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. 2024 Sep;67(9):1912-1929.
doi: 10.1007/s00125-024-06194-5. Epub 2024 Jun 14.

Non-invasive quantification of stem cell-derived islet graft size and composition

Väinö Lithovius  1 , Salla Lahdenpohja  2 , Hazem Ibrahim  3 , Jonna Saarimäki-Vire  3 , Lotta Uusitalo  2 , Hossam Montaser  3 , Kirsi Mikkola  2   4 , Cheng-Bin Yim  2 , Thomas Keller  2 , Johan Rajander  5 , Diego Balboa  3 , Tom Barsby  3 , Olof Solin  2   5   6 , Pirjo Nuutila  2   7   8 , Tove J Grönroos  2   4 , Timo Otonkoski  9   10
Affiliations

Non-invasive quantification of stem cell-derived islet graft size and composition

Väinö Lithovius et al. Diabetologia. 2024 Sep.

Abstract

Aims/hypothesis: Stem cell-derived islets (SC-islets) are being used as cell replacement therapy for insulin-dependent diabetes. Non-invasive long-term monitoring methods for SC-islet grafts, which are needed to detect misguided differentiation in vivo and to optimise their therapeutic effectiveness, are lacking. Positron emission tomography (PET) has been used to monitor transplanted primary islets. We therefore aimed to apply PET as a non-invasive monitoring method for SC-islet grafts.

Methods: We implanted different doses of human SC-islets, SC-islets derived using an older protocol or a state-of-the-art protocol and SC-islets genetically rendered hyper- or hypoactive into mouse calf muscle to yield different kinds of grafts. We followed the grafts with PET using two tracers, glucagon-like peptide 1 receptor-binding [18F]F-dibenzocyclooctyne-exendin-4 ([18F]exendin) and the dopamine precursor 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine ([18F]FDOPA), for 5 months, followed by histological assessment of graft size and composition. Additionally, we implanted a kidney subcapsular cohort with different SC-islet doses to assess the connection between C-peptide and stem cell-derived beta cell (SC-beta cell) mass.

Results: Small but pure and large but impure grafts were derived from SC-islets. PET imaging allowed detection of SC-islet grafts even <1 mm3 in size, [18F]exendin having a better detection rate than [18F]FDOPA (69% vs 44%, <1 mm3; 96% vs 85%, >1 mm3). Graft volume quantified with [18F]exendin (r2=0.91) and [18F]FDOPA (r2=0.86) strongly correlated with actual graft volume. [18F]exendin PET delineated large cystic structures and its uptake correlated with graft SC-beta cell proportion (r2=0.68). The performance of neither tracer was affected by SC-islet graft hyper- or hypoactivity. C-peptide measurements under fasted or glucose-stimulated conditions did not correlate with SC-islet graft volume or SC-beta cell mass, with C-peptide under hypoglycaemia having a weak correlation with SC-beta cell mass (r2=0.52).

Conclusions/interpretation: [18F]exendin and [18F]FDOPA PET enable non-invasive assessment of SC-islet graft size and aspects of graft composition. These methods could be leveraged for optimising SC-islet cell replacement therapy in diabetes.

Keywords: Beta cell mass; Cell replacement therapy; Congenital hyperinsulinism; PET; Positron emission tomography; Stem cell-derived islets; Transplantation; Type 1 diabetes.

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Figures

Fig. 1
Fig. 1
Study set-up and tracer cell type specificity. (a) Study set-up: cohort-1 consisted of comparison of different-sized SC-islet grafts in each leg; cohort-2 consisted of comparison of SC-islet grafts of different functional phenotypes in each leg. Immature SC-islets were differentiated as in the Balboa et al 2018 protocol [49], and functional SC-islets were differentiated as in the Barsby et al 2022 protocol [50]. Here, 1 mm3 corresponds to the volume of 566 IEQs (islets of 150 µm diameter) or 150 SC-islets (of 230 µm diameter). (b) PET tracers used in the study: [18F]exendin and [18F]FDOPA. (c–f) Tracer target expression in different cell types in SC-islet kidney subcapsular grafts at 6 months post implantation using single-cell RNA sequencing data from Balboa et al 2022 [2]. Red lines, median. The panels show GLP1R (c), SLC7A5 (d) and DDC (e) mRNA expression in the different cell types, and the percentages of different cell types expressing the tracers (f)
Fig. 2
Fig. 2
KCNJ11 mutant SC-islet phenotypic characterisation in vitro. (a) Simplified molecular mechanism and genome editing strategy underlining the hyper-, normo- and hypoactive phenotypes of SC-islet beta cells in cohort-2. KCNJ11−/− mutation leads to loss of KATP-channel expression, leading to constant depolarisation and insulin secretion. KCNJ11R201H/+ mutation prevents KATP-channel closure under high glucose, leading to inactive beta cells. nt, nucleotide; PAM, protospacer adjacent motif; ssODN, single-stranded oligonucleotide. (b) Percentage of total insulin content secreted in 2.8 mmol/l (G3) or 16.7 mmol/l (G17) glucose, or G17 and 100 nmol/l GBC, or G3 and 30 mmol/l KCl after 6 weeks of maturation culture. The horizontal line indicates the secretion rate in G3 in KCNJ11+/+ cells. (c) Same analysis as in (b) normalised against secretion in G3. (d, e) Immunohistochemistry (d) and quantification (e) of INS+ SC-beta cells and glucagon (GCG)+ SC-alpha cells at 6 weeks of maturation culture; scale bars, 100 µm. *p<0.05 by one-way ANOVA
Fig. 3
Fig. 3
Characterisation of the SC-islet grafts. (a) Skinned mouse hind leg at 5 months post implantation; arrow indicates graft. (b) Hoechst-stained muscle graft sections used for volume determination; examples of pure and impure grafts in the same scale (bar, 1000 µm). Sections are displayed as CellProfiler pipeline output images highlighting the total (blue) and cyst-free (yellow) areas. (c) Implanted SC-islet volume and histologically determined graft volume. Pink squares, ‘pure’ with >50% SYP+ endocrine cells out of all graft cells; black circles, ‘impure’ with <50% SYP+ endocrine cells. (d) Cyst proportion of the grafts, quantified from images such as (b). (e) Immunohistochemistry analysis of SYP, the SC-beta cell marker INS, the EC cell marker SLC18A1, the acinar cell marker trypsin and the ductal cell marker CK-19; scale bar, 100 µm; insets display ×4 magnification. Examples are of pure and impure KCNJ11+/+ grafts. (f) Quantification of immunohistochemistry as percentage of all cells in the graft including connective tissue. (g) Percentage of INS+ and SLC18A1+ cells in SYP+ endocrine cells. (h) Pearson’s correlation of the composition features. The scale bar displays the spectrum of colour associated with the spectrum of correlation coefficients from −1 to 1. Data in (c), (d), (f) and (g) are displayed as mean ± SD
Fig. 4
Fig. 4
Follow-up and quantification of SC-islet graft volume with PET. (a) Imaging follow-up schedule; crosses indicate imaging, ex vivo autoradiography and harvesting time points. aCohort-1 only. (b) Views of PET/CT and PET images (summed 30 min scans); arrows indicate uptake volume considered the graft; abladder, bkidneys. (c, d) Tracer detection performance per time point (N indicated on graph) (c) or according to actual graft volume (d) as a fraction of all imaged grafts detected. (e) Ex vivo autoradiography of muscle sections taken immediately after imaging with [18F]exendin or [18F]FDOPA and insulin immunohistochemistry from adjacent sections. (f) Linear regression between [18F]exendin and [18F]FDOPA uptake volume and the histologically determined graft volume (including cysts). Inset depicts low values. Values in parentheses are for [18F]FDOPA when three of the most cystic grafts are excluded (white-filled squares). (g) Example frame and VOI (blue) in [18F]exendin PET/CT highlighting low-uptake areas inside the VOI, and Hoechst-stained section of the same graft with corresponding cysts. (h) Cyst proportion determined by histology or PET (by drawing full and cyst-free uptake volumes). (i) Example of an elongated pattern in [18F]exendin PET/CT (abladder) and a Hoechst-stained section of the same graft. (j) Graft size follow-up with [18F]exendin PET, grouped by final purity and SC-islet differentiation protocol, with grafts with >50% of all cells SYP+ considered pure. n: Balboa impure = 17, Barsby impure = 11, Barsby pure = 13. *p<0.05 by unpaired Welch’s t test
Fig. 5
Fig. 5
Correlation of graft composition and genotype with tracer uptake. (a) Schematic and associated formulas for volume dilution correction to determine graft uptake concentration. C, tracer uptake concentration; A, radioactivity (Bq); V, volume (mm3); Bg, background; VOI1, uptake volume; VOI2, additional VOI encompassing the whole muscle. (b) Pearson’s correlation of graft tracer uptake with different composition parameters. (c, d) Linear regression of graft exendin uptake and graft SC-beta cell (c) and EC cell (d) proportions. (e) Linear regression of graft [18F]exendin uptake variability and cyst proportion. CV = uptake SD/graft uptake mean × 100. (f) Graft [18F]exendin and [18F]FDOPA uptake in cohort-2 grafts of different genotypes. A single KCNJ11+/R201H graft was detected with DOPA and is not plotted
Fig. 6
Fig. 6
Relationship between C-peptide and implanted dose or engrafted volume. (a) Linear regression between SC-islet dose implanted under the kidney capsule and the fasted C-peptide level at 1 month post implantation. Implanted dose as total volume of SC-islets for each graft was quantified from stereomicroscope images. Here, 1 mm3 corresponds to 566 IEQs (150 µm diameter) or around 150 SC-islets (around 230 µm diameter). (b) Pearson’s correlation heatmap between implanted dose or histologically determined engrafted volume (total including cysts) or SC-beta cell mass (BCM) and C-peptide parameters. (c, d) Follow-up of fasting glucose (c) and fasting human-specific C-peptide (d) in the kidney cohort. n: 1 mm3 = 4; 3 mm3 = 5; 6.3 mm3 = 5. Low and high limits of human fasting normoglycaemia are indicated by horizontal lines. Vertical lines separate random-fed and fasted measurements. (e, f) Glucose (e) and C-peptide (f) following i.p. injection of glucose (3 mg/g) at 5 months post implantation, after a 5 h fast. (g, h) Glucose (g) and C-peptide (h) following i.p. injection of an insulin analogue (Actrapid, Novo Nordisk, Denmark) (0.75 mIU/g) at 5.5 months post implantation, after a 5 h fast. The low limit of human normoglycaemia is indicated by the horizontal line. (i, j) Linear regression of C-peptide parameters and total volume of the kidney graft (i) or the cyst-free, beta cell fraction-corrected graft volume (j). The C-peptide parameters were C-peptide in the fasted state (mean of 2 days) or 30 min after glucose injection or 60 min after insulin injection. The inset graph displays the same data with the y-axis scale focused to values below 200 pmol/l. Data in (c–h) are grouped by implanted SC-islet dose. Data in (i) and (j) display the histologically determined engrafted volume
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