Journal: JCI Insight

Article Title: Modeling immune responses to autologous and allogeneic human stem cell–derived islet grafts in vivo

doi: 10.1172/jci.insight.200738

Figure Lengend Snippet: ( A and B ) Human immune system reconstitution in cohort 1 ( A ) and 2 ( B ); human chimerism is measured as the percentage of human CD45 + cells among total CD45 + cells. Each symbol represents an individual animal. In A , a hollow triangle represents an allogeneic mouse not injected with polyclonal T cells. ( C ) CD34 – nonadherent fetal liver cells reprogramming into iPSCs using Klf4, Oct3/4, Sox2, and c-Myc transcription factors delivered via the nonintegrating Sendai virus, with examples of iPSC colonies. Scale bar: 200 µm. ( D ) iPSC-to-β cell differentiations performed in the study. ( E ) Circulating C-peptide levels in autologous (cohort 1 and 2) and allogeneic grafts. In autologous cohort 1 and 2, n = 2/3 and n = 3/12 mice were injected with IAR-TCR + T cells that did not infiltrate the grafts, respectively, and n = 5/6 allogeneic mice were injected with additional in vitro activated polyclonal T cells from a mouse with the same HIS. In E , the asterisk indicates the level of significance (* P < 0.05) when comparing the percentage of circulating C-peptide in allogeneic and autologous cohort 2 at weeks 12 and 15 after β cell transplantation. ( F ) Correlation between percentage of C-peptide + cells in islet before transplant and circulating human C-peptide levels 12 and 15 weeks later. In A , B , and E , data show the mean ± SEM. In F , different groups are represented with the same symbols as E, and each symbol represents the mean of the group. Statistical analysis was performed using a mixed-effects model with Geisser-Greenhouse correction and Tukey’s post hoc test for E and a simple linear regression for F . C-1, cohort 1; C-2, cohort 2.

Article Snippet: Circulating human C-peptide was measured on 20 μL of plasma, isolated from whole blood, using the STELLUX Chemi Human C-peptide ELISA kit (Alpco).

Techniques: Injection, Virus, In Vitro, Transplantation Assay

Journal: JCI Insight

Article Title: Modeling immune responses to autologous and allogeneic human stem cell–derived islet grafts in vivo

doi: 10.1172/jci.insight.200738

Figure Lengend Snippet: ( A ) Endocrine cell populations found in the grafts, represented as percentage of total endocrine cells. ( B and C ) Comparison of C-peptide + ( B ) and C-peptide + NKX6.1 + cells ( C ) among cells found in the islet region between autologous (cohort 1, cohort 2) and allogeneic grafts. ( D ) Representative images of endocrine cell subpopulations in autologous grafts from cohort 1. Scale bar: 100 µm. ( E ) Representative images of endocrine cell subpopulations and CD57 expression in the grafts of autologous cohort 2. Scale bar: 100 µm. ( F ) Quantification of CD57 + cells in the grafts. ( G ) Quantification of CD57 expression among SC-islet cell subtypes. For D and E , markers used for analysis are indicated in the figure. In autologous cohorts 1 and 2, n = 2 and n = 3 mice were injected with IAR-TCR + T cells, respectively; n = 5 allogeneic mice were injected with additional in vitro activated polyclonal T cells (a hollow triangle represents the allogeneic mouse not injected with additional polyclonal T cells, and autologous mice injected with IAR-TCR + T cells are represented as solid circles). Mean ± SEM; 1-way ANOVA/Tukey’s post hoc test was used for B , C , and F . * P < 0.05, ** P < 0.01. C-1, cohort 1; C-2, cohort 2; SST, somatostatin.

Article Snippet: Circulating human C-peptide was measured on 20 μL of plasma, isolated from whole blood, using the STELLUX Chemi Human C-peptide ELISA kit (Alpco).

Techniques: Comparison, Expressing, Injection, In Vitro

Journal: JCI Insight

Article Title: Modeling immune responses to autologous and allogeneic human stem cell–derived islet grafts in vivo

doi: 10.1172/jci.insight.200738

Figure Lengend Snippet: ( A ) Quantification of CD45 + cell proliferation in autologous and allogeneic grafts. ( B ) Example of CD45 + cell proliferation in allogeneic grafts. Scale bar: 100 µm. ( C ) Quantification of CD3 + cell proliferation in autologous and allogeneic grafts. ( D and E ) Quantification of glucagon + α ( D ) and C-peptide + β ( E ) cell proliferation in autologous and allogeneic grafts. ( F ) Example of α and β cell proliferation in allogeneic grafts. Scale bar: 100 µm. ( G ) Example of CD45 + α and β cell proliferation in autologous grafts. n = 5 allogeneic mice were injected with additional in vitro activated polyclonal T cells (a hollow triangle represents the allogeneic mouse not injected with polyclonal T cells). Scale bar: 100 µm. Panel in F represents a lower-magnification view of the area shown in , visualized with different markers. Data shown as mean ± SEM; statistical analysis was performed with an unpaired 2-tailed t test for A and C – E . * P < 0.05. C-1, cohort 1; C-2, cohort 2.

Article Snippet: Circulating human C-peptide was measured on 20 μL of plasma, isolated from whole blood, using the STELLUX Chemi Human C-peptide ELISA kit (Alpco).

Techniques: Injection, In Vitro

Journal: JCI Insight

Article Title: Modeling immune responses to autologous and allogeneic human stem cell–derived islet grafts in vivo

doi: 10.1172/jci.insight.200738

Figure Lengend Snippet: ( A and B ) Human immune system reconstitution in cohort 1 ( A ) and 2 ( B ); human chimerism is measured as the percentage of human CD45 + cells among total CD45 + cells. Each symbol represents an individual animal. In A , a hollow triangle represents an allogeneic mouse not injected with polyclonal T cells. ( C ) CD34 – nonadherent fetal liver cells reprogramming into iPSCs using Klf4, Oct3/4, Sox2, and c-Myc transcription factors delivered via the nonintegrating Sendai virus, with examples of iPSC colonies. Scale bar: 200 µm. ( D ) iPSC-to-β cell differentiations performed in the study. ( E ) Circulating C-peptide levels in autologous (cohort 1 and 2) and allogeneic grafts. In autologous cohort 1 and 2, n = 2/3 and n = 3/12 mice were injected with IAR-TCR + T cells that did not infiltrate the grafts, respectively, and n = 5/6 allogeneic mice were injected with additional in vitro activated polyclonal T cells from a mouse with the same HIS. In E , the asterisk indicates the level of significance (* P < 0.05) when comparing the percentage of circulating C-peptide in allogeneic and autologous cohort 2 at weeks 12 and 15 after β cell transplantation. ( F ) Correlation between percentage of C-peptide + cells in islet before transplant and circulating human C-peptide levels 12 and 15 weeks later. In A , B , and E , data show the mean ± SEM. In F , different groups are represented with the same symbols as E, and each symbol represents the mean of the group. Statistical analysis was performed using a mixed-effects model with Geisser-Greenhouse correction and Tukey’s post hoc test for E and a simple linear regression for F . C-1, cohort 1; C-2, cohort 2.

Article Snippet: Circulating human C-peptide was measured on 20 μL of plasma, isolated from whole blood, using the STELLUX Chemi Human C-peptide ELISA kit (Alpco).

Techniques: Injection, Virus, In Vitro, Transplantation Assay

Journal: JCI Insight

Article Title: Modeling immune responses to autologous and allogeneic human stem cell–derived islet grafts in vivo

doi: 10.1172/jci.insight.200738

Figure Lengend Snippet: ( A ) Endocrine cell populations found in the grafts, represented as percentage of total endocrine cells. ( B and C ) Comparison of C-peptide + ( B ) and C-peptide + NKX6.1 + cells ( C ) among cells found in the islet region between autologous (cohort 1, cohort 2) and allogeneic grafts. ( D ) Representative images of endocrine cell subpopulations in autologous grafts from cohort 1. Scale bar: 100 µm. ( E ) Representative images of endocrine cell subpopulations and CD57 expression in the grafts of autologous cohort 2. Scale bar: 100 µm. ( F ) Quantification of CD57 + cells in the grafts. ( G ) Quantification of CD57 expression among SC-islet cell subtypes. For D and E , markers used for analysis are indicated in the figure. In autologous cohorts 1 and 2, n = 2 and n = 3 mice were injected with IAR-TCR + T cells, respectively; n = 5 allogeneic mice were injected with additional in vitro activated polyclonal T cells (a hollow triangle represents the allogeneic mouse not injected with additional polyclonal T cells, and autologous mice injected with IAR-TCR + T cells are represented as solid circles). Mean ± SEM; 1-way ANOVA/Tukey’s post hoc test was used for B , C , and F . * P < 0.05, ** P < 0.01. C-1, cohort 1; C-2, cohort 2; SST, somatostatin.

Article Snippet: Circulating human C-peptide was measured on 20 μL of plasma, isolated from whole blood, using the STELLUX Chemi Human C-peptide ELISA kit (Alpco).

Techniques: Comparison, Expressing, Injection, In Vitro

Journal: JCI Insight

Article Title: Modeling immune responses to autologous and allogeneic human stem cell–derived islet grafts in vivo

doi: 10.1172/jci.insight.200738

Figure Lengend Snippet: ( A ) Quantification of CD45 + cell proliferation in autologous and allogeneic grafts. ( B ) Example of CD45 + cell proliferation in allogeneic grafts. Scale bar: 100 µm. ( C ) Quantification of CD3 + cell proliferation in autologous and allogeneic grafts. ( D and E ) Quantification of glucagon + α ( D ) and C-peptide + β ( E ) cell proliferation in autologous and allogeneic grafts. ( F ) Example of α and β cell proliferation in allogeneic grafts. Scale bar: 100 µm. ( G ) Example of CD45 + α and β cell proliferation in autologous grafts. n = 5 allogeneic mice were injected with additional in vitro activated polyclonal T cells (a hollow triangle represents the allogeneic mouse not injected with polyclonal T cells). Scale bar: 100 µm. Panel in F represents a lower-magnification view of the area shown in , visualized with different markers. Data shown as mean ± SEM; statistical analysis was performed with an unpaired 2-tailed t test for A and C – E . * P < 0.05. C-1, cohort 1; C-2, cohort 2.

Article Snippet: Circulating human C-peptide was measured on 20 μL of plasma, isolated from whole blood, using the STELLUX Chemi Human C-peptide ELISA kit (Alpco).

Techniques: Injection, In Vitro

Journal: Islets

Article Title: Human and mouse regenerative macrophages enhance beta cell survival, function, and proliferation

doi: 10.1080/19382014.2026.2659457

Figure Lengend Snippet: Mouse M(IL-4, IL-13) macrophages enhance islet secretion of C-peptide. Intact mouse islets were co-cultured with M(−), M(LPS, IFNγ), and M(IL-4, IL-13) macrophages for 3 d, and then were subjected to a glucose-stimulated insulin secretion (GSIS) assay. (A–C) C-peptide released into the supernatants was measured by ELISA after stimulation with (A) 16.7 mM glucose and (B) KCl, and expressed as a percentage of total islet C-peptide content. (C) Total C-peptide content was measured from islet lysates at the end of the assay ( n = 6). (D–F) Similar measurements were performed using a Transwell co-culture system, where mouse islets were indirectly cultured with macrophages for 3 d and then stimulated with (D) 16.7 mM glucose, then (E) KCl. (F) Total islet C-peptide content was measured from lysates at the end of the assay ( n = 4). Bars indicate mean ± SEM. Statistical significance was calculated by one-way ANOVA. * p < 0.05. n indicates a different sample from an independent experiment or an individual mouse.

Article Snippet: C-peptide content from both the supernatants and islet pellets, extracted using acid ethanol, was measured using mouse C-peptide ELISA (Alpco) and human C-peptide ELISA (Mercodia).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

Journal: Islets

Article Title: Human and mouse regenerative macrophages enhance beta cell survival, function, and proliferation

doi: 10.1080/19382014.2026.2659457

Figure Lengend Snippet: Human M(IL-4, IL-13) enhances human beta cell survival and function. (A and B) Human monocyte-derived macrophages were left unpolarized or differentiated into M(IL-4, IL-13) macrophages. (A) Representative and averaged data showing CD206 expression on gated live CD14+CD11b+ cells ( n = 3). (B) Polarized cells were washed with media, incubated for 24 h, and then supernatants were collected to measure CCL17 ( n = 4). (C and D) Intact human islets were cultured alone or directly co-cultured with M(IL-4, IL-13) macrophages for 3 d. (C) Insulin+ cells were gated on CD45-Glucagon- cells, and the proportion of live (fixable viability dye [FVD] negative) Insulin+ cells was measured ( n = 4). Total number of insulin⁺ cells analyzed per condition—islets with -:781; M(IL-4, IL-13): 1904. (D) Representative data showing amount of EdU incorporation into insulin + beta cells after 3 d of incubation with harmine (10 μM, dissolved in 0.1% DMSO) and M(IL-4, IL-13) macrophages. EdU was added at 10 μM during the final 24 h. Representative and averaged data showing the percentage of EdU+ cells within insulin+cells, gated as live CD45-Glucagon- ( n = 3). Total number of live insulin⁺ cells analyzed per condition—islets with DMSO: 2545; Harmine: 2850; Harmine + M(IL-4, IL-13): 2265. (E-H) C-peptide secretion by islets cultured with or without M(IL-4, IL-13) macrophages for 3 d. (E) Islets were pre-incubated in low glucose (1.67 mM) before stimulation with (F) high glucose (16.7 mM), then (G) KCl. (H) The level of total C-peptide content in the islets was measured in lysates made at the end of the assay. The stimulation index (SI) was calculated as C-peptide secretion during high glucose and KCl incubations relative to the initial low glucose condition ( n = 3). Squares indicate obese donor samples. Bars indicate mean ± SEM. Statistical significance was calculated using a paired t -test for (A–C) and (E–H) or a one-way ANOVA test for (D). * p < 0.05 ** p < 0.01. n indicates a different sample from an independent experiment or an individual mouse. The number of islets cited for each experiment refers to a single representative experiment, corresponding to the panels shown in the figures.

Article Snippet: C-peptide content from both the supernatants and islet pellets, extracted using acid ethanol, was measured using mouse C-peptide ELISA (Alpco) and human C-peptide ELISA (Mercodia).

Techniques: Derivative Assay, Expressing, Incubation, Cell Culture

Journal: Stem Cells Translational Medicine

Article Title: Whole-genome CRISPR screening identifies genetic modifiers of stem cell–derived islet transplantation

doi: 10.1093/stcltm/szag012

Figure Lengend Snippet: Transplantation of CRISPR-activation of FCAMR , GSTM3 , and SIX3 . (A-C) Blood glucose of 10 million genetically engineered SC-islet cells transplanted into the subcutaneous space ( n = 5). (A) FCAMR overexpression and control SC-islet cells, (B) GSTM3 overexpression and control SC-islet cells, and (C) SIX3 overexpression and control SC-islet cells. * P < .05 by 2-way paired t -test comparing control and overexpression. (D - F) C-peptide measurements from week 5 and 9 after a 30-min 3 g/kg intraperitoneal glucose injection ( n = 5). (D) FCAMR overexpression and control SC-islet cells, (E) GSTM3 overexpression and control SC-islet cells, and (F) SIX3 overexpression and control SC-islet cells. NSG male mice were used for this experiment. Source Data can be found in the Source Data file.

Article Snippet: C-peptide was measured with the Ultrasensitive Human C-peptide ELISA (Mercodia, 10-1141-01).

Techniques: Transplantation Assay, CRISPR, Activation Assay, Over Expression, Control, Injection

Journal: Stem Cells Translational Medicine

Article Title: Whole-genome CRISPR screening identifies genetic modifiers of stem cell–derived islet transplantation

doi: 10.1093/stcltm/szag012

Figure Lengend Snippet: Kidney and muscle transplantations of SC-islet overexpressing FCAMR. (A, E) Schematic of SC-islet cells transplanted into two groups of mice ( N = 6–8): (A) 5 million into the kidney capsule. (E) 10 million intramuscularly. (B, F) Blood glucose measurements before inducing diabetes, 5-day injections of streptozotocin (STZ), and post-STZ. ** P < .01 by way of 2-way unpaired t -test comparing control (CTRL) and FCAMR overexpressing ( FCAMR OE) SC-islets in (B) kidney capsule or (F) intramuscular transplants. (C, G) C-peptide measurements after a 30-min 3 g/kg intraperitoneal glucose injection of SC-islets transplanted: (C) into the kidney capsule or (G) intramuscularly. (D, H) Bar graphs of weights of mice before transplantation (week 0), after STZ injection (week 6), and before nephrectomy (week 11) in (D) kidney transplant or (H) intramuscular transplanted mice. *** P < .001, ** P < .01, and * P < .05 by way of 2-way unpaired or paired t -test. (I) Immunohistochemistry of kidney grafts stained with human C-peptide (green), CHGA (red), and DAPI (blue) on the left, GCG (green), SST (red), and DAPI (blue) in the middle, and SYP (green), Ki67 (red), and DAPI (blue) on the right. Scale bar: 200 µM. Source Data can be found in the Source Data file.

Article Snippet: C-peptide was measured with the Ultrasensitive Human C-peptide ELISA (Mercodia, 10-1141-01).

Techniques: Control, Injection, Transplantation Assay, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: BLAST: A blue light-assisted secretion toolkit tunable by reversible protein-protein interactions

doi: 10.64898/2026.03.30.715452

Figure Lengend Snippet: Secretion of therapeutic proteins with a-BLAST and d-BLAST. (a) The human preproinsulin (preproINS) construct contains a signal peptide, B-chain (yellow), C-peptide (dark gray), and A-chain (yellow), with engineered Furin cleavage sites flanking the C-peptide for maturation. To facilitate efficient processing within the Golgi apparatus, Furin protease was co-transfected with the BLAST modules. (b) Kinetic profiling of light-induced insulin secretion. Summary graphs of secreted C-peptide levels, quantified by ELISA, as a proxy for insulin secretion from a-BLAST (left) and d-BLAST (right). Both systems exhibited significant, time-dependent insulin release starting from 2 h of illumination (8.2-fold for a-BLAST, 8.4-fold for d-BLAST), reaching maximal induction at 24 h (13.8-fold for a-BLAST, 19.3-fold for d-BLAST). (c) Plasmid configurations for IL-12 secretion. Schematic of the heterodimeric cytokine IL-12-a-BLAST (left) and d-BLAST-IL-12 (right) constructs. (d) Kinetic profiling of light-induced IL-12 secretion. Summary graphs showing IL-12 secretion levels measured by ELISA. Significant secretion was observed starting at 3 h for a-BLAST (2.5-fold) and 2 h for d-BLAST (2.2-fold). At the 24 h time point, d-BLAST (4.7-fold) demonstrated a slightly higher dynamic range compared to a-BLAST (4.5-fold). Open circles represent individual measurements from three biologically independent samples. Data are presented as means ± S.D. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test (ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Secreted human C-peptide levels in the supernatant were quantified using a Human C-peptide ELISA Kit (R&D Systems; Catalog #DICP00).

Techniques: Construct, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: BLAST: A blue light-assisted secretion toolkit tunable by reversible protein-protein interactions

doi: 10.64898/2026.03.30.715452

Figure Lengend Snippet: Secretion of therapeutic proteins with a-BLAST and d-BLAST. (a) The human preproinsulin (preproINS) construct contains a signal peptide, B-chain (yellow), C-peptide (dark gray), and A-chain (yellow), with engineered Furin cleavage sites flanking the C-peptide for maturation. To facilitate efficient processing within the Golgi apparatus, Furin protease was co-transfected with the BLAST modules. (b) Kinetic profiling of light-induced insulin secretion. Summary graphs of secreted C-peptide levels, quantified by ELISA, as a proxy for insulin secretion from a-BLAST (left) and d-BLAST (right). Both systems exhibited significant, time-dependent insulin release starting from 2 h of illumination (8.2-fold for a-BLAST, 8.4-fold for d-BLAST), reaching maximal induction at 24 h (13.8-fold for a-BLAST, 19.3-fold for d-BLAST). (c) Plasmid configurations for IL-12 secretion. Schematic of the heterodimeric cytokine IL-12-a-BLAST (left) and d-BLAST-IL-12 (right) constructs. (d) Kinetic profiling of light-induced IL-12 secretion. Summary graphs showing IL-12 secretion levels measured by ELISA. Significant secretion was observed starting at 3 h for a-BLAST (2.5-fold) and 2 h for d-BLAST (2.2-fold). At the 24 h time point, d-BLAST (4.7-fold) demonstrated a slightly higher dynamic range compared to a-BLAST (4.5-fold). Open circles represent individual measurements from three biologically independent samples. Data are presented as means ± S.D. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test (ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Secreted human C-peptide levels in the supernatant were quantified using a Human C-peptide ELISA Kit (R&D Systems; Catalog #DICP00).

Techniques: Construct, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: RGX-019-MMAE inhibits leukemia progression by targeting MER proto-oncogene tyrosine kinase (MERTK) in acute myeloid leukemia

doi: 10.1186/s13046-026-03657-y

Figure Lengend Snippet: The antibody-drug conjugate RGX-019-MMAE induced cytotoxic activity in MERTK-expressing AML cell lines. A MERTK expression in the indicated AML cell lines; leukemic cells (1 × 10 6 ) were stained with anti-MERTK-APC antibody, and the expression was measured by flow cytometry. Data are plotted as mean values with error bars representing standard error. B - F Bar graph showing the percentage of relative luminescence in Kasumi 1 ( B ), OCI-AML3 ( C ), MOLM-13 ( D ), MOLM-14 ( E ), and MV4-11 ( F ) cells treated with the indicated concentrations of RGX-019-MMAE and monoclonal antibody RGX-019. Data are plotted as mean values with error bars representing standard error (Student unpaired t -test). * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001. UT = untreated

Article Snippet: Mouse IgG1/kappa light chain MERTK-specific monoclonal antibodies were generated in mice by immunization with Fc-tagged human MERTK peptide (R&D Systems, Minneapolis, MN) USA).

Techniques: Activity Assay, Expressing, Staining, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: RGX-019-MMAE inhibits leukemia progression by targeting MER proto-oncogene tyrosine kinase (MERTK) in acute myeloid leukemia

doi: 10.1186/s13046-026-03657-y

Figure Lengend Snippet: The antibody-drug conjugate RGX-019-MMAE induced cytotoxic activity in MERTK-expressing AML primary cells. A - D Bar graphs show the percentage of relative luminescence in primary AML cells, RGX946 ( A ), RGX694 ( B ), RGX702 ( C ), and RGX470 ( D ) treated with the indicated concentrations of RGX-019-MMAE or the monoclonal antibody RGX-019. Data are plotted as mean values with error bars representing standard error (Student unpaired t -test). E Representative figures of colony-forming units for normal peripheral blood mononuclear cells in response to DMSO and indicated concentrations of RGX-019-MMAE and RGX-019. Data are plotted as mean values with error bars representing standard error (Student unpaired t -test) * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001

Article Snippet: Mouse IgG1/kappa light chain MERTK-specific monoclonal antibodies were generated in mice by immunization with Fc-tagged human MERTK peptide (R&D Systems, Minneapolis, MN) USA).

Techniques: Activity Assay, Expressing