PIPO
Conditions
Keywords
PIPO, CIPO, ACTG2, pseudo-obstruction, organoid, iPS, Pediatric Intestinal Pseudo-Obstruction, Chronic Intestinal Pseudo-obstruction
Brief summary
The primary objective of this study is to describe the transcriptional impact of R178, R257, R40 or A136 variants of the ACTG2 gene on iPS differentiation mechanisms up to organoids derived from PIPO patient samples versus those derived from control / WT patients (generation of IPS from cultured cell lines), at different stages of their experimental ex vivo development.
Detailed description
Recruited patients will be sampled during a consultation: a blood sample and a biopsy will be taken directly from the patient. Once these samples have been taken, they will be cultured to be reprogrammed into iPS cells, then grown and differentiated into intestinal organoids. Various experiments will be carried out (as described in the outcomes) to identify at molecular, cellular and tissue level the mechanisms altered in patients with an R178, R257, R40 or A136 variant, assessing their consequence(s) on the development and functionality of the digestive mesenchyme.
Interventions
PROCEDUREBiopsy
A skin biopsy and a blood sample will be taken to culture the iPS cells and intestinal organoids.
Sponsors
University Hospital, Grenoble
Study design
Allocation
NON_RANDOMIZED
Intervention model
FACTORIAL
Primary purpose
BASIC_SCIENCE
Masking
NONE
Intervention model description
Experimental, descriptive, prospective, non-randomized, controlled, comparative, monocentric in recruitment but multicentric study in procedures and analyses.
Eligibility
Sex/Gender
ALL
Age
4 Years to No maximum
Healthy volunteers
No
Inclusion criteria
PIPO Population: 1. Minor or adult patient ≥ 4 years of age 2. Patient with PIPO before age 18 3. Male or female 4. Patient with PIPO meeting at least 2 of the ESPGHAN criteria (Thapar et al 2018) and carrying the R178, R257, R40 or A136 mutation of the ACTG2 gene. 5. Patient whose assent has been obtained and whose legal guardians have given their written informed consent 6. Patient affiliated to the French Social Security system or benefiting from an equivalent plan WT population: \- iPS cell lines MS573 or WT8288 or 202CT or SD378M, from the Nantes University Hospital biological collection and generated from samples from control patients without POIC who have consented to donate their samples.
Exclusion criteria
PIPO population : 1. Patients with a history of radiotherapy treatment 2. Patient with lymphocyte lineage damage
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Description of the transcriptional impact of R178, R257, R40 or A136 variants of the ACTG2 gene | At different stages of their experimental ex vivo development (mesenchymal progenitors, determined smooth muscle cells, differentiated smooth muscle cells and 3D organization of smooth muscle) through study completion, an average of 5 years | Difference in transcript expression (%) in RNASeq transcriptomic analysis between samples carrying the R178, R257, R40 or A136 variants of the ACTG2 gene on the differentiation mechanisms of iPS up to organoids derived from PIPO patient samples versus those derived from control patients |
Secondary
| Measure | Time frame | Description |
|---|---|---|
| Evaluate the impact of R178, R257, R40 or A136 variants of the ACTG2 gene on gastrointestinal contractile function between mutant versus WT organoids. | At different stages of their experimental ex vivo development (mesenchymal progenitors, determined smooth muscle cells, differentiated smooth muscle cells and 3D organization of smooth muscle) an average of 5 years | Difference (%) in isometric contraction force between mutant versus WT organoids by electrical stimulation and specific agonist. |
| Evaluate the immunofluorescence labeling differential between mutant and WT cells from IPS and organoids | At different stages of their experimental ex vivo development (mesenchymal progenitors, determined smooth muscle cells, differentiated smooth muscle cells and 3D organization of smooth muscle) an average of 5 years | Difference in fluorescence (%) between mutant and WT cells as cell stages from iPS to organoids |
| Evaluate the impact of R178, R257, R40 or A136 mutations of the ACTG2 gene on the actin network of fibroblasts derived from skin samples of diseased patients versus those of WTpatients. | At different stages of their experimental ex vivo development (mesenchymal progenitors, determined smooth muscle cells, differentiated smooth muscle cells and 3D organization of smooth muscle) an average of 5 years | Difference in actin network labeling by immunofluorescence (%) between skin fibroblasts from PIPO and WT patients. |
| Evaluate the effect of reversion of the R178, R257, R40 or A136 mutation versus WT on functionality and contractility during differentiation of cells into organoids. | At different stages of their experimental ex vivo development (mesenchymal progenitors, determined smooth muscle cells, differentiated smooth muscle cells and 3D organization of smooth muscle) an average of 5 years | Difference in isometric contraction strength (%) between mutant versus WT versus revertant organoids via specific agonist and electrical stimulation. |
| Assessing the potential of a chemical library to correct the phenotype | At different stages of their experimental ex vivo development (mesenchymal progenitors, determined smooth muscle cells, differentiated smooth muscle cells and 3D organization of smooth muscle) an average of 5 years | Identification of molecules inducing a significant difference in isometric contraction force (%) between treated mutant organoids, untreated mutant organoids and treated and untreated WT |
Countries
France
Contacts
CONTACTJohn Rendu, Dr
CONTACTMandy Leger
Outcome results
None listed