Mesenchymal cell replacement corrects thymic hypoplasia in murine models of 22q11.2 deletion syndrome
Pratibha Bhalla, … , Antonio Baldini, Nicolai S.C. van Oers
Pratibha Bhalla, … , Antonio Baldini, Nicolai S.C. van Oers
Published September 22, 2022
Citation Information: J Clin Invest. 2022;132(22):e160101. https://doi.org/10.1172/JCI160101.
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Research Article Genetics Immunology

Mesenchymal cell replacement corrects thymic hypoplasia in murine models of 22q11.2 deletion syndrome

Abstract

22q11.2 deletion syndrome (22q11.2DS) is the most common human chromosomal microdeletion, causing developmentally linked congenital malformations, thymic hypoplasia, hypoparathyroidism, and/or cardiac defects. Thymic hypoplasia leads to T cell lymphopenia, which most often results in mild SCID. Despite decades of research, the molecular underpinnings leading to thymic hypoplasia in 22q11.2DS remain unknown. Comparison of embryonic thymuses from mouse models of 22q11.2DS (Tbx1neo2/neo2) revealed proportions of mesenchymal, epithelial, and hematopoietic cell types similar to those of control thymuses. Yet, the small thymuses were growth restricted in fetal organ cultures. Replacement of Tbx1neo2/neo2 thymic mesenchymal cells with normal ones restored tissue growth. Comparative single-cell RNA-Seq of embryonic thymuses uncovered 17 distinct cell subsets, with transcriptome differences predominant in the 5 mesenchymal subsets from the Tbx1neo2/neo2 cell line. The transcripts affected included those for extracellular matrix proteins, consistent with the increased collagen deposition we observed in the small thymuses. Attenuating collagen cross-links with minoxidil restored thymic tissue expansion for hypoplastic lobes. In colony-forming assays, the Tbx1neo2/neo2-derived mesenchymal cells had reduced expansion potential, in contrast to the normal growth of thymic epithelial cells. These findings suggest that mesenchymal cells were causal to the small embryonic thymuses in the 22q11.2DS mouse models, which was correctable by substitution with normal mesenchyme.

Authors

Pratibha Bhalla, Qiumei Du, Ashwani Kumar, Chao Xing, Angela Moses, Igor Dozmorov, Christian A. Wysocki, Ondine B. Cleaver, Timothy J. Pirolli, Mary Louise Markert, Maria Teresa de la Morena, Antonio Baldini, Nicolai S.C. van Oers

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Figure 7

The presence of minoxidil in RTOC cultures restores tissue growth for hypoplastic thymuses.

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The presence of minoxidil in RTOC cultures restores tissue growth for hy...
RTOC assays were performed using cell suspensions generated from E13–E13.5 fetal thymic lobes. Cells from either normal or Tbx1neo2/neo2 thymuses were reaggregated with equivalent starting clusters of approximately 30,000 cells/group. Cultures were maintained in media alone or supplemented with 3 μM minoxidil. (A) Live cell imaging revealed cell expansion after 10 days of culturing. Scale bars: 1 mm. (B) Thymopoiesis was compared using antibodies specific for CD4 and CD8. (C) Cell numbers, cell viability, and the percentage of DP cells are shown. Note that the number of cells in Tbx1neo2/neo2 thymuses was severely limited, as established in Figure 4, B, D, and E. n = 10, 10, 3, and 3 for the indicated groups, from left to right, in each panel. Statistical significance was determined by 1-way ANOVA. (D) Control FTOCs were grown in the absence or presence of minoxidil. On day 3 and day 4 after culturing, the cells were processed for qRT-PCR using probes detecting 2 Plod and 2 Col1a genes, along with GAPDH for normalization. Day 3, n = 5; day 4, n = 4. (E) Mesenchymal cells and TECs from E13–E13.5 embryonic thymuses from Tbx1+/+ or Tbx1neo2/neo2 embryos were flow sorted. Mesenchymal sorted cells were grown in MesenCult differentiation media. After 15 days of culturing, the cells were fixed, and live cell images were obtained. The well was from a 6-well tissue culture plate. Bottom image: A representative cluster of cells was imaged following crystal violet staining. Scale bars: 1 mm. (F) Total number of pixels in the images in E in conjunction with 5 additional independent experiments were calculated. These values were divided by the total number of mesenchymal cells seeded in each experiment and plotted as pixel area divided by the total cell number. This was compared with TECs grown in EpiCult. These cells were enumerated by cell counting, as shown. Statistical significance was determined by Student’s t test.