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 4

Tissue expansion is restored for hypoplastic thymuses by replacement of Tbx1neo2/neo2-derived mesenchymal cells with normal control cells.

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Tissue expansion is restored for hypoplastic thymuses by replacement of ...
(A) Depiction of RTOC using flow-sorted cells. Single-cell suspensions from E13–E13.5 fetal thymic lobes were prepared, and mesenchymal cells (Pdgfrα+), TECs (EpCam+) and the remaining unstained cells (Pdgfrα–Epcam–, which includes ETPs, other hematopoietic cells, and endothelial cells) were sorted by flow cytometry. These 3 subgroups were reaggregated at cell ratios established with control fetal thymuses and placed onto membranes and cultured. A minimum of 30,000 cells/aggregate was needed to sustain RTOC growth with normal cells (Supplemental Figure 5). The aggregates appear as a small dot in the yellow circled area. Endothelial cell replacements required sorting of CD31+ cells from the remaining cell subsets prior to reaggregate culturing. (B) Live cell imaging was used to visualize RTOCs after 10 days of culturing. The control corresponds to the 3 subgroups of cells from Tbx1+/+;+/neo2 thymic lobes. In the first column, control thymuses were a combination of cells from either Tbx1+/+ and/or Tbx1+/neo2 embryos. In the second column, 22q11.2DS hypoplastic thymuses were from Tbx1neo2/neo2 embryos. In the third column, normal mesenchymal cells were used as substitutes for those in the 22q11.2DS tissues (Sub Tbx1neo2/neo2 Mes). In columns 4 and 5, normal TECs or endothelial cells were used as substitutes for Tbx1neo2/neo2 TECs (Sub Tbx1neo2/neo2 TECs) or endothelial cells (Sub Tbx1neo2/neo2 Endo), respectively. Scale bars: 1 mm. (C) Cell viability (top row) and thymopoiesis (DN to DP and then SP progression, bottom row) are shown for the cells after 10 days of RTOC. (D) Cumulative cell numbers are shown for a representative RTOC experiment. (E–G) The fold increase in cell numbers following 10 days of RTOC along with cell viability and the percentage of DP cells developing over this period. n = 37, 28, 13, 8, and 5 experiments per group, respectively, for E–G. Statistical analyses done with 1-way ANOVA (Brown-Forsythe and Welch tests).