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Generation of Angiotensin-Converting Enzyme 2/Transmembrane Protease Serine 2-Double-Positive Human Induced Pluripotent Stem Cell-Derived Spheroids for Anti-Severe Acute Respiratory Syndrome Coronavirus 2 Drug Evaluation.

ABSTRACT: We newly generated two human induced pluripotent stem cell (hiPSC)-derived spheroid lines, termed Spheroids__4M^ACE2-TMPRSS2 and Spheroids__15M63^ACE2-TMPRSS2, both of which express angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2), which are critical for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Both spheroids were highly susceptible to SARS-CoV-2 infection, and two representative anti-SARS-CoV-2 agents, remdesivir and 5h (an inhibitor of SARS-CoV-2's main protease), inhibited the infectivity and replication of SARS-CoV-2 in a dose-dependent manner, suggesting that these human-derived induced spheroids should serve as valuable target cells for the evaluation of anti-SARS-CoV-2 activity. IMPORTANCE The hiPSC-derived spheroids we generated are more expensive to obtain than the human cell lines currently available for anti-SARS-CoV-2 drug evaluation, such as Calu-3 cells; however, the spheroids have better infection susceptibility than the existing human cell lines. Although we are cognizant that there are human lung (and colonic) organoid models for the study of SARS-CoV-2, the production of those organoids is greatly more costly and time consuming than the generation of human iPSC-derived spheroid cells. Thus, the addition of human iPSC-derived spheroids for anti-SARS-CoV-2 drug evaluation studies could provide the opportunity for more comprehensive interpretation of the antiviral activity of compounds against SARS-CoV-2.

SUBMITTER: Higashi-Kuwata N

PROVIDER: S-EPMC9769601 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Generation of Angiotensin-Converting Enzyme 2/Transmembrane Protease Serine 2-Double-Positive Human Induced Pluripotent Stem Cell-Derived Spheroids for Anti-Severe Acute Respiratory Syndrome Coronavirus 2 Drug Evaluation.

Higashi-Kuwata Nobuyo N Yabe Shigeharu G SG Fukuda Satsuki S Nishida Junko J Tamura-Nakano Miwa M Hattori Shin-Ichiro SI Okochi Hitoshi H Mitsuya Hiroaki H

Microbiology spectrum 20221031 6

We newly generated two human induced pluripotent stem cell (hiPSC)-derived spheroid lines, termed Spheroids_4MACE2-TMPRSS2 and Spheroids_15M63ACE2-TMPRSS2, both of which express angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2), which are critical for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Both spheroids were highly susceptible to SARS-CoV-2 infection, and two representative anti-SARS- ...[more]

PMID: 36314907

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Project description:Lower levels of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) in the nasal epithelium of children may be related to a lower incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, compared to adults. However, no direct evidence is available to support this hypothesis. In this study, we compared the transcript levels of ACE2 and TMPRSS2 in nasopharyngeal swab samples (n = 234) from children and adult family members within SARS-CoV-2-exposed families and assessed the association with SARS-CoV-2 infection status. Transcript levels for ACE2, but not TMPRSS2, were higher in adults than in children (n = 129 adults and 105 children; P < 0.05). The expression of the two genes was not significantly different between SARS-CoV-2 positive and SARS-CoV-2 negative patients within the same age groups. However, in families with one or more SARS-CoV-2 positive adult family members, expression of both genes was significantly higher in SARS-CoV-2 positive children than in SARS-CoV-2 negative children (P < 0.05). By multivariate analysis, ACE2 expression adjusted for age and sex was significantly associated with SARS-CoV-2 infection in the overall population (odds ratio [OR], 1.112 [95% confidence interval [CI], 1.012 to 1.229]; P < 0.05). The degree of this association was higher (OR, 1.172 [95% CI, 1.034 to 1.347]; P < 0.05) in the subgroup of families with only SARS-CoV-2 positive adult family members. Our results suggest that children with lower levels of nasal ACE2 and TMPRSS2 are more likely to remain SARS-CoV-2 negative despite being exposed to a SARS-CoV-2 positive adult family member. IMPORTANCE ACE2 and TMPRSS2 are well established in the literature as SARS-CoV-2 entry factors. Recent data suggest that lower levels of nasal ACE2 in children may be associated with their lower incidence of coronavirus disease 2019 (COVID-19). In this study, using data from nasopharyngeal swab specimens from adult and pediatric members of families in which one or more members of the family had laboratory-confirmed SARS-CoV-2 infection, we show that children with lower levels of ACE2 and TMPRSS2 are more likely to remain SARS-CoV-2 negative despite being exposed to a SARS-CoV-2 positive adult family member. These results provide new insights into the roles of nasopharyngeal ACE2 and TMPRSS2 in acquiring SARS-CoV-2 infection, and they show that the differential expression of these genes in adults versus children may contribute to differential rates of SARS-CoV-2 infection in these populations.

Project description:ObjectiveTo study messenger ribonucleic acid (mRNA) and protein expressions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry receptors (angiotensin 1-converting enzyme 2 [ACE2] and CD147) and proteases (transmembrane serine protease 2 [TMPRSS2] and cathepsin L [CTSL]) in human oocytes, embryos, and cumulus (CCs) and granulosa cells (GCs).DesignResearch study.SettingClinical in vitro fertilization (IVF) treatment center.PatientsPatients undergoing IVF were treated at the Colorado Center for Reproductive Medicine.InterventionsOocytes (germinal vesicle and metaphase II [MII]) and embryos (1-cell [1C] and blastocyst [BL]) were donated for research at the disposition by the patients undergoing IVF. Follicular cells (CC and GC) were collected from women undergoing egg retrieval after ovarian stimulation without an ovulatory trigger for in vitro maturation/IVF treatment cycles.Main outcome measuresPresence or absence of ACE2, CD147, TMPRSS2, and CTSL mRNAs detected using quantitative reverse transcription polymerase chain reaction and proteins detected using capillary Western blotting in human oocytes, embryos, and ovarian follicular cells.ResultsThe quantitative reverse transcription polymerase chain reaction analysis revealed high abundance of ACE2 gene transcripts in germinal vesicle and MII oocytes than in CC, GC, and BL. ACE2 protein was present only in the MII oocytes, and 1C and BL embryos, but other ACE2 protein variants were observed in all the samples. TMPRSS2 protein was present in all the samples, whereas mRNA was observed only in the BL stage. All the samples were positive for CD147 and CTSL mRNA expressions. However, CCs and GCs were the only samples that showed coexpression of both CD147 and CTSL proteins in low abundance.ConclusionsCCs and GCs are the least susceptible to SARS-CoV-2 infection because of lack of the required combination of receptors and proteases (ACE2/TMPRSS2 or CD147/CTSL) in high abundance. The coexpression of ACE2 and TMPRSS2 proteins in the MII oocytes, zygotes, and BLs demonstrated that these gametes and embryos have the cellular machinery required and, thus, are potentially susceptible to SARS-CoV-2 infection if exposed to the virus. However, we do not know whether the infection occurs in vivo or in vitro in an assisted reproductive technology setting yet.

Dataset Information

Generation of Angiotensin-Converting Enzyme 2/Transmembrane Protease Serine 2-Double-Positive Human Induced Pluripotent Stem Cell-Derived Spheroids for Anti-Severe Acute Respiratory Syndrome Coronavirus 2 Drug Evaluation.

Publications

Generation of Angiotensin-Converting Enzyme 2/Transmembrane Protease Serine 2-Double-Positive Human Induced Pluripotent Stem Cell-Derived Spheroids for Anti-Severe Acute Respiratory Syndrome Coronavirus 2 Drug Evaluation.

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