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Tissue and Cell 43 (2011) 331– 336

Contents lists available at ScienceDirect

Tissue and Cell

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ATP channels in mesenchymal stromal stem cells: Strong up-regulation of Kir6.2ubunits upon osteogenic differentiation

nke Diehlmanna, Simone Borka, Rainer Saffricha, Rüdiger W. Vehb,olfgang Wagnera,c,∗,1, Christian Derstb,∗∗,1

Department of Medicine V, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyInstitute for Integrative Neuroanatomy, Charité, Philippstr. 12, 10115 Berlin, GermanyRWTH Aachen Medical School, Stem Cell Biology and Cellular Engineering, Pauwelsstrasse 20, 52078 Aachen, Germany

r t i c l e i n f o

rticle history:eceived 3 November 2010eceived in revised form 30 May 2011ccepted 6 June 2011vailable online 5 August 2011

eywords:

ATP channel

a b s t r a c t

The promising use of mesenchymal stromal cells (MSC) in regenerative technologies accounts for neces-sity of detailed study of their physiology. Proliferation and differentiation of multipotent cells ofteninvolve changes in their metabolic state. In the present study, we analyzed the expression of ATP-sensitivepotassium (KATP) channels in MSC and upon in vitro differentiation. KATP channels are present in manycells and regulate a variety of cellular functions by coupling cell metabolism with membrane potential.Kir6.1, Kir6.2 and SUR2A were expressed in undifferentiated MSC, whereas SUR2B and SUR1 were notdetected on cDNA and protein level. Upon adipogenic differentiation Kir6.1 and SUR2A showed a sig-

esenchymal stromal cellsdipocytesteoblastulfonylurea receptorir6 channel

nificant reduction of the amount of mRNA by 84% and 95%, respectively, whereas Kir6.2 expression wasunchanged. Osteogenic differentiation strongly up-regulated Kir6.2 mRNA (28-fold) whereas Kir6.1 andSUR2A showed no significant change in expression. Quantitative Western blot analysis and immunofluo-rescence staining confirmed the elevated expression of Kir6.2 upon osteogenic differentiation. Takentogether, expression changes of KATP channels may contribute to in vitro differentiation of MSC andrepresent changes in the metabolic state of the developing tissue.

. Introduction

Human mesenchymal stromal cells (MSC, also named mes-nchymal stem cells) are non-hematopoietic stem cells withultilineage potential that can be isolated from bone marrow and

arious other tissues (Friedenstein et al., 1970; Pittenger et al.,999; Dominici et al., 2006). Usually, MSC are considered as pre-ursors of mesodermal cell types such as osteocytes, adipocytesnd chondrocytes whereas it is still under debate, if MSC can giveise to non-mesodermal cell types such as hepatocytes or neuronsPhinney and Prockop, 2007; Bianco et al., 2001; Caplan, 1991; Jiangt al., 2002). Due to the lack of a specific marker for MSC, they are

efined by plastic adherent growth, a panel of surface markers andheir in vitro differentiation ability (Wagner and Ho, 2007; Ho et al.,008; Abdallah and Kassem, 2008). Their differentiation and tissue

∗ Corresponding author at: RWTH Aachen Medical School, Stem Cell Biology andellular Engineering, Pauwelsstrasse 20, 52078 Aachen, Germany.el.: +49 2418088611.∗∗ Corresponding author. Tel.: +49 30450528369; fax: +49 30450528912.

E-mail addresses: wwagner@ukaachen.deW. Wagner), christian.derst@charite.de (C. Derst).

1 Authors contributed equally to this article.

040-8166/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.tice.2011.06.004

© 2011 Elsevier Ltd. All rights reserved.

regeneration potential has already been used in therapeutic clinicalapproaches involving tissue engineering and gene therapy (Satijaet al., 2009; Baksh et al., 2004).

In vitro differentiation of MSC requires the activation of spe-cific regulatory genes, transcription factors and signal cascades(Nakashima and de, 2003; Komori, 2006). Induction of adipogenesisresults in preadipocytes with cytoplasmatic accumulation of lipidvesicles and synthesis of extracellular matrix-associated proteins(MacDougald and Mandrup, 2002). The mechanisms of osteo-genesis leads to mature osteoblasts which produce mineralizedextracellular matrix, containing high levels of calcium phosphatewhich form hydroxyapatite crystals (Jaiswal et al., 1997). Sinceosteoblasts and adipocytes originate from a common MSC pre-cursor cell, it seems obvious that osteoblastic and adipocytedifferentiation pathways are regulated jointly (Rodriguez et al.,2008). Upon growth and differentiation, regulation of the metabolicstate is a major function of MSC. Especially, after differentiation thedeveloping new tissue may have a different metabolism than priorto differentiation. Indeed, recent analysis in cardiogenesis indicatesa major switch from low-energy requiring pluripotent stem cells

to high-energy consuming mature cardiac cells (Chung et al., 2008,2010). Besides differential regulation of major metabolic pathways,such as glycolysis and oxidative phosphorylation as functional roleof KATP channels has been demonstrated in local submembrane

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ompartments in close association with membrane ATPases andhosphotransfer enzymes (Chung et al., 2010; Selivanov et al.,004).

KATP channels are key molecules in metabolism by coupling theetabolic state to the resting potential of a cell. As octameric pro-

eins KATP channels possess four pore-forming Kir subunits (Kir6.1r Kir6.2) and four regulatory sulfonylurea receptor subunits (SUR1r SUR2). The Kir subunit is a typical inwardly rectifying potas-ium channel, the SUR subunit is structurally a member of the ABCransporter superfamily. Different combinations of Kir6 and SURubunits lead to tissue-specific KATP channels with typical physi-logical and pharmacological properties (Seino et al., 2000). KATPhannels are inhibited by physiological intracellular ATP and arectivated by increasing intracellular ADP/ATP ratio, e.g. as a resultf energy depletion, hypoxia or metabolic stress. As a consequence,+ efflux leads to hyperpolarization of the membrane, protecting

he cell from over-excitability and cell death (Miki et al., 2001).The aim of this present report was to study the expres-

ion of the different KATP channel subunits in undifferentiated,steogenic and adipogenic differentiated MSC. The identificationnd the differentiation-associated expression change of KATP chan-el subunits may represent an important characteristic of thehysiological and pharmacological properties of MSC.

. Materials and methods

.1. MSC from human bone marrow

Human MSC were obtained from healthy donors for allogeneicone marrow transplantation. In brief, bone marrow aspiratesere taken after written consent using guidelines approved by

he Ethic Committee on the Use of Human Subjects at the Uni-ersity of Heidelberg (Heidelberg, Germany). The mononuclear cellraction (MNC) was isolated by Biocoll density-gradient centrifuga-ion (Biochrom, Berlin, Germany). MNC were plated in expansion

edium as described before at a density of 105 cells/cm2 in tis-ue culture flasks (Nunc, Wiesbaden, Germany) (Reyes et al.,001; Wagner et al., 2005). Tissue culture flasks were coated with0 ng/ml fibronectin (Sigma, Hamburg, Germany). Upon subconflu-nt growth, cells were always reseaded at a density of 104 cells/cm2.n this study we used MSC of passages 2–5.

.2. In vitro differentiation

Differentiation of MSC into adipogenic and osteogenic lineagesas carried out as described (Wagner et al., 2005). To induce

steogenic differentiation, cells were cultured in DMEM with 10%CS (Invitrogen, Karlsruhe, Germany), 10 mM �-glycerophosphate,0−7 M dexamethasone and 0.2 mM ascorbic acid. The medium washanged every 3 days. After three weeks the MSC were harvestedrom the culture dish with a mild trypsin treatment and the concen-ration was adjusted to 1 × 106 cells per ml. To induce adipogenicifferentiation, cells were cultured after the second passage inMEM with 10% FCS, 0.5 mM isobutylmethylxanthine (IBMX),

�M dexamethasone, 10 �M insulin and 200 �M indomethacin.he medium was changed every 3 days.

.3. Preparation of membrane fractions

MSC were homogenized in homogenization buffer (10 mM TEAH 7.5, 250 mM sucrose containing 1 tablet Complete Protease

nhibitor Cocktail, Roche, Mannheim, Germany) and sonicated for min. After 10 min centrifugation at 1000 × g the supernatantas ultracentrifuged for 1 h at 200,000 × g at 4 ◦C. Pellets were

e-suspended in homogenization buffer, sonicated, and stored in

Cell 43 (2011) 331– 336

aliquots at −80 ◦C. The total protein concentration was determinedby bicinchoninic acid (BCA) assay.

2.4. Western blot analysis

100 �g protein lysate from membrane fractions were separatedelectrophoretically on a 10% SDS-PAGE. After transfer to a nitro-cellulose membrane unspecific binding sites were blocked by 5%low-fat milk powder in PBS containing 1% Tween-20 for 1 h. Thefollowing affinity-purified primary antibodies were used for overnight incubation (Skatchkov et al., 2002; Thomzig et al., 2005;Jons et al., 2006): anti-Kir6.1 (1:200 dilution), anti-Kir6.2 (1:200dilution), anti-SUR1 (1:200 dilution), anti-SUR2A (1:200 dilution),anti-SUR2B (1:200 dilution). Three PBS wash steps were followedby incubation with the secondary antibody (alkaline-phosphatase-conjugated anti-rabbit IgG, 4 h at room temperature, 1:10.000dilution). Immunosignal was detected using p-nitro blue tetra-zolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate(BCIP).

2.5. Quantitative RT-PCR

RNA was isolated using Trizol (Invitrogen, Karlsruhe, Germany)and reverse transcribed using Sensiscript reverse transcriptase(Qiagen, Hilden, Germany). Quantitative PCR was performed usingTaqMan assays under standard conditions (Applied Biosystems,Darmstadt, Germany). The following TaqMan assays for humantissues were used for amplification: Kir6.1 (Hs00958961 m1),Kir6.2 (Hs00265026 s1), SUR1 (Hs00165861 m1), SUR2A(Hs01072316 m1) and SUR2B (Hs02515384 s1). RUNX2(Hs00231692 m1) and ADIPOQ (Hs0065917 m1) were usedas additional controls for as osteogenic and adipogenic differentia-tion. All assays were performed as duplex reaction using a humanGAPDH VIC-MGB assay as endogenous control.

2.6. Immunofluorescence staining

MSC were seeded on cover slips in 12 multiwell plates andcultured in normal expansion medium or under osteogenic dif-ferentiation conditions as mentioned above. After 3 weeks oftreatment, cells were fixed with 4% paraformaldehyde and perme-abilized with 0.2% Triton X-100. MSC were rehydrated with Trisbuffered saline (TBS) and incubated in blocking solution (3% milkpowder in TBS). For Kir6.2 detection, a rabbit polyclonal anti-Kir6.2(Thomzig et al., 2005) was used in conjunction with a Cy3-labeledsecondary anti-rabbit antibody (Jackson Laboratories). Incubationfor 60 min was followed by three washing steps in PBS for 5 minfor each antibody. DAPI (4′,6-diamidino-2-phenylindol, Merck,Germany) was used for nuclear staining. Cells were mounted inDABCO/Mowiol and analyzed using an Olympus IX70 inverted flu-orescence microscope equipped with a digital image acquisitionand processing system (analySIS 3.2, Soft Imaging System, Münster,Germany).

2.7. Statistical analysis

Results are presented as mean ± standard error of mean. Sta-tistical analysis between undifferentiated and differentiated MSCwas performed with Student’s t-test. For data analysis Sigma Plot(Systat Software Inc.) was used.

3. Results

We examined numerous MSC populations derived from humanbone marrow of healthy donors aged 21–68. All isolated MSCpopulations displayed a spindle-shaped morphology (Fig. 1A) and

A. Diehlmann et al. / Tissue and Cell 43 (2011) 331– 336 333

Fig. 1. Differentiation of human MSC. MSC were isolated from bone marrow and cultured for two passages in growth medium. (A) MSC in culture are plastic adherent witha spindle-shaped morphology/appearance. (B) MSC cultured under osteogenic conditions for 21 days showing mineral deposits that indicate early stages of bone formation.Osteogenic differentiation was examined with the von Kossa method to label calcium deposits with silver. Cells are counterstained with hematoxylin. (C) MSC grown ina oplasmo toxyl

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dipose maintenance medium for 21 days accumulate lipid vacuoles within the cytf adipogenic differentiation. The adipocytes were stained with Oil Red O and hema

ere able to differentiate into osteocytes (Fig. 1B, accumulationf mineral deposits) and adipocytes (Fig. 1C, accumulation ofipid vesicles) under appropriate in vitro culture conditions. Theyad CD34−, CD45−, CD13+, CD29+, CD44+, CD73+, CD90+, CD105+,D146+ and CD166+ phenotype as described before (data nothown) (Wagner et al., 2005, 2008).

KATP channel expression was analyzed by Western blot in undif-erentiated MSC: a ∼48 kD band of Kir6.1 was always detectedn = 4; Fig. 2). Several additional bands at 60 kD, 80 kD and 200 kD

ay represent posttranslational modifications or high molecularomplexes (e.g. with SUR subunit). Kir6.2 was detected as a sin-le strong band at ∼60 kD (n = 3, Fig. 2). The calculated moleculareight for Kir6.2 is 43.5 kD and the discrepancy might be due toosttranslational modifications or aberrant electrophoretic mobil-

ty of the transmembrane proteins (Thomzig et al., 2005). For thehree known sulfonylurea subunits only SUR2A (n = 4, Fig. 2), butot SUR2B (Fig. 2) and SUR1 (not shown) could be detected. ForUR2A a strong band around 200 kD and a weaker band around70 kD were visible. This corresponds to the predicted moleculareight of 174.4 kD, with the higher band representing modifi-

ation or a strong association with a Kir6 subunit. To validatexpression of KATP subunits, we performed quantitative RT-PCR.RNA for Kir6.1, Kir6.2 and SUR2A mRNA was identified, whereas

UR1 mRNA was not detected and this is in line with the West-

rn blot results. Comparison of gene expression level relative toAPDH reference indicated, that Kir6.2 is expressed at higher levels

han SUR2 and Kir6.1 (�Ct-values = −0.5; −2.5 and −5.5, respec-ively).

. The bright round fat globules are seen in more and more cells over time as a signin as a counterstain. Scale bar is 50 �m, 10× objective.

Subsequently, we analyzed whether the expression of KATP sub-units changes upon adipogenic or osteogenic differentiation. Adi-pogenic differentiation resulted in a significant down-regulationof Kir6.1 (−84.6%, p < 0.0042) and SUR2 (−95.3%, p < 0.000076) ongene expression level, whereas Kir6.2 was not affected (Fig. 3A).Osteogenic differentiation resulted in a strong increase in Kir6.2expression by a factor of 28 (+2716%, p < 0.000007, Fig. 3A), whereasKir6.1 and SUR2 mRNA amount showed no significant change. Thestrong up-regulation of Kir6.2 upon osteogenic differentiation wasconfirmed by Western blot analysis: whereas in undifferentiatedand adipogenic differentiated cells only weak signals in mem-brane samples were found, rather strong bands were found inosteogenic differentiated cells (Fig. 3B and C). Immunocytochem-istry was performed to determine expression of Kir6.2 channels inMSC. A significant increase of KATP subunit Kir 6.2 expression uponosteogenic differentiation was observed compared to undifferen-tiated MSC (Fig. 4). These findings support the results gained fromWestern analysis.

4. Discussion

Human bone marrow mesenchymal stromal cells (MSC) havegenerated a great deal of interest in the last few years. Due to theiraccessibility, expandability and ability to differentiate, MSC hold

promise for clinical applications. A better understanding of theirbiological characteristics and differentiation may facilitate saferand more reliable medical treatment. There are currently four areasfor potential medical use under exploration: local implantation

334 A. Diehlmann et al. / Tissue and Cell 43 (2011) 331– 336

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Fig. 3. Quantitative KATP channel expression analysis after osteogenic and adi-pogenic differentiation. (A) Quantitative PCR analysis of undifferentiated anddifferentiated mesenchymal stem cells. The relative expression change (indicatedas ��Ct value or in percent) compared to undifferentiated cells is given for adi-pogenic (grey, black dots) and osteogenic (black, white dots) differentiated cells.SEM is given, and evaluation by Student’s t-test is indicated *p < 0.05, **p < 0.01,***p < 0.001 (8–9 samples from 3 different donors were used for data evaluation).(B) Western blot analysis of the expression of Kir6.2 in undifferentiated and adi-pogenic and osteogenic differentiated mesenchymal stem cells (samples from three

ig. 2. Western blot analysis of the expression of KATP channel subunits in undiffer-ntiated mesenchymal stem cells. A molecular weight marker (Dual color, Bio-Rad)nd the tested KATP channel subunit are indicated.

f MSC for localized diseases, systemic transplantation, combin-ng stem cell therapy with gene therapy and use of MSC in tissuengineering protocols.

ATP sensitive potassium channels reside in the plasma mem-rane of many cells types such as heart cells, skeletal muscle cellsnd neurons, where they contribute to membrane potential andlectrical excitability. KATP channels also play an important rolen glucose-mediated insulin secretion and several lines supporthe hypothesis that they modulate pancreatic islet cell differentia-ion. Mice lacking Kir6.2 show abnormal differentiation of islet cellsWinarto et al., 2001). Pathophysiologically, KATP channelopathiesn human has been shown to directly affect metabolic homeostasis,mplicated in epilepsy formation and the development of dilatedardiomyopathies. In addition, developmental delay and neona-al diabetes may indicate a more direct role of KATP channels inody growth and stem cell differentiation (Olson and Terzic, 2010).his study demonstrates that Kir6.2 (together with the sulfonylureaeceptor SUR2A) is the prevailing component of inwardly rectifyingotassium channels in MSC, whereas Kir6.1 is expressed at lower

evel. Furthermore, Kir6.2 channels are significantly up-regulatedy osteogenic differentiation. It is conceivable, that this channel is aegulator for osteogenic differentiation. Many experimental mod-ls in human cell culture have provided substantial evidence thatsteogenic differentiation is a process accompanied by coordinatedhanges in cell morphology, hormone sensitivity and gene expres-ion (Krause et al., 2010; Komori, 2006). Therefore bone has also

n important metabolic function for the body in balancing serumevels of Ca2+, K+ and phosphate ions (Rosen and Bouxsein, 2006).

Increase in potassium conductance impacts on cellular Ca2+

nflux by inactivation of voltage-activated Ca2+ channels due to

different donors). A molecular weight marker (Dual Color, Bio-Rad) is indicated.(C) Quantitative analysis of the Western blot shown in panel (B) using LabImagesoftware (lowest data point used as calibrator).

a stabilization of the membrane resting potential. Indeed, sev-eral voltage-sensitive Ca2+ channels of L-type (Cav1.2) and T-type(Cav3.2) have been described as the main component of osteoblastCa2+ permeability (Shao et al., 2005; Bergh et al., 2006; Jorgensenet al., 2003; Gu et al., 2001). Therefore, KATP channel subunits maybe a key regulator coupling metabolism to Ca2+ influx and boneformation by inactivating voltage-sensitive Ca2+ channels. Inter-estingly, a potent blocker of L-type Ca2+ channels has been shownto promote osteoblast differentiation (Nishiya et al., 2002). Thus,KATP channel subunits may provide alternative targets for pharma-cological intervention to control MSC differentiation.

Indeed recent evidence for the presence of KATP channels in

osteoblasts are accumulating. Especially the action of Calcitoningene-related peptide (CGRP) was shown to increase potassiumefflux through KATP channels (Kawase et al., 1996; Kawase andBurns, 1998). In addition a functional role of KATP channels sub-

A. Diehlmann et al. / Tissue and Cell 43 (2011) 331– 336 335

Fig. 4. Expression analysis of Kir6.2 in MSC. MSC populations were cultured until passage two and four, respectively. MSC without differentiation treatment were fixedimmediately and analyzed with Kir6.2 antibody (red). The other populations were grown in osteogenic differentiation medium for 21 days until mineralization appearedand then analyzed with Kir6.2 antibody. Nuclear staining is done with DAPI (blue). (A) Passage 2, undifferentiated MSC, (B) passage 2, osteogenic differentiated MSC, day2 t field5 end, t

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1, (C) passage 4, undifferentiated MSC, insert in C shows representatively the brigh �m, 100× objective. (For interpretation of the references to color in this figure leg

nits, mainly Kir6.2, has been suggested in mitochondria of humansteoblast-like cells (Butler et al., 2010). Other ion channels haveeen implicated with osteogenic differentiation in human andodent MSC. A direct role has been described for the chloridehannels ClC-3 (Wang et al., 2010) and CLIC1 (Yang et al., 2009)nd for the Transient Receptor Potential family member TRPM7Cheng et al., 2009). In addition several voltage-activated calciumZahanich et al., 2005), sodium and potassium currents (Li et al.,006) have been demonstrated.

In summary, KATP-channels play a central role in the couplingf the metabolic state to the membrane potential and they reg-late several physiological properties such as intracellular Ca2+

evels and excitability. Besides SUR2A, Kir6.2 is the main com-onent of KATP channels in undifferentiated cells and the Kir6.2xpression is highly up-regulated upon osteogenic differentiation.harmacological modulation of KATP-channels may therefore pro-ide a new perspective for regulation of MSC in cellular therapy and

issue engineering. However several limitations should be takennto account: (1) the transfer of our observations from cell cultureo native tissue is not easy. Therefore, according to the exper-ments described for cardiac development (Chung et al., 2010),

image of the cell (D) passage 4, osteogenic differentiated MSC, day 21. Scale bar ishe reader is referred to the web version of the article.)

similar analysis of metabolic shifts and electrophysiological prop-erties of osteoblasts in native bone tissue are needed. (2) Furtherexperimental limitations are bone marrow donor variability anddifferences in cell preparation, culturing methods and in vitro dif-ferentiation (Wagner and Ho, 2007). (3) Functional tests such asinvestigation of rodent models (e.g. knockout mice of KATP chan-nel subunits) would provide a better insight in the role of KATPchannels in osteogenic differentiation. Therefore the physiologicalrole of KATP channels in differentiation of MSC needs to be furtherelucidated.

Acknowledgements

This work was supported by the Heidelberg Academy of Sci-ences (WIN-Kolleg), by the German Ministry of Education andResearch (BMBF) within the supporting program “cell based regen-

erative medicine” (START-MSC2 and CB-HERMES) and by the StemCell Network North Rhine Westphalia. We are indebted to SemaÜnsal for technical help and to Annett Kaphahn for editorialhelp.

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