Three-dimensional heart valve-like tissues generated from human pluripotent stem cells may show enhanced maturation, reproduce features of inflammatory heart valve disease, and partially recapitulate molecular features of native human valves.
Researchers developed a directed differentiation protocol and tested it across three independent human pluripotent stem cell lines: PB522.3 induced pluripotent stem cells, PB10.5 induced pluripotent stem cells, and HES3 embryonic stem cells. After 15 days, 85% of the cells expressed the valve insterstitial cell lineage marker SOX9. The researchers then incorporated the cells into valve-engineered tissues (VET) in two formats: microVETs measuring approximately 1 mm and containing 60,000 cells each for disease modeling and drug screening as well as macroVETs measuring 3 to 5 cm and containing 20 to 50 million cells for biomechanical assessment and implantation studies.
Proteomic analysis comparing two-dimensional (2D) stem cell–derived valve cells with macroVETs cultured for 12 and 30 days showed that three-dimensional (3D) culture promoted a more mature valve-like molecular profile. Compared with 2D cells, about 12% of proteins were upregulated and 24% were downregulated in day-30 macroVETs. Extracellular matrix proteins COL1A1 and DCN and proteins associated with extracellular matrix remodeling were enriched. Two-dimensional cells also clustered separately from native and engineered 3D tissues in the proteomic analysis.
The researchers integrated three human valve proteomic datasets to generate a native-valve protein signature. They found that 99.7% of native proteins were expressed in macroVETs. Maturation-associated proteins such as TGFBI, LUM, and CILP2 increased in 3D culture, and macroVETs showed reduced Ki-67 positivity, consistent with valve maturation.
Single-cell RNA sequencing showed that valve interstitial cells accounted for 74% of cells in macroVETs, compared with 75% in fetal and 81% in adult human valves. Macrophages represented 25% of macroVET cells, compared with 4% in fetal and 6% in adult valves. By contrast, valve endothelial cells accounted for less than 1% of macroVET cells, compared with 19% in fetal and 11% in adult valves.
The researchers used microVETs to model inflammatory valve disease by exposing the tissues to interleukin (IL)-6, IL-8, and tumor necrosis factor–alpha profiled in patients with acute rheumatic fever. Cytokine stimulation increased passive tissue tension, pathologic extracellular matrix markers tenascin C and COL1A1, and Von Kossa staining, indicating tissue calcification.
The proteomic analysis identified 799 differentially expressed proteins, with upregulated proteins enriched in pathways associated with acute inflammatory and bacteria-related responses. Transcriptomic analysis identified 141 upregulated genes associated with immune and inflammatory responses. When the researchers compared the model with diseased clinical valve samples, both showed increases in several disease-associated proteins and shared a disease signature involving chronic inflammatory responses, macrophage activity, and endothelial and immune-cell activation.
In a proof-of-concept assessment, glutaraldehyde-treated macroVETs had inferior biomechanical properties compared with glutaraldehyde-treated human aortic valves. Investigators also implanted treated macroVETs subcutaneously in rats. Explanted tissues showed no evidence of degradation or calcification at 7 or 30 days. The researchers hope to further develop of the tissues’ biomechanical properties for longer-term assessments of safety, durability, and functional efficacy.
The study had several limitations. VETs did not fully reproduce native valve cellular composition, with valve endothelial cells underrepresented and macrophages overrepresented. The tissues also lacked the organized endothelial layer surrounding native valves and did not express high levels of elastin. The researchers suggested that additional factors, including mechanical stress from blood flow or hormonal cues may be required to reproduce postnatal valve maturation. The inferior biomechanical properties of the macroVETs and short-duration subcutaneous rat implantation experiments further limited conclusions about their potential as functional replacement valves.
The findings supported VETs as an experimental platform for investigating human valve development, maturation, inflammatory disease pathogenesis, and potential drug discovery. Their use as replacement valves remained a longer-term possibility requiring further development.
“VETs provide a platform to understand human valve development, maturation, disease pathogenesis, and drug discovery and, in the long term, may provide a basis for the development of [human induced pluripotent stem cells]–derived replacement valves,” wrote lead study author Holly K. Voges, of Murdoch Children’s Research Institute in Australia, and colleagues.
Full disclosures of the study authors can be found in the study.
Source: Cell Stem Cell
