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A Novel Approach for Profiling of Glycosphingolipid Glycosylation by xCGE-LIF Identifies Cell-Surface Markers of Human Pluripotent Stem Cells and Derived Cardiomyocytes Charlotte Rossdam, Sarah A. Konze, Astrid Oberbeck, Erdmann Rapp, Rita Gerardy-Schahn, Mark von Itzstein, and Falk F. R. Buettner Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01114 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 6, 2019

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Analytical Chemistry

1

A Novel Approach for Profiling of Glycosphingolipid

2

Glycosylation by xCGE-LIF Identifies Cell-Surface Markers

3

of

4

Cardiomyocytes

Human

Pluripotent

Stem

Cells

and

Derived

5 6

Charlotte Rossdama,b, Sarah A. Konzea,b, Astrid Oberbecka,b, Erdmann Rappc,d, Rita Gerardy-

7

Schahna,b, Mark von Itzsteine & Falk F. R. Buettnera,b, *

8 9

aInstitute

of Clinical Biochemistry, Hannover Medical School, Hannover, Germany

10

bREBIRTH

11

cMax

12

dglyXera

GmbH, Magdeburg, Germany

13

eInstitute

for Glycomics, Griffith University, Gold Coast Campus, Queensland, Australia

Cluster of Excellence, Hannover Medical School, Hannover, Germany

Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany

1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Application of human induced pluripotent stem cell-derived cardiomyocytes

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(hiPSC-CMs) as tissue transplants in regenerative medicine depends on cell-surface marker-

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based characterisation and/or purification. Glycosphingolipids (GSLs) are a family of highly

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diverse surface-exposed biomolecules that have been neglected as potential surface

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markers for hiPSC-CMs due to significant analytical challenges. Here, we describe the

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development of a novel and high-throughput-compatible workflow for the analysis of GSL-

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derived glycans based on ceramide glycanase digestion, APTS labelling and multiplexed

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capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF).

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GSL glycans were detected with highly reproducible migration times after repeated analysis

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by xCGE-LIF. We built up a migration time database comprising 38 different glycan species

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and we showed exemplarily that as few as 10 pg of fucosyl lactotetra were detectable. GSL

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glycan profiling could be performed with 105 human induced pluripotent stem cells and we

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quantitatively dissected global alterations of GSL glycosylation of hiPSCs and hiPSC-CMs by

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employing xCGE-LIF. In our study we observed a general switch from complex GSLs with

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lacto- and globo-series core structures comprising the well-known human pluripotent stem

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cell markers stage-specific embryonic antigen 3 (SSEA3) and SSEA4 in hiPSCs towards the

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simple gangliosides GM3 and GD3 in hiPSC-CMs. This is the first description of GM3 and

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GD3 being highly abundant GSLs on the cell-surface of stem cell-derived cardiomyocytes.


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INTRODUCTION

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As the human heart has a low regeneration capacity,1 tissue transplants made from human

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pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are considered a viable option.2

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These transplants must contain pure cardiomyocytes without contaminating stem cells that

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could lead to teratoma formation3 or affect the functionality of the graft. To date, the

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characterisation and purification of stem cell-derived cardiomyocytes has entirely relied on

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few cell-surface exposed proteins4-7 or N-glycans.8 While glycosphingolipid (GSL) glycans

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are commonly applied as markers for pluripotent stem cells9 or cancer cells,10,11 GSL

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glycosylation of hiPSC-CMs has not been assessed at a global level by analytical methods,

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but rather investigated with a particular focus on histo-blood group antigens using specific

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antibodies.12 Several hundred different glycan structures are known in humans and their

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expression is highly dynamic and strongly influenced by the developmental state and

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environmental conditions.13 Mass spectrometry-based14-18 as well as chromatographic19-21

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techniques have been developed for GSL analysis. Tetramethylrhodamine-labelled GSLs

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could be efficiently separated and detected at high sensitivity by capillary electrophoresis

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with laser-induced fluorescence detection.22 However to globally decipher GSL glycosylation

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at high-throughput there is a paucity of novel analytical technologies due to significant

49

technical challenges. Our interest in regenerative medicine using hiPSC-CMs8,23,24 led us to

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the development of multiplexed capillary gel electrophoresis coupled to laser induced

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fluorescence detection (xCGE-LIF8,25-31) for the analysis of GSL-derived glycans. xCGE-LIF

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has particular advantages over other glyco-analytical technologies including potential for

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high-throughput, isomer separation capacity, sensitivity and low cost per sample.32 To the

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best of our knowledge we here present the first application of xCGE-LIF in the

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characterisation of GSL-based cell surface markers for hiPSC-CMs.



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EXPERIMENTAL METHODS

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All experimental procedures can be found in the Supporting Information

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RESULTS AND DISCUSSION

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Glycan-based standards library development

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In the first instance we needed to establish a GSL-based standards library to be used in the

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xCGE-LIF protocol. We commenced this library development by using a leech-derived

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(Hirudo medicinalis) ceramide glycanase digestion33 of GSLs from commercially available

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GSL standards. This enzyme efficiently released the glycan head groups leading to free

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reducing-end glycans that could be subsequently labelled with the fluorescent dye 8-

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aminopyrene-1,3,6-trisulfonic acid (APTS) and analysed by xCGE-LIF. Specifically, we used

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six different commercially available GSLs including GM3 (aa), GD1a (ab), GD3 (ac), GD1b

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(ad), GD2 (ae) and GM1a (af) and demonstrated in a multi-replicate analysis that the entire

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workflow including ceramide glycanase digestion, fluorescent labelling with APTS and xCGE-

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LIF led to defined signals at distinct migration time units (MTUs, Figure 1) with minimal

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deviations below 0.5 MTU (Figure S1; Table S1). Variations between different runs could be

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efficiently minimized by internal calibration to a size standard that is detected at a different

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wave length. The measurements correlating a value for the migration time to a defined

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glycan were used to build up a database. However, in standards of GSLs with terminal sialic

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acids, we could also detect the respective desialylated species after analysis by xCGE-LIF

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(Figure 1 A-C). As we applied similar labelling conditions as for N-glycans, this is expected to

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be caused by partial loss of terminal sialic acids.29 We further expanded the repertoire of

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different glycan structures by analysing commercially available glycans that are known to be

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present as head groups on GSLs including Lewis x pentaose (Le x penta, ag), fucosyl

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lactotetraose / stage-specific embryonic antigen 5 (fucosyl Lc4 / SSEA5, ah), fucosyl

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neolactotetraose (fucosyl nLc4, ai), sialyl lactotetraose (sialyl Lc4, aj), sialyl globopentaose /

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stage specific embryonic antigen 4 (sialyl Gb5 /SSEA4, ak), fucosyl globopentaose (fucosyl 4 ACS Paragon Plus Environment

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Gb5 / globoH, al), globotriaose (Gb3, am) neolactotetraose (nLc4, an), globopentaose / stage

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specific embryonic antigen 3 (Gb5 / SSEA3, ao), blood group A antigen hexaose type 1 (A

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type 1 hexa, ap), Forssman antigen pentaose (Forssman penta, aq), isoforssman antigen

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pentaose (isoforssman penta, ar), globoA (as), fucosyl GM1-derived glycan (at), GT1c-

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derived glycan (au), GT1a-derived glycan (av), GM1b-derived glycan (aw), isoglobopentaose

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(isoGb5, ax), blood group B antigen hexaose type 2 (B type 2 hexa, ay), blood group B

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antigen hexaose type 1 (B type 1 hexa, az), blood group A antigen hexaose type 2 (A type 2

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hexa, ba), neolactohexaose (nLc6, bb, Figure S2; Figure S3; Table S2). Smaller glycans

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migrated faster than larger glycans, and addition of charged sialic acid residues significantly

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increased migration velocity during the electrophoresis. Importantly, using xCGE-LIF we

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could not only discriminate glycans differing in the number or type of monosaccharides but

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also clearly distinguish peaks of structural isomers that differ either in the positioning of

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identical monosaccharides (e.g. GD1a-derived glycan (ab) vs. GD1b-derived glycan (ad)) or

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even in the type of linkage between identical monosaccharides (e.g. fucosyl Lc4 / SSEA5

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(ah) having galactose bound to N-acetylglucosamine (GlcNAc) in β1-3-linkage vs. fucosyl

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nLc4 (ai) with galactose bound to GlcNAc in β1-4-linkage). Determination of the xCGE-LIF

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detection limit revealed that as little as 10 pg of fucosyl Lc4 is sufficient to give rise to a

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detectable signal (Figure S4). We completed our library development by treating

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commercially available (GSL-derived) glycans with different exoglycosidases. Thereby we

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obtained additional glycan structures including lactose (Lac, bc), gangliotriaose (Gn3, bd),

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gangliotetraose (Gn4, be), GM2-derived glycan (bf), lactotriaose (Lc3, bg), lactotetraose

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(Lc4, bh), globotetraose (Gb4, bi), isoglobotetraose (isoGb4, bj), isoglobotriaose (isoGb3, bk)

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and neolactopentaose (nLc5, bl) that were applied to expand our migration time database

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(Figure S5, Table S3). The applied exoglycosidases were shown to cleave their designated

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target glycans, but did not modify a panel of non-target glycans (Figure S6), underlining their

108

specificity. We ended up with a glycan library of 38 different structures covering a broad

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spectrum of GSL glycans present on human pluripotent stem cells and early derivatives

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thereof.9,34 5 ACS Paragon Plus Environment


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Characterisation of human induced pluripotent stem cell (hiPSC) GSL glycans

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The establishment of this novel analytical approach with various standards provided us the

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opportunity to analyse GSL glycans prepared from human induced pluripotent stem cells

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(hiPSCs). We obtained electropherograms with numerous clear baseline separated peaks

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(Figure S7a and b) that consistently appeared with similar MTUs (Table S4). However, the

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analysis of GSL glycans from hiPSCs by xCGE-LIF at different time points (hiPSC1 to 3 vs.

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hiPSC4 to 6, Table S4) revealed that MTU values are less diverging when samples are

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analysed in a short term interval as the lot or aging of the polymer slightly affects the

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migration times. Based on our database, 15 peaks could be assigned to specific glycans

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(Figure S7) comprising ganglio-, globo- and lacto-series glycans. Treatment of hiPSC-

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derived GSL glycans with different exoglycosidases (Figure S8) confirmed these peak

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assignments. Relative quantification of peak intensities based on their heights and

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comparison between different biological replicates underscored the reproducibility of the

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observed peak intensities (Figure S7c and d). For assessment of the detection limit of our

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novel analytical approach, we compared peak heights of 104, 105, and 106 hiPS cells. For

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inter-sample comparison, we added the same amount of a defined N-glycan standard (Man6)

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to each sample, enabling normalization of peak intensities of different electropherograms by

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adjustment to the intensity of the internal standard peak. This analysis revealed that the

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majority of peaks could still be detected from as few as 105 hiPS cells (Figure S9). For

131

comprehensive glycosphingolipidomics of biomaterials, broad substrate specificity of the

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applied ceramide glycanase is pivotal and we therefore compared our enzyme from Hirudo

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medicinalis with the known broad specific endoglycoceramidase from Rhodococcus

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triatomea.19 hiPSC-derived GSLs were digested in parallel with both enzymes and xCGE-LIF

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analysis revealed similarities and differences in the substrate specificity of both enzymes.

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Nevertheless, both enzymes seem comparably suited for digestion of a broad spectrum of

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GSLs (Figure S10; Table S6). Alternatively to enzymatic digestion, GSL glycans can be


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released by oxidation with bleach (NaClO) leading to free glycan nitriles.35 However,

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suitability of glycan nitriles for xCGE-LIF remains elusive.

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Determining changes in the glycosphingolipidome by cardiac differentiation of hiPSCs

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Using our established novel xCGE-LIF analytical approach for the determination of GSL-

143

derived glycans, we interrogated the changes in the glycosphingolipidome that are

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associated with cardiac differentiation of hiPSCs. Thus, hiPSCs were differentiated into

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cardiomyocytes applying the GiWi approach36,37 that led to the formation of 82+/-6% cardiac

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muscle troponin T (cTnT) positive cardiomyocytes on d10 of differentiation in three

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independent differentiation approaches (Figure S11). GSLs of hiPSCs and hiPSC-derived

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cardiomyocytes (hiPSC-CMs) were prepared and their released glycans were analysed by

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xCGE-LIF in the presence of an internal standard enabling comparison of peak heights

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between different samples (Figure 2, Table 1, Table S5). Thereby, we could confirm the

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human pluripotent stem cell characteristic pattern of GSL glycans in our hiPSCs including

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Gb5 / SSEA3 and sialyl Gb5 / SSEA4.9 These common stem cell markers as well as Gb4

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that belong to the GSL globo-series were strongly reduced in hiPSC-CMs compared to

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hiPSCs as was the lacto-series GSL glycan Lc4. On the other hand, simple ganglioside-

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derived glycans such as GM3-derived glycan and GD3-derived glycan were strongly

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augmented in hiPSC-CMs whereas complex ganglio-series GSL-derived glycans including

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GD1b-derived glycan and GM1a-derived glycan were reduced. These findings imply a

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general shift from globo- and lacto-series GSLs in hiPSCs to simple gangliosides in hiPSC-

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CMs. Interestingly, a similar switch of GSL core structures has been reported previously by

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Liang et al., who applied an undirected embryoid body-based differentiation into cell types of

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all three germ layers.34 Unlike Liang et al., we did directed differentiation into highly enriched

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hiPSC-CMs. The glycan lactose of the ubiquitous precursor for GSL synthesis,

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lactosylceramide, as well as the glycan Gb3, the precursor of globo-series GSLs, were found

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at almost similar levels in hiPSCs and hiPSC-CMs. Notably, the complex lacto-series and



Page 8 of 18

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neolacto-series glycans sialyl Lc4 and nLc4 were significantly increased in hiPSC-CMs,

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respectively (Figure 2).

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CONCLUSIONS

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Taken together, to the best of our knowledge, we here present the first description of xCGE-

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LIF for the analysis of GSL-derived glycans. This paved the way for an unprecedented

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comprehensive analysis of GSL glycans of hiPSC-CMs leading to the identification of GD3,

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GM3, sialyl Lc4 and nLc4 as novel cell-surface markers. These markers have the potential to

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be used in the isolation of pure stem cell-derived cardiomyocytes from residual stem cells

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that is in high demand for applications in regenerative medicine.


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ASSOCIATED CONTENT

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The Supporting Information is available free of charge on the ACS Publications website at

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DOI:

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Human induced pluripotent stem cell culture; cardiomyogenic differentiation; flow cytometry;

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extraction of glycolipids from cells; deglycosylation using endoglycoceramidases; fluorescent

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labelling and HILIC-solid phase extraction (SPE); xCGE-LIF; exoglycosidase digests; data

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processing and statistics; figures with additional xCGE-LIF analyses; tables with raw data of

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xCGE-LIF analyses

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AUTHOR INFORMATION

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Corresponding Author

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*E-Mail: [email protected]

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Phone: +49/511/532-8245. Fax: +49/511/532-8801.

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ORCID

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Falk F. R. Buettner: 0000-0002-8468-1223

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Rita Gerardy-Schahn: 0000-0002-5796-368X

191

Note

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The authors declare no competing financial interest.

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ACKNOWLEDGEMENTS

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We are grateful to Prof. Dr. Scheper (Institute of Technical Chemistry, Leibniz University of

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Hannover) for providing basic fibroblast growth factor, Rock Inhibitor Y-27632 and CHIR-

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99021 as well as Prof. Dr. Martin and Dr. Haase (LEBAO, MHH) for providing the human iPS

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cell

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Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence

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REBIRTH (From Regenerative Biology to Reconstructive Therapy, EXC 62/2). F.F.R.B. was

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funded by the DFG (FOR2509 BU 2920/2-1 and BU 2920/3-1).

line

“Phoenix”.

This

work

was

supported

by

funding

from

the

Deutsche



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oligosaccharides in human milk utilizing multiplexed capillary gel electrophoresis with laserinduced fluorescence detection. Electrophoresis 2013, 34 (16), 2323-2336.

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29. Ruhaak, L. R.; Hennig, R.; Huhn, C.; Borowiak, M.; Dolhain, R. J.; Deelder, A. M.; Rapp, E.; Wuhrer, M. Optimized workflow for preparation of APTS-labeled N-glycans allowing high-throughput analysis of human plasma glycomes using 48-channel multiplexed CGE-LIF. J. Proteome. Res. 2010, 9 (12), 6655-6664.

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30. Schwarzer, J.; Rapp, E.; Reichl, U. N-glycan analysis by CGE-LIF: profiling influenza A virus hemagglutinin N-glycosylation during vaccine production. Electrophoresis 2008, 29 (20), 4203-4214.

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31. Thiesler, C. T.; Cajic, S.; Hoffmann, D.; Thiel, C.; van Diepen, L.; Hennig, R.; Sgodda, M.; Weibetamann, R.; Reichl, U.; Steinemann, D.; Diekmann, U.; Huber, N. M.; Oberbeck, A.; Cantz, T.; Kuss, A. W.; Korner, C.; Schambach, A.; Rapp, E.; Buettner, F. F. Glycomic Characterization of Induced Pluripotent Stem Cells Derived from a Patient Suffering from Phosphomannomutase 2 Congenital Disorder of Glycosylation (PMM2-CDG). Mol. Cell Proteomics. 2016, 15 (4), 1435-1452.

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32. Huffman, J. E.; Pucic-Bakovic, M.; Klaric, L.; Hennig, R.; Selman, M. H.; Vuckovic, F.; Novokmet, M.; Kristic, J.; Borowiak, M.; Muth, T.; Polasek, O.; Razdorov, G.; Gornik, O.; Plomp, R.; Theodoratou, E.; Wright, A. F.; Rudan, I.; Hayward, C.; Campbell, H.; Deelder, A. M.; Reichl, U.; Aulchenko, Y. S.; Rapp, E.; Wuhrer, M.; Lauc, G. Comparative performance of four methods for high-throughput glycosylation analysis of immunoglobulin G in genetic and epidemiological research. Mol. Cell Proteomics. 2014, 13 (6), 1598-1610.

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Figure 1 xCGE-LIF analysis of glycans derived from GSL standards. Glycans were released

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by ceramide glycanase (CGase) from (A) GM3, (B) GD1a, (C) GD3, (D) GD1b, (E) GD2,

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(F) GM1a. The dominant peak in each electropherogram was assigned to the known glycan

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(lowercase letters and depicted structures) of the purchased GSL. Further intense peaks

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(indicated with lowercase letters in brackets) could be subsequently assigned to degradation

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products of the standard glycan (see Figure S5). Additionally, a sample containing CGase

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but no glycan (negative control) was measured and is depicted together with GM3 as relative

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fluorescence units (red line, A) to exclude possible contaminations from the enzyme. Symbol 13 ACS Paragon Plus Environment


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key: blue circle: glucose, yellow circle: galactose, yellow square: N-acetylgalactosamine,

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purple diamond: sialic acid. RFU: relative fluorescence units, nRFU: normalized RFU

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(normalized to the most intense peak).

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Figure 2 Comparison of GSL glycosylation of hiPSCs and hiPSC-derived cardiomyocytes by

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xCGE-LIF. (A) Structures of identified GSL glycans. Symbol key: blue circle: glucose, yellow

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circle: galactose, blue square: N-acetylglucosamine, yellow square: N-acetylgalactosamine,

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red triangle: fucose, purple diamond: sialic acid. (B) Overlay of xCGE-LIF electropherograms 15 ACS Paragon Plus Environment


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of APTS-labelled GSL-derived glycans of hiPSCs (black) and hiPSC-derived cardiomyocytes

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(blue). Peaks were annotated (lowercase letters) depending on migration time units matching

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the newly established database (see Figure 1, Figure S2, Figure S5) and exoglycosidase

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digests (see Figure S8 for hiPSCs). For comparison of peak heights between different

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samples, 0.083 ng of APTS-labelled Man6 standard was spiked into each sample and the

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intensity of this standard (#) was set to 1 RFU. (C) Comparison of relative signal intensities of

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major peaks (>1% relative signal intensity) of glycans derived from GSLs of hiPSCs and

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hiPSC-derived cardiomyocytes plotted against their mean migration time. Annotated glycans

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are depicted by lower case letters. Bar diagram shows mean + S.D. (n=6 for hiPSCs with cell

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numbers of 107, each and n=3 for hiPSC-derived cardiomyocytes with cell numbers of

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approx. 5x106, each). An unpaired Student’s t-test was performed between the peaks of the

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different cell lines and statistical significant differences are highlighted (* p

Approach for Profiling of Glycosphingolipid ... - ACS Publications

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