Improved Human Mesenchymal Stem Cells Isolation
Tzu-Min Chan1,2, Horng-Jyh Harn3,4, Hui-Ping Lin5, Pei-Wen Chou2,6, Julia Yi-Ru
Chen6, Hong-Meng Chuang2,4, Tzyy-Wen Chiou7, Bing-Chiang Liang8,9, Shinn-Zong
Lin1,10,11,12＊
1
2
3
Neuropsychiatry Center, China Medical University Hospital, Taichung, Taiwan, ROC
Everfront Biotech Inc., New Taipei City, Chinese Taipei, Taiwan, ROC
Department of Pathology, China Medical University Hospital, Taichung, Taiwan,
ROC
4
Department of Medicine, China Medical University, Taichung, Taiwan, ROC
5
Institute of Cellular and System Medicine, National Health Research Institutes,
Miaoli ,Taiwan, ROC
6
Guang Li Biomedicine, Inc., New Taipei City, Chinese Taipei, Taiwan, ROC
7
Department of Life Science and Graduate Institute of Biotechnology, National Dong
Hwa University, Hualien, Taiwan, ROC
8
Department of Emergency Medicine, Taipei Veterans General Hospital, Taiwan,
ROC
9
National Yang-Ming University School of Medicine, Taiwan, ROC
10
Department of Neurosurgery, China Medical University Beigan Hospital, Yunlin,
Taiwan, ROC
11
Department of Neurosurgery, Tainan Municipal An-Nan Hospital-China Medical
University, Tainan, Taiwan, ROC
12
Graduate Institute of Immunology, China Medical University, Taichung, Taiwan,
ROC
Address correspondence to Prof. Shinn-Zong Lin, MD., PhD.
＊
E-mail: shinnzong@yahoo.com.tw
key word: Human mesenchymal stem cells (hMSCs); improved isolation of hMSCs;
Vascular perfusion for liver progenitors; Mechanical dissociation lipoaspirate;
Isolation adipose-derived cell from blood/saline phase; Novel marrow filter device;
Clot Spots method; Stimulation BMMSCs mobilized into peripheral blood
ABSTRACT
Human mesenchymal stem cells (hMSCs) are currently available range of
applications and show much benefits to become a good material for regenerative
medicine, tissue engineering and disease therapy. Before ex vivo expansion, isolating
and characterizing primary hMSCs from peripheral tissues are the key steps for
obtaining adequate materials to clinical application. The proportion of peripheral stem
cells is very low in surrounding tissues or organs, thus, recovery ratio and quality will
be limiting factors. In this review, we summarized current common methods used to
isolate peripheral stem cells, and what new insights revealed to improve amount of
stem cells and their stemness. These strategies offered alternative ways to acquire
hMSCs in convenient and/or effective manners, which are important for commercial
purposes. Later mass-amplify primary hMSCs procedures, ensuring their stemness
until differentiate into tissue types cell for clinical usage. Enlarged qualified hMSCs
were more clinical applicability for therapeutic transplants, which may help people
live longer and better.
INTRODUCTION
Mesenchyme are derived from embryonic mesodermal progenitors, and we could
isolate hMSCs from tissues, such as bone marrow (BM), umbilical cord blood,
adipose, muscle, corneal stroma, tooth bud and so on (15,20,30,35,48,58,62,83). The
advantages of hMSCs, extracted from the individual, compared to general human
embryonic stem cells (hES) lines, having lower of immunorejection, pathogen
transmission and tumorigenesis (61,79,100). Besides, hMSCs can be handed easily in
their proliferation potential, and permitting differentiation into kinds of cell types
(74,86). MSCs are retained multipotent ability, which can proliferate into osteoblasts,
chondrocytes, adipocytes, neuron and hepatocyte like cells, and they have self-renew
capacity (50,58,64,68,97).
Expanding knowledge on salient features of hMSCs in regenerative and
immunosuppressive properties, which provided new strategies into clinical
applications (2,12,60,83). hMSCs have been largely used as potential therapeutic
strategies for diseases, involving hepatology, cardiology, neurology, orthopedics,
pancreatic, rheumatology and so on (14,27,51,60,67,79,86,105,108). hMSCs mainly
function as supplier to repair degenerative or defective organs (11,34). Combining
cures have been applied by treating with small molecular drugs, for example, valproic
acid (VPA) that stimulates stem cell proliferation and self-renewal (8,24,77). For
targeting deliver system, hMSCs have been considered to be the attractive vehicles to
carry therapeutic agents and effectively release them toward various tumor diseases
(1,36,57,84). In the neuron degenerative disease or injury issues, especially in
Parkinson's disease, amyotrophic lateral sclerosis, stroke and Alzheimer's disease,
stem cell-mediated transfer were widely used for gene therapy (5,28,47,56).
Regarding to transplant medicine, there are basic technologies in developing
separation from tissues and operating above these cells, but these are the key
procedures to success (6,102). hMSCs content are only 0.01 to 0.001% nucleated cells,
thus, isolation technologies become the important steps for clinical applications
(66,73). It would be an limited element about inadequate amounts and stemness of
hMSCs for clinical therapy; therefore, improved isolation of the stem cells is desired
(28,106). Clinical applications expected to be able to find a suitable in vitro isolation
conditions, to resolve the problem of limited cells and stemness maintaining. In this
review, we focused on issues that have been considered when manipulating hMSCs
techniques in varying isolation procedures. Further, we summarized improved
methods and new insights, theses may help people to improve hMSCs isolation and
provide more materials for transplant medicine.
ESTABLISHMENT OF hMSCs
hMSCs can be harvested in a relatively less invasive manner and easily isolated
from peripheral organs, and maintained their multipotentiality during passages
(15,72,88). It was worth noting the efficiency and quality of isolated hMSCs have
variable effects in different mediums, procedures, temperatures, and oxygen tension
(13,107). The simple method has been established for different kinds of hMSCs,
which is mainly relayed on their adherence properties to achieve isolation (21,45).
After plating of multipotent stem cells, extracted from umbilical cord and BM, most
of non-hMSCs could be separated through continuous cultures (81). For tissue types,
adipose and liver, of hMSC isolation were additional digested with collagenase
(38,54).
Following immunophenotypic analysis are necessary for identification above
adherent cells, however, to further separate non-multipotent or non-conformance cells
through their specific surface markers (59,76). It would be utmost importance to
identify hMSC lineage, and can easily obtainable to verify them through flow
cytometry (66,95). Although hMSCs were no confidence markers have been
consensus, the data showed that they still have some common cell surface markers. A
minimal phenotypic pattern requires expression of CD73, CD90, and CD105, but
shown negatively in CD34, CD45, HLA-DR and other molecules (43,66,76). For
specific immunophenotypic patterns, the variety tissues source of peripheral stem cell
could isolated by their lineage specific surface markers were summarized in Table 1
(68,109).
IMPROVED ISOLATION
Separating methods from tissues have been established, however, exist challenges
faced by investigators are that the primary hMSCs were decreased clinical
applicability (43,55). Obtaining primary hMSCs completely and rapidly, to explore a
optimized method for isolation and purification, have become a prerequisite for ex
vivo expansion (41). Improvement sections include lower segregation ratio of hMSCs,
and shorter times to maintain stem cell properties during subculture the cells (103).
Besides, the methods must be continuous developed to obtain sufficient qualities,
such as unified cell type and their multipotency (38,62). In current, improved isolation
methods have been concerned, and showed many new insights to acquire better
primary hMSCs for subsequent medical applications (25,62,97).
Vascular perfusion for liver progenitors
Traditional techniques, isolated from peripheral organs and tissues, yield only a
fewer available hMSCs and cause cell injuries with collagenase usage. This will be an
important subject to improve the quality of hMSCs, if investigators can reduce
collagenase exposure time to prevent cell from enzymatic damage (29,71).
Experimental fetal liver donated from therapeutic abortion, an alternative resource of
adult liver, have been used for isolating mesenchymal progenitor cells (49,78,93).
Further, a improved method proposed that vascular perfusion technique reduces the
exposure of the tissue to collagenase (26). The method mainly based on procurement
for perfusion via the portal vein with the adaptation of a 5-step portal vein in situ
perfusion method (26). They designed suitable solutions A to D and optimized other
conditions, to separate mesenchymal progenitor cells from liver, and cultured
hepatocytes in Williams’ E medium–based Heparmed Vito 143 (26). In contrast to the
static collagenase digestion, vascular perfusion obtained high viabilities of
mesenchymal progenitors and more cell numbers, and prolonged their stemness
(26,80).
Mechanical dissociation lipoaspirate
Methods to isolate and culture adipose-derived stem cells (ADSCs) were develop
extensively, however, little has been done to improve yields and multipotency
(3,23,38). An alternative method, mechanical dissociation, was used to isolate a
population of hMSCs from lipoaspirate without collagenase treatments. The aim of
previous study was to increase yields and cryopreservation them before ex vivo
expansion, maintaining multipotency, for therapeutic application. Adipose tissue
samples were incubate with ACK buffer solution, and then shake for preliminary red
blood cell lysis (3). Following subsequently culture to select adherent cells, and
isolate ADSCs through flow cytometry analysis. They further evidenced that treated
lipoaspirate samples can be stored without damage to ADSCs during mechanical
dissociation procedure at 4 ℃ (3). Mechanical dissociation offer a convenient
isolation method and allow large volume of adipose separation, which are more
suitable for commercial purposes (3,4,46,103).
Isolation adipose-derived cell from blood/saline phase
Compared with original isolation of ADSCs from adipose, spending 8-10 hour of
continuous intense effort in usually, another rapid separation method revealed in less
than 30 minutes (19,110). The basic principle relies on obtaining ADSCs from
blood/saline phase, containing rich adipose-derived cell due to perivascular origin,
easily than oil phase of adipose extracts (10,85). Defined simple 5-step process could
isolate ADSCs from more buoyant adipose tissue, and show a mesenchymal
morphology and immunophenotype (19). The method provides time-saving and
simple technique for ADSCs preparing, is critical for advancing transplant medicine
therapeutics.
Novel marrow filter device
A novel BM filter device has been explored to improve isolation methods, which
collects nucleated cells without continuous gradient centrifugation (70). The mainly
procedures are filtered BM deliver into the device and connect with a designed
nonwoven fabric filter in a closed system. Most of nucleated cells could be separated
through saline flow washed, removing red blood cells (RBCs) and platelets (PLTs),
and harvested in defined collection medium (70). Such closed workflow offered a
rapid purification duration times, and prevented a risk of contamination from
exposure operation. Besides, it permits decreased operator factors, influencing the cell
yields and qualities, and establish a standard protocol for clinical cell therapy trials
(31-33,69). It would be represent a major advance in BM by increasing the recovery
ratio, allowing for less ex vivo expansion, from freshly harvested peripheral stem cell
tissues (31,70).
Clot Spots method
Wharton's jelly mesenchymal stem cells (WJ-MSCs) within human umbilical cord,
is a noncontroversial source of hematopoietic stem cells (HSCs), which frequently
used in transplant medicine (18,65). The properties of WJ-MSCs considered
comparable with fetal rather than adult-derived hMSCs, thus, showed more
proliferative and immunosuppressive for therapeutically active (46). For more suitable
as a clinical application, a new approach displayed a improvement in isolating
WJ-MSCs and recognized it was a good source (39). Different to Rosset Sep method,
Clot Spot method use mesencult complete medium to culture primary human
umbilical cord blood (HUCB) (39,52). Semi-solid cord blood clots were explanted on
medium without disturbing the blood clots. Further, sub-cultured for adherent cells
selection, and then morphology identification and immunophenotyping. Compared to
Rosset Sep method, Clot Spot method demonstrated 3-fold increase of hMSCs from
WJ-MSCs (39).
Stimulation BMMSCs mobilized into peripheral blood
Since bone marrow mesenchymal stem cells (BMMSCs) found in the interior of
bones, are the most common source of hMSCs, the limitations are painful harvest and
surgical risks (18,104). To prevent invasive surgery, the in vivo mobilization of
BMMSCs into bloods seems a alternative method to harvest stem cells for
transplantation (44). Fibrin microbeads (FMB) could bind mononuclear cells and
isolate hMSCs from human peripheral blood that were mobilized with a granulocyte
colony-stimulating factor (G-CSF). G-CSF would alter homing niches in BM through
reduced VCAM-1, SDF-1, and SCF expression, caused marked down-regulation of
adhesion and released HSCs from BM into peripheral blood (53,99). This
mobilization procedure separates efficiently in HSCs isolation, and show significantly
lower contamination by other cell types (44,89). Advanced method were performed in
combinational treatment of G-CSF with plerixafor, reversibly blocks SDF-1 binding
to CXCR4, which can improve the collection of HSCs compared with G-CSF alone
(53,92).
CONCLUSION
To hMSCs used in regenerative medicine, it must be established the basis of core
technologies for preparing large amounts of suitable stem cells (87). With these key
technologies capabilities in order to provide an endless supply of stem cells, to
actually applied in cell therapy (9,74). The technologies involve acquiring source
from the peripheral tissues, isolation multipotent cells and further ex vivo expansion
(Fig 1) (74). However, enhancing isolation methods may be a critical issue, due to
inadequate source of peripheral stem cells, to acquire higher qualities of hMSCs
(3,43). In summary, improved separation technologies have following features: (1)
prevent potential contaminations during manipulation. (2) enhance recovery ratio of
hMSCs from limited source. (3) improve maintaining time of stemness during
passages. (4) make the procedures become more convenient and less cost, which are
important for commercialized purpose. Taking together, isolation strategies of hMSCs
were toward to increase the clinical applicability by improving primary stem cells to
reduce ex vivo expansion (Fig 1).
ACKNOWLEDGMENTS
Stem Cell and Regeneration Medicine Foundation, NSC 99-2320-B-039-008-MY3
and Everfront Biotech Inc.
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Table 1: Surface marker expression profiles of main MSCs types (updated from review article, Hakan
Orbay, 2012) (68).
MSCs
CD marker expression
CD9+ , CD10+, CD13+, CD29+, CD44+, CD49d+, CD49e+, CD54+, CD55+, CD71+,
ADSCs
CD73+, CD90+, CD105+, CD106+, CD146+, CD166+ and STRO-1+ (38,109).
CD13+, CD33+, CD44+, CD73+, CD90+,CD105+, CD166+, CD28+, HLA class I+
BMMSCs
(7,17,98)
PDLSCs
STRO-1+, CD13+, CD29+, CD44+, CD59+, CD90+, CD105+ (37)
TBMSCs
CD73+, STRO-1+, CD105+ (91)
SMMSCs
CD44+, CD73+, CD90+, CD105 + (16)
PMSCs
CD90+ (42)
MMSCs
CD34+, CD117+, Sca1+ (40,75)
CD105+, CD90+, CD73+, CD29+, CD13+, CD44+ CD59+, VCAM-1+, ICAM-1+,
SSCs
CD49+, CD166+, SH2+, SH4+, EGFR+, PDGFRa+, CD271+, Stro-1+, CD71+, CD133+,
CD166+, Keratin-19+ (63,82,94)
WJ-MSCs
CD13+, CD29+, CD44+, CD51+, CD73+, CD90+, CD105+, SH2, SH3 (22,96,101)
HSC
EpCAM+, E-cadherin+, CD133+, CD29 (90)+
Abbreviation: adipose-derived stem cells (ADSCs), bone marrow-derived-stem cells (BMMSCs),
periodontal ligament-derived stem cells (PDLSCs), trabecular bone-derived-stem cells (TBMSCs),
synovial membrane-derived stem cells (SMMSCs), periosteum-derived stem cells (PMSCs),
muscle-derived stem cells and satellite cells (MMSCs), skin stem cells (SSCs), wharton’s jelly
stem cells (WJ-MSCs), hepatic stem cells (HSC).
Figure 1. The relationship of hMSCs separation technology with potential clinical
application. Isolation methods would be influenced operating times, and the hMSCs
amounts and qualities that are important for transplant medicine.