Characterization of two populations of mesenchymal progenitor cells in umbilical cord blood

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Characterization of two populations of mesenchymal progenitor

cells in umbilical cord blood

Yu-Jen Chang

a,b

, Ching-Ping Tseng

b

, Lee-Feng Hsu

a

,

Tzu-Bou Hsieh

c

, Shiaw-Min Hwang

a,

*

a

Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu 300, Taiwan

b

Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 300, Taiwan

c

Department of Life Science, National Tsing Hua University, Hsinchu 300, Taiwan Received 20 June 2005; revised 5 November 2005; accepted 20 December 2005

Abstract

Umbilical cord blood (UCB) is a valuable source for hematopoietic progenitor cell therapy. Moreover, it contains another subset of

non-hematopoietic population referred to as mesenchymal progenitor cells (MPCs), which can be ex vivo expanded and differentiated into

osteo-blasts, chondrocytes and adipocytes. In this study, we successfully isolated the clonogenic MPCs from UCB by limiting dilution method. These

cells exhibited two different morphologic phenotypes, including flattened fibroblasts (majority) and spindle-shaped fibroblasts (minority). Both

types of MPCs shared similar cell surface markers except CD90 and had similar osteogenic and chondrogenic potentials. However, the

spindle-shaped clones possessed the positive CD90 expression and showed a greater tendency in adipogenesis, while the flattened clones were CD90

negative cells and showed a lower tendency in adipogenesis. The high number of flattened MPCs might be linked to the less sensitivity of

UCB-derived MPCs in adipogenic differentiation.

Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Keywords: Mesenchymal progenitor cells; Clonogenic; Differentiation

1. Introduction

During development, hematopoiesis is migratory, occurring

at several sites in the body of the developing fetus before

con-fining itself to the bone marrow. This implies that both

hema-topoietic progenitor cells and their stromal supporting cells

could exist in the circulatory system of prenatal fetus. Besides

hematopoietic stem/progenitor cells, umbilical cord blood

(UCB), similar to bone marrow, has been demonstrated to

con-tain mesenchymal stem cells/mesenchymal progenitor cells

(MPCs) (

Erices et al., 2000; Lee MW, et al., 2004

). MPCs

were initially referred to as plastic-adherent cells in bone

mar-row that formed fibroblastic colonies in vitro (

Friedenstein

et al., 1974

). Currently, MPCs are found in many different

tissues and can be expanded ex vivo in large quantities and

induced to differentiate into cells of mesodermal lineage,

such as osteoblasts, chondrocytes and adipocytes (

Barry and

Murphy, 2004; Pittenger et al., 1999; Erices et al., 2000;

Goodwin et al., 2001

).

Lee OK, et al. (2004)

reported that

UCB contained a more primitive population of multipotent

MPCs, which could differentiate into cells of three germ

layers. However, two different phenotypic clones of MPCs

are found in bone marrow and placenta, which are flattened

fibroblasts and spindle-shaped fibroblasts, and these

clono-genic MPCs have similar surface marker expression (

Muraglia

et al., 2000; Fukuchi et al., 2004

). It is not clear that if these

two types of clonogenic MPCs possess the same

mesenchyme-lineage differentiation capability. We are trying to explore

whether these two types of clonogenic MPCs exist in UCB

and assess their differentiation potentials in mesenchymal

lin-eages. In this study, we isolated two different types of MPCs

Abbreviations: UCB, umbilical cord blood; MPCs, mesenchymal progenitor cells.

* Corresponding author. Tel.:þ886 3 522 3191; fax: þ886 3 521 4016. E-mail address:hsm@firdi.org.tw(S.-M. Hwang).

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from UCB at clonal level, and their surface marker profiles

and differentiation potentials were comparatively analyzed

further.

2. Materials and methods

2.1. Clonogenic MPCs isolation and flow cytometric analysis

Term UCB was harvested with a standard 250-ml blood bag (Terumo, Shibuya-ku, Tokyo, Japan) with informed consent and processed within 24 h. MPCs were isolated by Ficoll-Paque density centrifugation (1.077 g/ml, Amersham, Uppsala, Sweden) and cultured in Minimum Essential Medium alpha-modification (a-MEM, Hyclone, Logan, UT) containing 20% fetal bovine serum (FBS, Hyclone), 4 ng/ml b-FGF (R&D Systems, Minneapolis, MN), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma, St. Louis, MO) according to the method described previously (Erices et al., 2000). To obtain single cell-derived MPCs, the first passage MPCs were cultured onto 96-well plate (Corning, Acton, MA) by limiting dilution (Lee OK, et al., 2004). The clonogenicity of the first passage MPCs samples was about 15%. The clonogenic MPCs were expanded at a split ratio 1:4 as follows. For surface markers analysis, cells at passage 6 were trypsinized and sus-pended in phosphate buffer saline (PBS, Gibco BRL). Primary antibodies against human antigens: CD26, CD29, CD31, CD34, CD44, CD45, CD90 (Thy-1), HLA-A, B, C, and HLA-DR were purchased from BectoneDickinson (San Jose, CA), and SH2, SH3 and SH4 were purified from respective hybrid-oma cells acquired from American Type Culture Collection (Manassas, VA). The non-specific mouse IgG (BectoneDickinson) was substituted for the primary antibodies as isotype control and anti-mouse IgG-FITC (Beckman Coulter, Brea, CA) was used as the secondary antibody for staining. Data were analyzed using a FACSscan flow cytometry system (BectoneDickinson).

2.2. In vitro differentiation

Clonogenic MPCs cells were cultured to confluence for osteogenic and adi-pogenic differentiations and over-confluence for chondrogenic differentiation for 3 weeks. The in vitro differentiations were performed by a-MEM supplemented with 10% FBS, 0.1 mM dexamethasone (Sigma), 10 mM b-glycerolphosphate (Sigma), 50 mM ascorbic acid (Sigma) for osteogenesis, a-MEM supplemented with 10% FBS, 1 mM dexamethasone (Sigma), 0.5 mM methyl-isobutylxan-thine (Sigma), 10 mg/ml insulin (Invitrogen, Carlsbad, CA), 100 mM indometh-acin (Sigma) for adipogenesis and a-MEM supplemented with 10 ng/ml TGF-b1 (PeproTech, Rocky Hill, NJ) for chondrogenesis. Osteogenic potential was assessed by von Kossa staining method, chondrogenic potential was eval-uated by the staining of proteoglycan with Safranin O (Sigma), and adipogenic potential was observed by staining with Oil Red O (Sigma). For quantification of adipogenic differentiation, ethanol was added to each well to extract the Oil Red O from the cells. The amount of Oil Red O released was determined spec-trophotometrically at 550 nm with a reference of 650 nm and compared to an Oil Red O standard titration curve (in ’t Anker et al., 2003). For detecting the mRNA expression, total RNA was isolated using Trizol reagent (MRC, Cin-cinnati, OH), and the complementary DNA (cDNA) was synthesized by ImPro-II reverse transcriptase (Promega, Madison, WI) with oilgo-dT primer. The primer sequences used were as follows: b-actin forward: 50_-TGTGGATCAGC

AAGCAGGAGTA-30_{, reverse: 5}0_{-CAAGAAAGGGTGTAACGCAACTAAG-3}0_;

PPARg2 forward: 50_{-CCAGAAAATGACAGACCTCAGACA-3}0_{, reverse: 5} 0_{-GCAGGAGCGGGTGAAGACT-3}0_{. The relative expression level of b-actin}

was used as an internal control to normalize PPARg2 gene expression in each sample. Real-time PCR was performed by ABI Prism 7000 Sequence De-tection System (Applied Biosystems, Foster City, CA) with SYBR Green PCR master mix (Applied Biosystems).

3. Results and discussions

Clonogenic MPCs with different phenotypes were observed

in human bone marrow and placenta (

Muraglia et al., 2000;

Fukuchi et al., 2004

). In this study, we successfully established

56 clones with high proliferation capability from 10 UCB

units. Among them, two different morphologic phenotypes

were observed: flattened fibroblastic clones (93%) and

spindle-shaped fibroblastic clones (7%) (

Fig. 1

A, B). The growth

rates were similar between flattened MPCs (28.6

3.4 h)

and spindle-shaped MPCs (30.4

2.5 h) calculated during

passages 4e6. Both types of clonogenic MPCs showed

a high proliferative capacity, which were passed over 10

pas-sages. Interestingly, the ratio of these two different

pheno-typic MPCs in UCB was significantly different from that in

bone marrow (no data for placenta). At the clonogenic level,

MPCs with spindle-shaped phenotype are highly abundant in

bone marrow, while flattened MPCs are rare (

Muraglia et al.,

2000

). The physiological interpretation of the difference

between these two types of MPCs is unclear, but it implies

that the differences of microenvironment might be an important

factor between UCB and bone marrow.

The cell surface markers of these two types of MPCs were

examined by FACS analysis. As shown in

Fig. 2

, both types of

MPCs were negative for CD34, CD26, CD31, CD45 and

HLA-DR. Both were positive for mesenchymal progenitor

cell markers SH2, SH3 and SH4, adherent molecules CD29,

CD44 and HLA-A, B, C. These surface marker profiles are

consistent with previously reported UCB- and bone

marrow-derived MPCs (

Goodwin et al., 2001; Pittenger et al., 1999

).

However, CD90 was differently expressed by these two cell

populations. Spindle-shaped clonogenic MPCs expressed

a high level of CD90, while flattened clonogenic MPCs showed

negative expression of CD90. These data might explain the

inconsistent results in CD90 expression of UCB-derived

MPCs in different reports (

Erices et al., 2000; Goodwin

et al., 2001; Bieback et al., 2004

). It suggests that different

levels of CD90 expression in UCB-derived MPCs may be

re-lated to the percentage of these two populations in

heteroge-neous culture condition. This result was consistent with the

findings in murine lung fibroblasts in which two populations

were identified, one was spindle-shaped and CD90 positive

fibroblasts, and the other was rounded and CD90 negative

fibroblasts (

Phipps et al., 1989; Penney et al., 1992

).

Further-more, CD90 has been known as a negative regulator for

hema-topoietic proliferation (

Mayani and Lansdrop, 1994

). It was

also reported that hematopoietic progenitor cells from UCB

possessed higher proliferation and expansion potential than

that from bone marrow (

Mayani and Lansdrop, 1998

). The

lower frequency of CD90

þ

MPCs might provide a more

beneficial environment for the proliferation of hematopoietic

progenitor cells in cord blood.

The differentiation potentials of different types of

clono-genic MPCs were investigated further. Results showed that

both types of clonogenic MPCs could differentiate into

osteo-genic and chondroosteo-genic lineages under appropriate conditions

(

Fig. 1

CeF). However, in adipogenic induction, the

spindle-shaped MPCs exhibited many typical neutral lipid vacuoles

within the cells as mature adipocytes (

Fig. 1

H), while the

flat-tened MPCs only contained sparsely small lipid droplets or

even no lipid droplets at all (

Fig. 1

G). We further quantified

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the intracellular triacylglycerol accumulation between both

types of clonogenic MPCs. As shown in

Fig. 3

A, the amount

of cell-bound Oil Red O in spindle-shaped MPCs was 5.3-fold

higher than that found in flattened MPCs during adipogenesis.

The adipogenic transcription factor, PPARg2, in

spindle-shaped MPCs was expressed higher than that expressed in

flattened MPCs by 1.6-fold (

Fig. 3

B). It was reported that

UCB-derived MPCs showed a reduced capability to undergo

adipogenesis (

Bieback et al., 2004

). Recently, we have also

found that UCB-derived MPCs have lower adipogenic

poten-tial than bone morrow-derived MPCs in vitro (

Chang et al.,

2006

). It was demonstrated that CD90 could serve as a marker

of preadipocytes in 3T3-L1 cells, and the CD90

þ

subpopula-tion was lipid-containing cells within lung fibroblasts (

Gagnon

et al., 2004; Phipps et al., 1989

). Our data suggested that high

number of flattened MPCs might actually be linked to the less

sensitivity of UCB-derived MPCs in adipogenic differentiation.

Although the nature of adipogenesis from MPCs was unknown

in vivo, the ratio between flattened MPCs and spindle-shaped

MPCs in different tissues, including UCB and adult bone

marrow, may account for their physiology in terms of

adipo-genic development.

Fig. 1. Morphology and differentiation potentials of two types of clonogenic MPCs from umbilical cord blood. Flattened fibroblastic phenotype (A) and spindle-shaped fibroblastic phenotype (B). Both types of MPCs were exposed in vitro to differentiation medium for 3 weeks. The osteogenic differentiation was assessed by von Kossa staining showing the presence of matrix mineralization (C, D), the chondrogenic differentiation was stained positively in proteoglycan using Safranin O (E, F), and adipogenic differentiation was assayed by Oil Red O staining at lipid vacuoles (G, H). The flattened clonogenic MPCs showed a low tendency in adipogenic differentiation. Bar scales: 50 mm.

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Fig. 2. Comparison of cell surface marker profiles between two types of clonogenic MPCs. Flattened fibroblastic MPCs (A) and spindle-shaped fibroblastic MPCs (B). Both types of MPCs at passage 6 were analyzed by flow cytometry with antibodies against the indicated antigens. The respective isotype control was shown in dotted line. The flattened clonogenic MPCs showed negative expression of CD90, while the spindle-shaped clonogenic MPCs expressed a high level of CD90.

Fig. 3. Adipogenic capacity of two types of clonogenic MPCs. The adipogenic capacity was represented by the extraction of cell-bound Oil Red O, which was normalized by the cell number in a panel of wells in parallel (A). The PPARg2 gene expression in both type of clonogenic MPCs was detected by real-time PCR at the third week of induction (B). The data were represented as fold changed in differentiated cells relative to the corresponding undifferentiated cells. Undiffer-entiated: white bar, Adipogenic induction at the third week: black bar . Results represented mean SD of three replicas and derived from at least two independent experiments. Asterisks indicate statistically significant difference (p < 0.05) compared to undifferentiated condition.

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Acknowledgment

This work was supported by the Ministry of Economic

Affairs, Taiwan (93-EC-17-A-17-R7-0525) and the

Founda-tion of Research and Development from Food Industry

Research and Development Institute, Taiwan.

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