Gene-editing enables non-invasive in vivo PET imaging of human induced pluripotent stem cell derived therapeutic liver organoids.

Hepatocyte transplantation (HTx) is an established, safe cell therapy for liver disease, but wider application is limited by primary cell availability and poor engraftment, partly due to cell quality. Hepatocyte-like cells (HLCs), differentiated from human induced pluripotent stem cells (hiPSCs), have clinical transplantation potential, aiming to provide a potentially unlimited, high quality hepatocyte supply. Preclinically, variations in transplantation site, cell format and stage of differentiation are reported with no standard protocol for HLC transplantation. Whole-body in vivo imaging would enable transplanted cell survival and/or expansion to be monitored non-invasively over time, allowing robust comparisons between technologies and transplant modalities. Radionuclide imaging is ideal for this purpose, offering high sensitivity compared to other imaging modalities, quantification of deep tissue signals and direct applicability to the clinic. The human sodium iodide symporter (hNIS) can be exploited as a radionuclide reporter gene for positron emission tomography (PET) or single photon emission computed tomography (SPECT)-afforded whole body in vivo cell tracking. Here we used transcription activator-like effector nuclease (TALEN) gene-editing to incorporate the hNIS-mGFP fusion ORF into the AAVS1 safe harbour locus of a cGMP compliant hiPSC line. Incorporation within the safe harbour locus ensures no downstream reporter silencing, enabling constitutive hNIS-mGFP expression in hiPSCs and their differentiated progeny. We first confirmed in vitro that incorporation of the reporter did not impact hiPSC pluripotency or HLC-differentiation capacity relative to parental cells. Immunostaining and qPCR showed comparable expression of the pluripotency genes Oct4, Sox2 and C-Myc. Cells differentiated into hepatic endoderm also demonstrated comparable expression of the hepatic endoderm genes TBX3, HNF1B, AFP and FOXA2 by qPCR, with retention of the reporter at this stage confirmed by qPCR and flow cytometry. After 33 days of differentiation AAVS1-hNIS-mGFP HLCs exhibited comparable expression of mature hepatocyte markers (Albumin, Hepatocyte Nuclear Factor 4ɑ, Cytokeratin-18 and Cytochrome P450 3A4) by qPCR. Results were verified by immunostaining (Albumin, HNF4A, CK18) and functional assays (CYP3A4 activity assay and ELISA analysis of secreted albumin). Retention of the reporter was also verified by qPCR, analysis of GFP expression by flow cytometry and 99mTcO4- uptake confirming functionality. Subsequently, AAVS1-hNIS-mGFP-hiPSCs were used to produce in vivo traceable multilineage liver bud organoids (LBs) by combining three differentiated liver progenitor populations in vitro (hepatic endoderm, endothelial cells and septum transverse mesenchyme), this yielded more mature hepatocytes than HLCs cultured without supporting cells. LBs were transplanted into kidney capsules of healthy NOD SCID and liver injured TK NOG mice and PET imaged in vivo at two timepoints post-transplantation. We demonstrated LB survival and traceability by [18F]BF4 PET and showed cells retained their differentiated function, as demonstrated by human albumin presence in mouse sera, immunostaining of human endothelial markers (CD31 and CD144) and hepatic markers (CK18 and albumin) in recovered tissue. This study is the first to show hNIS-expressing hiPSC progeny retain their differentiated function and can be tracked noninvasively in vivo by PET. LBs were chosen for tracking as scalability of these organoids to clinically relevant capacity has been previously demonstrated, their use with a clinically applicable radiotracer and imaging modality in this study highlights the potential for this tracking strategy to answer wider questions in the HLC field.