Glycome Diagnosis of Human Induced Pluripotent Stem Cells Using Lectin Microarray^*

Cells

Endometrium (UtE) (29), placental artery endothelium (PAE) (30), and amnion (AM) (31, 32) were independently established. UtE, AM, and MRC5 cells were maintained in the POWEREDBY10 medium (MED SHIROTORI CO., Ltd.). PAE cells were cultured in EGM-2MV BulletKit (Lonza) containing 5% FBS. Human iPSCs from UtE, PAE, and AM cells were generated according to the procedures described by Yamanaka and colleagues (1) with slight modification (27, 28, 33). The iPSCs derived from MRC5, UtE, PAE, and AM cells were maintained in iPSellon medium (Cardio Inc.) supplemented with 10 ng/ml recombinant human basic fibroblast growth factor (Wako Pure Chemical Industries, Ltd.) on irradiated MEF feeder cells. hiPS201B7 and hiPS253G1 cells were maintained in DMEM/F-12 medium (Invitrogen) supplemented with 20% KSR (Invitrogen), 0.1 mm 2-mercaptoethanol (Sigma-Aldrich), minimum essential medium non-essential amino acids (Invitrogen), and 5–10 ng/ml recombinant human basic FGF (Wako) on mitomycin C-treated mouse embryo fibroblast feeder cells. ESCs were generated and maintained as described previously (34).

Lectins

Lectins from natural sources (58 lectins) were purchased from J-OIL MILLS, Vector Laboratories, EY Laboratories, and Seikagaku Corp. (see the lectin list in supplemental Table S2). Recombinant lectins were prepared as follows. Briefly, genes of carbohydrate recognition domains were cloned into pET27b (Stratagene) and were overexpressed in the Escherichia coli BL21-CodonPlus (DE3)-RIL strain under the control of isopropyl-β-d-thiogalactopyranoside (Fermentas Hanover) at appropriate temperatures. All recombinant lectins were purified by affinity chromatography using appropriate sugar-immobilized Sepharose 4B-CL (GE Healthcare) based on the glycan binding specificity of each lectin. They were then dialyzed against diluted PBS (final concentration 2.5 mm phosphate buffer containing 0.015 m NaCl). The protein concentration was determined by a BCA protein assay (Bio-Rad). Lectins were freeze-dried and stored at 4 °C until use. The purity was checked by SDS-PAGE and gel filtration chromatography on Shodex PROTEIN KW-802.5 (Shodex). The glycan binding activity and specificity were analyzed by hemagglutinating activity using 4% rabbit erythrocytes, frontal affinity chromatography (35), and glycoconjugate microarray (36).

Lectin Microarray Production

The lectin microarray was produced as described previously with minor modifications (14, 15). Fifty-eight natural lectins and 38 recombinant lectins (see supplemental Table S2 for a lectin list) were dissolved at a concentration of 0.5 mg/ml in a spotting solution (Matsunami Glass), and spotted onto epoxysilane-coated glass slides (Schott) in triplicate using a non-contact microarray printing robot (MicroSys4000, Genomic Solutions). The glass slides were then incubated at 25 °C overnight to allow lectin immobilization. The lectin-immobilized glass slides were then washed with probing buffer (25 mm Tris-HCl, pH 7.5, 140 mm NaCl (TBS) containing 2.7 mm KCl, 1 mm CaCl₂, 1 mm MnCl₂, and 1% Triton X-100) and incubated with blocking reagent N102 (NOF Co.) at 20 °C for 1 h. Finally, the lectin-immobilized glass slides were washed with TBS containing 0.02% NaN₃ and stored at 4 °C until use. The spot quality and reproducibility of the produced microarrays were checked before use, using a Cy3-labeled test probe containing 250 μg/ml asialofetuin (Sigma-Aldrich), 25 ng/ml Siaα2–3Galβ1–4GlcNAc-BSA (Dextra), 10 ng/ml Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc-BSA (Dextra), 10 ng/ml βGlcNAc-BSA (Dextra), 10 ng/ml GalNAcα1–3(Fucα1–2)Gal-BSA (Dextra), 10 ng/ml Galα1–3Galβ1-4GlcNAc-BSA (Dextra), 10 ng/ml Manα1–3(Manα1–6)Man-BSA (Dextra), 10 ng/ml αFuc-BSA (Dextra), 10 ng/ml αGalNAc-BSA (Dextra), and 10 ng/ml Siaα2–6Galβ1–4Glc-BSA (Dextra) dissolved in probing buffer.

Lectin Microarray Analysis

Hydrophobic fractions were prepared using CelLytic minimum essential medium protein extraction (Sigma-Aldrich) in accordance with the manufacturer's procedures (25, 27). After protein quantification using a BCA assay (Thermo Fisher Scientific), hydrophobic fractions were fluorescently labeled with Cy3 monoreactive dye (GE Healthcare), and excess Cy3 was removed with Sephadex G-25 desalting columns (GE Healthcare). After adjusting the protein concentration to 2 μg/ml with PBST (10 mm PBS, pH 7.4, 140 mm NaCl, 2.7 mm KCl, 1% Triton X-100), the hydrophobic fraction was labeled with Cy3 NHS ester (GE Healthcare). After dilution with probing buffer at 0.5 μg/ml, the Cy3-labeled hydrophobic fraction was applied to the lectin microarray and incubated at 20 °C overnight. After washing with probing buffer, fluorescence images were acquired using an evanescent field-activated fluorescence scanner (GlycoStation^TM reader 1200; GP BioSciences). The fluorescence signal of each spot was quantified using Array Pro Analyzer version 4.5 (Media Cybernetics, Bethesda, MD), and the background value was subtracted. The background value was obtained from the area without lectin immobilization. The lectin signals of triplicate spots were averaged and normalized to the mean value of 96 lectins immobilized on the array. An inhibition assay was performed by incubating Cy3-labeled cell membrane fractions of MEF(#1) or MRC5-iPS#25(P22)(#13) with a lectin microarray either in the absence or presence of 100 μg/ml of Galα1–3Galβ1–4GlcNAc-PAA (catalog no. 01-079, Glycotech) or a negative control PAA (catalog no. 01-000, Glycotech).

Gene Expression Analysis

Total RNA was extracted from each sample by using ISOGEN (NipponGene). The global gene expression patterns were monitored using Agilent whole human genome microarray chips (G4112F) with one-color (cyanine 3) dye. This microarray covers 41,000 well characterized human genes and transcripts. Of the 41,000 probes, 16,483 representative probes corresponding to the microarray quality control unique genes were used for the following analyses (37).

Statistics

Unsupervised clustering was performed by employing the average linkage method using Cluster 3.0 software. The heat map with clustering was acquired using Java Treeview. Differences between the two arbitrary data sets were evaluated by Student's t test to each lectin signal using SPSS Statistics 19 (SPSS). Significantly different lectin signals or the glycosyltransferase expression were selected if they satisfied a familywise error rate (FWER) by the Bonferroni method of <0.001.

Immunocytochemistry

Immunocytochemical analysis was performed as described previously (29, 33, 38). Human iPSCs were fixed with 4% paraformaldehyde in PBS for 10 min at 4 °C. After washing with 0.1% Triton X-100 in PBS (PBST), the cells were prehybridized in blocking buffer for 1–12 h at 4 °C and then incubated for 6–12 h at 4 °C with the following primary antibodies: anti-SSEA4 (1:300 dilution; Chemicon), anti-TRA-1-60 (1:300; Chemicon), anti-Oct4 (1:50; Santa Cruz Biotechnology, Inc.), anti-Nanog (1:300; ReproCELL), and anti-Sox2 (1:300; Chemicon). The cells were then incubated with anti-rabbit IgG, anti-mouse IgG, or anti-mouse IgM conjugated with Alexa Fluor 488 or Alexa Fluor 546 (1:500; Molecular Probes) in blocking buffer for 1 h at room temperature. The cells were counterstained with DAPI and then mounted using the SlowFade light antifade kit (Molecular Probes).

Teratomas

Teratoma formation was performed as described previously (1, 2). The 1:1 mixtures of the human iPSC suspension and basement membrane matrix (BD Biosciences) were implanted subcutaneously at 1.0 × 10⁷ cells/site into immunodeficient, non-obese diabetic/severe combined immunodeficiency mice. Teratomas were surgically dissected out 8–12 weeks after implantation and were fixed with 4% paraformaldehyde in PBS and embedded in paraffin. Sections of 10-μm thickness were stained with hematoxylin-eosin.

Glycoconjugate Microarray Analysis

Glycoconjugate microarray production and analysis were performed as described previously (36). Briefly, glycoproteins and glycoside-polyacrylamide conjugates were dissolved in the Matsunami spotting solution at a final concentration of 0.5 and 0.1 mg/ml, respectively. After filtration, they were spotted on the Schott epoxy-coated glass slide using the Microsys non-contact microarray printing robot.

Cy3-labeled lectins dissolved in the probing solution (10 or 1 μg/ml) were applied to each chamber of the glycoconjugate microarray (100 μl/well) and were incubated at 20 °C overnight. After washing the chambers with the probing solution, fluorescent images were immediately acquired using an evanescent field-activated fluorescence scanner, the GlycoStation^TM Reader 1200, under Cy3 mode. Data were analyzed with the Array Pro analyzer version 4.5 (Media Cybernetics, Inc.). The net intensity value for each spot was determined by signal intensity minus background value. The lectin signals of triplicate spots were averaged and normalized to the highest signal intensity among 98 glycoconjugates immobilized on the array.

Development of High Density Lectin Microarray

In order to increase glycome coverage and the selection range of lectins suitable for stem cell evaluation, we first increased the number of immobilized lectins from 43 to 96, which is the largest number of immobilized lectins reported (39). For this purpose, lectins with defined structures were first categorized into lectin families with different protein scaffolds. We then selected lectins from various lectin families, intending to cover a wider range of glycan binding specificities. Especially, we increased lectins specific to terminal modifications, such as Sia and Fuc, which often change dramatically depending on cell properties. For production of recombinant lectins, the E. coli expression system was chosen to avoid glycosylation of the produced lectins, which might cause nonspecific binding to lectin-like molecules in the objective samples. The recombinant lectins thus produced were purified by affinity chromatography using the most appropriate sugar-immobilized Sepharose. The glycan-binding specificities of 96 lectins used in this study were analyzed by both glycoconjugate microarray (supplemental Fig. S1 and Table S1; also see “Experimental Procedures”) (36) and, more quantitatively, frontal affinity chromatography (see the Lectin Frontier Database Web page) (35, 40). Their basic specificities evaluated by the above two analytical methods are briefly summarized in supplemental Table S2. The 96 lectins were spotted onto epoxy-activated glass slides by a non-contact spotter (supplemental Fig. S2), and their quality was extensively assessed using a Cy3-labeled test probe (25). Lot-to-lot variance (coefficients of variation) of the developed high density lectin microarray was confirmed to be low (0.14) after mean normalization (25).

Transcription Factor-induced Reprogramming Leads to a Global Reversion Down to the Pluripotent State at a Cellular Glycome Level as Well

Using the developed lectin microarray, we have analyzed 135 cell samples in total, including 114 iPSCs, 11 SCs, and nine ESCs, all from human origins, as well as one mouse embryonic fibroblast (MEF). Human iPSCs were generated from four different SC lines: MRC5, AM, UtE, and PAE (supplemental Table S3) (28). We have also analyzed human iPSCs generated from human dermal fibroblasts with four (201B7) (1) and three transcription factors (253G1) (41) and three cell lines of human ESCs (42). All iPSCs used in this study were morphologically similar to ESCs, and their pluripotency was confirmed by staining with the established undifferentiation markers (SSEA4, Tra1–60, Oct4, Nanog, and Sox2) and DNA microarray (28).

Cell membrane hydrophobic fractions were prepared, and the extracted glycoproteins were then labeled with Cy3-N-hydroxysuccinimide ester and analyzed by lectin microarray (25). We have analyzed cell membrane fractions because they can be stored in a freezer until use and are easy to handle, allowing comprehensive analysis of a large number of samples (25, 26).

After being mean-normalized, the obtained data were first analyzed by unsupervised hierarchical clustering (Fig. 1). As a result, differentiated SCs and undifferentiated iPSCs/ESCs were clearly separated into two large clusters, whereas the four SCs were further separated according to their origins. This indicates that SCs (MRC5, AM, UtE, and PAE) with different glycan profiles have acquired profiles quite similar to one another and even to ESCs upon induction of pluripotency. Thus, transcription factor-induced reprogramming was found to lead to a global reversion down to the pluripotent state at a cellular glycome level as well (27, 28).

Characteristic Features of Glycome Alteration upon Induction of Pluripotency

We then examined in more detail how glycan structures altered during the induction of pluripotency. The mean-normalized data were processed by Student's t test to select significant probe lectins discriminating between SCs and iPSCs/ESCs (supplemental Table S4). As a result, 38 lectins were selected with FWER of <0.001. Among them, nine gave higher signals in iPSCs than SCs, whereas 29 exhibited lower signals. Among the 38 lectins, 35 lectins were then categorized into six groups based on their glycan binding specificities, from which glycan alterations having occurred upon induction of pluripotency were estimated (Fig. 2), whereas the three lectins with broader specificities (wheat germ agglutinin (WGA), a Sia binder; rRSIIL and aleuria aurantia lectin (AAL), broad Fuc binders) were not included in this categorization. Here, the lectins with higher signals in iPSCs/ESCs than SCs are shown below gray lines, whereas lower signals are shown below black lines. The characteristic features of glycan structures of undifferentiated iPSCs/ESCs relative to differentiated SCs are summarized as follows. 1) The signals of α2–6Sia-binding lectins (SNA, SSA, TJAI, and rPSL1a) were increased, whereas those of α2–3Sia-binding lectins (MAL, rACG, and ACG) were decreased correspondingly (28). This agrees well with the previous report that α2–6-sialylated glycan expression is higher in undifferentiated (human ESCs) than differentiated cells (embryoid body) (43). 2) In terms of fucosylation, the signals of α1–2Fuc-specific lectins (rGC2 and rBC2LCN) were increased, whereas those of α1–6Fuc-specific lectins (LCA, PSA, and rPTL) were decreased. This is consistent with the recent report that human ESCs are stained with anti-Globo H (Fucα1–2Galβ1–3GalNAcβ1–3Galα1–4Galβ1–4Glc) and anti-H type 1 (Fucα1–2Galβ1–3GlcNAc), whose antigens contain α1–2Fuc (9). 3) The signals of type 1 LacNAc (Galβ1–3GlcNAc)-binding lectins (BPL) were increased, whereas those of type 2 LacNAc (Galβ1–4GlcNAc)-binding lectins (rLSLN, ECA, RCA120, and rCGL2) were decreased. This agrees well with the recent finding that type 1 LacNAc is the glycan epitope recognized by the well known pluripotency markers Tra-1-60 and Tra-1-81 (7). 4) The lectin signals specific to bisecting GlcNAc (PHAE), tetra-antennary N-glycans (PHAL), high mannose type N-glycans (Heltuba, rRSL, VVAII, rHeltuba, Heltuba, rBanana, rGRFT, and CCA), and O-glycans (MPA, HEA, WFA, Jacalin, ABA, rABA, rSRL, rCNL, and rDiscoidin II) were decreased.

The expression profiles of glycosyltransferases synthesizing glycans agreed well with the results obtained by lectin microarray (Fig. 3 and supplemental Table S5); the expression of α2–6-sialyltransferases (ST6GAL1 and -2) (28), α1–2-fucosyltransferases (FUT1 and -2), the major glycosyltransferase involved in the synthesis of type 1 LacNAc (B3GALT5) (28), and MGAT5, a glycosyltransferase involved in the synthesis of tetra-antennary N-glycans, was increased, whereas that of α2–3-sialyltransferases (ST3GAL1, -3, -4, and -5), α1–6-fucosyltransferase (FUT8), and the major glycosyltransferase related to the synthesis of type 2 LacNAc (B4GalT1) was decreased correspondingly in iPSCs/ESCs relative to SCs. Based on the results obtained by lectin and DNA microarrays, it is conceivable that the expression of α2–6-sialylation, α1–2-fucosylation, and type 1 LacNAc is increased, whereas that of α2–3-sialylation and tetra-antennary N-glycans is decreased upon induction of pluripotency (Fig. 4).

Selection of the Best Lectin Probe to Discriminate Pluripotency

We then addressed the challenge to develop a lectin-based procedure to discriminate between differentiated SCs and undifferentiated iPSCs/ESCs, which could be utilized to monitor the state of differentiation. As described, rGC2, rBC2LCN, SNA, TJAI, SSA, rPSL1a, rRSIIL, BPL, and AAL gave significantly higher signals in iPSCs/ESCs than SCs with FWER < 0.001 (Table 1). Among them, rBC2LCN showed the best performance as a probe to detect only undifferentiated iPSCs/ESCs but never reacted with differentiated SCs and MEF, whereas other lectins also reacted with SCs (Table 1). Namely, although rGC2 showed a better score in terms of FWER (1 × 10⁻²¹) than rBC2LCN (2 × 10⁻¹⁹), the former reacted strongly with MEF (Fig. 5). Similarly, SNA (4 × 10⁻¹¹), a representative α2–6Sia-binding lectin, showed significant cross-reactivity with a part of SCs derived from PAE in addition to MEF (Fig. 5).

Monitoring the Contamination of the Xenoantigen, αGal Epitope

From a practical viewpoint, monitoring possible contamination by xenotransplantation antigens in iPSCs/ESCs is essential for their safe use in regenerative medicine. A recombinant MOA (rMOA) recognizes the xenotransplantation antigen Galα1–3Galβ1–4GlcNAc (44) present in most cells from New World monkeys and non-primate mammals, including mice, but not in humans. Indeed, rMOA strongly bound to MEFs but not to any human SCs (Fig. 6). Therefore, rMOA signals should not be detected in human iPSCs. However, triplicate samples of the two cell lines MRC5-iPS#25(P22)(#13–15) and UtE-iPSB05(P13)(#64–66) exhibited significant signals on rMOA. In order to validate whether the binding of rMOA is mediated by a carbohydrate recognition domain of rMOA, we then performed inhibition assay. As shown in Fig. 6B, the binding of rMOA to cell membrane fractions of MEF and MRC5-iPS#25(P22,#13) were abolished in the presence of 100 μg/ml Galα1–3Galβ1–4GlcNAc-PAA (Fig. 6B), but no inhibitory effect was observed for 100 μg/ml of a negative control (PAA without sugar moiety), indicating that the binding is due to specific interactions via the rMOA carbohydrate recognition domain. As expected, no inhibitory effect of Galα1-3Galβ1–4GlcNAc-PAA on a Fuc-binding lectin, rAAL, was observed. These data unambiguously reflect contamination by the xenoantigen αGal epitope, in the above two cell lines, which were most probably contaminated with MEF.