A NOTCH2 pathogenic variant and HES1 regulate osteoclastogenesis in induced pluripotent stem cells

Area of Science:

• Genetics and Developmental Biology
• Cell Biology
• Skeletal Biology

Background:

• Hajdu Cheney Syndrome (HCS) is a rare monogenic disorder linked to NOTCH2 pathogenic variants.
• Clinical manifestations include neurological, craniofacial, and skeletal abnormalities, with mouse models showing osteopenia.
• Understanding the cellular consequences of HCS-associated NOTCH2 variants in human cells is crucial.

Purpose of the Study:

• To investigate the in vitro cellular and molecular effects of a specific NOTCH2 pathogenic variant (NOTCH2^6949C>T) found in HCS.
• To determine the role of HES1, a Notch target gene, in osteoblastogenesis and osteoclastogenesis in the context of HCS.
• To utilize human induced pluripotent stem cells (iPSCs) to model HCS cellular phenotypes.

Main Methods:

• Generation of human iPSCs harboring a NOTCH2^6949C>T mutation or lacking HES1 alleles.
• Directed differentiation of iPSCs towards neural crest, mesenchymal, osteogenic, embryoid body, hematopoietic, and osteoclast lineages.
• Confirmation of cell phenotypes using cell surface markers and gene expression signatures.
• Analysis of Notch target gene expression, osteogenesis, and osteoclastogenesis markers.

Main Results:

• NOTCH2^6949C>T iPSCs exhibited a NOTCH2 gain-of-function phenotype with enhanced Notch target gene expression.
• A modest increase in osteogenesis was observed in NOTCH2^6949C>T cells, indicated by mineralized nodule formation and specific gene expression.
• NOTCH2^6949C>T cells showed enhanced osteoclastogenesis, with increased osteoclast numbers and transient upregulation of key osteoclast markers.
• HES1 deletion minimally impacted osteoblastogenesis but significantly impaired osteoclast differentiation.

Conclusions:

• A pathogenic NOTCH2 variant associated with HCS leads to modest increases in both osteoblastogenesis and osteoclastogenesis in human iPS cells.
• HES1 plays a critical role in osteoclast differentiation in human iPS cells.
• These findings provide in vitro cellular insights into the skeletal abnormalities observed in Hajdu Cheney Syndrome.