Fig 1.
(A) Schematic representation of the ARX gene and contained exons. Approximate protein domains and important functional regions of ARX including: octapeptide domain (OP), nuclear localization signals (NLS), poly-alanine expansion repeats (PA), Acidic domain (Acidic), prd-like homeodomain (prd-like HD), and Aristaless domain / C-peptide (AR). Approximate location of zinc finger nuclease mediated genomic editing induced deletion mutations in ARX knockout (ARX ko) hESCs and naturally occurring mutation of the XLAG pancreatic sample used in this study are also depicted. (B) Specific nucleotide deletions of ARX surrounding the high-specificity 36 bp ZFN recognition sequence within exon 1. ARX ko hESCs clones resulted in a frameshift mutation and premature stop codons in ARX. (C) Wild type (WT) and ARX ko clones 1 and 2 immunostained for pluripotency associated markers OCT4 (green) or SSEA3 (green) and counterstained for DNA with Hoechst 33342. Scale bar is 50 μm. (D) Immunostaining of ARX protein in agarose-embedded sections of 26-day differentiated WT or ARX ko pancreatic cells as well as human fetal pancreatic tissue at 13 weeks of gestation (13w HFP); ARX (green), chromogranin A (red), and DAPI (white). (E) 13w HFP immunostained for ARX (green), insulin (blue), glucagon (red), and DAPI (white) showing natural ARX expression patterns. Inset is a ~3x enlarged portion from the region indicated by the star. Scale bar for D and E is 100 μm.
Fig 2.
Pancreatic Differentiation of ARX ko hESCs.
(A) Schematic representation of the in vitro pancreatic endocrine differentiation method as previously described by Bruin et al. (2014). (B) Flow cytometry of wild type (WT), ARX knockout (ARX ko) clone 1 (C1), and ARX ko clone 2 (C2) at the end of definitive endoderm (CXCR4), foregut (PDX1), and pancreatic progenitor (NKX6.1) stages. (C-H) Media composition and cellular content analysis of differentiating wild type and ARX ko hESCs. Glucagon, C-peptide and somatostatin were assayed from un-stimulated media samples collected from days 11 to 26 (C, E, G; left column); a static sequential glucose stimulated hormone release assay was performed on day 24 of culture which included 1 hour treatments of low glucose (2 mM), high glucose (25 mM), and potassium chloride (KCl, 30 mM) (C, E, G; right column). (D, F, H) total hormone content measurements of day 26 WT and ARX ko cell lysate samples normalized for total protein content. (N = 4 independent differentiation trials for all data sets). * indicates p < 0.05 wild type vs ARX ko C1 and C2 at indicated time point or treatment based on a one-way ANOVA and Bonferroni post hoc test.
Fig 3.
Pancreatic Progenitors in ARX ko hESCs.
(A) Immunostaining of PDX1 (green) and NKX6.1 (red) in agarose-embedded wild type (WT) and ARX knockout (ARX ko) cell sheets from day 14, 17, 20, and 26 of culture. * indicates region of enlargement (right) which includes individual red and green channels and overlay with DAPI (blue) showing coexpression patterns. (B) Immunostaining of 13 week human fetal pancreatic tissue the same as A. (C) RT-qPCR of day 17 and day 26 whole population samples examining the expression of transcription factors believed to be involved in pancreatic progenitor induction. Wild type (black bars) and ARX ko clone 1 (red bars) cell samples relative to adult human islets expression levels. † indicates p < 0.05 within the genotype over time, * indicates p < 0.05 wild type vs ARX ko at given culture day, N = 4 per group. (D) Immunostaining of NKX6.1 (green), proliferating cell nuclear antigen (PCNA, red), and chromogranin A (blue) in 26 day differentiated wild type and ARX ko cells, 13 week human fetal pancreatic tissue, and adult human pancreatic tissue. Inset is a ~3x enlarged portion from the region indicated by the star. Scale bar is 100 μm for all panels.
Fig 4.
Pancreatic Endocrine Profile of ARX ko hESCs.
(A) RT-qPCR of day 17 and day 26 whole population samples examining the expression of the five major pancreatic hormones in wild type (WT; black bars) and ARX knockout clone 1 (ARX ko; red bars) relative to adult human islets expression levels. † indicates p < 0.05 within the genotype over time, * indicates p < 0.05 wild type vs ARX ko at given culture day. (B) 26-day differentiated cultures were stained in situ for insulin (blue), glucagon (green), somatostatin (red), and nuclei (white). (C) Image based cell counting was performed to determine the number of cells positive for any of the three hormones as a percentage of the total number of nuclei. (D) Single cell pancreatic endocrine population profile as a percentage of the total number of insulin, glucagon and somatostatin-positive cells. * indicates significant changes in the population between wild type and ARX ko (both clones). (E) Immunostaining of 13w HFP and 26-day differentiated wild type and ARX ko cells for chromogranin A (blue), somatostatin (red), ghrelin (green) and DAPI (white). (F) Single cell quantification of ghrelin-positive and ghrelin/somatostatin co-positive cells as a percentage of the total hormone (chromogranin A) positive fraction. (G) Immunostaining of pancreatic polypeptide-positive cells in day 26 cultures and 13w HFP with pancreatic polypeptide (green), somatostatin (red) and DAPI counterstain (white). Inset is a ~3x enlarged portion from the region indicated by the star. Scale bar is 100 μm for all panels.
Fig 5.
XLAG Pancreatic Endocrine Profile.
16-month old XLAG (ARX knockout) pancreatic tissue, age-matched control pancreatic tissue, 13w human fetal pancreatic tissue, and adult human pancreatic tissue were immunostained for insulin (blue), glucagon (green), somatostatin (red) and DAPI (white) in (A) and ghrelin (blue), pancreatic polypeptide (green), somatostatin (red) and DAPI (white) in (B). "Head" indicates specific sample from head region of the pancreas. Inset is a ~3x enlarged portion from the region indicated by the star. Scale bar is 100 μm for all panels.
Fig 6.
Pancreatic Fate specification in ARX ko hESCs.
(A) RT-qPCR of day 17 and day 26 whole population samples examining the expression of transcription factors believed to be involved in pancreatic endocrine fate specification. Wild type (WT; black bars) and ARX knockout clone 1 (ARX ko; red bars) cell samples relative to adult human islets expression levels. † indicates p < 0.05 within the genotype over time, * indicates p < 0.05 wild type vs ARX ko at given culture day, N = 4 per group. (A-E) Immunostaining of 26-day differentiated wild type and ARX ko cells, 13 week human fetal pancreatic tissue and adult human pancreatic tissue. Inset is a ~3x enlarged portion from the region indicated by the star. Scale bar is 100 μm for all panels.
Fig 7.
Expression of ISL1 in ARX ko hESCs and Human Tissue.
(A) Immunostaining of ISL1 (green), somatostatin (red), and insulin (blue) in 26 day differentiated wild type (WT) and ARX knockout cells (ARX ko), 13 week human fetal pancreatic tissue, and adult human pancreatic tissue. (B) Immunostaining of PAX6 (green), ISL1 (red), and NKX2.2 (blue) in the same sample series as above. Inset is a ~3x enlarged portion from the region indicated by the star. Scale bar is 100 μm for all panels. Extracellular immunoreactivitity of ISL1 is a staining artifact associated with the agarose which surrounds the embedded cell sheets.
Fig 8.
Expression of PAX6 in ARX ko mice.
(A) Immunostaining of PAX6 (green), somatostatin (red), and insulin (blue) in ARX knockout (ARX ko) and wild type (WT) E18.5 mouse pancreatic samples. Image on the right shows individual PAX6 channel showing nuclear PAX6 in wild type islet cells and lower levels of PAX6 in ARX ko tissue. (B) Immunostaining of PAX6 (green), ISL1 (red), and NKX2.2 (blue) in the same samples as in A. Inset is a ~3x enlarged portion from the region indicated by the star. Scale bar is 100 μm for all panels.
Fig 9.
Expression of PREP1, PBX1 and Processing of Somatostatin in ARX ko hESCs.
(A—D) Immunostaining of PREP1, PBX1a/1b, PC1/3, PC2 (all green), somatostatin (red), and insulin (blue) in 26 day differentiated wild type (WT) and ARX knockout (ARX ko) cells, and adult human pancreatic tissue. RT-qPCR of day 17 and day 26 whole population samples examining the expression of transcription factors believed to be involved expression and processing of somatostatin. Wild type (WT; black bars) and ARX ko clone 1 (ARX ko; red bars) cell samples relative to adult human islets expression levels. † indicates p < 0.05 within the genotype over time, * indicates p < 0.05 wild type vs ARX ko at given culture day, N = 4 per group. (E and F) Immunostaining of 13 week human fetal pancreatic tissue for expression of PC1/3 and PC2 (both green), somatostatin (red), and insulin (blue). DAPI is white in all images. Inset is a ~3x enlarged portion from the region indicated by the star. Scale bar is 100 μm for all panels.
Fig 10.
Adenoviral ARX Expression in Developing ARX ko hESCs.
(A) Schematic diagrams of adenoviral infection timeline during pancreatic differentiation stages and the adenoviral construct used to express human ARX under the constitutive EF1α promoter. (B) Immunostaining of ARX (green), somatostatin (red), and C-peptide (blue) in ARX knockout (ARX ko) cells differentiated to day 26 after transduction at day 13 or 19 with Ad GFP or Ad ARX. (C) RT-qPCR of day 26 whole population samples examining the expression of ARX in wild type (WT) (left graph) and ARX ko clone 1 (right graph) cells treated with Ad GFP (green bars) or Ad ARX (blue bars) at an MOI of 2 on day 13 (filled bars) or 19 (open bars). * indicates p < 0.05 Ad GFP vs Ad ARX from a given transduction (Tdxn) day. (D) Glucagon and C-peptide levels from 24-hour static media samples taken between days 13 and 26 comparing Ad ARX treatment in wild type cells (black lines) and ARX ko cells (red lines). Treatments are indicated by symbol colour in the figure. * indicates p < 0.05 Ad ARX on day 13 versus all other populations on day 22 and 26. (E—F) Immunostaining and single cell hormone analysis of Ad GFP and Ad ARX treated wild type and ARX ko day 26 cultures for insulin (blue), glucagon (green), and somatostatin (red). (E) Total number of hormone-positive cells (any combination of insulin, glucagon and or somatostatin) represented as a percentage of the total number of nuclei. Endocrine population breakdown of wild type and ARX ko cultures. * indicates p < 0.05 Ad ARX delivered on day 13 versus no virus (NON) and Ad GFP delivered on day 13 in ARX ko cells. N = 3 per group. Inset is a ~3x enlarged portion from the region indicated by the star. Scale bar is 100 μm for all panels.
Fig 11.
Adenoviral Infection Efficiency in ARX ko Cells.
(A) Brightfield and green channel (GFP) images of differentiating ARX knockout (ARX ko) cell cultures 72 hours post adenoviral infection at a MOI of 2. (B) GFP infection efficiency quantified by flow cytometry 72 hours after viral delivery as a percentage of the total population. (C) Transgene expression (GFP) in 26-day differentiated ARX ko hESCs based on immunostatining of GFP (green) and chromogranin A (red). Nuclei are counterstained with DAPI (white). Inset in C is a ~3x enlarged portion from the region indicated by the star. Scale bar is 200 μm for panel A and 100 μm for C.
Fig 12.
Fate Specification Factors in Ad ARX Treated ARX ko hESCs.
(A) RT-qPCR of day 26 whole population samples examining the expression of three hormones and key transcription factors in ARX ko clone 1 cells treated with Ad GFP (green bars) or Ad ARX (blue bars) at an MOI of 2 on day 13 (filled bars) or 19 (open bars). * indicates p < 0.05 Ad GFP vs Ad ARX from a given transduction (Tdxn) day. N = 3 per group. (B) Immunostaining of PAX6 (green), somatostatin (red), and C-peptide (blue) in ARX ko cells differentiated to day 26 after transduction at day 13 or 19 with Ad GFP or Ad ARX. Inset is a ~3x enlarged portion from the region indicated by the star. Scale bar is 100 μm for all panels.
Fig 13.
The Role of ARX in Pancreatic Endocrine Specificatin of hESCs.
NGN3 induction in hESC derived pancreatic progenitors (PDX+ NKX6.1+) initiates the endocrine lineage. (A) In wild type hESCs, pancreatic endocrine progenitors express both PAX4 and ARX that are mutually corepressive. High expression of ARX specifies and maintains glucagon positive cells while elevated PAX4 levels drives the insulin and somatostatin lineages in normal abundance. (B) In the absence of functional ARX (ARX ko), repression of PAX4 is disrupted leading to a bias toward the insulin/somatostatin lineage. Elevated expression of HHEX as well as altered expression of other transcription factors, leads to large numbers of somatostatin cells at the expense of both insulin and glucagon lineages. (C) Re-expression of ARX in ARX ko pancreatic progenitors temporarily rescues ARX expression resulting in recovery of PAX6 expression (and other factors) which restores the specification of insulin positive cells. Glucagon positive cells are not rescued due to poorly sustained ARX expression in subsequent endocrine cells.