ST8SIA4-Dependent Polysialylation is Part of a Developmental Program Required for Germ Layer Formation from Human Pluripotent Stem Cells

Abstract

Polysialic acid (PSA) is a carbohydrate polymer of repeating α-2,8 sialic acid residues that decorates multiple targets, including neural cell adhesion molecule (NCAM). PST and STX encode the two enzymes responsible for PSA modification of target proteins in mammalian cells, but despite widespread polysialylation in embryonic development, the majority of studies have focused strictly on the role of PSA in neurogenesis. Using human pluripotent stem cells (hPSCs), we have revisited the developmental role of PST and STX and show that early progenitors of the three embryonic germ layers are polysialylated on their cell surface. Changes in polysialylation can be attributed to lineage-specific expression of polysialyltransferase genes; PST is elevated in endoderm and mesoderm, while STX is elevated in ectoderm. In hPSCs, PST and STX genes are epigenetically marked by overlapping domains of H3K27 and H3K4 trimethylation, indicating that they are held in a "developmentally-primed" state. Activation of PST transcription during early mesendoderm differentiation is under control of the T-Goosecoid transcription factor network, a key regulatory axis required for early cell fate decisions in the vertebrate embryo. This establishes polysialyltransferase genes as part of a developmental program associated with germ layer establishment. Finally, we show by shRNA knockdown and CRISPR-Cas9 genome editing that PST-dependent cell surface polysialylation is essential for endoderm specification. This is the first report to demonstrate a role for a glycosyltransferase in hPSC lineage specification. Stem Cells 2016;34:1742-1752.

Keywords: Glycosylation; Neural cell adhesion molecule; Pluripotent stem cells; Polysialic acid.

Figure 1

Polysialic acid is expressed upon differentiation to all three germ layers. (A) Flow cytometry of hPSC differentiation to definitive endoderm showing surface expression of PSA along with endoderm marker CXCR4 (Top) and pluripotency marker SSEA3 (Bottom). (B) Immunoblot of PSA expression in hPSC and DE. (C) Flow cytometry of PSA expression (Top) in hPSC (Shaded gray), DE (Red), mesoderm (Blue), and NCC (Green). PSA⁺ fractions in each cell type are 2.9%, 79.7%, 72.4% and 81.1%, respectively. Isotype control is shown for comparison (Bottom). (D-H) Immunostaining of PSA expression in hPSC differentiation. Scale bar, 100μm. (D) hPSC: PSA and DE marker SOX17 (Top), PSA and Golgi marker α-mannosidase II (Bottom). (E) DE: PSA and SOX17 (Top), PSA and α-mannosidase II (Bottom). (F) Mesoderm: PSA and T (Top), PSA and NANOG (Bottom). (G) NCC: PSA and NANOG (Top), PSA and Golgi marker Giantin (Bottom). (H) Zoom of PSA and α-mannosidase II in DE. Arrows point to overlap of PSA and α-mannosidase II. (I) Time course immunoblot of PSA expression during hPSC differentiation to NCC (Top). CDK2 shown as loading control (Bottom). Abbreviations: hPSC, human pluripotent stem cell; DE, definitive endoderm; NCC, neural crest cell.

Figure 2

Expression of polysialyltransferases is lineage specific and NCAM is the substrate of polysialylation. (A-C) qPCR expression of PST, STX, and NCAM (Top) and lineage markers (Bottom) in hPSC differentiation to DE (A), mesoderm (B), and NCC (C). (D) Immunostaining of PSA expression in DE day 4 cells transduced with shRNA targeting PST (Right) compared to control shRNA (Left). Scale bar, 100μm. (E) Flow cytometry of PSA and NCAM surface expression in hPSC, DE, and mesoderm. (F) Immunostaining of shRNA knockdowns of PST and NCAM in DE day 4. Scale bar, 100μm. (G) Immunostaining of PSA and α-mannosidase II expression in DE cells transduced with NCAM shRNA. Arrows point to overlap in expression. Scale bar, 100μm. (H) Immunoblot of NCAM (Top) and PSA (Middle) in all three germ layers. β-Actin shown as loading control (Bottom). (I) Immunoprecipitation of NCAM and PSA in hPSC and DE. IP lysate was then immunoblotted for PSA (Left) and NCAM (Right). Lysate from COS-1 cells overexpressing PST, STX, and NCAM was used as positive control. Abbreviations: hPSC, human pluripotent stem cell; DE, definitive endoderm; CTL, control.

Figure 3

Polysialylation is bivalently regulated with PST under the control of the T/GSC network. (A-C) Graphical representation of H3K4me3, H3K27me3, and CpG islands at the PST (A), STX (B), and NCAM (C) genomic loci in WA01 hPSCs (Data from the UCSC genome browser database [33]). (D) Graphical illustration of gene kinetics during DE differentiation. (E) Immunostaining comparison of T-GR expression of PSA and SNAI1 after 4 day addition of Dex compared to −Dex. Scale bar, 100μm. (F) qPCR of genes in T-GR cells −Dex, +Dex 2d, and +Dex 4d. (G) Transcript analysis of GSC and PST over 4 day time course addition of Dex in T-GR cells. (H) Flow cytometry for surface PSA expression in hPSCs, hPSCs+GFP, and hPSCs+GSC-GFP 24h post electroporation. (I) Analysis of the percentage of GFP⁺/PSA⁺ cells from Fig. 4H. (J) qPCR of samples from Fig. 4H. (K) Luciferase assay of hPSCs transfected with GSC-GR, PST-LUC, and LUC-Control after Dex addition for 0, 6, and 12h. (L) GSC ChIP assay in hPSCs transfected with GSC-GFP. Genomic primers used probe the TSS of the PST promoter and the STX −5kb upstream region shown as negative control. (M) qPCR of DE d4 cells transduced with shRNA targeting GSC compared to control shRNA. (N) Diagram of proposed interaction of T, GSC, and PST. Abbreviations: hPSC, human pluripotent stem cell; Dex, dexamethasone; GR, glucocorticoid receptor; DE, definitive endoderm; LUC, luciferase; TSS, transcription start site; CTL, control.

Figure 4

PST is required for efficient hPSC differentiation to endoderm. (A) Immunostaining of PSA and SOX17 expression in DE cells transduced with control shRNA (Left) and PST shRNA (Right). Scale bar, 100μm. (B) Schematic of CRISPR gene editing approach of the PST first exon. The 300 bp region containing the PST TSS was cleaved and replaced by a BFP-IRES-Zeo cassette via homology directed repair. (C) qPCR of genes in WT and PST^−/− cells differentiated to DE. (D) Flow cytometry of CXCR4 and PSA expression in WT and PST^−/− cells differentiated to DE. (E) Immunoblot comparisons of WT and PST^−/− cells differentiated to DE. (F) Immunostaining of PSA with SOX17 (Top) and FOXA2 (Bottom) in WT and PST^−/− cells differentiated to DE. Abbreviations: CTL, control; hPSC, human pluripotent stem cell; DE, definitive endoderm; TSS, transcription start site; sgRNA, single guide RNA.