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. Author manuscript; available in PMC: 2019 Apr 19.
Published in final edited form as: Cell Chem Biol. 2018 Feb 8;25(4):439–446.e5. doi: 10.1016/j.chembiol.2018.01.010

Engineered glycocalyx regulates stem cell proliferation in murine crypt organoids

Sara H Rouhanifard 1,†, Aime Lopez Aguilar 2,†, Lu Meng 3, Kelley W Moremen 3, Peng Wu 1,2,*
PMCID: PMC5910180  NIHMSID: NIHMS934760  PMID: 29429899

Summary

At the base of the intestinal crypt, long-lived Lgr5+ stem cells are intercalated by Paneth cells that provide essential niche signals for stem-cell maintenance. This unique epithelial anatomy makes the intestinal crypt one of the most accessible models for the study of adult stem cell biology. The glycosylation patterns of this compartment are poorly characterized and the impact of glycans on stem cell differentiation remains largely unexplored. We found that Paneth cells, but not Lgr5+ stem cells, express abundant terminal N-acetyllactosamine (LacNAc). Employing an enzymatic method to edit glycans in cultured crypt organoids, we assessed the functional role of LacNAc in the intestinal crypt. We show that blocking access to LacNAc on Paneth cells leads to hyperproliferation of the neighbouring Lgr5+ stem cells, which is accompanied by the down-regulation of genes that are known as negative regulators of proliferation.

eTOC

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Rouhanifard et al present a method for glycan engineering in a crypt-organoid system and apply it to assess functions of N-acetyllactosamine (LacNAc) in stem cell biology. Results suggest that Paneth cells express high levels of LacNAc, and that blocking access to it leads to hyperproliferation of neighbouring Lgr5+ stem cells.

Introduction

The epithelial layer of the small intestine has a higher self-renewal rate (4–5 days) than any other mammalian tissue (Sato and Clevers, 2013). This renewal is driven by Lgr5+ (leucine-rich-repeat-containing G-protein-coupled receptor 5) stem cells that intercalate between Paneth cells at the base of the crypts. The continuous contact provides niche signals which maintain “stemness” and regulate differentiation (Sato et al., 2011). The rest of the crypt is occupied by daughter transit amplifying cells (TA)—an undifferentiated population in transition between stem cells and terminally differentiated cells—that divide rapidly and differentiate into the other cells types found in the small intestine: enterocytes, goblet cells, enteroendocrine cells, Paneth cells and tuft cells (Clevers, 2013).

The epithelium of the mammalian intestinal tract is heavily glycosylated (Sharma and Schumacher, 2001). The glycoprotein-polysaccharide layer, or glycocalyx, is the first point of contact for intestinal cells to their surrounding environment, and therefore plays a critical role in mediating cell-cell interactions and cell signalling (Furukawa et al., 2012; Stanley and Okajima, 2010; Varki et al., 2009). The potential of intestinal crypts in regenerative therapy for many glycan-related gastrointestinal diseases such as ulcerative colitis and microvillus inclusion disease has long been appreciated (Holmberg et al., 2017; Vogel et al., 2016), however the molecular function of glycosylation in these processes is just beginning to be understood. The crypt compartment harbours abundant mannose-rich glycoproteins whereas the mature villous zone contains mainly complex-type glycoproteins (Köttgen et al., 1982). Early studies found that Celiac Disease or glutensensitive enteropathy, a disease occurring in genetically susceptible individuals induced by the ingestion of wheat proteins, may be linked to gluten’s ability to bind to mannose-rich glycoproteins from the immature crypt cells (Köttgen et al., 1982). Recently, it was discovered that loss of core 1-derived O-glycans induced inverse shifts in the abundance of Bacteroidetes and Firmicutes in mouse gut microbiota, which play active roles in balancing intestinal inflammation and affects gut architecture (Sommer et al., 2014). To decipher the precise functions of glycans in these processes and to facilitate the development of diagnostic and therapeutic tools for gastrointestinal diseases new methods are required.

Crypt organoids are ex vivo cultured “epithelial mini guts” for studying crypt biology (Buczacki et al., 2013; Sato and Clevers, 2013; Yilmaz et al., 2012). Under the matrigel-based long-term culture conditions established by Clevers and coworkers, a single crypt undergoes multiple crypt fission events generating villus-like epithelial domains in which all differentiated cell types are present. This system has been used to study the interrelationship of intestinal stem cell types and cell fate determination. It also offers innovative ways to model human disease and screen drug candidates (Little, 2017). Currently, one of the main challenges of applying this technology to regenerative medicine is how to control the growth and differentiation of stems cells in the cultured organoids (Serakinci and Keith, 2006).

As demonstrated by Sackstein, Xia, and others, glycan engineering using glycosyltransferases (e.g. fucosyltransferase) was successfully applied to enhance cell engraftment and trafficking (Parmar et al., 2015; Sackstein et al., 2008; Xia et al., 2004). Following these pioneering studies, our laboratory developed a method for in situ fucosylation of cell-surface glycans that exploits the use of a recombinant Helicobacter pylori α1,3 fucosyltransferase (ɑ1,3 FucT) to transfer fucose or a fucose analogue from a GDP-Fucose donor onto type II N-acetyllactosamine (LacNAc) to generate the Lewis X (LeX) trisaccharide (Figure 1A) (Zheng et al., 2011). This technique has been used to modify cell-surface glycosylation patterns (Jiang et al., 2017) or to introduce a tag via a fucose analogue that can be further derivatized for visualizing the labeled LacNAc-containing glycoconjugates (Jiang et al., 2014; Zheng et al., 2011). This approach has also been translated into a histological protocol to characterize glycosylation patterns in tissue sections; in this form, the method is termed Chemoenzymatic Histology of Membrane Polysaccharides (CHoMP) (Rouhanifard et al., 2014).

Figure 1. A two-step chemoenzymatic approach for LacNAc detection on murine crypts.

Figure 1

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(A) An alkyne bearing fucose analogue is transferred from GDP-L-6-ethynylfucose (GDP-FucAl) to LacNAc-containing acceptors on the cell surface by H. pylori α1,3 fucosyltransferase (α1,3 FucT). The alkyne group is then reacted via CuAAC with a complementary azide-bearing biotin probe for detection. (B) 20× photomicrograph of 5 µm serial sections of formalin-fixed paraffin embedded (FFPE) C57BL/6 mouse small intestine stained for H&E or CHoMP labeling for LacNAc (green); DAPI nuclear staining (blue); scale: 200 µm. (C) 63× photomicrograph of crypt zone labeled with CHoMP LacNAc, arrowpoints show areas of high LacNAc (green) at the base of the crypt; scale: 50 µm. (D) LacNAc staining is co-localized with Paneth cells. UEA lectin (red) stains Paneth cells and goblet cells, and CHoMP labeling of LacNAc (green); scale: 50 µm. Inlet shows areas of signal colocalization on Paneth cells; Scale=10 µm. (E) Labeling of dissociated crypt epithelium of Lgr5-EGFP-IRES-CreER mice with Paneth cell marker (UEA) and chemoenzymatic LacNAc labeling. Samples were analysed by flow cytometry. The three cell populations were gated (EGFP+/UEA− = Lgr5+ stem cell, UEA+/EGFP−=Paneth cell, EGFP−/UEA−=other cell), then assessed for LacNAc labeling by individual population. Histogram is representative of cells isolated from 3 biological replicates, and values in the graph are depicted as mean MFI ± SEM.

Here we apply this in situ glycan editing method (Jiang et al., 2017) to crypt organoids to study the impact of glycosylation on stem cell proliferation and differentiation in a complex, multicellular system. Using this approach, we uncovered a pattern of high LacNAc expression on the surface of Paneth cells. We then in situ modified the glycocalyx of crypt cells in the ex vivo cultured crypt organoids, and observed that glycan editing significantly alters the timing of crypt budding and their proliferation. To our knowledge, this is the first report of in situ glycan editing for ex vivo cultured organoids. Competition assays and gene profiling on stem cells suggest that the observed phenotype results from reduced access to LacNAc on the surface of Paneth cells with a consequential deregulation of the stem cell cycle and differentiation pathways, as well as a down-regulation of multiple negative regulators of proliferation.

Results

Paneth cells express high levels of LacNAc

The small intestine contains abundant galectin binding glycan epitopes among which type II LacNAc is the most prevalent. We utilized formalin-fixed, paraffin-embedded small intestinal tissue sections from C57BL/6J mice to characterize LacNAc expression in the intestinal epithelium via our previously reported CHoMP method (Rouhanifard et al., 2014). We observed two distinct patterns: a progressive increase in LacNAc expression on enterocytes from the base to the tip of the villi (Figure 1B) and high expression of LacNAc in the crypts (Figure 1C). Staining the same tissue sections with lectin UEA (Ulex europaeus agglutinin) which is known to label predominantly Paneth cells and some populations of goblet cells in the murine intestine (Falk et al., 1994), revealed that regions of high LacNAc expression overlapped with Paneth cells at the base of the crypt (Figure 1D). UEA preferentially binds to α1,2- and α1,4-linked fucosides rather than α1,3-linked ones (Liener et al., 1986; Molin et al., 1986; Sugii and Kabat, 1982). To ensure the CHoMp-based labelling does not interfere with the UEA staining, we performed UEA labeling prior to CHoMP and observed the same colocalization pattern (Figure S1A).

To confirm this observation, we isolated crypts from Lgr5-EGFP-IRES-CreER reporter mice. This strain expresses a GFP tag in Lgr5+ stem cells (Lgr5 is a well-established marker of adult intestinal stem cells capable of generating all cell lineages of the small intestinal epithelium) (Muñoz et al., 2012). A single-cell suspension of the isolated crypts was prepared and stained with lectin UEA (Falk et al., 1994). The cells were further fluorescently labeled using the chemoenzymatic method for tagging LacNAc (Zheng et al., 2011) and analysed by flow cytometry to assess LacNAc levels for each population: stem cells (EGFP+/UEA−), Paneth cells (EGFP−/UEA+), and transit amplifying cells (EGFP−/UEA−). Paneth cells were found to express abundant LacNAc. By contrast, no significant levels of LacNAc were detected in the other two cell types (Figure 1E). To assess whether the chemoenzymatically manipulated cells were apoptotic, a single-cell suspension of treated and untreated organoids were compared using annexin/propidium iodide staining and found that the treated organoids were equal to or less apoptotic than untreated organoids (Figure S2A and S2B).

Glycan editing on the cell surface of crypt organoids

We hypothesized that the glycocalyx of Paneth cells, and specifically LacNAc, contributes to the regulation of stem cell behaviour because these cells are in continual contact with stem cells and are known to provide niche signals to regulate stemness and differentiation (Sato et al., 2011). To test this hypothesis, we used in situ glycan editing to modify the glycocalyx of cells within the crypt organoid culture system (Figure 2A) (Sato et al., 2009). We exploited the fucosylation method mentioned above (Figure 1A) to introduce a fucose to the 3'OH of the GlcNAc residue in the terminal LacNAc on the cell surface of the cultured organoids; alternatively, we used a recombinant human α-2,6 sialyltransferase ST6Gal1 to transfer a sialic acid from a CMP-Sialic acid donor onto the 6'OH of the Gal residue of cell-surface LacNAc (Mbua et al., 2013; Meng et al., 2013).

Figure 2. Glycan editing in mini-gut organoid culture system.

Figure 2

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(A) Workflow for in situ fucose or sialic acid editing on the cell surface of organoids cultured in matrigel. (B) Flow cytometry analysis confirms the modified glycans in dissociated organoids. Single cell suspensions from fucosylated organoids were stained with anti-CD15 antibody (anti-LeX), cells from sialylated organoids were stained with SNA lectin. Values are depicted as mean MFI ± SEM from three independent experiments. **P≤0.01, ***P≤0.001 versus untreated.

We first sought to verify whether in situ glycan editing could be completed in a matrigel-based organoid culture system. Toward this end, we treated the matrigel-embedded organoids with GDP-Fucose and α1,3 FucT or CMP-Sialic acid and ST6Gal1 for 30 min. After washing away the labeling reagents, the system was replenished with the culture medium. This procedure was conducted once per day for four days, and the treated organoids were then dissociated into single cells and stained with either CD15 antibody (which recognizes LeX), or Sambuca Nigra (SNA) lectin (which recognizes Sia-α2,6-Gal). Flow cytometry analysis confirmed the presence of LeX or Sia-α2,6-Gal in organoids grown in matrigel, showing the success of the enzymatic modifications (Figure 2B). A titration of the nucleotide sugar donor GDP-Fucose or CMP-Sialic acid was conducted in the presence of 10 µg each of the corresponding glycosyltransferase to determine the concentration required to achieve the saturation level by glycan engineering. A single cell suspension of glycosylated organoids was prepared immediately after the first day of treatment and analysed by flow cytometry for CD15 or SNA staining (Figure S3). The fucosylation reaction achieved maximum binding to CD15 at 500 µM GDP-Fucose, while the sialylation reaction reached maximum signal for SNA binding at 250 µM of CMP-Sialic acid. This suggests that at 500 µM of donor concentration, both glycosylation reactions saturate available LacNAc.

In situ glycan editing leads to higher frequency of organoid budding

Having confirmed that in situ glycan editing can be realized in the matrigel culture system, we analysed its impact on the phenotype of the treated crypt organoids (Figure 3A). In situ fucosylation or sialylation was performed daily (using 500 µM of each donor) for 4 days and organoids were counted and analysed daily for survival, buds, and size. The treatment did not appear to have a significant impact on organoid survival (P = 0.1970; Figure 3B, right panel). Nevertheless, we observed significantly more budding organoids on treated cultures as compared with untreated controls on day 2–4 (P = 0.0017; Figure 3A, 3B, S4). The differences became less significant as compared to the no enzyme controls after day 4.

Figure 3. Phenotypes of glycan edited organoids.

Figure 3

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(A) Time course of single crypt organoid embedded in matrigel and either left untreated (top), treated with in situ fucose editing (middle) or in situ sialic acid editing (bottom). Cartoon diagram shown to the left of the panel (blue square=N-acetylglucosamine, yellow circle=galactose, red triangle=fucose, and purple diamond=sialic acid); Scale: 50 µm. (B) 200-normalized organoids with buds (left) and total organoids (right) counts. Values are depicted as mean ± SEM from a minimum of three biological replicates. *P≤0.05, **P≤0.002 versus untreated. (C) Summary of Figure 3B on day 4 of organoid culture. (D) Fold-change in budding patterns as a function of nucleotide sugar controls.

By day 4, ~60% of fucosylated or sialylated organoids had buds, while only 27% of untreated organoids had produced new crypts (Figure 3C). The highest effect of glycan editing peaked on day two of culture, with a 1.8-fold increase in budding organoids in fucosylated samples, and a 4.2-fold increase in sialylated organoids as compared to the negative controls (nucleotide donors without glycosyltransferases; Figure 3D). We also performed a saturation analysis to determine the concentration of nucleotide sugar donor required to observe the hyperproliferation. We found that the donor effect saturates at ~100 µM concentration (Figure S5A). As shown in Figure S3, in the presence of 100 µM of nucleotide sugar donors LacNAc labeling was ~50% saturated. Combined together, the above observations suggested that as little as ~50% LacNAc modification can lead to the observed phenotype.

Glycan editing results in hyperproliferation but normal differentiation

Budding is an indicator of proliferation and/or differentiation. To further explore the effects of in situ glycan editing on intestinal stem cell proliferation and differentiation, we subjected organoids to in situ fucose or sialic acid editing for seven days and then dissociated them into a single-cell suspension for flow cytometry analysis using Ki-67 as a marker of proliferation. We observed an increase in Ki-67 signal in the total cell population of glycosylated organoids when compared to untreated or just nucleotide donor-treated controls (P = 0.0043; Figure 4A). This result shows that the observed increase in size and budding frequency on glycosylated cultures is likely due to hyperproliferation as a consequence of cell-surface glycan editing.

Figure 4. LacNAc engineering affects proliferation but not differentiation in organoid cultures.

Figure 4

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(A) Organoids were subjected to in situ fucose or sialic acid editing for 7 days, and then dissociated into single cell suspensions and stained for Ki-67. Graph represents percentage Ki-67 positive cells from the total population. Values are depicted as mean ± SEM from a minimum of three independent experiments. *P≤0.05. (B) Flow cytometry analysis of single cell suspensions from untreated and fucosylated organoids from Lgr5-EGFP-IRES-CreER mice. Cells were counterstained with anti-CD24 antibody and assessed for total numbers of each cell population. Values are depicted as the mean from three independent experiments representing a minimum of 6 biological replicates.

To examine the effects of crypt glycan editing on differentiation, we characterized the ratio of Paneth cells to Lgr5+ stem cells using single-cell suspensions from fucosylated and untreated crypts from Lgr5-EGFP-IRES-CreER mice. The dissociated crypts from both groups were stained with anti-CD24 antibody to identify Paneth cells, and analysed by flow cytometry. Untreated organoids were found to have comparable numbers of Lgr5+ stem cells and Paneth cells, forming approximately 10% of the total cell population (Figure 4B). In fucosylated organoids, the number of Lgr5+ stem cells increased slightly, albeit non-significantly, while the percentage of Paneth cells remained stable. Based on these data, we infer that the higher frequency of crypt formation is caused mainly by hyperproliferation of stem cells, followed by normal differentiation, keeping the proportion of cells in the crypts constant.

Blocking access to LacNAc was responsible for the observed hyperproliferation phenotype

Our observation that similar levels of hyperproliferation was observed upon either in situ fucose or sialic acid editing suggests that the effect of glycan editing was caused by capping LacNAc rather than the formation of new glycan epitopes (i.e. LeX or sLacNAc). To verify whether blocking access to LacNAc was responsible for the observed hyperproliferation effect, free LacNAc was added to the organoid culture medium to compete with cell-surface LacNAc for interactions with LacNAc-binding proteins. The addition of free LacNAc would result in the disruption of the interactions of LacNAc-binding proteins with their glycosylated targets, affecting their downstream effects. Free LacNAc was titrated into the media for four days at various concentrations. The budding frequency increased along with increasing the concentration of LacNAc from 10 µM to 1 mM (Figure S5B). At a high concentration of free LacNAc (10 mM LacNAc), crypts began to resemble the cysts associated with stem cell hyperproliferation without differentiation,(Sato et al., 2011) with subsequent death by day four.

Supplementing culture media with 1 mM LacNAc generated a budding frequency that resembled the growth pattern observed when organoids were subjected to in situ glycan editing (Figure 5A), supporting the hypothesis that the observed hyperproliferation is related to the disrupted interaction of LacNAc-binding proteins with their targets. In control experiments, the effects of adding an unrelated disaccharide, sucrose, or trisaccharides, LeX and sialyl LacNAc, were also tested. Although we observed that all free sugar supplements conferred a survival advantage to the organoids (Figure 5C), presumably due to their nutritional characteristics, none of them produced budding frequency at a comparable level as that of LacNAc (Figure 5B). This result suggests that the observed effect is specific for LacNAc-binding proteins and it is related to blocking access to LacNAc rather than creating new glycan epitopes.

Figure 5. Effects of addition of free glycans to organoid cultures.

Figure 5

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(A) Time course of organoid growth after addition of 1 mM LacNAc to the culture media on day 1. (B) Organoids with buds (*P≤0.05) and (C) total organoids counted daily after the addition of 1 mM LacNAc. Values are depicted as mean ± SEM from a minimum of three biological replicates, normalized to 200.

Glycan editing leads to changes in gene expression

In an effort to provide additional insights into the changes generated by blocking access to LacNAc on the surface of Paneth cells, Lgr5+ stem cells were sorted from dissociated organoids that had been treated with in situ fucose editing for 4 days. RNA was isolated and analysed by qRT-PCR with an RT2 profiler array for mouse stem cells (Qiagen). The average fold-change of gene expression in stem cells was analysed for untreated vs. glycan modified organoids from six biological replicates for 84 genes related to the identification, growth and differentiation of stem cells. Figure 6 summarizes the genes which fall under a quantitatively reliable range, clustered by function. These genes were then analysed using the Ingenuity knowledge database.

Figure 6. Gene expression changes in Lgr5+ stem cells from fucosylated organoids vs. untreated organoids.

Figure 6

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Summary of validated gene changes clustered by function (red: up-regulated genes, green: downregulated genes; lighter shades indicate fold changes < 1.5).

We observed down-regulation of multiple negative regulators of proliferation, such as Rb1, Apc and Fgf3. Additionally, IPA (Ingenuity Pathway Analysis) revealed that the Notch signalling pathway and the canonical Wnt pathway were directly affected by glycan editing (Figure S6). In the Wnt signalling pathway, gene expression of Bmp1, Bmp2, Bmp3, and Btrc was increased with a concomitant decrease in the expression of Wnt receptor Fxd1 as well as the ligand Wnt1. In the Notch pathway, Dll1 and Jag1 were upregulated, while the expression of Notch1, Kat2a and Dtx2 was decreased.

Discussion

Organoid cultures have opened up numerous opportunities to recapitulate the developmental processes of mammalian tissues from the early differentiation of specific cell types to organogenesis or adult stage. This technology is well suited to the study of biological phenomena that require a closed epithelial structure with a physiological, polarized topology. However, its application to the study of glycan’s functions has never been explored.

Our study demonstrated the feasibility of in situ glycan editing in crypt organoids grown in culture. We observed that in situ fucose or sialic acid editing of organoids selectively modified the glycocalyx of Paneth cells, leaving stem cells and TA cells mostly unmodified. This modification caused an increase in the frequency of budding organoids that correlated with an increase in proliferation. We observed significant differences between the modified and unmodified organoids up to day four. However, beyond day four the organoids that were treated with nucleotide sugars alone were not significantly different from the ones modified by in situ glycan editing. Presumably by that time point significant amount of glycosyltransferases are released from accumulated dead cells, which transfer fucose and sialic acid to the cell surface, generating the observed phenotype. Results from competition experiments indicate that fucose or sialic acid editing caps terminal LacNAc residues. We screened a panel of recombinant galectins—lectins bind to β-galactose-containing glycoconjugates—on the organoids and observed a similar growth phenotype (Figure S2C). This observation suggests that the galectins also block cell-surface LacNAc from participating extracellular interactions.

The hyperproliferation phenotype is consistent with a previous report of β1,4-galactosyltransferase knockout in mice which resulted in an enlarged crypt phenotype, presumed to be due to the deregulation of stem cell proliferation as a result of LacNAc perturbation; the β1,4-galactosyltransferase knockout dramatically reduces LacNAc expression (Asano et al., 1997; Vanhooren et al., 2013). Our gene expression analysis supported the observed hyperproliferation phenotype with genes involved in the Wnt and Notch signalling pathways deregulated. We also observed a deregulation of negative regulators of proliferation. Interestingly, by applying CHoMP to human sections of the small intestine we observe a similar LacNAc expression pattern with abundant LacNAc found in Paneth cells (Figure S1B) suggesting that LacNAc capping may result in hyperproliferation in human crypts as well.

In situ fucose editing of LacNAc generates LeX, which may confer new cellular functions. A characterization of the natural expression of this glycan epitopes on a single-cell suspension of Lgr5-EGFP-IRES-CreER dissociated crypts revealed that both stem cells (Lgr5-EGFP+) and Paneth cells (CD24+) express LeX de novo (Figure S1C). There is a possibility that a coordinated cycling between unblocked LacNAc and “blocked” LacNAc, i.e. LeX, is responsible for the observed hyperproliferation.

Analysis of total cell numbers in dissociated organoids revealed that differentiation in the treated cultures maintains a constant ratio of Lgr5+ cells and Paneth cells. This phenomenon is distinct from a scenario of injury-like proliferation and differentiation where Lgr5+ -label retaining cells differentiate directly into Paneth cells, thus increasing the ratio of Paneth cells per crypt (Buczacki et al., 2013).

Significance

Adult epithelial stem cells are heralded as an attractive tool for regenerative medicine due to their unique abilities to proliferate, differentiate, and self-renew. However, these same properties may lead to tumor formation if they are not tightly controlled. A thorough understanding of the niche environment, including glycosylation, is critical for guiding and controlling stem cell growth to fully achieve their therapeutic potential. The current work lays the foundation for the systematic analysis of glycans in stem cell biology, and the model system presented here can be applied to the study of other types of glycans that can be enzymatically modified in situ.

STAR Methods

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Peng Wu (peng.wu@scripps.edu)

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animals

All studies were conducted in 8–12-week-old male C57BL/6J or Lgr5-EGFP-IRES-CreER mice. Mice were originally obtained from Jackson Labs and were subsequently bred and housed at the Animal facility at the Albert Einstein College of Medicine. All studies were carried out under a protocol approved by the Institutional Animal Care and Use Committee at the Albert Einstein College of Medicine. Animals were housed in standard cages (<5 per cage). All animals were allowed free access to a standard commercial diet and water during the study. The holding room was maintained under standard conditions.

Organoid culture conditions

Crypt isolation and organoid preparation were prepared as described below. Organoids were maintained in crypt culture medium (Advanced DMEM/F12, 2 mM GlutaMax, 10 mM Hepes, 1× PenStrep, 1× N2, 1× B27, 1 µM N-acetylcysteine, 50ng/ml EGF, 100 ng/ml Noggin, 1 µg/ml R-spondin). EGF, Noggin and Rspondin were replenished every 2 days, and the full media was changed every 4 days. For each organoid culture condition, 3–6 technical replicates were utilized from the same mice as well as a minimum of n = 3 biological replicates from different mice.

METHOD DETAILS

Nucleotide sugar donor synthesis

Synthesis of GDP-Fucose and GDP-Fucose-alkyne (GDP-FucAl) was carried out as described previously (Wang et al., 2009). In summary, a reaction mixture (5 mL) of L-fucose or fucose-alkyne (10 mM), ATP (10 mM), GTP (10 mM), MgSO4 (10 mM), Tris-HCl buffer (100 mM, pH 7.5), inorganic pyrophosphatase (90 units or 168 mg/L), and FKP (9 units or 600 mg/L) was incubated for 6 hrs at 37°C in a shaker (225 rpm). CMP-Sialic acid was synthetized according to a published protocol (Yu et al., 2004). Briefly, a reaction mixture containing 10mM ManNAc or mannose-alkyne, 50 mM sodium pyruvate, 100 mM Tris-HCl pH 8.8, 20 U of E. coli K12 Aldolase, 20 mM MgSO4, N. Meningitidis CMP-Sialic acid synthetase, and 20 mM CTP was incubated for 2 hrs at 37°C in a shaker (225 rpm). The reactions were monitored by TLC analysis (10 mM tetrabutylammonium hydroxide in 80% aqueous acetonitrile). When the reactions had gone to completion, 5 mL of cold ethanol was added and the mixtures were incubated for 30 minutes on ice to quench the reaction and precipitate the enzymes. The precipitate was removed by centrifugation (5,000 × g for 30 minutes) and the supernatant concentrated using a rotary evaporation to remove ethanol. The reaction products were purified by Bio-Gel P2 gel filtration chromatography (1.5 × 75 cm) and eluted with 50 mM ammonium bicarbonate buffer. Fractions containing the nucleotide sugar donors were lyophilized, and the products were characterized by NMR and LC/MS.

Expression and Purification of Fucosyltransferase

ɑ1,3 fucosyltransferase was prepared as described previously (Zheng et al., 2011). In summary ɑ1,3 FucT clonal populations of BL21(DE3) E.coli containing the plasmid were incubated in 1 L of LB broth with Kanamycin (50 mg/mL) with shaking at 37°C until OD600 = 0.8. Fucosyltransferase expression was induced with 100 mM isopropyl IPTG, and the temperature was lowered to 25°C. After 4 hours, the bacteria were harvested and resuspended in 40 mL of lysis buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10 mM imidazole, 1 mM DTT, 2 U/mL DNAse, 1 Complete EDTA-free protease inhibitor cocktail tablet) and sonicated. Cell debris was pelleted (8000 × g, 30 min) and 1 mL of Ni-Sepharose resin (Ni Sepharose 6 Fast Flow, GE Healthcare) was added to the supernatant. After an hour incubation at 4°C, the mixture was applied to a polypropylene column, and the resin was washed with wash buffer (20 mM Tris-HCl, pH 7,5, 500 mM NaCl, 50 mM imidazole, 0.5 mM DTT, 5% glycerol). The His6-tagged protein was eluted with elution buffer (20 mM Tris-HCl, pH 7,5, 500 mM NaCl, 500 mM imidazole, 0.5 mM DTT, 5% glycerol). Protein fractions were collected and dialyzed overnight in dialysis buffer (20 mM Tris-HCl, pH 7,5, 250 mM NaCl, 1 mM DTT, 10% glycerol).

CHoMP labeling

CHoMP labeling was performed according to published protocols (Rouhanifard et al., 2014). In summary, tissue sections were prepared with a Leica microtome at 5 µm. Slides were immersed sequentially for 5 min each in 100% HistoClear (2 times), 100% EtOH (2 times), 70% EtOH (2 times), 50% EtOH (2 times), and water. A hydrophobic barrier was drawn around the tissue samples using a PAP pen. Tissues were immersed in 50 mL of TBS + 0.1% Tween-20 (TBST) for 10 minutes. For the enzymatic reaction, slides were placed in a humidified chamber and 50–500 µl of enzyme solution were added (volume depending on tissue area). The enzyme solution contained: 200 µg/mL of ɑ1,3 FucT, 1 mM GDP-FucAl, and 5 mM MgCl2 in TBST. The slides were incubated for 1h at 37°C. The slides were then washed 3 times for 5 min in 50 mL of TBST in coplin jars. The slides were again placed in a humidified chamber and an accelerated biotin probe (Jiang et al., 2014) was then ‘clicked’ to the Fucose-alkyne using a solution of 100 µM accelerated biotin-azide, 75 µM CuSO4 premixed with 150 µM BTTP ligand (Wang et al., 2011) and 2.5 mM sodium ascorbate in TBST for 30 min at RT. Following three washes in TBST, the tissues were blocked for 10 min in 0.3% hydrogen peroxide diluted in TBS in coplin jars at RT, and washed 3 times in TBST to remove the H2O2. The slides were then placed in a humidified chamber and incubated with Neutravidin-HRP (1:100 in TBST) for 1h at RT, then subsequently washed 3 times for 5 min with TBST in coplin jars. Finally, the slides were placed in a humidified chamber and incubated with TSA-Plus FITC reagent according to manufacturer's protocol (1:50 dilution for 10 min at RT, protected from light), then washed 3 times for 5 min each in TBST in coplin jars and mounted with Prolong anti-fade gold with DAPI. Photomicrographs were acquired on a Zeiss AxioObserver.

CHoMP co-staining with UEA lectin or anti-lysozyme antibody

CHoMP labeling was performed as described above. Prior to mounting, tissues were incubated for 1 hr at RT with 1:100 rabbit anti-lysozyme antibody in TBST or Rhodamine-tagged UEA lectin (1:100). The slides were then washed 3 times for 5 min each in TBST in coplin jars. The lysozyme-tagged tissues were incubated with an anti-rabbit-HRP antibody (1:100 in TBST) for 1h at RT, then subsequently washed 3 times for 5 min with TBST in coplin jars, and finally the slides were placed in a humidified chamber and incubated with TSA-Plus Cy3 reagent according to manufacturer's protocol (1:50 dilution for 10 min at RT, protected from light), and then washed 3 times for 5 min each in TBST in coplin jars and mounted with Prolong anti-fade gold with DAPI. Tissues labeled with lectin were directly mounted with Prolong anti-fade gold with DAPI.

Crypt Isolation

Crypts from mice were prepared as published (Sato et al., 2009). In short, the small intestine was extracted and flushed with ice cold PBS (-Ca, -Mg). Intestines were cut into 3 inch pieces and opened longitudinally. The exposed surfaces were scraped using a glass coverslip to remove most of the villi, then the intestines were cut into 2–4 mm pieces and transferred to a 50 mL Falcon tube filled with 10 mL of ice cold PBS. The pieces were swirled, decantedm and the PBS replaced until the supernatant was almost clear. Samples were incubated in 20 mL of 2 mM EDTA/PBS at 4°C on a rotator for 45 minutes to dissociate the crypts. The EDTA solution was removed and the tissues were resuspended in 10 mL of cold PBS. The tubes were shaken vigorously 20 times, and the supernatant collected. This step was repeated 5 times, and the fractions were collected and checked under a microscope. Fractions 3 and 4 usually contain the crypts. Crypt fractions were passed through a 70 µm strainer into bovine serum albumin (BSA)-coated 50 mL Falcon tube. Cells were spun down at 300 × g for 5 minutes at 4°C.

Preparation of crypts single-cell suspension

Crypts were prepared as described before. The supernatant was removed and the crypts were resuspended in 2 mL of pre-warmed single-cell dissociation media (Advanced DMEM/F12 media, 2 mM GlutaMax, 10 mM Hepes, 1× PenStrep, 1× N2, 1× B27, 10 µM Y-27632) for 45 minutes at 37°C. While in the single-cell dissociation media, cells were pipetted up and down approximately every 10 minutes. After 45 minutes, the samples were passed through a 40 µm strainer and 10 mL of cold basal culture media (Advanced DMEM/F12, 2 mM GlutaMax, 10 mM Hepes, and 1× PenStrep) was added. This washing step was repeated twice and single cells were used without further treatment.

Flow cytometry of single-cell suspension of LacNAc labeled crypt cells

Single cell suspensions from isolated crypts were performed as described above from an Lgr5-EGFP-IRESCreER mice and C57BL/6J mouse as a negative control. The single-cell suspension was first subject to in situ fucose editing using GDP-FucAl. Cells were added to a 96 well plate and 100 µl of enzyme solution was added. The enzyme solution contained: 20 µg/mL of ɑ1,3 FucT, 500 µM GDP-FucAl, and 20 µM MgCl2 in HBSS/2% BSA. The cells were incubated for 10 min at 37°C, spun down at 300 × g for 3 min, and washed 3× with HBSS/BSA. An azide-functionalized biotin-probe (Jiang et al., 2014) was then ‘clicked’ to the FucAl in a reaction mixture containing 100 µM biotin-azide probe, 75 µM CuSO4 premixed with 375 µM BTTPS ligand (Wang et al., 2011), and 2.5 mM sodium ascorbate in HBSS/BSA for 10 min at RT. After 3 washes, the cells were incubated with Streptavidin-Alexa 647 (1:200 in HBSS/BSA) for 10 min at RT, then subsequently washed 3× with HBSS/BSA. Either CD24-PE (BD Pharmingen) or Rhodamine-tagged Ulex europaeus agglutinin (UEA), both markers for Paneth cells, were added at a 1:500 dilution in HBSS/BSA for 30 minutes on ice. The cells were spun down at 300 × g for 3 min, and washed 3× with HBSS/BSA. Samples were resuspended in 500 µL of FACS buffer (HBSS/BSA) with DAPI as a live/dead stain, and analysed by flow cytometry. Flow cytometry analysis was performed in either a BD LSR II Flow Cytometer or an iCyt Flow Cytometer (Sony). Sorting experiments were completed in a BD FACS Aria III Cell Sorter.

Crypt organoid culture preparation

Crypt isolation was performed as described above. After the initial centrifugation, crypts were gently resuspended instead in 10 mL of basal culture medium (Advanced DMEM/F12 media, 2 mM GlutaMax, 10 mM Hepes, 1× PenStrep), and centrifuged at 150 × g for 2 minutes at 4°C to remove single cells. This washing step was repeated twice. The total number of crypts was determined using a haemocytometer and crypts were resuspended with Matrigel to achieve a concentration of ~200 crypts per 50 µL. A 50 µL droplet was placed in the middle of each well of a prewarmed, 24-well cell culture dish. After plating, the plate was transferred into a 37°C incubator for 10 minutes, followed by the addition of 500 µL of crypt culture medium (basal culture medium including 1× N2, 1× B27, 1 µM N-acetylcysteine, 50 ng/ml EGF, 100 ng/ml Noggin, 1 µg/ml R-spondin).

In situ glycan editing

In situ glycan editing was performed once every 24 hours. Media was removed and saved. 100 µL of enzymatic fucosylation reaction mixture (20 mM MgCl2, 10 µg of ɑ1,3 FucT and 500 µM GDP-Fucose in HBSS) was added to the Matrigel-based culture and incubated for 20 minutes at 37°C, then washed gently with 500 µL HBSS. For in situ sialic acid editing, the reaction mixture (20 mM MgCl2, 10 µg of ST6Gal1 and 500 µM CMP-Sialic acid in HBSS) was added and incubated for 30 minutes at 37°C, then washed gently with 500 µL of HBSS.

Flow cytometry of dissociated organoids

Organoids were subjected to in situ fucose or sialic acid editing daily for 4 days. A single cell suspension of organoids was generated by vigorous pipetting and resuspended in single-cell dissociation media for 45 minutes at 37°C or alternatively the plates were incubated at 4°C for 1 hr in Cell Recovery Solution, followed by vigorous pipetting. Single cells were stained with anti-CD15-PE antibody (1:100 dilution in PBS/0.1% FBS) or SNA-biotin lectin (2 µg/ml in PBS/0.1%FBS). The antibody and lectin were each incubated for 30 minutes on ice with the cell mixture, and washed 3 times for 5 minutes in PBS/0.1%FBS. The cells were counterstained with FITC conjugated CD24 antibody and washed 3 times for 5 minutes in PBS/0.1%FBS. The cells treated with SNA-biotin were then incubated with Streptavidin-PE on ice for 30 minutes, then washed 3 times for 5 minutes in PBS/0.1%FBS. Cells were washed and resuspended in HBSS/BSA with DAPI, and analysed by flow cytometry.

Ki-67 Cell proliferation assay

Treated or control organoids were grown in culture for 6 days, then broken up by vigorous pipetting and resuspended in single-cell dissociation media for 45 minutes at 37°C. Samples were stained with Ghost dye (Tonbo Biosciences) for live vs. dead cell discrimination with 1 µL of ghost dye in 1 mL of PBS for 30 minutes on ice. The samples were then washed 3× with PBS, and fixed/permeabilized using a Transcription Factor Staining Buffer Set (eBioscience). Samples were incubated with 80 µL of 1× Fixation/Permeabilization buffer for 10 minutes on ice, then washed 3× with PBS. Samples were then incubated with 1× permeabilization buffer for 10 minutes on ice, washed 3× with PBS then incubated with Ki-67-APC antibody (eBioscience) diluted 1:100 in Permeabilization buffer for 30 minutes on ice. Following antibody staining, the cells were washed 3× with PBS, and resuspended in HBSS/BSA for flow cytometry analysis.

Free oligosaccharide competition assay

Organoids were prepared, grown and analysed as described above. Free LacNAc, sucrose, Lewis X or Sialyl LacNAc were added to the media at various concentrations on day 1 and day 4 or organoid culture, when the culture media was changed.

RNA isolation and gene expression profiling

Organoid cultures from Lgr5-EGFP-IRES-CreER mice were prepared and subjected to in situ fucose editing as described above. On day 4, media was replaced by a 1 mg/mL dispase solution in PBS and incubated at 37°C for 30 min, until the matrigel dissolved. Crypts were pipetted vigorously to dissociate into single cell suspension and washed twice with PBS. Lgr5+ cells (~30,000/sample) were sorted by flow cytometry into RNA Protect Reagent (Qiagen). RNA was isolated using QIAShredder columns and a RNeasy Micro kit (Qiagen) according to manufacturer's instructions. RNA was reverse transcribed and amplified with a RT2 PreAMP kit (Qiagen) for analysis with the RT2 Profiler Array for Mouse Stem Cells (Qiagen - PAMM-405Z) according to manufacturer’s instructions. qRT-PCR was completed in an ABI 7300 Real Time PCR system, data was uploaded to and analysed with GeneGlobe, and results were analysed with the Ingenuity Pathway Analysis software.

QUANTIFICATION AND STATISTICAL ANALYSIS

For each organoid culture condition, 3–6 technical replicates were utilized from the same mice as well as a minimum of n = 3 biological replicates from different mice. This information is indicated in individual figure legends. All results are presented as means ± SEM unless otherwise indicated. Flow cytometry data analysis was completed using FlowJo analysis software. All images were processed using ImageJ software. Statistical analysis and graph generation was completed using GraphPad Prism 7.0 software.

DATA AND SOFTWARE AVAILABILITY

The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE108512 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE108512).

Supplementary Material

supplement

Highlights.

  • LacNAc is abundant on Paneth cells and can be edited in crypt organoids

  • LacNAc blocking on Paneth cells leads to hyperproliferation of Lgr5+ stem cells

Acknowledgments

This work was supported by the NIH (R01GM113046 to P.W. and P41GM103390 and P01GM107012 to K.W.M.). We thank Prof. Hung-Lin Chen from the Glycocore at Academia Sinica, Taiwan for providing the recombinant galectin panel and Prof. Leonard H. Augenlicht at the Albert Einstein College of Medicine for providing tissue sections of human intestine. We also thank the Albert Einstein College of Medicine Histotechnology and Comparative Pathology facility as well as Dr. Winfried Edelmann, Dr. Johanna Van Oers, and Dr. Halley Ketchum Pierce for advice and support, and Prof. Pamela Stanley for valuable discussions.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Author Contributions

S.H.R, A.L.A. and P.W. designed the experiments. S.H.R, A.L.A. and L.M performed experiments. S.H.R, A.L.A. and P.W. prepared the manuscript and everyone edited it.

Declaration of Interests

The authors declare no competing interests.

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Supplementary Materials

supplement
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