research article increased haematopoietic supportive...

Hindawi Publishing CorporationStem Cells InternationalVolume 2013 Article ID 985285 12 pageshttpdxdoiorg1011552013985285

Research ArticleIncreased Haematopoietic Supportive Function ofUSSC from Umbilical Cord Blood Compared to CB MSC andPossible Role of DLK-1

Simone Maria Kluth Teja Falk Radke and Gesine Koumlgler

Institute for Transplantation Diagnostics and Cell Therapeutics Heinrich Heine University Medical Center40225 Duesseldorf Germany

Correspondence should be addressed to Simone Maria Kluth kluthitzuni-duesseldorfde

Received 11 October 2012 Revised 25 January 2013 Accepted 7 March 2013

Academic Editor David Allan

Copyright copy 2013 Simone Maria Kluth et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Multipotent stromal cells can be isolated from a variety of different tissues in the body In contrast to stromal cells from the adultbone marrow (BM) or adipose tissue cord blood (CB) multipotent stromal cells (MSC) are biologically younger Since first beingdescribed by our group delta like 1 homologue (DLK-1) was determined as a discriminating factor between the distinct cord blood-derived subpopulations the unrestricted somatic stromal cells (USSC) which lack adipogenic differentiation capacity and theBM MSC-like CB MSC In this study experiments assessing the haematopoiesis-supporting capacity and molecular biologicalanalyses were conducted and clearly confirmed different properties Compared to CB MSC USSC lead to a higher expansion ofhaematopoietic cells and in addition express significantly higher levels of insulin-like growth factor binding protein 1 (IGFBP1) butlower levels of IGF2 The data presented here also indicate that DLK-1 might not be the sole factor responsible for the inhibitionof adipogenic differentiation potential in USSC but nevertheless indicates a biological diversity among cord blood-derived stromalcells

1 Introduction

Within the last two decades cord blood (CB) has been wellestablished as a valuable source of haematopoietic stem andprogenitor cells applied in the treatment of blood-relateddiseases Besides haematopoietic cells CB also contains cellswith stromal properties [1 2] While initially all these cellswere termed unrestricted somatic stem cells further researchrevealed significant differences between the cell lines such asthe competence for adipogenic differentiation and expressionof the preadipocyte marker delta like 1 homologue (DLK-1)[3] and expression of the homeobox (HOX) genes [4]

As of today CB-derived cells have been distinguishedby these characteristics into unrestricted somatic stromalcells (USSC) and cord blood multipotent stromal cells (CBMSC) USSC do not respond to adipogenic induction andpossess a HOX-negative expression pattern while CB MSCform lipid vacuoles in adipogenic differentiation assays andexpress a variety of the 39 humanHOX-genes similar to bone

marrow (BM) derivedMSCThis classification of CB-derivedstromal cells was also confirmed on a clonal level thereforeexcluding an effect of heterogeneity of the bulk cultures [3]Althoughmultiple groups confirmed the generation of USSCandCBMSC fromcord blood [5ndash7] the questionwhether therespective cells are already present in cord blood or if they arecell culture artifacts is still not finally answered and discussedcontroversially For example Karagianni et al proposed thatall native CB-derived cells are USSC rather than MSC-like cells and that they gain their ability for adipogenesisby extrinsic factors such as dexamethasone [8] Howeverexperiments confirming this hypothesis are missing

The missing differentiation into adipocytes is the mostobvious yet functional difference between USSC and CBMSC and by current classification [9] excludes USSCfrom being regarded as MSC-like cell Though it has tobe mentioned that the acronym MSC might be misleadingOriginally introduced by Caplan [10] as abbreviation forldquomesenchymal stem cellsrdquo this term has been discussed

2 Stem Cells International

controversially within the last years [11] Although manycells of different origin are referred to as MSC characterizedby similar properties like morphology immunophenotypeandmandatory basic differentiation potential (chondrogenicosteogenic and adipogenic) subtle differences can be notedIn this context a potential relation of different MSC-likecells has to be discussed especially since evidence arose thatUSSC can be transformed into cells with full adipogenicdifferentiation capacity (manuscript in revision Liedtkeet al 2013) Therefore although less specific MSC as abbre-viation for ldquomultipotent stromal cellsrdquo might be more appro-priate for these cells which in turn would include nativeUSSC

The discrimination of USSC fromCBMSC by the expres-sion ofDLK-1 and the inverse correlation with the adipogenicdifferentiation capacity was the first evidence for two distinctstromal cell populations in cord blood [3] The function ofDLK-1 during adipogenic differentiation is well described[12ndash14] Besides the inhibitory effect on adipogenesis DLK-1is known to play a major role during differentiation of hepa-toblasts and considered a potent regulator of haematopoiesis[15ndash17]

The aim of this study was to further assess the functionof DLK-1 and its soluble form known as fetal antigen1 (FA1) [18] in regard to a potential biological diversitybetween USSC and CB MSC which might have impact onfuture clinical useTherefore the haematopoiesis-supportingcapacity of USSC andCBMSC in vitrowas compared directlyby co-culture experiments Additionally the expression ofhaematopoiesis relevant cytokines on molecular biologicallevel was analyzed

2 Materials and Methods

21 Generation and Expansion of CB Stromal Cells CB wascollected from umbilical cord vein with informed consentof the mother The bulk cell lines (USSC 119899 = 61 CB MSC119899 = 29) used in this study were isolated from 90 differentdonors USSC and CB MSC were generated by the samemethod as described previously [3] a classification intothe two subtypes was only possible after examination ofthe adipogenic differentiation potential and HOX expres-sion profile of each cell line Briefly mononuclear cells(MNC) of one cord blood were obtained by Ficoll gradi-ent separation (density 1077 gcm3 Biochrom AG BerlinGermany) followed by lysis of remaining erythrocytes withammonium chloride (pharmacy of the university medicalcentre Duesseldorf) 5ndash7 lowast 106 CB MNCmL were culturedin DMEM low glucose (Lonza Inc Allendale NJ USA)with 30 FCS (PerbioFisher Scientific Bonn Germany) 1lowast10minus7Mdexamethasone (Sigma-Aldrich StLouisMO USA)

penicillinstreptomycin and L-glutamine (PSG all LonzaInc) until detection of adherent growing coloniesThese cellswere expanded without dexamethasone in a closed systemapplying cell stacks (Corning Inc Corning NY USA)incubated at 37∘C in 5CO

2in a humidified atmosphere and

defined as one single polyclonal bulk cell line Reaching 80confluence cells were washed with phosphate buffered saline

(PBS Serag-Wiessner KG Naila Germany) detached with025 trypsin (Lonza Inc) and replated 1 3

22 Generation of Bone Marrow Stromal Cells BM MSClines were generated from bone marrow of healthy donorsas described previously [1] MNC were isolated and plated inDMEM low glucose with 30 FCS and PSG until detectionof adherent growing colonies

23 Biological Controls Human skin fibroblasts (NHDF)were provided by the Department of Dermatology HeinrichHeineUniversity Duesseldorf HepG2 and nTERA-2 cell lineswere purchased from ATCC and cultured according to themanufacturerrsquos instructions

24 Generation of Cell Clones Using the AVISO CellCelectorClonal populations were obtained from established cell linesin early passages applying the AVISO CellCelector (ALSGmbH Jena Germany) as described previously [3] Brieflycells of one single bulk cell line were plated at low density(166 cellscm2) in 6-well cell culture plates (Corning Inc)and after allowing the cells to get adherent again distinctsingle cells were selected picked and transported to a defineddestination well of a 96-well cell culture plate (Corning Inc)and cultured with preconditioned medium Pictures weretaken before and after each picking process to documentsuccessful single cell selection The term ldquoUSSC 1 clone 1rdquodefines the first clonal population isolated fromUSSC cell line1 this nomenclature also applies to CB MSC-derived clonalpopulations All cell lines were used between passage 5 and 8

25 In Vitro Differentiation into Adipocytes Differentiationinto adipocytes was performed as described previously [2]Adipogenic differentiation was induced applying DMEMhigh glucose (45 gL Lonza Inc) 10 FCS PSG 1 lowast 10minus6Mdexamethasone (water soluble cell culture tested) 02mMindomethacin 01mgmL insulin and 1mM 3-Isobutyl-methylxanthine (all Sigma-Aldrich) Media was changedtwice a week alternating with cultivation media containing001mgmL insulin Lipid vacuoles were stained with Oil RedO (Sigma Aldrich) after 21 days As negative control the cellswere cultured in DMEM low glucose (1 gL Lonza Inc) 10FCS and PSG

26 Co-Cultures of CD34-Selected Cells on Stromal FeederLayers Stromal feeders were prepared in 24-well plates byseeding 5 lowast 104 cells (USSC CB MSC) in standard culturemedia After 24ndash48 h when density was app 80ndash90 agrowth arrest was induced by irradiation (30 gy cobaltsource)

Cord blood-derived CD34+-cells were isolated from thefraction of the mononucleated cells following Ficoll-gradientcentrifugation and erythrocyte lysis by ammonium chlorideusing a CD34 progenitor kit for magnetic-activated cell sort-ing (MACS) and Midi-MACS magnets with LS columns (allMiltenyi Biotec Bergisch Gladbach Germany) according tothe manufacturerrsquos instructions Isolated CD34+-cells were

Stem Cells International 3

seeded on the irradiated adherent feeders at 1lowast1053mLwellin freshmedia for each day examined CD34+-cells and feedercells were clearly distinguishable microscopically in regard tosize and formOndays 3 7 10 and 14 non-adherent cells wereharvested enumerated and assayed by flow cytometry as wellas seeded in colony-forming unit assays

27 RNA Isolation and Real Time PCR Total RNA of cellswas isolated utilizing the RNeasy Mini Kit (Qiagen HildenGermany) 1 120583g RNAwas reverse transcribed applying Super-ScriptIII (InvitrogenLife Technologies Carlsbad Ca USA)according to the manual

Real time PCR was performed with SYBR Green PCRMastermix (Applied BiosystemsLife Technologies CarlsbadCa USA primer see Table 1) Evaluation of Taq Man GeneExpression Assays (Applied BiosystemsLife Technologies)was performed utilizing the SDS23 program and expressionnormalized to GAPDH As comparison total fetal liver RNAextract was obtained from Stratagene (StratageneAgilentTechnologies La Jolla CA USA)

28 Immunofluorescence For immunofluorescence detec-tion 10000 cellscm2 were plated on chamber slides (Lab-TekChamber Slide 2 Well Glass Slide Thermo ScientificWaltham MA USA) Cells were fixed with 4 PFA for 15minutes Primary antibody (dilution 1 250 Table 2) incuba-tion was performed at 4∘C overnight Secondary antibody(Table 2) was applied in 1 1000 dilutions Nuclei were stainedwith ProLong Gold antifade reagent with DAPI (Invitrogen)

29 Flow Cytometric Analysis and Colony Forming Unit AssayCell count was performed applying a Neubauer chamber05ndash10 lowast 105 cells were resuspended in 100120583L PBS andstained with CD45-FITC and CD34-PE antibodies (5 120583Leach both BD Biosciences Franklin lake NJ USA) Afterincubation for 15 minutes at 4∘C samples were washedwith PBS and after centrifugation analyzed for percentage ofCD34highCD45low cells using a FACSCanto flow cytometer(BD Biosciences) Additionally cells were seeded at adjustedconcentration (ranging from 100mL on day 0 to 10000mLon day 14) in semi-solid growth-factor enriched methylcellulose (Methocult GF H4434 STEMCELL TechnologiesInc Vancouver Canada) and after 14 days of incubation(37∘C 5 CO

2 humidified air) scored for the formation of

colonies as described before [1]For detection of DLK-1-expression on stromal cells1lowast105 cells in 100120583LPBS (SeragWiessner)were incubated for

30 minutes with 1120583L DLK-1 antibody (clone PF299-1 Enzolife sciences Farmingdale NY USA) and washed with PBSAs secondary antibody 15 120583L of FITC-conjugated goat-anti-mouse antibody (Jackson ImmunoResearch IncWest groovePA USA) was applied and incubated for another 20 minutesfollowed bywashingwith PBS and subsequent centrifugation

For intracytoplasmatic staining cells were fixed andpermeabilized utilizing the BD CytofixCytoperm kit (BDBiosciences) according to the manufacturerrsquos instructionsprior to addition of antibodies As fixative 4 paraformalde-hyde (PFA Otto Fischar GmbH Saarbruecken Germany)

was added after washing removal of PBS and resuspensionresulting in a final concentration of app 2 PFA

Finally flow cytometry analysis was performed on aFACSCanto flow cytometer (BD Biosciences) employingthe FACSDIVA software (version 503) for recording andWinMDI 28 (Freeware Joseph Trotter) for analysis

210 Culture with DLK-1 Enriched Supernatant CB MSCoverexpressing DLK-1 were cultured in standard expansionmedium for 4 to 5 days until reaching 80 confluence Thesupernatant was collected and filtered using a syringe filter(Thermo Scientific) Concentration of soluble DLK-1 wasdetermined applying DLK-1 ELISA

CB MSC were plated on 6 well cell culture plates andcultivated for 24 hours to allow adherence Subsequentlyincubation with DLK-1 positive medium was carried out for30minutes 4 hours and 24 hours respectivelyThe cells werewashed twice with PBS and lysed using RIPA cell lyse buffer(InvitrogenLife Technologies) for western blot analyses orcell lyse buffer (Qiagen) for PCR approaches

211 DLK-1 ELISA To perform enzyme-linked immuno-sorbend assay (ELISA) analysis of the supernatants CBMSCand USSC were cultured under standard conditions (HumanPref-1DLK-1FA1 Quantikine ELISA Kit RampD Systems IncMinneapolis MN USA) After 3 days the supernatant wascollected filtered and stored at minus80∘C until further proce-dures Subsequently the manufacturerrsquos instructions werefollowed Maternal plasma was employed as positive control

212 Western Blot Analysis Total protein was analyzed inWestern Blot analysis (NuPAGE System InvitrogenLifeTechnologies) For detection ECL PLUS Western BlottingDetection Reagents (GE Healthcare Buckinghamshire UK)was applied in accordance with the manufacturerrsquos instruc-tions

213 DLK-1 Overexpression As described previously [3]full-length human DLK-1 was amplified using PhusionTaq Polymerase (New England Biolabs) Insert and vector(pCL7Egwo) were digested employing Fast Digest EcoRIand Fast Digest XhoI (both Fermentas) with the eGFPgene replaced by the DLK-1 gene Products were thenelectrophoresed on 2 agarose gels and purified using theQIAquick Gel Extraction Kit (QIAGEN) Insert and vectorwere ligated at a 3 1-ratio applying T4 DNA Ligase (NewEngland Biolabs) Transformed electrocompetent Top10 bac-teria (Invitrogen) were plated on LB agar containing ampi-cillin (500120583gL) overnight and DLK-1 positive colonies wereexpanded in 100mLLBmedia Vectors were isolated applyingMaxiPrep Kit (QIAGEN) and 293T cells were transfectedwith pCL7Egwo DLK-1 GalvTM and CDNL-BH utilizingFuGene HD Transfection Reagent CB MSC were thentransfected with the resulting virus loaded supernatant

214 Cell-Sorting for DLK-1-Expression by Fluorescent Acti-vated Cell Sorting (FACS) CB MSC overexpressing DLK-1


Table 1 Primer applied in this study

Gene Primer Sequence 51015840-31015840 Product (bp) Annealingtemperature

Angiopoetin-like 3 (ANGPTL-3) NM 0144952

ForwardTGGGAGGCTTGATGGAGAATReverseGTAGCGTATAGTTGGTTTCG

164 bp 60∘C

CCAAT enhancer binding proteinalpha (CEBP120572) NM 0043643

ForwardGAGTCACACCAGAAAGCTAGReverseGATGGACTGATCGTGCTTC

184 bp 60∘C

Delta like-1 homologue (DLK-1) NM 0038365

ForwardAGCACCTATGGGGCTGAATReverseGTCACGCACTGGTCACAAAG

119 bp 60∘C

Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) NM 0020464

ForwardGAGTCAACGGATTTGGTCGTReverseTTGATTTTGGAGGGATCTCG

238 bp 60∘C

Insulin-like growth factor 2 (IGF-2)NM 0006124

NM 0010071394NM 0011275981

ForwardATTGCTGCTTACCGCCCCAReverseCACTCCTCAACGATGCCACG

143 bp 60∘C

Insulin-like growth factor-bindingprotein 1 (IGFBP-1) NM 0005962

ForwardCAAAAAATGGAAGGAGCCCReverseGATGTCTCACACTGTCTGC

153 bp 60∘C

Peroxisome proliferators activatorgamma 2 (PPAR1205742) NM 0158694

ForwardTCCATGCTGTTATGGGTGAAReverseTCAAAGGAGTGGGAGTGGTC

193 bp 60∘C

Ribosomal protein L13a (RPL13a) NM 0012704911NM 0124233

ForwardGAGGTATGCTGCCCCACAAAReverseTTCAGACGCACGACCTTGAG

136 bp 60∘C

Stem cell factor (SCF) NM 0039945NM 0008994

ForwardGGATAAGCGAGATGGTAGTAReverseCTTCAGGAGTAAAGAGCC

210 bp 55∘C

Stromal-derived factor 1 (SDF-1)

NM 1991683NM 0006095

NM 0010338862NM 0011781341

ForwardCTCAACACTCCAAACTGTGCReverseGCTTTCTCCAGGTACTCC

110 bp 60∘C

Thrombopoietin (THPO)NM 0004602NM 0011775971NM 0011775981

ForwardTCACCCTTTGCCTACACCReverseGTCACTGCTCCCAGAATGT

108 bp 60∘C

were labelled with antibodies according to the protocoldescribed above and sorted at theCore FlowCytometry Facil-ity of the University Medical Center Duesseldorf applyinga MoFlo XDP cell sorter (Beckman Coulter) with SummitSoftware (510)

215 Statistics Statistics were evaluated with the Graph PadPRISM software (version 501 GraphPad Software Inc LaJolla CA USA) A Studentrsquos 119905-test was used and all valuesgiven as mean plusmn SD if not stated otherwise

3 Results

31 DLK-1 as Marker to Distinguish USSC from CB MSCThe presence of distinct stromal cell populations in cordblood (CB) has been described before [3 4 19] Unrestrictedsomatic stromal cells (USSC) and CB multipotent stromalcells (MSC) share the same immunophenotype as well asosteogenic and chondrogenic differentiation potential invitro The two subpopulations can be classified by analyzingthe adipogenic differentiation capacity and the HOX-geneexpression profile In contrast to CB MSC (119899 = 21) and MSC


Table 2 Antibodies utilized in this study

Primary Antibody Clone Company

DLK-1 PF299-1Enzo life Science

Farmingdale NY USA

ERK12 16ERK BD TransductionLaboratories

p-ERK12 E4 Santa Cruz Dallas TXUSA

120573-Actin AC-74 Sigma-AldrichCD34-PE 8G12 BD BioscienceCD45-FITC 2D1 BD BioscienceSecondary AntibodyImmunofluorescenceRhodamineRed-X-conjugated goatantimouse IgG

Dianova HamburgGermany

Flow cytometryFITC-conjugated goatantimouse

JacksonImmunoResearch IncWest grove PA USA

Western blotHRP-conjugated goatantimouse IgG

sc-2005 Santa Cruz

from bone marrow (BMMSC) (119899 = 44) USSC (119899 = 69) lackthe adipogenic differentiation potential (Figure 1(a)) and asa general feature express high levels of DLK-1 on transcriptlevel

To evaluate the impact of heterogeneity of these sub-populations USSC- (119899 = 29) and CB MSC-derived (119899 =58) clonal populations were reanalyzed USSC-derived clonalpopulations never exhibited the adipogenic differentiationpotential though a heterogeneous DLK-1 expression wasdetected (Figure 1(b)) questioning the inhibitory function ofDLK-1 in USSC

32 DLK-1 in USSC and Correlation with the Lack of Adi-pogenic Differentiation Capacity DLK-1 is a transmembraneprotein containing a soluble domain [20] The cleavageof DLK-1 into a transmembrane and a soluble protein(termed PREF-1 in mice and FA1 in humans) is describedas being mandatory to inhibit the adipogenic differentiationof stromal cells [20] Overexpression or addition of DLK-1in murine stromal cells results in an activation of ERK12thereby downregulating the expression of adipogenesis-associated genes and inhibiting the adipogenic differentiation[21]

To analyze the DLK-1 protein level in USSC immunoflu-orescence assays were performed USSC (119899 = 4) exhibiteda DLK-1 protein expression while CB MSC (119899 = 3) wereDLK-1 negative (Figure 2(a)) as described before [3] In orderto validate these data the DLK-1 expression was additionallyanalyzed by flow cytometry applying a monoclonal antibodydetecting the extracellular domain of DLK-1FA1 Surpris-ingly no extracellular expression of DLK-1 protein in USSCcould be verified (Figure 2(b)) yet in some of the USSC lines

examined intracellular antibody labeling revealed a small butclearly DLK-1-positive subpopulation of USSC suggestingintracellular localization of DLK-1FA1

In vivo DLK-1 is released into the circulation [18] AnELISA-test detecting DLK-1FA1 was applied to address thequestion whether the missing extracellular expression ofDLK-1 in USSC might be ascribed to a high cleavage rateHowever secretion of DLK-1FA1 was detected neither insupernatant of USSC cultures (0014 plusmn 0064 ngmL) norof CB MSC cultures (0058 plusmn 0042 ngmL) (Figure 2(c))Maternal plasma and supernatant of DLK-1-overexpressingcells served as positive controls with an approximately 40-and 80-fold higher expression respectively (2112 and 4910each 119899 = 1)

In addition the DLK-1 expression of USSC and CBMSC did not correlate with expression of the adipogenictranscription factors 119862119864119861119875120572 (CB MSC 119899 = 4 CB MSC-derived clones 119899 = 3 USSC 119899 = 2 USSC-derived clones119899 = 2) and PPAR1205742 (CB MSC 119899 = 3 USSC 119899 = 4) Herethe analysis of quantitative 119862119864119861119875120572 and PPAR1205742 expressionby real time PCR did not reveal any significant differencesbetween the two subpopulations but instead demonstratedvery heterogeneous expression levels already between thedifferent cell lines analyzed (Figures 2(d) and 2(e))

To evaluate the phosphorylation status of ERK12 CBMSC were treated with cell culture supernatant of CB MSCoverexpressing DLK-1 (with extracellular expression of DLK-1 and secreting soluble functional DLK-1 as confirmed byELISA Figures 2(b) and 2(c) and ERK12 expression wasanalyzed For western blot analysis different time points (30minutes 4 hours and 24 hours) were applied to cover theexpression kinetics of the protein Osteogenic induced BMMSC (day 3 after induction) served as positive control for theERK12 activation (Figure 3) since the activation of ERK12is required to induce the expression of matrix producinggenes such as collagens bone morphogenetic proteins (BMPs)or transforming growth factors (TGFs) Surprisingly whilethe non-phosphorylated ERK12 was expressed under allconditions in CB MSC a DLK-1-specific phosphorylation ofERK12 was not detected However it has to be noted that areduced adipogenic differentiation was observed in the CBMSC overexpressing the full-length DLK-1 [3]

33 DLK-1 and Its Possible Role in Haematopoiesis-SupportingCapacity Besides the inhibitory role on adipogenic differen-tiation of stromal cell populations DLK-1 is also an importantregulator of haematopoeisis [16 17] In 2010 data of Chouand Lodish clearly demonstrated that a DLK-1-positive sub-population of murine fetal liver cells had a high impact onhaematopoietic support [15] Stromal cells like USSC CBMSC or BM MSC are suitable feeder layers for CD34+ cellco-culture ex vivo As published in 2005 expansion of cordblood-derived CD34+-cells on likewise CB-derived stromalcells leads to higher expansion rates compared to BM MSC[1] while in vivo cotransplantation of USSC into immuno-deficient mice resulted in improved engraftment [22]

However with regard to the discrimination into USSCand CBMSC here we demonstrate for the first time that only


USSCCB MSC BM MSCAd

ipog

enic

100 120583m 100 120583m 100 120583m

(a)

CB M

SC 1

CB M

SC 1

clon

e 1CB

MSC

1 cl

one 2

CB M

SC 1

clon

e 3CB

MSC

1 cl

one 4

CB M

SC 1

clon

e 5CB

MSC

1 cl

one 6

CB M

SC 2

CB M

SC 3

CB M

SC 4

CB M

SC 5

USS

C 1

USS

C 1

clone

1U

SSC

1 clo

ne 2

USS

C 1

clone

3U

SSC

1 clo

ne 4

USS

C 1

clone

5U

SSC

1 clo

ne 6

USS

C 1

clone

7U

SSC

2U

SSC

3U

SSC

4U

SSC

5nT

ERA-

2N

HD

F

0

Relat

ive

DLK

-1

002004

12345

20

mRN

A ex

pres

sion

times10minus3

(b)

Figure 1 DLK-1 is a marker to distinguish USSC from CB MSC in cord blood (a) Differentiation capacity of CB-derived stromal cellsBasal characterization of CB-derived stromal cells revealed the adipogenic differentiation as discriminating characteristic between CB MSC(119899 = 29) and USSC (119899 = 61) CB MSC exhibited a high adipogenic differentiation potential comparable to BM MSC while USSC neverformed lipid vacuoles following adipogenic induction as demonstrated by Oil Red O staining after 21 days of differentiation (b) DLK-1expression inversely correlated with the adipogenic differentiation potential DLK-1mRNA expression was significantly higher in USSC andclonal USSC lines compared to CB MSC and corresponding clonal populations The DLK-1 expressing teratocarcinoma cell line nTERA-2was tested as positive control dermal fibroblasts (NHDF) served as biological negative control

USSC possess an increased expansion capacity (Figure 4(a))while CB MSC lead to expansion rates comparable tothose achieved on BM MSC-feeder (data not shown) Toevaluate the impact of DLK-1 co-culture experiments werealso performed with CB MSC overexpressing DLK-1 (CBMSCDLK-1) In detail application of USSC-feeders resultedin higher maximum fold-expansion rates than CB MSC-feeders with regard to total cell count (3505 plusmn 375 versus978 plusmn 052 119899 = 6 each day 14) as well as CD34+ cellcount (306 plusmn 008 versus 158 plusmn 0037 119899 = 3 each day 14)Additionally an efficient expansion of colony-forming units(CFU) was observed only on USSC-feeder (439plusmn150 119899 = 2day 10)

Surprisingly no differences in regard to expansion of totalcells CD34+ cells and CFU were detectable between thenative CB MSC and the CB MSCDLK-1

The DLK-1+ fetal liver subpopulation as describedby Chou and Lodish expressed various haematopoieticgrowth factors such as insulin-like growth factor-2 (IGF-2)

insulin-like growth factor binding protein-1 (IGFBP-1)thrombopoietin (THPO) angiopoetin like-3 (ANGPTL-3)stem cell factor (SCF) and stromal-derived factor-1 (SDF-1)Therefore the expression of these cytokines was evaluated inUSSC CBMSC and BMMSC by quantitative real time PCRThe relative expression of IGF-2 SCF and SDF-1 was higherin CBMSC while USSC expressed significantly higher levelsof IGFBP-1 (357 lowast 10minus3 plusmn 080 lowast 10minus3 119899 = 3) than CB MSC(021 lowast 10minus3 plusmn 016 lowast 10minus3 119899 = 3 119875 = 00147) and BM MSC(004 lowast 10minus3 plusmn 002 lowast 10minus3 119899 = 3 119875 = 00117) THPO andANGPTL-3 were not expressed in any of the cell types tested(Figure 4(b))

Furthermore even when exemplarily enriching the over-expressing CB MSC for the strongest presence of DLK-1 (CBMSCDLK-1 high) by fluorescent activated cell sorting (FACS)no significant differences were observed (Figure 5(a)) Inaccordance with these findings DLK-1 overexpression alsodid not have an impact on the expression levels of theanalyzed haematopoietic cytokines (Figure 5(b))


75 micro m 75 micro m

HepG2

USSCCB MSC

75120583m 50120583m 25120583m

(a)

00

64

64

128

128

192

192

256

256

R1 R1

100

100

100101

101

100 101

101

102

102

102

102

103

103

103

103

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104

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105

00

64

64

128

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192

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101

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102

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100100

101

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R3 R3 R3

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200

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400

400

600

600

800

800

1000

10000

0

200

200

400

400

600

600

800

800

1000

1000

Anti IgG1 Anti DLK-1Positive control

DLK-1 FITC

DLK-1 FITCForward scatter Forward scatterIgG1 FITC

Side

r sca

tter

Intr

acel

lula

rEx

trac

ellu

lar

10A Aminus1

10minus1

100101

100 101

102

102

103

103

10minus1

10minus1

(b)

BM M

SC 1

BM M

SC 2

CB M

SC D

LK-1

+M

ater

nal p

lasm

a

USS

C 1

USS

C 2

USS

C 3

USS

C 4

CB M

SC 1

CB M

SC 2

CB M

SC 3

0

1

2

3

4

5

DLK

-1 (n

gm

L)

(c)

CB M

SC 1

CB M

SC 1

clon

eCB

MSC

4CB

MSC

4 cl

one

CB M

SC 5

CB M

SC 6

CB M

SC 6

clon

e 1U

SSC

1 su

bclo

neU

SSC

2U

SSC

2 clo

neU

SSC

4BM

MSC

Ad

ipoc

ytes

0

Rela

tive

CEBP

am

RNA

expr

essio

n

12345

204060times10minus4

(d)

CB M

SC 1

CB M

SC 4

CB M

SC 5

USS

C 1

USS

C 2

USS

C 3

USS

C 6

Adip

ocyt

es0

Relat

ive

PPA

Rg2

mRN

A ex

pres

sion

12345

50100100times10minus5

(e)

Figure 2 DLK-1 in USSC inhibitor of adipogenesis (a) Representative immunofluorescence staining of CB MSC (119899 = 3) USSC (119899 = 4)and HepG2 USSC revealed a clear DLK-1 expression detected by immunofluorescence while CB MSC were completely negative Thehepatocarcinoma cell line HepG2 served as biological positive control (b) DLK-1 flowcytometry analysis was applied to clearly distinguishbetween intracellular and extracellular expression of DLK-1 protein No DLK-1 expression could be determined in USSC by extracellularstaining After permeabilization a small DLK-1 positive subpopulation was detected in the USSC lines by intracellular staining DLK-1overexpressing CB MSC were used a positive control for extracellular staining (c) By ectodomain shedding the soluble and adipogenicinhibitory DLK-1 protein is cleaved To validate whether USSC express the functional DLK-1 an ELISA was performedThe supernatant of 3days standard cultures of USSC (119899 = 4) and CB MSC (119899 = 3) was analyzed maternal plasma was used as positive control No specific DLK-1release was detected in any of the analyzed USSC or CBMSC questioning function of DLK-1 expression in USSC However a clear secretionof DLK-1 was detected in the CB MSC overexpressing DLK-1 (d) A heterogeneous expression of CEBP120572 (CB MSC 119899 = 4 CB MSC-derivedclones 119899 = 3 USSC 119899 = 2 USSC-derived clones 119899 = 2) could be detected in different USSC and CBMSC cell lines analyzed by real time PCRanalysis (e) Real time PCR analysis revealed a heterogenous expression of PPAR1205742 expression (CB MSC 119899 = 3 USSC 119899 = 4) in the USSCand CB MSC cell lines


CB MSC 1

d0 co

ntro

l

30 m

in D

LK-1

4 h D

LK-1

24 h

DLK

-1

24 h

cont

rol

d0 D

LK-1

+

BM M

SC d

3 os

teog

enic

BM M

SC d

7 os

teog

enic

ERK12

p-ERK12

120573-actin

Figure 3 Regulations of DLK-1 in human and mice are not thesame To evaluate mechanisms induced by DLK-1 in human CBMSC upon exposure to DLK-1Pref1 Western Blot analysis ofERK12 and p-ERK12 was performed (119899 = 3 experiments) CBMSC were treated with conditioned media of either the control cellsor the DLK-1 overexpressing CB MSC CB MSC (d0 control) andDLK-1 overexpressing CB MSC (d0 DLK+) were lysed and westernblot analysis was performed applying full protein lysates after 30minutes 4 hours and 24 hours of incubation with conditionedmedia respectively ERK12 was already highly expressed in the nontreated cells and remained expressed also in the cells treated NoDLK-1Pref1 specific upregulation of p-ERK12 was detected in theCB MSC As biological positive control osteogenic differentiated(day 3 after induction) BMMSCwere used Internal loading control120573-actin

4 Discussion

CB MSC and USSC are two distinct stromal cell populationsin cord blood as confirmed by the different expression profilesin regard to DLK-1 HOX genes and CD146 [4 19]

To further analyze the differences between these cell linesprevious experiments of our group were reanalyzed Thesedata verify a higher haematopoiesis-supporting capacity inUSSC than in CB MSC or BM MSC as well as differences inthe expression of various genes influencing haematopoiesisUnexpectedly the expression of SCF which is regarded asone of the most potent haematopoietic cytokines [23 24]was higher in CB MSC than in USSC or BM MSC Sinceexpansion rates by co-culture on feeder layers of CB MSCwere comparable to those on BM MSC and lower than onUSSC a beneficial effect by SCF possibly is counteracted byanother factor Here as a potential antagonist SDF-1 has tobe discussed Althoughmostly known as chemoattractant forhaematopoietic stem cells [25] it can also induce quiescenceand therefore reduce proliferation in HSC [26] In summarythese results confirm that DLK-1-positive USSC have anincreased haematopoiesis-supporting capacity Whether thisis directly related to DLK-1 as it was described by Moore etal [16] or to other factors (such as IGFBP-1 or SCFSDF-1either secreted or presented via cell-cell contacts) still has tobe clarified In regard to the fact that USSC lack the secretionof the soluble form and overexpression in CB MSC did not

show an effect on the expansion of CD34+-cells a functionalinfluence of DLK-1 could not be verified

Moreover the lack of extracellular expression and secre-tion of DLK-1 by USSC questions what is known about itsfunction in adipogenesis in humans In the murine model(3T3-L1 cells) the soluble part of DLK-1 (Pref1) is thefunctional adipogenic inhibitory protein [20] and the humanform FA1 is highly abundant in serum [27] Therefore it hasbeen postulated that native cord blood-derived stromal cellsmay homogenously lack adipogenic differentiation capacitydue to inhibition by DLK-1 present in the plasma and thatthe adipogenic differentiation potential is artificially evokeddue to dexamethasone in the culture media However sinceall cell lines are generated by the same protocol this wouldnot explain the occurrence of both cell types which hasalso been confirmed by other groups [5] Additionally bothsubpopulations can also be generated from the same cordblood by application of cloning cylinders

It has to be noted that DLK-1 belongs to the Delta-Notchfamily [28] where regulation of transmembrane proteins byendosomal trafficking is one of the key signal mechanismsThis has been studied in detail (for review see Le Borgne et al2005) [29] but whether the intracellular localization of DLK-1 observed in this study can be ascribed to this mechanismremains to be analyzed Another possible explanation forthe lack of extracellular expression of DLK-1 might bean alternative splice variant of the DLK-1 protein since 4alternatively spliced DLK-1 Pref1 variants have already beendescribed in murine 3T3-L1 cells with only the 50 kDa formbeing functional [21] So far studies of humanDLK-1 cleavagerevealed only one soluble form with different molecularweights due to different stages of glycosylation [18 30]

However the molecular changes induced by DLK-1Pref1in human MSC analyzed here are not consistent with themechanisms described for several murine model systems(namely PPAR1205742 CEBP120572 expression and ERK12 phospho-rylation) and relativize the hypothesis for DLK-1 to be themain regulator of adipogenesis in human cord blood-derivedstromal cells

Interestingly IGFBP-1 which is higher expressed inUSSC is also known to be involved in the regulation ofadipogenesis Briefly IGFs were shown to be equipotent toinsulin in regulating adipogenic differentiation [31 32] andthe binding of IGF1 and IGBFPs leads to stable complexesthat reduce the availability of free IGF1 As described byNueda et al [33] this complex can interact with the protease-target region of DLK-1 and further minimize the adipogenicpotential of a cell by activation of ERK12

5 Conclusion

Thedata presented here allow further characterization of cordblood-derived stromal cells USSC and CB MSC based on atotal of 90 established and characterized cell lines (as of todaya total of 394 cell lines has been generated out of 1009 cordblood units)

According to their distinct properties most prominentlythe presence of longer telomeres associated with a higher


Total cells (fold)

0 1 2 3 4 5 6 7 8 9 10 11121314150

10

20

30

40

CB MSCUSSC

(days) (days) (days)

0 1 2 3 4 5 6 7 8 9 10 1112131415 0 1 2 3 4 5 6 7 8 9 10 1112131415

Tota

l cel

ls (fo

ld)

0

1

2

3

CFU (fold)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)

CD34+

-cel

ls (fo

ld)

CB MSCDLKCB MSCUSSC

CB MSCDLKCB MSCUSSC

CB MSCDLK

(a)

Gene expression in relation to GAPDH

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

IGF-2 IGFBP-1 SCF SDF-1 THPO ANGPTL-3

Relat

ive g

ene e

xpre

ssio

n

0

5

10

1550


lowast

lowast

(b)

Figure 4 Haematopoiesis-supporting capacity of USSC and CB MSC (a) Representative co-culture of CD34+-cells on feeders of USSC CBMSC and CB MSC overexpressing DLK-1 (CB MSCDLK) in multiple independent wells (119899 = 6) With regards to total cell count expansionof CD34+-cells and colony-forming units (CFU) co-culture on a USSC-feeder resulted in higher expansion rates than on CB MSC-feederWithin 14 days total cell count increased significantly stronger on USSC-feeder (by factor 3505 plusmn 375) than on CB MSC-feeder (factor1009 plusmn 037 119875 lt 00001) Expansion of total CD34+-cells also resulted in significantly higher expansion on USSC (306 plusmn 008-fold) than onCB MSC (157 plusmn 002 fold 119875 lt 00001) For colony-forming units cells cultured on USSC-feeder showed a higher expansion rate on day 10and 14 (up to 439 plusmn 150-fold versus 106 plusmn 003-fold 119875 = 01562) Overexpression of DLK-1 in the CBMSC line did not have an influence onexpansion rates as the results for CB MSC and CB MSCDLK were approximately identical (b) Expression levels of haematopoietic cytokines(IGF-2 IGFBP-1 SCF SDF-1 THPO ANGPTL-3) in USSC CB MSC and BM MSC were analyzed by real time PCR The expression ofinsulin-like growth factor binding protein (IGFBP-1) was highest in USSC (119899 = 3) while stromal-derived factor 1 (SDF-1) expression washigher in CB MSC (119899 = 3) and BM MSC (119899 = 3) compared to USSC Thrombopoietin (THPO) and angiopoietin-like 3 (ANGPTL-3) werenot detectable

proliferative potential and aHOX-negative expression profileUSSC are regarded as being of a biologically younger stateAnother functional difference is the lacking adipogenic dif-ferentiation potential of USSC which was discussed as beingpossibly related to the expression of DLK-1 [3 4]

In addition to these features the data provided heredemonstrate that USSC also can be distinguished fromCB MSC and BM MSC by their higher haematopoiesis-supporting capacity in co-culture experiments In contrastCBMSC show properties similar to stromal cells found in the

adult bone marrow for example lack of DLK-1 expression inassociation with a high adipogenic capacity and HOX-geneexpression (although they still possess a higher proliferativepotential and have longer telomeres than BMMSC)

These features substantiate the assumption that amongneonatal stromal cells CB MSC are closer related to bonemarrow stromal cells than USSC As an amendment to ourdefinition of USSC DLK-1 remains a useful marker to distin-guishUSSC fromCBMSCbut since the data presented in thisstudy confirm the absence of extracellular DLK-1 expression


Total cells (fold)

0 1 2 3 4 5 6 7 8 9 1011121314150

10

20

30

40

CB MSC


0 1 2 3 4 5 6 7 8 9 101112131415 0 1 2 3 4 5 6 7 8 9 101112131415

Tota

l cel

ls (fo

ld)

CFU (fold)

0

1

2

3

CD34+

-cel

ls (fo

ld)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)

CB MSCDLK highCB MSCCB MSCDLK high

CB MSCCB MSCDLK high

(a)

Gene expression in CB MSC overexpressing DLK-1

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

0


Gen

e exp

ress

ion

in re

latio

n to

RPL

13a

01

02

035

101520

2004006008001000times10minus2

(b)

Figure 5 Effect of DLK-1-overexpression on the haematopoiesis-supporting capacity of CBMSC (a) Exemplarily co-culture of CD34+-cellson feeders of CB MSC and FACS-sorted CB MSC strongly overexpressing DLK-1 (CB MSCDLK high) in multiple independent wells (119899 = 4)Within 14 days overexpression of DLK-1 did not have a beneficial effect on expansion rates of total cells CD34+-cells or CFU In accordanceto former results for native CB MSC only a weak expansion of total cells was observed while amounts of total CD34+-cells as well as CFUslightly declined over time (b) Expression levels of haematopoietic cytokines (IGF-2 IGFBP-1 SCF SDF-1THPOANGPTL-3) were analyzedby real time PCR exemplarily for a CB MSC line in native form after induced overexpression of DLK-1 (CB MSCDLK) as well as after furtherFACS-sorting for cells with high DLK-1 expression (CB MSCDLK high) respectively No significant differences in expression levels could bedetected and results were in accordance with former data as presented in Figure 4(b)

and the lack of protein release into the supernatant its specificrole remains unclear Accordingly an adipogenic inhibitoryfunction based on DLK-1 alone in USSC is unlikely althougha functional interaction with other factors such as IGFBP1needs to be analyzed The same accounts for the effect ofDLK-1 on haematopoiesis since overexpression of DLK-1 inCB MSC did not result in an increased supportive capacitycomparable to USSC

Therefore further analyses of USSC are required to testwhether they have yet unknown qualities beyond those of

ldquoclassicrdquo MSC and if this is related to a different origin ofUSSC and CBMSC respectively Additionally whether regu-lations during adipogenesis in human andmice are subject tothe same molecular mechanisms especially in regard to theimportance of DLK-1 has to be clarified

Conflict of Interests

There is no financial interest to disclose


Authorsrsquo Contribution

S M Kluth and T F Radke contributed equally

Acknowledgments

The authors would like to thank Dr Stefanie Liedtke andEvaMaria Freytag for reading the paper and Katharina RabaCore Flow Cytometry Facility of the University MedicalCenter Duesseldorf for technical support in fluorescenceactivated cell sorting

References

[1] G Kogler T F Radke A Lefort et al ldquoCytokine productionand hematopoiesis supporting activity of cord blood-derivedunrestricted somatic stem cellsrdquo Experimental Hematology vol33 no 5 pp 573ndash583 2005

[2] G Kogler S Sensken J A Airey et al ldquoA new human somaticstem cell from placental cord blood with intrinsic pluripotentdifferentiation potentialrdquo Journal of ExperimentalMedicine vol200 no 2 pp 123ndash135 2004

[3] S M Kluth A Buchheiser A P Houben et al ldquoDLK-1 asa marker to distinguish unrestricted somatic stem cells andmesenchymal stromal cells in cord bloodrdquo Stem Cells andDevelopment vol 19 no 10 pp 1471ndash1483 2010

[4] S Liedtke A Buchheiser J Bosch et al ldquoThe HOX Code as aldquobiological fingerprintrdquo to distinguish functionally distinct stemcell populations derived from cord bloodrdquo Stem Cell Researchvol 5 no 1 pp 40ndash50 2010

[5] Y J Chang D T B Shih C P Tseng T B Hsieh D C Leeand S M Hwang ldquoDisparate mesenchyme-lineage tendenciesin mesenchymal stem cells from human bone marrow andumbilical cord bloodrdquo Stem Cells vol 24 no 3 pp 679ndash6852006

[6] D T Covas R A Panepucci A M Fontes et al ldquoMultipo-tent mesenchymal stromal cells obtained from diverse humantissues share functional properties and gene-expression profilewith CD146+ perivascular cells and fibroblastsrdquo ExperimentalHematology vol 36 no 5 pp 642ndash654 2008

[7] S E Yang C W Ha M H Jung et al ldquoMesenchymalstemprogenitor cells developed in cultures from UC bloodrdquoCytotherapy vol 6 no 5 pp 476ndash486 2004

[8] M Karagianni I Brinkmann S Kinzebach et al ldquoA compara-tive analysis of the adipogenic potential in humanmesenchymalstromal cells from cord blood and other sourcesrdquo Cytotherapyvol 15 no 1 pp 76ndash88 2012

[9] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006

[10] A I Caplan ldquoMesenchymal stem cellsrdquo Journal of OrthopaedicResearch vol 9 no 5 pp 641ndash650 1991

[11] P Bianco P G Robey I Saggio andM Riminucci ldquolsquoMesenchy-malrsquo stem cells in human bone marrow (Skeletal Stem Cells) acritical discussion of their nature identity and significance inincurable skeletal diseaserdquo Human Gene Therapy vol 21 no 9pp 1057ndash1066 2010

[12] C M Smas and H S Sul ldquoCharacterization of pref-1 andits inhibitory role in adipocyte differentiationrdquo InternationalJournal of Obesity vol 20 supplement 3 pp S65ndashS72 1996

[13] C M Smas and H S Sul ldquoMolecular mechanisms of adipocytedifferentiation and inhibitory action of pref-1rdquo Critical Reviewsin Eukaryotic Gene Expression vol 7 no 4 pp 281ndash298 1997

[14] Y Wang and H S Sul ldquoPref-1 regulates mesenchymal cell com-mitment and differentiation through Sox9rdquo Cell Metabolismvol 9 no 3 pp 287ndash302 2009

[15] S Chou and H F Lodish ldquoFetal liver hepatic progenitorsare supportive stromal cells for hematopoietic stem cellsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 17 pp 7799ndash7804 2010

[16] K A Moore B Pytowski L Witte D Hicklin and I R Lemis-chka ldquoHematopoietic activity of a stromal cell transmembraneprotein containing epidermal growth factor-like repeat motifsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 94 no 8 pp 4011ndash4016 1997

[17] N Ohno A Izawa M Hattori R Kageyama and T Sudoldquodlk inhibits stem cell factor-induced colony formation ofmurine hematopoietic progenitors Hes-1-independent effectrdquoStem Cells vol 19 no 1 pp 71ndash79 2001

[18] C H Jensen T N Krogh P Hojrup et al ldquoProtein structureof fetal antigen 1 (FA1)mdasha novel circulating human epidermal-growth-factor-like protein expressed in neuroendocrine tumorsand its relation to the gene products of dlk and pG2rdquo EuropeanJournal of Biochemistry vol 225 no 1 pp 83ndash92 1994

[19] J Bosch A P Houben T F Radke et al ldquoDistinct differentia-tion potential of ldquoMSCrdquo derived from cord blood and umbilicalcord are cord-derived cells true mesenchymal stromal cellsrdquoStem Cells and Development vol 21 no 11 pp 1977ndash1988 2012

[20] C M Smas L Chen and H S Sul ldquoCleavage of membrane-associated pref-1 generates a soluble inhibitor of adipocytedifferentiationrdquoMolecular and Cellular Biology vol 17 no 2 pp977ndash988 1997

[21] B Mei L Zhao L Chen and H S Sul ldquoOnly the large solubleform of preadipocyte factor-1 (Pref-1) but not the small solubleand membrane forms inhibits adipocyte differentiation role ofalternative splicingrdquo Biochemical Journal vol 364 part 1 pp137ndash144 2002

[22] K S Jeltsch T F Radke S Laufs et al ldquoUnrestricted somaticstem cells Interaction with CD34+ cells in vitro and in vivoexpression of homing genes and exclusion of tumorigenicpotentialrdquo Cytotherapy vol 13 no 3 pp 357ndash365 2011

[23] D Kent M Copley C Benz B Dykstra M Bowie and CEaves ldquoRegulation of hematopoietic stem cells by the steelfactorKIT signaling pathwayrdquo Clinical Cancer Research vol 14no 7 pp 1926ndash1930 2008

[24] K Kita J O Lee C C Finnerty and D N Herndon ldquoCordblood-derived hematopoietic stemprogenitor cells currentchallenges in engraftment infection and ex vivo expansionrdquoStem Cells International vol 2011 Article ID 276193 8 pages2011

[25] R Mohle F Bautz S Rafii M A S Moore W Bruggerand L Kanz ldquoThe chemokine receptor CXCR-4 is expressedon CD34+ hematopoietic progenitors and leukemic cells andmediates transendothelial migration induced by stromal cell-derived factor-1rdquo Blood vol 91 no 12 pp 4523ndash4530 1998

[26] Y Nie Y C Han and Y R Zou ldquoCXCR4 is required forthe quiescence of primitive hematopoietic cellsrdquo Journal ofExperimental Medicine vol 205 no 4 pp 777ndash783 2008

[27] BMAbdallahMDing CH Jensen et al ldquoDlk1FA1 is a novelendocrine regulator of bone and fat mass and its serum level ismodulated by growth hormonerdquo Endocrinology vol 148 no 7pp 3111ndash3121 2007


[28] S J Bray S Takada E Harrison S C Shen andA C Ferguson-Smith ldquoThe atypical mammalian ligand Delta-like homologue1 (Dlk1) can regulate Notch signalling in Drosophilardquo BMCDevelopmental Biology vol 8 article 11 2008

[29] R Le Borgne ldquoRegulation of Notch signalling by endocytosisand endosomal sortingrdquo Current Opinion in Cell Biology vol18 no 2 pp 213ndash222 2006

[30] B M Abdallah C H Jensen G Gutierrez R G Q Leslie TG Jensen and M Kassem ldquoRegulation of human skeletal stemcells differentiation byDlk1Pref-1rdquo Journal of Bone andMineralResearch vol 19 no 5 pp 841ndash852 2004

[31] S Guller M Sonenberg K Y Wu P Szabo and R E CorinldquoGrowth hormone-dependent events in the adipose differenti-ation of 3T3-F442A fibroblasts modulation of macromolecularsynthesisrdquo Endocrinology vol 125 no 5 pp 2360ndash2367 1989

[32] P J Smith L S Wise R Berkowitz C Wan and C S RubinldquoInsulin-like growth factor-I is an essential regulator of thedifferentiation of 3T3-L1 adipocytesrdquo The Journal of BiologicalChemistry vol 263 no 19 pp 9402ndash9408 1988

[33] M L Nueda J J Garcıa-Ramırez J Laborda and V Baladronldquodlk1 specifically interacts with insulin-like growth factor bind-ing protein 1 to modulate adipogenesis of 3T3-L1 cellsrdquo Journalof Molecular Biology vol 379 no 3 pp 428ndash442 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


controversially within the last years [11] Although manycells of different origin are referred to as MSC characterizedby similar properties like morphology immunophenotypeandmandatory basic differentiation potential (chondrogenicosteogenic and adipogenic) subtle differences can be notedIn this context a potential relation of different MSC-likecells has to be discussed especially since evidence arose thatUSSC can be transformed into cells with full adipogenicdifferentiation capacity (manuscript in revision Liedtkeet al 2013) Therefore although less specific MSC as abbre-viation for ldquomultipotent stromal cellsrdquo might be more appro-priate for these cells which in turn would include nativeUSSC

The discrimination of USSC fromCBMSC by the expres-sion ofDLK-1 and the inverse correlation with the adipogenicdifferentiation capacity was the first evidence for two distinctstromal cell populations in cord blood [3] The function ofDLK-1 during adipogenic differentiation is well described[12ndash14] Besides the inhibitory effect on adipogenesis DLK-1is known to play a major role during differentiation of hepa-toblasts and considered a potent regulator of haematopoiesis[15ndash17]

The aim of this study was to further assess the functionof DLK-1 and its soluble form known as fetal antigen1 (FA1) [18] in regard to a potential biological diversitybetween USSC and CB MSC which might have impact onfuture clinical useTherefore the haematopoiesis-supportingcapacity of USSC andCBMSC in vitrowas compared directlyby co-culture experiments Additionally the expression ofhaematopoiesis relevant cytokines on molecular biologicallevel was analyzed

2 Materials and Methods

21 Generation and Expansion of CB Stromal Cells CB wascollected from umbilical cord vein with informed consentof the mother The bulk cell lines (USSC 119899 = 61 CB MSC119899 = 29) used in this study were isolated from 90 differentdonors USSC and CB MSC were generated by the samemethod as described previously [3] a classification intothe two subtypes was only possible after examination ofthe adipogenic differentiation potential and HOX expres-sion profile of each cell line Briefly mononuclear cells(MNC) of one cord blood were obtained by Ficoll gradi-ent separation (density 1077 gcm3 Biochrom AG BerlinGermany) followed by lysis of remaining erythrocytes withammonium chloride (pharmacy of the university medicalcentre Duesseldorf) 5ndash7 lowast 106 CB MNCmL were culturedin DMEM low glucose (Lonza Inc Allendale NJ USA)with 30 FCS (PerbioFisher Scientific Bonn Germany) 1lowast10minus7Mdexamethasone (Sigma-Aldrich StLouisMO USA)

penicillinstreptomycin and L-glutamine (PSG all LonzaInc) until detection of adherent growing coloniesThese cellswere expanded without dexamethasone in a closed systemapplying cell stacks (Corning Inc Corning NY USA)incubated at 37∘C in 5CO

2in a humidified atmosphere and

defined as one single polyclonal bulk cell line Reaching 80confluence cells were washed with phosphate buffered saline

(PBS Serag-Wiessner KG Naila Germany) detached with025 trypsin (Lonza Inc) and replated 1 3

22 Generation of Bone Marrow Stromal Cells BM MSClines were generated from bone marrow of healthy donorsas described previously [1] MNC were isolated and plated inDMEM low glucose with 30 FCS and PSG until detectionof adherent growing colonies

23 Biological Controls Human skin fibroblasts (NHDF)were provided by the Department of Dermatology HeinrichHeineUniversity Duesseldorf HepG2 and nTERA-2 cell lineswere purchased from ATCC and cultured according to themanufacturerrsquos instructions

24 Generation of Cell Clones Using the AVISO CellCelectorClonal populations were obtained from established cell linesin early passages applying the AVISO CellCelector (ALSGmbH Jena Germany) as described previously [3] Brieflycells of one single bulk cell line were plated at low density(166 cellscm2) in 6-well cell culture plates (Corning Inc)and after allowing the cells to get adherent again distinctsingle cells were selected picked and transported to a defineddestination well of a 96-well cell culture plate (Corning Inc)and cultured with preconditioned medium Pictures weretaken before and after each picking process to documentsuccessful single cell selection The term ldquoUSSC 1 clone 1rdquodefines the first clonal population isolated fromUSSC cell line1 this nomenclature also applies to CB MSC-derived clonalpopulations All cell lines were used between passage 5 and 8

25 In Vitro Differentiation into Adipocytes Differentiationinto adipocytes was performed as described previously [2]Adipogenic differentiation was induced applying DMEMhigh glucose (45 gL Lonza Inc) 10 FCS PSG 1 lowast 10minus6Mdexamethasone (water soluble cell culture tested) 02mMindomethacin 01mgmL insulin and 1mM 3-Isobutyl-methylxanthine (all Sigma-Aldrich) Media was changedtwice a week alternating with cultivation media containing001mgmL insulin Lipid vacuoles were stained with Oil RedO (Sigma Aldrich) after 21 days As negative control the cellswere cultured in DMEM low glucose (1 gL Lonza Inc) 10FCS and PSG

26 Co-Cultures of CD34-Selected Cells on Stromal FeederLayers Stromal feeders were prepared in 24-well plates byseeding 5 lowast 104 cells (USSC CB MSC) in standard culturemedia After 24ndash48 h when density was app 80ndash90 agrowth arrest was induced by irradiation (30 gy cobaltsource)

Cord blood-derived CD34+-cells were isolated from thefraction of the mononucleated cells following Ficoll-gradientcentrifugation and erythrocyte lysis by ammonium chlorideusing a CD34 progenitor kit for magnetic-activated cell sort-ing (MACS) and Midi-MACS magnets with LS columns (allMiltenyi Biotec Bergisch Gladbach Germany) according tothe manufacturerrsquos instructions Isolated CD34+-cells were
























164 bp 60∘C



184 bp 60∘C



119 bp 60∘C



238 bp 60∘C


NM 0010071394NM 0011275981


143 bp 60∘C



153 bp 60∘C



193 bp 60∘C



136 bp 60∘C



210 bp 55∘C


NM 1991683NM 0006095

NM 0010338862NM 0011781341


110 bp 60∘C



108 bp 60∘C



3 Results






Farmingdale NY USA








sc-2005 Santa Cruz












USSCCB MSC BM MSCAd

ipog

enic

100 120583m 100 120583m 100 120583m

(a)

CB M

SC 1

CB M

SC 1

clon

e 1CB

MSC

1 cl

one 2

CB M

SC 1

clon

e 3CB

MSC

1 cl

one 4

CB M

SC 1

clon

e 5CB

MSC

1 cl

one 6

CB M

SC 2

CB M

SC 3

CB M

SC 4

CB M

SC 5

USS

C 1

USS

C 1

clone

1U

SSC

1 clo

ne 2

USS

C 1

clone

3U

SSC

1 clo

ne 4

USS

C 1

clone

5U

SSC

1 clo

ne 6

USS

C 1

clone

7U

SSC

2U

SSC

3U

SSC

4U

SSC

5nT

ERA-

2N

HD

F

0

Relat

ive

DLK

-1

002004

12345

20

mRN

A ex

pres

sion

times10minus3

(b)









HepG2

USSCCB MSC

75120583m 50120583m 25120583m

(a)

00

64

64

128

128

192

192

256

256

R1 R1

100

100

100101

101

100 101

101

102

102

102

102

103

103

103

103

104

104

105

105

00

64

64

128

128

192

192

256

256 100100

101

101

102

102

103

103

104

104

105

105

100100

101

101

102

102

103

103

104

104

105

105

R3 R3 R3

00

200

200

400

400

600

600

800

800

1000

10000

0

200

200

400

400

600

600

800

800

1000

1000


DLK-1 FITC


Side

r sca

tter

Intr

acel

lula

rEx

trac

ellu

lar

10A Aminus1

10minus1

100101

100 101

102

102

103

103

10minus1

10minus1

(b)

BM M

SC 1

BM M

SC 2

CB M

SC D

LK-1

+M

ater

nal p

lasm

a

USS

C 1

USS

C 2

USS

C 3

USS

C 4

CB M

SC 1

CB M

SC 2

CB M

SC 3

0

1

2

3

4

5

DLK

-1 (n

gm

L)

(c)

CB M

SC 1

CB M

SC 1

clon

eCB

MSC

4CB

MSC

4 cl

one

CB M

SC 5

CB M

SC 6

CB M

SC 6

clon

e 1U

SSC

1 su

bclo

neU

SSC

2U

SSC

2 clo

neU

SSC

4BM

MSC

Ad

ipoc

ytes

0

Rela

tive

CEBP

am

RNA

expr

essio

n

12345

204060times10minus4

(d)

CB M

SC 1

CB M

SC 4

CB M

SC 5

USS

C 1

USS

C 2

USS

C 3

USS

C 6

Adip

ocyt

es0

Relat

ive

PPA

Rg2

mRN

A ex

pres

sion

12345


(e)



CB MSC 1

d0 co

ntro

l

30 m

in D

LK-1

4 h D

LK-1

24 h

DLK

-1

24 h

cont

rol

d0 D

LK-1

+

BM M

SC d

3 os

teog

enic

BM M

SC d

7 os

teog

enic

ERK12

p-ERK12

120573-actin


4 Discussion








5 Conclusion




Total cells (fold)

0 1 2 3 4 5 6 7 8 9 10 11121314150

10

20

30

40

CB MSCUSSC


0 1 2 3 4 5 6 7 8 9 10 1112131415 0 1 2 3 4 5 6 7 8 9 10 1112131415

Tota

l cel

ls (fo

ld)

0

1

2

3

CFU (fold)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)

CD34+

-cel

ls (fo

ld)

CB MSCDLKCB MSCUSSC

CB MSCDLKCB MSCUSSC

CB MSCDLK

(a)


USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC


Relat

ive g

ene e

xpre

ssio

n

0

5

10

1550


lowast

lowast

(b)







Total cells (fold)

0 1 2 3 4 5 6 7 8 9 1011121314150

10

20

30

40

CB MSC


0 1 2 3 4 5 6 7 8 9 101112131415 0 1 2 3 4 5 6 7 8 9 101112131415

Tota

l cel

ls (fo

ld)

CFU (fold)

0

1

2

3

CD34+

-cel

ls (fo

ld)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)



(a)


CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

0


Gen

e exp

ress

ion

in re

latio

n to

RPL

13a

01

02

035

101520

2004006008001000times10minus2

(b)










Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























164 bp 60∘C



184 bp 60∘C



119 bp 60∘C



238 bp 60∘C


NM 0010071394NM 0011275981


143 bp 60∘C



153 bp 60∘C



193 bp 60∘C



136 bp 60∘C



210 bp 55∘C


NM 1991683NM 0006095

NM 0010338862NM 0011781341


110 bp 60∘C



108 bp 60∘C



3 Results






Farmingdale NY USA








sc-2005 Santa Cruz












USSCCB MSC BM MSCAd

ipog

enic

100 120583m 100 120583m 100 120583m

(a)

CB M

SC 1

CB M

SC 1

clon

e 1CB

MSC

1 cl

one 2

CB M

SC 1

clon

e 3CB

MSC

1 cl

one 4

CB M

SC 1

clon

e 5CB

MSC

1 cl

one 6

CB M

SC 2

CB M

SC 3

CB M

SC 4

CB M

SC 5

USS

C 1

USS

C 1

clone

1U

SSC

1 clo

ne 2

USS

C 1

clone

3U

SSC

1 clo

ne 4

USS

C 1

clone

5U

SSC

1 clo

ne 6

USS

C 1

clone

7U

SSC

2U

SSC

3U

SSC

4U

SSC

5nT

ERA-

2N

HD

F

0

Relat

ive

DLK

-1

002004

12345

20

mRN

A ex

pres

sion

times10minus3

(b)









HepG2

USSCCB MSC

75120583m 50120583m 25120583m

(a)

00

64

64

128

128

192

192

256

256

R1 R1

100

100

100101

101

100 101

101

102

102

102

102

103

103

103

103

104

104

105

105

00

64

64

128

128

192

192

256

256 100100

101

101

102

102

103

103

104

104

105

105

100100

101

101

102

102

103

103

104

104

105

105

R3 R3 R3

00

200

200

400

400

600

600

800

800

1000

10000

0

200

200

400

400

600

600

800

800

1000

1000


DLK-1 FITC


Side

r sca

tter

Intr

acel

lula

rEx

trac

ellu

lar

10A Aminus1

10minus1

100101

100 101

102

102

103

103

10minus1

10minus1

(b)

BM M

SC 1

BM M

SC 2

CB M

SC D

LK-1

+M

ater

nal p

lasm

a

USS

C 1

USS

C 2

USS

C 3

USS

C 4

CB M

SC 1

CB M

SC 2

CB M

SC 3

0

1

2

3

4

5

DLK

-1 (n

gm

L)

(c)

CB M

SC 1

CB M

SC 1

clon

eCB

MSC

4CB

MSC

4 cl

one

CB M

SC 5

CB M

SC 6

CB M

SC 6

clon

e 1U

SSC

1 su

bclo

neU

SSC

2U

SSC

2 clo

neU

SSC

4BM

MSC

Ad

ipoc

ytes

0

Rela

tive

CEBP

am

RNA

expr

essio

n

12345

204060times10minus4

(d)

CB M

SC 1

CB M

SC 4

CB M

SC 5

USS

C 1

USS

C 2

USS

C 3

USS

C 6

Adip

ocyt

es0

Relat

ive

PPA

Rg2

mRN

A ex

pres

sion

12345


(e)



CB MSC 1

d0 co

ntro

l

30 m

in D

LK-1

4 h D

LK-1

24 h

DLK

-1

24 h

cont

rol

d0 D

LK-1

+

BM M

SC d

3 os

teog

enic

BM M

SC d

7 os

teog

enic

ERK12

p-ERK12

120573-actin


4 Discussion








5 Conclusion




Total cells (fold)

0 1 2 3 4 5 6 7 8 9 10 11121314150

10

20

30

40

CB MSCUSSC


0 1 2 3 4 5 6 7 8 9 10 1112131415 0 1 2 3 4 5 6 7 8 9 10 1112131415

Tota

l cel

ls (fo

ld)

0

1

2

3

CFU (fold)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)

CD34+

-cel

ls (fo

ld)

CB MSCDLKCB MSCUSSC

CB MSCDLKCB MSCUSSC

CB MSCDLK

(a)


USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC


Relat

ive g

ene e

xpre

ssio

n

0

5

10

1550


lowast

lowast

(b)







Total cells (fold)

0 1 2 3 4 5 6 7 8 9 1011121314150

10

20

30

40

CB MSC


0 1 2 3 4 5 6 7 8 9 101112131415 0 1 2 3 4 5 6 7 8 9 101112131415

Tota

l cel

ls (fo

ld)

CFU (fold)

0

1

2

3

CD34+

-cel

ls (fo

ld)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)



(a)


CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

0


Gen

e exp

ress

ion

in re

latio

n to

RPL

13a

01

02

035

101520

2004006008001000times10minus2

(b)










Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Farmingdale NY USA








sc-2005 Santa Cruz












USSCCB MSC BM MSCAd

ipog

enic

100 120583m 100 120583m 100 120583m

(a)

CB M

SC 1

CB M

SC 1

clon

e 1CB

MSC

1 cl

one 2

CB M

SC 1

clon

e 3CB

MSC

1 cl

one 4

CB M

SC 1

clon

e 5CB

MSC

1 cl

one 6

CB M

SC 2

CB M

SC 3

CB M

SC 4

CB M

SC 5

USS

C 1

USS

C 1

clone

1U

SSC

1 clo

ne 2

USS

C 1

clone

3U

SSC

1 clo

ne 4

USS

C 1

clone

5U

SSC

1 clo

ne 6

USS

C 1

clone

7U

SSC

2U

SSC

3U

SSC

4U

SSC

5nT

ERA-

2N

HD

F

0

Relat

ive

DLK

-1

002004

12345

20

mRN

A ex

pres

sion

times10minus3

(b)









HepG2

USSCCB MSC

75120583m 50120583m 25120583m

(a)

00

64

64

128

128

192

192

256

256

R1 R1

100

100

100101

101

100 101

101

102

102

102

102

103

103

103

103

104

104

105

105

00

64

64

128

128

192

192

256

256 100100

101

101

102

102

103

103

104

104

105

105

100100

101

101

102

102

103

103

104

104

105

105

R3 R3 R3

00

200

200

400

400

600

600

800

800

1000

10000

0

200

200

400

400

600

600

800

800

1000

1000


DLK-1 FITC


Side

r sca

tter

Intr

acel

lula

rEx

trac

ellu

lar

10A Aminus1

10minus1

100101

100 101

102

102

103

103

10minus1

10minus1

(b)

BM M

SC 1

BM M

SC 2

CB M

SC D

LK-1

+M

ater

nal p

lasm

a

USS

C 1

USS

C 2

USS

C 3

USS

C 4

CB M

SC 1

CB M

SC 2

CB M

SC 3

0

1

2

3

4

5

DLK

-1 (n

gm

L)

(c)

CB M

SC 1

CB M

SC 1

clon

eCB

MSC

4CB

MSC

4 cl

one

CB M

SC 5

CB M

SC 6

CB M

SC 6

clon

e 1U

SSC

1 su

bclo

neU

SSC

2U

SSC

2 clo

neU

SSC

4BM

MSC

Ad

ipoc

ytes

0

Rela

tive

CEBP

am

RNA

expr

essio

n

12345

204060times10minus4

(d)

CB M

SC 1

CB M

SC 4

CB M

SC 5

USS

C 1

USS

C 2

USS

C 3

USS

C 6

Adip

ocyt

es0

Relat

ive

PPA

Rg2

mRN

A ex

pres

sion

12345


(e)



CB MSC 1

d0 co

ntro

l

30 m

in D

LK-1

4 h D

LK-1

24 h

DLK

-1

24 h

cont

rol

d0 D

LK-1

+

BM M

SC d

3 os

teog

enic

BM M

SC d

7 os

teog

enic

ERK12

p-ERK12

120573-actin


4 Discussion








5 Conclusion




Total cells (fold)

0 1 2 3 4 5 6 7 8 9 10 11121314150

10

20

30

40

CB MSCUSSC


0 1 2 3 4 5 6 7 8 9 10 1112131415 0 1 2 3 4 5 6 7 8 9 10 1112131415

Tota

l cel

ls (fo

ld)

0

1

2

3

CFU (fold)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)

CD34+

-cel

ls (fo

ld)

CB MSCDLKCB MSCUSSC

CB MSCDLKCB MSCUSSC

CB MSCDLK

(a)


USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC


Relat

ive g

ene e

xpre

ssio

n

0

5

10

1550


lowast

lowast

(b)







Total cells (fold)

0 1 2 3 4 5 6 7 8 9 1011121314150

10

20

30

40

CB MSC


0 1 2 3 4 5 6 7 8 9 101112131415 0 1 2 3 4 5 6 7 8 9 101112131415

Tota

l cel

ls (fo

ld)

CFU (fold)

0

1

2

3

CD34+

-cel

ls (fo

ld)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)



(a)


CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

0


Gen

e exp

ress

ion

in re

latio

n to

RPL

13a

01

02

035

101520

2004006008001000times10minus2

(b)










Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


USSCCB MSC BM MSCAd

ipog

enic

100 120583m 100 120583m 100 120583m

(a)

CB M

SC 1

CB M

SC 1

clon

e 1CB

MSC

1 cl

one 2

CB M

SC 1

clon

e 3CB

MSC

1 cl

one 4

CB M

SC 1

clon

e 5CB

MSC

1 cl

one 6

CB M

SC 2

CB M

SC 3

CB M

SC 4

CB M

SC 5

USS

C 1

USS

C 1

clone

1U

SSC

1 clo

ne 2

USS

C 1

clone

3U

SSC

1 clo

ne 4

USS

C 1

clone

5U

SSC

1 clo

ne 6

USS

C 1

clone

7U

SSC

2U

SSC

3U

SSC

4U

SSC

5nT

ERA-

2N

HD

F

0

Relat

ive

DLK

-1

002004

12345

20

mRN

A ex

pres

sion

times10minus3

(b)









HepG2

USSCCB MSC

75120583m 50120583m 25120583m

(a)

00

64

64

128

128

192

192

256

256

R1 R1

100

100

100101

101

100 101

101

102

102

102

102

103

103

103

103

104

104

105

105

00

64

64

128

128

192

192

256

256 100100

101

101

102

102

103

103

104

104

105

105

100100

101

101

102

102

103

103

104

104

105

105

R3 R3 R3

00

200

200

400

400

600

600

800

800

1000

10000

0

200

200

400

400

600

600

800

800

1000

1000


DLK-1 FITC


Side

r sca

tter

Intr

acel

lula

rEx

trac

ellu

lar

10A Aminus1

10minus1

100101

100 101

102

102

103

103

10minus1

10minus1

(b)

BM M

SC 1

BM M

SC 2

CB M

SC D

LK-1

+M

ater

nal p

lasm

a

USS

C 1

USS

C 2

USS

C 3

USS

C 4

CB M

SC 1

CB M

SC 2

CB M

SC 3

0

1

2

3

4

5

DLK

-1 (n

gm

L)

(c)

CB M

SC 1

CB M

SC 1

clon

eCB

MSC

4CB

MSC

4 cl

one

CB M

SC 5

CB M

SC 6

CB M

SC 6

clon

e 1U

SSC

1 su

bclo

neU

SSC

2U

SSC

2 clo

neU

SSC

4BM

MSC

Ad

ipoc

ytes

0

Rela

tive

CEBP

am

RNA

expr

essio

n

12345

204060times10minus4

(d)

CB M

SC 1

CB M

SC 4

CB M

SC 5

USS

C 1

USS

C 2

USS

C 3

USS

C 6

Adip

ocyt

es0

Relat

ive

PPA

Rg2

mRN

A ex

pres

sion

12345


(e)



CB MSC 1

d0 co

ntro

l

30 m

in D

LK-1

4 h D

LK-1

24 h

DLK

-1

24 h

cont

rol

d0 D

LK-1

+

BM M

SC d

3 os

teog

enic

BM M

SC d

7 os

teog

enic

ERK12

p-ERK12

120573-actin


4 Discussion








5 Conclusion




Total cells (fold)

0 1 2 3 4 5 6 7 8 9 10 11121314150

10

20

30

40

CB MSCUSSC


0 1 2 3 4 5 6 7 8 9 10 1112131415 0 1 2 3 4 5 6 7 8 9 10 1112131415

Tota

l cel

ls (fo

ld)

0

1

2

3

CFU (fold)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)

CD34+

-cel

ls (fo

ld)

CB MSCDLKCB MSCUSSC

CB MSCDLKCB MSCUSSC

CB MSCDLK

(a)


USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC


Relat

ive g

ene e

xpre

ssio

n

0

5

10

1550


lowast

lowast

(b)







Total cells (fold)

0 1 2 3 4 5 6 7 8 9 1011121314150

10

20

30

40

CB MSC


0 1 2 3 4 5 6 7 8 9 101112131415 0 1 2 3 4 5 6 7 8 9 101112131415

Tota

l cel

ls (fo

ld)

CFU (fold)

0

1

2

3

CD34+

-cel

ls (fo

ld)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)



(a)


CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

0


Gen

e exp

ress

ion

in re

latio

n to

RPL

13a

01

02

035

101520

2004006008001000times10minus2

(b)










Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



HepG2

USSCCB MSC

75120583m 50120583m 25120583m

(a)

00

64

64

128

128

192

192

256

256

R1 R1

100

100

100101

101

100 101

101

102

102

102

102

103

103

103

103

104

104

105

105

00

64

64

128

128

192

192

256

256 100100

101

101

102

102

103

103

104

104

105

105

100100

101

101

102

102

103

103

104

104

105

105

R3 R3 R3

00

200

200

400

400

600

600

800

800

1000

10000

0

200

200

400

400

600

600

800

800

1000

1000


DLK-1 FITC


Side

r sca

tter

Intr

acel

lula

rEx

trac

ellu

lar

10A Aminus1

10minus1

100101

100 101

102

102

103

103

10minus1

10minus1

(b)

BM M

SC 1

BM M

SC 2

CB M

SC D

LK-1

+M

ater

nal p

lasm

a

USS

C 1

USS

C 2

USS

C 3

USS

C 4

CB M

SC 1

CB M

SC 2

CB M

SC 3

0

1

2

3

4

5

DLK

-1 (n

gm

L)

(c)

CB M

SC 1

CB M

SC 1

clon

eCB

MSC

4CB

MSC

4 cl

one

CB M

SC 5

CB M

SC 6

CB M

SC 6

clon

e 1U

SSC

1 su

bclo

neU

SSC

2U

SSC

2 clo

neU

SSC

4BM

MSC

Ad

ipoc

ytes

0

Rela

tive

CEBP

am

RNA

expr

essio

n

12345

204060times10minus4

(d)

CB M

SC 1

CB M

SC 4

CB M

SC 5

USS

C 1

USS

C 2

USS

C 3

USS

C 6

Adip

ocyt

es0

Relat

ive

PPA

Rg2

mRN

A ex

pres

sion

12345


(e)



CB MSC 1

d0 co

ntro

l

30 m

in D

LK-1

4 h D

LK-1

24 h

DLK

-1

24 h

cont

rol

d0 D

LK-1

+

BM M

SC d

3 os

teog

enic

BM M

SC d

7 os

teog

enic

ERK12

p-ERK12

120573-actin


4 Discussion








5 Conclusion




Total cells (fold)

0 1 2 3 4 5 6 7 8 9 10 11121314150

10

20

30

40

CB MSCUSSC


0 1 2 3 4 5 6 7 8 9 10 1112131415 0 1 2 3 4 5 6 7 8 9 10 1112131415

Tota

l cel

ls (fo

ld)

0

1

2

3

CFU (fold)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)

CD34+

-cel

ls (fo

ld)

CB MSCDLKCB MSCUSSC

CB MSCDLKCB MSCUSSC

CB MSCDLK

(a)


USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC


Relat

ive g

ene e

xpre

ssio

n

0

5

10

1550


lowast

lowast

(b)







Total cells (fold)

0 1 2 3 4 5 6 7 8 9 1011121314150

10

20

30

40

CB MSC


0 1 2 3 4 5 6 7 8 9 101112131415 0 1 2 3 4 5 6 7 8 9 101112131415

Tota

l cel

ls (fo

ld)

CFU (fold)

0

1

2

3

CD34+

-cel

ls (fo

ld)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)



(a)


CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

0


Gen

e exp

ress

ion

in re

latio

n to

RPL

13a

01

02

035

101520

2004006008001000times10minus2

(b)










Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


CB MSC 1

d0 co

ntro

l

30 m

in D

LK-1

4 h D

LK-1

24 h

DLK

-1

24 h

cont

rol

d0 D

LK-1

+

BM M

SC d

3 os

teog

enic

BM M

SC d

7 os

teog

enic

ERK12

p-ERK12

120573-actin


4 Discussion








5 Conclusion




Total cells (fold)

0 1 2 3 4 5 6 7 8 9 10 11121314150

10

20

30

40

CB MSCUSSC


0 1 2 3 4 5 6 7 8 9 10 1112131415 0 1 2 3 4 5 6 7 8 9 10 1112131415

Tota

l cel

ls (fo

ld)

0

1

2

3

CFU (fold)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)

CD34+

-cel

ls (fo

ld)

CB MSCDLKCB MSCUSSC

CB MSCDLKCB MSCUSSC

CB MSCDLK

(a)


USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC


Relat

ive g

ene e

xpre

ssio

n

0

5

10

1550


lowast

lowast

(b)







Total cells (fold)

0 1 2 3 4 5 6 7 8 9 1011121314150

10

20

30

40

CB MSC


0 1 2 3 4 5 6 7 8 9 101112131415 0 1 2 3 4 5 6 7 8 9 101112131415

Tota

l cel

ls (fo

ld)

CFU (fold)

0

1

2

3

CD34+

-cel

ls (fo

ld)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)



(a)


CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

0


Gen

e exp

ress

ion

in re

latio

n to

RPL

13a

01

02

035

101520

2004006008001000times10minus2

(b)










Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Total cells (fold)

0 1 2 3 4 5 6 7 8 9 10 11121314150

10

20

30

40

CB MSCUSSC


0 1 2 3 4 5 6 7 8 9 10 1112131415 0 1 2 3 4 5 6 7 8 9 10 1112131415

Tota

l cel

ls (fo

ld)

0

1

2

3

CFU (fold)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)

CD34+

-cel

ls (fo

ld)

CB MSCDLKCB MSCUSSC

CB MSCDLKCB MSCUSSC

CB MSCDLK

(a)


USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC

USS

CCB

MSC

BM M

SCU

SSC

CB M

SCBM

MSC


Relat

ive g

ene e

xpre

ssio

n

0

5

10

1550


lowast

lowast

(b)







Total cells (fold)

0 1 2 3 4 5 6 7 8 9 1011121314150

10

20

30

40

CB MSC


0 1 2 3 4 5 6 7 8 9 101112131415 0 1 2 3 4 5 6 7 8 9 101112131415

Tota

l cel

ls (fo

ld)

CFU (fold)

0

1

2

3

CD34+

-cel

ls (fo

ld)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)



(a)


CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

0


Gen

e exp

ress

ion

in re

latio

n to

RPL

13a

01

02

035

101520

2004006008001000times10minus2

(b)










Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Total cells (fold)

0 1 2 3 4 5 6 7 8 9 1011121314150

10

20

30

40

CB MSC


0 1 2 3 4 5 6 7 8 9 101112131415 0 1 2 3 4 5 6 7 8 9 101112131415

Tota

l cel

ls (fo

ld)

CFU (fold)

0

1

2

3

CD34+

-cel

ls (fo

ld)

0

2

4

6

8

CFU

(fol

d)

CD34+-cells (fold)



(a)


CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

CB M

SCCB

MSC

DLK

-1CB

MSC

DLK

-1 h

igh

0


Gen

e exp

ress

ion

in re

latio

n to

RPL

13a

01

02

035

101520

2004006008001000times10minus2

(b)










Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article increased haematopoietic supportive...

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