Bats have evolved with unique features equivalent to laryngeal echolocation and flight, with some able to tolerating viruses equivalent to severe acute respiratory syndrome coronaviruses (SARS-CoVs), Middle East respiratory syndrome CoVs (MERS-CoVs), in addition to Marburg and Nipah viruses. Developing robust cell-based bat models could provide a greater understanding of bat viral handling and biology.

In a recent study published on the bioRxiv* preprint server, researchers generated induced pluripotent stem cells (iPSCs) from Rhinolophus ferrumequinum bats using the modified Yamanaka protocol to ascertain bats as a novel in vivo model study species.

Study: Bat pluripotent stem cells reveal unique entanglement between host and viruses. Image Credit: Jezper / Shutterstock.com

Concerning the study

In the current study, researchers investigate whether bats could possibly be suitable for viral production.

The Yamanaka reprogramming approach was used based on reprogramming aspects equivalent to 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 the amounts of reprogramming aspects altered to activate and block several signaling pathways. As well as, immunostaining and ribonucleic acid (RNA) sequencing (RNA-seq) analyses were also performed.

Derivation of pluripotent bat stem cells. (A) Illustration of the bat pluripotent stem cell derivation strategy. 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) Immunofluorescent detection of Oct4 in BiPS cells. (D) MA plot of RNA-seq data illustrating the transcriptional differences between bat embryonic fibroblast (BEF) and pluripotent stem cells (BiPS). Chosen genes with known functions within the establishment or maintenance of pluripotency are highlighted. (E) Kmean cluster evaluation of ATAC-seq signals obtained from BEF or BiPS cells. C, cluster. (F), Density plot of RRBS results obtained from BEF and BiPS cells. PCC, Pearson correlation coefficient. (G) Scatter plots of histone 3 methylation status at K4 (activating chromatin modification) or K27 (repressing chromatin modification) after ChIP-seq from BEF or BiPS cells as indicated. (H)Scatter plot of H3K4me3 and H3K27me3 in BiPS cells illustrating the occurrence of bivalent chromatin sites in BiPS cells. (I) RNA-seq, ATAC-seq and H3K4me3 or H3K27me3 ChIP-seq signals of chosen genes with known roles in reprogramming which can be activated (Nanog, Kit) or repressed (Thy1) in BiPS compared to BEF cells.

The impact of the modified reprogramming method on bat epigenetic molecules and chromatin was assessed using the assay for transposase-accessible chromatin with sequencing (ATAC-seq). Deoxyribonucleic acid (DNA) methylome mapping evaluation and chromatin immunoprecipitation and sequencing (ChIP-seq) analyses were also performed. Protocols were optimized to enable bat SC differentiation into the three germ layers, whereas the embryoid body (EB) differentiation assay was performed to evaluate for pluripotency.

Bat iPSCs (BiPSCs) were subsequently injected into immunosuppressed mice, and embryo-like structures were created from the BiPS. The study protocol was validated by developing BiPS cells of the evolutionally distant Myotis myotis bats.

Comparative transcriptional gene profiling and principal component evaluation (PCA) were performed on the Rhinolophus bat species and phylogenetically different mammalian species of mice, humans, dogs, pigs, and marmoset.

Gene ontology evaluation was performed to evaluate the leading-edge gene enrichment for specific biological pathways. Novel pipelines were developed based on the metagenomic classification of stem cell ribonucleic acid (RNA)-sequenced (RNA-seq) data, de novo putative retroviral contig assembly, and genomic mapping for identifying true retroviral reads. As well as, antigen markers related to RNA viruses were explored.

Study findings

A selected reprogramming factor ratio, in addition to added 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 tight bat colonies appearing inside 14 to 16 days.

BiPSCs expressed the Oct4 pluripotency factor, with a proliferation rate equivalent to the human PSC proliferation rate. Most cells contained 56 chromosomes and replicated without exogenous reprogramming aspects and morphological alterations.

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

Nevertheless, the genetic profile didn’t entirely match one pluripotency state. As a substitute, naive pluripotent state aspects equivalent to Klf4 and 17, estrogen-related receptor beta protein (Essrb), transcription factor E3 (Tfe3), and transcription Factor CP2 Like 1 (Tfcp2l1)] were expressed. Co-expressed Tfcp2l1/zinc finger protein (Zic2) and orthodenticle homeobox 2 (Otx2)/Tfe3 and primed/naïve aspects were observed.

Chromatin configurational and CpG 191 methylation alterations were observed across the bat genome. ChiP-seq findings showed overlapping between human and bat bivalency genes, although some genes were species-specific.

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

The ERAS gene was downregulated, whereas hyaluronidases and ADP ribosylation aspects (ARFs) genes were indistinguishable between the groups. The Rhinolophus blastoids showed embryonic structures attached to a flattened trophoblastic epithelial outgrowth and inner cell mass expansion. Myotis bat findings indicated that the study protocol could possibly be applied across different bat species.

PCA evaluation showed a definite group of bat stem cells.Nevertheless, only eight leading-edge genes showed significant positive selection in R. ferrumequinum, with most genes belonging to unexpected categories. Furthermore, CoV disease was essentially the most significantly enriched category in Kyoto encyclopedia of genes and genomes (KEGG) pathways.

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

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

Several retroviral integration sites that were homologous to viruses equivalent to the Mason-Pfizer monkey virus, Koala virus, and Jaagsiekte sheep retrovirus were identified. The genome was homologous to volepox, variola, squirrel pox, monkeypox, and White spot syndrome viruses.

Conclusions

BiPSC sequences were just like viral genome sequences. Thus, the transcriptionally permissive pluripotency state of bats could possibly be exploited for locating novel bat virus sequences involved in bat physiology and their virus hosting abilities.

*Essential notice

bioRxiv publishes preliminary scientific reports that aren’t peer-reviewed and, due to this fact, mustn’t be thought to be conclusive, guide clinical practice/health-related behavior, or treated as established information.