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?:abstract
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Objective: Determine whether various stresses change the expression in embryonic stem cells (ESC) and their differentiated lineages, of a large set of viral receptors and susceptibility genes Design: Experimental Materials and Methods: ESC were tested by bulk or single cell (sc) RNAseq after 72 hr exposures to 0-300 mM hyperosmotic sorbitol (with stemness-maintaining Leukemia Inhibitory Factor, LIF) to quantify stress-forced differentiation Controls for normal stemness were LIF+ and normal differentiation were LIF- RNA was isolated by RNAeasy lysis or 10XGenomics Dropseq, RNA quality was checked by Agilent TapeStation, cDNA was synthesized using Lexogen’s QuantSeq library kit, and barcoded, multiplexed and sequenced by Illumina NovaSeq 6000 Data were demultiplexed by CASAVA software and FC expression was compared between conditions In triplicate experiments, significant fold change (FC) genes (FC≥2;FDR≤0 05, P<0 05) identified affected pathways Proliferation or death were assayed by Hoechst staining or Trypan blue staining, respectively Validating studies including qPCR Results: Using bulk RNAseq it was shown that VIM and TMPRSS2, two genes necessary for Covid-19 cell uptake are up-regulated significantly as measured by P-value and FDR VIM is up-regulated 5 6 fold higher at 300 mM sorbitol compared with normal differentiation and TMPRSS2 is also up-regulated 3 1 fold higher by high sorbitol despite LIF presence Havcr1 the hepatitis A virus receptor was up-regulated 55 fold over normal ESC differentiation with significance by p-Value and FDR Pvrl1 the Herpes simplex 1 (HSV1) virus receptor was up-regulated 4 5 fold compared with ESC undergoing normal differentiation by LIF removal Conclusions: These are proof-of-principle studies that suggest that some viruses have high susceptibility when ESC and their progeny differentiated lineages undergo hyperosmotic stress HSV1 is known to cross the placenta, and recent data suggest that Covid-19 may cross the placenta and induce IgM detected in cord blood We intend to extend these tests using Phthalates and cortisol and to test human embryonic stem cells for similar stress-enhanced susceptibility
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