Stem Cells International

Stem Cells International / 2017 / Article

Research Article | Open Access

Volume 2017 |Article ID 8749751 | 16 pages | https://doi.org/10.1155/2017/8749751

Involvement of WNT Signaling in the Regulation of Gestational Age-Dependent Umbilical Cord-Derived Mesenchymal Stem Cell Proliferation

Academic Editor: Zhaohui Ye
Received17 Apr 2017
Revised22 Jun 2017
Accepted04 Jul 2017
Published12 Sep 2017

Abstract

Mesenchymal stem cells (MSCs) are a heterogeneous cell population that is isolated initially from the bone marrow (BM) and subsequently almost all tissues including umbilical cord (UC). UC-derived MSCs (UC-MSCs) have attracted an increasing attention as a source for cell therapy against various degenerative diseases due to their vigorous proliferation and differentiation. Although the cell proliferation and differentiation of BM-derived MSCs is known to decline with age, the functional difference between preterm and term UC-MSCs is poorly characterized. In the present study, we isolated UC-MSCs from 23 infants delivered at 22–40 weeks of gestation and analyzed their gene expression and cell proliferation. Microarray analysis revealed that global gene expression in preterm UC-MSCs was distinct from term UC-MSCs. WNT signaling impacts on a variety of tissue stem cell proliferation and differentiation, and its pathway genes were enriched in differentially expressed genes between preterm and term UC-MSCs. Cell proliferation of preterm UC-MSCs was significantly enhanced compared to term UC-MSCs and counteracted by WNT signaling inhibitor XAV939. Furthermore, WNT2B expression in UC-MSCs showed a significant negative correlation with gestational age (GA). These results suggest that WNT signaling is involved in the regulation of GA-dependent UC-MSC proliferation.

1. Introduction

Mesenchymal stem cells (MSCs) are a heterogeneous cell population that has a potential to proliferate and differentiate into trilineage mesenchymal cells: adipocytes, osteocytes, and chondrocytes. MSCs were initially isolated and characterized from the bone marrow (BM) [1, 2] and subsequently derived from almost all tissues including adipose tissue (AT), synovium, skin, dental pulp, umbilical cord blood (UCB), placenta, and umbilical cord (UC) [3]. Due to the ability to home to sites of injury, undergo differentiation, suppress immune responses, and modulate angiogenesis, MSCs are paid an increasing attention as a source for cell therapy against various degenerative diseases. Currently, MSCs from different sources have been tested in clinical studies for treatment of graft-versus-host disease, myocardial infarction, cerebral infarction, and so on [4, 5].

Although BM is the most well-characterized source of MSCs, it has certain limitations with the invasive BM aspiration and the decline in MSC proliferation and differentiation capacity with age. In contrast, fetal MSCs obtained from UCB, placenta, and UC have advantages with the noninvasive sampling during newborn delivery and the vigorous proliferation and differentiation capacity for cell therapy [6, 7]. Especially, human UC starts to develop at 4–8 weeks of gestation, continues to grow until 50–60 cm in length, and is usually discarded as medical waste after newborn delivery. Taken together, UC-derived MSCs (UC-MSCs) will become a promising source for cell therapy [8, 9].

Various genes and signaling pathways are known to regulate MSC proliferation and differentiation. WNT signaling serves as a key regulator that influences various stages of embryonic development as well as tissue homeostasis in adulthood [10]. It affects the proliferation, self-renewal, and differentiation of various tissue stem cells and controls the various tissue renewal and regeneration in response to disease, trauma, and ageing [11]. WNT ligands, which comprise a family of 19 members in human, are evolutionally conserved, are lipid modified, and secreted glycoproteins. They can activate either β-catenin-dependent (canonical) or β-catenin-independent (noncanonical) pathways by acting on transmembrane receptor FZD and its coreceptors LRP5/LRP6. The canonical pathway inhibits the β-catenin destruction complex, associates the transcriptional coactivator β-catenin with the transcriptional factor complex TCF/LEF, and induces WNT target gene transcription. The noncanonical pathway is independent of β-catenin and mainly associates with Ca2+-dependent and JNK-dependent signaling pathways, which can impact on cell migration, cell polarity, and cytoskeletal organization. The molecular events occurring these noncanonical pathways are far less defined than the canonical pathway [12].

Early studies showed the profound impacts of WNT signaling on a variety of tissue stem cell proliferation and differentiation [1315]. In MSCs, both stimulatory and inhibitory roles for WNT signaling in cell proliferation and differentiation into trilineage mesenchymal cells were documented [16]. The adipogenic differentiation of AT-derived MSCs (AT-MSCs) was inhibited by WNT signaling activation [17]. The potential of WNT signaling on osteogenic differentiation of MSCs was controversial, with both stimulatory and inhibitory effects being reported [18, 19]. An inhibitory effect of WNT signaling on chondrogenic differentiation was demonstrated in AT-MSCs [20].

Although UC can be obtained from a wide range of gestational age (GA) newborn as a result of preterm, term, and postterm delivery, their functional differences are poorly characterized [21]. An understanding of the molecular mechanisms controlling UC-MSC proliferation and differentiation is crucial to determining the drivers and effectors of the functional difference between different GA UC-MSCs as well as the most suitable use of UC-MSCs for cell therapy against degenerative diseases. In the present study, we isolated UC-MSCs from 23 infants delivered at 22–40 weeks of gestation and analyzed their gene expression and cell proliferation.

2. Materials and Methods

2.1. Patients and Samples

Human UCs were obtained from 23 infants delivered at 22–40 weeks of gestation with parental written consent. This study was approved by the Ethics Committee at Kobe University Graduate School of Medicine (approval number 1370) and Hyogo Prefectural Kobe Children’s Hospital (approval numbers 24-25) and conducted in accordance with the approved guidelines.

2.2. Preparation of UC-MSC

The umbilical cord (2-3 g weight) was collected, cut into 2-3 mm pieces, enzymatically dissociated with Liberase DH Research Grade (Roche, Mannheim, Germany) in PBS for 45–60 min at 37°C followed by the addition of 10% fetal bovine serum (FBS; Sigma, St. Louis, MO) to inhibit enzyme activity, and filtered through a 100 μm cell strainer (BD Bioscience, Bedford, MA). The resulting cells derived from all compartments of the umbilical cord (whole UC) were cultured at 37°C (5% CO2 and 95% air) in MEM-α (Wako Pure Chemical, Osaka, Japan) containing 10% FBS and 1% antibiotic-antimycotic solution (Invitrogen, Carlsbad, CA) until confluent primary cultures were established. The cells were then disassociated with trypsin-EDTA (Wako Pure Chemical), and the trypsinized cells were seeded into fresh dishes and passaged to confluence. Serial passaging was carried out until the tenth passage. The cells at fifth to eighth passages were used in the present experiments.

2.3. Cell Surface Marker Analysis

UC-MSCs were dissociated with 0.25% trypsin-EDTA for 10 minutes, washed with PBS and suspended at ~1 × 106 cells/ml in FCM buffer containing 1 × PBS, 2 mM EDTA, and 10% Block Ace (Dainippon Pharmaceutical, Osaka, Japan). The cells were incubated with phycoeryhrin- (PE-) conjugated mouse primary antibodies against CD14, CD19, CD34, CD45, CD73, CD90, CD105, or HLA-DR (BD Bioscience, Franklin Lakes, NJ) for 45 min on ice, washed with PBS, incubated with Fixable Viability Stain 450 (BD Bioscience) for 15 min at room temperature, washed with PBS, and filtered through a 70 μm cell strainer (BD Bioscience). PE-conjugated mouse IgG1 k, IgG2a k, or IgG2b k isotype control (BD Bioscience) was used as a negative control for each primary antibody. Flow cytometric analysis was performed using FACSAria III carrying a triple laser (BD Bioscience) and FACSDiva software (BD Bioscience).

2.4. Cell Differentiation

To verify the multipotency of UC-MSCs, the cells were induced to differentiate into the adipogenic, osteogenic, and chondrogenic lineages. Adipogenic differentiation was induced in STEMPRO adipogenesis differentiation medium (Invitrogen) for 2-3 weeks and stained, and the differentiation was investigated by staining lipid vesicles with Oil Red O (Sigma). Osteogenic differentiation was induced in STEMPRO osteogenesis differentiation medium (Invitrogen) or STK-3 (DS Pharma Biomedical, Osaka, Japan) for 1-2 weeks, and the differentiation was examined by staining with Arizarin Red S (Sigma) reacting to calcium cation. Chondrogenic differentiation was induced by forming cell aggregates in micromass culture in STEMPRO chondorogenesis differentiation medium (Invitrogen) for 1 week, and the differentiation was assessed by staining anionic glycoconjugates with Toluidine Blue (Sigma). Cell images were acquired using a BZ-X700 microscope (Keyence, Osaka, Japan).

2.5. RNA Extraction

Total RNA from UC-MSCs and fibroblasts was extracted with a TRIZOL Plus RNA purification kit (Life Technologies) according to the manufacturer’s instructions. RNA integrity was evaluated by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) using RNA 6000 nanokit (Agilent Technologies) according to the manufacturer’s instructions.

2.6. Gene Expression Microarray Analysis

Total RNA from three term and five preterm UC-MSCs (Table 1) was subjected to global gene expression analysis using the Low Input Quick Amp Labeling Kit One-Color (Agilent Technologies) and SurePrint G3 Human Gene Expression v3 8 × 60 K Microarray Kit (Agilent Technologies) according to the manufacturer’s instruction. Briefly, double-stranded cDNA was synthesized from 100 ng of total RNA by AffinityScript-RT using T7 promoter-incorporated Oligo-dT primer. Cyanine 3- (Cy3-) CTP-incorporated RNA (cRNA) was generated using the second strand cDNA as a template via an in vitro transcription reaction. The amplified cRNA was purified with the RNeasy mini kit (Qiagen, Valencia, CA) and quantified cRNA by the NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA). 600 ng of Cy3-labeled cRNA was hybridized to the microarray slides at 65°C for 17 hr with rotation at 10 rpm. After hybridization, the slides were washed and scanned by the SureScan (Agilent Technologies), the images were subsequently extracted using the Feature Extraction Software (Agilent Technologies). Extracted data with good QC metrics were normalized (percentile shift to the 75th percentile) and filtered by gene expression (20.0–100.0 percentile), flags for signals and error for CV in the GeneSpring GX (v 14.5) (Agilent Technologies). The processed data were subjected to statistical analysis (moderated T-test with Benjamini-Hochberg FDR), and the corrected value <0.05 was determined to be significant (). The following analyses were performed for further data interpretation: principal component analysis (PCA), clustering analysis, GO (gene ontology) analysis, and pathway analysis with curated datasets of WikiPathways (413 pathways) and KEGG (10 pathways). A gene-set list associated with human WNT signaling pathway (150 genes, 04310 from KEGG pathways) was obtained from a public database (https://www.stemformatics.org/).


SampleGestational age (weeks)Birth weight (g)SexApgar score 1 minApgar score 5 minPaternal age (years)Maternal age (years)GravidityParityPerinatal historyMaternal complicationUsage in the present study

Pre-124530Female13423822Cesarean section due to non-reassuring fetal status involved with abruption of placentaPregnancy-induced hypertensionFigures 2, 3, 5, and 6, S
Pre-225656Male48263100Cesarean section due to active premature laborFigures 2, 3, 5, and 6, S
Pre-326338Male22373611Cesarean section due to active premature labor involved with placental hematomaFigures 2, 3, 4, 5, and 6, S
Pre-422550Male14262511Cesarean section due to active premature laborFigures 2, 3, 5, and 6, S
Pre-523530Female24323111Vaginal delivery due to active premature laborFigures 2, 3, 5, and 6, S
Pre-623478Female15323221Cesarean section due to active premature laborFigures 5 and 6, S
Pre-724642Male13161700Cesarean section due to active premature laborFigures 1, 5, and 6, S
Pre-826750Female59393821Cesarean section due to advancing pregnancy-induced hypertensionPregnancy-induced hypertension Graves’ diseaseFigures 4, 5, and 6, S
Pre-926568Female47383621Cesarean section due to non-reassuring fetal statusLow-lying placentaFigure 4
Int-1301546Female78444200Cesarean section due to non-reassuring fetal statusFigure 6, S
Int-2311170Male47272811Cesarean section due to non-reassuring fetal statusPregnancy-induced hypertensionFigure 6, S
Int-3342062Female79484232Cesarean section due to non-reassuring fetal statusPregnancy-induced hypertensionFigure 6, S
Term-1382550Male88373400Cesarean section due to placental previaFigures 2, 3, and 6, S
Term-2402546Male26404411Cesarean section due to non-reassuring fetal statusFigures 2, 3, and 6, S
Term-3383314Male88403300Cesarean section due to breech presentationFigures 2, 3, 5, and 6, S
Term-4372750Female89363300Cesarean section due to placental previaFigures 5 and 6, S
Term-5373062Male910353511Repeated cesarean sectionFigure 5
Term-6372776Female89424521Repeated cesarean sectionFigures 5 and 6, S
Term-7383390Male910322611Normal vaginal deliveryFigures 5 and 6, S
Term-8382960Male89383911Repeated cesarean sectionFigures 1, 5, and 6, S
Term-9383144Female88393911Repeated cesarean sectionFigure 4
Term-10382666Female89282800Cesarean section due to breech presentationFigure 4
Term-11392892Female99373110Cesarean section due to breech presentationFigure 4

2.7. Quantitative RT-PCR (RT-qPCR)

cDNA was synthesized from 1 μg of total RNA from UC-MSCs by using a QuantiTect reverse transcription kit (Qiagen). Real-time PCR analysis was performed with an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA) using FastStart Universal SYBR Green master mix (Roche) with 0.5 μM sense and antisense primers and cDNA (corresponding to 12.5 ng total RNA) according to the manufacturer’s instructions. Each cDNA was amplified with a precycling hold at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 60 sec, and one cycle at 95°C for 15 sec, 60°C for 60 sec, 95°C for 15 sec, and 60°C for 15 sec. Relative expression of each transcript was calculated based on the ΔΔCt method using β-actin (ACTB) as an endogenous reference for normalization. Primer sequences for WNT2, WNT2B, WNT3A, WNT4, WNT5B, WNT6, SFRP1, and ACTB were shown in Table 2. All sample measurements were repeated at least three times, and the results were expressed as the mean ± SE.


Forward primerReverse primer

WNT2tttggcagggtcctactcccctggtgatggcaaatacaa
WNT2Baacttacataataaccgctgtggtcactcacgccatggcactt
WNT3Aaactgcaccaccgtccacaaggccgactccctggta
WNT4gcagagccctcatgaacctcacccgcatgtgtgtcag
WNT5Bgcgagaagactggaatcaggcagagcagccgtgaacag
WNT6agagtgccagttccagttccgaacacgaaggccgtctc
SFRP1gctggagcacgagaccattggcagttcttgttgagca
ACTBccaaccgcgagaagatgaccagaggcgtacagggatag

2.8. Ki-67 Staining

Cell suspensions of UC-MSCs were centrifuged at 3000 rpm for 5 min, and two smears were immediately prepared. Slides were fixed in 95% ethanol for immunostaining or fixed in 20% formalin and 80% methanol and stained with hematoxylin and eosin (H&E), respectively. Immunostaining was performed with antibody against Ki-67 (Clone MIB-1, Dako, Santa Clara, CA) using Leica Bond-Max automation and Bond Polymer Refine detection kit (Leica Biosystems, Nussloch, Germany) according to manufacturer’s instructions. IHC cytology protocol included primary antibody incubation for 15 min, post primary for 8 min, polymer for 8 min, peroxide block for 5 min, mixed DAB refine for 10 min, and followed by 5 min hematoxylin counterstaining.

2.9. MTS Assay

UC-MSCs were seeded at the density of 12,000 cells/well in a 12-well plate, incubated in 1 ml of MEM-α with 10% FBS in the presence or absence of 10 μM XAV939 (Selleck Chemicals, Houston, TX) at 37°C (5% CO2 and 95% air) for 24, 48, or 72 h. Cell proliferation was then determined by the CellTiter 96H AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instruction. Briefly, 200 μl of MTS reagent (a tetrazolium compound) was added into each well and incubated at 37°C (5% CO2 and 95% air) for 4 h. The absorbance at 490 nm was measured using an EnSpire Microplate Reader (Perkin Elmer, Poland, OR). All experiments were repeated at least three times, and the results were expressed as the mean ± SE.

2.10. Statistical Analysis

Pearson’s correlation coefficients were determined, and the Mann–Whitney U test was used to compare two independent datasets, using Excel software (Microsoft, Redmond, WA) and Excel Statistics (Statcel 3; Social Survey Research Information, Tokyo, Japan). Differences were considered statistically significant for .

3. Results

3.1. UC-MSCs Isolated from Infants Delivered at 22–40 Weeks of Gestation

We first obtained UCs from infants delivered at 22–40 weeks of gestation and then isolated the plastic-adherent cells from these UCs (Table 1). The cells exhibited a spindle-like shape (Figure 1(a)). Their cell surface markers were positive for MSC signature markers CD73, CD90, and CD105 but negative for hematopoietic, macrophage, and endothelial markers CD14, CD19, CD34, CD45, and HLA-DR by flow cytometric analysis (Figure 1(b)). There were no statistically significant differences in the percentages of MSC signature marker-positive cells (CD73: 99.9 ± 0.1% and 99.6 ± 0.4%, CD90: 99.9 ± 0.1% and 99.5 ± 0.5%, and CD105: 99.7 ± 0.3% and 99.5 ± 0.5%) between preterm and term UCs.

Under standard in vitro differentiation conditions, both preterm and term UC-MSCs were induced to differentiate into osteocytes, adipocytes, and chondrocytes (Figure 1(c)). Preterm UC-MSCs did not qualitatively differ from term UC-MSCs in their capacity to differentiate into trilineage mesenchymal cells. Taken together, the resulting cells fulfilled the criteria defined by the ISCT position paper [22] and were defined as UC-MSCs.

3.2. Differentially Expressed Genes between Preterm and Term UC-MSCs

To get an insight into the functional difference between preterm and term UC-MSCs, we extracted total RNA from five preterm and three term UC-MSCs (Table 1) and performed microarray analysis. Principal component analysis (PCA) for global gene expression revealed that preterm UC-MSC samples were clustered together and were separated from term UC-MSC samples (Figure 2(a)). In total, 5578 unique genes (4272 upregulated and 1306 downregulated) showed greater than twofold-expression changes between preterm and term UC-MSCs with a corrected value less than 0.05 (Figure 2(b), Supplementary Table S available online at https://doi.org/10.1155/2017/8749751). The pathway analysis of all differentially expressed genes identified significant enrichment of signaling pathways for immune/inflammatory reactions, cell-cell/cell-extracellular matrix interactions, glucose/lipid metabolism, and cell proliferation and differentiation (Table 3). Among these signaling pathways, we focused WNT signaling pathway that was previously implicated in the regulation of MSC proliferation and differentiation. Noticeably, 32/150 of WNT signaling pathway genes were overlapped with differentially expressed genes between preterm and term UC-MSCs (Figures 2(c) and 2(d)).


Pathway valueMatched
entities
Pathway entities

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Hs_GPCR_downstream_signaling_WP1824_833011.85E−07171919
Hs_Interferon_gamma_signaling_WP1836_832348.41E−0632170
Hs_NRF2_pathway_WP2884_830413.20E−0536143
Hs_Immunoregulatory_interactions_between_a_Lymphoid_and_a_non-Lymphoid_cell_WP1829_831645.33E−0532332
Hs_Gastrin-CREB_signalling_pathway_via_PKC_and_MAPK_WP2664_832661.55E−0442180
Hs_Focal_Adhesion_WP306_803082.25E−0445191
Hs_GPCR_ligand_binding_WP1825_833462.99E−0485438
Hs_Regulation_of_beta-cell_development_WP3513_834073.98E−041128
Hs_Glycerophospholipid_biosynthesis_WP2740_833414.59E−042696
Hs_Allograft_Rejection_WP2328_785545.49E−0424100
Hs_Extracellular_matrix_organization_WP2703_831066.47E−042278
Hs_Selenium_Micronutrient_Network_WP15_827056.56E−042384
MAPK signaling pathway7.53E−0455257
Hs_MicroRNAs_in_cardiomyocyte_hypertrophy_WP1544_752587.89E−0423104
Hs_DNA_Damage_Response_(only_ATM_dependent)_WP710_799748.29E−0429114
Hs_Parkin-Ubiquitin_Proteasomal_System_pathway_WP2359_721219.16E−042073
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Hs_Insulin_Signaling_WP481_827310.008487700534161
Hs_L1CAM_interactions_WP1843_830820.0088329672192
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Hs_G1_to_S_cell_cycle_control_WP45_800010.0109317751768
Hs_Prostate_Cancer_WP2263_804390.01103618325117
Hs_Wnt_Signaling_Pathway_and_Pluripotency_WP399_794740.01132130123101
Hs_Human_Complement_System_WP2806_830050.01132130123136
Hs_Assembly_of_collagen_fibrils_and_other_multimeric_structures_WP2798_832310.0113662705929
Hs_DSCAM_interactions_WP1808_833720.0118092755511
Hs_Arrhythmogenic_Right_Ventricular_Cardiomyopathy_WP2118_712650.0120062861878
Hs_Endothelin_Pathways_WP2197_748520.01215774751033
Hs_Nuclear_Receptors_WP170_710830.0125465461138
Hs_Metapathway_biotransformation_WP702_735160.01343192834188
Hs_Potassium_Channels_WP2669_832720.01391017251559
Hs_Myometrial_Relaxation_and_Contraction_Pathways_WP289_810780.01439557932156
Hs_Collagen_biosynthesis_and_modifying_enzymes_WP2725_831300.0144584771454
Hs_Cardiac_Hypertrophic_Response_WP2795_785440.0144584771454
Hs_IL-3_Signaling_Pathway_WP286_785830.0149117171349
Hs_Hematopoietic_Stem_Cell_Differentiation_WP2849_830390.0153188281198
Hs_Telomere_Maintenance_WP1928_830970.0162105361561
Hs_Signaling_Pathways_in_Glioblastoma_WP2261_811970.0167991983
Glycolysis/Gluconeogenesis0.0177754051666
Hs_Reversible_hydration_of_carbon_dioxide_WP2770_831760.017944276512
Hs_Transport_of_inorganic_cations-anions_and_amino_acids-oligopeptides_WP1936_832670.018519322197
Hs_Neural_Crest_Differentiation_WP2064_792630.0192203222101
Hs_Signaling_by_the_B_Cell_Receptor_(BCR)_WP2746_831580.02034601424247
Hs_Adipogenesis_WP236_802090.0214582727131
Hs_Secretion_of_Hydrochloric_Acid_in_Parietal_Cells_WP2597_784850.02190148835
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Hs_Transport_of_vitamins,_nucleosides,_and_related_molecules_WP1937_832070.0271708821038
Hs_S_Phase_WP2772_833950.0276941325123
Hs_Integrin_cell_surface_interactions_WP1833_831810.0284216011566
Hs_GABA_synthesis,_release,_reuptake_and_degradation_WP2685_830900.030757299619
Hs_Transport_of_glucose_and_other_sugars,_bile_salts_and_organic_acids,_metal_ions_and_amine_compounds_WP1935_831320.031666321100
Hs_Class_I_MHC_mediated_antigen_processing_&_presentation_WP3577_834670.03238653849330
Hs_Formation_of_Fibrin_Clot_(Clotting_Cascade)_WP1818_831430.0323938581039
Hs_Parkinsons_Disease_Pathway_WP2371_797660.0323938581071
Hs_ErbB_Signaling_Pathway_WP673_802020.0323957841355
Hs_Interferon_type_I_signaling_pathways_WP585_802010.0323957841354
Hs_Metabolism_of_water-soluble_vitamins_and_cofactors_WP1857_830830.0335786531993
Hs_Primary_Focal_Segmental_Glomerulosclerosis_FSGS_WP2572_799470.0339473151674
Hs_Glycolysis_and_Gluconeogenesis_WP534_785850.0344274981249
Hs_Interleukin-11_Signaling_Pathway_WP2332_795250.036419721144
Hs_Sleep_regulation_WP3591_838610.03828181039
Hs_Regulation_of_toll-like_receptor_signaling_pathway_WP1449_811720.0394049827149
Hs_PI_Metabolism_WP2747_831600.039729611251
Hs_Signal_regulatory_protein_(SIRP)_family_interactions_WP1909_831900.0397491411
Hs_Neurotoxicity_of_clostridium_toxins_WP2665_833210.0397491422
Hs_Overview_of_nanoparticle_effects_WP3287_829260.039779507622
Hs_Protein_folding_WP1892_831030.039869573934
Hs_Platelet_homeostasis_WP1885_831920.0412754271570
Hs_Synthesis_of_DNA_WP1925_831440.041895852098
Hs_Pathogenic_Escherichia_coli_infection_WP2272_785940.0424287131364
Hs_Mitotic_G1-G1-S_phases_WP1858_833150.04292910525128
Hs_Synaptic_Vesicle_Pathway_WP2267_785950.045577751251
Hs_Spinal_Cord_Injury_WP2431_803430.04672784423117
Hs_Gamma_carboxylation,_hypusine_formation_and_arylsulfatase_activation_WP2762_833880.04724196936
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Hs_Binding_and_Uptake_of_Ligands_by_Scavenger_Receptors_WP2784_832170.04884686711195
Hs_Extracellular_vesicle-mediated_signaling_in_recipient_cells_WP2870_795550.04923168830

We then confirmed a subset of these WNT signaling pathway gene expressions by RT-qPCR using cDNA from the same five preterm and three term UC-MSCs as a template. A subset included secreted WNT ligands and modulators: WNT2, WNT2B, WNT3A, WNT4, WNT5B, WNT6, and SFRP1. Consistent with microarray analysis, upregulated WNT2, WNT2B, WNT3A, WNT4, and WNT6 showed increased expression in preterm UC-MSCs compared to term UC-MSCs by RT-qPCR (Table 4, Figure 3). Decreased expression of downregulated WNT5B and SFRP1 was also detected by RT-qPCR (Table 4, Figure 3). Collectively, these results suggested that WNT signaling pathway gene expression in preterm UC-MSCs was distinct from term UC-MSCs.


GeneFC (pre versus term) (Corr)

Ligands
WNT24.079170.01631
WNT2B2.647910.02698
WNT3A2.560170.02584
WNT62.355310.00938
WNT42.116230.00510
WNT5B−3.935330.01317
Receptors
FZD93.478200.04035
TCF7L22.292220.01187
Extracellular modulators
DKK43.790290.01143
DKK2−2.653270.01720
SFRP1−3.133360.00621
Intracellular signaling molecules
CCND23.498920.02933
DAAM22.712890.00467
CER12.401600.03446
MAPK82.360910.00501
NFATC42.256870.01089
APC22.217740.04864
PPP2R5B2.209560.00501
PRKCB2.117800.00504
CAMK2A2.034430.02825
APC−2.018610.00578
FOSL1−2.068720.03346
PRKACA−2.164030.02705
PPP2R1A−2.197190.04119
CCND3−2.207270.01748
RUVBL1−2.244270.02646
AXIN1−2.478320.02670
RAC2−2.518800.00902
TBL1X−2.732440.01261
DVL1−2.772990.00726
NFATC3−2.825610.02238
CCND1−3.285800.03436

3.3. Cell Proliferation of Preterm and Term UC-MSCs

To examine the function of WNT signaling pathway genes in preterm and term UC-MSCs, we isolated UC-MSCs from nine preterm (22–26 weeks of gestation) and nine term (37–39 weeks of gestation) infants (Table 1) and analyzed their cell proliferation. We first evaluated the expression of Ki-67, a marker of proliferating cells expressed in all active phases of the cell cycle (G1, S, G2, and M), by immunocytochemistry [23]. The percentages of Ki-67-positive cells were markedly increased in preterm UC-MSCs as compared to term UC-MSCs, albeit not statistically significant (Figure 4).

We then analyzed cell proliferation of preterm and term UC-MSCs by MTS assay. Although both preterm and term UC-MSCs showed vigorous proliferation, the proliferation rate of preterm UC-MSCs measured at 72 h was significantly faster than term UC-MSCs (Figure 5(a)). Next, we examined the effect of WNT signaling inhibition on the growth of preterm and term UC-MSCs using a small molecule XAV939. XAV939 is a potent inhibitor of Tankyrase1 and Tankyrase2, and this inhibition stabilizes Axin1 and Axin2, the concentration-limiting component of the WNT pathway transcription factor β-catenin destruction complex. Increased levels of Axin1 and Axin2 stimulate β-catenin degradation and thereby inhibit β-catenin-mediated transcription [24]. Treatment of preterm UC-MSCs with 10 μM XAV939 resulted in significant inhibition of cell proliferation (Figure 5(b)). Term UC-MSC proliferation was also reduced by 10 μM XAV939, but there was no statistical significance (Figure 5(c)). These results suggest that WNT signaling is involved in the enhanced cell proliferation of preterm UC-MSCs compared to term UC-MSCs.

3.4. Gestational Age-Dependent Expression of WNT Signaling Pathway Genes

We further analyzed WNT2, WNT2B, WNT3A, WNT4, WNT5B, WNT6, and SFRP1 expressions in UC-MSCs isolated from other 10 infants delivered at 22–40 weeks of gestation by RT-qPCR. Expression of these WNT signaling pathway genes tended to decrease or increase with gestational age. Among them, WNT2B expression showed a statistically significant negative correlation with gestational age (Figure 6, Supplementary Figure S).

4. Discussion

In the present study, we isolated UC-MSCs from 23 infants delivered at 22–40 weeks of gestation and obtained the following findings. (1) Global gene expression in preterm UC-MSCs was distinct from term UC-MSCs. (2) WNT signaling pathway genes were enriched in differentially expressed genes between preterm and term UC-MSCs. (3) Preterm UC-MSC proliferation was faster than term UC-MSCs. (4) WNT signaling inhibitor XAV939 significantly inhibited the cell proliferation of preterm but not term UC-MSCs. (5) WNT2B expression in UC-MSCs showed a significant negative correlation with GA.

MSCs are isolated from a variety of tissues and result in so heterogeneous population of cells, and not all of them express the same phenotypic markers. In the case of BM-MSCs, younger donor-derived BM-MSCs showed greater proliferative and differentiative potential than older counterparts and may have more potential for cell therapy [25, 26]. Although fetal MSCs could be isolated from newborns delivered at a wide range of GA as a result of preterm, term, and postterm delivery, their GA-dependent function remained poorly characterized [8, 9]. With regard to UCB-MSCs, the MSC population in UCB was significantly higher in preterm newborn compared to term newborn [27, 28]. In the case of UC-MSCs, MSCs were isolated from different UC compartments including cord lining, perivascular region (PV), Wharton’s jelly (WJ), and whole UC [2931]. Preterm UCs were shown to contain more perivascular cells (PVCs), identical to MSCs, than term UCs [32]. Preterm PVCs/UC-MSCs isolated from fetuses aborted at 8–12 weeks of gestation were reported to exhibit a greater proliferative potential, a more efficient differentiation into chondrogenic and adipogenic cell lineages, and a differential gene expression profile compared to term PVCs/UC-MSCs isolated from newborns delivered at 37–40 weeks of gestation [33]. Although we isolated UC-MSCs from the whole UC and preterm newborns delivered at 22–26 weeks of gestation, the present study and others supported that proliferative capacity of UC-MSCs declined with GA.

Global gene expression analysis identified 5578 differentially expressed genes between preterm and term UC-MSCs (Figure 2(a), Table S). The pathway analysis revealed significant enrichment of 111 signaling pathways (Table 3). Immune/inflammatory reaction-associated signaling pathways were top-ranked among the list (Table 3). The upregulation of interferon (IFN) signaling pathways in preterm UC-MSCs may be interpreted as the consequence of preterm delivery that has inherent fetal and/or maternal indications (Table 1, Table 3). Although cell cycle and senescence-associated secretory phenotype pathways were also expected to affect the growth rate and GA-dependent changes of UC-MSCs, these pathways were not included in the list (Table 3).

WNT signaling is a key regulator of stem cell functions in development, renewal, and regeneration of multiple tissues [1315]. In the case of MSCs, mRNA expression of a subset of WNT signaling pathway genes including WNT2, WNT4, WNT5A, WNT11, WNT16, SFRP2, SFRP3, and SFRP4 was detected in BM-MSCs [34]. WNT2, WNT2B, WNT4, WNT5A, WNT5B, SFRP1, and SFRP4 were also highly expressed in AT-MSCs under hypoxic stress conditions [35]. Comparison of BM-MSCs with UC-MSCs revealed lower differentiation capacity toward osteocytes and adipocytes along with the downregulation of WNT3A, WNT5A, WNT5B, WNT7B, WNT8A, SFRP1, and SFRP4 in UC-MSCs compared to BM-MSCs [36]. Consistent with these observations, the present study revealed a significant enrichment of WNT2, WNT2B, WNT3A, WNT4, WNT5B, WNT6, and SFRP1 in differentially expressed genes between preterm and term UC-MSCs (Figure 2(c)). Noticeably, WNT2, WNT2B, WNT4, WNT5B, WNT6, and SFRP1 were associated with a noncanonical WNT pathway, as opposed to only WNT3A with a canonical WNT pathway among these WNT ligands and modulators in UC-MSCs [12]. In contrast, the enhanced cell proliferation of preterm UC-MSCs was abolished by XAV939, which selectively decreased β-catenin expression through Tankyrase1 and Tankyrase2 inhibition and increased Axin1 and Axin2 expression (Figure 5) [24]. Accumulating evidence indicates that noncanonical WNT signaling can inhibit canonical WNT signaling [37, 38] and that activation of either canonical or noncanonical WNT signaling is highly dependent on the cell type and on specific receptors expressed by the cells [39, 40]. Further understanding of how WNT signaling pathway controls the GA-dependent proliferation of UC-MSC will be crucial to develop UC-MSC-based cell therapy.

In summary, preterm UC-MSC proliferation is significantly faster than term UC-MSCs, and WNT signaling is involved in the regulation of this GA-dependent proliferation of UC-MSCs.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research (C) (Grant no. 25461644) and Young Scientists (B) (Grant no. 26860845) of JSPS KAKENHI. The authors thank Drs. Shohei Ohyama, Sachiyo Fukushima, Oshi Tokuda, Kaori Maeyama, and Miwako Nagasaka for collecting the umbilical cord samples.

Supplementary Materials

Table S1: List of differentially expressed genes between preterm and term UC-MSCs. Figure S1: Gestational age-dependent expression of WNT signaling pathway genes. Figure S1 legend: Relative expression of WNT2, WNT3A, WNT4, WNT5B, WNT6, and SFRP1 mRNA in UC-MSCs isolated from 18 infants delivered at 22–40 weeks of gestation was analyzed by RT-qPCR. The mean of all UC-MSCs was defined as 1.

  1. Supplementary material
  2. Supplementary material

References

  1. A. J. Friedenstein, K. V. Petrakova, A. I. Kurolesova, and G. P. Frolova, “Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues,” Transplantation, vol. 6, no. 2, pp. 230–247, 1968. View at: Google Scholar
  2. A. I. Caplan, “Mesenchymal stem cells,” Journal of Orthopaedic Research, vol. 9, no. 5, pp. 641–650, 1991. View at: Publisher Site | Google Scholar
  3. M. Crisan, S. Yap, L. Casteilla et al., “A perivascular origin for mesenchymal stem cells in multiple human organs,” Cell Stem Cell, vol. 3, no. 3, pp. 301–313, 2008. View at: Publisher Site | Google Scholar
  4. P. Bianco, P. G. Robey, and P. J. Simmons, “Mesenchymal stem cells: revisiting history, concepts, and assays,” Cell Stem Cell, vol. 2, no. 4, pp. 313–319, 2008. View at: Publisher Site | Google Scholar
  5. P. Bianco, ““Mesenchymal” stem cells,” Annual Review of Cell and Developmental Biology, vol. 30, no. 1, pp. 677–704, 2014. View at: Publisher Site | Google Scholar
  6. D. Baksh, R. Yao, and R. S. Tuan, “Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow,” Stem Cells, vol. 25, no. 6, pp. 1384–1392, 2007. View at: Google Scholar
  7. S. Manochantr, Y. U-pratya, P. Kheolamai et al., “Immunosuppressive properties of mesenchymal stromal cells derived from amnion, placenta, wharton's jelly and umbilical cord,” Internal Medicine Journal, vol. 43, no. 4, pp. 430–439, 2013. View at: Publisher Site | Google Scholar
  8. D.-C. Ding, Y.-H. Chang, W.-C. Shyu, and S.-Z. Lin, “Human umbilical cord mesenchymal stem cells: a new era for stem cell therapy,” Cell Transplantation, vol. 24, no. 3, pp. 339–347, 2015. View at: Publisher Site | Google Scholar
  9. I. Arutyunyan, A. Elchaninov, A. Makarov, and T. Fatkhudinov, “Umbilical cord as prospective source for mesenchymal stem cell-based therapy,” Stem Cells International, vol. 2016, Article ID 6901286, 17 pages, 2016. View at: Publisher Site | Google Scholar
  10. H. Clevers and R. Nusse, “Wnt/β-catenin signaling and disease,” Cell, vol. 149, no. 6, pp. 1192–1205, 2012. View at: Publisher Site | Google Scholar
  11. H. Clevers, K. M. Loh, and R. Nusse, “Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control,” Science, vol. 346, no. 6205, article 1248012, 2014. View at: Publisher Site | Google Scholar
  12. H. A. Baarsma, M. Königshoff, and R. Gosens, “The Wnt signaling pathway from ligand secretion to gene transcription: molecular mechanisms and pharmacological targets,” Pharmacology & Therapeutics, vol. 138, no. 1, pp. 66–83, 2013. View at: Publisher Site | Google Scholar
  13. V. Korinek, N. Barker, P. Moerer et al., “Depletion of epithelial stem-cell compartments in the small intestine of mice lacking tcf-4,” Nature Genetics, vol. 19, no. 4, pp. 379–383, 1998. View at: Publisher Site | Google Scholar
  14. U. Gat, R. DasGupta, L. Degenstein, and E. Fuchs, “De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin,” Cell, vol. 95, no. 5, pp. 605–614, 1998. View at: Google Scholar
  15. R. DasGupta and E. Fuchs, “Multiple roles for activated lef/tcf transcription complexes during hair follicle development and differentiation,” Development, vol. 126, no. 20, pp. 4557–4568, 1999. View at: Google Scholar
  16. M. Visweswaran, S. Pohl, F. Arfuso et al., “Multi-lineage differentiation of mesenchymal stem cells - to Wnt, or not Wnt,” The International Journal of Biochemistry & Cell Biology, vol. 68, pp. 139–147, 2015. View at: Google Scholar
  17. J. R. Park, J.-W. Jung, Y.-S. Lee, and K.-S. Kang, “The roles of Wnt antagonists dkk1 and sfrp4 during adipogenesis of human adipose tissue-derived mesenchymal stem cells,” Cell Proliferation, vol. 41, no. 6, pp. 859–874, 2008. View at: Publisher Site | Google Scholar
  18. C. A. Gregory, W. G. Gunn, E. Reyes et al., “How Wnt signaling affects bone repair by mesenchymal stem cells from the bone marrow,” Annals of the New York Academy of Sciences, vol. 1049, no. 1, pp. 97–106, 2005. View at: Google Scholar
  19. J. de Boer, R. Siddappa, C. Gaspar, A. van Apeldoorn, R. Fodde, and C. van Blitterswijk, “Wnt signaling inhibits osteogenic differentiation of human mesenchymal stem cells,” Bone, vol. 34, no. 5, pp. 818–826, 2004. View at: Google Scholar
  20. S. Luo, Q. Shi, Z. Zha et al., “Inactivation of Wnt/β-catenin signaling in human adipose-derived stem cells is necessary for chondrogenic differentiation and maintenance,” Biomedicine & Pharmacotherapy, vol. 67, no. 8, pp. 819–824, 2013. View at: Publisher Site | Google Scholar
  21. D. Zhu, E. M. Wallace, and R. Lim, “Cell-based therapies for the preterm infant,” Cytotherapy, vol. 16, no. 12, pp. 1614–1628, 2014. View at: Publisher Site | Google Scholar
  22. M. Dominici, K. Le Blanc, I. Mueller et al., “Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement,” Cytotherapy, vol. 8, no. 4, pp. 315–317, 2006. View at: Publisher Site | Google Scholar
  23. J. Gerdes, H. Lemke, H. Baisch, H. H. Wacker, U. Schwab, and H. Stein, “Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody ki-67,” Journal of Immunology, vol. 133, no. 4, pp. 1710–1715, 1984. View at: Google Scholar
  24. S.-M. A. Huang, Y. M. Mishina, S. Liu et al., “Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling,” Nature, vol. 461, no. 7264, pp. 614–620, 2009. View at: Publisher Site | Google Scholar
  25. K. Mareschi, I. Ferrero, D. Rustichelli et al., “Expansion of mesenchymal stem cells isolated from pediatric and adult donor bone marrow,” Journal of Cellular Biochemistry, vol. 97, no. 4, pp. 744–754, 2006. View at: Google Scholar
  26. D. M. Choumerianou, G. Martimianaki, E. Stiakaki, L. Kalmanti, M. Kalmanti, and H. Dimitriou, “Comparative study of stemness characteristics of mesenchymal cells from bone marrow of children and adults,” Cytotherapy, vol. 12, no. 7, pp. 881–887, 2010. View at: Publisher Site | Google Scholar
  27. M. J. Javed, L. E. Mead, D. Prater et al., “Endothelial colony forming cells and mesenchymal stem cells are enriched at different gestational ages in human umbilical cord blood,” Pediatric Research, vol. 64, no. 1, pp. 68–73, 2008. View at: Publisher Site | Google Scholar
  28. A. Jain, N. Mathur, M. Jeevashankar et al., “Does mesenchymal stem cell population in umbilical cord blood vary at different gestational periods?” Indian Journal of Pediatrics, vol. 80, no. 5, pp. 375–379, 2013. View at: Publisher Site | Google Scholar
  29. M. T. Conconi and R. Di Liddo, “Phenotype and differentiation potential of stromal populations obtained from various zones of human umbilical cord: an overview,” The Open Tissue Engineering and Regenerative Medicine Journal, vol. 4, pp. 6–20, 2011. View at: Google Scholar
  30. C. Mennan, K. Wright, A. Bhattacharjee, B. Balain, J. Richardson, and S. Roberts, “Isolation and characterisation of mesenchymal stem cells from different regions of the human umbilical cord,” BioMed Research International, vol. 2013, Article ID 916136, 8 pages, 2013. View at: Publisher Site | Google Scholar
  31. A. Subramanian, C. Y. Fong, A. Biswas, and A. Bongso, “Comparative characterization of cells from the various compartments of the human umbilical cord shows that the wharton’s jelly compartment provides the best source of clinically utilizable mesenchymal stem cells,” PLoS One, vol. 10, no. 6, article e0127992, 2015. View at: Publisher Site | Google Scholar
  32. T. Montemurro, G. Andriolo, E. Montelatici et al., “Differentiation and migration properties of human foetal umbilical cord perivascular cells: potential for lung repair,” Journal of Cellular and Molecular Medicine, vol. 15, no. 4, pp. 796–808, 2011. View at: Publisher Site | Google Scholar
  33. S.-H. Hong, L. Maghen, S. Kenigsberg et al., “Ontogeny of human umbilical cord perivascular cells: molecular and fate potential changes during gestation,” Stem Cells and Development, vol. 22, no. 17, pp. 2425–2439, 2013. View at: Publisher Site | Google Scholar
  34. S. L. Etheridge, G. J. Spencer, D. J. Heath, and P. G. Genever, “Expression profiling and functional analysis of Wnt signaling mechanisms in mesenchymal stem cells,” Stem Cells, vol. 22, no. 5, pp. 849–860, 2004. View at: Google Scholar
  35. O. O. Udartseva, E. R. Andreeva, and L. B. Buravkova, “Wnt-associated gene expression in human mesenchymal stromal cells under hypoxic stress,” Doklady Biochemistry and Biophysics, vol. 465, no. 1, pp. 354–357, 2015. View at: Publisher Site | Google Scholar
  36. A. K. Batsali, C. Pontikoglou, D. Koutroulakis et al., “Differential expression of cell cycle and Wnt pathway-related genes accounts for differences in the growth and differentiation potential of Wharton’s jelly and bone marrow-derived mesenchymal stem cells,” Stem Cell Research & Therapy, vol. 8, no. 1, p. 102, 2017. View at: Publisher Site | Google Scholar
  37. M. J. Nemeth, L. Topol, S. M. Anderson, Y. Yang, and D. M. Bodine, “Wnt5a inhibits canonical Wnt signaling in hematopoietic stem cells and enhances repopulation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 39, pp. 15436–15441, 2007. View at: Publisher Site | Google Scholar
  38. A. J. Mikels and R. Nusse, “Purified Wnt5a protein activates or inhibits beta-catenin-tcf signaling depending on receptor context,” PLoS Biology, vol. 4, no. 4, article e115, 2006. View at: Google Scholar
  39. R. van Amerongen, A. Mikels, and R. Nusse, “Alternative Wnt signaling is initiated by distinct receptors,” Science Signaling, vol. 1, no. 35, article re9, 2008. View at: Publisher Site | Google Scholar
  40. G. Liu, A. Bafico, and S. A. Aaronson, “The mechanism of endogenous receptor activation functionally distinguishes prototype canonical and noncanonical Wnts,” Molecular and Cellular Biology, vol. 25, no. 9, pp. 3475–3482, 2005. View at: Publisher Site | Google Scholar

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