mesenchymal stem cells from infants born to obese mothers ...kristen e. boyle,1 zachary w....

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Kristen E. Boyle, 1 Zachary W. Patinkin, 1 Allison L.B. Shapiro, 2 Peter R. Baker II, 3 Dana Dabelea, 2 and Jacob E. Friedman 4 Mesenchymal Stem Cells From Infants Born to Obese Mothers Exhibit Greater Potential for Adipogenesis: The Healthy Start BabyBUMP Project Diabetes 2016;65:647659 | DOI: 10.2337/db15-0849 Maternal obesity increases the risk for pediatric obesity; however, the molecular mechanisms in human infants remain poorly understood. We hypothesized that mes- enchymal stem cells (MSCs) from infants born to obese mothers would demonstrate greater potential for adipo- genesis and less potential for myogenesis, driven by dif- ferences in b-catenin, a regulator of MSC commitment. MSCs were cultured from the umbilical cords of infants born to normal-weight (prepregnancy [pp] BMI 21.1 6 0.3 kg/m 2 ; n = 15; NW-MSCs) and obese mothers (ppBMI 34.6 6 1.0 kg/m 2 ; n = 14; Ob-MSCs). Upon differentiation, Ob-MSCs exhibit evidence of greater adipogenesis (+30% Oil Red O stain [ORO], +50% peroxisome prolif- eratoractivated receptor (PPAR)-g protein; P < 0.05) compared with NW-MSCs. In undifferentiated cells, total b-catenin protein content was 10% lower and phosphor- ylated Thr41Ser45/total b-catenin was 25% higher (P < 0.05) in Ob-MSCs versus NW-MSCs (P < 0.05). Coupled with 25% lower inhibitory phosphorylation of GSK-3b in Ob-MSCs (P < 0.05), these data suggest greater b-catenin degradation in Ob-MSCs. Lithium chloride in- hibition of GSK-3b increased nuclear b-catenin content and normalized nuclear PPAR-g in Ob-MSCs. Last, ORO in adipogenic differentiating cells was positively corre- lated with the percent fat mass in infants (r = 0.475; P < 0.05). These results suggest that altered GSK- 3b/b-catenin signaling in MSCs of infants exposed to maternal obesity may have important consequences for MSC lineage commitment, fetal fat accrual, and off- spring obesity risk. Nearly one in ve children in the U.S. is obese (1), and obesity during pregnancy is increasingly recognized as an important contributor to obesity risk in the next generation (2,3). Birth weight and neonatal fat mass are positively associated with maternal BMI (4,5), though neonatal fat-free mass (FFM) is not, suggesting that maternal BMI has a preferential impact on infant adiposity (6). Epidemiological data suggest that these relationships between maternal obesity and offspring adiposity are not conned to neonatal life but affect off- spring across the life span, independent of postnatal life- style factors (7). However, surprisingly little is known about how maternal obesity might inuence obesity risk in the human neonate. Adipocytes as well as myocytes, osteocytes, and several other cell types all differentiate from the multipotent fetal mesenchymal stem cell (MSC) population. MSC differentiation is regulated by the canonical wingless type (Wnt)/glycogen synthase kinase (GSK)-3b/b-catenin path- way. In the absence of Wnt signaling, cytosolic b-catenin is constitutively phosphorylated by GSK-3b, targeting it for proteasomal degradation (8). When activated, Wnt signal transduction leads to sequestration of GSK-3b, allowing for b-catenin accumulation, nuclear translocation, and initiation of target gene transcription, including myo- genic factors such as myogenin (8,9). Alternatively, GSK-3b inhibitory phosphorylation at Ser9 leads to b-catenin accumulation and nuclear translocation, independent of Wnt signaling (10,11). In vitro studies suggest that b-catenin 1 Section of Nutrition, Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO 2 Colorado School of Public Health, Aurora, CO 3 Section of Clinical Genetics and Metabolism, Department of Pediatrics, Univer- sity of Colorado School of Medicine, Aurora, CO 4 Section of Neonatology, Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO Corresponding author: Kristen E. Boyle, [email protected]. Received 19 June 2015 and accepted 20 November 2015. Clinical trial reg. no. NCT02273297, clinicaltrials.gov. This article contains Supplementary Data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi:10.2337/db15-0849/-/DC1. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for prot, and the work is not altered. Diabetes Volume 65, March 2016 647 OBESITY STUDIES

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Page 1: Mesenchymal Stem Cells From Infants Born to Obese Mothers ...Kristen E. Boyle,1 Zachary W. Patinkin,1 Allison L.B. Shapiro,2 Peter R. Baker II,3 Dana Dabelea,2 and Jacob E. Friedman4

Kristen E. Boyle,1 Zachary W. Patinkin,1 Allison L.B. Shapiro,2 Peter R. Baker II,3

Dana Dabelea,2 and Jacob E. Friedman4

Mesenchymal Stem Cells From InfantsBorn to Obese Mothers Exhibit GreaterPotential for Adipogenesis: The HealthyStart BabyBUMP ProjectDiabetes 2016;65:647–659 | DOI: 10.2337/db15-0849

Maternal obesity increases the risk for pediatric obesity;however, the molecular mechanisms in human infantsremain poorly understood. We hypothesized that mes-enchymal stem cells (MSCs) from infants born to obesemothers would demonstrate greater potential for adipo-genesis and less potential for myogenesis, driven by dif-ferences in b-catenin, a regulator of MSC commitment.MSCs were cultured from the umbilical cords of infantsborn to normal-weight (prepregnancy [pp] BMI 21.1 6

0.3 kg/m2; n = 15; NW-MSCs) and obese mothers (ppBMI34.66 1.0 kg/m2; n = 14; Ob-MSCs). Upon differentiation,Ob-MSCs exhibit evidence of greater adipogenesis(+30% Oil Red O stain [ORO], +50% peroxisome prolif-erator–activated receptor (PPAR)-g protein; P < 0.05)compared with NW-MSCs. In undifferentiated cells, totalb-catenin protein content was 10% lower and phosphor-ylated Thr41Ser45/total b-catenin was 25% higher (P <

0.05) in Ob-MSCs versus NW-MSCs (P < 0.05). Coupledwith 25% lower inhibitory phosphorylation of GSK-3bin Ob-MSCs (P < 0.05), these data suggest greaterb-catenin degradation in Ob-MSCs. Lithium chloride in-hibition of GSK-3b increased nuclear b-catenin contentand normalized nuclear PPAR-g in Ob-MSCs. Last, OROin adipogenic differentiating cells was positively corre-lated with the percent fat mass in infants (r = 0.475;P < 0.05). These results suggest that altered GSK-3b/b-catenin signaling in MSCs of infants exposed tomaternal obesity may have important consequencesfor MSC lineage commitment, fetal fat accrual, and off-spring obesity risk.

Nearly one in five children in the U.S. is obese (1), andobesity during pregnancy is increasingly recognized asan important contributor to obesity risk in the nextgeneration (2,3). Birth weight and neonatal fat massare positively associated with maternal BMI (4,5),though neonatal fat-free mass (FFM) is not, suggestingthat maternal BMI has a preferential impact on infantadiposity (6). Epidemiological data suggest that theserelationships between maternal obesity and offspringadiposity are not confined to neonatal life but affect off-spring across the life span, independent of postnatal life-style factors (7). However, surprisingly little is knownabout how maternal obesity might influence obesity riskin the human neonate.

Adipocytes as well as myocytes, osteocytes, and severalother cell types all differentiate from the multipotentfetal mesenchymal stem cell (MSC) population. MSCdifferentiation is regulated by the canonical wingless type(Wnt)/glycogen synthase kinase (GSK)-3b/b-catenin path-way. In the absence of Wnt signaling, cytosolic b-cateninis constitutively phosphorylated by GSK-3b, targeting itfor proteasomal degradation (8). When activated, Wntsignal transduction leads to sequestration of GSK-3b,allowing for b-catenin accumulation, nuclear translocation,and initiation of target gene transcription, including myo-genic factors such as myogenin (8,9). Alternatively, GSK-3binhibitory phosphorylation at Ser9 leads to b-cateninaccumulation and nuclear translocation, independent ofWnt signaling (10,11). In vitro studies suggest that b-catenin

1Section of Nutrition, Department of Pediatrics, University of Colorado School ofMedicine, Aurora, CO2Colorado School of Public Health, Aurora, CO3Section of Clinical Genetics and Metabolism, Department of Pediatrics, Univer-sity of Colorado School of Medicine, Aurora, CO4Section of Neonatology, Department of Pediatrics, University of Colorado Schoolof Medicine, Aurora, CO

Corresponding author: Kristen E. Boyle, [email protected].

Received 19 June 2015 and accepted 20 November 2015.

Clinical trial reg. no. NCT02273297, clinicaltrials.gov.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-0849/-/DC1.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

Diabetes Volume 65, March 2016 647

OBESITYSTUDIES

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accumulation can also inhibit adipogenesis via downregu-lation of CCAAT/enhancer binding protein (C/EBP)-aand peroxisome proliferator–activated receptor (PPAR)-g(12,13). Studies of pregnant ewes have shown that fetusesexposed to obesity in utero have less skeletal musclemass, less b-catenin content, and less inhibitory phos-phorylation of GSK-3b, whereas PPAR-g expressionis higher when compared with offspring from controldams (14–16). These data suggest a mechanism wherebymaternal obesity could induce a shift in MSC commit-ment from the myocyte to the adipocyte lineage viadisruption of b-catenin signaling. Such a shift in MSCdifferentiation could have profound effects on fetal tis-sue development, in particular because there is a largewindow of overlap for adipogenesis and myogenesisduring midgestation, when secondary fetal myogenesisis at its peak (17,18). However, whether this mechanismis evident in MSCs from infants born to mothers withobesity is not known.

We hypothesized that fetal MSCs from infants born toobese mothers would have greater potential for adipo-genesis and less potential for myogenesis. Likewise, wehypothesized that differences in the b-catenin pathwaycould be a potential early determinant of differences inMSC lineage commitment. Last, we investigated whethermarkers of MSC lineage commitment were correlatedwith measures of infant body composition at birth.

RESEARCH DESIGN AND METHODS

Ethics StatementThis study used umbilical cord tissue samples and otherdata collected by the Healthy Start study (R01DK076648,ClinicalTrials.gov identifier NCT02273297). Approval forthis study was obtained from the Colorado MultipleInstitutional Review Board at the University of ColoradoHospital. Written informed consent was obtained from allparticipants at enrollment.

SubjectsThe Healthy Start longitudinal prebirth cohort studyenrolled 1,410 pregnant women, ages 16 and older andat #23 weeks’ gestation, recruited from the obstet-rics clinics at the University of Colorado Hospital dur-ing 2010–2014. Women were excluded if they hadprior diabetes, prior premature birth, serious psychi-atric illness, or a current multiple pregnancy. Pregnantwomen were evaluated twice (at median weeks 17 and27) for demographics, tobacco use, height, and weight.Fasting blood samples were drawn for measures of glu-cose, insulin, triglycerides, and free fatty acids (FFAs).Prepregnancy BMI was obtained through medical recordabstraction (84%) or self-report at the first research visit(16%). Gestational weight gain was defined as the dif-ference between the mother’s prepregnancy weight andher weight at delivery. Infant birth weight was obtainedfrom medical records, and infant weight, length, andbody composition (fat mass [FM], FFM; whole-body air

plethysmography [PEA POD; COSMED, Inc.]) were mea-sured within 72 h after birth.

To investigate the biology of intrauterine metabolicprogramming (BabyBUMP), umbilical cord tissue wasobtained at birth from a convenience sample of 165infants and was used to culture MSCs as part of theancillary Healthy Start BabyBUMP Project. From thissubsample, additionally excluding women younger than18 years of age upon enrollment and women who developedgestational diabetes mellitus or preeclampsia during thestudy, 15 obese women (prepregnancy BMI .30 kg/m2)were frequency matched with 15 normal-weight (NW)women (prepregnancy BMI ,25 kg/m2) for maternalage, gestational age at delivery, infant sex, and MSC cul-ture time to confluence. During data analysis we discov-ered that one obese mother had had gestational diabetesmellitus, and this dyad was excluded at that time, leaving29 dyads for the sample included in this report. Sampleselection methods are summarized by flowchart inSupplementary Fig. 1.

MSC Isolation and CultureMSCs were cultured from fresh umbilical cord tissueexplants, as described elsewhere (19) but with slight mod-ification. Briefly, at delivery, a 4-inch section of the um-bilical cord was cut below the clamp (infant side) andstored at 4°C in antibiotic-supplemented PBS until pro-cessing (#12 h). Umbilical cord tissue explants were dis-sected free from visible blood vessels and plated,Wharton’s jelly side down, onto tissue culture–treatedplastic. Low-glucose DMEM supplemented with MSCgrowth media (Lonza, Walkersville, MD) was added andthe explants were maintained at 37°C in 5% CO2. Atconfluence, MSCs were harvested for cryogenic storageuntil experimentation. The number of days to reach cryo-preservation (passage 2) was recorded as the culture timeto confluence. All experiments were performed on cells inpassages 3–4.

Immunophenotype and Cellular DifferentiationAnalysis by Flow CytometryUndifferentiated MSCs were harvested for MSC immu-nophenotype and CD13/aminopeptidase N (APN) anal-ysis. For all tests, at least 1 3 105 cells were used forstaining. All antibodies were titered before the MSC ex-periments using a pool of primary MSCs and lysed wholeblood. For MSC characterization, antibodies (CD73, CD90,CD105, CD34, CD45, and CD19; Supplementary Table 1)were measured in separate aliquots from the same pool ofcells (5–10 subjects/pool). This was repeated on four sep-arate pools of cells with mixed representation of MSCsfrom NW mothers (NW-MSCs) and from obese mothers(Ob-MSCs) (separate experiment days). On each experi-ment day appropriate IgG isotype controls were measuredfrom the same pool of cells for each fluorescent marker.Mean fluorescence intensity (MFI) was measured for IgGand markers of interest. For CD13/APN expression anal-ysis, MSCs from each subject were tested individually. For

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CD13/APN measures, the change in MFI (DMFI) was cal-culated as the difference in MFI from IgG to CD13/APN forall cells.

Adipogenic and myogenic differentiating cells wereanalyzed by flow cytometry at day 7 of differentiation.Side-scatter area was determined by flow cytometry andcompared between undifferentiated and adipogenic dif-ferentiating cells as a marker of differentiation induction.Side-scatter signals are largely influenced by the refractiveindex of intracellular components such as lipids (20). Dif-ferentiation efficiency was estimated by the percent ofcells positive for BODIPY 493/503 neutral lipid stain(Life Technologies, Carlsbad, CA) at day 7 of adipogenicdifferentiation and the percent of cells positive for intra-cellular myogenin protein content at day 7 of myogenicdifferentiation. These measures were compared betweengroups in a subset of subjects (n $ 5 per group for allmeasures). BODIPY 493/503 stain and myogenin anti-body were titered before the MSC experiments using apool of undifferentiated and differentiating MSCs.

Samples were acquired on the Beckman Coulter GalliosFlow Cytometer (Beckman Coulter, Brea, CA) using Kaluza1.0 data acquisition software. Data were analyzed usingKaluza Analysis 1.3 software. The setup of the flowcytometer is described in Supplementary Table 2. Thegating tree was set as follows (Supplementary Fig. 2): lightscatter gate (side-scatter area/forward scatter area), live/dead gate (DAPI-negative cells), singlet gate (forward scat-ter height/forward scatter area). For BODIPY 493/503and intracellular myogenin measures, cells were fixed be-fore staining; thus the live/dead gate was omitted.

Adipogenic InductionMSC adipogenesis was induced as described previously(21), with slight modification. Adipogenesis was inducedfor up to 21 days with three cycles of adipogenic induc-tion medium (low-glucose DMEM, 5% FBS, 1 mM dexa-methasone, 200 mmol/L indomethacin, 500 mmol/L3-isobutyl-1-methylxanthine, 170 pmol/L insulin, and0.13 penicillin/streptomycin [pen/strep]) and adipogenicmaintenance medium (low-glucose DMEM, 5% FBS, 170pmol/L insulin, and 0.13 pen/strep) for 3 days each, afterwhich adipogenic maintenance medium was used for theremainder of the experiment. For adipogenesis in thepresence of lithium chloride (LiCl), adipogenic inductionwas performed as described above for 3 days with10 mmol/L LiCl or without (control) (n = 9 per group;from subjects listed in Table 1, matched to remain repre-sentative of each group).

Myogenic InductionMSC myogenesis was induced as described previously(22), with slight modification. MSCs were subculturedon collagen-coated (5 mg/cm2 Collagen I; BD Biosciences,San Jose, CA) dishes, and myogenesis was induced at 90–100% confluence using myogenic induction medium (low-glucose DMEM, 10% FBS, 5% horse serum, 0.1 mmol/Ldexamethasone, 50 mmol/L hydrocortisone, and 0.13pen/strep) for 7 or 21 days.

RNA Isolation and AnalysisMyosin heavy chain (MyHC) mRNA content was mea-sured from day 0 to day 5 of myogenic induction, as werenanog and Oct4, identified markers of mesenchymal

Table 1—Subject characteristics

NW(n = 15)

Obese(n = 14) P value

Maternal characteristicsAge (years) 28.0 6 1.5 26.7 6 1.9 0.60Prepregnancy BMI (kg/m2) 21.1 6 0.3 34.6 6 1.0 ,0.001*Primiparous, n (%) 6 (40.0) 6 (42.9) 0.88Glucose (mg/dL) 74.2 6 1.2 76.0 6 1.7 0.39Insulin (mU/mL) 7.7 6 0.5 12.5 6 1.7 0.01*HOMA-insulin resistance 2.1 6 0.3 3.1 6 0.4 0.07Triglycerides (mg/dL) 129.3 6 15.3 138.7 6 12.9 0.64FFA (mg/dL) 334.9 6 28.0 471.8 6 44.2 0.01*Gestational weight gain (kg) 14.2 6 0.9 10.2 6 2.2 0.10Gestational age at delivery (weeks) 39.9 6 0.2 39.7 6 0.3 0.52Cesarean delivery, n (%) 2 (13.3) 2 (14.3) 0.94

Infant characteristicsSex, n (female/male) 7/8 5/9 0.57Birth weight (g) 3,316.8 6 94.7 3,325.9 6 102.5 0.95Birth length (cm) 49.8 6 0.5 49.3 6 0.5 0.49FM (g) 270.0 6 31.8 358.3 6 34.7 0.07FM (%) 8.43 6 0.9 11.1 6 0.9 0.05*FFM (g) 2,877.8 6 62.3 2,836.3 6 92.8 0.71FFM (%) 91.6 6 0.9 88.9 6 0.9 0.05*

MSC characteristicsCulture time to confluence (days) 26.3 6 1.2 26.7 6 1.9 0.84

Data are mean 6 SEM, unless otherwise stated. *Significant independent t test, P # 0.05.

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stem cell pluripotency (23). Gene names and primer se-quences are listed in Supplementary Table 3. Cells wererinsed twice with PBS, then harvested in Buffer RLT(Qiagen, Valencia, CA). Total RNA was isolated using anRNeasy Plus Mini Kit (Qiagen). cDNA was transcribedfrom 200 ng total RNA using an iScript cDNA SynthesisKit (Bio-Rad). Quantitative PCR was performed usingprimer sets for genes of interest, RPL13a and ubiquitinC as reference genes, and iQ SYBR Supermix (Bio-Rad,Hercules, CA), as described elsewhere (24). Reactions wererun in duplicate on an iQ5 Real-Time PCR Detection System(Bio-Rad), along with a no-template control per gene. RNAexpression data were normalized to reference genes usingthe comparative threshold cycle method.

Lipid AccumulationFollowing 21 days of adipogenic induction, cells wererinsed with PBS and fixed with 4% formaldehyde. Cellswere stained for 1 h with 0.2% Oil Red O stain (ORO) inpropylene glycol, rinsed twice with fresh 85% propyleneglycol, then rinsed twice with deionized water. CellularORO stain was solubilized with isopropanol for 5 min andtransferred to a clean 96-well plate; the degree of stainingwas determined spectrophotometrically (at 520 nm). Rep-resentative photographs were taken using phase-contrastmicroscopy with a 103 objective.

Measures of Protein ContentCells were rinsed twice with PBS and harvested in cell lysisbuffer (CelLytic MT; Sigma-Aldrich, St. Louis, MO)supplemented with protease and phosphatase inhibitorcocktails (Sigma-Aldrich). Subcellular fractionation wasperformed as described by Abcam. Total protein wasdetermined by bicinchoninic acid assay. Protein contentof b-actin, PPAR-g, fatty acid binding protein (FABP) 4,C/EBP-b, myogenin, MyHC, b-catenin, phosphorylatedb-catenin (Thr41/Ser45), phosphorylated b-catenin(Ser552), GSK-3b, phosphorylated GSK-3b (Ser9), extra-cellular signal–related kinases (ERK) 1/2, phosphory-lated ERK1/2 (Thr202/Tyr204), phosphorylated Akt(Ser473), phosphorylated signal transducer and activatorof transcription 3 (Tyr705), and phosphorylated AMPK(Thr172) were determined by either Western blot as de-scribed previously (25) or by simple Western size-basedprotein assay (ProteinSimple, Santa Clara, CA) followingthe manufacturer’s protocol. Nuclear fractions were usedto determine content of b-catenin and PPAR-g duringLiCl experiments, which represent active protein. Resultsfrom Western size-based protein assay were analyzedusing ProteinSimple Compass software. All antibodieswere optimized in house for this system; antibody spe-cifics and assay conditions are listed in SupplementaryTable 4.

Statistical AnalysesD’Agostino and Pearson tests were used to assess thenormality of the data. Levene tests were used to assessunequal variance. For testing comparisons between

NW-MSCs and Ob-MSCs, independent t tests or Mann-Whitney U tests were used, where appropriate. For theLiCl experiments, independent t tests were used forcomparison of NW-MSCs versus Ob-MSCs under controlconditions, followed by paired t tests for comparison ofcontrol versus LiCl conditions for NW-MSCs and Ob-MSCs combined. All data are expressed as mean 6 SEM.Differences between groups were considered statisticallysignificant at P # 0.05.

Pearson correlations were used to identify relation-ships between MSC measures. To identify relationshipsbetween MSC markers of differentiation and infant bodycomposition, correlation analyses were performed asfollows. First, bivariate Pearson correlations were usedto identify significant zero-order correlations betweeninfant birth weight, FM (absolute and percent), andFFM (absolute and percent) and MSC measures of OROstaining and FABP4, PPAR-g, myogenin, and MyHC pro-tein content. Only those factors with significant zero-ordercorrelations between MSC and infant measures were sub-jected to further analysis; partial correlations were per-formed with infant sex and maternal prepregnancy BMIincluded as control variables.

RESULTS

Subject CharacteristicsCharacteristics for 29 mother–infant pairs included inthis substudy are listed in Table 1. By design, the obesemothers had higher BMI but similar maternal age, ges-tational age at delivery, infant sex, and time to conflu-ence for initial MSC culture. Maternal metabolic markerswere measured at a median of 17 weeks’ gestation (mid-pregnancy). At midpregnancy, obese mothers had higherinsulin and FFA concentrations (P , 0.05) and tended tohave higher HOMA-insulin resistance scores (P = 0.07),though none of these women had preexisting diabetes ordeveloped gestational diabetes mellitus during the indexpregnancy. At birth, compared to the infants of NWmothers, the infants of the obese mothers weighed thesame but had higher percent FM and lower percent FFM(P # 0.05).

Umbilical Cord Cells Express MSC Markers andDifferentiate to Adipocytes and Myocytes In VitroThe umbilical cord cells were .98% positive for stainingof MSC markers CD74, CD105, and CD90 and negativefor hematopoietic stem cell or lymphocyte markersCD34, CD45, and CD19 (Fig. 1). Adipogenic differenti-ating cells were increasingly positive for adipogenicmarkers from day 0 to day 7 (C/EBP-b, PPAR-g, andORO stain; Fig. 2A and B), indicating induction of adipo-genesis. Side-scatter area increased by 30% from day0 to day 7 of adipogenesis (Fig. 2C), reflecting a largechange in the cellular refractive index. Adipogenic dif-ferentiating cells were 97% positive for BODIPY stain atday 7 of differentiation, whereas myogenic differentiat-ing cells were 85% positive for Myogenin Alexa Fluor

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488 at day 7 of differentiation (Fig. 2D and E). From day0 to day 5 of myogenesis, cells expressed less of thestem cell markers nanog and Oct4 in mRNA (Fig. 2Fand G), whereas mRNA expression of MyHC increased(Fig. 2H). Likewise, from day 0 to day 21 of myogenesis,cells expressed more myogenin and MyHC protein (Fig.2I), indicating induction of myogenesis. Representativephotographs of undifferentiated, adipocyte differentiat-ing, and myocyte differentiating cells show that undif-ferentiated cells are characteristically spindle-shaped,whereas 21-day adipogenic differentiating cells appearmore rounded. Myogenic differentiating cells have be-gun to fuse together and form tubes (Fig. 2J–L).

Adipogenesis Is Greater in Ob-MSCs, but Markers ofMyogenesis Are Not DifferentWe measured CD13/APN, a potential novel marker ofadipogenic potential (26), in undifferentiated cells usingflow cytometry. The percent of cells gated at eachstep was similar for all subjects, indicating no overt dif-ferences in cell size, density, or viability (Fig. 3A–C).CD13/APN is a recognized MSC marker and, as expected,.90% of cells were positive for CD13/APN, with no dif-ferences observed between groups (P = 0.80; Fig. 3D).However, the DMFI was over twofold greater in theMSCs from offspring of obese mothers (P , 0.05; Fig.3E and F). Because there were no differences in cell size,density, or viability, or in the number of cells positive forCD13/APN, these data indicate that greater CD13/APN

fluorescence intensity is not the result of differences incellular morphology or immunophenotype, but representsmore cellular protein content.

Once adipogenesis was induced, there were no differ-ences in the percent of cells positive for BODIPY 493/503neutral lipid stain at day 7 of differentiation (P = 0.47;Fig. 4A). At day 3 of adipogenesis, FABP4 protein contentwas 35% higher (P # 0.05; Fig. 4B), whereas PPAR-gprotein content was 50% higher (P # 0.05; Fig. 4C) andORO staining was 30% higher in Ob-MSCs compared withNW-MSCs at day 21 (P # 0.05; Fig. 4D and E).

Myogenic differentiating cells were 80–85% positivefor myogenin protein at day 7 of differentiation, withno differences between groups (P = 0.67; Fig. 4F). Meangroup differences in myogenin protein content at day 7and MyHC protein content at day 21 of myogenesis wereconsistent, though these results did not reach statisticalsignificance (216%, P = 0.14 and 215%, P = 0.23 inOb-MSCs versus NW-MSCs, respectively, for myogeninand MyHC; Fig. 4G and H). Despite the fact that therewere not significant differences in specific myogenicmarkers, MyHC protein content at day 21 of differentia-tion was inversely correlated with PPAR-g protein contentin the adipogenic differentiating MSCs (P , 0.05; Fig. 4I).

Ob-MSCs Have Less b-Catenin Content and GSK-3bInhibitory PhosphorylationTotal b-catenin protein content was 10% lower in undif-ferentiated Ob-MSCs compared with NW-MSCs, whereas

Figure 1—Umbilical cord–derived cells exhibit MSC markers. Undifferentiated cells were pooled (more than five subjects) and stainedfor MSC markers (CD73, CD105, and CD90) and hematopoietic and lymphocyte markers (CD34, CD45, and CD19). Representative plotsare shown for MSC expression of CD73 (A), CD105 (B), and CD90 (C ), gated as described in RESEARCH DESIGN ANDMETHODS. Correspondinghistograms show gated cells for IgG isotype controls (white) and the marker of interest (gray). The MFI data summary (D) indicates thatcells are positive for MSC markers and negative for hematopoietic and lymphocyte markers. APC, allophycocyanin; FITC, fluoresceinisothiocyanate; SS, side scatter.

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phosphorylated/total b-catenin (Thr41/Ser45) was 25%higher (P , 0.05; Fig. 5A). Upstream, this correspondedto 25% lower inhibitory phosphorylation of GSK-3b (phos-phorylated Ser9/total) in the Ob-MSCs versus the NW-MSCs

(P , 0.05; Fig. 5B). There were no difference in stabilizingphosphorylation of b-catenin at Ser552 (1.00 6 0.07 and1.07 6 0.11 in NW-MSCs and Ob-MSCs, respectively; P =0.59) or AMPK phosphorylation at Thr172 (1.00 6 0.21

Figure 2—MSCs differentiate to adipocytes and myocytes in culture. Adipogenesis was induced for 2 to 7 days and showed increasedprotein expression of adipogenic markers c/EBP-b and PPAR-g (A) and increased ORO staining (B). Side-scatter area increased inadipogenic differentiating cells by day 7 of differentiation (C ), and cells were 97% positive for BODIPY 493/503 at day 7 of differentiation(D). Myogenesis was induced for 3–21 days. Cells were 85% positive for myogenin at day 7 of differentiation (E). From day 0 to day 3, cellsshowed decreased mRNA content of stem cell markers nanog and Oct4 (F and G) and increased mRNA content of the myogenic markerMyHC (H) from days 0 to 5 of myogenic differentiation. Protein content of myogenin and MyHC was increased from 0 to 21 days ofmyogenesis (I). Representative photographs are shown for undifferentiated (J), 21-day adipogenic differentiating (K), and 21-day myogenicdifferentiating cells (L). Data are expressed as mean 6 SEM. d, day; SS, side scatter.

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and 1.06 6 0.28 in NW-MSCs and Ob-MSCs, respec-tively; P = 0.86), which is shown to phosphorylate b-cateninat Ser552 (27). Phosphorylation of Akt at Ser473, up-stream of GSK-3b, was not different between the groups

(1.00 6 0.18 and 0.93 6 0.17 in NW-MSCs and Ob-MSCs, respectively; P = 0.78). However, phosphorylated/total ERK1/2 (Thr202/Tyr204) tended to be lower in Ob-MSCs versus NW-MSCs (220%; P = 0.11; Fig. 5C) and

Figure 3—CD13/APN content is greater in Ob-MSCs. Undifferentiated cells from each subject were stained for CD13/APN. Data shown arefor the allophycocyanin (APC) IgG isotype control (A) and representative NW-MSC (B) and Ob-MSC (C) flow plots for CD13 APCs. For A–C,graphs are the light scatter gate (first graph), live/dead gate (second graph), singlet gate (third graph), and APC fluorescence intensity(fourth graph) showing that NW-MSCs and Ob-MSCs have similar size, density, and viability. D: The percent of cells positive for CD13/APNwas similar for NW-MSCs and Ob-MSCs (black bars and white bars, respectively), though the CD13/APN APC fluorescence intensity,shown from representative subjects (E) and a data summary (F ), is higher in Ob-MSCs. Data are expressed as mean 6 SEM. *Significantdifference from NW-MSCs (P # 0.05). FS, forward scatter; SS, side scatter.

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Figure 4—Ob-MSCs exhibit greater potential for adipogenesis but no difference in myogenesis. MSCs from offspring of NW andobese mothers underwent adipogenic or myogenic differentiation for 7 or 21 days, and standard markers were measured. NW-MSCsand Ob-MSCs are black bars and white bars, respectively. At day 7 of adipogenesis, there were no differences in the percent of cells positivefor BODIPY 493/503 (A), though the Ob-MSCs expressed more FABP4 (B), PPAR-g (C), and ORO staining (D) than NW-MSCs at day 21 ofadipogenesis. E: Representative photographs of ORO staining at 203 magnification are shown for NW-MSCs and Ob-MSCs. At day 7 ofmyogenesis, there were no differences in the percent of cells positive for myogenin (F) or for total myogenin protein content (G). Therewere no differences in MyHC at day 21 of myogenesis (H). MyHC protein content in myogenic differentiating cells was inversely correlatedwith PPAR-g protein content in adipogenic differentiating cells (I). Data are expressed as mean 6 SEM. *Significant difference from NW-MSCs (P # 0.05). d, day.

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Figure 5—GSK-3b/b-catenin signaling is lower in Ob-MSCs. Amounts of phosphorylated (phospho or P) and total protein was measuredin undifferentiated MSCs. A: Phosphorylated/total b-catenin (Thr41/Ser45) was increased and total b-catenin content was decreased inOb-MSCs compared with NW-MSCs (white and black bars, respectively). Upstream of b-catenin, inhibitory Ser9 phosphorylation of GSK-3b waslower in Ob-MSCs, as was phosphorylated/total GSK-3b protein content (B). Upstream of GSK-3b, there were no differences in phosphorylated/totalERK1/2 (C), though this was correlated with phosphorylated/total GSK-3b (D). b-Catenin content in undifferentiated MSCs was positively correlatedwithMyHC protein content in the 21-daymyogenic differentiating cells (E), whereas phosphorylated/total b-catenin was correlated with ORO stainingin the adipogenic differentiating cells (F). Data are expressed as mean 6 SEM. *Significant difference from NW-MSCs (P # 0.05). d, day.

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was correlated with GSK-3b phosphorylation (P , 0.05;Fig. 5D). Last, b-catenin protein content was positivelycorrelated with MyHC protein content in the 21-day myo-genic differentiating cells (P , 0.05; Fig. 5E), whereasphosphorylated/total b-catenin (Thr41/Ser45) was posi-tively correlated with ORO staining (P , 0.05; Fig. 5F)and tended to be positively correlated with PPAR-g pro-tein content (Pearson r = 0.41; P = 0.066) in 21-day adi-pogenic differentiating cells.

Inhibition of GSK-3b Reduces Nuclear PPAR-g in Ob-MSCsTo investigate the role of GSK-3b in MSC adipogenesis,we incubated cells with LiCl, a common inhibitor of GSK-3b. At day 3 of adipogenesis, LiCl increased GSK-3bphosphorylation and nuclear content of b-catenin (P ,0.05; Fig. 6A and B), while reducing nuclear content ofPPAR-g (P, 0.05; Fig. 6C). Additionally, nuclear contentof b-catenin was lower in Ob-MSCs versus NW-MSCs inthe control condition (Fig. 6B).

MSC Adipogenesis Is Correlated With Infant AdiposityPearson correlations showed that MSC ORO content inadipogenic differentiating cells was positively correlatedwith infant FM and percent FM (P , 0.05; Table 2). By

nature of the two-compartment body composition mea-sure, MSC ORO was also inversely correlated with per-cent FFM. The MSC ORO relationships with percent FMand percent FFM remained evident after controlling formaternal prepregnancy BMI and infant sex (P , 0.05;Table 2).

DISCUSSION

We have shown that umbilical cord–derived MSCs frombabies of obese mothers exhibit greater potential foradipogenesis, as evidenced by larger amounts of threeclassic markers of adipogenesis. Additionally, we demon-strated that inhibition of GSK-3b by LiCl led to increasednuclear content of b-catenin and repression of nuclearPPAR-g protein content. These results are the first evi-dence that fetal MSCs from infants born to obese mothersexhibit differences in GSK-3b/b-catenin signaling, includ-ing less nuclear content of b-catenin, which may drivesubsequent stem cell fate. In addition, we have demon-strated that MSCs’ potential for adipogenesis is positivelycorrelated with infant percent FM at birth, suggestingthat differences in MSC differentiation may be one mech-anism by which babies of obese mothers accrue more fatin utero.

Figure 6—Inhibition of GSK-3b reduces adipogenesis in Ob-MSCs. MSCs underwent 3 days of adipogenesis with or without LiCl in-cubation. LiCl induced greater phosphorylation of GSK-3b in both NW-MSCs and Ob-MSCs (A) and normalized differences in nuclearb-catenin content (B), which represents active protein. Nuclear content of PPAR-g also was markedly reduced with LiCl incubation (C),indicating lower adipogenic induction. Data are expressed as mean 6 SEM. *Significant difference from NW-MSCs in control (CTRL)conditions (P # 0.05). #Significant effect of LiCl incubation for NW-MSCs and Ob-MSCs combined (P # 0.05). OB, obese.

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b-Catenin signaling induces myogenic differentiationof MSCs (8–10) and also has been shown to inhibitadipogenesis (12,13). In the absence of Wnt signaling,cytosolic b-catenin is constitutively targeted for degra-dation by GSK-3b–mediated phosphorylation at severalserine/threonine residues. In undifferentiated MSCs weobserved greater phosphorylation targeting b-cateninfor degradation and less total b-catenin content inOb-MSCs versus NW-MSCs. We also observed lowerinhibitory phosphorylation of GSK-3b upstream in theOb-MSCs. Our results are consistent with animalmodels of maternal obesity, where b-catenin content is10–15% lower in the fetal skeletal muscle of animals ex-posed to obesity in utero, as is GSK-3b phosphorylation(15). It is important to note that Wnt signaling inhibitsGSK-3b by sequestration rather than phosphorylation(10,28). Thus, there are at least two mechanisms bywhich b-catenin accumulation may be regulated: oneby Wnt signaling and another by inhibition of GSK-3bby upstream kinases such as ERK/mitogen-activatedprotein kinase or Akt (11,29). Indeed, direct inhibitionof GSK-3b reduces adipogenesis and accelerates myo-genesis in vitro (30,31). We observed the same effectwith regard to adipogenesis when cells were incubatedwith LiCl, which increased GSK-3b phosphorylation andnuclear b-catenin content and reduced nuclear contentof PPAR-g, a fundamental regulator of adipogenesis.Upstream, ERK1/2 functions to prime GSK-3b for in-hibitory phosphorylation at Ser9 (31). While we did not

observe differences in the phosphorylation of either Aktor signal transducer and activator of transcription 3, wedid observe a trend for lower ERK1/2 phosphorylation inthe Ob-MSCs, which was correlated with phosphorylatedGSK-3b. Overall, these data show one mechanism wherebyless GSK-3b phosphorylation in Ob-MSCs could have im-portant consequences for stem cell adipogenesis.

Studies using a sheep model of maternal obesity havelinked deficits in b-catenin with poor offspring skeletalmuscle development and greater adipogenesis, as evi-denced by morphological differences in fetal skeletal mus-cle tissue from offspring of obese dams (14,15,32). Lowerb-catenin content and GSK-3b phosphorylation in skele-tal muscle of these offspring correspond to deficits inMyoD and myogenin protein content, as well as to lowerAMPK and Akt phosphorylation and higher PPAR-g pro-tein content (14,15,32). Although we did not observe sta-tistically significant differences in myogenic markers ateither day 7 or day 21 of myogenesis, the 15% deficitsin myogenin and MyHC content are consistent in bothdirection and magnitude with myogenic markers in fetalskeletal muscle from animals exposed to maternal obesity(15). In addition, we did find that MyHC protein contentin myogenic differentiating cells was inversely correlatedwith PPAR-g content in adipogenic differentiating cellsand positively correlated with b-catenin content in un-differentiated cells. Likewise, we found that b-cateninphosphorylation was correlated with lipid accumulationand tended to be correlated with PPAR-g content in the

Table 2—Correlations of MSC differentiation markers with infant body composition

ORO FABP4 PPAR-g Myogenin MyHC

Bivariate zero-order correlationsBirth weight (kg)r 20.004 20.012 0.299 0.330 20.096P 0.983 0.966 0.147 0.093 0.633

Birth FM (kg)r 0.427 0.259 0.326 0.237 20.236P 0.037* 0.351 0.12 0.243 0.247

Birth FM (%)r 0.474 0.320 0.249 0.152 20.221P 0.019* 0.244 0.241 0.458 0.278

Birth FFM (kg)r 20.045 20.121 0.303 0.280 20.061P 0.833 0.668 0.150 0.167 0.769

Birth FFM (%)r 20.474 20.320 20.249 20.152 0.221P 0.019* 0.244 0.241 0.458 0.278

Partial correlations†Birth FM (kg)r 0.402 — — — —

P 0.064 — — — —

Birth FM (%)r 0.475 — — — —

P 0.026* — — — —

Birth FFM (%)r 20.475 — — — —

P 0.026* — — — —

*Significant result (P , 0.05). †Controlled for infant sex and maternal prepregnancy BMI.

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adipogenically differentiated cells. Taken together, theserelationships suggest that small differences in inherentGSK-3b/b-catenin signaling in the undifferentiatedMSCs may ultimately influence both adipogenic and myo-genic pathways.

Our results showing greater adipogenesis in Ob-MSCsversus NW-MSCs are consistent with animal models of adultobesity, where MSCs derived from bone marrow, adiposetissue, or skeletal muscle tissue of genetically obese orstreptozotocin-treated rodents exhibit greater adipogenesiscompared with MSCs derived from their control counter-parts (33–35). Independent of adipogenesis, greater lipidaccumulation in the Ob-MSCs may also reflect differencesin lipid trafficking or metabolism; regardless of the mecha-nism, however, greater capacity for adipocyte lipid storage inearly postnatal life may be somewhat protective against in-flammatory infiltration into adipose tissue depots or “lipidspillover” into other tissues (36).

With regard to maternal obesity and fetal programmingevents, our results are also consistent with greater adipo-genesis in human placental amnion–derived MSCs, whereCD13/APN content is elevated and necessary for greateradipogenesis in MSCs from offspring of obese mothersand CD13/APN overexpression is sufficient for inducinggreater adipogenesis in MSCs from offspring of NW moth-ers (26). While CD13/APN is considered a common MSCmarker that has been linked to cancer and T-cell prolifer-ation via ERK/mitogen-activated protein kinase signaling(37) and inflammatory and angiogenic mechanisms invitro (38), very few groups have investigated obesity-related differences in CD13/APN content or function.The contribution of CD13/APN to these processes in vivoor its potential role in stem cell biology remains to bedetermined.

Maternal obesity increases the risk for neonataladiposity in humans, independent of diabetic status(4). While circulating maternal glucose and lipid concen-trations are associated with neonatal adiposity (6,39),the pathways/mechanisms responsible for greater fetalfat accrual in babies of obese mothers are not well un-derstood. Our study did not address whether the differ-ences we observed in the MSCs were the result ofgenetic differences passed down from the parents, in-trauterine exposure to excess lipids, or other factorsassociated with the obesity-related milieu. Nevertheless,our results show that MSCs of infants born to obesemothers not only exhibit baseline differences in pro-teins linked with adipogenesis but also respond morerobustly to experimentally induced differentiation, in-dicating greater inherent propensity for adipogenesis.Experimental inhibition of GSK-3b revealed that thismay be one mechanism for greater adipogenesis instem cells from infants of obese mothers. Further in-vestigation into the molecular pathways that differ inthese cells on the basis of maternal body size, includingthe potential genetic and epigenetic regulation of thesedifferences, is an important area of future research and

may help us to understand better how obesity duringpregnancy increases susceptibility to obesity in the chil-dren of these women.

Acknowledgments. The authors thank M. Martinez (the Healthy StartStudy project coordinator, Colorado School of Public Health, University ofColorado) and the Healthy Start team for their hard work and dedication. Theauthors also thank S. Ryan (Department of Pediatrics, University of ColoradoSchool of Medicine) for her assistance with flow cytometry protocols.Funding. This work was supported by the Obesity Society by an Early CareerResearch Grant 2013 (to K.E.B.) and the University of Colorado Center for Women’sHealth Research by the Junior Faculty Seed Grant (to K.E.B.). Costs for flowcytometry analyses were offset by the National Cancer Institute (Cancer CenterSupport grant no. P30/CA046934). K.E.B. was supported by the Building Inter-disciplinary Research Careers in Women’s Health (BIRCWH) Program from theNational Institute of Child Health and Human Development (BIRCWH K12/HD057022, to principal investigator J. Regensteiner). The Healthy Start BabyBUMPProject is supported by grants from the American Heart Association (predoctoralfellowship 14PRE18230008 to A.L.B.S.) and the Colorado Nutrition and ObesityResearch Center (National Institute of Diabetes and Digestive and Kidney Diseasesgrant no. P30 DK048520 to principal investigator J. Hill) and by the parent HealthyStart study (to D.D.). The Healthy Start study was supported by the National Instituteof Diabetes and Digestive and Kidney Diseases (R01/DK076648 to D.D.) and theNational Institutes of Health/National Center for Advancing Translational SciencesColorado Clinical and Translational Sciences Award grant no. UL1 TR001082.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.The contents of this article are the sole responsibility of the authors and do not

necessarily represent official NIH views.Author Contributions. K.E.B. conceived and designed the mesenchymalstem cell study, carried out experiments, analyzed data, and wrote and edited themanuscript. Z.W.P. designed and carried out experiments, analyzed data, andwrote and edited the manuscript. A.L.B.S. carried out experiments and edited themanuscript. P.R.B. assisted with stem cell collection and edited the manuscript.D.D. conceived, designed, and carried out the Healthy Start study and edited themanuscript. J.E.F. designed experiments and edited the manuscript. All authorsapproved the final version of the manuscript. K.E.B. and D.D. are the guarantorsof this work and, as such, had full access to all the data in the study and takeresponsibility for the integrity of the data and the accuracy of the data analysis.Prior Presentation. Data from this article were presented at the 75thScientific Sessions of the American Diabetes Association, Boston, MA, 5–9 June2015, and at the Obesity Society ObesityWeek, Los Angeles, CA, 4 November 2015.

References1. Flegal KM, Carroll MD, Kit BK, Ogden CL. Prevalence of obesity and trendsin the distribution of body mass index among US adults, 1999-2010. JAMA 2012;307:491–4972. Gluckman PD, Hanson MA. Developmental and epigenetic pathways toobesity: an evolutionary-developmental perspective. Int J Obes 2008;32(Suppl.7):S62–S713. Heerwagen MJR, Miller MR, Barbour LA, Friedman JE. Maternal obesity andfetal metabolic programming: a fertile epigenetic soil. Am J Physiol Regul IntegrComp Physiol 2010;299:R711–R7224. Sewell MF, Huston-Presley L, Super DM, Catalano P. Increased neonatal fatmass, not lean body mass, is associated with maternal obesity. Am J ObstetGynecol 2006;195:1100–11035. Starling AP, Brinton JT, Glueck DH, et al. Associations of maternal BMI andgestational weight gain with neonatal adiposity in the Healthy Start study. AmJ Clin Nutr. 2015;101:302–3096. Shapiro ALB, Schmiege SJ, Brinton JT, et al. Testing the fuel-mediatedhypothesis: maternal insulin resistance and glucose mediate the association

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Page 13: Mesenchymal Stem Cells From Infants Born to Obese Mothers ...Kristen E. Boyle,1 Zachary W. Patinkin,1 Allison L.B. Shapiro,2 Peter R. Baker II,3 Dana Dabelea,2 and Jacob E. Friedman4

between maternal and neonatal adiposity, the Healthy Start study. Diabetologia2015;58:937–9417. Pirkola J, Pouta A, Bloigu A, et al. Risks of overweight and abdominalobesity at age 16 years associated with prenatal exposures to maternal pre-pregnancy overweight and gestational diabetes mellitus. Diabetes Care 2010;33:1115–11218. von Maltzahn J, Chang NC, Bentzinger CF, Rudnicki MA. Wnt signaling inmyogenesis. Trends Cell Biol 2012;22:602–6099. Shang Y-C, Wang S-H, Xiong F, et al. Wnt3a signaling promotes pro-liferation, myogenic differentiation, and migration of rat bone marrow mesen-chymal stem cells. Acta Pharmacol Sin 2007;28:1761–177410. Armstrong DD, Esser KA. Wnt/beta-catenin signaling activates growth-control genes during overload-induced skeletal muscle hypertrophy. Am J PhysiolCell Physiol 2005;289:C853–C85911. Sharma M, Chuang WW, Sun Z. Phosphatidylinositol 3-kinase/Akt stimu-lates androgen pathway through GSK3beta inhibition and nuclear beta-cateninaccumulation. J Biol Chem 2002;277:30935–3094112. Ross SE, Hemati N, Longo KA, et al. Inhibition of adipogenesis by Wntsignaling. Science 2000;289:950–95313. Kang S, Bennett CN, Gerin I, Rapp LA, Hankenson KD, MacDougald OA. Wntsignaling stimulates osteoblastogenesis of mesenchymal precursors by sup-pressing CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma. J Biol Chem 2007;282:14515–1452414. Yan X, Huang Y, Zhao J-X, et al. Maternal obesity-impaired insulin signalingin sheep and induced lipid accumulation and fibrosis in skeletal muscle of off-spring. Biol Reprod 2011;85:172–17815. Tong JF, Yan X, Zhu MJ, Ford SP, Nathanielsz PW, Du M. Maternal obesitydownregulates myogenesis and beta-catenin signaling in fetal skeletal muscle.Am J Physiol Endocrinol Metab 2009;296:E917–E92416. Zhu MJ, Han B, Tong J, et al. AMP-activated protein kinase signallingpathways are down regulated and skeletal muscle development impaired infetuses of obese, over-nourished sheep. J Physiol 2008;586:2651–266417. Barbet JP, Thornell LE, Butler-Browne GS. Immunocytochemical charac-terisation of two generations of fibers during the development of the humanquadriceps muscle. Mech Dev 1991;35:3–1118. Poissonnet CM, Burdi AR, Garn SM. The chronology of adipose tissue ap-pearance and distribution in the human fetus. Early Hum Dev 1984;10:1–1119. Majore I, Moretti P, Stahl F, Hass R, Kasper C. Growth and differentiationproperties of mesenchymal stromal cell populations derived from whole humanumbilical cord. Stem Cell Rev 2011;7:17–3120. Shapiro HM. Parameters and probes. In Practical Flow Cytometry. 3rd ed.Hoboken, NJ, John Wiley & Sons, Inc, 2005, p. 273–41021. Janderová L, McNeil M, Murrell AN, Mynatt RL, Smith SR. Human mes-enchymal stem cells as an in vitro model for human adipogenesis. Obes Res2003;11:65–7422. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adiposetissue: implications for cell-based therapies. Tissue Eng 2001;7:211–22823. Tsai C-C, Su P-F, Huang Y-F, Yew T-L, Hung S-C. Oct4 and Nanog directlyregulate Dnmt1 to maintain self-renewal and undifferentiated state in mesen-chymal stem cells. Mol Cell 2012;47:169–182

24. Boyle KE, Newsom SA, Janssen RC, Lappas M, Friedman JE. Skeletalmuscle MnSOD, mitochondrial complex II, and SIRT3 enzyme activities are de-creased in maternal obesity during human pregnancy and gestational diabetesmellitus. J Clin Endocrinol Metab 2013;98:E1601–E160925. Boyle KE, Hwang H, Janssen RC, et al. Gestational diabetes is characterizedby reduced mitochondrial protein expression and altered calcium signalingproteins in skeletal muscle. PLoS One 2014;9:e10687226. Iaffaldano L, Nardelli C, Raia M, et al. High aminopeptidase N/CD13levels characterize human amniotic mesenchymal stem cells and drive theirincreased adipogenic potential in obese women. Stem Cells Dev 2013;22:2287–229727. Zhao J, Yue W, Zhu MJ, Sreejayan N, Du M. AMP-activated protein kinase(AMPK) cross-talks with canonical Wnt signaling via phosphorylation of beta-catenin at Ser 552. Biochem Biophys Res Commun 2010;395:146–15128. Metcalfe C, Bienz M. Inhibition of GSK3 by Wnt signalling–two contrastingmodels. J Cell Sci 2011;124:3537–354429. Bikkavilli RK, Feigin ME, Malbon CC. p38 mitogen-activated protein kinaseregulates canonical Wnt-beta-catenin signaling by inactivation of GSK3beta.J Cell Sci 2008;121:3598–360730. Ohashi K, Nagata Y, Wada E, Zammit PS, Shiozuka M, Matsuda R. Zincpromotes proliferation and activation of myogenic cells via the PI3K/Akt and ERKsignaling cascade. Exp Cell Res 2015;333:228–23731. Ding Q, Xia W, Liu J-C, et al. Erk associates with and primes GSK-3betafor its inactivation resulting in upregulation of beta-catenin. Mol Cell 2005;19:159–17032. Zhu MJ, Han B, Tong J, et al. AMP-activated protein kinase signallingpathways are down regulated and skeletal muscle development impaired infetuses of obese, over-nourished sheep. J Physiol 2008;586:2651–266433. Vanella L, Sanford C Jr, Kim DH, Abraham NG, Ebraheim N. Oxidative stressand heme oxygenase-1 regulated human mesenchymal stem cells differentiation.Int J Hypertens 2012;2012:89067134. Chuang CC, Yang RS, Tsai KS, Ho FM, Liu SH. Hyperglycemia enhancesadipogenic induction of lipid accumulation: involvement of extracellular signal-regulated protein kinase 1/2, phosphoinositide 3-kinase/Akt, and peroxisomeproliferator-activated receptor gamma signaling. Endocrinology 2007;148:4267–427535. Scarda A, Franzin C, Milan G, et al. Increased adipogenic conversion ofmuscle satellite cells in obese Zucker rats. Int J Obes 2010;34:1319–132736. Rutkowski JM, Stern JH, Scherer PE. The cell biology of fat expansion.J Cell Biol 2015;208:501–51237. Lendeckel U, Kähne T, Arndt M, Frank K, Ansorge S. Inhibition of alanylaminopeptidase induces MAP-kinase p42/ERK2 in the human T cell line KARPAS-299. Biochem Biophys Res Commun 1998;252:5–938. Rahman MM, Ghosh M, Subramani J, Fong G-H, Carlson ME, ShapiroLH. CD13 regulates anchorage and differentiation of the skeletal musclesatellite stem cell population in ischemic injury. Stem Cells 2014;32:1564–157739. Harmon KA, Gerard L, Jensen DR, et al. Continuous glucose profiles inobese and normal-weight pregnant women on a controlled diet: metabolic de-terminants of fetal growth. Diabetes Care 2011;34:2198–2204

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