중간엽 줄기세포(Mesenchymal stem cells)의 특성과 임상적 적용

[문형철]

권강범교수에게 분자생물학의 용어를 공부하고 나서 읽는 첫번째 논문 ㅎㅎ

The stem cell division can increase dramatically, when new cells are needed urgently!!

침치료, 침도, 가열식화침, 인대약침, 재생운동법 등은 모두 

재생의학이다. 

우리 몸 구석구석에 존재하는 줄기세포의 재생을 극대화하는 방법을 찾는 것이 부작용없고, 효과적인 재생의학이다. 

panic bird...

Mesenchymal stem cells- characteristics and clinical applications.pdf

Mesenchymal stem cells: characteristics and clinical applications

Abstract

Mesenchymal stem cells (MSCs) are bone marrow populating cells, different from hematopoietic stem cells,which possess an extensive proliferative potential and ability to differentiate into various cell types, including: osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes and neurons. 

- 중간엽 줄기세포는 골수에 거주하는 세포로 조혈 줄기세포로부터 분화됨. 이는 광범위한 재생가능한 세포인 osteocyte, adipocyte, chondrocyte, myocyte, cardiomyocyte and neurons로 분화함. 

MSCs play a key role in the maintenance of bone marrow homeostasis and regulate the maturation of both hematopoietic and non-hematopoietic cells. The cells are characterized by the expression‎ of numerous surface antigens, but none of them appears to be exclusively expressed on MSCs. Apart from bone marrow, MSCs are located in other tissues, like: adipose tissue, peripheral blood, cord blood, liver and fetal tissues. 

- 중간엽 줄기세포는 골수 항상성을 유지하고, 조혈과 비조혈 세포사이의 성숙을 조절함. ... 중간엽 줄기세포는 지방세포, 말초혈액, 간,  fetal 조직에 위치함. 

MSCs have been shown to be powerful tools in gene therapies, and can be effectively transduced with viral vectors containing a therapeutic gene, as well as with cDNA for specific proteins, expression‎ of which is desired in a patient. Due to such characteristics, the number of clinical trials based on the use of MSCs increase. 

- 중간엽 줄기세포는 유전자치료에서 가장 중요한 도구임. 그래서 치료적 유전자...

These cells have been successfully employed in graft versus host disease (GvHD) treatment, heart regeneration after infarct, cartilage and bone repair, skin wounds healing, neuronal regeneration and many others. Of special importance is their use in the treatment of osteogenesis imperfecta (OI), which appeared to be the only reasonable therapeutic strategy. MSCs seem to represent a future powerful tool in regenerative medicine, therefore they are particularly important in medical research.

- 이 세포는 ...

Introduction

Mesenchymal stem cells (MSCs) are non-hematopoietic cells, which reside in the bone marrow together with better known and characterized class of stem cells - hematopoietic stem cells. They were first described by Fridenstein et al. in 1976, as the clonal, plastic adherent cells, being a source of the osteoblastic, adipogenic and chondrogenic cell lines [38]. The interest in MSCs rapidly grows with expanding knowledge about their exceptional characteristics and usefulness in the clinic. This review describes the latest data about MSC biology and behavior in vitro, as well as in vivo. It presents also molecular features of MSCs and their broad use in various clinical settings.

Sources of MSCs

The main source of MSCs is the bone marrow. These cells constitute, however, only a small percentage of the total number of bone marrow populating cells. Pittenger et al. showed that only 0.01% to 0.001% of mononuclear cells isolated on density gradient (ficoll/percoll) give rise to plastic adherent fibroblastlike colonies [96]. The number of MSCs isolated from this tissue may vary in terms of the yield and the quality,even when the cells are obtained from the same donor [95].

Apart from the bone marrow, MSCs are also located in other tissues of the human body. There is an increasing number of reports describing their presence in adipose tissue [43], umbilical cord blood, chorionic villi of the placenta [54], amniotic fluid [122], peripheral blood [133], fetal liver [11], lung [57], and even in exfoliated deciduous teeth [85]. 

The amount of MSCs decreases with age [36] and infirmity [56]. The greatest number of MSCs is found in neonates, than it is reduced during the lifespan to about one-half at the age of 80 [36]. As for circulating fetal MSCs, the highest number is detected in the first trimester and declines during the second trimester to about 0.0001% and further to 0.00003% of nucleated cells in cord blood [11].

Surface markers on MSCs

MSCs constitute a heterogeneous population of cells, in terms of their morphology, physiology and expression‎ of surface antigens. Up to now, no single specific marker has been identified. MCSs express a large number of adhesion molecules, extracellular matrix proteins, cytokines and growth factor receptors, associated with their function and cell interactions within the bone marrow stroma [28]. They also express a wide variety of antigens characteristic for other cell types, as confirmed by advanced molecular techniques, including serial analysis of gene expression‎ [111] and DNA microarray [61]. 

The population of MSCs isolated from bone marrow express: CD44, CD105 (SH2; endoglin), CD106 (vascular cell adhesion

molecule; VCAM-1), CD166, CD29, CD73 (SH3 and SH4), CD90 (Thy-1), CD117, STRO-1 and Sca-1 [5, 7, 21, 26, 44, 160]. Interestingly, the observations made by Bonyadi et al. [8] present late-onset osteoporosis in mice lacking Sca-1. Parallelly, MSCs do not possess markers typical for hematopoietic and endothelial cell lineages: CD11b, CD14, CD31, CD33, CD34, CD133 and CD45 [96]. The absence of CD14, CD34 and CD45 antigens on their surface create the basis to distinguish them from the hematopoietic precursors [5]. In Figure 1 we present the phenotype characteristic of the 2nd passage BM-MSCs. This data from our laboratory confirm‎ the standard description of these cells.

MSCs are also known to express a set of receptors

associated with matrix- and cell-to-cell adhesive interactions,

like integrins αVβ3 and αVβ5, ICAM-1,

ICAM-2, LFA-3 and L-selectin [21, 96, 7].

There have been studies to find an accurate combination

of a limited number of antigens in order to isolate

pure population of MSCs from a tissue. From the

data available up to now, several options have been

proposed in this context. One of them suggests that the

co-expression‎ of CD105 and CD73 could be sufficient

[96]. Another one implies that the expression‎ of

CD166 and CD105 makes it possible to separate the

earliest precursors of MSCs from more mature cells

[2]. In turn, examination of the CFU-F obtained from

bone marrow stroma demonstrated that the MSCs fraction

may by identified by several markers, including

STRO-1, Thy-1, CD49a, CD10, Muc18/CD146, as

well as with the antibodies to receptors for PDGF

(platelet derived growth factor) and EGF (epithelial

growth factor) [5, 8, 26, 44, 96, 98].

Although MSCs have been described by a subset of

surface antigens, little is known about fresh or nonexpanded

MSCs, mostly because of their very low frequency

in adult bone marrow [7]. The findings by

Boiret et al. [7] showed that the most discriminative

markers for MSCs examined after short time of adherence

(1-3 days) were: CD73 and CD49a, as all the

CFU-F-colonies (100%) were CD73- and most

(95.2%) were CD49a- positive. Interestingly, these

data did not confirm‎ that CD105 and CDw90 could be

selective markers for MSCs, as only 45.4% and 49%

of the CFU-F were positive for these molecules,

respectively [7]. Furthermore, the authors checked the

surface protein expression‎ on freshly isolated bone

marrow MSCs, showing, as found previously, that

CD73 and CD49a were the most extensively expressed

antigens in CFU-F-enriched subset. These results

stand in opposition with the popular description of

MSC phenotype, which postulated the STRO-1 antigen

to be exclusively expressed by primitive mesenchymal

precursors [26, 44].

However, the presence of some antigens may

change in vitro, due to specific culture conditions and

the duration prior to individual passages [22]. Interestingly,

some antigens may be found on freshly isolated

MSCs, but their expression‎ disappears in culture. Such

a phenomenon was observed in case of CD34 antigen.

This molecule was expressed by MSCs obtained from

mouse fetal lungs, but could not be found in in vitro

cultures of MSCs [36]. This would suggest that the

expression‎ of that molecule vanishes during the maturation

process. Similar results were obtained in case of

chemokine receptor expression‎ on human MSCs [49].

The second passage BMSCs expressed: CCR1, CCR7,

CCR9, CXCR4, CXCR5 and CXCR6. At the 12-16th

passage, there was no expression‎ of any of those molecules,

which was also confirmed by a disability of the

cells to migrate towards specific chemokine attractants.

Moreover, the loss of these receptors' expression‎

was accompanied by a decrease in the expression‎ of

adhesion molecules - ICAM-1, ICAM-2, VCAM-1

and CD157. Moreover, the alteration in BM-MSCs

phenotype was associated with increasing cell cycle

arrest and induction of the apoptotic pathway [49].

The change in antigen expression‎ has been also

described for MSCs undergoing differentiation

process. As an example, the CD166 antigen (activated

leukocyte cell adhesion molecule) has been presented

on undifferentiated MSCs but was absent from the

cells that underwent osteogenic differentiation [10].

Furthermore, the cell clones derived from different tissues

may slightly differ in terms of cell surface molecules.

A survey investigating the antigen profile on

MSCs isolated from adipose tissue revealed that in

majority these cells are very much alike as bone marrow-

derived MSCs [64]. However, in a small number of surface proteins, the two populations differ. The adipose

tissue MSCs were shown to possess additionallyCD49d [64], CD62e and CD31 [43].

Basic biology and functions of MSCs

Human MSCs are known to constitute a heterogeneous population of cells and their properties and functionality depend on the environmental characteristics. MSCs can be expanded in culture were they give rise to fibroblastic colonies (CFU-F). The CFU-F units are well documented to possess an extended proliferative potential in vitro [22]. Studies in rodents with 3[H]-thymidine labeling demonstrated that CFU-F are essentially in a noncycling state in vivo [133]. The number of colonies obtained from bone marrow aspirates differs among species, as well as throughout the culture conditions used in each individual experiment. Colony formation by MSCs derived from adult human BM is feeder cell independent, while the rodent cells require a source of irradiated feeder cells to achieve maximal plating efficiency [9, 97].

The cultures of MSCs are, however, not completely explored. Former studies claimed that MSCs isolated from bone marrow comprise a single phenotypic population forming symmetric, spindle-shaped colonies (homology up to 98%) [96]. More recent studies, however, indicate that single-cell derived colonies are morphologically heterogeneous, containing at least two different cell types: small spindleshaped cells and large cuboidal or flattened cells [9,55]. 

In terms of proliferative potential, the cells have been also described as small rapidly-renewing, and large slowly-renewing [102]. Contrastingly, the work performed by Colter et al. [19] describes the population of small and agranular cells (RS-1) within stationary culture of MSCs with a low capacity to generate colonies and non-reactive to the cell cycle-specific antigen Ki-67. That cell subpopulation was shown, however, to be responsible for the capacity of the whole population of MSCs to expand in culture. Furthermore, it was speculated that RS cells may cycle under stimulation by factors secreted by the more mature MSCs. These cells were, thus, proposed to represent an ex vivo subset of recycling uncommitted mesenchymal stem cells [19].

Nevertheless, the latest findings show that MSC colonies contain as much as three types of cells. The third fraction was described to be composed of very small rapidly self-renewing cells [20], which are reported as the earliest progenitors and possess the greatest potential for multilineage differentiation. The examination of these cells revealed that they were about 7 μm in diameter and had a high nucleus-tocytoplasm ratio. They could be also distinguished from more mature cells by the presence of specific surface epitopes and expressed proteins, like vascular endothelial growth factor receptor-2, tyrosine kinase receptor, transferrin receptor and annexin II (lipocortin 2). Some of the rapidly renewing cells contained also other markers, like c-kit (CD117), multidrug resistance epitope and epithelial membrane antigen. 

Interestingly, these cells were negative for STRO-1, an antigen originally considered as a marker for MSCs [26]. MSCs play a significant role in bone marrow microenvironment. The major function of these cells is to create a tissue framework, which assures a mechanical support for hematopoietic cell system. They secrete a number of extracellular matrix proteins, including fibronectin, laminin, collagen and proteoglycans 

[28]. Moreover, MSCs produce hematopoietic and non-hematopoietic growth factors, chemokines and cytokines, thereby participating in the regulation of hemopoiesis. MSCs secrete: IL-1a, IL-1b, IL-6, IL- 7, IL-8, IL-11, IL-14, IL-15, macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-SCF), leukemia inhibitory factor, stem cell factor (SCF), fetal liver tyrosine kinase-3, thrombopoietin and hepatocyte growth factor (HGF)[7, 20, 22, 44, 64]. Some of these proteins are produced by quiescent cells, whereas the others after stimulation.

The involvement of MSCs in hematopoiesis is additionally consolidated by their presence in fetal liver and bone marrow just prior to the onset of definitive hemopoiesis at those sites [11, 80]. An animal model study confirmed that human MSCs marked with

GFP and transplanted into the tibia of NOD/SCID mice, integrated into the functional components of hematopoietic microenvironment and actively participated in the hematopoietic cell development [86]. During 4 to 10 weeks after transplantation, GFP-MSCs differentiated into pericytes, myofibroblasts, stromal

cells, osteocytes and endothelial cells. This led to the

increase in the number of functionally and phenotypically

primitive human hematopoietic cells in murine

bone marrow microenvironment. The engrafted cells

supported human hematopoiesis via secreted factors

and by physical interactions with primitive hematopoietic

cells [86]. Other studies showed that cotransplantation

of human MSCs and HSCs resulted in increased

chimerism or/and accelerated hematopoietic recovery

in animal models and in humans [36, 67, 71]. Moreover,

MSCs are known to produce a variety of

cytokines that are involved in homing (stromal derived

factor-1 - SDF-1) or proliferation and differentiation of

hematopoietic cells (GM-CSF, SCF, IL-6) [48]. It has

been proposed that several chemokine axes are

involved in maintaining bone marrow homeostasis,

and that some chemokines, which MSCs possess the

receptors for, like CCR9 and CXCR4 may operate in

an autocrine manner, similarly as it is in case of HSCs

[49].

Among other well known biological activities of

MSCs, it is worth to emphasize their immunomodulatory

functions. These cells are able to inhibit responses

of alloreactive T lymphocytes. They express neither

MHC class II molecules nor costimulatory receptors

(CD80, CD86) on their surface, therefore they do not

exhibit antigen-presenting cell activities [3, 36]. The

addition of interferon-γ (IFN-γ) to the cultures of

MSCs enhances the expression‎ of MHC class I and

triggers the expression‎ of MHC class II, but not of the

costimulatory molecules. [36]. It has been well established

that MSCs from various species can exert profound

immunosupression by inhibiting T-cell responses

to polyclonal stimuli [29] and to their cognate peptide

[69]. The inhibition did not seem to be antigen

specific and targeted both primary and secondary Tcell

responses [69]. The inhibitory effect was shown to

be directed mostly at the level of cell proliferation. T

cells stimulated in the presence of MSCs were arrested

in the G1 phase as a result of cyclin D downregulation

[41]. The suppression, however, was not apoptotic

and could be reversed. In the absence of MSCs and

with appropriate stimuli, T cells continue to proliferate

[29]. The precise mechanism by which MSCs modulate

immunological response is still to be clarified, but

overall data suggest that soluble factors as well as cell

contact mediated mechanisms are involved. Blocking

experiments with the use of neutralizing monoclonal

antibodies against transforming growth factor-β

(TGF-β) and HGF suggest that these factors are at

least in part responsible for the inhibitory effects

caused by MSCs [29]. Moreover, MSCs can affect

other cells participating in immune response like B

cells [41] and dendritic cells [63].