notch signaling enhances osteogenic differentiation while inhibiting adipogenesis in primary human...

Experimental Hematology 2009;37:867–875

Notch signaling enhances osteogenicdifferentiation while inhibiting adipogenesis

in primary human bone marrow stromal cells

Fernando Ugartea, Martin Rysera,c, Sebastian Thiemea,c,Fernando A. Fierrob, Katrin Navratiela, Martin Bornhauserb,c, and Sebastian Brennera,c

aDepartment of Pediatrics, University Clinic Dresden, Dresden, Germany; bMedical Clinic I, University Clinic Dresden, Dresden,

Germany; cDFG Research Center and Cluster of Excellence for Regenerative Therapies Dresden, Technical University Dresden, Dresden, Germany

(Received 1 December 2008; revised 12 March 2009; accepted 16 March 2009)

Offprint requests to

University Clinic Dr

E-mail: Sebastian.Bre

0301-472X/09 $–see

doi: 10.1016/j.exph

Objective. The Notch signaling pathway has been shown to play a role in bone marrowLder-ived stromal cell differentiation, however, the precise outcome of Notch activation remainscontroversial. The aim of this study was to evaluate the effect of Notch signaling in primaryhuman bone marrowLderived stromal cells (hBMSCs).

Materials and Methods. hBMSCs were transduced to O90% with lentiviral vectors containingeither human notch1 intracellular domain (NICD), jagged1, or dominant negative mastermind1.Cells were exposed to adipogenic and osteogenic differentiation stimuli and differentiationwas quantified by oil red or alizarin red staining, alkaline phosphatase liver/bone/kidney(ALPL) activity and expression of adipogenic or osteogenic marker genes.

Results. NICD and jagged1 transgeneLexpressing hBMSCs demonstrated enhanced mineral-ization, nodule formation, and ALPL activity in osteogenic differentiation media. These find-ings correlated with increased gene expression of bone morphogenetic protein 2 and ALPL. Incontrast, NICD or jagged1 transgene expression strongly inhibited adipocyte formation andreduced peroxisome proliferator-activated receptor-g, fatty acid binding protein 4, and adiponec-tin precursor gene expression. Co-overexpression of dominant negative mastermind1 andNICD or jagged1 led to a partial rescue of the differentiation phenotypes. In addition, highendogenous jagged1 expression levels were observed in hBMSCs samples with strong ALPLactivity compared to a group of samples with low ALPL activity.

Conclusion. In summary, our data suggest that induction of Notch signaling enhances theosteogenic differentiation of hBMSCs while inhibiting the adipogenic fate. � 2009 ISEH -Society for Hematology and Stem Cells. Published by Elsevier Inc.

Bone marrow contains stromal cells (BMSCs) (also calledmesenchymal stem cells) that give rise to mesenchymaltissues such as bone, cartilage, fat, and stromal cells [1,2].Apart from their great potential to regenerate damaged tissues[3], BMSCs play an important supportive role in hematopoi-esis [4–6]. It has been shown that differentiation of BMSCs toone determined lineage is regulated by bone morphogeneticproteins (BMP), Wnt/b-catenin, and Notch signaling, amongothers [7–9]. During recent years, a lot of effort has gone intoexplaining the action of these pathways in regulation ofBMSC differentiation, leading to a fairly complex picture

: Sebastian Brenner, M.D., Department of Pediatrics,

esden, Fetscherstr. 74, 01307 Dresden, Germany;

[email protected]

front matter. Copyright � 2009 ISEH - Society for Hemat

em.2009.03.007

in consideration of all possible extracellular signal eventsand signaling crosstalk.

Notch signaling is a highly conserved signaling pathwayassociated with cell-fate determination, self-renewal poten-tial, and apoptosis [10]. Notch receptors and their ligands(jagged and delta) are families of transmembrane proteinswith large extracellular domains. Both are expressed inBMSCs. The Notch receptor is activated when bound to itsligand, leading to g-secretase�dependent cleavage of theNotch intracellular domain (NICD). The NICD then translo-cates to the nucleus, where it interacts with the CSL family oftranscriptional regulators and forms part of a Notch targetgene�activating complex. Inhibitors of g-secretases disruptNotch signaling, leading to impaired proliferation anddifferentiation of BMSCs [11].

ology and Stem Cells. Published by Elsevier Inc.

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868 F. Ugarte et al./ Experimental Hematology 2009;37:867–875

Using murine stromal cell lines, it has been suggested thattransient activation of Notch stimulates osteogenic differenti-ation [12]. Jagged- or delta-mediated activation of Notch wasshown to augment BMP2-mediated activation of osteogenesisin the murine cell lines MC3T3-E1 and C2C12, and appears tobe essential for proper BMP2 signaling [13]. However, thesedata stand in contrast to results published in the year 2006,which suggest that osteogenic differentiation is reduced byNotch activation in Kusa, MC3T3, and ST-2 cell lines[14,15]. The described inhibitory effect of Notch for osteogen-esis was, in part, attributed to expression of hairy and enhancerof split 1 (HES1), a Notch target gene, which also opposed theeffect of Wnt/b-catenin signaling [16]. It was later revealedthat HEY1, another Notch target gene, may collaborate inthis inhibitory effect of osteogenesis by repressing runt-relatedtranscription factor 2 (Runx2/Cbfa1, core binding factoralpha1) activity [17]. Originally, Cbfa1 was highlighted asa master regulator of osteogenesis, and its disruption showscomplete lack of bone formation [18,19]. In addition, two invivo studies demonstrate the important role of Notch in osteo-blastogenesis and bone homeostasis, acting indirectly as anregulator of osteoclastogenesis by modulating expression ofreceptor activator of nuclear factor kB ligand and osteoprote-gerin in osteoblasts [20,21].

Adipogenic differentiation of BMSCs also appears to bea complex process in which many transcriptional regulatorsand extracellular signals play a role, with peroxisome prolifer-ator-activated receptor� g (PPAR-g) being the master regu-lator [22]. Initially, Notch signaling was thought to benecessary for adipogenesis in the 3T3-L1 preadipocyte cellline [23]. In contrast, Ross et al. [24] suggested that Notch acti-vation reduces adipogenesis using the same model, and thatHES1 is the main downstream candidate of Notch action.Experiments performed with embryonic stem cells and fibro-blasts derived from knockout mice for Notch receptors org-secretases further suggested that Notch signaling is not neces-sary for proper adipogenic differentiation of these cells [25].

In light of the previously mentioned observations, it isimportant to note that most studies were performed in vitrousing predifferentiated murine cell lines, which may not reflectphysiological conditions [26]. In these studies, osteogenicdifferentiation was achieved mainly by BMP2 stimulation[27], while human-derived BMSCs have failed to be activatedby BMP2 in the same way [28–30]. These numerous studiessuggest that Notch signaling plays a role in osteogenic aswell as in adipogenic differentiation of BMSCs, however,the evidence is contradictory and the precise role of Notchsignaling remains to be elucidated. We have used a primaryhuman BMSCs model to allow studies under more physiolog-ical conditions. Furthermore, we not only used NICD to mimicNotch activation, but also studied jagged1-ligand mediatedactivation of Notch as well as inhibition of signaling byoverexpressing a mastermind1 dominant negative form(dnMAML1), which efficiently blocks the binding of theNICD-CSL�activated complex to DNA [31].

Materials and methods

Cell culturePrimary human bone marrow stromal cells (hBMSCs) were ob-tained from bone marrow aspirates of healthy donors after informedconsent to the research protocol, which had been approved by theInstitutional Review Board of the University Hospital of Dresden.Briefly, mononuclear cells were separated using Ficoll gradientand seeded at 0.5� 106cells/cm2 in Dulbecco’s modified Eagle’smedium 1.0 g/l D-glucose (PAA Laboratories, Pasching, Austria),200 mM L-glutamine, and 10% fetal bovine serum at 37�C and5% CO2. hBMSCs were then selected by their plastic adherenceand expanded to 90% confluence before passaging. The hBMSCsimmunophenotype was characterized by flow cytometric analyses[CD73þ, CD90þ, CD105þ, CD146þ, CD166þ, CD14� CD34�,CD45� (see Suppl. Fig. 1)], and their capacity to differentiate intoosteoblasts, adipocytes, and chondriocytes was evaluated. OnlyhBMSCs samples with no hematopoietic cell contamination wereused for further analyses. 293 T cells (ATCC 11268) were culturedin Iscove’s modified Dulbecco’s medium (Biochrom, Berlin,Germany) supplemented with 200 mM L-glutamine, 100 U/mLpenicillin, 100 mg/mL streptomycin, and 10% fetal bovine serumat 37�C and 5% CO2.

Generation of lentivirus vectorconstructs and transduction of primary hBMSCsLentiviral transfer vectors were created with the human ORF ofjagged1, NICD (1753-2256 AA), and dnMAML1 (13-74 AA).Transgenes were amplified from a human complementary DNAlibrary (MegaMan; Stratagene, La Jolla, CA, USA) and inserteddirectionally into the ClaI-SalI cloning sites of thepRRL.PPT.SF.GFPpre lentivector [32]da kind gift of ChristopherBaum and Luigi Naldinidto replace green fluorescent protein(GFP). For jagged1 and NICD, an internal ribosome entry si-te�GFP fragment was added by overlapping polymerase chainreaction (PCR) downstream of the transgene. dnMAML1 wascloned as a C-terminal fusion protein with a mCherry reportergene. Virus vector particles were obtained by transient transfectionof 293 T cells with transfer vector and packaging plasmids asdescribed previously [33]. Primary hBMSCs were transducedonce they had reached confluency, with 1:20 diluted neat virusvector supernatant, including protamine (1 mg/mL) at 37�C over-night. Transduction efficiency was quantified by flow cytometryusing a FACSCalibur Cytometer (BD, Franklin Lakes, NJ, USA).

Osteogenic and adipogenic differentiation of hBMSCsAll differentiation experiments were performed with hBMSCspassaged only once to avoid any variations due to aging of the cells.Twelve hours after transduction, the media was changed and supple-mented with either adipogenic differentiation medium containingdexamethasone (50 mM), 3-isobutyl-1-methylxanthine (0.5 mM),insulin (10 mg/mL), and indomethacin (100 mM); or osteogenicdifferentiation medium containing 1a,25-dihydroxy-vitamin D3

(25 mg/mL), b-glycerophosphate (10 mM) and dexamethasone(100 nM). Cells were cultured at 37�C and differentiation mediumwas replaced every 3 days. Mineralization was quantified at giventime points by alizarin red staining, as described previously [17].Briefly, cells were washed twice with phosphate-buffered saline,fixed with 3.7% formaldehyde, washed, and stained with alizarinred solution (40 mM) for 30 minutes at room temperature. Then

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869F. Ugarte et al./ Experimental Hematology 2009;37:867–875

cells were washed three times with water to remove any excess dye.Alkaline phosphatase activity was measured in cell extracts usingthe pNPP Liquid Substrate System (Sigma-Aldrich, St Louis, MO,USA) and Infinite m200 Elisa reader (TECAN, Crailsheim,Germany). Adipogenic differentiation was quantified 21 days afterdifferentiation by counting the number of adipocytes per cm2 underphase contrast using oil red staining as described previously [25].Briefly, cells were washed twice with phosphate-buffered saline,fixed with 3.7% formaldehyde, washed, and stained with oil redsolution (0.5 g oil red, 70 mL isopropanol, 30 mL H2O) for30 minutes at room temperature. Excess stain was removed by threewashes with water and adipocytes were visualized under the micro-scope (Axiovision 200 M; Zeiss, Gottingen, Germany).

Quantitative reverse transcription (RT)-PCRTotal RNA was isolated (RNAeasy kit, Qiagen, Hilden, Germany)and complementary DNA was prepared (Superscript II; Invitrogen,Carlsbad, CA, USA). Expression levels of notch1, jagged1, jagged130UTR (to quantify endogenous jagged1 expression), alkalinephosphatase type liver/bone/kidney (ALPL), peroxisome prolifera-tor-activated receptor-gamma (PPAR-g), adiponectin precursor(ADIPOQ), fatty acid binding protein 4 (FABP4), osteocalcin(BGLAP), bone morphogenetic protein 2 (BMP2), hairy/enhancer-of-split related with YRPW motif 1 (HEY1), and hairy and enhancerof split 1 (HES1) (see Table 1 for list of primers) were quantified byTaqMan assay (Applied Biosystems, Foster City, CA, USA). Rela-tive expression was determined according to the housekeeping geneglyceraldehyde-3- phosphate dehydrogenase.

Statistical analysisResults were reported as mean 6 standard error of mean. Linear modelof variance analyses with repeated measurements followed by Tukeyadjusted multiple comparison test were performed using GraphPadPrism version 5.01 for Windows, GraphPad Software. Statistical signif-icance was established at *p ! 0.05, **p ! 0.01, ***p ! 0.001.

Results

Highly efficient lentiviral vector-mediatedexpression of jagged1 and NICD in hBMSCsenables functional analysis of Notch signalingWe used a lentiviral vector system to efficiently overexpressour genes of interest in primary hBMSCs to levels O90%,as was quantified by GFP or mCherry expression (Fig. 1A).In addition, expression of jagged1-, NICD-, and dnMAML1-transgenes was confirmed by real-time PCR (see Suppl.Fig. 2). Activation of Notch signaling was assayed bygene expression levels of HEY1 and HES1d two knownNotch target genesd using quantitative RT-PCR(Fig. 1B). Detected expression levels demonstrated thatHEY1 is more sensitive to Notch activation (O100-foldincrease) through overexpression of NICD than HES1 (5-fold increase). Jagged1 ligand�mediated Notch activationis also able to increase expression of these target genesup to 15-fold for HEY1 and 5-fold for HES1. Co-overex-pression experiments demonstrated that dnMAML1partially blocked activation of HEY1 and HES1 caused byNICD or jagged1. Incomplete inhibition of dnMAML1 in

Figure 1. Overexpression of jagged1, notch1 intracellular domain (NICD), and mastermind1 dominant negative form (dnMAML1) in human bone marrow

stromal cells (hBMSCs) and activation of Notch target genes. (A) To overexpress jagged1, NICD, and dnMAML1 in hBMSCs, the transgenes were cloned

into a lentiviral vector. Lentivirus vector particles were generated and hBMSCs were transduced with efficiencies O90% as quantified by flow cytometry. (B)

Reverse transcription polymerase chain reaction of Notch target genes HES1 and HEY1, demonstrates upregulated expression in jagged1 and NICD transgene

expressing cells compared to the GFP control-transduced cells. Significance is denoted by asterisk (*p ! 0.05, **p ! 0.01, ***p ! 0.001). Coexpression of

dnMAML1 significantly reversed the effect caused by jagged1 or NICD as is shown by horizontal bars. Results are presented as the mean 6 standard error of

mean of four different donors.


co-expression conditions is probably due to unequal levelsof dnMAML1 expression compared to NICD or jagged1.

Notch activation in hBMSCsenhances mineralization, increases alkalinephosphatase levels, and inhibits adipocyte formationTo study their differentiation capacity, transduced hBMSCswere exposed to adipogenic and osteogenic differentiationstimuli. In osteogenic differentiation medium, mineraliza-tion deposits by NICD and jagged1 transgene overexpressing

hBMSCs was greatly enhanced compared to GFP control-transduced cells, as determined by alizarin red staining(Fig. 2A). Calcium phosphate accumulation was strongestin NICD cells and nodule structure formation was alreadyobserved after 7 days. Compared to the GFP control-trans-duced cells, jagged1 overexpression had a strong, however,less pronounced effect than NICD on mineralization(Fig. 2A). With jagged1 overexpression, nodule formationwas not observed. Increased mineralization by jagged1 andnotch1 was partially rescued by coexpression with

Figure 2. Notch induces enhanced mineralization and alkaline phosphatase activity, and inhibits adipocyte formation of hBMSCs. (A) Alizarin red staining

of hBMSCs, 7 days after initiation of osteogenic culture conditions illustrates enhanced mineralization in response to Notch signaling. (B) Mastermind1

dominant negative form (dnMAML1) hBMSCs show reduced mineralization compared to GFP control-transduced hBMSCs after 2 weeks of osteogenic

differentiation. (C) A significant (p ! 0.05) increase in alkaline phosphatase type liver/bone/kidney (ALPL) activity is observed in notch1 intracellular

domain (NICD) and jagged1 transgene positive hBMSCs. (D) Counting of adipocytes after oil red staining illustrates complete inhibition of adipocyte forma-

tion upon NICD overexpression (p ! 0.001), 3 weeks after initiation of adipogenic culture conditions. ALPL activity and the number of adipocytes were

compared to the GFP control-transduced cells and the significance was denoted by asterisk (*p ! 0.05, **p ! 0.01, ***p ! 0.001). Coexpression of

dnMAML1 significantly reversed the effect caused by jagged1 or NICD as is shown by horizontal bars in (B) and (C), respectively. All data are presented

as mean 6 standard error of mean of three different donors.


dnMAML1, indicating that the effect is a result of activeNotch signaling. Interestingly, following 2 weeks of osteo-genic differentiation, cells overexpressing dnMAML1 aloneshowed less mineralization than the GFP control-transducedcells (Fig. 2B). Results were concordant with the increasedlevels of ALPL in NICD- and jagged1-transduced hBMSCscompared to GFP control-transduced cells at day 7 (Fig. 2C).

After 14 days of adipogenic differentiation, on the otherhand, we observed complete inhibition of adipocyte forma-tion in NICD-transduced hBMSCs (p ! 0.001), as well asa significant reduction of the number of adipocytes injagged1 transduced cells (p ! 0.001; Fig. 2D). By cotrans-duction of cells with NICD and dnMAML1, inhibition ofadipocyte formation was reversed and cells formed signifi-cant numbers of adipocytes (p ! 0.05). Cotransduction of

cells with dnMAML1 and jagged1 also increased the numberof adipocytes twofold, compared to jagged1 only transducedhBMSCs. dnMAML1 on its own did not affect the number ofadipocytes, suggesting that endogenous Notch signalingmight not be necessary for proper adipogenic differentiation.Transgene overexpression (GFP, jagged1, NICD,dnMAML1) did not affect proliferation of hBMSCs asmeasured by cell cycle analysis, however, jagged1 andNICD transgenes enhanced the metabolic activity/viabilityof hBMSCs as measured by MTT assay (data not shown).

Effect of Notch activation on theexpression of adipogenic and osteogenic marker genesTo further investigate the effect of Notch signaling on thehBMSCs differentiation process, we analyzed expression

Figure 3. Expression of adipogenic and osteogenic marker genes upon Notch activation. (A) Kinetics of osteogenic marker gene expression in human bone

marrow stromal cells (hBMSCs). Notch1 intracellular domain (NICD) transgene positive cells demonstrate significantly enhanced alkaline phosphatase type

liver/bone/kidney (ALPL) (p ! 0.05) and bone morphogenetic protein 2 BMP2 (p ! 0.05) expression compared to Mastermind1 dominant negative form

(dnMAML1) transduced cells with a denoted peak at day 5, while showing complete inhibition of Osteocalcin expression (p ! 0.001). (B) Expression of

adipogenic marker genes peroxisome proliferator-activated receptor� g (PPAR-g), fatty acid binding protein 4 (FABP4), and adiponectin precursor (ADI-POQ) showed significantly reduced expression upon jagged1 or NICD overexpression when compared to the GFP control-transduced cells (*p ! 0.05,

**p ! 0.01, ***p ! 0.001). Coexpression of dnMAML1 significantly reversed the effect caused by jagged1- or NICD-transduced cells as is shown by

respective horizontal bars. The results are presented as the mean 6 standard error of mean of three different donors.


of genes described to be part of the osteogenic and adipo-genic phenotypes. It is important to note that the maturationprocess occurs in several stages with different gene expres-sion signatures [34], we, therefore, analyzed their expres-sion at various time points during differentiation. Underconditions that induce osteogenic differentiation, weobserved overall higher expression of ALPL and BMP2 inNICD-transduced cells compared to dnMAML1-transducedcells (p ! 0.05). Interestingly, there is a defined peak ofexpression in NICD-transduced hBMSCs at day 5, withan 11-fold increase in ALPL and a 20-fold increase inBMP2 compared to control (Fig. 3A). Afterward, expres-sion of ALPL drops to levels similar to the control, whereasBMP2 expression continues to be about eight times higherin NICD transduced cells after 14 days. Jagged1-transducedcells showed threefold increase in expression of ALPLand BMP2 compared to GFP control-transduced cellsthroughout the differentiation experiment. There was nosignificant difference observed in ALPL or BMP2 expres-sion for dnMAML1 cells compared to the GFP control.Analysis of expression of the osteoblast late marker osteo-calcin (BGLAP, bone g-carboxyglutamate [gla] protein)

showed strong downregulation by NICD or jagged1, andslight upregulation by dnMAML1 compared to GFPcontrol-transduced cells (see Fig. 3A). At day 7 of differen-tiation, there is a 4-fold and 100-fold increase in osteocalcinexpression in dnMAML1-transduced cells compared tocells transduced with jagged1 and NICD, respectively(p ! 0.05).

In thecaseofadipogenicdifferentiation,weobserveda three-to fourfold downregulation of PPAR-g, fatty acid bindingprotein 4, and adiponectin precursor expression levels(p ! 0.05) in jagged1-transduced cells compared to GFPcontrol-transduced cells (Fig. 3B). Expression of the samegenes was hardly detectable for NICD-transduced cells(p ! 0.001), while their expression was restored by co-overex-pression with dnMAML1 to similar levels as the GFP control(p ! 0.05). Overexpression of dnMAML1 on its own did notsignificantly affect expression of any of these genes.

Significant difference in endogenousjagged1 expression levels according to ALPL activityTo validate our previous data using lentiviral vector overex-pression of Notch-related transgenes, we compared alkaline

Table 2. Clinical data from donor samples

Sample number Gender Age (y) ALPL activity (mU/mL)

hBMSCs 137 Male 29 48.8



hBMSCs 204 Female 18 66.0

















ALPL 5 alkaline phosphatase type liver/bone/kidney; hBMSCs 5 human

bone marrow stromal cells.


phosphatase activity levelsdas an indicator for osteogenicdifferentiation potentialdwith the endogenous expressionlevels of notch1 and jagged1 in two groups of primaryhBMSCs. Of 250 primary hBMSCs samples, we selectedthe 10 samples with highest ALPL activity (mean activity:516 mU/mL; mean age: 31.7 years) and the 10 with the lowestALPL activity [(mean activity: 72.9 mU/mL; mean age: 26.9years), (Table 2)] and compared them to their endogenousnotch1 and jagged1 expression levels. ALPL enzymaticactivity was confirmed by ALPL gene expression levels(data not shown). Quantitative RT-PCR analyses demon-strated high jagged1 expression in hBMSCs samples withstrong ALPL activity, and low jagged1 expression inhBMSCs samples with low ALPL activity (Fig. 4A). Thedifference in jagged1 expression between the two groupswas statistically significant (p 5 0.044). In contrast, notch1expression was equally expressed within the groups ofhBMSCs samples with high ALPL and low ALPL activity.We did not analyze ALPL or jagged1 expression on a singlecell level, thus cannot exclude that hBMSCs show heteroge-neous expression and thus certain clones might have contrib-uted differently to the overall expression level. A possibleexplanation for the observed differences in jagged1 expres-sion and not of notch1 expression with ALPL activity isthat the transmission of signaling between neighboring cellsis regulated through ligand expression (jagged1) and notthrough receptor expression (notch1) [35]. Supporting thishypothesis, we found that cells transduced with NICD induceexpression of endogenous jagged1 up to 10-fold (p ! 0.001),3 days after transduction (Fig. 4B), suggesting a positivefeedback loop. In contrast, jagged1 overexpression did notresult in upregulation of endogenous notch1 (data not shown).

DiscussionNotch signaling has been implicated in the process ofBMSCs differentiation, however, results reported in theliterature, mainly based on murine BMSCs, are highlycontradictory. Moreover, Hilton et al. [21] demonstratedthe opposing effects of Notch signaling on bone formationduring different stages of mouse development. While Notchdepletion during early stages of development-inducedformation of dense bone structures, the ablation of Notchin later adult stages led to deficient bone structures.

Our study demonstrates the role of Notch signaling in thedifferentiation of primary human bone marrow�derivedstromal cells. We show that activation of Notch signaling inthese primary hBMSCs strongly enhances their osteogenicdifferentiation while inhibiting their adipocyte-formationcapacity. Furthermore, our data suggest that cell-fate determi-nation is proportional to the level of Notch signaling activa-tion. While high signaling activity due to constitutivelyactive NICD induces strong osteogenic differentiation andcomplete inhibition of adipogenesis in hBMSCs, jagged1overexpression has a rather moderate effect (Fig. 2A and

D). The rather moderate differentiation effect seen withjagged1 compared to NICD overexpression might be due tothe rate-limiting amount of endogenous Notch receptors.These Notch receptors, expressed on the surface of neigh-boring cells, are targeted by the abundant overexpression ofthe jagged1 ligand to transmit the differentiation effect. More-over, blocking Notch signaling by dnMAML1 in the NICD orjagged1 overexpressing hBMSCs reverses the induced osteo-genic or adipogenic differentiation phenotype and confirmsthe specific influence of Notch signaling in hBMSCs.

The described osteogenic differentiation experiments re-vealed high mineralization capacity and increased ALPL levelsupon Notch activation. As described previously, Core bindingfactor alpha1 (Cbfa1) is a transcription factor known to beessential in the process of osteogenesis [18,19]. Recent datasuggest that Notch signaling (through NICD as well as Hey1)influences osteogenic differentiation through regulation ofCbfa1 expression [20,21]. We did not observe any differencein Cbfa1 expression levels in our transduced hBMSCs (datanot shown). Notably, we found increased BMP2 expressionlevels, a fundamental protein for bone formation, that hasbeen used, albeit ineffectively, to induce differentiation ofhuman cells [28]. Our data are in line with the observations ofNobta et al. [13], who described the requirement of Notchsignaling for BMP2-induced osteogenesis in C2C12 andMC3T3-E1 mouse cell lines. Despite the enhanced osteogenicphenotype induced by Notch, we found inhibited osteocalcinexpression in NICD-positive hBMSCs, and this effect wasreversed by dnMAML1 coexpression (Fig. 3A). Osteocalcinwas published as a late marker for osteoblast maturation [34].Furthermore, dnMAML1 overexpression on its own slightlyenhanced expression of osteocalcin compared to GFP

Figure 4. Differences in endogenous jagged1 expression levels in human

bone marrow stromal cells (hBMSCs) samples with high and low alkaline

phosphatase type liver/bone/kidney (ALPL) activity. (A) Of 250 primary

hBMSCs samples, we selected the 10 samples with highest ALPL activity

and the 10 with the lowest ALPL activity, and analyzed their endogenous

notch1 and jagged1 expression levels by quantitative reverse transcription

polymerase chain reaction. Results showed high endogenous jagged1

expression in hBMSCs samples with strong ALPL activity. These results

were also confirmed checking gene expression of ALPL (data not shown).

Data are presented as mean 6 standard error of mean and statistical anal-

ysis was performed using unpaired t-test. (B) Overexpression of Notch1

intracellular domain (NICD) significantly induced expression of endoge-

nous jagged1, as a possible positive feedback mechanism. Data are pre-

sented as mean 6 standard error of mean of six different donors.


control-transduced cells. Similar observations were made byseveral groups [12–14] that failed to see induction of osteocalcinupon Notch activation under conditions that induce osteogenicdifferentiation. The strong accumulation of calcium deposits incells overexpressing NICD or jagged1 might be comparable tothe formation of woven bone in vivo, which is a premature anddisorganized structure that forms early in bone development.Therefore, according to our results, it seems reasonable thatenhanced Notch activation in human BMSCs induces earlystages of osteoblast differentiation, yet prevents formation ofa mature functional osteoblast. In support of this notion, Enginet al. [20] observed that activation of Notch signaling in vivoinduces formation of highly mineralized bone structures asa result of increased proliferation of immature osteoblast precur-sors. Comparable results were described by Oldershaw et al.[36], who suggested that Notch activation is necessary for initi-ation of chondrogenesis in human BMSCs, but must beswitched-off to complete the maturation process. In summary,activation of Notch signaling in hBMSCs induces early andstrong osteogenic differentiation, however, it prevents forma-tion of mature osteoblasts.

Unlike the induction of osteogenic differentiation, activa-tion of Notch signaling by NICD or jagged1 strongly inhibitsformation of adipocytes and downregulates expression ofimportant adipocyte-specific marker genes. PPAR-g is animportant transcription factor for proper adipogenic differen-tiation [22]. Reduced adipogenic formation seen in NICD-and jagged1-transduced hBMSCs might thus be due to theobserved reduction of PPAR-g expression in these samples.It appears then that activation of Notch favors the osteogenicover the adipogenic fate, and that the decision for one cell fateexcludes the other. Hong et al. [37] described a similar situa-tion in which the transcriptional coactivator TAZ regulatesthe decision of murine BMSCs to assume an osteogenic oradipogenic fate. TAZ regulates this cell-fate decision by acti-vating Cbfa1 while repressing PPAR-g. Whether Notch regu-lates expression of TAZ and subsequentially regulatesdifferentiation of human BMSCs remains to be studied. Ac-cording to our data, overexpression of dnMAML1 alonedoes not significantly affect adipogenesis in human BMSCs.Therefore, Notch signaling might not be required for properadipocyte formation, as Nichols et al. [25] described formurine embryonic stem cells.

In addition, a significant difference in endogenous jagged1expression levels was observed between two groups ofhBMSCs samples with high and low ALPL activity, respec-tively. Further studies are needed to investigate whether theendogenous jagged1 expression levels determine the osteo-genic differentiation potential of human BMSCs in vivo. Wedemonstrate that Notch activation by NICD is able to induceexpression of the Notch ligand jagged1 in human BMSCs,creating a positive feedback regulation via jagged1. Thesedata suggest that Notch signaling within hBMSCs neighboringcells is regulated by its ligand expression (lateral specifica-tion), as has been described previously in NIH-3T3 cells [35].


In summary, using primary hBMSCs, we present furtherevidence for the significance of Notch signaling inhBMSCs differentiation. Questions remain as to how Notchsignaling participates in diseases displaying impaired bonehomeostasis. Elucidation of these issues will guide ustoward a better understanding of hBMSC differentiationand their optimized use in regenerative medicine.

AcknowledgmentsSupported by the Deutsche Forschungsgemeinschaft (SFB 655 toS.B. and M.B.) and the Deutsche Krebshilfe research grant no.106169 (S.B.). We thank Katrin Muller for expansion and charac-terization of hBMSCs and we thank Rainer Koch for advice instatistical analysis. No financial interest/relationships with finan-cial interest relating to the topic of this article have been declared.

References1. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of

adult human mesenchymal stem cells. Science. 1999;284:143–147.

2. Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting

history, concepts, and assays. Cell Stem Cell. 2008;2:313–319.

3. Caplan AI. Adult mesenchymal stem cells for tissue engineering

versus regenerative medicine. J Cell Physiol. 2007;213:341–347.

4. Wilson A, Trumpp A. Bone-marrow haematopoietic-stem-cell niches.

Nat Rev Immunol. 2006;6:93–106.

5. Calvi LM, Adams GB, Weibrecht KW, et al. Osteoblastic cells regulate

the haematopoietic stem cell niche. Nature. 2003;425:841–846.

6. Sacchetti B, Funari A, Michienzi S, et al. Self-renewing osteoprogeni-

tors in bone marrow sinusoids can organize a hematopoietic microen-

vironment. Cell. 2007;131:324–336.

7. Gregory CA, Ylostalo J, Prockop DJ. Adult bone marrow stem/proge-

nitor cells (MSCs) are preconditioned by microenvironmental

‘‘niches’’ in culture: a two-stage hypothesis for regulation of MSC

fate. Sci STKE. 2005;2005. e37.

8. Nuttall ME, Gimble JM. Controlling the balance between osteoblasto-

genesis and adipogenesis and the consequent therapeutic implications.

Curr Opin Pharmacol. 2004;4:290–294.

9. Satija NK, Gurudutta GU, Sharma S, et al. Mesenchymal stem cells:

molecular targets for tissue engineering. Stem Cells Dev. 2007;16:7–23.

10. Bray SJ. Notch signaling: a simple pathway becomes complex. Nat

Rev Mol Cell Biol. 2006;7:678–689.

11. Vujovic S, Henderson SR, Flanagan AM, Clements MO. Inhibition of

gamma-secretases alters both proliferation and differentiation of

mesenchymal stem cells. Cell Prolif. 2007;40:185–195.

12. Tezuka K, Yasuda M, Watanabe N, et al. Stimulation of osteoblastic

cell differentiation by Notch. J Bone Miner Res. 2002;17:231–239.

13. Nobta M, Tsukazaki T, Shibata Y, et al.Critical regulation of bone morpho-

genetic protein-induced osteoblastic differentiation by Delta1/Jagged1-

activated Notch1 signaling. J Biol Chem. 2005;280:15842–15848.

14. Shindo K, Kawashima N, Sakamoto K, et al. Osteogenic differentia-

tion of the mesenchymal progenitor cells, Kusa is suppressed by Notch

signaling. Exp Cell Res. 2003;290:370–380.

15. Sciaudone M, Gazzerro E, Priest L, Delany AM, Canalis E. Notch 1 impairs

osteoblastic cell differentiation. Endocrinology. 2003;144:5631–5639.

16. Deregowski V, Gazzerro E, Priest L, Rydziel S, Canalis E. Notch 1 over-

expression inhibits osteoblastogenesis by suppressing Wnt/beta-catenin

but not bone morphogenetic protein signaling. J Biol Chem. 2006;281:

6203–6210.

17. Zamurovic N, Cappellen D, Rohner D, Susa M. Coordinated activa-

tion of notch, Wnt, and transforming growth factor-beta signaling

pathways in bone morphogenic protein 2-induced osteogenesis. Notch

target gene Hey1 inhibits mineralization and Runx2 transcriptional

activity. J Biol Chem. 2004;279:37704–37715.

18. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/Cbfa1: a tran-

scriptional activator of osteoblast differentiation. Cell. 1997;89:747–754.

19. Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfa1

results in a complete lack of bone formation owing to maturational

arrest of osteoblasts. Cell. 1997;89:755–764.

20. Engin F, Yao Z, Yang T, et al. Dimorphic effects of Notch signaling in

bone homeostasis. Nat Med. 2008;14:299–305.

21. Hilton MJ, Tu X, Wu X, et al. Notch signaling maintains bone marrow

mesenchymal progenitors by suppressing osteoblast differentiation.

Nat Med. 2008;14:306–314.

22. Rosen ED, MacDougald OA. Adipocyte differentiation from the inside

out. Nat Rev Mol Cell Biol. 2006;7:885–896.

23. Garces C, Ruiz-Hidalgo MJ, Font de MJ, et al. Notch-1 controls the

expression of fatty acid-activated transcription factors and is required

for adipogenesis. J Biol Chem. 1997;272:29729–29734.

24. Ross DA, Rao PK, Kadesch T. Dual roles for the Notch target gene

Hes-1 in the differentiation of 3T3-L1 preadipocytes. Mol Cell Biol.

2004;24:3505–3513.

25. Nichols AM, Pan Y, Herreman A, et al. Notch pathway is dispensable

for adipocyte specification. Genesis. 2004;40:40–44.

26. Leis HJ, Hulla W, Gruber R, et al. Phenotypic heterogeneity of oste-

oblast-like MC3T3-E1 cells: changes of bradykinin-induced prosta-

glandin E2 production during osteoblast maturation. J Bone Miner

Res. 1997;12:541–551.

27. Leboy PS. Regulating bone growth and development with bone

morphogenetic proteins. Ann N Y Acad Sci. 2006;1068:14–18.

28. Diefenderfer DL, Osyczka AM, Reilly GC, Leboy PS. BMP respon-

siveness in human mesenchymal stem cells. Connect Tissue Res.

2003;44(Suppl 1):305–311.

29. Friedman MS, Long MW, Hankenson KD. Osteogenic differentiation

of human mesenchymal stem cells is regulated by bone morphogenetic

protein-6. J Cell Biochem. 2006;98:538–554.

30. Osyczka AM, Leboy PS. Bone morphogenetic protein regulation of

early osteoblast genes in human marrow stromal cells is mediated by

extracellular signal-regulated kinase and phosphatidylinositol 3-kinase

signaling. Endocrinology. 2005;146:3428–3437.

31. Maillard I, Weng AP, Carpenter AC, et al. Mastermind critically regu-

lates Notch-mediated lymphoid cell fate decisions. Blood. 2004;104:

1696–1702.

32. Schambach A, Bohne J, Chandra S, et al. Equal potency of gammar-

etroviral and lentiviral SIN vectors for expression of O6-methylgua-

nine-DNA methyltransferase in hematopoietic cells. Mol Ther. 2006;

13:391–400.

33. Dull T, Zufferey R, Kelly M, et al. A third-generation lentivirus vector

with a conditional packaging system. J Virol. 1998;72:8463–8471.

34. Lian JB, Stein GS, Stein JL, van Wijnen AJ. Osteocalcin gene

promoter: unlocking the secrets for regulation of osteoblast growth

and differentiation. J Cell Biochem. 1998;30–31(Suppl):62–72.

35. Ross DA, Kadesch T. Consequences of Notch-mediated induction of

Jagged1. Exp Cell Res. 2004;296:173–182.

36. Oldershaw RA, Tew SR, Russell AM, et al. Notch signaling through

Jagged-1 is necessary to initiate chondrogenesis in human bone

marrow stromal cells but must be switched off to complete chondro-

genesis. Stem Cells. 2008;26:666–674.

37. Hong JH, Hwang ES, McManus MT, et al. TAZ, a transcriptional

modulator of mesenchymal stem cell differentiation. Science. 2005;

309:1074–1078.

Supplementary data associated with the article can befound, in the online version, at doi:10.1016/j.exphem.2009.03.007.

http://dx.doi.org/doi:10.1016/j.exphem.2009.03.007

http://dx.doi.org/doi:10.1016/j.exphem.2009.03.007

Supplementary Figure 1. Flow cytometric immunophenotyping of isolated human bone marrow stromal cells (hBMSCs). hBMSCs were positive for CD73,

CDw90, CD105, CD166, CD146, and were negative for CD14, CD34, CD45 demonstrating no hematopoietic cell contamination. A representative analysis is

shown.

Supplementary Figure 2. Expression of notch1 and jagged1 in human bone marrow stromal cells (hBMSCs). Transduction of Notch1 intracellular domain

(NICD) (open squares) and jagged1 (circles) transgenes resulted in strong expression of respective transcripts in hBMSCs. Green fluorescent protein (GFP)

control-transduced hBMSCs show minimal endogenous notch1 or jagged1 expression levels (filled circles and squares). Gene expression was analyzed by

quantitative reverse transcription polymerase chain reaction normalized to glyceraldehyde-3-phosphate dehydrogenase.

875.e1 F. Ugarte et al./ Experimental Hematology 2009;37:867–875

notch signaling enhances osteogenic differentiation while inhibiting adipogenesis in primary human...

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