scholarly journals B3GALT5 knockout alters glycosphingolipid profile and facilitates transition to human naïve pluripotency

2020 ◽  
Vol 117 (44) ◽  
pp. 27435-27444
Author(s):  
Ruey-Jen Lin ◽  
Ming-Wei Kuo ◽  
Bei-Chia Yang ◽  
Hsiu-Hui Tsai ◽  
Kowa Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
X Chromosome ◽  
Human Embryonic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cloning Efficiency ◽  
Dna Hypomethylation ◽  
Intracellular Ca ◽  
Cells Cultured

Conversion of human pluripotent stem cells from primed to naïve state is accompanied by altered transcriptome and methylome, but glycosphingolipid (GSL) profiles in naïve human embryonic stem cells (hESCs) have not been systematically characterized. Here we showed a switch from globo-(SSEA-3, SSEA-4, and Globo H) and lacto-series (fucosyl-Lc4Cer) to neolacto-series GSLs (SSEA-1 and H type 2 antigen), along with marked down-regulation of β-1,3-galactosyltransferase (B3GALT5) upon conversion to naïve state. CRISPR/Cas9-generatedB3GALT5-knockout (KO) hESCs displayed an altered GSL profile, increased cloning efficiency and intracellular Ca2+, reminiscent of the naïve state, while retaining differentiation ability. The altered GSLs could be rescued through overexpression of B3GALT5.B3GALT5-KO cells cultured with 2iLAF exhibited naïve-like transcriptome, global DNA hypomethylation, and X-chromosome reactivation. In addition,B3GALT5-KO rendered hESCs more resistant to calcium chelator in blocking entry into naïve state. Thus, loss of B3GALT5 induces a distinctive state of hESCs displaying unique GSL profiling with expression of neolacto-glycans, increased Ca2+, and conducive for transition to naïve pluripotency.

189 SMALL MOLECULE INHIBITORS PD0325901 AND CHIR99021 CAUSE REDUCED EXPRESSION OF PLURIPOTENCY GENES IN PUTATIVE PORCINE INDUCED PLURIPOTENT STEM CELLS

2014 ◽  
Vol 26 (1) ◽  
pp. 209
Author(s):  
S. Petkov ◽  
H. Niemann
Keyword(s):  
Stem Cells ◽  
Small Molecule ◽  
Human Embryonic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Small Molecule Inhibitors ◽  
Embryonic Stem ◽  
Egfp Expression ◽  
Induced Pluripotent ◽  
Cells Cultured ◽  
Pluripotency Genes

Small molecule inhibitors acting on the MEK and Wnt signalling pathways (PD0325901 and CHIR99021, respectively) have been used successfully for the maintenance of murine and rat pluripotent stem cells in the in vitro culture. The effects of these compounds in other species have not been conclusively determined, and some reports suggest that they may actually cause loss of pluripotency in human embryonic stem cells and decrease of OCT4 expression levels in porcine induced pluripotent stem cells (piPSC). In our experiments, putative piPSC lines derived from transgenic porcine fetal fibroblasts (pFF) and adipose mesenchymal stem cells (pAMSC) that harbor the OCT4-EGFP reporter construct were maintained in piPSC culture medium [DMEM supplemented with 10% Knockout Serum Replacement (Life Technologies, Carlsbad, CA, USA), 10% fetal bovine serum, nonessential amino acids, L-glutamine, penicillin-streptomycin, and 1000 U mL–1 murine leukemia inhibitory factor (LIF)], with or without addition of 1 μM PD0325901 and 3 μM CHIR99021. After five passages in the two experimental culture conditions, three pFF-derived putative piPSC lines were evaluated morphologically and the expression of various pluripotency genes was analysed by real-time quantitative PCR. The results showed decrease of OCT4-EGFP expression and loss of compact colony morphology of the cells cultured in the presence of PD0325901 and CHIR99021. Moreover, the transcriptional expression levels of OCT4, SOX2, NANOG, REX1, UTF1, and TDH were reduced by 12-, 9-, 10-, 3-, 5-, and 20-fold, respectively, compared with the cells cultured without these inhibitors. When putative piPSC derived from pAMSC were cultured in medium supplemented with small molecule inhibitors, the OCT4-EGFP expression was completely lost within a few days. The cells also lost their iPSC-like colony morphology and were further propagated as single, mesenchymal-like cells. The same effects were observed when the cells were cultured with CHIR99021 alone, whereas there were no such changes when we used only PD0325901. This suggests that, similarly to human embryonic stem cells (ESC), the activation of the Wnt pathway in pAMSC-derived iPSC may lead to differentiation in these cells. The effects of the CHIR99021 alone on the pFF-derived piPSC-like cells are still to be determined. In conclusion, the results of our preliminary investigations call into question the effectiveness of PD0325901 and CHIR99021 in the maintenance of piPSC in culture.

A New Feeder-Free Technique to Expand Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

2009 ◽  
Vol 1 (1) ◽  
pp. 76-82 ◽  
Author(s):  
Mark Denham ◽  
Jessie Leung ◽  
Cheryl Tay ◽  
Raymond C.B. Wong ◽  
Peter Donovan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Induced Pluripotent

Cyclosporin A promotes invasion and migration of extravillous trophoblast cells derived from human induced pluripotent stem cells and human embryonic stem cells

2020 ◽  
Author(s):  
Jiaxing Wang ◽  
Ping Long ◽  
Shengnan Tian ◽  
Weihua Zu ◽  
Jing Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Extravillous Trophoblast ◽  
Invasion And Migration ◽  
And Migration ◽  
Induced Pluripotent

Abstract Background Extravillous trophoblast (EVT) cells play an essential role in the maternal-fetal interaction. Although abnormal development and function of EVT cells, including impaired migration and invasion capability, are believed to be etiologically linked to severe pregnancy disorders including pre-eclampsia (PE), the associated molecular mechanisms are not clear ascribed to the lack of an appropriate cell model in vitro. Cyclosporine A (CsA) is a macrolide immunosuppressant and is also used in clinic to improve pregnancy outcomes. However, whether CsA has any effects on the function of EVT cells has not been well investigated. Methods In this study, we induced differentiation of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) into EVT cells (hiPSC-EVT and hESC-EVT cells, respectively) by Y27632, NRG1, A83-01 and matrigel, and collected these derived EVT cells by flow cytometry for sorting cells positive for double HLA-G and KRT7, which are EVT markers. We then investigated the effects of CsA on the invasion and migration of these derived EVT cells. Results We found that the hiPSC-EVT and hESC-EVT cells expressed high levels of the EVT markers such as KRT7, ITGA5 and HLA-G but low levels of OCT4, a stem cell marker, and that CsA significantly promoted the invasion and migration of hiPSC-EVT and hESC-EVT cells. Conclusions We successfully generated hiPSC/hESC-derived human EVT cells, which may be applicable for investigating the remodeling process of spiral arteries remodeling and the possible mechanisms of EVT-related diseases in vitro. Furthermore, our findings provide direct evidence that CsA regulates the function of EVT cells and molecular basis by which CsA may be used to treat pregnancy complications in clinic associated with deficient EVT function.

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