Generation of macrophages with altered viral sensitivity from genome-edited rhesus macaque iPSCs to model human disease

Because of their close biological similarity to humans, non-human primate (NHP) models are very useful for the development induced pluripotent stem cell (iPSC)-based and regenerative organ transplantation therapies. However, knowledge on establishment, differentiation, genetic modification NHP-iPSCs, especially rhesus macaque iPSCs, is limited. We succeeded in establishing iPSCs from peripheral blood macaques (Rh-iPSCs) by combining Yamanaka reprograming factors two inhibitors (GSK-3 inhibitor [CHIR 99021] MEK1/2 [PD0325901]) differentiated cells into functional macrophages through hematopoietic progenitor cells. To confirm feasibility Rh-iPSC-derived as a platform bioassays model diseases, we knocked out TRIM5 gene Rh-iPSCs CRISPR-Cas9, which species-specific HIV resistance factor. knockout (KO) had same differentiation potential did Rh-iPSCs, but showed gain sensitivity infection vitro. 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These results indicate multipotential HPCs.Table 1Summary (confluent 6-cm dish)Total (×106)CD34+ (%)CD34+ (×106)R1863#78.69 ± 0.10731.467 5.1472.63 0.009R1887#115.07 0.3025.567 3.13.92 0.110R1889#318.91 0.85623.267 6.7163.48 0.068Data shown mean SE independent experiments. Open table new tab Data Next, HPCs macrophages, target HIV/SIV (simian virus), method our group.5Higaki After co-cultured C3H10T1/2 feeder 10 presence macrophage colony-stimulating (M-CSF) granulocyte-macrophage CSF (GM-CSF), adherent seeded low-adsorption plate cultured 3A). Starting confluent dish, 1–2 × 107 obtained 34. 34, obvious growth. FACS analysis CD11b+/CD14+/CD68+/CD86+/CD163−. They CCR5, co-receptor 3B S1A). phenotypes resembled monocyte-derived S1B). phagocytosis h co-culturing Alexa Fluor 594-conjugated Escherichia coli bioparticles 3C). stimulated lipopolysaccharide (LPS) produced inflammatory cytokines necrosis (TNF) interleukin (IL)-6 3D). SIV infection. Macrophage tropic SIVmac316 SIVmac239 p27 protein measured ELISA 1, 4, 7. co-culture suggest protocol. next edit Rh. PRYSPRY its C-terminal connected long link N-terminal, includes motifs: RING domain, B-box 2 coiled-coil domain. Candidate sequences single guide (sgRNA) selected CRISPOR (http://crispor.tefor.net/crispor.py). sequence Figure 4A. sgRNA Cas9 electroporation picked up 24 manually without drug selection. An revealed mutations 7 clones, 3 showing homozygous 4B). in-frame mutation allele. randomly selecting heterozygous performing additional able create TRIM5αKO stop codon frameshift 4C). considered important controlling infection.21Nakayama existed exon3 downstream translated. Thus, When mRNA qPCR, level decreased strains, caused nonsense mutation-dependent degradation (NMD)35Garneau N.L. Wilusz highways byways decay.Nat. Mol. 113-126Crossref (880) Scholar,36Lykke-Andersen Jensen T.H. Nonsense-mediated decay: intricate machinery shapes transcriptomes.Nat. 665-677Crossref (376) 4D). reference parental Parental equivalent efficiencies 4E 4F). levels CD86 different TRIM5αKO. One report found dendritic (DCs) lacking TRIM5α-mediated retroviral upregulate expression.37Portilho D.M. Fernandez Ringeard Machado A.K. Boulay Mayer Müller-Trutwin Beignon Kirchhoff Nisole Arhel N.J. Endogenous regulated SUMOylation nuclear sequestration sensing 14: 355-369Abstract (23) hypothesize increased mechanism. off-target sites RNAs (gRNAs), identified software Sanger sequencing (Table 2). From these results, compromise potential.Table 2Results top five candidates determined CFD score gRNAPositionSequence (5′→3′)No. mismatchesCFD scoreKO #1KO #2KO #3TRIM5 on-target sitechr14:67658119–110054350CTACGACAAAACCAACGTCT CGG–1.0000−14 bp, −14 bp−5 bp−14 bpPotential siteschr4:103904324–103904346CTATAATAAAACCAACATCT TGG40.525778WTWTWTchr2:76073316–76073338CTAAGGCAAAAACAACATCT AGG40.401003WTWTWTchr2:98763243–98763265CTGCCACAAACCCAACATCT TGG40.179259WTWTWTchr7:42594298–42594320ATAAGATAAAACCAACGTCT GAG30.177388WTWTWTchr9:104127964–104127986CTACAAAGAAACCAACTTCT AGG40.119167WTWTWTCFD, cutting frequency determination; WT, wild-type. CFD, order functionally infected (vesicular stomatitis glycoprotein G [VSV-G]-pseudotyped lentivirus expressing luciferase [NL43-Luci/VSV-G]38Taya Nakayama Moderate macrophage-tropic SAMHD1 macrophages.PLoS ONE. 9: e90969Crossref (4) Scholar). (NL43-Luci/VSV-G) Luminescence (2, 3, 4), suggesting early 5A). determine TRIM5α, degrades reverse transcription stage, tested transcriptase (nevirapine [NVP]), it suppressed 5B). possible TRIM5. As first step creating evaluating genome-edited generate PBMCs, induce HPCs/macrophages, concept, deleted adding (PD0325901) (i.e., 2i) collected aseptically relatively easily. originally maintain mouse (ESCs),31Ying since