Original Research
Dynamic Formation of Microvillus Inclusions During Enterocyte Differentiation in Munc18-2–Deficient Intestinal Organoids

https://doi.org/10.1016/j.jcmgh.2018.08.001Get rights and content
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Background & Aims

Microvillus inclusion disease (MVID) is a congenital intestinal malabsorption disorder caused by defective apical vesicular transport. Existing cellular models do not fully recapitulate this heterogeneous pathology. The aim of this study was to characterize 3-dimensional intestinal organoids that continuously generate polarized absorptive cells as an accessible and relevant model to investigate MVID.

Methods

Intestinal organoids from Munc18-2/Stxbp2-null mice that are deficient for apical vesicular transport were subjected to enterocyte-specific differentiation protocols. Lentiviral rescue experiments were performed using human MUNC18-2 variants. Apical trafficking and microvillus formation were characterized by confocal and transmission electron microscopy. Spinning disc time-lapse microscopy was used to document the lifecycle of microvillus inclusions.

Results

Loss of Munc18-2/Stxbp2 recapitulated the pathologic features observed in patients with MUNC18-2 deficiency. The defects were fully restored by transgenic wild-type human MUNC18-2 protein, but not the patient variant (P477L). Importantly, we discovered that the MVID phenotype was correlated with the degree of enterocyte differentiation: secretory vesicles accumulated already in crypt progenitors, while differentiated enterocytes showed an apical tubulovesicular network and enlarged lysosomes. Upon prolonged enterocyte differentiation, cytoplasmic F-actin–positive foci were observed that further progressed into classic microvillus inclusions. Time-lapse microscopy showed their dynamic formation by intracellular maturation or invagination of the apical or basolateral plasma membrane.

Conclusions

We show that prolonged enterocyte-specific differentiation is required to recapitulate the entire spectrum of MVID. Primary organoids can provide a powerful model for this heterogeneous pathology. Formation of microvillus inclusions from multiple membrane sources showed an unexpected dynamic of the enterocyte brush border.

Keywords

Microvillus Atrophy
Disease Modeling
Brush Border Formation
Apical Vesicular Transport

Abbreviations used in this paper

3D
3-dimensional
DAPI
4′,6-diamidino-2-phenylindole
EGFP
enhanced green fluorescent protein
FHL5
familial hemophagocytic lymphohistiocytosis type 5
IWP-2
inhibitor of WNT production-2
KO
knock-out
MVIs
microvillus inclusions
MVID
microvillus inclusion disease
PBS
phosphate-buffered saline
STXBP2
syntaxin binding protein 2
Stx3
syntaxin 3
TEM
transmission electron microscopy
VPA
valproic acid
WT
wild-type

Cited by (0)

Author contributions Ophélie Nicolle and Sophia Maschalidi contributed equally to this work; Mohammed H. Mosa, Ophélie Nicolle, Sophia Maschalidi, Aurelien Bidaud-Meynard, and Henner F. Farin performed the experiments; Fernando E. Sepulveda, Constantin Menche, and Birgitta E. Michels provided technical support; Mohammed H. Mosa, Ophélie Nicolle, Aurelien Bidaud-Meynard, Grégoire Michaux, and Henner F. Farin analyzed the data; Grégoire Michaux, Geneviève de Saint Basile, and Henner F. Farin conceived the study, supervised the experiments, and obtained funding; and Geneviève de Saint Basile and Henner F. Farin drafted the manuscript. All authors discussed the results and commented on the manuscript.

Conflicts of interest The authors disclose no conflicts.

Funding Supported by the French National Institute of Health and Medical Research (INSERM), the Agence Nationale de la Recherche Hemophagocytic lymphohistiocytosis-Cytotox/ANR-12-BSV1-0020-01 (G.d.S.B.), the LOEWE Center for Cell and Gene Therapy Frankfurt, Hessisches Ministerium für Wissenschaft und Kunst (H.F.F.), and by the Centre national de la recherche scientifique and the Ligue Régionale Contre le Cancer (22/29/35/72) (G.M.).

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Authors share co-senior authorship.