Experimental Hematology 35 (2007) 385–393

Bcl-2 and Bcl-XL are indispensable for the late phaseof mast cell development from mouse embryonic stem cells

Christine Mollera, Mats Karlberga, Magnus Abrinkb,Keiichi I. Nakayamac, Noboru Motoyamad, and Gunnar Nilssona

aDepartment of Medicine, Karolinska Institutet, Stockholm, Sweden;bDepartment of Molecular Biosciences, Swedish University of Agricultural Sciences,

Uppsala, Sweden; cDivision of Cell Biology, Department of Molecular and Cellular Biology,

Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; dDepartment of Geriatric Research,

National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan

(Received 3 May 2006; revised 16 November 2006; accepted 16 November 2006)

Objective. The aim of this study was to determine the importance of the prosurvival factorsBcl-2 and Bcl-XL for mast cell development and survival.

Methods. bcl-x�/� and bcl-2�/� mouse embryonic stem cells were maintained in medium sup-plemented with either interleukin (IL)-3 or IL-3 in combination with stem cell factor (SCF) tofavor mast cell development. The development of Bcl-2 family deficient embryonic stemcell-derived mast cells (ESMCs) was monitored and Bcl-2 family gene expression and cellnumbers were analyzed.

Results. Deficiency in either bcl-x or bcl-2 totally inhibited the development of ESMCs whenIL-3 alone was used as a mast cell growth factor. Intriguingly, when IL-3 was used in combi-nation with SCF, the ESMCs developed normally the first 2 weeks but thereafter the cell num-bers dropped drastically. The remaining ESMCs express mouse mast cell protease 1,suggesting a mucosal-like phenotype. ESMCs lacking bcl-x or bcl-2 exhibited strong expres-sion of A1, another prosurvival Bcl-2 family member.

Conclusion. For the first time we provide direct evidence that both bcl-x and bcl-2 are indis-pensable for mast cell survival during the late phase of their development. � 2007 Interna-tional Society for Experimental Hematology. Published by Elsevier Inc.

Cell functions are regulated by soluble factors and cell–cellinteractions dictating the outcome of the signals (e.g., se-cretion, proliferation, differentiation, and apoptosis/cell sur-vival). One way of regulating apoptosis is through the Bcl-2family, which consists of both prosurvival (e.g., Bcl-2, A1/Bfl-1, Bcl-XL, and Mcl-1) and prodeath (e.g., Bcl-XS, Bad,Bim, Bax, Bik, PUMA, and Noxa) members. These pro-teins may either serve as sensors of apoptotic stimuli or reg-ulators of the release of apoptotic factors [1–3]. The ratioand interactions between the anti- and proapoptotic mem-bers determine whether a cell will live or die.

Mast cells are inflammatory cells with a long life spanand the capacity to survive immunoglobulin (Ig) E-medi-ated activation during an allergic reaction [4]. Stem cell

Offprint requests to: Christine Moller, Ph.D., Karolinska Institutet,

Department of Medicine, Clinical Immunology and Allergy Unit, KS

L2:04, SE-171 76 Stockholm, Sweden; E-mail: Christine.Moller@ki.se

0301-472X/07 $–see front matter. Copyright � 2007 International Society for

doi: 10.1016/j.exphem.2006.11.008

factor (SCF) is the most important growth and differentia-tion factor for mast cells and also functions as a survivalfactor for mast cells [5,6]. SCF acts as a survival factorfor mast cells by repressing programmed cell death throughdownregulation and phosphorylation of the proapoptoticprotein Bim [7]. In contrast, activation-induced survivalof mast cells via FceRI is dependent on the prosurvivalgene A1/Bfl-1 [8]. To date, the role for other Bcl-2 familymembers in regulating mast cell survival is not clear.

Bcl-XL and Bcl-2 are two Bcl-2 family members thathave antiapoptotic responses and can suppress cell deathto a variety of apoptotic stimuli such as g-irradiation andgrowth factor deprivation [9,10]. However, the bcl-x genecan also be alternatively spliced into Bcl-XS, which pos-sesses proapoptotic functions [9]. Treatment of mast cellswith SCF increases the protein levels of Bcl-XL and Bcl-2 [11], whereas withdrawal of SCF from the cells leadsto a reduced expression of these prosurvival proteins [12].These results suggest, but do not prove, that SCF regulates

Experimental Hematology. Published by Elsevier Inc.

386 C. Moller et al. / Experimental Hematology 35 (2007) 385–393

Bcl-2 and Bcl-XL expression and thereby mast cell sur-vival. Furthermore, bone marrow mast cells of patientswith systemic mastocytosis have been shown to highly ex-press Bcl-XL [13]. In addition, Bcl-2-deficient mice havereduced numbers of mucosal mast cells compared withwild-type [14], and neutralization of Bcl-2 intracellularlyinhibits its antiapoptotic activity, rendering bone marrow–derived cultured mast cells (BMCMCs) susceptible to apo-ptosis [15]. Furthermore, mast cells derived from vav-bcl-2transgenic mice are not sensitive to apoptosis induced bygrowth factor withdrawal [16]. Thus, Bcl-2 and Bcl-XLmight play a role in dictating mast cell numbers in differentdiseases.

To determine the importance of Bcl-2 and Bcl-XL in theearly and late phases of mast cell development, we usedembryonic stem (ES) cells, which provide a unique systemfor studying the effects of mutations in genes expressed inany cell line, including mast cells. bcl-2- and bcl-x-deficientES cells [17,18] were cultured in the presence of interleukin(IL)-3 and SCF or IL-3 alone to favor mast cell development[19,20]. ES cells were used for the development of mast cellsbecause mice that lack bcl-x die at about embryonic day 13 asa result of massive apoptotic cell death during the develop-ment of the nervous and hematopoietic systems [17]. More-over, bcl-2 mutant mice are smaller but viable, althoughabout half of them die by 6 weeks of age [21].

Here we show that the prosurvival Bcl-2 family mem-bers Bcl-XL and Bcl-2 have an essential role for mastcell survival during their development in vitro. In the ab-sence of functional bcl-2 or bcl-x genes, the number ofES cell-derived mast cells (ESMCs) developed is highly re-duced compared with wild-type (wt) ESMC cultures. Themagnitude of the decrease of ESMCs is dependent on thecytokines added during ESMC development. In addition,the absence of Bcl-XL and Bcl-2 leads to an increase ofA1, another antiapoptotic Bcl-2 family member. Thus, theantiapoptotic family members seem to compensate for theloss of other proteins in the family to maintain cell survival.

Materials and methods

Mast cell culturesE14 ES cells derived from strain 129/Ola were used to produce wt,bcl-x�/�, and bcl-2�/� ES cell lines, as described previously[17,18]. The cells were maintained undifferentiated in cell culturedishes in ready-made Dulbecco’s Modified Eagle’s Medium con-taining glucose, glutamax-1, and sodium pyruvate (Invitrogen,Paisley, Scotland, UK), supplemented with 20% ES cell qualifiedfetal calf serum (FCS; Invitrogen), 0.1 mM nonessential aminoacids, 450 mM monothioglycerol (Sigma, St. Louis, MO, USA),and 1000 U/mL recombinant mouse leukemia inhibitory factor(Chemicon International, Temecula, CA, USA). To induce devel-opment of ESMCs, we used a modified protocol, previously pub-lished by Tsai et al. [22]. Instead of using a semisolid medium togrow embryonic bodies (EBs) in hanging droplets, 2000 ES cells

were plated in bacterial dishes in 100 mL liquid Iscove’s ModifiedDulbecco’s Medium (Sigma) droplets containing 10% FCS, 2 mML-glutamine, 5 ng/mL IL-11, 50 ng/mL SCF (PeproTech, London,England), 50 mg/mL G-418, and 450 mM monothioglycerol toenable the formation of EBs. Seven days later IMDM containing10% FCS, 2 mM L-glutamine, 30 ng/mL IL-6, 30 ng/mL IL-3,50 ng/mL SCF, 50 mg/mL G-418, and 450 mM monothioglycerolwas added. To induce the development ESMCs from EBs, 30EBs from each clone were let to attach to the plastics of tissue cul-ture dishes containing DMEM (Sigma) supplemented with 10%FCS, 2 mM L-glutamine, and 450 mM monothioglycerol, 50 ng/mL SCF, and/or 30% IL-3 containing WEHI-3 cell-conditionedmedium. Once a week all suspensional cells were transferredfrom the mother plates (i.e., the plates with attached EBs) intoempty cell culture dishes and new medium was added to thenew dishes and to the mother plates. Each week the newly devel-oped suspensional cells were removed from the mother plates(containing attached EBs) and ESMC numbers were determinedby toluidine blue staining. BMCMCs were obtained as describedpreviously [7].

Flow cytometryThe cells developed from the EBs were analyzed by flow cytom-etry for cell surface expression of FceRI (anti-mouse FceRI-FITCeBiosciences, San Diego, CA, USA) and c-Kit/CD117 (anti-mouse CD117-FITC, Pharmingen, San Diego, CA, USA). Iso-type-matched mouse antibodies IgG1 (eBiosciences) and IgG2B

(Pharmingen) were used to detect nonspecific backgroundfluorescence.

Cell countThirty EB from the wt, bcl-x�/�, and bcl-2�/� clones were trans-ferred to tissue culture dishes allowing the EBs to attach to theplastics and start developing suspensional ESMCs. Each weeknewly developed cells were transferred from the mother plates(i.e., the plates containing attached EBs) and the number of livingsuspensional was determined by trypan blue exclusion. For statis-tical analysis, we used the Student’s t test.

Analysis of RNA expression by reverse-transcriptasepolymerase chain reaction and RNAse protection assayTotal RNA was extracted by using TriPure isolation reagent(Roche, Mannheim, Germany). For reverse-transcriptase polymer-ase chain reaction (RT-PCR), 1 mg of RNA was reverse-transcribedin a 20-mL reaction by oligo-p(dT)15 priming using the first-standcDNA synthesis kit for RT-PCR (Roche) according to manufac-turer’s protocol. The primers used for fragment amplification ofmouse mast cell protease 1 (mMCP-1) were 50-GGAAAACTGGAGAGAAAGAACCTAC-30 (upper) and 50-GACAGCTGGGGACAGAATGGGG-30 (lower) spanning a region of 460 bp [23];for mMCP-5 50-AGGAGCCCATAACAAAACAT-30 (upper) and50-TATTCCAGTTCCAGATTTCC-30 (lower) spanning a regionof 562 bp; for carboxypeptidase A (CPA) 50-CTCAGACCATCCAGTCAACCT-30 (upper) and 50-GCCACAGTCCATAAAGATAGCC3-30 (lower) spanning a region of 299 bp; for A1 50-AATTCCAACAGCCTCCAGATATG-30 (upper) and 50-GAAACAAAATATCTGCAACTCTGG-30 (lower) spanning a region of743 bp [24]; and for Mcl-1 50-GCTCCGGAAACTGGACATTA-30

(upper) and 50-CCCAGTTTGTTACGCCATCT-30 (lower) span-ning a region of 92 bp [25]. PCR Core Kit (Roche) was used

387C. Moller et al./ Experimental Hematology 35 (2007) 385–393

for the amplification steps involving denaturation for 5 minutes at94�C followed by 30 cycles of denaturation for 30 seconds at94�C; annealing for 45 seconds at 53�C for mMCP-5, at 56�Cfor Mcl-1, at 57�C for A1, at 62�C for CPA, and at 63�C formMCP-1; extension for 1 minute at 72�C, and a final extensionfor 7 minute at 72�C. The PCR products were analyzed on a 2%agarose gel.

The RNA was also analyzed by an RNAse protection assay(RPA) using a Bcl-2 family-customized template set (Pharmingen)as described elsewhere [26]. Quantification of mRNA expressionwas determined by using a PhosphorImaging device and the levelsof each gene transcript were quantified by MacBasV2.2 (FujiPhoto Film Co. Ltd., Japan).

Results

Cell growth from wt, bcl-x�/�, and bcl-2�/� ES cellsES cells of wt, bcl-x�/�, and bcl-2�/� genotype were ana-lyzed for their ability to develop into ESMCs in the pres-ence or absence of SCF. ES cells of all the genotypesproliferated and differentiated similarly into EBs (data notshown). After 2 weeks, the EBs were plated onto cell

culture dishes, which allowed them to attach and spreadon the dishes. To promote mast cell development, theEBs were maintained in medium supplemented with IL-3 6

SCF [19,20]. In the presence of both IL-3 and SCF, theEBs of all genotypes had already given rise to cells insuspension at week 2 (Fig. 1), although the bcl-2�/� cul-tures had proliferated to a much lesser extent than the wtand the bcl-x�/� cultures. In the absence of SCF (i.e., onlyIL-3), hardly any cells in suspension had developed evenat week 6 from the bcl-x�/� and bcl-2�/� cells (Fig. 1). Incontrast, wt cultures grown in IL-3 alone were unaffectedby the lack of SCF (Fig. 1). Thus, cells can develop fromES cells mutated for either bcl-x or bcl-2, but the cellnumbers are considerably decreased compared to wt.This decrease in cell numbers depended on both the lossof the prosurvival genes and the presence or absence ofSCF.

Mast cells develop from ES cellswith disrupted bcl-x or bcl-2 genesTo determine if the cells that developed from the EBs of wt,bcl-x�/� and bcl-2�/� genotypes were ESMCs, they were

Figure 1. Differentiation of wt, bcl-2�/�, and bcl-x�/� ES cells via EBs into ESMC. Two weeks after the EBs had been plated onto culture dishes, many cells

in suspension were generated from the wt and bcl-x�/� cultures, although only few emerged in bcl-2�/� cultures maintained in SCF- and IL-3-supplemented

medium. Even 6 weeks after the EBs had been plated onto culture dishes, limited numbers of suspensional cells developed in bcl-x�/� and bcl-2�/� cultures

maintained in only IL-3-supplemented medium. In contrast, in wt cultures numerous amounts of cells developed each week. Similar results were seen in four

independent experiments. Original magnification �40.

388 C. Moller et al. / Experimental Hematology 35 (2007) 385–393

stained every week with toluidine blue. The cells of allgenotypes cultured in IL-3 and SCF and wt cells culturedin IL-3 alone exhibited morphologic characteristics similarto those of BMCMCs produced in IL-3 (Fig. 2). By week 2the number of ESMCs in the wt, bcl-x�/�, and bcl-2�/� cul-tures produced in IL-3 and SCF were 90.1 6 2.2, 92.7 6

1.8, and 92.3 6 2.0, respectively. By week 6, 94.7 6 2.0of the cells in the wt culture produced in IL-3 alone wereESMCs. Furthermore, all of these cells expressed mRNAfor the mouse mast cell proteases mMCP-1, mMCP-5,and CPA (Fig. 3), thus rendering the ESMCs as mucosal-like mast cells.

Compared to mouse BMCMCs [16] and wt ESMCs thathad been produced in IL-3, the ESMCs of wt and bcl-x�/�

genotypes produced in IL-3 and SCF expressed similarlevels of the high-affinity receptor for IgE, FceRI, and c-Kit on the cell surface (Fig. 4). bcl-2�/� ESMCs producedin IL-3 and SCF had to some degree lower expression ofFceRI and c-Kit (Fig. 4). Thus, our data suggest that cellsof all genotypes produced in IL-3 and SCF and wt cellsproduced in IL-3 alone are ESMCs.

ESMC numbers are not only affectedby loss of prosurvival bcl-x or bcl-2 butalso by the growth factors used during maturationThirty EBs from each of the different clones were transferredto tissue culture dishes in order to enable the formation ofESMCs. Once a week the newly produced suspensionalESMCs were transferred from the mother plate (i.e., the platecontaining the attached ESMC-producing EBs) into new cul-ture dishes and the ESMC numbers were counted. AttachedEBs of all genotypes cultured in the presence of both IL-3and SCF gave rise to suspensional ESMCs after 2 weeks in

culture (Fig. 5A). However, the number of ESMCs generatedfrom the bcl-x�/� and bcl-2�/� ES cells peaked at week 2(45.9 � 105 6 1.35 � 105, and 74.6 � 105 6 4.33 � 105,respectively) and then declined. In contrast, in cultureswith wt cells the number of newly developed ESMCs contin-ued to increase until weeks 5 to 8 (Fig. 5A), when cell devel-opment reached a plateau at approximately 90 � 105 cells.

The bcl-x�/� and bcl-2�/� cultures maintained in mediumsupplemented with only IL-3 gave rise to almost no ESMCsduring the entire 9-week culture period studied (Fig. 5B). Incontrast, the wt cultures launched massive cell production atweek 5 that peaked at week 8 (130.3 � 105 6 75 � 105;Fig. 5B). The onset of ESMC development from wt culturesgenerated in IL-3 was delayed compared with wt ESMCgenerated in both IL-3 and SCF (Fig. 5A and B). The firstwt ESMCs were determined in the IL-3 cultures at week 5compared with week 2 for wt cells generated in IL-3 andSCF. Thus, wt ES cells or ES cells with bcl-2 or bcl-xmutated genotype give rise to ESMCs, though with differentonset, magnitude, and decline of ESMC production depend-ing on both genotype and culturing conditions.

The prosurvival A1 gene expressionis induced in ESMCs deficient in bcl-x or bcl-2To explore whether the expression levels of the pro- or anti-apoptotic Bcl-2 family members in ESMCs are affected bythe loss of functional bcl-2 or bcl-x genes, total RNA wasprepared from ESMCs, and gene expression levels wereexamined by RPA (Fig. 6A). At all time points explored,the antiapoptotic Bcl-2 family member A1 was upregulatedin the bcl-x�/� and bcl-2�/� ESMCs produced in IL-3 andSCF compared with wt (Fig. 6A and data not shown). Byweek 6, the expression of prosurvival Bcl-w had declined

Figure 2. Similar toluidine blue staining patterns among ESMCs of all genotypes as well as BMCMCs. Cytocentrifuged preparations of wt, bcl-x�/�, and

bcl-2�/� ESMCs developed after 2 weeks in cultured in SCF and IL-3, wt ESMCs developed after 6 weeks in culture in IL-3 alone, and BMCMCs cultured

for 4 weeks in IL-3 alone were stained with toluidine blue. Comparable results were obtained in at least three separate experiments.

389C. Moller et al./ Experimental Hematology 35 (2007) 385–393

Figure 3. ESMCs developed in vitro express mast cell specific proteases.

RT-PCR analysis of CPA, mMCP-1, and mMCP-5 gene expression on wt,

bcl-x�/�, and bcl-2�/� ESMCs developed in SCF and IL 3 or wt ESMCs

developed in IL-3 alone. Comparable results were obtained in three sepa-

rate experiments.

in all cells produced in IL-3 and SCF except for wt cells.A further increase of antiapoptotic A1 and Bcl-XL between2 and 6 weeks of culture was seen only in bcl-2�/� ESMCs,although proapoptotic Bax and Bad increased in all ESMCs(Fig. 6A and B). The expression of Bcl-2 family membersin wt ESMC produced in IL-3 alone did not differ from wtESMC produced in both IL-3 and SCF (Fig. 6A and datanot shown).

The balance between pro and antiapoptotic Bcl-2 familymembers is thought to determine whether a cell will live ordie. The prosurvival factor Mcl-1, which shares severalsimilarities with A1, is not included in the RPA templateset and we therefore analyzed its expression by RT-PCR.We confirmed the induced expression of A1 in bcl-x�/�

and bcl-2�/� ESMCs by RT-PCR (Fig. 7). In contrast toinduced expression of A1, we found that Mcl-1 mRNAwas constitutively expressed in all ESMCs and thus nodifference was detectable among wt, bcl-x�/�, or bcl-2�/�

ESMCs produced in IL-3 and SCF (Fig. 7). Thus, antiapop-totic A1 was the only prosurvival gene analyzed that wasupregulated in bcl-x�/� and bcl-2�/� ESMCs comparedwith wt ESMCs in which no A1 mRNA was detectable.

DiscussionIn the present study the importance of the bcl-x and bcl-2genes for mouse mast cell development and survival was in-vestigated using ES cells. We not only showed that the pro-survival genes bcl-x and bcl-2 are important for mast celldevelopment and survival but also that the culturing

Figure 4. Wt and bcl-x�/� ESMCs express FceRI and c-Kit to an extent similar to that of BMCMCs. By flow cytometric analysis, the receptor expression of

c-Kit and FceRI was analyzed on wt, bcl-x�/�, and bcl-2�/� ESMCs generated after 2 weeks in IL-3 and SCF and wt ESMCs and BMCMCs generated after 6

weeks in IL-3 alone. Filled peaks 5 isotype-matched controls. Upper panel: empty peaks 5 FceRI expression. Lower panel: empty peaks 5 c-Kit expression.

The results shown represent three independent experiments.

390 C. Moller et al. / Experimental Hematology 35 (2007) 385–393

conditions during cell development affect the outcome.When SCF is added, ES cells with disrupted bcl-x or bcl-2 genes can differentiate into ESMCs, and the developmentis accompanied by an induction of A1 expression.

Figure 5. Different cell numbers in ESMC cultures produced in IL-3 and

SCF or IL-3 alone. Once a week newly developed cells in suspension were

removed from the mother cell culture dishes with attached EBs, and the

total number of collected cells (A) produced in IL-3 and SCF or (B) pro-

duced in IL-3 alone was counted separately each week. The experiment

was repeated three times and the data shown correspond to the mean 6

standard error of the mean of three the experiments. Significant effects

compared with bcl-2�/� were obtained as indicated (*p ! 0.05, **p !0.01, ***p ! 0.001).

Bcl-XL, Bcl-2, and other Bcl-2 family members havepreviously been described to be expressed in mast cellsand therefore suggested to regulate mast cell survival andapoptosis [8,11,13,16,27,28]. However, there is a discrep-ancy in the literature on the role of different Bcl-2 familymembers in the regulation of cell survival. This differencemay be the result of the experimental setup, the use ofmast cells from different species, and the fact that mastcells do not represent a homogeneous population. Theycan have different phenotypes and functions, even in thesame tissue, which are dictated by the influence of the sur-rounding microenvironment and the cytokine milieu [29].

SCF and IL-3 are two major growth, differentiation, andsurvival factors for mast cells. SCF is a prerequisite for

Figure 6. A1 mRNA expression is upregulated in bcl-x�/�, and bcl-2�/�

ESMCs compared with wt cells. (A): An RPA was performed to investigate

the expression levels of indicated genes on total RNA from ESMC popu-

lations arising 2 and 6 weeks after the EBs had been maintained in IL-3

and SCF or 6 to 9 weeks after maintained in IL-3 alone. (B): The changes

in gene expression levels are shown as the ratio of week 6 to week 2, rel-

ative to the expression levels of the house keeping genes L32 and GAPDH.

The results shown are representative of three independent experiments.

391C. Moller et al./ Experimental Hematology 35 (2007) 385–393

mast cell development in vivo [30,31], whereas IL-3 con-tributes to the expansion of the mast cell population uponinfection [32]. However, if injected into mast cell-deficientmice, IL-3 stimulates the development of mast cells [33].Both IL-3 and SCF have been used in protocols to developESMCs [19,20]. In our study, we found that differentiationof ESMCs in the presence of SCF and IL-3 is independentof Bcl-2 and Bcl-XL expression during the first 2 weeks ofdevelopment, whereas the later stage depends on a func-tional expression of Bcl-2 and Bcl-XL. However, in thepresence of only IL-3, ESMCs do not develop in the ab-sence of bcl-2 or bcl-x genes. Thus, SCF, but not IL-3 alone,can promote survival and differentiation of ESMCs defi-cient in either bcl-2 or bcl-x genes during the first 2 weeksof their development.

It appears that IL-3 and SCF promotes cell survival bydistinct mechanisms. IL-3 regulates the expression ofprosurvival Bcl-2 family members [34,35], and SCF sup-presses the levels of proapoptotic Bim [7]. Human stemcells need at least two signals for their survival, one pro-vided by Bcl-2 and one by SCF [36], which seems to besimilar to our findings. IL-3-dependent BMCMCs can berescued by SCF when IL-3 is withdrawn [5]. The combina-tion of both IL-3 and SCF has been shown to be essentialfor the development and survival of BMCMCs fromSTAT5�/� mice; cultures in either cytokine alone lead toreduced expression of Bcl-2 and Bcl-XL and increasedapoptosis [37].

In vivo, bcl-2 deficiency affects mast cell numbers dif-ferently depending on mast cell type (i.e., connective tissue

Figure 7. Mcl-1 mRNA levels are not affected by the loss of functional

bcl-x or bcl-2. RT-PCR was performed to study the mRNA expression of

pro-survival Mcl-1 and A1. Mcl-1 was equally expressed in ESMCs of dif-

ferent genotypes developed in IL-3 and SCF or IL-3 alone, although A1

was induced in bcl-x�/� and bcl-2�/� ESMCs. The results shown are rep-

resentative of two independent experiments.

or mucosal mast cells) [14]. Bcl-2�/� mice have about 50%fewer mucosal mast cells in the stomach mucosa comparedto wt, although the number of connective tissue mast cellsin skin is unaffected [14]. The developed ESMCs in thisstudy had a phenotype resembling mucosal-like mast cells.The diminished development and survival of bcl-2- orbcl-x-deficient ESMCs thus resembles the in vivo results.We are currently exploring the possibility to developESMCs with a connective tissuelike mast cell phenotype,to investigate the impact of bcl-2 and bcl-x on the survivaland development of these cells.

Cell fate is thought to be dictated by the balancebetween the antiapoptotic and proapoptotic Bcl-2 familymembers. However, up to now there have been no reportsaddressing whether Bcl-2 family members can compensatefor the loss of other family members and sustain cellhomeostasis and apoptosis/survival. Our studies show forthe first time that the antiapoptotic Bcl-2 family memberA1 is induced in ESMCs that lack Bcl-2 or Bcl-XL.Thus, A1 might compensate for the loss of other antiapop-totic family members in the early development of ESMCs.However, increased A1 expression is not enough to sustainESMC development for a longer time because the numberof newly developed ESMCs declines over time even thoughA1 expression remains high.

In contrast to A1, we could not observe any changes in theexpression levels of Mcl-1. Mcl-1 has several functional sim-ilarities with A1, and we have recently described that Mcl-1is upregulated together with Bfl-1 (the human counterpart ofA1) upon FceRI-mediated activation-induced mast cell sur-vival [38]. All ESMCs exhibited constitutive expression ofMcl-1 independent of genotype. Sustained Mcl-1, Bcl-2,and Bcl-XL expression has previously been reported forhematopoietic progenitor cells in the presence of IL-3 [35].

The decrease in cell development, even in the presenceof high A1 expression, can be explained by another inter-esting finding. Although A1 was at a consistently high levelall the time, the mRNA level for prosurvival Bcl-w declinedby week 6 in bcl-2�/� and bcl-x�/� ESMCs. In anotherstudy, SCF has been reported to support germ cell survivalduring spermatogenesis by upregulating the prosurvivalBcl-2 family proteins Bcl-w and Bcl-XL and downregulat-ing proapoptosis Bcl-2 family proteins [39]. Therefore, thereduction of ESMC development over time can result fromthe fact that SCF cannot maintain Bcl-w expression fora longer time period in the absence of bcl-2 or bcl-x. How-ever, A1 expression, which we previously have shown to beunaffected by SCF or IL-3 stimulation [8], did not changeover time. Thus, the high expression levels of A1 appar-ently are not enough to compensate for the loss of yetanother prosurvival factor.

None of the proapoptotic Bcl-2 family members investi-gated in the current work were induced at the time point ofreduced ESMC development and cannot explain the drop incell number at this time point. Though an increase of

392 C. Moller et al. / Experimental Hematology 35 (2007) 385–393

prodeath Bax, Bim and Bad mRNA could be detected atweek 6 in bcl-2�/� and bcl-x�/� ESMCs developed inSCF and IL-3. However, SCF is previously known to phos-phorylate Bad and Bim, thus inhibiting their proapoptoticfunctions [7,40,41]. The role of different Bcl-2 familymembers during the early stages of ESMC developmentand the specific events regulating the survival of ESMCaround week 2 will be further investigated.

AcknowledgmentsWe would like to thank Uppsala University Transgenic Facility forthe help in growing ES cells. This study was supported by grantsfrom the Swedish Research Council-Medicine, the Swedish Can-cer Foundation, the Swedish Cancer and Allergy Fund, ConsulTh C Berg’s Foundation, Ollie and Elof Ericsson’s Foundation,King Gustaf V’s 80-Years Foundation, Ellen, Walter and LennartHesselman’s Foundation, Swedish Animal Welfare Agency, andKarolinska Institutet.

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