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Skeletogenic phenotype of human Marfan ESCs faithfully phenocopied by patient-specific iPSCs

From PNAS
By Stuart P. Atkinson

Marfan syndrome (MFS) is a heritable dominant disorder caused by mutations in the FBN1 gene (Dietz et al and Pereira et al) affecting the skeletal, ocular and cardiovascular systems. FBN1 itself is an extracellular matrix (ECM) glycoprotein and although the molecular pathogenesis was originally thought to be due to resultant defects in the ECM, other results suggested that altered TGFβ signalling may be the main cause of pathogenic abnormalities in MFS (Dietz et al and Liu et al). Now, researchers from the laboratory of Michael Longlaker at the Standford University School of Medicine have succeeded in deriving FBN1 mutant human embryonic stem cells (hESCs) and also producing human induced pluripotent stem cells (hiPSCs) from FBN1 mutant fibroblasts (Quarto et al). Importantly, during this study of FBN1 mutations in hESCs, they also found that mutant hESCs and hiPSCs give rise to differentiated cells which demonstrate the same phenotype, demonstrating that iPSCs can provide complementary and powerful tools to gain further insights into human molecular pathogenesis.

Human MFS-ESCs were derived from a human blastocyst carrying a FBN1 mutation via standard derivation conditions on mouse embryonic fibroblast feeder cells, while MFS-iPSCs were generated from fibroblasts from patients with a FBN1 splice-site mutation and a separate patient with a frame shift mutation. During osteogenic differentiation of MFS-ESCs, CD73+ cells, representative of cells differentiating toward both osteogenic and chondrogenic fates (Bühring HJ et al, Liu H et al and Tuli R et al), were analysed and it was shown that there was an impairment in the osteogenic capabilities of MFS cells compared to wild type hESCs. QPCR analysis also found a downregulation in levels of the osteogenic markers RUNX2, ALPL, and osteocalcin (BGLAP). Osteogenesis may be impaired due to enhanced TGFβ activity, and indeed it was found that SMAD2 was highly phosphorylated in MFS cells compared to wild type, while markers known to be induced in response to TGFB-signalling (PAI-1 and COL1A1) were also upregulated. It was also found that MFS-ESCs were engaged in active autocrine TGFβ signalling, as medium conditioned by MFS-ESCs led to TGFβ-mediated SMAD2 phosphorylation upon addition of this medium to wild type ESCs. However, these differences were abrogated by addition of a selective inhibitor of TGFβ (SB431542) or a TGFβ-neutralizing antibody.   SB431542 also rescued the deficit in osteogenesis observed in the MFS-ESCs collectively suggesting that they are unable to differentiate along the osteogenic lineage because of an enhanced autocrine TGFβ signaling. TGFβ signaling has previously been shown to promote mesenchymal cell differentiation toward chondrocytes (Johnstone et al, Kawaguch et al and Mello and Tuan) and expression analysis found that the MFS-ESCs could differentiate efficiently towards a chondrogenic fate even in absence of exogenous TGFβ, whereas chondrogenesis was greatly impaired in wild type cells in the absence of exogenous TGFβ. Further, SB431542 treatment impeded the chondrogenic ability of MFS-ESCs in the absence of exogenous TGFβ, thus strengthening the role of active TGFβ signaling in determining phenotypic differences between MFS and wild type ESCs.

MFS-iPSCs were generated using retroviral vectors that express OCT4, SOX2, KLF4 and MYC after selection of SSEA3+ cells to aid the reprogramming process. MFS-iPSCs recapitulated the osteogenic phenotype of the MFS-ESCs, with a reduced level of osteogenic differentiation observed, rescued by SB431542 exposure. Further, TGFβ signalling was also enhanced, high levels of phosphorylated SMAD2 and high expression of PAI-1 and COL1A1 observed and high levels of autocrine TGFβ were found in medium conditioned by MFS-iPSCs. The similarity of the ESC and iPSC cells extended to their chondrogenic differentiation capabilities in the absence of TGFβ and inhibition of chondrogenesis upon SB431542 treatment.

This paper, the authors suggest, is the first report demonstrating that disease-specific iPSCs can phenocopy disease-specific hESCs (Also see another related article on the Stem Cell Portal - The Living Dead (of the iPSC world): Autopsy donor-derived iPSCs; which discusses iPSC derivation from autopsy donor-derived cells). This suggests that the study of iPSCs generated from disease-specific fibroblasts, at least for simple monogenic disease, will be useful tools in place of disease specific ESCs, being more difficult to obtain, allowing for the elucidation of  mechanisms underlying clinically observed pathological variability and helping to pave the way to personalized therapeutic interventions.

 

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