Skeletogenic phenotype of human Marfan embryonic stem cells faithfully phenocopied by patient-specific induced-pluripotent stem cells Natalina Quartoa,b,1, Brian Leonardc,d,2,3, Shuli Lia,2, Melanie Marchandc, Erica Andersonc, Barry Behrd, Uta Franckee, Renee Reijo-Perac,d, Eric Chiaoc,d,1,3, and Michael T. Longakera,c,1 aDepartment of Surgery, Hagey Laboratory for Pediatric Regenerative Medicine, dDepartment of Obstetrics and Department of Gynecology, and eDepartment of Genetics and Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305; bDipartimento di Scienze Chirurgiche, Anestesiologiche-Rianimatorie e dell’Emergenza “Giuseppe Zannini,” Universita’ degli Studi di Napoli Federico II, 80131 Naples, Italy; and cInstitute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305 Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved November 17, 2011 (received for review August 18, 2011) Marfan syndrome (MFS) is a heritable connective tissue disorder and Loeys-Dietz syndrome, and myosin heavy chain (MYH)11 caused by mutations in the gene coding for FIBRILLIN-1 (FBN1), an and actin/alpha2 smooth muscle/aorta (ACTA2) in familial tho- extracellular matrix protein. MFS is inherited as an autosomal racic aortianeurysms and dissections (21, 22). dominant trait and displays major manifestations in the ocular, To date, by necessity most knowledge of MFS has been skeletal, and cardiovascular systems. Here we report molecular obtained by extrapolation of studies in the mouse Fbn1 null/ and phenotypic profiles of skeletogenesis in tissues differentiated transgenic models (2, 23–27). However, with the derivation of from human embryonic stem cells and induced pluripotent stem human embryonic stem cells carrying a common FBN1 mutation, cells that carry a heritable mutation in FBN1. We demonstrate that, as well as human induced pluripotent-stem (iPS) cells from MFS as a biological consequence of the activation of TGF-β signaling, patients, we now have a unique opportunity to examine key osteogenic differentiation of embryonic stem cells with a FBN1 features of this syndrome on a human genome background. mutation is inhibited; osteogenesis is rescued by inhibition of Moreover, we can address whether phenotypes observed fol- BIOLOGY TGF-β signaling. In contrast, chondrogenesis is not perturbated lowing reprogramming of somatic cells to pluripotency are le- DEVELOPMENTAL and occurs in a TGF-β cell-autonomous fashion. Importantly, skel- gitimately reflected in pluripotent stem cells directly obtained etal phenotypes observed in human embryonic stem cells carrying from human MFS embryos. Below, we describe our studies that the monogenic FBN1 mutation (MFS cells) are faithfully phenocop- used human MFS embryonic stem cells and iPS cells to unveil ied by cells differentiated from induced pluripotent-stem cells de- a unique skeletogenic phenotype featuring impaired osteogenic rived independently from MFS patient fibroblasts. Results indicate differentiation and the ability to undergo chondrogenesis in the a unique phenotype uncovered by examination of mutant plurip- absence of exogenous TGF-β. Importantly, our study demon- otent stem cells and further demonstrate the faithful alignment of strates that phenotypes observed in MFS embryonic stem cells phenotypes in differentiated cells obtained from both human em- are phenocopied reliably in MFS reprogrammed iPS cells. bryonic stem cells and induced pluripotent-stem cells, providing complementary and powerful tools to gain further insights into Results human molecular pathogenesis, especially of MFS. Derivation of Human Marfan Embryonic Stem Cells and iPS Cells from an MFS-Specific Patient. In the routine clinical practice of in vitro arfan syndrome (MFS) is a heritable dominant disorder of fertilization, embryos are sometimes tested via preimplantation Mfibrous connective tissue, caused by mutations in the gene genetic diagnosis for common disorders; genetic testing occurs at encoding fibrillin-1 on chromosome 15 (1, 2). MFS shows strik- the eight-cell stage before blastocyst formation. We obtained ing pleiotropism and clinical variability (3, 4). Cardinal patho- a human blastocyst carrying a FBN1 mutation, following pre- logical features occur in three systems—skeletal, ocular, and implantation genetic diagnosis, and derived a human embryonic cardiovascular (4–8)—and share overlapping features with con- stem cell line (referred to as MFS cells) via standard derivation genital contractural arachnodactyly, which is caused by a muta- conditions on mouse embryonic fibroblast feeder cells. The tion in the FIBRILLIN-2 (FBN2) gene (9). FBN1 mutations are embryos and the MFS cells were both shown to carry a frame- detected in the majority of the patients fulfilling the clinical shift mutation (c.1747delC) in the 5′ region (exon 14) of the criteria, but also in incomplete phenotypes, referred to as type 1 FBN1 gene that results in a stop codon (in exon 15) at the amino fibrillinopathies (10). FBN1 is an extracellular matrix glycopro- acid position 624 (Fig. 1A). tein containing 43 calcium-binding EGF-like domains and 78 Control WT human embryonic stem cells (referred to as WT cysteine-containing TB motifs (11, 12). Mutations in FBN1 are cells) were derived from a blastocyst that does not carry FBN1 the etiology of many phenotypes observed in MFS. The most mutation donated for research. common mutations found in FBN1 in MFS are missense muta- tions (56%), mainly substituting or creating a cysteine in a cal- cium-binding EGF-like domain. Other mutations are frame- Author contributions: N.Q., M.M., and E.C. designed research; N.Q., B.L., S.L., and E.A. shift, splice, and nonsense mutations (13). There are only a few performed research; N.Q., B.L., B.B., U.F., and E.C. contributed new reagents/analytic reports of patients with marfainoid features and a molecularly tools; N.Q., B.L., S.L., U.F., R.R.-P., and M.T.L. analyzed data; and N.Q., R.R.-P., and M.T.L. proven complete deletion of a FNB1 allele (14–16). Most of wrote the paper. FBN1 deletions are associated with a severe or classical Marfan The authors declare no conflict of interest. phenotype (17–20). Although the molecular pathogenesis of This article is a PNAS Direct Submission. MFS was initially attributed to a structural weakness of the 1To whom correspondence may be addressed. E-mail: [email protected], eric.chiao@roche. fibrillin-rich microfibrils within the ECM, more recent results com, or [email protected]. have documented that many of the pathogenic abnormalities in 2B.L. and S.L. contributed equally to this work. MFS are the result of alterations in TGF-β signaling (18, 19). 3Present address: Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110. Mutations in other genes have been reported to cause MFS-re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lated disorders, such as TGF-β receptor-I and -II in MFS type 2 1073/pnas.1113442109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1113442109 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Fig. 1. Characterization of MFS and MFSiPS cells. (A) DNA sequencing analysis of MFS cells showing a mutation in FBN1 exon 14. (B) Cell morphology of representative MFS and iPS clones by phase con- trast (bright field), alkaline phosphatase (AP) staining, and immunofluorescence staining for pluripotent markers: NANOG, OCT-4, TRA-1–60, TRA-1–81, and SSEA-4. (Insets) Nuclear counter- staining performed with DAPI. (Scale bars, 100 μm.) (C) qPCR for the expression of exogenous and en- dogenous SOX2, KLF4, OCT4, and C-MYC genes. (D) Differentiation of MFS and MFSiPS cells, teratomas containing cells from three germ layers (endoderm, mesoderm, and ectoderm) developed from MFSiPS and MFS cells injected into the dorsal flank of nude mice. Endoderm (gut epithelium), mesoderm, (car- tilage), and ectoderm (neuroectoderm) are in- dicated by white asterisks. (Scale bars, 100–250 μm.) (E) Spectral karyotyping analysis of MFSiPS cells. TransFib, parental transduced fibroblasts. Human iPS cells (MFSiPS cells) were generated from fibroblasts Enhanced Activation of TGF-β Signaling in MFS Cells Inhibits obtained from a patient with MFS that harbored a FBN1 splice-site Osteogenic Differentiation. A potential explanation for the im- mutation (c.3839–1g> t) that causes skipping of exon 31, (proband pairment of osteogenesis might be enhanced activation of TGF-β FB1121), leading to a severe neonatal clinical phenotype (28). A and downstream signaling. Therefore, we investigated the extent second MFSiPS cell line was generated from fibroblasts obtained of SMAD2 phosphorylation in MFS cells and corresponding WT from a different MFS patient (proband FB1592) harboring a FBN1 controls. Immunoblotting and immunofluorescence analyses frame-shift mutation (c.1642del3ins20bp) previously characterized revealed a greater endogenous phosphorylated SMAD2 in MFS (29) (Fig. S4). The iPS cell derivation was as previously described (30). cells (Fig. 3 A and B), which could be blocked by treatment with MFS fibroblasts and control WT fibroblasts from healthy male pan–TGF-β–neutralizing antibody (Fig. 3C). Moreover, a higher (WTiPS) were transduced with the retroviral vectors harboring than normal activation of TGF-β signaling in the MFS cells was the human reprogramming genes SOX2, OCT4, KLF4, and further indicated by the up-regulation of TGF-β1–induced ECM c-MYC. Two clones, collectively referred to as MFSiPS cells, were markers PAI-1 and collagen (COL1A1)(35–38) in MFS cells then selected for detailed analysis, with both giving similar results. compared with WT controls (Fig. 3D). These differences were MFSiPS cells exhibited morphology similar to MFS embryonic abrogated by treatment with either SB431542, a selective in- stem cells, were alkaline phosphatase-positive (ALPL), and were hibitor of endogenous TGF-β signaling with no effect on bone immunoreactive for NANOG, OCT4, TRA1.60, TRA1.81, and morphogenetic protein (BMP) signaling (39), or a pan–TGF-β– SSEA4 (Fig. 1B). Expression levels of endogenous and exogenous neutralizing antibody (Fig. 3 D and E).