Agonist (P1) Antibody Converts Stem Cells into Migrating Beta-Like Cells in Pancreatic Islets

Figures

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  Fig. 1. Scheme for the in vivo phenotype selection of the P1 antibody that induces differentiation and migration of mouse HSCs. Genes from a human ScFv phage library (108 members) were cloned into a lentiviral vector to make a lentiviral membrane-tethered library in which antibody molecules are attached to the plasma membrane and displayed on the cell surface. Total mouse bone marrow cells were infected with the antibody library in vitro and transplanted into lethally irradiated C57BL/6J mice. The system is autocrine based because each cell has a different antibody and the putative target. After 7 days, the mouse pancreatic tissues were harvested and analyzed by PCR to identify any antibody genes in cells that traffic to the islet.

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  Fig. 2. Antibody induced differentiation of HSCs into beta-like cells. (A) Scheme of the in vivo migration of cells from the bone marrow to the islet. tdTomato+ bone marrow cells were adoptively transplanted into lethally irradiated wildtype mice. P1 Ab (50 μg/mouse, i.p., two times/week) was injected into transplanted wild-type mice for 3 weeks. The mice were perfused, harvested, and analyzed by fluorescence microscope. (B-C) Significantly more tdTomato+ cells were identified by fluorescence microscope in the treated tissues as compared to controls (untreated tdTomato+ cells). These assays were repeated 3 times. The white box indicates the region magnified in the image to the right. Scale bars = 50 μm.

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  Fig. 3. Antibody induced differentiation of bone marrow cells into beta-like cells. P1 Ab (50 μg/mouse, i.p., two times/week) was injected into lethally irradiated wild-type mice. After 3 weeks, the mice were perfused with PBS prior to harvesting the islets for immuno-fluorescence histochemistry. Islets were stained with DAPI, anti-Insulin, PDX-1 (beta cell marker), and glucagon (alpha cell marker). (A) Significantly more Insulin+ and PDX-1+ staining was identified in the P1 Abtreated dTomato+ tissues as compared to controls, suggesting that the beta-like cells migrated from the bone marrow to the islet. (B) Glucagon was not detected on the dTomato+ Insulin+ cells. Scale bars = 50 μm.

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  Fig. 4. An agonist antibody induces beta-like cell differentiation. (A) Beta-like cells induced by the P1 Ab. Human CD34+ cells were incubated with P1 Ab (10 μg/ml) for 2 weeks. The red arrow in the image on the left indicates beta-like cells, which are magnified in the image to the right. (B) Insulin levels in the culture media were measured, and treatment with P1 Ab increased levels of insulin. . ***p-value, 0.0005; Student’s t-test. (C) Human CD34+ hematopoietic stem cells were incubated with P1 antibody and harvested islets were analyzed by flow cytometry. Cells were then stained with anti-Insulin, PDX-1, and Nkx-6.1. Scale bars = 50 μm.

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  Fig. 5. Identification of a novel antigen recognized by the P1 Ab. (A) The P1 Ab recognized human POSTN protein and murine bone marrow lysates when analyzed by western blotting. (B) Beta-like cell gene expression as determined by RNA sequencing analysis. Highly expressed beta cell markers from the RNAseq analysis (n = 3) are summarized as Transcripts Per Million mapped reads (TPM). (C) Human CD34+ cells were treated with P1 Ab or control isotype antibody, and cell lysates were assessed by western blotting using antibodies against phosphorylated (p-) AKT and ERK.