RESEARCH ARTICLE

Molecular Reproduction & Development (2014)

Characterization and In Vitro Differentiation Potency ofEarly-Passage Canine Amnion- and Umbilical Cord-DerivedMesenchymal Stem Cells as Related to Gestational Age

MANUEL FILIOLI URANIO,* MARIA ELENA DELL’AQUILA, MICHELE CAIRA, ANTONIO CIRO GUARICCI,MARIO VENTURA, CLAUDIA RITA CATACCHIO, NICOLA ANTONIO MARTINO, AND LUISA VALENTINI

Veterinary Clinics and Animal Productions Section, Department for Emergency and Organ Transplantation, University of BariAldo Moro, Valenzano, Italy

SUMMARY

Fetal adnexa are a non-controversial source of mesenchymal stem cells (MSCs) thathave high plasticity, a high proliferation rate, and the ability to differentiate towardsmultiple lineages. MSC populations have been characterized for their stemness anddifferentiation capabilities; more recent work has focused on MSC selection and onestablishing predictable elements to discriminate the cells with the most potential forregenerative medicine. In this study, we cytogenetically and molecularly character-ized and followed the in vitro proliferation and differentiation potential of early-passage canine amniotic membrane MSCs (AM-MSCs) and umbilical cord matrixMSCs (UCM-MSCs) isolated from fetuses at early (35�40 days) and late (45�55days) gestational ages. We found that cells from both fetal gestational ages showedsimilar features. In all examined cell lines, the morphology of proliferating cellstypically appeared fibroblast-like. Population doublings, passaged up to 10 times,increased significantly with passage number. In both cell types, cell viability andchromosomal number and structure were not affected by gestational age at earlypassages. Passage-3 AM- and UCM-MSCs from both gestational phases alsoexpressed embryonic (POU5F1) and mesenchymal (CD29, CD44) stemnessmarkers, whereas hematopoietic and histocompatibility markers were never foundin anysample.Passage-3 cell populations of eachcell typewerealsomultipotential asthey could differentiate into neurocytes and osteocytes, based on cell morphology,specific stains, and molecular analysis. These results indicated that MSCs retrievedfrom the UCMand AM in the early and late fetal phases of gestation could be used forcanine regenerative medicine.

Mol. Reprod. Dev. 2014. � 2014 Wiley Periodicals, Inc.

Received 14 May 2013; Accepted 15 March 2014

�Corresponding author:Veterinary Clinics and AnimalProduction Section, Department forEmergency and OrganTransplantation, University of Bari AldoMoro, Str Prov Casamassima, km 3,Valenzano, Bari 70010, Italy.E-mail: m.filioliuranio@gmail.com

Grant sponsor: Fondi d’Ateneo Project,University of Bari Aldo Moro;Grant sponsor: Futuro in RicercaProject, MIUR; Grant sponsor: PONProject, MIUR

Published online in Wiley Online Library(wileyonlinelibrary.com).DOI 10.1002/mrd.22322

INTRODUCTION

Pluripotent embryonic stem cells (ESCs) can be derivedfrom the inner cell mass of the preimplantation embryo, bydestroying the blastocyst, or isolated from the primordialgerm-cell pool from an early fetus��but both methods haveethical concerns (Abdulrazzak et al., 2010). Furthermore,

Abbreviations: AM, amniotic membrane; MSC, mesenchymal stem cells;P#, passage number; UCM, umbilical cord matrix; BGLAP, bone gamma-carboxyglutamate protein (alias, osteocalcin); CD29, integrin beta 1 [ITGB1];CD45, protein tyrosine phosphatase, receptor type C [PTPRC]; DLA, majorhistocompatibility complex antigen genes; GFAP, glial fibrillar acidic protein;HPRT1, hypoxanthine phosphoribosyltransferase 1; NES, nestin; RUNX2,runt-related transcription factor 2

� 2014 WILEY PERIODICALS, INC.

ESCs are reported to have high tumorigenicity when usedin cell therapy (Abdulrazzak et al., 2010).

Mesenchymal stem cells (MSCs), on the other hand, aremultipotent cells that can differentiate into various celltypes, both in vitro and in vivo, under controlled conditions(Abdulrazzaket al., 2010). The possibility of isolatingMSCsfrom adult tissues, including fat, skin, brain, and othertissues (Hipp and Atala, 2008), is well established in hu-mans as well as in several animal species. The mostcommon source of MSCs is bone marrow, from wherethese cells were first isolated (Jiang et al., 2002; Marcusand Woodbury, 2008), but important alternative sourcesinclude fetal adnexa, such as the amniotic membrane(or amnion; AM) and umbilical cord matrix (UCM), asdemonstrated in human studies (for AM, see: Ilancheranet al., 2007; Gucciardo et al., 2009; Pappa and Anagnou,2009; for UCM, see: Karahuseyinolu et al., 2007; Troyerand Weiss, 2008; Pappa and Anagnou, 2009). Althoughstem cells from human adult tissues are promising forclinical applications, limitations can be found in some casesin terms of access to, and use of, these sources (Sonciniet al., 2007). Further, some adult-derived stem cells arecharacterized by restricted plasticity and limited prolifer-ative capacity (Abdulrazzak et al., 2010). MSCs isolatedfrom fetal adnexa, however, circumvent all of these limita-tions because the adnexa are routinely discarded at partu-rition, and, thanks to their extracorporeal nature, thesefetal-derived MSC populations are easy to obtain andavailable in large supply (Soncini et al., 2007; Marcusand Woodbury, 2008).

Human MSCs isolated from amniotic fluid in the second(Tsai et al., 2004) and the third trimester (You et al., 2008) ofpregnancy have been subjected to molecular and pheno-typic characterization by flow cytometry and by differentia-tion. The isolated cells expressed the pluripotency markerPOU5F1 and various mesenchymal genes, such as CD29,CD73, CD90, and CD105 (Tsai et al., 2004; You et al.,2008). The absence of expressed, polymorphic class-I and-II HLA antigens (Weiss et al., 2008) strongly supports thepossible use of MSCs in cell therapy (Soncini et al., 2007).These cells also expressed, albeit at low levels, co-stimu-latory molecules such as CD40, CD80, and CD86, whichinduce tolerogenic effects (Weiss et al., 2008). Thesefindings suggest that MSCs derived from fetal adnexamay be immunogenically inert, and thus may have a re-duced risk of rejection upon transplantation (Ilancheranet al., 2007). Moreover, these cells were found to be char-acterized bya very high expansion potential and bya lackoftumorigenicity, which makes them an attractive option forregenerative medicine in cell therapy and tissue engineer-ing settings (Abdulrazzak et al., 2010). Finally, the isolatedcells could differentiate into adipocytes, osteocytes, andneuronal cells (Tsai et al., 2004).

In order to establish suitable criteria for selectingthe most promising cells populations, various differentobstetric parameters (e.g., baby’s gender, mother’s age,gestational age) have been analyzed (Penolazzi et al.,2009). UCM-MSCs obtained from full-term babies showthe greater capacity to differentiate, whereas no differ-

ences were found between the baby’s gender or for themother’s age (Penolazzi et al., 2009). In a recent study, Jainand collaborators (2013) also analyzed the number ofMSCs that can be isolated from fetal adnexa recoveredat different gestational ages, and reported a significantlyhigher MSC population in infants born pre-term than inthose born at term.

MSCs derived from theAMandUCMhave been isolatedand characterized fromvarious animalmodels aswell, suchas horse (Hoynowski et al., 2007; Schuh et al., 2009), cattle(Raoufi et al., 2011), sheep (Mauro et al., 2010), dog (Seoet al., 2009; Filioli Uranio et al., 2011), and others (Cremo-nesi et al., 2011). The caninemodel has helped to evaluatethe biology of stem cells and their role in regenerativemedicine for pets and in pre-clinical medical trials(de Bakker et al., 2013). One advantage of using the caninemodel is the ability to perform MSC transplantation inlarge animals (Seo et al., 2009), although scientific useof the dog as a research model to study human diseaseshas been increasing exponentially (Ostrander et al., 2000;Songsasen and Wildt, 2007; Travis et al., 2009) as morethan 370 canine genetic disorders have been reported,most of which resemble human diseases and dysfunctions(Songsasen and Wildt, 2007). Mapping of the canine ge-nome, alongwith theadvantages conferredbyhigh levels ofdisease penetrance and numerous canine genetic isolateswith breed-specific diseases, make dogs invaluable forstudying the mechanism and etiology of human geneticdiseases, especially rare recessive disorders with complexinheritance that are difficult to study in human populations(Ostrander et al., 2000; Patterson, 2000). The isolation ofcanine MSCs from fetal adnexa has been reported severaltimes, and their stemness and capacity to differentiate havebeenconfirmed (Seoet al., 2009; Zucconi et al., 2010; FilioliUranio et al., 2011). Fernandes et al. (2012) isolated andcharacterized progenitor/stem cells from allantoic and am-niotic fluids of fetuses at late gestational age, a period that isreportedly comparable with the third trimester of humangestation. The majority of allantoic- and amniotic-fluid cellsthat they isolated expressed stem cell markers (vimentin[VIM], nestin [NES], andCK18), and could be differentiatedinto osteogenic, adipogenic, chondrogenic, and neurogen-ic lineages. The authors thus concluded that allantoic- andamniotic-fluid cells derived at this gestational age may bequalified as progenitor stem cells (Fernandes et al., 2012).

Given these benefits, it is of high interest to characterizethe potential differences in stemness among fetal adnexa-derived MSCs, particularly as each population may bebetter-suited for different purposes, such as proliferationtrials, characterization studies, drug/toxicant testing,auto-/allo-/xenogenic transplantation, or for molecularanalysis of pathologies or intrauterine therapy (Abdulraz-zak et al., 2010; De Santis et al., 2011; Shaw et al., 2011).Even though differences in fetal development have beenreported between humans and dogs, 35�40 and 45�55days of canine pregnancy are comparable to the secondand third trimester of human gestation, respectively, whenusing the beginning of organ maturation and the structuraldefinition of the fetus as a reference point. Therefore, the

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aim of the present study was to isolate and to analyze theproliferative capacity, specific gene expression, and in vitrodifferentiation potential of AM-MSCs and UCM-MSCs iso-lated from canine fetuses at early and late phases ofgestation.

RESULTS

Cell Morphology, Population Doubling Time, andCell Viability

Cell cultures were established from both AM and UCMisolated from fetuses at early and late gestational ages. Themorphology of proliferating cells typically appeared fibro-blast-like, and clusters of rapidly expanding cells wereobserved (Fig. 1), as previously described (Filioli Uranioet al., 2011).

Cells were cultured up to passage 10 (P10), and it wasobserved that the doubling time increased with passagenumber (Fig. 2A). Cells isolated from the fetal AM at earlyand late gestational ages showed similar behavior. Thedoubling times of UCM-derived cultures remained constant

for the first four passages and, from P5, increased signifi-cantly (cultures from: early UCM, P1 versus P5�P10,P< 0.001; lateUCM,P1 versusP5,P< 0.05 andP1 versusP6�P10, P< 0.001). Similarly, AM-derived culture dou-bling time remained constant for the first three passagesand, starting from P4, increased significantly (culture from:early AM, P1 versus P4�P10, P< 0.001; late AM, P1versus P4, P< 0.05 and P1 versus P5�P10, P< 0.001).No statistical differences in doubling time were observedbetween cells obtained from fetuses at early and lategestational age for either cell type.

Cell viability did not differ among biological-replicate cellcultures of either cell type isolated from fetuses of early andlate gestational ages. Within cultures derived from thetissue source, however, cell viability did exhibit differencesacross culture/passage age (Fig. 2B). The viability of UCM-MSCs from fetuses at early gestational age remainedconstant for all passages (mean: 71.40� 17.3%) whereasthe viability of late-derived cultures decreased startingfrom P5 (P1 vs. P5�P10, P< 0.001). In AM-MSCs fromearly fetuses, on the other hand, cell viability significantlydecreased starting from P4 (P1 vs. P4�P8 and P1 vs. P10,

Figure 1. Photomicrographs of undifferentiated MSCs in culture. In both cell lines, a spindle-shaped fibroblast-like appearance can be observed:(A,C) AM- and (B,D) UCM-MSCswere derived from canine fetuses in (A,B) early (35�40 days) and (C,D) late (45�55 days) phases of gestation.Cells were observed on Day 3 of P2. Scale bars, 50mm.

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P< 0.001) whereas those from late ages remainedconstant for four passages (mean: 92.83� 3.89%) anddecreased starting from P5 (P1 vs. P5 or P8, P< 0.05;P1 vs. P6, P7, P9, or P10, P< 0.001).

Based on these proliferation and viability profiles, ali-quots of fresh samples at P3 were used to test the differ-entiation potential. Aliquots of fresh samples at P4 wereused for karyotype analysis.

KaryotypeThe karyotype of stem cells has been reported to

undergo changes during passaging, and that they mayshow chromosomal abnormalities (Buzzard et al., 2004),so we performed karyotype analysis on all our samples atP4. All metaphase spreads of AM-MSCs (n¼ 25; Fig. 3)and UCM-MSCs (n¼ 25; data not shown) from both gesta-tional ages were normal in chromosomal number andstructure, thus confirming the chromosomal stability ofthe cells in culture.

mRNA Expression of Embryonic, Mesenchymal,and Hematopoietic Cell Markers

Stem cell-marker expression was evaluated in bothcell lines from each gestational age (Fig. 4). At P3, the

expression of embryonic (POU5F1) and mesenchymal(CD29, CD44) markers was confirmed in cells isolatedfrom the fetal UCM and AM at either gestational age.On the other hand, NANOG expression was found onlyin late-derived UCM-MSCs. CD184 expression was foundin both early- and late-derived UCM-MSC cultures, where-as it only occurred in early-derived AM-MSC populations.Hematopoietic markers (CD34 and CD45) and major-histocompatibility-complex markers (DLA-DRA, DLA-DQA1, DLA-79, and DLA-88) were never found in theseMSC cultures. Canine subcutaneous fibroblasts wereused as negative controls for both embryonic and mesen-chymalmarker assessment, and theydidnot expressanyofthese target genes. Dog leukocytes were used as positivecontrols for hematopoietic and major-histocompatibility-complex markers, but did not express embryonic or mes-enchymal stemness markers.

Multipotentiality by In Vitro DifferentiationWhen induced todifferentiate toaneuronal lineage, cells

from the AM and UCM from both gestational ages showedneuron-like morphology and the presence of Nissl bodies(Fig. 5). By real-time reverse-transcriptase PCR analysis,all cultures analyzed and differentiated along a neuronallineage expressed glial fibrillary acidic protein (GFAP),

Figure 2. Proliferation study of canine fetal adnexa-derived AM- and UCM-MSCs. (A) Doubling time (DT), expressed as number of days requiredfor cell number doubling. Doubling times increasedwith passages forUCM- (A1) andAM-MSCs (B1) derived fromboth phasesof gestation. (B) Cellviability, expressedaspercentageof living (unstained) cells after Trypanblue staining. In bothUCM- (A2) andAM-MSCs (B2), cell viability remainedat considerably high values. Data are expressed as mean� standard deviation of values obtained by four determinations. Student’s t-test: a,bP<0.05; a,c P<0.001.

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whereas control cells did not. NES transcripts were moreabundant in cells cultured under differentiation-inducingconditions compared to controls (Fig. 6). In particular,early-derived AM-MSCs contained 15.13-fold more NEStranscript than undifferentiated control cells, while late-derived AM-MSCs contained only 3.74-fold more than

controls. In contrast, differentiated UCM-MSCs isolatedat an early gestational age expressed 2.71-fold moreNES than controls, whereas UCM-MSCs collected at alate phase of pregnancy expressed 6.65-fold more NES.

Calcium-phosphate mineralization was not observed inany cultures induced to differentiate along an osteogeniclineage, although cells took on a typical osteocyte-likemorphology (Fig. 5). Real-time PCR analysis revealedthat these differentiating cells expressed all the osteogenicgenes tested for. In particular, differentiated AM-MSCsisolated at an early gestational age expressed RUNX2,

Figure 3. Representative images of a Quinacrine-band karyogram ofAM-MSCs from fetuses at early (A) and late (B) phases of gestation.Cellswere analyzed at P4. All examined cells, retrieved from fetuses atlate or early phasesof gestation, showedanormal, diploid karyotypeof78 chromosomes.

Figure 4. Reverse transcriptase-polymerase chain reaction (RT-PCR) data comparing canine AM-MSCs and UCM-MSCs with differ-entiated canine subcutaneous fibroblasts (negative controls, NC) anddog leucocytes (hematopoietic controls, HC). Cells at P3 were exam-ined for expression of markers for embryonic pluripotent cells(NANOG, 185 bp; POU5F1, 200 bp), markers for MSCs (CD44,268bp; CD184, 114bp; CD29, 188 bp), markers for hematopoieticstem cells (CD34, 255bp; CD45, 159bp), and markers for majorhistocompatibility complex (DLA-DRA, 246bp; DLA-DQ1, 163 bp;DLA-79, 333bp; DLA-88, 355 bp). All cell lines expressed at leastone embryonic and MSC markers (see text for details). Expression ofthe hematopoietic markers and dog leucocyte antigens were neverfound in MSC cultures. All cell lines expressed the housekeeping geneHPRT1 (144bp).

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SP7, andbonegamma-carboxyglutamateprotein (BGLAP)at 10.58-, 2.17-, and 2.83-fold higher levels than in controlcells, while differentiated AM-MSCs from a late phase ofgestation contained RUNX2, SP7, and BGLAP at 6.14-,34.43-, and 1.41-fold more transcripts than controls.Differentiation of early-derived UCM-MSCs resulted in1.30-, 14.32-, and 2.07-foldmoreRUNX2,SP7, andBGLAPthan in control cells, while late-derived UCM-MSCs had1.40-, 4.06-, and 2.24-fold more RUNX2, SP7, and BGLAPtranscripts than controls, respectively (Fig. 7).

DISCUSSION

In the present study, we analyzed proliferation, specificgene expression, and in vitro differentiation capacity ofcanine AM- and UCM-derived stem cells to understand

how fetal age affected these characteristics, focusing ontwogestational ages that are comparable to the secondandthe third trimester of human pregnancy. In dogs, the fetalperiod starts from 35 days of gestation, when embryonicorganogenesis is completed, and lasts to parturition, whenthe fetus can be recognized as belonging to the caninespecies (Pretzer, 2008). Starting fromDay 40 until birth, thestructure of the whole canine fetus is defined and it con-tinues to grow (Hyttel et al., 2010). In humans, organogen-esis starts during the first trimester of pregnancy (from the3rd to the 8th week of development), and the organscontinue their maturation throughout the second trimester(from the 3rd to the 6th month of development). In the3rd trimester (7th month to birth), the structure of thewhole human fetus is defined, and it continues to grow(Sadler, 2011).

Figure 5. In vitro differentiation of canine AM-MSCs at P3 to neurogenic and osteogenic lineages. Representative photomicrographs of specificstaining (A,C) and cell morphologies (B,D) of differentiated cells in neurogenic (A,B) and osteogenic (C,D) lineages. Images are from AM-MSCsrecovered from fetuses at a late fetal phase of gestation. (A) Nissl bodies can be observed after 7 days of neurogenic differentiation. (C) Very lowcalcium-phosphate mineralization can be seen after 14 days of osteogenic differentiation, using Von Kossa staining. Similar results were obtainedwith UCM-MSCs, and for cultures derived from early and late gestational ages. Scale bars, 30mm (A, B, and D); 100mm (C).

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In both cell types and phases of gestation, MSCs werespindle-shaped, adherent, and able to be cultured in vitro.The results of the proliferation study showed that AM- andUCM-MSCs behaved similarly, in amanner that is indepen-dent of the gestational period theywere isolated from. Cellswere cultured up to P10, and their doubling times, whichremained low in early passages, increased starting fromP4/P5. This could be due to senescence of fibroblastscontained in the early population of fetal adnexa-derivedMSCs (Ha et al., 2012) and/or to spontaneous differentia-tion (Foudah et al., 2012) during in vitro culture. Theseproliferation profiles are in line with those reported inprevious studies for canine fetal adnexa-derived MSCs,such as from umbilical cord blood, allantoic fluid, the AM,and the UCM (Seo et al., 2009; Filioli Uranio et al., 2011).Most term placenta and fetal membrane-derived humanstem cells, however, are in the quiescent G0/G1 phase ofthe cell cycle (Abdulrazzaket al., 2010). Invitro, human fetaladnexa-derived MSCs exhibit higher proliferation ratescompared to adult-derived stem cells (Fauza, 2004;Witkowska-Zimny and Wr�obel, 2011).

The cell viability observed in the present study de-creased with passage number, although absolute valueswere high at all observed passages. These results are inline with those reported in previous studies for dogs(Songsasen and Wildt, 2007; Filioli Uranio et al., 2011)

and other species (Lovati et al., 2011; Rutigliano et al.,2013). Based on our proliferation rate and cell viabilityobservations, we performed all the subsequent analysesat early passages (P3 and P4).

The cytogenetic analysis revealed that gestational agedoes not affect the cells’ chromosomal stability during earlypassages. To our knowledge, this is the first study reportingthe karyotype analysis in fetal adnexa-derived stemcells, isolated in early and late fetal stages, for any animal.At P3, the molecular expression of embryonic (POU5F1)and mesenchymal (CD29, CD44, and CD184) stem-cellmarkers confirmed the stemness of MSCs isolated fromthe AM and UCM of early and late gestational ages. Theabsence of hematopoietic markers, such as CD34 andCD45, confirmed that the isolated cells did not belong tothe hematopoietic lineages and, similar to previously re-ports (Seo et al., 2009; Zucconi et al., 2010; Filioli Uranioet al., 2011), that there was no contamination with hemato-poietic cells during the isolation and culturing procedures,Major-histocompatibility-complex gene expression wasalso not detected in these MSC cultures, which is in linewith those reported previously for dogs (Seo et al., 2009;Zucconi et al., 2010; Filioli Uranio et al., 2011), horses(Lange-Consiglio et al., 2013a), cats (Rutigliano et al.,2013), and humans (review by Abdulrazzak et al., 2010).This finding confirmed the low immunogenicity of these

Figure 6. Relative expression ofNES by real-time PCR, with reference toHPRT1, in in vitro-differentiated cells compared with controls. Analyseswere performed after 6 days of neurogenic differentiation for AM- (A,C) and UCM-MSCs (B,D) recovered in early (A,B) and late (C,D) phases ofgestation. For each gene and for each cell line, the control is shown as 1.0.

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populations of cells, and supports their use for allogenictransplantation, as reported for horses (Lange-Consiglioet al., 2013b; Broeckx et al., 2014) and humans (Houet al., 2013). Lange-Consiglio et al. (2013b), for example,demonstrated that AM-derived mesenchymal cells couldbe used in allogenic transplantations to promote tendonrepair in the horse. To our knowledge, only one work hasreported the use of allogenic bone MSCs to evaluate theimmune response and differentiation capacity after subcu-taneous implantation in dogs without using immunosup-pressive therapy (Xie et al., 2014). In this canine study,osteo-induced, allogeneic bone MSCs enhanced ectopicbone formation (Xie et al., 2014). This work further showedno differences in the number of CD4- and CD8-positive Tcells or in the CD4-/CD8-positive T-cell ratios among dogstreated with allogenic MSCs, dogs treated with autogenicMSCs, and control groups, thus indicating no immuneresponseagainst the grafted cells (Xie et al., 2014).Despitethese data, no uniform characterization criteria are avail-able for MSCs, thus critical basic knowledge is still requiredto encourage the therapeutic use of these cells in caninetherapy (de Bakker et al., 2013).

Our results regarding fetal-adnexia MSC differentiationare in line with those previously reported for dogs (Seoet al., 2009; Zucconi et al., 2010; Filioli Uranio et al., 2011).

At early passages, MSCs were able to differentiate intoneuronal cell lines (ectodermal lineages), as demonstratedby the presence of the intracellular Nissl bodies, GFAPexpression, and NES overexpression��although NES wasexpressed in both undifferentiated and differentiated cells.Recently, it has been reported that NES is expressed indifferentiated and/or differentiating, committed cells (Nagelet al., 2013; Yin et al., 2013) and also in undifferentiatedstem cells (Ramalho-Santos et al., 2002) and cancer cells(He et al., 2013). In particular, Yin et al. (2013) found thatNES expression in neural stem cells decreased with in-creasing gestational age. Differentiation into osteogeniclineages (mesodermal origins), on the other hand, wasscarcely evident by Von Kossa staining and morphologicalanalysis. Extracellular calcium phosphate mineralizationwas poor, possibly due to low cell density in culture. Ex-pression of osteogenic genes (RUNX2, SP7, and BGLAP),however, was highly enriched in differentiated culturescompared to undifferentiated control cells.

No differences were observed between cell culturesisolated from early or late fetal phases of gestation. Incontrast, it was reported that human MSCs obtainedfrom the UCM of full-term babies showed a bias towardsosteoblast differentiation, expressed as alkaline phospha-tase activity and expression of RUNX2, compared with

Figure7. RelativeexpressionofRUNX2,SP7, andBGLAPby real-timePCR,with reference toHPRT1, in invitro-differentiatedcells comparedwithcontrols. Analyseswere performed after 14 days of osteogenic differentiation for AM- (A,C) andUCM-MSCs (B,D) recovered in early (A,B) and late(C,D) phases of gestation. For each gene and for each cell line, the control is shown as 1.0.

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those recovered between the 29th and 37th week of preg-nancy (Penolazzi et al., 2009). Thus, our data demonstratethe possibility to isolate MSCs, with the same proliferativeand differentiation capabilities, from canine fetuses at dif-ferent gestational ages that are comparable to the secondand the third trimester of human gestation. Similar char-acteristics were reported for cells from canine allantoic andamniotic fluids isolated at 50 days of gestational age,which expressed stemness markers and were able todifferentiate into various mesodermal (adipogenic, osteo-genic, and chondrogenic) and ectodermal (neurogenic)lineages (Fernandes et al., 2012).

In conclusion, cells from the canine AM and UCM ofearly and late fetal phases of gestation were successfullyisolated and comparatively characterized for the first time,and their expression and differentiation profiles suggestthat these MSCs can be used in regenerative-medicineapproaches. Moreover, canine fetal adnexa-derived MSCscould be very important for comparative genetic studiesdue to their emerging role as a fundamental model forstudying human hereditary disease.

MATERIALS AND METHODS

Cell Isolation and ExpansionSampleswere recoveredafter elective ovary-hysterectomy

in four bitches at 35�40 days of gestational age (early fetalstage) and in four bitches at 45�55 days of gestational age(late fetal stage). The AM and UCM of canine fetuses wereused for the isolation of mononuclear cells, as previouslydescribed (Filioli Uranio et al., 2011). In brief, pieces of theAM and UCM were placed in two different Petri disheswith sterile, pre-warmed phosphate-buffered saline(PBS; P4417, Sigma, Milan, Italy) with antibiotics (penicillin100U/ml, streptomycin 100mg/ml solution; P0781, Sigma).After three flushing with PBS, pieces were cultured at atemperature of 38.58C with 5% CO2 in culture medium,consisting of Dulbecco Modified Eagle’s Medium (DMEM;D5546, Sigma); 10% fetal bovine serum (FBS; F3018,Sigma); 2mM L-Glutamine (G7513, Sigma); penicillin(100U/ml) streptomycin (100mg/ml) and amphotericin(0.25mg/ml) solution (A5955, Sigma); and 10ng/ml epider-mal growth factor (EGF; E4127, Sigma). After the initialplating and attachment, remaining tissue pieces were re-moved and cells were cultured until the population expand-ed. Plated cells were monitored during the first expansionperiod (P0)and,every3days, themediumwaschanged.OnDay 10 of culture, well-developed colonies of fibroblast-likecells were clear. Cultures were then harvested with 0.05%trypsin-0.02% EDTA (ECM0920D, Euroclone, Milan, Italy),and re-plated in six-well plates (3516, Corning, New York,NY). Four cell lines were established and evaluated pertissue type (AM or UCM). Cells were maintained for at least10 passages to perform growth and proliferation studies.

Cell Proliferation AnalysisCells were counted at each passage using a Bürker

chamber by dilution (1:1) in Trypan Blue (T8154, Sigma),

starting at P1. Cell viability was expressed as the percent-age of unstained cells.

Cell lines were seeded in six-well plates at a density of104 cells/cm2, and doubling-time analysis was performed.Culture passages were performed every 3 days, and cellcounts were performed for each culture passage. Valueswere used to calculate the doubling time using theformula: Doubling time ¼ Culture time

Cell doubling where ‘‘Culture time’’is the time between passage ‘‘n’’ and passage ‘‘nþ 1’’ andCell doubling ¼ lnðnf=niÞ=ln 2 where ‘‘nf’’ is the harvestedcell number and ‘‘ni’’ is the seeded cell number (FilioliUranio et al., 2011). At each passage, aliquots of eachcell linewere cryopreserved for molecular characterization.

KaryotypingKaryotype analyses of early and late-derived AM-MSCs

and UCM-MSCs at P4 were performed using standard Q-banding procedures. Briefly, the cell line was processedwith colcemid (0.1mg/ml in culturemedium;D1925;Sigma)for 4 hr, followed by three washes in fixative (3 methanol: 1acetic acid). Chromosomes were banded by immersion inquinacrine mustard (Q2876; Sigma), fully washed with tapwater, and mounted in 1� PBS. Metaphase spreads wereacquired by CCD camera using a Leica fluorescence mi-croscope. Chromosomes were counted, and the bandingpattern was compared to the standard Canis familiariskaryotype, according to Breen et al. (1999).

Neurogenic DifferentiationTwenty-four hours prior to neuronal induction, culture

medium was replaced with pre-induction media consistingofDMEM,20%FBS, and1mMb-mercaptoethanol (M7522,Sigma; Seo et al., 2009). The next day, the pre-inductionmedium was removed and cells at P3 were washed withPBS and transferred to neuronal inductionmedium consist-ing of DMEM, 20% FBS, 2% dimethyl sulfoxide (DMSO;D5879, Sigma), and 200mM butylated hydroxyanisole(B1253, Sigma;Woodbury et al., 2000). Cells were culturedfor 5 days and, after differentiation, Nissl staining wasperformed as reported by Hou et al. (2003) and Lu et al.(2010). Molecular analysis of NES and GFAP expressionwas performed to confirm differentiation neuronal lineage.

Osteogenic DifferentiationCell lines at P3 were cultured for 16 days in a differenti-

ation medium consisting of DMEM, 10% FBS, penicillin(100U/ml), streptomycin (100mg/ml), and amphotericin(0.25mg/ml) solution, 0.1mM dexamethasone, 0.25mML-ascorbic acid 2-phosphate (A8960, Sigma), and 10mMb-glycerophosphate (50020, Sigma). The medium waschanged twice weekly. Differentiation was evaluated byVon Kossa staining (Ilancheran et al., 2007; Mauroet al., 2010), with some modifications (1% silver nitrateand 5% sodium thiosulfate) to detect phosphate deposits.Molecular analysis was performed to detect RUNX2, SP7,and BGLAP expression.

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Reverse-Transcriptase PCR Analysis of StemnessMarker Expression in Undifferentiated Cells

Total mRNA was isolated from P3 AM-MSCs and UCM-MSCs, before in vitro differentiation, by using NucleoSpinRNA II (FC140955L, Macherey-Nagel, Düren, Germany).This total mRNA was converted into cDNA using anEnhanced Avian HS RT-PCR kit (HSRT100-1KT, Sigma).The cDNA was then amplified in the following reaction:cDNA (100 ng), 1� PCR buffer (5ml), dNTP mix (10mMeach; 1ml), PCR primers (mixture of 50mM each primer;1ml), 1 U Hot Master Taq DNA Polymerase (2200320,5Prime, Milan, Italy), and nuclease-free water for a finalvolume of 50ml.

Expression of the following set of canine genes wasevaluated for molecular characterization before in vitrodifferentiation: nanog homeobox (NANOG); POU class 5homeobox 1 (POU5F1, alias OCT4); CD44; chemokine(C-X-C motif) receptor 4 (CXCR4, alias CD184); integrinbeta 1 (ITGB1, alia CD29); CD34; protein tyrosine phos-phatase, receptor type C (PTPRC, alias CD45), and majorhistocompatibility complexes (MHC) class II DR alphachain (DLA-DRA) and DQ alpha 1 (DLA-DQA1), class Ib(DLA-79), and class I DLA-88 (DLA-88). Canine hypoxan-thine phosphoribosyltransferase 1 (HPRT1) was used asan internal control forRNAextraction, reverse-transcription,and PCR amplification. All oligonucleotide primers werespanned exon junctions. Table 1 shows primer sequences,and the expected length of corresponding amplifiedfragments.

TheRNA templates of all analyzedgeneswere amplifiedfor 35 cycles of 45 sec at 948C (denaturation), 45 sec at598C (annealing), 40 sec at 728C (elongation), followed byone cycle at 728C for 5min;CD45 andDLA-88 used a 578Cannealing temperature. Negative controls (PCR withouttemplate and RNA without reverse transcription) wereperformed in each experiment (data not shown). Subcuta-neous fibroblasts were used as negative controls forembryonic, mesenchymal, and hematopoietic markers.Dog leucocytes, isolated by Ficoll gradient separation,were used as positive controls for hematopoietic andmajor-histocompatibility-complex marker expression.PCR products were visualized with ethidium bromide ona 2% agarose gel.

Real-Time Reverse-Transcriptase PCR Analysis ofDifferentiation-Marker Expression

Real-timePCRwasperformed usingReal TimeTaqMantechnology, and analyzed on an automated StepOne Sys-tem (Applied Biosystems, Monza, Milan, Italy) to confirmcell differentiation. Nestin (NES; Cf02706322_g1; AppliedBiosystem) and glial fibrillary acidic protein (GFAP;Cf02655694_m1; Applied Biosystems). Genes wereused to confirm neurogenic differentiation. Runt-relatedtranscription factor 2 (RUNX2; Applied Biosystem), SP7transcription factor (SP7; Cf02679117_s1; Applied Biosys-tems), and bone gamma-carboxyglutamate (gla) protein(BGLAP, alias osteocalcin; Cf02623891_g1; Applied Bio-systems) genes were used to confirm osteogenic differen-tiation. The exon-junction spanning primers and probes ofall tested genes were designed using Primer Express 3.0(Applied Biosystems) based on sequences from the caninegenome (http://www.ncbi.nlm.nih.gov/genome?term¼canis%20familiaris). TaqMan canine hypoxanthine phos-phoribosyltransferase 1 (HPRT1), already available fromApplied Biosystems (Cf02626256_m1), was used as endo-genous control.

Total RNAwas isolated, according to themanufacturer’sinstructions, using the Total RNA Purification kit (cat. #17200, Norgen�Biotek Corp., Ontario, Canada). Afterquantification, RNA was reverse-transcribed into cDNAusing the High Capacity cDNA Reverse Transcription kit(Applied Biosystems, Foster City, CA).

Samples were run in duplicate on a MicroAmp FastOptical 48-well Reaction Plate (Applied Biosystems), using20-ml reactions for eachwell containing: 11ml TaqMangeneexpression Master Mix 2� (Applied Biosystems), 1ml Taq-Mangeneexpressionassay, 100 ngcDNA, 5andml RNase-Free H2O. Cycling parameters were: 2min at 508C, 10minat 958C followed by 47 cycles of 15 sec at 958Cand 1min at608C. Data were collected using OneStep Software, andrelative quantification was performed by using a compara-tive method after determining the Ct (threshold cycle)values for the reference (HPRT1) and the target gene ineach sample, according to the 2�DDCt method as describedby the manufacturer. Changes in mRNA expression levelswere calculated after normalization to HPRT1. The pro-gram calculates the DCt and DDCt valueswith the formulas

TABLE 1. Expected Length of Amplification Product and Primer Sequences of Analyzed Markers

Gene Amplification product (bp) Forward primer Reverse primer

NANOG 185 50-GGCCCCCAAGCACCCAACTC 50-ACCCGCGGACTGGTGGAAGAPOU5F1 200 50-TCAGGCGGATGTGGGGCTCA 50-GGGCCTGCACGAGGGTCTCTCD44 268 50-GCCCTGAGCGTGGGCTTTGA 50-TCTGGCTGTAGCGGGTGCCACD184 114 50-GCGGGCGAGCGGTTACCAT 50-CCTCCCGGAAGCAGGGTTCCTTCD29 188 50-TGGGCTTGCGTTGCTGCTGA 50-CACCGGCAACTTAGAGACCAGCCD34 255 50-GCCTGCTCAGTCTGCTGCCC 50-TGGTCCCAGGCGTTAGGGTGACD45 159 50-CTCACGCACACAGGCTCGCA 50-CCCACCCACTGGCACTGCTGDLA-DRA 246 50-CGCTCCAACCACACCCCGAA 50-GGCTGAGGGCAGGAAGGGGADLA-DQA1 163 50-GCACTGGGGCCTGGATGAGC 50-ACCTGAGCGCAGGCCTTGGADLA-79 333 50-CTTCCTGGAGGGCAGGTGCT 50-GGCTTGGGCAGGCTCTTGTGDLA-88 355 50-GTGTGAGACCAGCTGCCTATGG 50-ACCTCATCAGCCTCACATCCCAHPRT1 144 50-CGCAGCCTTGGCGTCGTGAT 50-TCGAGCAAGCCGCTCAGTCC

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below:DCt¼Ct_Mean (HPRT1)�Ct_Mean (gene);DDCt¼DCt�DCt_Mean, so that the gene expression level¼2�DDCt. Changes in gene expression were reported aspercentage changes relative to controls.

Statistical AnalysisOne-way ANOVA and Student’s t-test were used to

evaluate the statistical significance of the results. Valuesof P< 0.05 were considered statistically different.

ACKNOWLEDGMENTS

This research was funded by Fondi d’Ateneo project,University of Bari Aldo Moro (title: ‘‘Isolamento, propaga-zione in vitro e caratterizzazione di cellule staminali canineed equine prelevate da annessi fetali (amnios, liquidoamniotico, cordone ombelicale) in diverse epoche gesta-zionali’’; cod. ORBA10KSV7; sci. resp. Prof. Luisa Valen-tini) and by Futuro in ricerca project MIUR (2010; cod.RBFR103CE3; sci. resp. Prof. Mario Ventura). M.F.U.was supported by ONEV (Omiche e Nanotecnologie pergli Esseri Viventi��Uomo, Animali e Piante per la diagnosidi malattie) Project MIUR (PONa3_00134-n.254/R&C 18/05/2011; sci. resp. RU on germ cells OMICS Prof. MariaElenaDell’Aquila). The authors thankAnthonyGreen of theDepartment of Veterinary Medicine, University of Bari AldoMoro for language revision and assistance.

REFERENCES

AbdulrazzakH,MoschidouD, JonesG,Guillot PV. 2010.Biologicalcharacteristics of stem cells from foetal, cord blood and extra-embryonic tissues. J R Soc Interface 7:S689�S706.

Breen M, Bullerdiek J, Langford CF. 1999. The DAPI bandedkaryotype of the domestic dog (Canis familiaris) generatedusing chromosome-specific paint probes. Chromosome Res7:401�406.

Broeckx S, Zimmerman M, Crocetti S, Suls M, Marin T, FergusonSJ, Chiers K, Duchateau L, Franco-Obreg�on A, Wuertz K,Spaas JH. 2014. Regenerative therapies for equine degenera-tive joint disease: a preliminary study. PLoS ONE 9:e85917.

Buzzard JJ, Gough NM, Crook JM, Colman A. 2004. Karyotype ofhuman ES cells during extended culture Nat Biotechnol22:381�382.

Cremonesi F, Corradetti B, Lange Consiglio A. 2011. Fetal adnexaderived stem cells from domestic animal: Progress and per-spectives. Theriogenology 75:1400�1415.

de Bakker E, Van Ryssen B, De Schauwer C, Meyer E. 2013.Canine mesenchymal stem cells: state of the art, perspectivesas therapy for dogs and as amodel for man. Vet Q 33:225�233.

De Santis M, De Luca C, Mappa I, Cesari E, Quattrocchi T,Spagnuolo T, Visconti D, Noia G, Caruso A. 2011. In-uterostem cells transplantation: Clinical use and therapeutical poten-tial. Minerva Ginecol 63:387�398.

Fauza D. 2004. Amniotic fluid and placental stem cells. Best PractRes Clin Obstet Gynaecol 18:877�891.

Fernandes RA,Wenceslau CV, Reginato AL, Kerkis I, Miglino MA.2012. Derivation and characterization of progenitor stem cellsfrom canine allantois and amniotic fluids at the third trimester ofgestation. Placenta 33:640�644.

Filioli Uranio M, Valentini L, Lange-Consiglio A, Caira M,Guaricci AC, L’Abbate A, Catacchio CR, Ventura M, CremonesiF, Dell’Aquila ME. 2011. Isolation, proliferation, cytogenetic,and molecular characterization and in vitro differentiation po-tency of canine stem cells from foetal adnexa: A comparativestudy of amniotic fluid, amnion, and umbilical cord matrix. MolReprod Dev 78:361�373.

Foudah D, Redondo J, Caldara C, Carini F, Tredici G, Miloso M.2012. Expression of neural markers by undifferentiated ratmesenchymal stem cells. J Biomed Biotechnol 2012:820821.

Gucciardo L, Lories R, Ochsenbein-K€olble N, Don�e E, Zwijsen A,Deprest J. 2009. Fetal mesenchymal stem cells: Isolation,properties and potential use in perinatology and regenerativemedicine. BJOG 116:166�172.

Ha JW, Kim JA, Ha CW. 2012. Do the fibroblasts contained in earlypassage MSC population adversely affect the characteristics ofstem cell population obtained from human placenta? Int J StemCells 5:89�95.

HeQZ, LuoXZ, ZhouQ,WangK, Li SX, Li Y, ZhuHT,DuanT. 2013.Expression of nestin in ovarian serous cancer and its clinico-pathologic significance. Eur Rev Med Pharmacol Sci 17:2896�2901.

Hipp J, Atala A. 2008. Sources of stem cells for regenerativemedicine. Stem Cell Rev 4:3�11.

Hou L, Cao H, Wang D, Wei G, Bai C, Zhang Y, Pei X. 2003.Induction of umbilical cord blood mesenchymal stem cells intoneuron-like cells in vitro. Int J Hematol 78:256�261.

Hou ZL, Liu Y, Mao XH,Wei CY, MengMY, Liu YH, Zhuyun Yang Z,Zhu H, Short M, Bernard C, Xiao ZC. 2013. Transplantation ofumbilical cord and bone marrow-derived mesenchymal stemcells in a patient with relapsing-remitting multiple sclerosis. CellAdh Migr 7:404�407.

Hoynowski SM, Fry MM, Gardner BM, Leming MT, Tucker JR,Black L, Sand T, Mitchell KE. 2007. Characterization and differ-entiation of equine umbilical cord-derived matrix cells. BiochemBiophys Res Commun 362:347�353.

Hyttel P, Sinowatz F, Vejlsted M. 2010. Chapter 19: Comparativelisting of developmental chronology. In: EdwardsRRodenhuis J,editors. Essentials of domestic animal embryology. London:Saunder Elsevier. pp 330�337. ISBN 978-0-7020-2899-1.

Ilancheran S, Michalska A, Peh G, Wallace EM, Pera M, Manuel-pillai U. 2007. Stem cells derived from human fetal membranesdisplay multilineage differentiation potential. Biol Reprod77:577�588.

Jain A, Mathur N, Jeevashankar M, Mukhopadhyay A, Agarwal R,Deorari AK, Paul VK. 2013. Does mesenchymal stem cell

CANINE MESENCHYMAL STEM CELLS AND GESTATIONAL AGE

Mol. Reprod. Dev. (2014) 11

population in umbilical cord blood vary at different gestationalperiods? Indian J Pediatr 80:375�379.

Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD,Ortiz-Gonz�alez XR, ReyesM, Lenvik T, Lund T, BlackstadM, DuJ, Aldrich S, Lisberg A, LowWC, LargaespadaDA, Verfaille CM.2002. Pluripotency of mesenchymal stem cells derived fromadult marrow. Nature 418:41�49.

Karahuseyinolu S, CinarO, Kilic E, Kara F, AkayGG,Demiralp DO,Tukun A, Uckan D, Can A. 2007. Biology of stem cells in humanumbilical cord stroma: In situ and in vitro surveys. Stem Cells25:319�331.

Lange-Consiglio A, Tassan S, Corradetti B, Meucci A, Perego R,Bizzaro D, Cremonesi F. 2013a. Investigating the efficacy ofamnion-derived compared with bone marrow-derived mesen-chymal stromal cells in equine tendon and ligament injuries.Cytotherapy 15:1011�1020.

Lange-Consiglio A, Rossi D, Tassan S, Perego R, Cremonesi F,Parolini O. 2013b. Conditioned medium from horse amnioticmembrane-derived multipotent progenitor cells: Immunomodu-latory activity in vitro and first clinical application in tendon andligament injuries in vivo. Stem Cells Dev 22:3015�3024.

Lovati AB, Corradetti B, LangeConsiglio A, Recordati C, BonacinaE, Bizzaro D, Cremonesi F. 2011. Comparison of equine bonemarrow-, umbilical cord matrix and amniotic fluid-derived pro-genitor cells. Vet Res Commun 35:103�121.

Lu YP, Liu SY, Sun H, Wu XM, Li JJ, Zhu L. 2010. Neuroprotectiveeffect of astaxanthin on H2O2-induced neurotoxicity in vitro andon focal cerebral ischemia in vivo. Brian Res 1360:40�48.

Marcus AJ, Woodbury D. 2008. Fetal stem cells from extra-embryonic tissues: Do not discard. J Cell Mol Med 12:730�742.

MauroA, TurrianiM, Ioannoni A, RussoV,Martelli A, DiGiacintoO,Nardinocchi D, Berardinelli P. 2010. Isolation, characterization,and in vitro differentiation of ovine amniotic stem cells. Vet ResCommun 34:S25�S28.

Nagel S, Rohr F, Weber C, Kier J, Siemers F, Kruse C, Danner S,Brandenburger M, Matthiessen AE. 2013. Multipotent nestin-positive stem cells reside in the stroma of human eccrine andapocrine sweat glands and can be propagated robustly in vitro.PLoS ONE 8:e78365.

Ostrander EA, Galibert F, Patterson DF. 2000. Canine geneticscomes of age. Trends Genet 16:117�124.

Pappa KI, Anagnou NP. 2009. Novel sources of fetal stem cells:Where do they fit on the developmental continuum?RegenMed4:423�433.

Patterson DF. 2000. Companion animal medicine in the age ofmedical genetics. J Vet Intern Med 14:1�9.

Penolazzi L,Vecchiatini R,Bignardi S, Lambertini E, Torreggiani E,Canella A, Franceschetti T, Calura G, Vesce F, Piva R. 2009.Influence of obstetric factors on osteogenic potential of umbilicalcord-derived mesenchymal stem cells. Reprod Biol Endocrinol7:106.

Pretzer SD. 2008. Canine embryonic and fetal development:A review. Theriogenology 70:300�303.

Ramalho-SantosM,YoonS,Matsuzaki Y,MulliganRC,MeltonDA.2002. ‘‘Stemness’’: Transcriptional profiling of embryonic andadult stem cells. Science 298:597�600.

Raoufi MF, Tajik P, Dehghan MM, Eini F, Barin A. 2011. Isolationand differentiation of mesenchymal stem cells from bovineumbilical cord blood. Reprod Domest Anim 46:95�99.

Rutigliano L, Corradetti B, Valentini L, Bizzaro D, Meucci A,Cremonesi F, Lange-Consiglio A. 2013. Molecular characteri-zation and in vitro differentiation of feline progenitor-like amni-otic epithelial cells. Stem Cell Res Ther 4:133. DOI: 10.1186/scrt344

Sadler TW. 2011. Chapter 6: Third month to birth: The fetusand placenta. In: Sadler TW, editor. Langman’s medical embry-ology. Philadelphia: Lippincott Williams & Wilkins. pp 117�148.ISBN-10: 0781790697, ISBN-13: 978-0781790697.

Schuh EM, Friedman MS, Carrade DD, Li J, Heeke D, OysermanSM, Galuppo LD, Lara DJ, Walker NJ, Ferraro GL, Owens SD,Borjesson DL. 2009. Identification of variables that optimizeisolation and culture of multipotent mesenchymal stem cellsfrom equine umbilical-cord blood. Am J Vet Res 70:1526�1535.

Seo MS, Jeong YH, Park JR, Park SB, Rho KH, Kim HS, Yu KR,Lee SH, Jung JW, Lee YS, Kang KS. 2009. Isolation andcharacterization of canine umbilical cord blood-derived mesen-chymal stem cells. J Vet Sci 10:181�187.

Shaw SW, David AL, De Coppi P. 2011. Clinical applicationsof prenatal and postnatal therapy using stem cellsretrieved from amniotic fluid. Curr Opin Obstet Gynecol 23:109�116.

Soncini M, Vertua E, Gibelli L, Zorzi F, Denegri M, Albertini A,Wengler GS, Parolini O. 2007. Isolation and characterization ofmesenchymal cells from human fetal membranes. J Tissue EngRegen Med 1:296�305.

Songsasen N, Wildt DE. 2007. Oocyte biology and challenges indeveloping in vitro maturation systems in the domestic dog.Anim Reprod Sci 98:2�22.

Travis AJ, Kim Y, Meyers-Wallen V. 2009. Development of newstem cell-based technologies for carnivore reproduction re-search. Reprod Dom Anim 44:22�28.

Troyer DL, Weiss ML. 2008. Concise review: Wharton’s jelly-derived cells are a primitive stromal cell population. Stem Cells26:591�599.

Tsai MS, Lee JL, Chang YJ, Hwang SM. 2004. Isolation of humanmultipotent mesenchymal stem cells from second-trimesteramniotic fluid using a novel two-stage culture protocol. HumReprod 19:1450�1456.

Weiss ML, Anderson C, Medicetty S, Seshareddy KB, Weiss RJ,VanderWerff I, TroyerD,McIntoshKR. 2008. Immunepropertiesof human umbilical cord Wharton’s jelly-derived cells. StemCells 26:2865�2874.

Molecular Reproduction & Development FILIOLI URANIO ET AL.

12 Mol. Reprod. Dev. (2014)

Witkowska-Zimny M, Wr�obel E. 2011. Perinatal sources of mes-enchymal stem cells: Wharton’s jelly, amnion and chorion. CellMol Biol Lett 16:493�514.

Woodbury D, Schwarz EJ, Prockop DJ, Black IB. 2000. Adultrat and human bone marrow stromal cells differentiate intoneurons. J Neurosci Res 61:364�370.

Xie F, Teng L, Wang Q, Sun XJ, Cai L, Zeng HF, Xiao R. 2014.Ectopic osteogenesis of allogeneic bone mesenchymal stemcells loading on b-tricalcium phosphate in canines. Plast Re-constr Surg 133:142e�153e.

Yin X, Li L, Zhang X, Yang Y, Chai Y, Han X, Feng Z. 2013.Development of neural stem cells at different sites of fetus brainof different gestational age. Int J Clin Exp Pathol 6:2757�2764.

YouQ,Cai L, Zheng J, TongX, ZhangD,ZhangY. 2008. Isolation ofhuman mesenchymal stem cells from third-trimester amnioticfluid. Int J Gynaecol Obstet 103:149�152.

Zucconi E, Vieira NM, Bueno DF, Secco M, Jazedje T, AmbrosioCE, Passos-Bueno MR, Miglino MA, Zatz M. 2010. Mesenchy-mal stem cells derived from canine umbilical cord vein - A novelsource for cell therapy studies. Stem Cells Dev 19:395�402.

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