Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics.

BACKGROUND: DNA variants in APOL1 associate with kidney disease, but the pathophysiologic mechanisms remain incompletely understood. Model organisms lack the APOL1 gene, limiting the degree to which disease states can be recapitulated. Here we present single-cell RNA sequencing (scRNA-seq) of genome-edited human kidney organoids as a platform for profiling effects of APOL1 risk variants in diverse nephron cell types. METHODS: We performed footprint-free CRISPR-Cas9 genome editing of human induced pluripotent stem cells (iPSCs) to knock in APOL1 high-risk G1 variants at the native genomic locus. iPSCs were differentiated into kidney organoids, treated with vehicle, IFN-γ, or the combination of IFN-γ and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared with isogenic G0 controls. RESULTS: Both G0 and G1 iPSCs differentiated into kidney organoids containing nephron-like structures with glomerular epithelial cells, proximal tubules, distal tubules, and endothelial cells. Organoids expressed detectable APOL1 only after exposure to IFN-γ. scRNA-seq revealed cell type-specific differences in G1 organoid response to APOL1 induction. Additional stress of tunicamycin exposure led to increased glomerular epithelial cell dedifferentiation in G1 organoids. CONCLUSIONS: Single-cell transcriptomic profiling of human genome-edited kidney organoids expressing APOL1 risk variants provides a novel platform for studying the pathophysiology of APOL1-mediated kidney disease.

On-Demand Intraoperative 3-Dimensional Printing of Custom Cranioplastic Prostheses.

BACKGROUND: Currently, implantation of patient-specific cranial prostheses requires reoperation after a period for design and formulation by a third-party manufacturer. Recently, 3-dimensional (3D) printing via fused deposition modeling has demonstrated increased ease of use, rapid production time, and significantly reduced costs, enabling expanded potential for surgical application. Three-dimensional printing may allow neurosurgeons to remove bone, perform a rapid intraoperative scan of the opening, and 3D print custom cranioplastic prostheses during the remainder of the procedure. OBJECTIVE: To evaluate the feasibility of using a commercially available 3D printer to develop and produce on-demand intraoperative patient-specific cranioplastic prostheses in real time and assess the associated costs, fabrication time, and technical difficulty. METHODS: Five different craniectomies were each fashioned on 3 cadaveric specimens (6 sides) to sample regions with varying topography, size, thickness, curvature, and complexity. Computed tomography-based cranioplastic implants were designed, formulated, and implanted. Accuracy of development and fabrication, as well as implantation ability and fit, integration with exiting fixation devices, and incorporation of integrated seamless fixation plates were qualitatively evaluated. RESULTS: All cranioprostheses were successfully designed and printed. Average time for design, from importation of scan data to initiation of printing, was 14.6 min and average print time for all cranioprostheses was 108.6 min. CONCLUSION: On-demand 3D printing of cranial prostheses is a simple, feasible, inexpensive, and rapid solution that may help improve cosmetic outcomes; significantly reduce production time and cost-expanding availability; eliminate the need for reoperation in select cases, reducing morbidity; and has the potential to decrease perioperative complications including infection and resorption.