Fig 2: Characterization of PTPRF+/+ and PTPRF−/− H1-hESC-derived β-like cells after implantation(A) Schematics showing the protocol for studying glucose homeostasis and H1-hESC-derived β-like islet cell function in implanted mice by glucose tolerance test (GTT) and glucose-stimulated insulin secretion (GSIS).(B) Blood glucose levels and circulating human C-peptide during GTT-GSIS of fasted PTPRF+/+ and PTPRF−/− implanted mice pre-STZ treatment at 16 weeks post-implantation (fasted blood glucose levels in the implanted mice decreased to human levels (4 mM) after 16 weeks), representative result of two independent experiments.

Fig 3: Detailed procedure for the implantation of PTPRF+/+ and PTPRF−/− H1-hESC-derived β-like cells under the kidney capsule of NOD/SCID Mice(A) Schematic representation showing the data points and sampling procedure post-implantation of H1-hESC-derived β-like islet cells under the kidney capsule of non-obese diabetic severe combined immunodeficient (NOD/SCID) mice.(B–T) Implantation of differentiated β-like cells under the kidney capsule. Preparation and loading of spheroids in polyethylene (PE) tubing (B-F). Preparation of the implantation PE tubing with the gel-loading tip (black arrow points toward the spheroids) (B). A pipette is used to push the spheroid cell suspension inside the PE tubing from the gel-loading tip (the black arrow points toward the spheroids) (C). PE tubing containing spheroid cell suspensions is placed inside syringe barrels within 50 mL tubes for centrifugation of both wild-type (WT) and knockout (KO) samples (D). PE tubing with the compact spheroid mass at the bottom (black arrow) (E). Close-up view of the PE tubing with the compact spheroid mass at the bottom (black arrow) (F). Surgical procedure (G-T). Anesthetized mouse positioned for surgery (G). Incision made at the surgical site to access the kidney (H-J). Application of DPBS on externalized kidney and exposed kidney for implantation (K-L). The needle tip is used to make a small incision on the kidney capsule (M). Using a gel-loading pipette tip, a space below the kidney capsule is created (N). PE tubing containing the spheroids is inserted in the space below the kidney capsule (O). The surgical assistant carefully pushes the spheroids using a Hamilton pipette (P). Close-up view of the implanted kidney (black arrow points toward the spheroids) (Q). Sutures securing the abdominal incision (R). Surgical area post skin-suturing (S). Mouse recovering post-surgery with a protective covering over the surgical site (T).

Fig 4: Nephrectomy and analysis of implanted kidney with PTPRF+/+ and PTPRF−/− H1-hESC-derived β-like cells(A–P) Nephrectomy of the kidney implanted with differentiated β-like cells under the kidney capsule. Isolation and positioning the implanted kidney for the nephrectomy procedure (A–C). Kidney fully exteriorized, ready for surgery (D). Hydration of the kidney with DPBS to maintain tissue viability (E). Stabilization of the kidney using forceps (F). Isolation of the renal artery, renal vein, and ureter from other tissues (G). Clamping the renal artery, vein, and ureter with bulldog forceps (H–J). Ligation of the renal artery, vein, and ureter using absorbable sutures (K). Ensuring tight knots to prevent bleeding (L). Cutting the renal artery, vein, and ureter between the kidney and knot (M). Excised kidney showing the implantation site (N). Suturing the abdominal incision post-nephrectomy (O). Final sutures securing the surgical site, ensuring proper closure and promoting healing (P).(Q) Blood glucose pre- and post-implanted kidney removal in random-fed STZ-induced diabetic mice; normoglycemia in S7-SC-β-like cell spheroid implants was lost upon graft removal, representative result of several independent experiments.(R) Immunofluorescence image showing insulin-positive (INS+), glucagon-positive (GCG+), and somatostatin-positive (SST+) cells at 20 weeks post-implantation (Scale bar = 100 μm).

Fig 5: Characterization of PTPRF+/+ and PTPRF−/− H1-hESC during in vitro differentiation(A) Schematic of PTPRF+/+ and PTPRF−/− H1 SC-β-like cell differentiation protocol. The first four stages (S1-S4) are conducted as monolayers in planar culture (2D). The subsequent three stages (S5-S7) are performed in static microwells (3D).(B and C) Representative bright-field images showing the morphology and immunofluorescence images of differentiation stage-specific markers for PTPRF+/+ and PTPRF−/− clones: SOX17+ and OCT4- cells for definitive endoderm (B) and NKX6.1+ and PDX1+ for pancreatic endoderm (C) (Scale bar = 100 μm).(D) Percentage viability of total cells at the end of Stage 4 (S4) and Stage 7 (S7) (n = 2, mean ± SEM).(E) Representative bright-field image showing the morphology and size of Stage 5 PTPRF+/+ and PTPRF−/− aggregates differentiating into β-like cells in static microwells (Scale bar = 100 μm).(F) Relative gene expression levels of PDX1 and NGN3 at Stage 5 (S5) and INS, GCG, and SST at Stage 6 (S6) and Stage 7 (S7). β-actin was used as an endogenous housekeeping gene to calculate ΔCt and normalized to undifferentiated PTPRF+/+ H1-hESC to calculate ΔΔCt values and relative gene expression (n = 2, mean ± SEM).(G) Representative immunofluorescence staining of differentiation stage β-like cells (S7) specific markers showing insulin-positive (INS+), glucagon-positive (GCG+), and somatostatin-positive (SST+) cells in PTPRF+/+ and PTPRF−/− in dispersed β-like cell spheroids (Scale bar = 50 μm). The number of INS+, GCG+, and SST+ cells is quantified with CellProfiler as a percentage of total cells. Nuclei are visualized with DAPI.