hiPSC Modeling of Lineage-Specific Smooth Muscle Cell Defects Caused by
TGFBR1

A230T

Variant, and Its Therapeutic Implications for Loeys-Dietz Syndrome
Authors: Zhou Dong , Feng Hao , Yang Ying , Huang Tingting , Qiu Ping , Zhang Chengxin , Olsen Timothy R. , Zhang Jifeng , Chen Y. Eugene , Mizrak Dogukan , Yang Bo 
Published: August 2021

Background:
Loeys-Dietz syndrome (LDS) is an inherited disorder predisposing individuals to thoracic aortic aneurysm and dissection. Currently, there are no medical treatments except surgical resection. Although the genetic basis of LDS is well-understood, molecular mechanisms underlying the disease remain elusive, impeding the development of a therapeutic strategy. In addition, aortic smooth muscle cells (SMCs) have heterogenous embryonic origins, depending on their spatial location, and lineage-specific effects of pathogenic variants on SMC function, likely causing regionally constrained LDS manifestations, have been unexplored.

Methods:

We identified an LDS family with a dominant pathogenic variant in the
TGFBR1
gene (
TGFBR1

A230T

) causing aortic root aneurysm and dissection. To accurately model the molecular defects caused by this mutation, we used human induced pluripotent stem cells from a subject with normal aorta to generate human induced pluripotent stem cells carrying
TGFBR1

A230T

, and corrected the mutation in patient-derived human induced pluripotent stem cells using CRISPR-Cas9 gene editing. After their lineage-specific SMC differentiation through cardiovascular progenitor cell (CPC) and neural crest stem cell lineages, we used conventional molecular techniques and single-cell RNA sequencing to characterize the molecular defects. The resulting data led to subsequent molecular and functional rescue experiments using activin A and rapamycin.

Results:

Our results indicate the
TGFBR1

A230T

mutation impairs contractile transcript and protein levels, and function in CPC-SMC, but not in neural crest stem cell–SMC. Single-cell RNA sequencing results implicate defective differentiation even in
TGFBR1

A230T/+

CPC-SMC including disruption of SMC contraction and extracellular matrix formation. Comparison of patient-derived and mutation-corrected cells supported the contractile phenotype observed in the mutant CPC-SMC.
TGFBR1

A230T

selectively disrupted SMAD3 (SMAD family member 3) and AKT (AKT serine/threonine kinase) activation in CPC-SMC, and led to increased cell proliferation. Consistently, single-cell RNA sequencing revealed molecular similarities between a loss-of-function SMAD3 mutation (
SMAD3

c.652delA/+

) and
TGFBR1

A230T/+

. Last, combination treatment with activin A and rapamycin during or after SMC differentiation significantly improved the mutant CPC-SMC contractile gene expression and function, and rescued the mechanical properties of mutant CPC-SMC tissue constructs.

Conclusions:

This study reveals that a pathogenic
TGFBR1
variant causes lineage-specific SMC defects informing the etiology of LDS-associated aortic root aneurysm. As a potential pharmacological strategy, our results highlight a combination treatment with activin A and rapamycin that can rescue the SMC defects caused by the variant.