Glycosyltransferase ST6GAL1 contributes to the regulation of pluripotency in human pluripotent stem cells

Abstract

Many studies have suggested the significance of glycosyltransferase-mediated macromolecule glycosylation in the regulation of pluripotent states in human pluripotent stem cells (hPSCs). Here, we observed that the sialyltransferase ST6GAL1 was preferentially expressed in undifferentiated hPSCs compared to non-pluripotent cells. A lectin which preferentially recognizes α-2,6 sialylated galactosides showed strong binding reactivity with undifferentiated hPSCs and their glycoproteins, and did so to a much lesser extent with differentiated cells. In addition, downregulation of ST6GAL1 in undifferentiated hPSCs led to a decrease in POU5F1 (also known as OCT4) protein and significantly altered the expression of many genes that orchestrate cell morphogenesis during differentiation. The induction of cellular pluripotency in somatic cells was substantially impeded by the shRNA-mediated suppression of ST6GAL1, partially through interference with the expression of endogenous POU5F1 and SOX2. Targeting ST6GAL1 activity with a sialyltransferase inhibitor during cell reprogramming resulted in a dose-dependent reduction in the generation of human induced pluripotent stem cells (hiPSCs). Collectively, our data indicate that ST6GAL1 plays an important role in the regulation of pluripotency and differentiation in hPSCs, and the pluripotent state in human cells can be modulated using pharmacological tools to target sialyltransferase activity.

Figure 1. ST6GAL1 is preferentially expressed and active in hPSCs.

(a) Heatmap representation of ST6GAL1 transcript expression, as well as other glycosyltransferases, measured by global gene expression analysis in hPSCs and non-pluripotent cell samples. The expression of ST6GAL1 in each cell sample, detected by two independent probes targeting different regions of the ST6GAL1 transcript is shown. Black asterisk: WA07 hESCs and their isogenic differentiated derivatives. Purple asterisk: HDF51 cells and hiPSCs generated from HDF51 cells. (b) Upper panel: Western blotting analysis of ST6GAL1 in cell samples showed that undifferentiated hPSCs had higher ST6GAL1 protein expression, compared to differentiated cells. Lane 1: HMi-506_Mel Diff¹⁶, 2: HDF68i-505_Mel Diff¹⁶, 3: HM (HEMl), 4: HDF68, 5: HDF51, 6: WA09, 7: HDF68i-505¹⁶, 8: HMi-506¹⁶, 9: TSRI001i-HDF⁸. Lower panel: SNA-mediated blotting showed that protein samples extracted from undifferentiated hPSCs had higher reactivity to SNA binding. Lane 1: HM, 2: HDF68i-505_Mel Diff, 3: HDF51, 4: HDF68, 5: WA09, 6: HMi-506, 7: TSRI001i-HDF, 8: HDF68i-505. (c) SNA-mediated blotting showed that protein samples extracted from derivatives of undirected differentiation (embryoid bodies, EBs) had less reactivity to SNA, compared to those from paired, undifferentiated hPSCs. Lane 1: WA09, 2: WA07, 3: TSRI001i-HDF, 4: HMi-506, 5: WA09_EBs, 6: WA07_EBs, 7: TSRI001i-HDF_EBs, 8: HMi-506_EBs. (d) Flow cytometric analysis demonstrated that live human cells in the pluripotent state were more reactive to SNA at the cellular level.

Figure 2. Downregulation of ST6GAL1 in hPSCs has an impact on signaling networks involved in pluripotency regulation and embryogenesis.

(a) Left Panel: Western blotting analysis showed that two independent shRNA sequences (shRNA2 and shRNA5) that target ST6GAL1 transcripts led to effective downregulation of ST6GAL1 protein 72 hours after transduction. While the protein level of NANOG was relatively unaffected, the protein level of POU5F1 was decreased in hPSCs that received shRNA2 and shRNA5. Right Panel: SNA-mediated blotting showed that protein samples extracted from hPSCs which received shRNA2 had lower reactivity to SNA, indicating a decreased amount of α-2,6 sialylated glycoconjugates in the cells. pLKO1: the empty factor control for the transduction of shRNA expression vectors. (b) Global gene expression profiling followed by differential gene expression analysis revealed a group of genes (~400 genes) that were differentially expressed (P < 0.01, F-test) in multiple lines of hPSCs with and without ST6GAL1 knockdown. (c) Quantitative RT-PCR was used to validate the expression of a selection of differentially expressed genes, confirming the upregulation of the PITX2 gene and the downregulation of KATA6A and ST6GAL1 genes due to ST6GAL1 knockdown. D13: 13 days after the beginning of puromycin selection (14 days post transduction). (d) The expression level of POU5F1 transcripts reflected by normalized fluorescence units in expression array analysis indicated that the POU5F1 gene expression was relatively unchanged at the transcriptional level in multiple lines of hPSCs with ST6GAL1 knockdown.

Figure 3. ST6GAL1 knockdown impedes cellular reprogramming and establishment of induced pluripotency in human somatic cells.

(a) Schematic illustration of the experimental strategy to examine the influence of ST6GAL1 knockdown on cellular reprogramming. Twenty-four hours after HDFs received POU5F1, SOX2, KLF4, and MYC with or without ST6GAL1 shRNA, the transduced HDFs were seeded on X-ray irradiated feeder cells (mouse embryonic fibroblasts, MEFs). Fourteen days later, alkaline phosphatase (AP) staining was used to examine hiPSC colonies formed by transduced HDFs on the feeder cells. (b) AP staining showed that dramatically fewer hiPSC colonies with AP activity were obtained from cellular reprogramming under ST6GAL1 knockdown mediated by shRNA2 and shRNA5. (c) Quantitative analysis of NANOG expressing cells in the reprogrammed cell population showed that shRNA2 and shRNA5 both led to a significant reduction in NANOG expressing cells in the analyzed cell populations. Left Panel: The histogram representation of flow cytometry analysis. Right panel: the quantitative result of flow cytometry analysis (n = 3; *P < 0.05, t-test). (d) The expression of endogenous POU5F1, SOX2, KLF4 and MYC genes in cells that underwent reprogramming with or without ST6GAL1 knockdown was measured by qRT-PCR with primer sets that target untranslated regions of the endogenous gene transcripts. The induction of endogenous POU5F1 and SOX2 gene expression at the early stage of cellular reprogramming in HDFs was substantially suppressed by shRNA-mediated ST6GAL1 knockdown (n = 3; *P < 0.05, t-test). Day 6: cell samples collected at 6 days after the initial transduction (2 days after the beginning of puromycin selection). Day 12: cell samples collected at 12 days after the initial transduction (8 days after the beginning of puromycin selection).

Figure 4. Treatment of a sialyltransferase inhibitor suppresses cellular reprogramming and establishment of induced pluripotency in human somatic cells.

(a) AP staining showed that fewer hiPSC colonies with AP activity were obtained from cellular reprogramming under the treatment of 3F_ax-peracetyl Neu5Ac. (b) The numbers of AP-positive colonies with a size equal or larger than 1.0 mm in diameter were counted at the end of reprogramming experiments, which showed a dose-dependent reduction of hiPSC formation due to the treatment of 3F_ax-peracetyl Neu5Ac (n = 3; *P < 0.05, t-test). (c) The percentages of cells with enhanced SNA reactivity in reprogrammed HDF51 cells were significantly reduced by the treatment of 3F_ax-peracetyl Neu5Ac for 6 days in a dose-dependent manner (n = 3; *P < 0.05, t-test), indicating decreased sialylation on the cell surface. (d) The cell counts of reprogrammed HDF51 cells at different time points over 6 days were marginally but significantly reduced by the treatment of 3F_ax-peracetyl Neu5Ac in a dose-dependent manner (n = 3; *P < 0.05, t-test), indicating a slight suppression of cell proliferation. (e) The number of apoptotic cells in reprogrammed HDF51 cells was slightly increased by the treatment of 500 μM 3F_ax-peracetyl Neu5Ac for 6 days.