Human endogenous retroviruses (HERVs) are remnants of retroviruses occupying 8% of human genome. Transcription of HERVs is generally suppressed by epigenetic mechanisms; but recent studies, including ours, have provided evidence that LTR5_Hs, the human-specific HERV species, is specifically reactivated in human Primordial Germ Cells as well as its human pluripotent stem cell (hPSC)-derived cell culture model hPGCLCs (human Primordial Germ Cell-Like Cells). Currently available evidence obtained from our preliminary studies as well as presented in very recent literature supports that LTR5_Hs is required for conversion of hPSCs to hPGCLCs and that hPGCLCs (but not hPSCs) release Virus-Like Particles (VLPs) from plasma membrane. Taking advantage of our recent success in Long-Term Culture hPGCLCs (LTC-hPGCLCs), this project will examine (1) whether the reactivation of LTR5_Hs creates new, hPGCLC-specific boundaries of the topologically associated domains (TADs), which may support germline differentiation of hPSCs; and (2) whether VLPs released from hPGCLCs can convey viral and/or non-viral molecules, including the retroviral reverse transcriptase, from LTC-hPGCLCs to other human cells. Our Specific Aim 1 will use the CARGO (chimeric array of gRNA oligos)-CRISPRi and the piggyBac vector for comprehensive suppression of the 697 known copies of LTR5_Hs and random insertion of exogenous copies of LTR5_Hs from/to the genome of hPSCs and LTC-hPGCLCs. The engineered cells will be subjected to deep sequencing analyses of TADs (Hi- C seq), transcriptomes (RNA-seq), and epigenomes (ChIP-seq). Efficiencies of (A) in vitro conversion of hPSCs to hPGCLCs or cells representing the three germ layers, and (B) male LTC-hPGCLC differentiation to gonocytes in the xrTestis xenogeneic reconstituted testis organoid culture system, will be evaluated by single cell RNA-seq as well as immunofluorescence staining. Specific Aim 2 will attempt to establish the LTR5_Hs origin of VLPs released from LTC-hPGCLCs by immunogold staining and enrich the VLPs for further analyses of their components such as RNA species, proteins, and enzymatic activities derived from viral or host cells. Attempts will be further made to label the VLPs with peptide antigen tags and/or fluorescence proteins to determine whether the VLPs can fuse into, and possibly internalized in, other human cells surrounding hPGCLCs in human embryoids generated in a microfluidics system as well as other types of hiPSC-derived embryonic cells. If the proposed experiments are successfully completed, we expect to obtain important mechanistic insights into how retrovirus-derived, human-specific genomic elements contribute to development of germline cells, potentially creating a novel paradigm that highlights the physiological importance of exapted HERVs in human reproduction. Our experiments may also support the novel concept that VLPs function as an intercellular conveyer of viral and/or host cell molecules, potentially rev...