography (143) contained vesicular material and retained MSC activity.
Accordingly, MSC-derived EVs provide a promising therapeutic agent, as recently reviewed (144).
EVs are released from cells under various physiological
and pathological conditions, in particular during cellular activation, and have been detected in all body fluids (145–149).
Based on their biogenesis, diameter, and molecular markers,
they have been classified into endosome-derived exosomes
(30–150 nm), plasma membrane-derived microvesicles (100–
1,000 nm; also referred to as microparticles or ectosomes),
and apoptotic bodies (1,000–3,000 nm). Since an exact discrimination of these vesicle types is hampered by a lack of reliable separation and characterization methods, and as there are
considerable overlaps in their size and their phenotypical
characteristics (150), the general term extracellular vesicles
will be used here to refer to both, microvesicles and exosomes.
A number of studies have indicated the potential of
MSC-derived EVs to counteract tissue damage and organ failure (151–153). MSC-derived EVs have been shown to support
kidney regeneration (154–156), and their potential to treat
ischemic cardiovascular injuries has been reported (157,158).
Furthermore, the regenerative potential of MSC-derived EVs
has been demonstrated in conditions such as liver fibrosis
(159), neurogenesis and traumatic brain injury (160), Parkinson’s disease, glioma, and schwannoma (161), as well as in
wound healing (162). Recent studies have provided evidence
for the presence of antimicrobial peptides in MSC-derived
EVs, and it has been proposed that MSC might release EVs
containing specific combinations of antimicrobial peptides
depending on particular stimuli in their microenvironment
(163).
7
Review Article
As to the different sources and methods used for stem
cell isolation, the question arises whether the tissue source or
the isolation protocol influence the bioactivity of stem cell
derived EVs. While literature data on these effects are still
scarce, EVs secreted by MSC from different cell sources have
recently been shown to exhibit differential effects on glioblastoma cells (164). EVs derived from bm-MSC and ucMSC
decreased cell proliferation and induced apoptosis, whereas
adMSC-derived EVs enhanced tumor cell proliferation, providing evidence that the tissue source may influence the bioactivity of MSC-derived EVs. As to the impact of culture
conditions, or, generally speaking, the influence of the microenvironment on MSC-derived EV bioactivity, proteomic analysis of EVs derived from BM-MSC revealed significantly
increased expression of proteins associated with angiogenic
signaling pathways under ischemic conditions (165). Along
this line, the release of EVs containing pro-angiogenic mRNA
by cord blood MSC was strongly enhanced in an in vitro
model of renal damage (166). Likewise, EVs released by
human adMSC triggered macrophage polarization toward an
anti-inflammatory M2 phenotype, and hypoxic preconditioning induced an intensified release of EVs enriched in miRNAs
involved in wound healing (167).
CHARACTERIZATION OF EVS
There is consensus that the heterogeneity of EVs requires
a spectrum of methods to achieve EV characterization and to
generate comparable data (168–173), and that preanalytical
parameters influence EV isolation and characterization(150,174). In the following, we will focus on the flow
cytometric characterization of EVs and on cytometry-based
approaches to study their interaction with target cells.
Flow cytometry has found widespread application to
characterize EVs in body fluids and in cell culture supernatants (175–177). Flow cytometric analysis is fast and may be
suited to track EVs as prognostic parameters during the clinical course of disease. Beyond the quantification of vesicles,
flow cytometry allows for the determination of their cellular
origin based on surface markers derived from their parent
cells. Regarding the characterization of MSC-derived EVs, it
has recently been shown that MSC surface markers CD44,
CD73, and CD90 can identify hMSC-derived EVs in flow
cytometry (178). As a limitation, the current lower detection
limit of flow cytometry is in the range of 200 nm, precluding
the characterization of smaller vesicles.
Flow cytometric characterization of EVs has evolved rapidly and has undergone profound developments over the past
years. While the size-based characterization of EVs using calibration with fluorescent microbeads of defined size is widely
used, this approach may be associated with drawbacks originating from differences in the refractive index of calibration
beads and EVs. Polystyrene based beads, in particular, have a
higher refractive index as compared to EVs, resulting in an
underestimation of EV size (179,180). Silica beads appear
favorable in this respect due to their lower refractive index,
which is closer to that of biological material (179).
8
Alternatively, fluorescence triggering using fluorochromelabeled Annexin V (Anx5) which binds to phosphatidylserine
exposed on EVs has been suggested to have advantages over
size-based EV detection (175) with respect to sensitivity. EVs
lacking phosphatidylserine exposure, however, (181), will
remain undetected when using fluorochrome-linked Anx5 as
triggering signal. In general, the question arises whether an
exact determination of EV size is required from a biological,
immunological, or hematological point of view, or whether
their characterization should rather focus on their cellular origin, their functional characteristics, their molecular cargo, and
their interaction with different cell populations.
Regarding the impact of preanalytical parameters, flow
cytometric analysis of EVs requires a number of precautions
during sample preparation and staining (182–184). Plasma is
generally recommended for the analysis of EVs in the circulation, but centrifugation of whole blood precludes the analysis
of EV subfractions adhering to blood cells, whereas the quantification and characterization of EVs directly in whole blood
yield information on adhesion of EVs to different populations
of blood cells (184). Venous blood should be collected without
application of a tourniquet using a needle size of 21 gauges or
lower to avoid or minimize platelet activation during blood
sampling. Anticoagulation as well as storage conditions, such
as temperature and agitation have been shown to have significant impact on postsampling vesicle release, as well. Antibodies used for EV staining have to be centrifuged
immediately before use to remove antibody aggregates, and
samples have to be diluted prior to analysis to reduce coincidence effects and swarm detection, a well-known issue in flow
cytometry whenever complexes or aggregates of cells do not
reflect the true number of particles, or when a number of
vesicles pass the laser beam at the same time, resulting in
underestimation of EV concentrations. The flow rate during
EV measurement represents another critical parameter, as
increasing the flow rate expands the sample stream and may
generate swarm effects, as well. With respect to the characterization of EVs in cell culture supernatants, cell culture medium
components such as fetal bovine serum (FBS) may represent a
source of extracellular vesicles and may, therefore, interfere
with EV detection and characterization both in flow cytometry and in functional assays, mandating appropriate control
experiments, and the use of sterile filtered FBS or serum-free
media. Conversely, the depletion of EVs from cell culture
media has been shown to alter the proliferation and differentiation capacity of cultured cells, suggesting roles for circulating
EVs in supporting cell growth and survival in vivo (185,186).
As described above, the characterization of EVs using
flow cytometry is limited with respect to size. Most flow
cytometers are unable to discriminate particles smaller than
500 nm, while newer cytometers permit the measurement of
particles down to a size of around 200–250 nm, and newest
technical advances even allow for the detection of particles
down to a size of around 100 nm or below. The so-called
nanoscale flow cytometry (nanoFCM) method has recently
been published. The experiments were partly performed on a
cell sorter equipped with a special high resolution
hMSC
Review Article
Figure 2. Visualization of EV-leukocyte aggregates using imaging flow cytometry. Cells in human whole blood were stained with antiCD45-PB and anti-CD14-PE, and EVs were traced with lactadherin (LA)-FITC and anti-CD41-PC7 for platelet origin as well as anti-CD235aAPC for red blood cell origin and analyzed using imaging flow cytometry (ImageStreamx MkII cytometer, Amnis, EMD Millipore, Seattle,
WA). Monocytes (upper panel) and granulocytes (lower panel), and lymphocytes (not shown) were identified based on their expression
pattern of CD45 and CD14 as described in Ref. (189). EVs appeared as clear spots on the surface of monocytes and granulocytes, while no
extracellular vesicles could be detected on the surface of lymphocytes. (Reproduced from Ref. 189, Elsevier Inc. RightsLink license
#4114450370981). [Color figure can be viewed at wileyonlinelibrary.com]
configuration allowing for the detection of exosomes exclusively by size (187).
Another approach combined particle fluorescence staining
with powerful laser excitation. Gaudin et al. demonstrated that a
60 nm and a 150 nm fraction of infectious Junin virus (JUNV)
could be discriminated after staining with the Alexa Fluor 647conjugated antibody LD05 using a combination of a 300 mW
high power 488 nm laser for forward scatter size detection and a
640 nm laser for Alexa Fluor 647 fluorescence detection (188).
These examples show the continuous progress of flow
cytometry. The advantages of nanoFCM high-resolution size
distribution in combination with multicolor staining make this
method highly suited for the characterization of EVs, for example, for disease monitoring and sorting for content analysis. Further developments are required to reduce background noise and
to design highly fluorescent dyes to increase the effectiveness of
fluorescence staining. As a future perspective, the possibility of
detecting even smaller quantities of EV-bound antibodies will
make particles visible which cannot be detected yet, allowing for
the analysis of the whole range of circulating vesicles.
IMAGING FLOW CYTOMETRY TO VISUALIZE INTERACTIONS
OF EVS WITH CELLS
EVs may be present in body fluids in free form or may
interact with and adhere to cells. Imaging flow cytometry
(IFC) represents a versatile method to characterize EV-cell
interactions. It allows for the evaluation of morphological and
fluorescent data at a single-cell as well as at a population level
and combines the statistical advantage of flow cytometry with
the ability to identify each single event based real images
(189,190). EVs can be visualized using fluorescently labeled
antibodies and can be identified based on their phosphatidylserine exposure using fluorescently labeled lactadherin or
Anx5 and additional markers to track their cellular origin
(184) as shown in Figure 2 for EVs on blood cells, and their
cellular uptake can be displayed using IFC, as well.
CONCLUSION
The flexible potential of MSC to differentiate into several
tissues and their ability to expand for extended periods of
Cytometry Part A 00A: 00 00, 2017
time without losing their original characteristics highlights
them for use in stem cell-based therapies and translational
research. Although it is obvious that even small variations in
the isolation and culture protocol, such as centrifugation
speed, media composition, and serum concentration can significantly influence the yield, quality, and composition of the
isolated cell population, their ability to differentiate into specialized cells makes them distinct from other stem cells. Still,
their self-renewal and differentiation mechanisms still remain
to be fairly understood. Coupled to a lack of standardized
protocols, differences in isolation and purification of MSC
may result in EV fractions showing varied immunomodulatory properties. To identify MSC-EV fractions with optimized
therapeutic potential, appropriate functional assays need to be
set up and their mode of action needs to be unraveled. A better understanding of MSC self renewal properties together
with the biology of MSC-EVs, their isolation and storage
methods, molecular characterization, as well as establishing
potency assays will greatly enhance the future therapeutic
applications of MSC and MSC-derived EVs.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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