Pluripotent bat stem cells as a model for studying novel viruses

Pluripotent bat stem cells as a model for studying novel viruses

Bats have evolved with unique features such as laryngeal echolocation and flight, with some able to tolerate viruses such as severe acute respiratory syndrome coronaviruses (SARS-CoVs), Middle East respiratory syndrome CoVs (MERS-CoVs), and Marburg and Nipah viruses. The development of robust cell-based bat models could lead to a better understanding of bat virus management and their biology.

In a recently published study on the subject bioRxiv * Preprint server: Researchers generated induced pluripotent stem cells (iPSCs) from Rhinolophus ferrumequinum bats using the modified Yamanaka protocol to establish bats as a novel in vivo model study species.

Study: Bat pluripotent stem cells reveal unique host-virus interconnection.Photo credit: Jezper / Shutterstock.com

This news article was a review of a preliminary scientific report that had not been peer-reviewed at the time of publication. Since its initial publication, the scientific report has now been peer-reviewed and accepted for publication in an academic journal. Links to the preliminary and peer-reviewed reports can be found in the Sources section at the end of this article. View sources

About the study

In the present study, researchers are investigating whether bats could be suitable for virus production.

The Yamanaka reprogramming approach was used based on reprogramming factors such as the sex-determining region Y-box-2 gene, octamer-binding transcription factor 4 (Oct4), cMyc and Kruppel-like factor 4 (Klf4).

Bat embryonic fibroblast (BEF) cells were isolated from R. ferrumequinum with levels of reprogramming factors altered to activate and block multiple signaling pathways. In addition, immunostaining and ribonucleic acid (RNA) sequencing (RNA-seq) analyzes were also performed.

Obtaining pluripotent bat stem cells. (A) Illustration of the strategy for obtaining pluripotent bat stem cells. BEF, embryonic fibroblasts; OSMK, Oct4, Sox2, cMyc, Klf4; FB, fibroblast medium; PSC, pluripotent stem cell medium; PSC+, PSC with additives. (B) Morphology of established BiPS cell colonies grown on mouse embryonic fibroblasts. (C) Immunofluorescence detection of Oct4 in BiPS cells. (D) MA plot of RNA-seq data illustrating the transcriptional differences between bat embryonic fibroblasts (BEF) and pluripotent stem cells (BiPS). Selected genes with known functions in establishing or maintaining pluripotency are highlighted. (E) Kmean cluster analysis of ATAC-seq signals obtained from BEF or BiPS cells. C, clusters. (F), Density plot of RRBS results obtained from BEF and BiPS cells. PCC, Pearson correlation coefficient. (G) Scatterplots of histone 3 methylation status at K4 (activating chromatin modification) or K27 (repressive chromatin modification) after ChIP-seq from BEF or BiPS cells, as indicated. (H) Scatter plot of H3K4me3 and H3K27me3 in BiPS cells illustrating the presence of bivalent chromatin sites in BiPS cells. (I) RNA-seq, ATAC-seq, and H3K4me3 or H3K27me3 ChIP-seq signals of selected genes with known roles in reprogramming that are activated (Nanog, Kit) or repressed (Thy1) in BiPS compared to BEF cells.

The effects of the modified reprogramming method on bat epigenetic molecules and chromatin were assessed using the transposase-accessible chromatin with sequencing (ATAC-seq) assay. Deoxyribonucleic acid (DNA) methylome mapping analyzes and chromatin immunoprecipitation and sequencing (ChIP-seq) analyzes were also performed. Protocols were optimized to allow SC differentiation of bats into the three germ layers, while embryoid body (EB) differentiation assay was performed to assess pluripotency.

Bat iPSCs (BiPSCs) were then injected into immunosuppressed mice and embryonic structures were created from the BiPSCs. The study protocol was validated by developing BiPS cells from the evolutionarily distant bat Myotis myotis.

Comparative transcriptional gene profiling and principal component analyzes (PCA) were performed in the bat species Rhinolophus and phylogenetically diverse mammalian species of mice, humans, dogs, pigs, and marmosets.

Gene ontology analysis was performed to assess state-of-the-art gene enrichment for specific biological pathways. Novel pipelines have been developed based on metagenomic classification of stem cell ribonucleic acid (RNA)-sequenced (RNA-seq) data, de novo putative retroviral contig assembly, and genomic mapping to identify bona fide retroviral reads. In addition, antigen markers associated with RNA viruses were examined.

Study results

A specific reprogramming factor ratio as well as the addition of fibroblast growth factor-2 (Fgf-2), stem cell factor (Scf), leukemia inhibitory factor (Lif) and forskolin to the culture medium enabled uninhibited BiPSC growth, with homogeneous and dense bat colonies appearing within 14 to 16 days.

BiPSCs expressed Oct4 pluripotency factor at a proliferation rate identical to the proliferation rate of human PSCs. Most cells contained 56 chromosomes and replicated without exogenous reprogramming factors and morphological changes.

BiPSCs differentiated into the three germ layers and subsequently formed EBs and organoids. RNA-seq analysis showed induced endogenous expression of canonical pluripotency-related genes such as SRY-2, Nanog and Oct4.

However, the genetic profile was not completely consistent with a pluripotency state. Instead, naïve pluripotent state factors such as Klf4 and 17, estrogen-related receptor beta protein (Essrb), transcription factor E3 (Tfe3), and transcription factor CP2 Like 1 (Tfcp2l1) were expressed. Coexpressed Tfcp2l1/zinc finger protein (Zic2) and Orthodenticle Homeobox 2 (Otx2)/Tfe3 as well as primed/naïve factors were observed.

Changes in chromatin configuration and CpG 191 methylation were observed throughout the bat genome. ChiP-seq results showed overlap between human and bat bivalence genes, although some genes were species specific.

BiPSCs were transcriptionally and epigenetically reprogrammed. BIPSCs were positive for paired box protein (Pax6), 213T, and alpha-fetoprotein (AFP) markers for ectoderm, mesoderm, and endoderm.

The ERAS gene was downregulated, whereas the genes for hyaluronidases and ADP-ribosylation factors (ARFs) were indistinguishable between groups. The Rhinolophus blastoids showed embryonic structures bound to a flattened trophoblastic epithelial outgrowth and internal cell mass expansion. The Myotis bat results suggested that the study protocol could be applied to different bat species.

PCA analysis revealed a distinct group of bat stem cells. However, only eight top genes showed significant positive selection in R. ferrumequinum, with most genes belonging to unexpected categories. Furthermore, CoV disease was the most significantly expanded category in the Kyoto Encyclopedia of Genes and Genomes (KEGG).

The collagen type III alpha 1 (Col3a1) and mucin 1 (Muc1) genes were detected in BiPSCs, indicating bat-specific genetic adaptations. The reprogramming revealed endogenous retrovirus (ERV) sequences.

BiPSCs contained multiple virus-associated endogenized sequences with regions homologous to human herpesvirus 4, human respiratory syncytial virus, and a SARS-CoV-2 isolate. The genomic sequences of R. ferrumequinum were similar to those of human CoV 229E and human CoV OC43.

Several retroviral integration sites were identified that were homologous to viruses such as Mason-Pfizer monkey virus, koala virus, and Jaagsiekte sheep retrovirus. The genome was homologous to volepox, variola, squirrelpox, monkeypox and whitepox syndrome viruses.

Conclusions

BiPSC sequences were similar to viral genome sequences. Thus, the transcriptionally permissive pluripotency state of bats could be exploited to discover novel bat virus sequences involved in bat physiology and their virus-hosting abilities.

This news article was a review of a preliminary scientific report that had not been peer-reviewed at the time of publication. Since its initial publication, the scientific report has now been peer-reviewed and accepted for publication in an academic journal. Links to the preliminary and peer-reviewed reports can be found in the Sources section at the end of this article. View sources

References:

• Vorläufiger wissenschaftlicher Bericht.
  Dejosez, M., Marin, A., Hughes, GM, et al. (2022). Pluripotente Stammzellen von Fledermäusen offenbaren einzigartige Verflechtung zwischen Wirt und Viren. bioRxiv. doi:10.1101/2022.09.23.509261. https://www.biorxiv.org/content/10.1101/2022.09.23.509261v1.
• Von Experten begutachteter und veröffentlichter wissenschaftlicher Bericht.
  Déjosez, Marion, Arturo Marin, Graham M. Hughes, Ariadna E. Morales, Carlos Godoy-Parejo, Jonathan L. Gray, Yiren Qin, et al. 2023. „Pluripotente Stammzellen von Fledermäusen offenbaren ungewöhnliche Verflechtung zwischen Wirt und Viren.“ Zelle 186 (5): 957-974.e28. https://doi.org/10.1016/j.cell.2023.01.011. https://linkinghub.elsevier.com/retrieve/pii/S0092867423000417.

Article revisions

• 15. Mai 2023 – Das vorab gedruckte vorläufige Forschungspapier, auf dem dieser Artikel basiert, wurde zur Veröffentlichung in einer von Experten begutachteten wissenschaftlichen Zeitschrift angenommen. Dieser Artikel wurde entsprechend bearbeitet und enthält nun einen Link zum endgültigen, von Experten begutachteten Artikel, der jetzt im Abschnitt „Quellen“ angezeigt wird.