The Cell Surface Glycosphingolipids SSEA-3 and SSEA-4 Are Not Essential for Huma - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 01:01 |只看该作者 |倒序浏览 |打印

作者：Sandii N. Brimblea, Eric S. Sherrera, Elizabeth W. Uhlb, Elaine Wangc, Samuel Kellyc, Alfred H. Merrill, Jr.c, Allan J. Robinsa, Thomas C. Schulza


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      【摘要】
   Pluripotent cells can be isolated from the human blastocyst and maintained in culture as self-renewing, undifferentiated, human ESCs (hESCs). These cells are a valuable model of human development in vitro and are the focus of substantial research aimed at generating differentiated populations for cellular therapies. The extracellular markers that have been used to characterize hESCs are primarily carbohydrate epitopes on proteoglycans or sphingolipids, such as stage-specific embryonic antigen (SSEA)-3 and -4. The expression of SSEA-3 and -4 is tightly regulated during preimplantation development and on hESCs. Although this might imply a molecular function in undifferentiated cells, it has not yet been tested experimentally. We used inhibitors of sphingolipid and glycosphingolipid (GSL) biosynthesis to block the generation of SSEA-3 and -4 in hESCs. Depletion of these antigens and their precursors was confirmed using immunostaining, flow cytometry, and tandem mass spectroscopy. Transcriptional analysis, immunostaining, and differentiation in vitro and in teratomas indicated that other properties of pluripotency were not noticeably affected by GSL depletion. These experiments demonstrated that the GSLs recognized as SSEA-3 and -4 do not play critical functional roles in maintaining the pluripotency of hESCs, but instead suggested roles for this class of molecules during cellular differentiation. - E3 h6 D  n+ q
      【关键词】 Cell surface markers Oct- expression levels Human embryonic stem cells Embryoid body Differentiation
   INTRODUCTION

Human ESCs (hESCs) are pluripotent cells derived from the inner cell mass of the blastocyst , this does not necessarily indicate functional involvement in the control of self-renewal.

Although hESCs grow in epithelial-like colonies and exhibit poor clonal plating efficiency . These studies rely on SSEA-3 expression being linked to the undifferentiated state but do not address the function of this marker. Similarly, no function associated with pluripotency has yet been ascribed to these other cell surface antigens.

SSEA-3 and SSEA-4 are epitopes on related glycosphingolipids (GSLs), termed GL-5 and GL-7  have important function(s) in early embryogenesis.0 b4 Y0 ]9 r) D; E* q% l
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To examine the potential functions of SSEA-3 and -4 in hESCs, we inhibited the biosynthesis of these antigens using PDMP or (2S),(3R),(4R),6E-2-amino-3,4-dihydroxy-2-hydroxymethyl-14-oxo-6-eicosenoic acid (ISP-1, also called myriocin). ISP-1 is a potent inhibitor of serine palmitoyltransferase (SPT) (supplemental online Fig. 1)  and blocks the synthesis of sphingolipids and ceramides as well as GSLs. These inhibitors caused the depletion of SSEA-3 and -4 and their biosynthetic precursors in undifferentiated hESCs. Cells grown in the presence of PDMP or ISP-1 could proliferate and maintain expression of other cell surface markers. PDMP treatment did not substantially alter the gene expression profile of hESCs or alter their capacity to differentiate in vitro. GSL-depleted hESCs differentiated to ectodermal, endodermal, and mesodermal lineages in vivo. Therefore, GSL depletion had no apparent effect on the pluripotent characteristics of hESCs, and the SSEA-3 and -4 GSLs do not have a critical function in the maintenance of the undifferentiated state.

MATERIALS AND METHODS: W6 g- J6 x7 U, f: I0 Y4 w; {2 T

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There are D- or L-, threo-, or erythro- enantiomers of PDMP, but only D-threo-PDMP inhibits GCS. D,L-threo-PDMP (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) was used in some experiments as indicated, whereas purified D-threo-PDMP (Matreya, Pleasant Gap, PA, www.matreya.com) was used for the majority of the study. ISP-1 (myriocin) was purchased from Sigma-Aldrich. PDMP was dissolved in ethanol, and ISP-1 was dissolved in dimethyl sulfoxide (DMSO) for inhibition experiments.  T4 z* n0 S+ S

Cell Culture and Differentiation% ?* z. ^& L" [& N; p8 w2 c9 U
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BG01, -02, and -03 cells . For depletion experiments, PDMP, ISP-1, or carrier was added to hESC cultures and changed daily for the course of the experiment.

Embryoid body (EB) differentiations were carried out as described . Animal research protocols (A2002-10203) were reviewed and approved by the University of Georgia (Athens, GA), and experiments were conducted according to institutional guidelines./ d6 F6 M* d$ b
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Immunocytochemistry and Gene Expression Analysis+ d9 o$ j' M% U# ^4 e2 T
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Immunostaining of hESC cultures for markers of pluripotency was performed as described previously . Briefly, cells were fixed in 4% paraformaldehyde, washed, blocked, and assayed with monoclonal antibodies specific for SSEA-3 (MC631, 1:100), SSEA-4 (MC813-70, 1:100), TRA-1-60 (1:100), TRA-1-81 (1:100) (all from Chemicon International, Temecula, CA, http://www.chemicon.com), or OCT4 (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com). Triton X-100 (0.3%) was used to permeabilize cell membranes for OCT4 staining. AlexaFluor-488-conjugated goat anti-mouse immunoglobulin M (IgM), IgG, or anti-rat IgM secondary antibodies were used (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Nuclei were stained with DAPI (4,6-diamidino-2-phenylindole). Equivalent image capture and processing were used for images within a comparative series./ `6 Q5 I$ }& \" R+ ?

Transcriptional profiling was performed using the GEArray S Series Human Stem Cell Gene Array (catalog number Hs-601.2; SuperArray Bioscience Corporation, Frederick, MD, http://www.superarray.com) in accordance with the manufacturer's instructions. RNA was prepared using the Trizol reagent (Invitrogen). Probes were generated using the GEArray Ampolabeling-LPR Kit (catalog number L-03; SuperArray Bioscience Corporation), and hybridization was detected using chemiluminescence (catalog number D-01; SuperArray Bioscience Corporation). The layout of the focused array is indicated in supplemental online Table 1. Comparative data analysis was performed using the GEArray Expression Analysis Suite (SuperArray Bioscience Corporation). Spots were detected, quantified, and normalized using the average of the three control glyceraldehyde-3-phosphate dehydrogenase and three ACTB spots on each array. Low-intensity spots were confirmed visually.
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Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed using Assays-On-Demand (Applied Biosystems, Foster City, CA, https://www2.appliedbiosystems.com) prequalified TaqMan primer sets (supplemental online Table 1). Amplification and detection used Micro-Fluidic cards in the ABI PRISM 7900HT sequence detection system (Applied Biosystems), as described previously . Relative expression levels were determined using the CT method.

Flow Cytometry

Expression of cell surface antigens was monitored using flow cytometry. Briefly, single-cell suspensions were prepared using 0.05% trypsin/EDTA and fixed in 4% paraformaldehyde on ice for 10 minutes. The cells were then washed twice with 5% fetal bovine serum (FBS)/phosphate-buffered saline (PBS) and resuspended at 2 x 106 cells per milliliter. Antibody stainings were carried out on 50 µl (105 cells) per well in 96-well trays. Antibodies to CD9, SSEA-3, SSEA-4, SSEA-1, TRA-1-81, and TRA-1-60 (Chemicon International) were used at a 1:100 dilution. Anti-isotype (anti-rat IgM, diluted 1:10 ) alone were used as negative controls. The cells were incubated with antibody for 30 minutes at 4¡ãC, washed three times with cold 5% FBS/PBS, incubated with the appropriated secondary antibody for 30 minutes at 4¡ãC, washed three times with cold 5% FBS/PBS, and resuspended in 100 µl of 5% FBS/PBS. Samples were analyzed in duplicate using the Cytomics CXP software on a Beckman Coulter cytomics FC500 (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). The t test was used for statistical analyses." {8 O1 P7 r8 C9 r
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Quantiation of Sphingolipid Metabolites by Mass Spectrometry
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The methods used for sample preparation, high-performance liquid chromatography (HPLC), and tandem mass spectrometry (MS/MS) analysis of sphingolipid metabolites have been reported in detail . For each sample, 106 cells were harvested and analyzed in quadruplicate. The level of each sphingolipid metabolite was quantified by comparison with a relevant internal standard. The t test was used for statistical analyses.% E* r0 T, i2 {8 h1 H* f

RESULTS

Depletion of the SSEA-3 and -4 Antigens in hESCs/ x! p$ R/ S0 O! [! _
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The National Institutes of Health (NIH)-approved BG01, -02, -03, and BG01v hESC lines were used in these experiments , BG01v cells are not transformed in the classic sense that EC cells are, as they can differentiate spontaneously in vitro without the requirement for induction with agents such as retinoic acid. These observations demonstrate that the BG01v cell line accurately models key aspects of normal hESCs and is useful for experimental analysis due to ease of culturing.. U  c1 ?0 D  c2 \% R8 O

BG01v cells cultured in 5¨C50 µM D,L-PDMP for 2 weeks exhibited normal morphology and maintenance of undifferentiated cells expressing OCT4 and TRA-1-60 (supplemental online Fig. 2). This demonstrated that D,L-PDMP did not cause overt differentiation of hESCs. SSEA-3 and -4 immunoreactivity was depleted by treatment with D,L-PDMP, which was first observed with 5 or 20 µM D,L-PDMP, respectively. D-PDMP was then used to deplete SSEA-3 and -4 in normal hESCs. BG03 cells were maintained in 20 µM D-PDMP for more than 2 months, and treated cells grew as undifferentiated colonies (Fig. 1A), with tightly packed cells exhibiting a characteristic high nucleus/cytoplasm ratio and distinct nucleoli (Fig. 1B). Expression of OCT4 and TRA-1-81 was apparently unaffected by 20 µM D-PDMP, whereas SSEA-3 and -4 immunoreactivity was substantially reduced compared with an untreated control culture (Fig. 1C). Similar results were observed with BG02 cells cultured in 20 µM D-PDMP for 5 days (not shown). GSL depletion did not markedly affect the morphology or proliferation of hESCs over prolonged culture periods, as BG01 and 02 cultures were also maintained in 20 µM D,L-PDMP for more than 2 months and BG01v cultures in D,L- or D-PDMP for more than 4 months or more than 2 months, respectively (not shown). Higher concentrations of PDMP slowed the proliferation of hESCs, and lower split ratios were used to enable simultaneous passaging of all conditions (supplemental online Table 2). This slower proliferation may be related to the inhibition of 1-O-acylceramide synthase by D-PDMP .

Figure 1. Depletion of stage-specific embryonic antigen (SSEA)-3 and -4 in human ESCs (hESCs). (A, B): Low- and high-magnification phase-contrast images of BG03 cells grown in 20 µM D-PDMP for more than 2 months. Normal colony morphology and tight packing of characteristic undifferentiated cells were exhibited. (C): Immunofluorescent staining of BG03 cells and cells grown in 20 µM D-PDMP for 4 days or in 1 µM ISP-1 (myriocin) for 8 days. Expression of OCT4 and TRA-1-81 was unaffected by these inhibitors, whereas SSEA-3 and -4 expression was depleted or extinguished. (D): Persistence, despite overall depletion, of foci of SSEA-4 staining (arrowheads) in glycosphingolipid-depleted hESCs. BG01v cells were treated with dimethyl sulfoxide (DMSO) or 1 µM ISP-1, immunostained for SSEA-4, and imaged using a x60 objective. Scale bars = 1 mm (A, C), 100 µm (B), 50 µm (D). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; D-PDMP, D-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; ISP-1, (2S),(3R), (4R),6E-2- amino-3,4-dihydroxy-2-hydroxymethyl-14-oxo-6-eicosenoic acid.3 B* o% t& e1 h; }* G' {: f

GSL depletion was also examined in hESCs using ISP-1 (myriocin), an inhibitor of SPT. BG01v cells were cultured in 1 nM to 1 µM ISP-1 for more than 2 weeks without any apparent affect on growth rate (supplemental online Table 2) or morphology (not shown). SSEA-3 and -4 immunoreactivity was depleted by 1 µM ISP-1 compared with a DMSO-treated control (supplemental online Fig. 2B). Expression of OCT4 (not shown) and TRA-1-81 (supplemental online Fig. 2B) was maintained. BG03 cells treated with 1 µM ISP-1 for 8 days maintained expression of OCT4 and TRA-1-81, but SSEA-3 and -4 immunoreactivity was depleted (Fig. 1C). Similar results were observed with BG02 cells cultured in 1 µM ISP-1 for 5 days (not shown).; ^* w* R0 t" p% B3 ~; T
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Other observations were made during this analysis. Depletion of SSEA-3 consistently occurred with lower concentrations of PDMP than observed for SSEA-4, and some SSEA-4 immunoreactivity remained (Fig. 1C), even at higher concentrations of inhibitor (50 µM D,L-, 20 µM D-PDMP). Although ISP-1 appeared to deplete SSEA-4 more effectively than PDMP, some SSEA-4 immunoreactivity also persisted when depleted cultures were examined under higher magnification (Fig. 1D). This was observed as localized punctate staining on some cells within PDMP- or ISP-treated cultures. These observations were not consistent with the biosynthetic relationship of GL-5 and GL-7 or with the demonstrated ability of the MC631 antibody (SSEA-3) to detect both of these molecules . Therefore, cells that were negative for SSEA-3 should also have been negative for SSEA-4. These apparent discrepancies could be explained by the presence of epitopes recognized by MC813-70 on additional nonsphingoid molecules in hESCs.
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Cytometric Analyses of SSEA-3 and -4 Depletion in hESC Cultures

Flow cytometry was used for more detailed analyses of the changes to SSEA-3 and -4 levels in BG01v cultures treated with 5¨C50 µM D,L-PDMP or 1 nM to 1 µM ISP-1 (Fig. 2). Firstly, the relative intensity of the SSEA-3  and SSEA-4  cells in the different treatments was examined by tracking the peak of the fluorescence distribution profile (termed X-Mode) (Fig. 2A, 2B). The intensity of SSEA-3 or SSEA-4 staining was significantly reduced in 5 µM D,L-PDMP (p 87%) or TRA-1-81 (>91%) (Fig. 2C, 2D). Conversely, the proportion of SSEA-3  cells decreased from 70.2% ¡À 0.2% in the ethanol control cultures to 9.5% ¡À 0.3% in 5 µM D,L-PDMP (p * P; W/ n; I  c

Figure 2. Flow cytometry analysis of cell surface antigens in BG01v cultures treated with PDMP or ISP-1. (A, B): Dose-responsive reduction in the peak of the fluorescence distribution (X-Mode) for stage-specific embryonic antigen (SSEA)-3 and -4 in BG01v cells grown in 5¨C50 µM D,L-PDMP for more than 1 month or in 1 nM-1 µM ISP-1 for 2 weeks, compared with ethanol (EtOH) or dimethyl sulfoxide (DMSO) carrier controls. (C, D): Percentages of cells expressing CD9, SSEA-1, -3, -4, or TRA-1-81 in these cultures. (E, F): Five-day time course of the depletion of SSEA-3 caused by 20 µM D-PDMP or 1 µM ISP-1, respectively. The controls demonstrated maintenance of TRA-1-81 expression in all treatments and maintenance of SSEA-3 expression in EtOH and DMSO carrier-treated cultures. Abbreviations: ISP-1, (2S),(3R),(4R),6E-2-amino-3,4-dihydroxy-2-hydroxymethyl-14-oxo-6-eicosenoic acid; PDMP, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol.3 Y2 H: m# ]/ Q' ?1 ^7 ^
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Quantitation of Sphingolipid Intermediates in hESCs
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HPLC-MS/MS was used to quantify components of the sphingolipid pathway in hESCs. Untreated BG01v cells were first compared with mouse embryonic fibroblasts (MEFs), a heterogeneous mix of embryonic day-12.5 differentiated mouse tissue. BG01v cells exhibited a profile of ceramides and sphingolipids that was distinct from that of MEFs (Table 1). Whereas the levels of sphinganine, sphinganine-1-phosphate, ceramide, and ceramide-1-phosphate (C1P) appeared relatively consistent, BG01v cells exhibited significantly lower levels of glucosylceramide (GC), sphingosine, sphingosine-1-phosphate (S1P), and sphingomyelin (SM) than did MEFs.
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Table 1. Sphingolipids in MEFs and human embryonic stem cells5 a. c% S. K1 R) t" t3 u
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HPLC-MS/MS was then used to examine the effects of 20 µM D-PDMP or 1 µM ISP-1 on sphingolipid intermediates in BG01v cultures, compared with the appropriate carrier controls (Fig. 3; Table 2). Because D-PDMP does not cause irreversible inhibition of GCS, a culture grown in the presence, then absence, of 20 µM D-PDMP was also examined ( /¨CPDMP). Restoration of uniform cell surface SSEA-4 was confirmed in a  /¨CPDMP culture by immunostaining (supplemental online Fig. 2C). D-PDMP treatment led to an approximately 90% reduction in GC, which was restored after withdrawal of inhibitor. Ceramide and SM levels were raised slightly by D-PDMP treatment, possibly reflecting an accumulation caused by inhibiting GCS. Conversely, ISP-1 treatment reduced ceramide content by approximately 96%, GC by approximately 98%, and SM by approximately 82%. This analysis confirmed that D-PDMP and ISP-1 inhibited the synthesis of GC, a mandatory biosynthetic intermediate in the generation of SSEA-3 and -4, without apparent affect on the maintenance of these cells. Several additional effects of D-PDMP and ISP-1 were identified (Table 2). Treatment with ISP-1 reduced the levels of all eight sphingolipid intermediates that were assayed. D-PDMP caused an increase in C1P as well as ceramide, but a decrease in sphinganine. The relative flux of these sphingolipid pathway intermediates in response to treatment with inhibitors is indicated in supplemental online Figure 1.

Figure 3. High-performance liquid chromatography-tandem mass spectrometry analysis of sphingolipid pathway intermediates in D-PDMP- or ISP-1-treated cultures. BG01v cells were grown in 20 µM D-PDMP or 1 µM ISP-1 for 19 days and compared with ethanol (EtOH)- or dimethyl sulfoxide (DMSO)-treated control cultures, respectively. A culture grown in the presence (19 days), then absence (8 days), of 20 µM D-PDMP was also examined ( /¨CPDMP). Quantitation of ceramide, glucosylceramide, and sphingomyelin is expressed as picomoles per 106 BG01v cells. Abbreviations: ISP-1, (2S),(3R),(4R),6E-2-amino-3,4-dihydroxy-2-hydroxymethyl-14-oxo-6-eicosenoic acid; PDMP, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol.; Q# J6 n( A: g$ `9 s. ]7 ^2 k

Table 2. Changes in sphingolipids caused by D-PDMP or ISP-16 }* O" S6 N+ f3 _8 c7 k3 Q" r# D# t

Gene Expression Analysis in GSL-Depleted hESCs) [* v( s/ s6 ~& `# u. N3 X8 K

Because the depletion of GSLs did not appear to affect hESC self-renewal, in vitro differentiation and gene expression analyses were used to determine whether other properties of pluripotency were unaltered. A stem cell-focused cDNA array (layout shown in supplemental online Table 1) was used to examine gene expression in undifferentiated BG01v cells (ethanol control), BG01v cells treated with 50 µM D,L-PDMP for 2 weeks, and in EB populations derived from these starting populations (Fig. 4A).
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Figure 4. Gene expression analysis of PDMP-treated human ESCs (hESCs) and differentiated populations. (A): BG01v cells were maintained in ethanol carrier (A1) or 50 µM D,L-PDMP (A2) for 4 weeks and were differentiated in embryoid bodies (EBs) for 14 days in the absence of inhibitor (A3 and A4, respectively). Parallel BG01v cultures were maintained in 50 µM D,L-PDMP and differentiated in EBs in the presence of 20 µM D,L-PDMP (A5). RNA from these populations was used to probe stem cell-focused arrays, the layout of which is shown in supplemental online Table 1. The comparisons performed are summarized on the right and are shown in detail in supplemental online Table 3. Markers that were either expressed outside of a twofold range or only detected in one sample are listed. Negative controls and spots for normalization are indicated (bottom row, A5). (B): Immunostaining of differentiated EBs derived from BG01v cells treated with 20 µM D-PDMP for 10 days, followed by differentiation in EBs in the absence of inhibitor. Differentiated cells expressing ßIII-tubulin, -fetoprotein, or smooth muscle actin, were detected, representing ectoderm, endoderm, and mesoderm, respectively. Scale bar = 50 µm. (C): Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) analysis of gene expression in untreated BG01 cells and cells treated with 20 µM D-PDMP for 8 weeks. Stem cell-related and other markers that fell within a twofold range of expression are listed, as are the four markers that were downregulated in the PDMP sample. The data are presented in more detail in supplemental online Table 3. Abbreviations: EtOH, ethanol; PDMP, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol.- B: l7 O- S) A: U
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GSL depletion did not cause substantial alteration to the gene expression of hESCs (Fig. 4A1, 4A2), and this profile was consistent with other hESC lines examined with this array . Of the markers detected in the hESC samples, 20 were within a twofold range of normalized expression and included known markers of hESCs: DNM3TB, GDF3, POU5F1 (OCT4), UTF1, and ZPF42. Nine additional low-level markers were observed in both samples (listed in supplemental online Table 3). Visual inspection revealed only three markers that varied appreciably in the undifferentiated cell samples, but all were detected at low levels: TGFB3, EGR2, and SOX3.4 L! x; E( \; E
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As expected, the markers expressed in the EB populations were markedly different than in hESCs (Fig. 4A3, 4A4). Expression of POU5F1 (OCT4) and five other markers, including FGF2 and UTF1, were downregulated with differentiation, indicating depletion of undifferentiated cells within the EB population. Sixteen markers were upregulated in the EB population derived from control hESCs. This included several integrins and confirmed the generation of differentiated lineages within the EB population. The expression profiles of the EBs generated from control and PDMP-treated hESCs were highly similar (Fig. 4A3, 4A4; supplemental online Table 3). This demonstrated that depletion of GSLs did not overtly affect the gene expression of undifferentiated hESCs or their capacity to differentiate in vitro. This was also examined by immunostaining of EB populations derived from D-PDMP-treated hESCs, which confirmed the differentiation of cells expressing markers of differentiated lineages (Fig. 4B).7 Z+ y' `2 n( Y' s: M5 a. P. ?

To assess whether GSLs might have important functions during the process of cellular differentiation, viable EBs were generated from GSL-depleted BG01v cells by aggregating and differentiating in the presence of 20 µM D,L-PDMP. EBs grown in the presence or absence of PDMP exhibited markedly different gene expression profiles (Fig. 4A4, 4A5). Twenty markers showed differences between these EBs, demonstrating that GSL inhibition substantially altered the transcriptional profile of this population. Although there are limitations with this population-wide approach, it suggests that the lineages formed during differentiation might be substantially different in the absence of GSLs. The gene expression differences observed in this study are summarized in Figure 4A, and full comparisons are shown in supplemental online Table 3.
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To determine whether depletion of GSLs affected the gene expression of normal hESCs, untreated BG01 cells and cells treated with 20 µM D-PDMP were compared by real-time quantitative RT-PCR. This analysis showed that 39 of 43 markers that were detected in BG01 cells fell within twofold expression levels and were therefore not obviously affected by GSL depletion (Fig. 4C, data shown in supplemental online Table 3). This confirmed that GSL depletion did not appear to substantially affect the gene expression in normal hESCs, consistent with the ability to culture these cells for long periods in these inhibitors.
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Differentiation of GSL-Depleted hESCs in Teratomas
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BG01v cells that had been depleted of GSLs with either 20 µM D-PDMP or 1 µM ISP-1 (parallel samples to Figure 3) were implanted to severe combined immune deficient (SCID)/beige mice to formally demonstrate the maintenance of pluripotency. Complex teratomas, which exhibited differentiated representatives of endoderm, ectoderm, and mesoderm lineages, were formed from hESCs treated with either D-PDMP or ISP-1 (Fig. 5). Small nests of undifferentiated OCT4  cells persisted in these BG01v-derived teratomas (not shown), as observed previously . This demonstrated that the depletion of GC and downstream GSLs with D-PDMP and ISP-1, as well as other sphingolipid intermediates, did not noticeably impact the capacity of BG01v cells to differentiate in teratomas.

Figure 5. Differentiation of D-PDMP- and ISP-1-treated BG01v cells in teratomas. BG01v cultures were treated with 20 µM D-PDMP or 1 µM ISP-1 for 19 days (parallel cultures to those analyzed in Figure 3) and implanted into the hind limb of severe combined immune deficient/beige mice. Immunohistochemical analysis of the tissue types generated in teratomas was performed after 11¨C14 weeks. (A¨CC): Pancytokeratin (PC) /p63¨C endodermal villi with goblet cells and (D¨CF) endodermal glands. (G, H): Mesodermal cartilage and bone. (I, J): Ectodermal PC  (not shown)/p63  skin. (K¨CM): Ectodermal p63 /PC  skin and hair follicles. (N¨CQ): Neuron-specific enolase (NSE)  and S100  neural lineages. Other lineages detected included presumptive glomeruli and ducts (not shown). D-PDMP- and ISP-1-treated samples are indicated. The images from (B, C), (D¨CF), (I, J), and (K¨CM) were from matched sections. Scale bars = 200 µm (A¨CM, O), 50 µm (N, P, Q). Abbreviations: ISP-1, (2S),(3R),(4R),6E-2-amino-3,4-dihydroxy-2-hydroxymethyl-14-oxo-6-eicosenoic acid; PDMP, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol." k, W$ i0 }; P/ c

DISCUSSION! o3 s3 Z9 }# a# z: ?3 u

SSEA-3 and -4 were originally identified as cell surface antigens exhibited at specific stages of mouse preimplantation embryogenesis and, subsequently, as markers of undifferentiated human EC cells and hESCs . The SSEA-3 and -4 GSLs do not therefore appear to play critical roles in regulating human pluripotent cells.
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Monitoring of cell surface expression by immunostaining and flow cytometry revealed that SSEA-3 was depleted more rapidly than SSEA-4 with these inhibitors. This was similar to reports of SSEA-3 being extinguished more rapidly during hESC differentiation ., x5 p) T) u( Y
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Although hundreds of different GSLs have been identified and are highly related (http://www.sphingomap.org), only SSEA-3 and -4 have been studied in detail in hESCs. ISP-1 and D-PDMP would lead to the depletion of all GSLs downstream of ceramide and GC, respectively. It is therefore unlikely that there are other, currently unrecognized, GSLs with critical functions in regulating pluripotency. This is consistent with previous studies in the mouse, in which inner cell mass and primitive ectoderm cells in GCS-null embryos appeared to develop normally . Interestingly, when BG01v EBs were differentiated in the presence of D/L-PDMP, marked differences in gene expression were detected using the focused cDNA array compared with control EBs. This suggested that different populations were generated when GSL synthesis was inhibited during differentiation, consistent with important functions for GSLs during this process.

D-PDMP and ISP-1 altered the levels of other sphingolipid intermediates in BG01v cells, including ceramide (supplemental online Fig. 1). Despite the known bioactivities of some of these metabolites , altering the flux of these endogenous intermediates did not appear to have serious consequences for cell viability. This was consistent with the ability to derive and maintain viable mouse ESCs from GCS-null embryos and suggests that strict control of these metabolites is not critical in undifferentiated cells. An alternate interpretation is that these changes compensated for the loss of a GSL function and that some of these molecules may have opposing effects on pluripotency versus differentiation which are cancelled out using "global" inhibition. This seems unlikely, however, because hESCs are particularly sensitive to changes in their environment and readily differentiate or die in suboptimal conditions. Exposure of hESCs to PDMP or ISP-1, which inhibit different steps of the pathway and caused different changes to these other components, did not elicit any apparent alteration to growth rate, morphology, or cell death.4 T" o5 g. J6 Q8 `% b

In this study, we used multiple approaches to inhibit and confirm the depletion of the SSEA-3 and -4 GSLs and other sphingolipid metabolites and demonstrate the maintenance of self-renewal and pluripotency in hESCs. These experiments generated an SSEA-3- and -4-depleted hESC population that could not be distinguished from untreated cells using other markers but that retained the capacity to resynthesize GSLs. Although expression of these antigens clearly marks undifferentiated cells, downregulation of SSEA-3 and -4 does not necessarily indicate differentiation, and caution should therefore be exercised when using them for this purpose . This study did not address the possibility that SSEA-3 and -4 must be downregulated to enable differentiation of pluripotent cells, but a function(s) for complex GSLs during cellular differentiation was suggested. The demonstration that the SSEA-3 and -4 GSLs do not have obvious roles in maintaining pluripotency indicates the importance of functional assessment of current hESC markers and the development of new markers that are involved in the mechanism of self-renewal." {/ G7 }, c9 h3 i1 j
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DISCLOSURES

S.B., E.S., A.R., and T.S. have financial interests in BresaGen Inc. (Athens, GA). They are employees of BresaGen Inc.+ u" N: q7 L6 w$ Z" O; b# M! {

ACKNOWLEDGMENTS

We thank Clifton Baile and Diane Hartzel and the Animal Facility of the University of Georgia Animal and Dairy Science Department for SCID experiments, and Melissa Carpenter for critical comments. This work was supported by NIH grant R24RR021313-05 (T.S.) and the NIH Integrated Technology Resource for Medical Glycomics (PA-02-132, M. Pierce, PI).
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