Myh11 Lineage Corneal Endothelial Cells and ASCs Populate Corneal Endothelium

Abstract

PURPOSE. To establish Myh11 as a marker of a subset of corneal endothelial cells (CECs), and to demonstrate the feasibility of restoring the corneal endothelium with Myh11-lineage (Myh11-Lin[þ]) adipose-derived stromal cells (ASCs). METHODS. Intraperitoneal administration of tamoxifen and (Z)-4-hydroxytamoxifen eyedrops were used to trace the lineage of Myh11-expressing cells with the Myh11-Cre-ER T2-flox-tdTomato mouse model. Immunostaining and Western blot characterized marker expression and spatial distribution of Myh11-Lin(þ) cells in the cornea, and administration of 5-ethynyl-2 0-deoxyuridine labeled proliferating cells. ASCs were isolated from epididymal adipose Myh11þ mural cells and treated with cornea differentiation media to evaluate corneal endothelial differentiation potential. Differentiated ASCs were injected into the anterior chamber to test for incorporation into corneal endothelium following scratch injury. RESULTS. A subset of CECs express Myh11, a marker previously thought restricted to only mural cells. Myh11-Lin(þ) CECs marked a stable subpopulation of cells in the cornea endothelium. Myh11-Lin(þ) ASCs undergo CEC differentiation in vitro and incorporate into injured corneal endothelium. CONCLUSIONS. Dystrophy and dysfunction of the corneal endothelium accounts for almost half of all corneal transplants, the maintenance of the cornea endothelium is poorly understood, and there are a lack of mouse models to study specific CEC populations. We establish a mouse model that can trace the cell fate of a subpopulation of CECs based on Myh11 expression. A subset of ASCs that share this Myh11 transcriptional lineage are capable of differentiating into CECs that can incorporate into injured corneal endothelium, revealing a potential cell source for creating engineered transplant material.

Myh11+ microvascular mural cells and derived mesenchymal stem cells promote retinal fibrosis

“…This severe injury also creates a highly inflammatory environment as demonstrated by substantial upregulation of cytokines, chemokines, and growth factors in the affected retinal tissue. Surprisingly, Myh11-derived myofibroblasts maintain expression of Myh11, which challenges the claimed specificity of Myh11 to vSMCs and PCs 41 , but adds to our recent observations that Myh11 is also expressed by corneal endothelial cells 42 . Whether myofibroblast Myh11 remains of functional importance will require determining Myh11 protein turnover and synthesis in the Myh11-derived myofibroblasts, and assessing its role in myofibroblast contractility.…”

“…As previously mentioned, TGFβ1 may assume a larger role in initiating off-vessel transition of mural cells, while subsequent fibrotic transformation of off-vessel mural cells may be initiated through additional fibrotic regulatory pathways, including YAP/TAZ, BMP, MRTF, and WNT 67 . In fact, we recently reported dual SMAD inhibition in Myh11-derived MSCs can promote a corneal endothelial cell differentiation in vitro 42 . Thus, future studies will require a more systematic approach to regulate the molecular milieu and multiple potential fibrotic signaling pathways of Myh11-derived MSCs.…”

A New Autosomal Myh11-CreER T2 Smooth Muscle Cell Lineage Tracing and Gene Knockout Mouse Model—Brief Report

“…To validate and compare the activity of Myh11-CreER T2 -RAD with Myh11-CreER T2 -Off , mice were crossed to either tdTom or eYFP reporter mice as shown in the schematic in Figure 1A and compared to Myh11-CreER T2 -Off mice crossed to the same reporter mice. 21,31 Following tamoxifen administration, reporter gene recombination and subsequent fluorescent protein expression in the brachiocephalic artery (BCA) was assessed by confocal microscopy or in aortic cells by flow cytometry as shown in the schematic in Figure 1B. Tamoxifen diet was used in lieu of intraperitoneal injected tamoxifen based on our previous finding that intraperitoneal injection of tamoxifen in peanut oil caused autofluorescence of adipose tissue macrophages causing false identification as eYFP + cells.…”

“…For the generation of SMC lineage tracing mice, Myh11-CreER T2 29,30 The Myh11-CreER T2 -Off/ROSA26-STOP flox -eYFP and Myh11-CreER T2 -Off/ROSA26-STOP flox -tdTom using the Offermanns Myh11-CreER T2 (The Jackson Laboratory Strain 019079) have been previously described. 21,31 All experimental mice using the Myh11-CreER T2 -RAD lines were Cre +/to avoid any unanticipated effects from transgene insertion. Male Cre + and female Creexperimental mice from the Myh11-CreER T2 -Off lines were used due to the transgene location on the Y chromosome.…”

“Cre”ating New Tools for Smooth Muscle Analysis

“…BAC indicates bacterial artificial chromosome; Chr, chromosome; and GI, gastrointestinal. Figure created with BioRender.com 23,24.…”