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. 2022 Oct;163(4):1053-1063.e7.
doi: 10.1053/j.gastro.2022.06.083. Epub 2022 Jul 6.

Using Human Induced Pluripotent Stem Cell-Derived Organoids to Identify New Pathologies in Patients With PDX1 Mutations

Mansa Krishnamurthy  1 , Daniel O Kechele  2 , Taylor Broda  2 , Xinghao Zhang  2 , Jacob R Enriquez  2 , Heather A McCauley  2 , J Guillermo Sanchez  2 , Kyle McCracken  2 , Joseph Palermo  3 , Anas Bernieh  4 , Margaret H Collins  4 , Inas H Thomas  5 , Haley C Neef  6 , Amer Heider  7 , Andrew Dauber  8 , James M Wells  9
Affiliations

Using Human Induced Pluripotent Stem Cell-Derived Organoids to Identify New Pathologies in Patients With PDX1 Mutations

Mansa Krishnamurthy et al. Gastroenterology. 2022 Oct.

Abstract

Background & aims: Two patients with homozygous mutations in PDX1 presented with pancreatic agenesis, chronic diarrhea, and poor weight gain, the causes of which were not identified through routine clinical testing. We aimed to perform a deep analysis of the stomach and intestine using organoids derived from induced pluripotent stem cells from PDX1188delC/188delC patients.

Methods: Gastric fundic, antral, and duodenal organoids were generated using induced pluripotent stem cell lines from a PDX1188delC/188delC patient and an isogenic induced pluripotent stem cell line where the PDX1 point mutation was corrected.

Results: Patient-derived PDX1188delC/188delC antral organoids exhibited an intestinal phenotype, whereas intestinal organoids underwent gastric metaplasia with significant reduction in enteroendocrine cells. This prompted a re-examination of gastric and intestinal biopsy specimens from both PDX1188delC/188delC patients, which recapitulated the organoid phenotypes. Moreover, antral biopsy specimens also showed increased parietal cells and lacked G cells, suggesting loss of antral identity. All organoid pathologies were reversed upon CRISPR-mediated correction of the mutation.

Conclusions: These patients will now be monitored for the progression of metaplasia and gastrointestinal complications that might be related to the reduced gastric and intestinal endocrine cells. This study demonstrates the utility of organoids in diagnosing uncovered pathologies.

Keywords: CRISPR/Cas9; Metaplasia; Organoids; PDX1.

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Conflict of interest statement

Conflict Of Interest Statement: The authors declare that no conflict of interest exists.

Figures

Figure 1.
Figure 1.. Loss of PDX1 results in intestinal or gastric metaplasia in the stomach and intestine, respectively.
(A-B) Immunofluorescence and (C-D) quantification of (A) control, PDX1188delC/188delC, and PDX1188delC/188delC CRISPR Corrected antral and post-transplantation intestinal organoids and (B) antral and duodenal biopsies from control, PDX1188delC/188delC patient A and PDX1188delC/188delC patient B biopsies stained with gastric epithelial marker CLDN18 and intestinal epithelial markers CDH17 and VIL1. (E) Histological staining of antral and duodenal biopsies with regions of metaplasia encircled and inflammatory foci marked with asterisk. Sydney classification of PDX1188delC/188delC patient A and B antral and intestinal biopsies compared with controls diagnosed with intestinal and gastric metaplasia respectively. All sections counterstained with nuclear DAPI. Scale bars represent 100 μm.
Figure 2.
Figure 2.. Loss of PDX1 results in loss of antral identity in the stomach.
(A) Immunofluorescence and (B) quantification of control antral, PDX1188delC/188delC A and PDX1188delC/188delC B biopsies stained with parietal cell marker ATP4B, transcription factor PDX1, pepsinogens PGA3 and PGC and hormones GAST and GHRL. (C) Quantitative pCR of control antral, PDX1188delC/188delC antrum and control fundic organoids for markers GAST, GHRL, ATP4B and PGA5. (D) Immunofluorescence and (E) quantification of PDX1188delC/188delC and PDX1188delC/188delC CRISPR corrected fundic organoids, antral organoids, antral organoids with PD03, BMP4, CHIRON with ATP4B. (F) Immunofluorescence and (G) quantification of control, PDX1188delC/188delC, PDX1188delC/188delC CRISPR corrected post-transplantation intestinal organoids and control, PDX1188delC/188delC A and PDX1188delC/188delC B patient duodenal biopsies with gastric marker ATP4B and epithelial marker CDH1. All sections counterstained with nuclear DAPI. Scale bars represent 100 μm.
Figure. 3.
Figure. 3.. PDX1 controls parietal cell number in the fundus.
(A-B) Immunofluorescence and (C-D) quantification of control PDX1188delC/188delC, and PDX1188delC/188delC CRISPR Corrected fundic organoids and control, PDX1188delC/188delC A and PDX1188delC/188delC B biopsies stained with gastric epithelial marker CLDN18 and intestinal epithelial markers CDH17 and VIL1. (E) Immunofluorescence and (F) quantification of control, PDX1188delC/188delC A and PDX1188delC/188delC B fundic biopsies with ATP4B. (G) Immunofluorescence (H) quantification and qPCR of fundic organoids without and with doxycycline treatment on d20–34 with transcription factor PDX1 and parietal cell marker ATP4B.
Figure. 4.
Figure. 4.. Loss of PDX1 decreases enteroendocrine cells in the intestine.
(A) Immunofluorescence and (B) quantification of control, PDX1188delC/188delC, and PDX1188delC/188delC CRISPR corrected post-transplantation intestinal organoids and control, PDX1188delC/188delC A and PDX1188delC/188delC B duodenal patient biopsies stained for secretory protein CHGA and enteroendocrine hormones SST, PYY, GHRL, GIP, CCK, 5-HT and GAST. All sections counterstained with nuclear DAPI. Scale bars represent 100 μm.
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