Bats have evolved with unique features such as laryngeal echolocation and flight, with some capable of tolerating viruses such as severe acute respiratory syndrome coronaviruses (SARS-CoVs), Middle East respiratory syndrome CoVs (MERS-CoVs), as well as 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, 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.
Results
A particular reprogramming factor ratio, as well as 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 within 14 to 16 days.
BiPSCs expressed the Oct4 pluripotency factor, with a proliferation rate identical to the human PSC proliferation rate. Most cells contained 56 chromosomes and replicated without exogenous reprogramming factors and morphological alterations.
BiPSCs differentiated into the three germ layers, subsequently forming EBs and organoids. RNA-seq analysis showed induced endogenous expression of canonical pluripotency-related genes like SRY-2, Nanog, and Oct4.
However, the genetic profile did not entirely match one pluripotency state. Instead, naive 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. Co-expressed Tfcp2l1/zinc finger protein (Zic2) and orthodenticle homeobox 2 (Otx2)/Tfe3 and primed/naïve factors 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 factors (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 be applied across different bat species.
PCA analysis showed a distinct group of bat stem cells.However, only eight leading-edge genes showed significant positive selection in R. ferrumequinum, with most genes belonging to unexpected categories. Moreover, CoV disease was 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 such as 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.
Conclusion
BiPSC sequences were similar to viral genome sequences. Thus, the transcriptionally permissive pluripotency state of bats could be exploited for discovering novel bat virus sequences involved in bat physiology and their virus hosting abilities.
Source:
https://www.news-medical.net/news/20220929/Pluripotent-bat-stem-cells-as-a-model-to-study-novel-viruses.aspx