- Email: [email protected]
stromal cells
Cytotherapy, 2015; 17: 1723–1731
DECIDUAL AND UMBILICAL CORD STROMAL CELLS
The effect of fibroblast growth factor on distinct differentiation potential of cord blood–derived unrestricted somatic stem cells and Wharton’s jelly–derived mesenchymal stem/stromal cells
SEUNGOK LEE1,2,*, BYUNG-JOON PARK3,*, JI YEON KIM1, DONGWOOK JEKARL1,2, HYUN YOO CHOI1, SEONG YEOUN LEE1, MYUNGSHIN KIM2, YONGGOO KIM2 & MOON-SEO PARK4 1
Department of Laboratory Medicine, Incheon St. Mary’s Hospital, College of Medicine,The Catholic University of Korea, Incheon, South Korea, 2Department of Laboratory Medicine, College of Medicine,The Catholic University of Korea, Seoul, South Korea, 3Department of Obstetrics and Gynecology, Incheon St. Mary’s Hospital, College of Medicine,The Catholic University of Korea, Incheon, South Korea, and 4Institute of Catholic Integrative Medicine, Incheon St. Mary’s Hospital, College of Medicine,The Catholic University of Korea, Incheon, South Korea Abstract Background aims. Perinatal tissues are considered an attractive source of mesenchymal stem/stromal cells (MSCs) and have unique characteristics depending on their origin. In this study, we compared the basic characteristics of unrestricted somatic stem cells isolated from cord blood (CB-USSCs) and MSCs isolated from Wharton’s jelly of umbilical cords (WJ-MSCs). We also evaluated the effect of basic fibroblast growth factor (bFGF) supplementation on the growth and differentiation of these cells. Methods. CB-USSCs and WJ-MSCs were isolated from the same individual (n = 6), and their morphology, cell surface antigens, proliferation, expression of stemness markers and adipogenic, osteogenic and chondrogenic differentiation potentials were evaluated. Their morphology, proliferation and differentiation potentials were then also compared in the presence of bFGF supplementation (10 ng/mL). Results. Overall, CB-USSCs expressed DLK-1 and negative for all the HOX gene markers. The expression of cell surface antigen CD90, growth capacity and adipogenic differential potential of CB-USSCs were lower than those of WJ-MSCs. WJ-MSCs showed higher growth capacity, but the expression of CD73 and CD105 and their osteogenic differentiation potential were lower than those of CB-USSCs. The spindle morphology of both CB-USSCs and WJ-MSCs and the growth and adipogenic differentiation of CB-USSCs were improved by bFGF supplementation. However, the bFGF supplement did not have any positive effect on the tri-lineage differentiation potentials of WJ-MSCs. Conclusions. CB-USSCs and WJ-MSCs each had distinct characteristics including different growth capacity, distinguishable cell surface markers and distinct adipogenic and osteogenic potentials. bFGF supplementation improved the growth capacity and adipogenic differentiation of CB-USSCs. Key Words: basic fibroblast growth factor, mesenchymal stem/stromal cells, umbilical cord blood, unrestricted somatic stem cells, Wharton’s jelly
Introduction Mesenchymal stem/stromal cells (MSCs) are multipotent precursor cells with self-renewal ability and have characteristics that vary depending on their origin in many adult and fetal tissues [1–3]. Ideally, for stem cells to be effective in regenerative medical
applications, stem cells should be abundant and obtainable through a minimally invasive procedure to ensure donor safety [4]. Bone marrow (BM) is a standard source for the isolation of MSCs, but the procedure for BM harvest is highly invasive. Perinatal tissues, such as umbilical cord blood (CB), Wharton’s jelly (WJ), amnion and
*These authors contributed equally to this work. Correspondence: Myungshin Kim, MD, PhD, Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Secho-gu, Seoul 137-701, South Korea. E-mail: [email protected] (Received 31 March 2015; accepted 18 September 2015) ISSN 1465-3249 Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2015.09.007
1724
S. Lee et al.
chorion, are considered to be attractive sources of MSCs because they can be obtained by less invasive methods without harm to mothers or neonates [1,4–9]. To date, results from isolations of CB-MSCs from CB (CB-MSCs) have been inconsistent because of the low frequency and viability in CB after the donor’s birth. Consequently, a large CB volume and a rapid isolation procedure after collection are needed to increase the success rate [5,8].Therefore, MSCs derived from CB have not yet been widely used in clinical applications. CB-MSCs may also be obtained as functionally distinct heterogenous populations [5,10] in a nonselective manner by same isolation procedure. Recently, an unrestricted somatic stem cell population in CB (CB-USSCs) was identified with lower adipogenic differentiation potential that correlated with the expression of the δ-like 1 (DLK-1) gene [11]. These cells can be differentiated from the CBMSCs population because of their lack of expression of the HOX genes, including HOXA9, HOXB7, HOXC10 and HOXD8 [10]. WJ is a connective tissue of the umbilical cord, and is histologically composed of myofibroblast-like stromal cells, collagen fibers, and proteoglycans [4,7,12]. Recently,WJ-MSCs have become known as an additional good source of stem cells. MSCs derived from WJ (WJMSCs) can be obtained more easily because the success rate is high (100%) and isolation is a less time-limited procedure than for CB-MSCs [3].They are embryonic and somatic in origin, highly homogenous, have a greater expansion capacity than BM-MSCs and may have the potential to differentiate into several lineages, such as adipose cells, chondrocytes, osteoblasts, neuronal cells, hepatocyte-like cells and endothelial cells in vitro [3,4,7,13]. However, a minority of reports has questioned the stemness nature of WJ-MSCs because their differentiation potential into the tri-lineages has not been achieved in vitro [2]. However,WJ-MSC meet two requirements of the minimal criteria of the International Society for CellularTherapy (2006), which state that MSCs should be plastic adherent when maintained in standard culture condition; express CD73, CD90 and CD105; lack expression of CD14, CD45 and CD34; and differentiate into adipoblasts, osteoblasts and chondrocytes in vitro [14]. For clinical applications using MSCs, growth factors such as basic fibroblast growth factor (bFGF), known as FGF-2, have been widely added to in vitro culture systems in stem cell research [15]. bFGF promotes population growth by increasing bFGF receptor signaling and affects differentiation potential into several lineages of adult stem cells, such as human BM and adipose MSCs [15–18]. However, not much is known about the effect of bFGF on the multi-potency of CBUSSCs or WJ-MSCs.
In this study, we first compared the basic characteristics of CB-USSCs and WJ-MSCs derived from the same individual (n = 6), with BM-MSCs. Second, we evaluated the effects of bFGF supplementation on morphology, proliferation and the adipogenic, osteogenic and chondrogenic differentiation potentials of CB-USSCs and WJ-MSCs. Methods Isolation of CB-USSCs This study was performed in accordance with the ethical standards of the Declaration of Helsinki and was approved by the institutional review board of our institution. Healthy women with full-term pregnancies and without underlying disease or obstetrical complications and their uneventful newborn were included in the study.Three of six infants were male and the others female; all infants fell within normal range of parameters for healthy infants. Paired CB and umbilical cord were collected at delivery with informed consent of the mother at Incheon St. Mary’s Hospital, the Catholic University of Korea. CB-USSCs were isolated from CB using the same protocol used for harvesting CB-MSCs, according to the methods of Lee et al., Zhang et al. and Phuc et al. with modification [8,19,20]. The procedure was performed within 2 hours of CB collection. Briefly, CB was diluted in phosphate buffer saline (PBS) at a ratio of 1:1 and 15 mL of this diluted blood was gently loaded onto an equal amount of Ficoll Hypaque solution (1.077 g/mL, Sigma-Aldrich) in a 50-mL tube. After centrifugation at 1600 rpm for 20 min, mononuclear cells (MNCs) were obtained from the interphase layer and washed twice with PBS [20]. MNCs were counted by an automated cell analyzer XE-2100 (Sysmex). MNCs were plated in culture at a density of 1 × 106/ cm2 into six-well culture plates (BD falcon 353046) in culture medium and incubated at 37°C in a 5% CO2 incubator [8]. Standard culture medium in this study consisted of α-modified minimum essential medium (α-MEM; Hyclone) and 20% fetal bovine serum (FBS; Hyclone), supplemented with 10 ng/mL bFGF (Gibco), 1% penicillin-streptomycin (Gibco) and Glutamax (1×; Gibco) [19]. Cells were allowed to adhere for 2 days and nonadherent cells were washed off with a medium change.The medium was changed twice weekly for 4 weeks.A colony of suspected MSCs was harvested using 0.25% trypsin-EDTA (1×; Gibco). For expansion, the cells were seeded at a density of 1 × 104/cm2 [8]. Isolation ofWJ-MSCs MSCs were isolated from WJ according to the methods of Bakhshi et al. and Hsieh et al., with modification [13,21].The procedure was performed within 24 h after
Effect of bFGF on cord blood USSC and Wharton’s jelly MSC delivery. Briefly, a portion of the umbilical cord was cut into approximately 5-cm-long segments, and these were placed immediately into PBS. Each segment was dissected longitudinally.WJ were isolated from the segments and then digested with collagenase II (Gibco) for 1 h and trypsin/EDTA for 30 min. Cells were washed twice with PBS and cultured in a T25 flask (BD 353108) at 37°C in 5% CO2 incubator. After approximately 4 days to 1 week, fibroblast-like adherent cells were detached using 0.25% trypsin/0.5 mmol/L EDTA and reseeded in fresh culture medium for further expansion [13]. MSCs from BM were obtained commercially from Texas A&M Institute for Regenerative Medicine. Expansion was performed according to the manufacturer’s instructions. Cell morphology and proliferation assay The morphology of the pairs of second passage CBUSSCs and WJ-MSCs, maintained in standard culture medium either without or with bFGF supplementation (10 ng/mL), were examined under a phase contrast microscope by two laboratory experts. Cell proliferation rates of the pairs of CB-USSCs and WJ-MSCs (n = 6) and BM-MSCs (n = 2), without and with bFGF supplemented medium (10 ng/mL), were measured from days 1 to 9 after plating 8 × 102 cells in a 96-well plate. Cell viability was measured by the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega), according to the manufacturer’s instructions [17]. All measurements were performed in triplicate. Flow cytometry Flow cytometry was performed for pairs of CBUSSCs and WJ-MSCs (n = 6), and BM-MSCs (n = 2) at the third or fourth passage. To evaluate cellsurface antigen expression, cell suspensions of 1 × 106 cells were incubated for 15 min at room temperature in the dark with phycoerythrin-conjugated monoclonal antibodies specific for the human markers associated with mesenchymal and hematopoietic lineages.The antibodies used were CD73, CD90, CD105, CD146, HLA DR, CD56 and CD45 (BD Biosciences). The samples were fixed using fixation buffer and analyzed on a FACSCanto II cytometer and the resulting data was processed using FACSDiva software (BD Biosciences). Reverse transcriptase polymerase chain reaction for the identification of CB-USSCs Reverse transcriptase polymerase chain reaction (PCR) for HOX markers (HOXA9, HOXB7, HOXC10, HOXD8) and DLK-1 was carried out on CB-USSCs
1725
together withWJ-MSCs and BM-MSCs [10,11]. HOXnegative and DLK-positive cells were identified as CBUSSCs, according to the previous studies by Liedtke et al. and Klute et al. [10,11]. In vitro differentiation MSCs from pairs of CB-USSCs andWJ-MSCs (n = 6), and BM-MSCs (n = 2) were differentiated into adipocytes, osteoblasts and chondrocytes after three passages, as previously described but with modification [3]. Briefly, for adipogenic and osteogenic differentiation, MSCs were seeded at a concentration of 4 × 104 for CB-USSCs, 3 × 104 for WJ-MSCs and 3.5 × 104 for BM-MSCs per 12-well plate, respectively, in the expansion medium without and with bFGF supplementation (10 ng/mL) for 3 days. Then the expansion medium was replaced by adipogenic or osteogenic induction medium (Gibco), without and with the bFGF supplement (10 ng/mL) for 3 weeks. Control cells were maintained in standard culture medium with 10% FBS during the same time period. After 3 weeks, cells were fixed in 4% formaldehyde. To assess adipogenic differentiation, lipid droplets of differentiated cells were stained with oil red O (SigmaAldrich). To assess osteogenic differentiation, cells were stained with alizarin red S (Sigma-Aldrich). For chondrogenic differentiation, 2.5 × 10 5 USSCs, WJ-MSCs or BM-MSCs per 15-mL conical tube (BD Biosciences) were centrifuged at 150 g for 5 min and subsequently cultivated in chondrogenic induction medium (Gibco) with or without the bFGFsupplement for 6 weeks. Control cells were maintained in standard culture medium with 10% FBS during the same time period. To assess chondrogenic differentiation, cell pellets were fixed in 4% formaldehyde and embedded in paraffin. Sections were stained with Alcian Blue (Sigma-Aldrich) and hematoxylin-eosin (H&E; Sigma-Aldrich). Real-time quantitative PCR Cell differentiation was also assessed using differentiation gene markers evaluated by real-time quantitative PCR, carried out with SYBR Green master mix (Takara Bio Inc.) on an ABI 7500 Detection System (Applied Biosystems) [10]. For adipogenic differentiation, fatty acid-binding protein 4 (FABP4), peroxisome proliferator-activated receptor gamma-2 (PPARγ) and lipoprotein lipase (LPL) markers were assessed at day 0 and 1 week [13]. For osteogenic differentiation, alkaline phosphatase (ALP), collagen I and runt-related transcription factor 2 (Runx2) markers were assessed at day 0 and 1 week [2,22]. For chondrogenic differentiation, SRY-Box 9 (Sox-9) was assessed at day 0 and 1 week [23].
1726
S. Lee et al.
The relative mRNA expression of the genetic markers was estimated with the ΔΔCt method using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control [9,10]. The relative expression of genetic markers 1 week after induction was compared with the relative expression at day 0 in each cell populations, and the results are illustrated as log2 scales as in this study.
expansion medium with bFGF (10 ng/mL), compared with cells propagated in medium lacking bFGF (supplementary Figure S2). The proliferation rate of cells in medium lacking bFGF was highest in WJMSCs, followed by CB-USSCs and BM-MSCs (P > 0.05; Figure 1). bFGF supplementation increased the mean proliferation rate significantly in CBUSSCs from days 6 to 9 (P < 0.05) and in BMMSCs from days 6 to 9 (P < 0.05).
Statistical analysis Experimental data are presented as mean ± SD. Statistical analyses were performed using a paired sample t-test for two paired groups, unpaired t-test for two independent groups or one-way analysis of variance for three independent groups.Two-way analysis of variance was performed to estimate the proliferation effect resulting from bFGF supplementation in each cell populations. A P < 0.05 was considered statistically significant. Results Identification of CB-USSCs based on HOX and DLK-1 expressivity All six MSC-like cell populations isolated from CB were negative for HOXA9, HOXB7, HOXC10 and HOXD8 and positive for DLK-1, findings that are typical of CB-USSCs, according to previous studies. In contrast, all WJ-MSCs were positive for all the HOX gene markers and negative for DLK-1 [10,11]. Representative PCR analysis is shown in supplementary Figure S1. CB-USSCs showed lower growth capacity than WJ-MSCs; spindle shape and cell growth were facilitated by bFGF supplementation The spindle morphology of CB-USSCs and WJMSCs was more distinct in cells propagated in
Cell surface antigen expression in CB-USSCs and WJ-MSCs showed distinct differences in fluorescence intensity and in the positive population The overall expression of cell surface antigens on CBUSSCs and WJ-MSCs was consistent with the minimal criteria of the International Society for Cellular Therapy (2006) [14] (Figure 2, supplementary Figure S3, supplementary Table SI and supplementary Table SII). Compared with those of BM-MSCs, both the mean fluorescence intensity (FI) and % of positive population (% Pos) of CD90, CD105 and CD146 in CBUSSCs were lower (FI, P < 0.001, all; % Pos, P = 0.002, P = 0.04, P = 0.001, respectively, by unpaired t-test). Interestingly, four of six CB-USSCs showed a CD90weak positive population (FI, 16,741 ± 7,982; % Pos, 53.6 ± 21.3). The FI and % Pos of CD73, CD105 and CD146 in WJ-MSCs were lower than those in BM MSCs (FI, P < 0.001, P < 0.001, P = 0.003; % Pos, P < 0.001, P = 0.001, P = 0.003, respectively). Interestingly, the FI and % Pos of CD73 and CD105 in WJMSCs were lower than those in CB-USSCs (FI, P < 0.001, P = 0.001; % Pos, P < 0.001, P = 0.01, respectively, by paired t-test), whereas the FI and % Pos of CD90 in CB-USSCs were lower than those in WJMSCs (FI, P < 0.001; % Pos, P = 0.001), suggesting that these may serve as good discriminatory markers. CBUSSCs and WJ-MSCs were negative for CD56, CD45 and HLA-DR.
Figure 1. Growth rates of CB-USSCs, WJ-MSCs and BM-MSCs. The fold change in proliferation of CB-USSCs (A, red line; n = 6), WJ-MSCs (B, blue line; n = 6) and BM-MSCs (C, green line; n = 2) was plotted for cells maintained in media without bFGF (dotted line, −bFGF; n = 2) and with bFGF (solid line, +bFGF). All graphs show the mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05.
Effect of bFGF on cord blood USSC and Wharton’s jelly MSC
1727
Figure 2. Flow cytometric evaluation of the expression of cell surface markers on CB-USSCs, WJ-MSCs, and BM-MSCs. Mean FI (A, C, E, G) and % of positive population (% Pos, B, D, F, H) of CD73 (A), CD90 (B), CD105 (C) and CD146 (D) in CB-USSCs (n = 6) and WJ-MSCs (n = 6) compared with BM-MSCs (n = 2). All graphs show the mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05.
The adipogenic differentiation potentials of CB-USSCs andWJ-MSCs CB-USSCs and WJ-MSCs showed distinct adipogenic differentiation potential after 3 weeks of adipogenic induction. Fat vacuoles, characteristic of adipocyte-like cells, were most abundant in BM-MSCs (n = 2), followed by in WJ-MSCs (n = 6) in conditions without and with bFGF.The formation of fat vacuoles was not facilitated by the bFGF supplement (Figure 3). Adipogenesis was unsuccessful in CB-USSCs (n = 6) lacking bFGF. However, it was stimulated in four of six CB-USSCs by bFGF supplementation. Without bFGF, FABP4, PPARγ and LPL were expressed at low levels in CB-USSC (all Ps < 0.001, all), compared with their expression levels in BM-MSCs. On the other hand, expression levels of FABP4 and LPL were low in WJ-MSCs, compared with the levels expressed in BM-MSCs (all Ps < 0.001). Of note, PPARγ, a prime inducer of adipogenesis [24], was highly expressed in WJ-MSCs when compared with CB-USSCs (P < 0.001 by paired t-test), indicating that PPARγ may have been a good adipogenic discrimination marker in this study (Figure 3, supplementary Table SIII). The relative expression levels of adipogenic markers in CB-USSCs displayed a similar pattern 1 week after adipogenic induction with the bFGF supplement. However, FABP4 and PPARγ were down-regulated in
WJ-MSCs 1 week after adipogenic induction by bFGF supplementation (P < 0.001, P = 0.001, respectively), and all adipogenic markers (FABP4, PPARγ and LPL) were also down-regulated in BM-MSCs by the bFGF supplement (P < 0.001, P = 0.008, P = 0.004, respectively; supplementary Table SIV). The osteogenic differentiation potentials of CB-USSCs andWJ-MSCs After 3 weeks of osteogenic induction, CB-USSCs and WJ-MSCs showed distinct differentiation potentials. The degree of mineralization was assessed as ALP precipitates by alizarin red S staining and was found to be highest in five of six CB-USSCs. bFGF supplementation caused osteogenic differentiation to be slightly suppressed in five of six CB-USSCs and a more pronounced suppression was seen in bFGF-treated BM-MSCs (Figure 4). However, osteogenesis was unsuccessful in WJ-MSCs lacking bFGF, which was also not facilitated by the bFGF supplementation (n = 6). Under conditions lacking bFGF, the relative expression of all osteogenic markers (ALP, Collagen I and Runx2) was lower in CB-USSCs 1 week after induction, compared with those of BM-MSCs (P = 0.03, P > 0.05 and P > 0.05, respectively). These changes did not correlate with the morphology of the ALP precipitates visualized by alizarin red S staining (Figure 4,
1728
S. Lee et al.
Figure 3. Adipogenic differentiation of CB-USSCs, WJ-MSCs and BM-MSCs in vitro. In vitro adipogenic differentiation was detected by oil red O staining in CB-USSCs (A-a, A-d), WJ-MSCs (A-b, A-e) and BM-MSCs (A-c, A-f) after 3 weeks in medium without bFGF (A-a–c, −FGF) and with bFGF (A-d–f, +FGF). Difference in adipogenic gene expression (B), FABP4 (B-a), PPARr (B-b) and LPL (B-c) in CB-USSCs, WJ-MSCs and BM-MSCs in medium without bFGF (−FGF) and with bFGF (+FGF) were evaluated by reverse transcriptase PCR 7 days after induction. All graphs show the mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05.
supplementary Table SIII). Because the low osteogenic differential potential of WJ-MSCs correlates with the low expression of only ALP (P < 0.001), this may be a good osteogenic discrimination marker. Interestingly, ALP was down-regulated by the bFGF supplement in BM-MSCs 1 week after induction (P = 0.01; supplementary Table SIV); a finding that correlates with morphology. Runx2 was also down-regulated 1 week after induction in all three cell populations by the bFGF supplement; however, this finding was not statistically significant (P > 0.05). The chondrogenic differentiation potentials of CB-USSCs andWJ-MSCs After 6 weeks of chondrogenic induction, chondrogenesis was seen in cryosections of pellets from CBUSSCs and WJ-MSCs stained with Alcian blue and H&E (Figure 5). Expression of Sox9 was lower in WJMSCs than in CB-USSCs under conditions without bFGF (P < 0.001 by paired t-test; Figure 5, Supplementary Table SIII). Chondrogenesis was also not facilitated by the bFGF supplement in CB-USSCs and WJ-MSCs, a finding that correlates with the down-
regulation of Sox9 in our study (CB-USSCs, P = 0.05; WJ-MSCs, P > 0.05 by paired t-test; Figure 5, Supplementary Table SIV). Discussion In this study, we compared the morphology, proliferation and adipogenic, osteogenic and chondrogenic differentiation potentials of six populations of CBUSSCs and WJ-MSCs, which were derived from the same individual and therefore independent of the interpersonal genetic background. WJ-MSCs had a greater expansion capacity than did CB-USSCs in the absence of bFGF. The growth of CB-USSCs was more enhanced by bFGF, indicating that bFGF supplementation can be used more effectively to enhance the growth of cells with limited expansion capacities. The spindle-shaped morphology of CB-USSCs and WJ-MSCs became more prominent, and growth of all cell types was facilitated by 10 ng/mL bFGF. These findings are consistent with previous reports that a wide range of bFGF concentrations has stimulatory effects on the growth of various types of MSCs, such as human adipose-, CB-, WJ- or BM-MSCs [15,17,18,25].
Effect of bFGF on cord blood USSC and Wharton’s jelly MSC
1729
Figure 4. Osteogenic differentiation of CB-USSCs, WJ- and BM-MSCs in vitro. In vitro osteogenic differentiation was detected by alizarin red S staining in CB-USSCs (A-a, A-d), WJ-MSCs (A-b, A-e) and BM-MSCs (A-c, A-f) after 3 weeks in medium without bFGF (A-a–c, −FGF) and with bFGF (A-d–f, +FGF). Differences in osteogenic gene expression (B), ALP (B-a), Collagen I (B-b) and Runx2 (B-c) in CB-USSCs, WJ-MSCs and BM-MSCs in medium without bFGF (−FGF) and with bFGF (+FGF) were detected by reverse transcriptase PCR 7 days after induction. All graphs show the mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05.
The cell surface expression (both mean FI and % Pos ) of CD73 (ecto-5′-nuclease) and CD105 (endoglin) on WJ-MSCs was lower than on CBUSSCs, whereas the expression of CD90 (Thy-1 membrane glycoprotein) on CB-USSCs was lower than on WJ-MSCs in medium lacking bFGF. CD90 expression was particularly variable in CB-USSCs, which is inconsistent with the previous report that CBUSSCs are positive for CD90 [26]. All WJ-MSCs showed less inter-donor variability than the CBUSSCs in this study, a finding that is consistent with that reported by Wegener et al. [3]. CB-USSCs and WJ-MSCs each had distinct trilineage differentiation potentials. CB-USSCs in our study showed low adipogenic potential that correlated with a lack of HOX expression and expression of DLK-1, findings that are consistent with those of previous reports [10,13,26]. The adipogenic potential of CB-USSCs was facilitated by bFGF supplementation (four of six cases), whereas in all WJMSCs, bFGF supplementation did not induce osteogenic potential. Overall in this study, PPARγ, ALP and Sox9 were good discriminatory markers for trilineage differentiation in CB-USSCs and WJ-MSCs.
Various previous studies have concluded that bFGF has a positive effect on the growth of MSCs; however, the effect of bFGF on the differentiation potentials of MSCs remains controversial [18,27]. Some studies reported that bFGF enhanced the chondrogenic potential of human BM-MSCs [18,28,29] or adiposeMSCs [17], and the osteogenic and chondrogenic differentiation of embryonic stem cells [23]. In this study, bFGF supplementation did not have a positive effect on the osteogenic or chondrogenic differential potentials of either human CB-USSCs or WJMSCs. Our results are in line with previous reports that bFGF inhibited osteogenesis of mouse adiposeMSCs by antagonizing BMPR-IB expression in response to retinoic acid [30] or inhibited tri-lineage differentiation of mouse BM-MSCs by inducing Twist2 and Spy4 mRNAs, and repressing Sfrp2 and Runx2 mRNAs [27]. Furthermore, low osteogenic potential was shown to be correlated with low expression of Runx2 and Twist2 in human placenta-MSCs [31]. In summary, CB-USSCs and WJ-MSCs displayed individual characteristics including differences in growth, distinguishable cell surface markers and distinct adipogenic and osteogenic potentials.
1730
S. Lee et al.
Figure 5. Chondrogenic differentiation of CB-USSCs, WJ- and BM-MSCs in vitro. In vitro chondrogenic differentiation was detected by Alcian blue and H&E staining in CB-USSCs (A-a, A-d, A-g, A-j), WJ-MSCs (A-b, A-e, A-h, A-k) and BM-MSCs (A-c, A-f, A-i, A-l) after 6 weeks in medium without bFGF (A-a–c, A-g–i, −FGF) and with bFGF supplement (A-d–f, A-j–l, +FGF). Scale bar: 100 μm. Differences in chondrogenic gene expression (B-a), Sox9 in CB-USSCs, WJ-MSCs and BM-MSCs in medium without bFGF (−FGF) and with bFGF (+FGF) were detected by reverse transcriptase PCR 7 days after induction. All graphs show the mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05.
CB-USSCs had a low growth capacity and a low adipogenic differential potential. However, their growth and adipogenesis were improved by bFGF supplementation. Therefore, bFGF supplementation could be useful in overcoming these difficulties in CBUSSCs used for biomedical engineering. On the other hand, WJ-MSCs were considered to be good candidates for biomedical engineering studies because of their high growth capacity, despite their low osteogenic differentiation potential. bFGF supplementation did not have a positive effect on the tri-lineage differentiation potential ofWJ-MSCs in this study. Further studies of the effects of bFGF on the differentiation ofWJ-MSCs into other lineages will be needed. Gaining a better understanding of the characteristics of these perinatal tissue specific-MSCs could be helpful in biomedical engineering and for their successful use in clinical applications. Acknowledgments This study was supported by the Clinical Research Foundation of Incheon St. Mary’s Hospital, The
Catholic University of Korea (S. Lee) and a grant (14172MFDS974) from Ministry of Food and Drug Safety in 2014 (M. Kim). Disclosure of interest: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References [1] Kern S, Eichler H, Stoeve J, Klüter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006;24:1294–301. [2] Bosch J, Houben AP, Radke TF, Stapelkamp D, Bünemann E, Balan P, et al. Distinct differentiation potential of “MSC” derived from cord blood and umbilical cord: are cord-derived cells true mesenchymal stromal cells? Stem Cells Dev 2012;21:1977–88. [3] Wegmeyer H, Bröske AM, Leddin M, Kuentzer K, Nisslbeck AK, Hupfeld J, et al. Mesenchymal stromal cell characteristics vary depending on their origin. Stem Cells Dev 2013; 22:2606–18. [4] Witkowska-Zimny M, Wrobel E. Perinatal sources of mesenchymal stem cells:Wharton’s jelly, amnion and chorion. Cell Mol Biol Lett 2011;16:493–514.
Effect of bFGF on cord blood USSC and Wharton’s jelly MSC [5] Markov V, Kusumi K, Tadesse MG, William DA, Hall DM, Lounev V, et al. Identification of cord blood-derived mesenchymal stem/stromal cell populations with distinct growth kinetics, differentiation potentials, and gene expression profiles. Stem Cells Dev 2007;16:53–73. [6] Mitchell KE, Weiss ML, Mitchell BM, Martin P, Davis D, Morales L, et al. Matrix cells from Wharton’s jelly form neurons and glia. Stem Cells 2003;21:50–60. [7] Troyer DL, Weiss ML. Wharton’s jelly–derived cells are a primitive stromal cell population. Stem Cells 2008;26:591– 9. [8] Zhang X, Hirai M, Cantero S, Ciubotariu R, Dobrila L, Hirsh A, et al. Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue. J Cell Biochem 2011;112:1206–18. [9] Koo BK, Park IY, Kim J, Kim JH, Kwon A, Kim M, et al. Isolation and characterization of chorionic mesenchymal stromal cells from human full term placenta. J Korean Med Sci 2012;27:857–63. [10] Liedtke S, Buchheiser A, Bosch J, Bosse F, Kruse F, Zhao X, et al. The HOX Code as a “biological fingerprint” to distinguish functionally distinct stem cell populations derived from cord blood. Stem Cell Res 2010;5:40–50. [11] Kluth SM, Buchheiser A, Houben AP, Geyh S, Krenz T, Radke TF, et al. DLK-1 as a marker to distinguish unrestricted somatic stem cells and mesenchymal stromal cells in cord blood. Stem Cells Dev 2010;19:1471–83. [12] Kobayashi K, Kubota T, Aso T. Study on myofibroblast differentiation in the stromal cells of Wharton’s jelly: expression and localization of alpha-smooth muscle actin. Early Hum Dev 1998;51:223–33. [13] Hsieh JY, Fu YS, Chang SJ, Tsuang YH, Wang HW. Functional module analysis reveals differential osteogenic and stemness potentials in human mesenchymal stem cells from bone marrow and Wharton’s jelly of umbilical cord. Stem Cells Dev 2010;19:1895–910. [14] Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315–17. [15] Park SB, Yu KR, Jung JW, Lee SR, Roh KH, Seo MS, et al. bFGF enhances the IGFs-mediated pluripotent and differentiation potentials in multipotent stem cells. Growth Factors 2009;27:425–37. [16] Rider DA, Dombrowski C, Sawyer AA, Ng GH, Leong D, Hutmacher DW, et al. Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-derived mesenchymal stem cells. Stem Cells 2008;26:1598–608. [17] Yoo GS, Lee E, Jang J, Lee E, Son Y. The chondrogenic stimulating effect of fibroblast growth factor-2 on adiposederived stem cells. Tissue Eng Regen Med 2010;7:93– 100. [18] Hagmann S, Moradi B, Frank S, Dreher T, Kämmerer PW, Richter W, et al. FGF-2 addition during expansion of human bone marrow-derived stromal cells alters MSC surface marker distribution and chondrogenic differentiation potential. Cell Prolif 2013;46:396–407.
1731
[19] Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 2004;103:1669–75. [20] Phuc PV, Ngoc VB, Lam DH, Tam NT, Viet PQ, Ngoc PK. Isolation of three important types of stem cells from the same samples of banked umbilical cord blood. Cell Tissue Bank 2012;13:341–51. [21] Bakhshi T, Zabriskie RC, Bodie S, Kidd S, Ramin S, Paganessi LA, et al. Mesenchymal stem cells from the Wharton’s jelly of umbilical cord segments provide stromal support for the maintenance of cord blood hematopoietic stem cells during long-term ex vivo culture. Transfusion 2008;48:2638–44. [22] Shafiee A, Seyedjafari E, Soleimani M, Ahmadbeigi N, Dinarvand P, Ghaemi N. A comparison between osteogenic differentiation of human unrestricted somatic stem cells and mesenchymal stem cells from bone marrow and adipose tissue. Biotechnol Lett 2011;33:1257–64. [23] Lee TJ, Jang J, Kang S, Jin M, Shin H, Kim DW, et al. Enhancement of osteogenic and chondrogenic differentiation of human embryonic stem cells by mesodermal lineage induction with BMP-4 and FGF2 treatment. Biochem Biophys Res Commun 2013;430:793–7. [24] Takada I, Kouzmenko AP, Kato S. Wnt and PPARgamma signaling in osteoblastogenesis and adipogenesis. Nat Rev Rheumatol 2009;5:442–7. [25] Nekanti U, Mohanty L, Venugopal P, Balasubramanian S, Totey S, Ta M. Optimization and scale-up of Wharton’s jelly-derived mesenchymal stem cells for clinical applications. Stem Cell Res 2010;5:244–54. [26] Bosch J, Houben AP, Hennicke T, Deenen R, Köhrer K, Liedtke S, et al. Comparing the gene expression profile of stromal cells from human cord blood and bone marrow: lack of the typical “bone” signature in cord blood cells. Stem Cells Int 2013;2013:631984. [27] Lai WT, Krishnappa V, Phinney DG. Fibroblast growth factor 2 (Fgf2) inhibits differentiation of mesenchymal stem cells by inducing Twist2 and Spry4, blocking extracellular regulated kinase activation, and altering Fgf receptor expression levels. Stem Cells 2011;29:1102–11. [28] Solchaga LA, Penick K, Porter JD, Goldberg VM, Caplan AI, Welter JF. FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow–derived mesenchymal stem cells. J Cell Physiol 2005;203:398–409. [29] Cheng T, Yang C, Weber N, Kim HT, Kuo AC. Fibroblast growth factor 2 enhances the kinetics of mesenchymal stem cell chondrogenesis. Biochem Biophys Res Commun 2012;426:544–50. [30] Quarto N, Wan DC, Longaker MT. Molecular mechanisms of FGF-2 inhibitory activity in the osteogenic context of mouse adipose-derived stem cells (mASCs). Bone 2008;42:1040–52. [31] Ulrich C, Rolauffs B, Abele H, Bonin M, Nieselt K, Hart ML, et al. Low osteogenic differentiation potential of placentaderived mesenchymal stromal cells correlates with low expression of the transcription factors Runx2 and Twist2. Stem Cells Dev 2013;22:2859–72.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.jcyt.2015.09.007.
DECIDUAL AND UMBILICAL CORD STROMAL CELLS
The effect of fibroblast growth factor on distinct differentiation potential of cord blood–derived unrestricted somatic stem cells and Wharton’s jelly–derived mesenchymal stem/stromal cells
SEUNGOK LEE1,2,*, BYUNG-JOON PARK3,*, JI YEON KIM1, DONGWOOK JEKARL1,2, HYUN YOO CHOI1, SEONG YEOUN LEE1, MYUNGSHIN KIM2, YONGGOO KIM2 & MOON-SEO PARK4 1
Department of Laboratory Medicine, Incheon St. Mary’s Hospital, College of Medicine,The Catholic University of Korea, Incheon, South Korea, 2Department of Laboratory Medicine, College of Medicine,The Catholic University of Korea, Seoul, South Korea, 3Department of Obstetrics and Gynecology, Incheon St. Mary’s Hospital, College of Medicine,The Catholic University of Korea, Incheon, South Korea, and 4Institute of Catholic Integrative Medicine, Incheon St. Mary’s Hospital, College of Medicine,The Catholic University of Korea, Incheon, South Korea Abstract Background aims. Perinatal tissues are considered an attractive source of mesenchymal stem/stromal cells (MSCs) and have unique characteristics depending on their origin. In this study, we compared the basic characteristics of unrestricted somatic stem cells isolated from cord blood (CB-USSCs) and MSCs isolated from Wharton’s jelly of umbilical cords (WJ-MSCs). We also evaluated the effect of basic fibroblast growth factor (bFGF) supplementation on the growth and differentiation of these cells. Methods. CB-USSCs and WJ-MSCs were isolated from the same individual (n = 6), and their morphology, cell surface antigens, proliferation, expression of stemness markers and adipogenic, osteogenic and chondrogenic differentiation potentials were evaluated. Their morphology, proliferation and differentiation potentials were then also compared in the presence of bFGF supplementation (10 ng/mL). Results. Overall, CB-USSCs expressed DLK-1 and negative for all the HOX gene markers. The expression of cell surface antigen CD90, growth capacity and adipogenic differential potential of CB-USSCs were lower than those of WJ-MSCs. WJ-MSCs showed higher growth capacity, but the expression of CD73 and CD105 and their osteogenic differentiation potential were lower than those of CB-USSCs. The spindle morphology of both CB-USSCs and WJ-MSCs and the growth and adipogenic differentiation of CB-USSCs were improved by bFGF supplementation. However, the bFGF supplement did not have any positive effect on the tri-lineage differentiation potentials of WJ-MSCs. Conclusions. CB-USSCs and WJ-MSCs each had distinct characteristics including different growth capacity, distinguishable cell surface markers and distinct adipogenic and osteogenic potentials. bFGF supplementation improved the growth capacity and adipogenic differentiation of CB-USSCs. Key Words: basic fibroblast growth factor, mesenchymal stem/stromal cells, umbilical cord blood, unrestricted somatic stem cells, Wharton’s jelly
Introduction Mesenchymal stem/stromal cells (MSCs) are multipotent precursor cells with self-renewal ability and have characteristics that vary depending on their origin in many adult and fetal tissues [1–3]. Ideally, for stem cells to be effective in regenerative medical
applications, stem cells should be abundant and obtainable through a minimally invasive procedure to ensure donor safety [4]. Bone marrow (BM) is a standard source for the isolation of MSCs, but the procedure for BM harvest is highly invasive. Perinatal tissues, such as umbilical cord blood (CB), Wharton’s jelly (WJ), amnion and
*These authors contributed equally to this work. Correspondence: Myungshin Kim, MD, PhD, Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Secho-gu, Seoul 137-701, South Korea. E-mail: [email protected] (Received 31 March 2015; accepted 18 September 2015) ISSN 1465-3249 Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2015.09.007
1724
S. Lee et al.
chorion, are considered to be attractive sources of MSCs because they can be obtained by less invasive methods without harm to mothers or neonates [1,4–9]. To date, results from isolations of CB-MSCs from CB (CB-MSCs) have been inconsistent because of the low frequency and viability in CB after the donor’s birth. Consequently, a large CB volume and a rapid isolation procedure after collection are needed to increase the success rate [5,8].Therefore, MSCs derived from CB have not yet been widely used in clinical applications. CB-MSCs may also be obtained as functionally distinct heterogenous populations [5,10] in a nonselective manner by same isolation procedure. Recently, an unrestricted somatic stem cell population in CB (CB-USSCs) was identified with lower adipogenic differentiation potential that correlated with the expression of the δ-like 1 (DLK-1) gene [11]. These cells can be differentiated from the CBMSCs population because of their lack of expression of the HOX genes, including HOXA9, HOXB7, HOXC10 and HOXD8 [10]. WJ is a connective tissue of the umbilical cord, and is histologically composed of myofibroblast-like stromal cells, collagen fibers, and proteoglycans [4,7,12]. Recently,WJ-MSCs have become known as an additional good source of stem cells. MSCs derived from WJ (WJMSCs) can be obtained more easily because the success rate is high (100%) and isolation is a less time-limited procedure than for CB-MSCs [3].They are embryonic and somatic in origin, highly homogenous, have a greater expansion capacity than BM-MSCs and may have the potential to differentiate into several lineages, such as adipose cells, chondrocytes, osteoblasts, neuronal cells, hepatocyte-like cells and endothelial cells in vitro [3,4,7,13]. However, a minority of reports has questioned the stemness nature of WJ-MSCs because their differentiation potential into the tri-lineages has not been achieved in vitro [2]. However,WJ-MSC meet two requirements of the minimal criteria of the International Society for CellularTherapy (2006), which state that MSCs should be plastic adherent when maintained in standard culture condition; express CD73, CD90 and CD105; lack expression of CD14, CD45 and CD34; and differentiate into adipoblasts, osteoblasts and chondrocytes in vitro [14]. For clinical applications using MSCs, growth factors such as basic fibroblast growth factor (bFGF), known as FGF-2, have been widely added to in vitro culture systems in stem cell research [15]. bFGF promotes population growth by increasing bFGF receptor signaling and affects differentiation potential into several lineages of adult stem cells, such as human BM and adipose MSCs [15–18]. However, not much is known about the effect of bFGF on the multi-potency of CBUSSCs or WJ-MSCs.
In this study, we first compared the basic characteristics of CB-USSCs and WJ-MSCs derived from the same individual (n = 6), with BM-MSCs. Second, we evaluated the effects of bFGF supplementation on morphology, proliferation and the adipogenic, osteogenic and chondrogenic differentiation potentials of CB-USSCs and WJ-MSCs. Methods Isolation of CB-USSCs This study was performed in accordance with the ethical standards of the Declaration of Helsinki and was approved by the institutional review board of our institution. Healthy women with full-term pregnancies and without underlying disease or obstetrical complications and their uneventful newborn were included in the study.Three of six infants were male and the others female; all infants fell within normal range of parameters for healthy infants. Paired CB and umbilical cord were collected at delivery with informed consent of the mother at Incheon St. Mary’s Hospital, the Catholic University of Korea. CB-USSCs were isolated from CB using the same protocol used for harvesting CB-MSCs, according to the methods of Lee et al., Zhang et al. and Phuc et al. with modification [8,19,20]. The procedure was performed within 2 hours of CB collection. Briefly, CB was diluted in phosphate buffer saline (PBS) at a ratio of 1:1 and 15 mL of this diluted blood was gently loaded onto an equal amount of Ficoll Hypaque solution (1.077 g/mL, Sigma-Aldrich) in a 50-mL tube. After centrifugation at 1600 rpm for 20 min, mononuclear cells (MNCs) were obtained from the interphase layer and washed twice with PBS [20]. MNCs were counted by an automated cell analyzer XE-2100 (Sysmex). MNCs were plated in culture at a density of 1 × 106/ cm2 into six-well culture plates (BD falcon 353046) in culture medium and incubated at 37°C in a 5% CO2 incubator [8]. Standard culture medium in this study consisted of α-modified minimum essential medium (α-MEM; Hyclone) and 20% fetal bovine serum (FBS; Hyclone), supplemented with 10 ng/mL bFGF (Gibco), 1% penicillin-streptomycin (Gibco) and Glutamax (1×; Gibco) [19]. Cells were allowed to adhere for 2 days and nonadherent cells were washed off with a medium change.The medium was changed twice weekly for 4 weeks.A colony of suspected MSCs was harvested using 0.25% trypsin-EDTA (1×; Gibco). For expansion, the cells were seeded at a density of 1 × 104/cm2 [8]. Isolation ofWJ-MSCs MSCs were isolated from WJ according to the methods of Bakhshi et al. and Hsieh et al., with modification [13,21].The procedure was performed within 24 h after
Effect of bFGF on cord blood USSC and Wharton’s jelly MSC delivery. Briefly, a portion of the umbilical cord was cut into approximately 5-cm-long segments, and these were placed immediately into PBS. Each segment was dissected longitudinally.WJ were isolated from the segments and then digested with collagenase II (Gibco) for 1 h and trypsin/EDTA for 30 min. Cells were washed twice with PBS and cultured in a T25 flask (BD 353108) at 37°C in 5% CO2 incubator. After approximately 4 days to 1 week, fibroblast-like adherent cells were detached using 0.25% trypsin/0.5 mmol/L EDTA and reseeded in fresh culture medium for further expansion [13]. MSCs from BM were obtained commercially from Texas A&M Institute for Regenerative Medicine. Expansion was performed according to the manufacturer’s instructions. Cell morphology and proliferation assay The morphology of the pairs of second passage CBUSSCs and WJ-MSCs, maintained in standard culture medium either without or with bFGF supplementation (10 ng/mL), were examined under a phase contrast microscope by two laboratory experts. Cell proliferation rates of the pairs of CB-USSCs and WJ-MSCs (n = 6) and BM-MSCs (n = 2), without and with bFGF supplemented medium (10 ng/mL), were measured from days 1 to 9 after plating 8 × 102 cells in a 96-well plate. Cell viability was measured by the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega), according to the manufacturer’s instructions [17]. All measurements were performed in triplicate. Flow cytometry Flow cytometry was performed for pairs of CBUSSCs and WJ-MSCs (n = 6), and BM-MSCs (n = 2) at the third or fourth passage. To evaluate cellsurface antigen expression, cell suspensions of 1 × 106 cells were incubated for 15 min at room temperature in the dark with phycoerythrin-conjugated monoclonal antibodies specific for the human markers associated with mesenchymal and hematopoietic lineages.The antibodies used were CD73, CD90, CD105, CD146, HLA DR, CD56 and CD45 (BD Biosciences). The samples were fixed using fixation buffer and analyzed on a FACSCanto II cytometer and the resulting data was processed using FACSDiva software (BD Biosciences). Reverse transcriptase polymerase chain reaction for the identification of CB-USSCs Reverse transcriptase polymerase chain reaction (PCR) for HOX markers (HOXA9, HOXB7, HOXC10, HOXD8) and DLK-1 was carried out on CB-USSCs
1725
together withWJ-MSCs and BM-MSCs [10,11]. HOXnegative and DLK-positive cells were identified as CBUSSCs, according to the previous studies by Liedtke et al. and Klute et al. [10,11]. In vitro differentiation MSCs from pairs of CB-USSCs andWJ-MSCs (n = 6), and BM-MSCs (n = 2) were differentiated into adipocytes, osteoblasts and chondrocytes after three passages, as previously described but with modification [3]. Briefly, for adipogenic and osteogenic differentiation, MSCs were seeded at a concentration of 4 × 104 for CB-USSCs, 3 × 104 for WJ-MSCs and 3.5 × 104 for BM-MSCs per 12-well plate, respectively, in the expansion medium without and with bFGF supplementation (10 ng/mL) for 3 days. Then the expansion medium was replaced by adipogenic or osteogenic induction medium (Gibco), without and with the bFGF supplement (10 ng/mL) for 3 weeks. Control cells were maintained in standard culture medium with 10% FBS during the same time period. After 3 weeks, cells were fixed in 4% formaldehyde. To assess adipogenic differentiation, lipid droplets of differentiated cells were stained with oil red O (SigmaAldrich). To assess osteogenic differentiation, cells were stained with alizarin red S (Sigma-Aldrich). For chondrogenic differentiation, 2.5 × 10 5 USSCs, WJ-MSCs or BM-MSCs per 15-mL conical tube (BD Biosciences) were centrifuged at 150 g for 5 min and subsequently cultivated in chondrogenic induction medium (Gibco) with or without the bFGFsupplement for 6 weeks. Control cells were maintained in standard culture medium with 10% FBS during the same time period. To assess chondrogenic differentiation, cell pellets were fixed in 4% formaldehyde and embedded in paraffin. Sections were stained with Alcian Blue (Sigma-Aldrich) and hematoxylin-eosin (H&E; Sigma-Aldrich). Real-time quantitative PCR Cell differentiation was also assessed using differentiation gene markers evaluated by real-time quantitative PCR, carried out with SYBR Green master mix (Takara Bio Inc.) on an ABI 7500 Detection System (Applied Biosystems) [10]. For adipogenic differentiation, fatty acid-binding protein 4 (FABP4), peroxisome proliferator-activated receptor gamma-2 (PPARγ) and lipoprotein lipase (LPL) markers were assessed at day 0 and 1 week [13]. For osteogenic differentiation, alkaline phosphatase (ALP), collagen I and runt-related transcription factor 2 (Runx2) markers were assessed at day 0 and 1 week [2,22]. For chondrogenic differentiation, SRY-Box 9 (Sox-9) was assessed at day 0 and 1 week [23].
1726
S. Lee et al.
The relative mRNA expression of the genetic markers was estimated with the ΔΔCt method using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control [9,10]. The relative expression of genetic markers 1 week after induction was compared with the relative expression at day 0 in each cell populations, and the results are illustrated as log2 scales as in this study.
expansion medium with bFGF (10 ng/mL), compared with cells propagated in medium lacking bFGF (supplementary Figure S2). The proliferation rate of cells in medium lacking bFGF was highest in WJMSCs, followed by CB-USSCs and BM-MSCs (P > 0.05; Figure 1). bFGF supplementation increased the mean proliferation rate significantly in CBUSSCs from days 6 to 9 (P < 0.05) and in BMMSCs from days 6 to 9 (P < 0.05).
Statistical analysis Experimental data are presented as mean ± SD. Statistical analyses were performed using a paired sample t-test for two paired groups, unpaired t-test for two independent groups or one-way analysis of variance for three independent groups.Two-way analysis of variance was performed to estimate the proliferation effect resulting from bFGF supplementation in each cell populations. A P < 0.05 was considered statistically significant. Results Identification of CB-USSCs based on HOX and DLK-1 expressivity All six MSC-like cell populations isolated from CB were negative for HOXA9, HOXB7, HOXC10 and HOXD8 and positive for DLK-1, findings that are typical of CB-USSCs, according to previous studies. In contrast, all WJ-MSCs were positive for all the HOX gene markers and negative for DLK-1 [10,11]. Representative PCR analysis is shown in supplementary Figure S1. CB-USSCs showed lower growth capacity than WJ-MSCs; spindle shape and cell growth were facilitated by bFGF supplementation The spindle morphology of CB-USSCs and WJMSCs was more distinct in cells propagated in
Cell surface antigen expression in CB-USSCs and WJ-MSCs showed distinct differences in fluorescence intensity and in the positive population The overall expression of cell surface antigens on CBUSSCs and WJ-MSCs was consistent with the minimal criteria of the International Society for Cellular Therapy (2006) [14] (Figure 2, supplementary Figure S3, supplementary Table SI and supplementary Table SII). Compared with those of BM-MSCs, both the mean fluorescence intensity (FI) and % of positive population (% Pos) of CD90, CD105 and CD146 in CBUSSCs were lower (FI, P < 0.001, all; % Pos, P = 0.002, P = 0.04, P = 0.001, respectively, by unpaired t-test). Interestingly, four of six CB-USSCs showed a CD90weak positive population (FI, 16,741 ± 7,982; % Pos, 53.6 ± 21.3). The FI and % Pos of CD73, CD105 and CD146 in WJ-MSCs were lower than those in BM MSCs (FI, P < 0.001, P < 0.001, P = 0.003; % Pos, P < 0.001, P = 0.001, P = 0.003, respectively). Interestingly, the FI and % Pos of CD73 and CD105 in WJMSCs were lower than those in CB-USSCs (FI, P < 0.001, P = 0.001; % Pos, P < 0.001, P = 0.01, respectively, by paired t-test), whereas the FI and % Pos of CD90 in CB-USSCs were lower than those in WJMSCs (FI, P < 0.001; % Pos, P = 0.001), suggesting that these may serve as good discriminatory markers. CBUSSCs and WJ-MSCs were negative for CD56, CD45 and HLA-DR.
Figure 1. Growth rates of CB-USSCs, WJ-MSCs and BM-MSCs. The fold change in proliferation of CB-USSCs (A, red line; n = 6), WJ-MSCs (B, blue line; n = 6) and BM-MSCs (C, green line; n = 2) was plotted for cells maintained in media without bFGF (dotted line, −bFGF; n = 2) and with bFGF (solid line, +bFGF). All graphs show the mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05.
Effect of bFGF on cord blood USSC and Wharton’s jelly MSC
1727
Figure 2. Flow cytometric evaluation of the expression of cell surface markers on CB-USSCs, WJ-MSCs, and BM-MSCs. Mean FI (A, C, E, G) and % of positive population (% Pos, B, D, F, H) of CD73 (A), CD90 (B), CD105 (C) and CD146 (D) in CB-USSCs (n = 6) and WJ-MSCs (n = 6) compared with BM-MSCs (n = 2). All graphs show the mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05.
The adipogenic differentiation potentials of CB-USSCs andWJ-MSCs CB-USSCs and WJ-MSCs showed distinct adipogenic differentiation potential after 3 weeks of adipogenic induction. Fat vacuoles, characteristic of adipocyte-like cells, were most abundant in BM-MSCs (n = 2), followed by in WJ-MSCs (n = 6) in conditions without and with bFGF.The formation of fat vacuoles was not facilitated by the bFGF supplement (Figure 3). Adipogenesis was unsuccessful in CB-USSCs (n = 6) lacking bFGF. However, it was stimulated in four of six CB-USSCs by bFGF supplementation. Without bFGF, FABP4, PPARγ and LPL were expressed at low levels in CB-USSC (all Ps < 0.001, all), compared with their expression levels in BM-MSCs. On the other hand, expression levels of FABP4 and LPL were low in WJ-MSCs, compared with the levels expressed in BM-MSCs (all Ps < 0.001). Of note, PPARγ, a prime inducer of adipogenesis [24], was highly expressed in WJ-MSCs when compared with CB-USSCs (P < 0.001 by paired t-test), indicating that PPARγ may have been a good adipogenic discrimination marker in this study (Figure 3, supplementary Table SIII). The relative expression levels of adipogenic markers in CB-USSCs displayed a similar pattern 1 week after adipogenic induction with the bFGF supplement. However, FABP4 and PPARγ were down-regulated in
WJ-MSCs 1 week after adipogenic induction by bFGF supplementation (P < 0.001, P = 0.001, respectively), and all adipogenic markers (FABP4, PPARγ and LPL) were also down-regulated in BM-MSCs by the bFGF supplement (P < 0.001, P = 0.008, P = 0.004, respectively; supplementary Table SIV). The osteogenic differentiation potentials of CB-USSCs andWJ-MSCs After 3 weeks of osteogenic induction, CB-USSCs and WJ-MSCs showed distinct differentiation potentials. The degree of mineralization was assessed as ALP precipitates by alizarin red S staining and was found to be highest in five of six CB-USSCs. bFGF supplementation caused osteogenic differentiation to be slightly suppressed in five of six CB-USSCs and a more pronounced suppression was seen in bFGF-treated BM-MSCs (Figure 4). However, osteogenesis was unsuccessful in WJ-MSCs lacking bFGF, which was also not facilitated by the bFGF supplementation (n = 6). Under conditions lacking bFGF, the relative expression of all osteogenic markers (ALP, Collagen I and Runx2) was lower in CB-USSCs 1 week after induction, compared with those of BM-MSCs (P = 0.03, P > 0.05 and P > 0.05, respectively). These changes did not correlate with the morphology of the ALP precipitates visualized by alizarin red S staining (Figure 4,
1728
S. Lee et al.
Figure 3. Adipogenic differentiation of CB-USSCs, WJ-MSCs and BM-MSCs in vitro. In vitro adipogenic differentiation was detected by oil red O staining in CB-USSCs (A-a, A-d), WJ-MSCs (A-b, A-e) and BM-MSCs (A-c, A-f) after 3 weeks in medium without bFGF (A-a–c, −FGF) and with bFGF (A-d–f, +FGF). Difference in adipogenic gene expression (B), FABP4 (B-a), PPARr (B-b) and LPL (B-c) in CB-USSCs, WJ-MSCs and BM-MSCs in medium without bFGF (−FGF) and with bFGF (+FGF) were evaluated by reverse transcriptase PCR 7 days after induction. All graphs show the mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05.
supplementary Table SIII). Because the low osteogenic differential potential of WJ-MSCs correlates with the low expression of only ALP (P < 0.001), this may be a good osteogenic discrimination marker. Interestingly, ALP was down-regulated by the bFGF supplement in BM-MSCs 1 week after induction (P = 0.01; supplementary Table SIV); a finding that correlates with morphology. Runx2 was also down-regulated 1 week after induction in all three cell populations by the bFGF supplement; however, this finding was not statistically significant (P > 0.05). The chondrogenic differentiation potentials of CB-USSCs andWJ-MSCs After 6 weeks of chondrogenic induction, chondrogenesis was seen in cryosections of pellets from CBUSSCs and WJ-MSCs stained with Alcian blue and H&E (Figure 5). Expression of Sox9 was lower in WJMSCs than in CB-USSCs under conditions without bFGF (P < 0.001 by paired t-test; Figure 5, Supplementary Table SIII). Chondrogenesis was also not facilitated by the bFGF supplement in CB-USSCs and WJ-MSCs, a finding that correlates with the down-
regulation of Sox9 in our study (CB-USSCs, P = 0.05; WJ-MSCs, P > 0.05 by paired t-test; Figure 5, Supplementary Table SIV). Discussion In this study, we compared the morphology, proliferation and adipogenic, osteogenic and chondrogenic differentiation potentials of six populations of CBUSSCs and WJ-MSCs, which were derived from the same individual and therefore independent of the interpersonal genetic background. WJ-MSCs had a greater expansion capacity than did CB-USSCs in the absence of bFGF. The growth of CB-USSCs was more enhanced by bFGF, indicating that bFGF supplementation can be used more effectively to enhance the growth of cells with limited expansion capacities. The spindle-shaped morphology of CB-USSCs and WJ-MSCs became more prominent, and growth of all cell types was facilitated by 10 ng/mL bFGF. These findings are consistent with previous reports that a wide range of bFGF concentrations has stimulatory effects on the growth of various types of MSCs, such as human adipose-, CB-, WJ- or BM-MSCs [15,17,18,25].
Effect of bFGF on cord blood USSC and Wharton’s jelly MSC
1729
Figure 4. Osteogenic differentiation of CB-USSCs, WJ- and BM-MSCs in vitro. In vitro osteogenic differentiation was detected by alizarin red S staining in CB-USSCs (A-a, A-d), WJ-MSCs (A-b, A-e) and BM-MSCs (A-c, A-f) after 3 weeks in medium without bFGF (A-a–c, −FGF) and with bFGF (A-d–f, +FGF). Differences in osteogenic gene expression (B), ALP (B-a), Collagen I (B-b) and Runx2 (B-c) in CB-USSCs, WJ-MSCs and BM-MSCs in medium without bFGF (−FGF) and with bFGF (+FGF) were detected by reverse transcriptase PCR 7 days after induction. All graphs show the mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05.
The cell surface expression (both mean FI and % Pos ) of CD73 (ecto-5′-nuclease) and CD105 (endoglin) on WJ-MSCs was lower than on CBUSSCs, whereas the expression of CD90 (Thy-1 membrane glycoprotein) on CB-USSCs was lower than on WJ-MSCs in medium lacking bFGF. CD90 expression was particularly variable in CB-USSCs, which is inconsistent with the previous report that CBUSSCs are positive for CD90 [26]. All WJ-MSCs showed less inter-donor variability than the CBUSSCs in this study, a finding that is consistent with that reported by Wegener et al. [3]. CB-USSCs and WJ-MSCs each had distinct trilineage differentiation potentials. CB-USSCs in our study showed low adipogenic potential that correlated with a lack of HOX expression and expression of DLK-1, findings that are consistent with those of previous reports [10,13,26]. The adipogenic potential of CB-USSCs was facilitated by bFGF supplementation (four of six cases), whereas in all WJMSCs, bFGF supplementation did not induce osteogenic potential. Overall in this study, PPARγ, ALP and Sox9 were good discriminatory markers for trilineage differentiation in CB-USSCs and WJ-MSCs.
Various previous studies have concluded that bFGF has a positive effect on the growth of MSCs; however, the effect of bFGF on the differentiation potentials of MSCs remains controversial [18,27]. Some studies reported that bFGF enhanced the chondrogenic potential of human BM-MSCs [18,28,29] or adiposeMSCs [17], and the osteogenic and chondrogenic differentiation of embryonic stem cells [23]. In this study, bFGF supplementation did not have a positive effect on the osteogenic or chondrogenic differential potentials of either human CB-USSCs or WJMSCs. Our results are in line with previous reports that bFGF inhibited osteogenesis of mouse adiposeMSCs by antagonizing BMPR-IB expression in response to retinoic acid [30] or inhibited tri-lineage differentiation of mouse BM-MSCs by inducing Twist2 and Spy4 mRNAs, and repressing Sfrp2 and Runx2 mRNAs [27]. Furthermore, low osteogenic potential was shown to be correlated with low expression of Runx2 and Twist2 in human placenta-MSCs [31]. In summary, CB-USSCs and WJ-MSCs displayed individual characteristics including differences in growth, distinguishable cell surface markers and distinct adipogenic and osteogenic potentials.
1730
S. Lee et al.
Figure 5. Chondrogenic differentiation of CB-USSCs, WJ- and BM-MSCs in vitro. In vitro chondrogenic differentiation was detected by Alcian blue and H&E staining in CB-USSCs (A-a, A-d, A-g, A-j), WJ-MSCs (A-b, A-e, A-h, A-k) and BM-MSCs (A-c, A-f, A-i, A-l) after 6 weeks in medium without bFGF (A-a–c, A-g–i, −FGF) and with bFGF supplement (A-d–f, A-j–l, +FGF). Scale bar: 100 μm. Differences in chondrogenic gene expression (B-a), Sox9 in CB-USSCs, WJ-MSCs and BM-MSCs in medium without bFGF (−FGF) and with bFGF (+FGF) were detected by reverse transcriptase PCR 7 days after induction. All graphs show the mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05.
CB-USSCs had a low growth capacity and a low adipogenic differential potential. However, their growth and adipogenesis were improved by bFGF supplementation. Therefore, bFGF supplementation could be useful in overcoming these difficulties in CBUSSCs used for biomedical engineering. On the other hand, WJ-MSCs were considered to be good candidates for biomedical engineering studies because of their high growth capacity, despite their low osteogenic differentiation potential. bFGF supplementation did not have a positive effect on the tri-lineage differentiation potential ofWJ-MSCs in this study. Further studies of the effects of bFGF on the differentiation ofWJ-MSCs into other lineages will be needed. Gaining a better understanding of the characteristics of these perinatal tissue specific-MSCs could be helpful in biomedical engineering and for their successful use in clinical applications. Acknowledgments This study was supported by the Clinical Research Foundation of Incheon St. Mary’s Hospital, The
Catholic University of Korea (S. Lee) and a grant (14172MFDS974) from Ministry of Food and Drug Safety in 2014 (M. Kim). Disclosure of interest: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References [1] Kern S, Eichler H, Stoeve J, Klüter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006;24:1294–301. [2] Bosch J, Houben AP, Radke TF, Stapelkamp D, Bünemann E, Balan P, et al. Distinct differentiation potential of “MSC” derived from cord blood and umbilical cord: are cord-derived cells true mesenchymal stromal cells? Stem Cells Dev 2012;21:1977–88. [3] Wegmeyer H, Bröske AM, Leddin M, Kuentzer K, Nisslbeck AK, Hupfeld J, et al. Mesenchymal stromal cell characteristics vary depending on their origin. Stem Cells Dev 2013; 22:2606–18. [4] Witkowska-Zimny M, Wrobel E. Perinatal sources of mesenchymal stem cells:Wharton’s jelly, amnion and chorion. Cell Mol Biol Lett 2011;16:493–514.
Effect of bFGF on cord blood USSC and Wharton’s jelly MSC [5] Markov V, Kusumi K, Tadesse MG, William DA, Hall DM, Lounev V, et al. Identification of cord blood-derived mesenchymal stem/stromal cell populations with distinct growth kinetics, differentiation potentials, and gene expression profiles. Stem Cells Dev 2007;16:53–73. [6] Mitchell KE, Weiss ML, Mitchell BM, Martin P, Davis D, Morales L, et al. Matrix cells from Wharton’s jelly form neurons and glia. Stem Cells 2003;21:50–60. [7] Troyer DL, Weiss ML. Wharton’s jelly–derived cells are a primitive stromal cell population. Stem Cells 2008;26:591– 9. [8] Zhang X, Hirai M, Cantero S, Ciubotariu R, Dobrila L, Hirsh A, et al. Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue. J Cell Biochem 2011;112:1206–18. [9] Koo BK, Park IY, Kim J, Kim JH, Kwon A, Kim M, et al. Isolation and characterization of chorionic mesenchymal stromal cells from human full term placenta. J Korean Med Sci 2012;27:857–63. [10] Liedtke S, Buchheiser A, Bosch J, Bosse F, Kruse F, Zhao X, et al. The HOX Code as a “biological fingerprint” to distinguish functionally distinct stem cell populations derived from cord blood. Stem Cell Res 2010;5:40–50. [11] Kluth SM, Buchheiser A, Houben AP, Geyh S, Krenz T, Radke TF, et al. DLK-1 as a marker to distinguish unrestricted somatic stem cells and mesenchymal stromal cells in cord blood. Stem Cells Dev 2010;19:1471–83. [12] Kobayashi K, Kubota T, Aso T. Study on myofibroblast differentiation in the stromal cells of Wharton’s jelly: expression and localization of alpha-smooth muscle actin. Early Hum Dev 1998;51:223–33. [13] Hsieh JY, Fu YS, Chang SJ, Tsuang YH, Wang HW. Functional module analysis reveals differential osteogenic and stemness potentials in human mesenchymal stem cells from bone marrow and Wharton’s jelly of umbilical cord. Stem Cells Dev 2010;19:1895–910. [14] Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315–17. [15] Park SB, Yu KR, Jung JW, Lee SR, Roh KH, Seo MS, et al. bFGF enhances the IGFs-mediated pluripotent and differentiation potentials in multipotent stem cells. Growth Factors 2009;27:425–37. [16] Rider DA, Dombrowski C, Sawyer AA, Ng GH, Leong D, Hutmacher DW, et al. Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-derived mesenchymal stem cells. Stem Cells 2008;26:1598–608. [17] Yoo GS, Lee E, Jang J, Lee E, Son Y. The chondrogenic stimulating effect of fibroblast growth factor-2 on adiposederived stem cells. Tissue Eng Regen Med 2010;7:93– 100. [18] Hagmann S, Moradi B, Frank S, Dreher T, Kämmerer PW, Richter W, et al. FGF-2 addition during expansion of human bone marrow-derived stromal cells alters MSC surface marker distribution and chondrogenic differentiation potential. Cell Prolif 2013;46:396–407.
1731
[19] Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 2004;103:1669–75. [20] Phuc PV, Ngoc VB, Lam DH, Tam NT, Viet PQ, Ngoc PK. Isolation of three important types of stem cells from the same samples of banked umbilical cord blood. Cell Tissue Bank 2012;13:341–51. [21] Bakhshi T, Zabriskie RC, Bodie S, Kidd S, Ramin S, Paganessi LA, et al. Mesenchymal stem cells from the Wharton’s jelly of umbilical cord segments provide stromal support for the maintenance of cord blood hematopoietic stem cells during long-term ex vivo culture. Transfusion 2008;48:2638–44. [22] Shafiee A, Seyedjafari E, Soleimani M, Ahmadbeigi N, Dinarvand P, Ghaemi N. A comparison between osteogenic differentiation of human unrestricted somatic stem cells and mesenchymal stem cells from bone marrow and adipose tissue. Biotechnol Lett 2011;33:1257–64. [23] Lee TJ, Jang J, Kang S, Jin M, Shin H, Kim DW, et al. Enhancement of osteogenic and chondrogenic differentiation of human embryonic stem cells by mesodermal lineage induction with BMP-4 and FGF2 treatment. Biochem Biophys Res Commun 2013;430:793–7. [24] Takada I, Kouzmenko AP, Kato S. Wnt and PPARgamma signaling in osteoblastogenesis and adipogenesis. Nat Rev Rheumatol 2009;5:442–7. [25] Nekanti U, Mohanty L, Venugopal P, Balasubramanian S, Totey S, Ta M. Optimization and scale-up of Wharton’s jelly-derived mesenchymal stem cells for clinical applications. Stem Cell Res 2010;5:244–54. [26] Bosch J, Houben AP, Hennicke T, Deenen R, Köhrer K, Liedtke S, et al. Comparing the gene expression profile of stromal cells from human cord blood and bone marrow: lack of the typical “bone” signature in cord blood cells. Stem Cells Int 2013;2013:631984. [27] Lai WT, Krishnappa V, Phinney DG. Fibroblast growth factor 2 (Fgf2) inhibits differentiation of mesenchymal stem cells by inducing Twist2 and Spry4, blocking extracellular regulated kinase activation, and altering Fgf receptor expression levels. Stem Cells 2011;29:1102–11. [28] Solchaga LA, Penick K, Porter JD, Goldberg VM, Caplan AI, Welter JF. FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow–derived mesenchymal stem cells. J Cell Physiol 2005;203:398–409. [29] Cheng T, Yang C, Weber N, Kim HT, Kuo AC. Fibroblast growth factor 2 enhances the kinetics of mesenchymal stem cell chondrogenesis. Biochem Biophys Res Commun 2012;426:544–50. [30] Quarto N, Wan DC, Longaker MT. Molecular mechanisms of FGF-2 inhibitory activity in the osteogenic context of mouse adipose-derived stem cells (mASCs). Bone 2008;42:1040–52. [31] Ulrich C, Rolauffs B, Abele H, Bonin M, Nieselt K, Hart ML, et al. Low osteogenic differentiation potential of placentaderived mesenchymal stromal cells correlates with low expression of the transcription factors Runx2 and Twist2. Stem Cells Dev 2013;22:2859–72.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.jcyt.2015.09.007.