Induced pluripotent stem cell model for cardiofaciocutaneous syndrome and uses thereof

10174,288

15/103271

December 11, 2013

The present invention relates to an induced pluripotent stem cell (iPS) model for cardiofaciocutaneous (CFC) syndrome, a method for producing the model, and uses of the iPS model in the analysis of neural development in CFC syndrome. Specifically, the CFC syndrome-derived iPS and generation and differentiation of an embryonic body were induced from the fibroblasts of a CFC syndrome patient, and the CFC syndrome-derived iPS and embryonic body were confirmed to exhibit broken embryonic body shapes and no differentiation into neurons. When a CFC syndrome-derived embryonic body was induced by treating with p-ERK and p-SMAD1 inhibitors, the embryonic body exhibited a normal embryonic body shape and effectively differentiated into neurons. Thus, the CFC syndrome patient-derived stem cell model of the invention can be effectively used in the research for neural development in cardiofaciocutaneous syndrome.

Korea Advanced Institute of Science and Technology (Daejeon, KR)

Korea Advanced Institute of Science and Technology (Daejeon, KR)

1. A method of using induced pluripotent stem cells (iPSC) as a cardiofaciocutaneous syndrome (CFC syndrome) model comprising the following steps: i) inducing dedifferentiation of fibroblasts separated from a CFC syndrome patient into induced pluripotent stem cells (iPSC) in vitro, wherein the fibroblasts comprise a BRAF gene which encodes a Gln257Arg BRAF protein mutation; ii) dedifferentiating the iPSC into an embryonic body and analyzing characteristics of the embryonic body, wherein said characteristics comprise the following a)-c): a) broken embryonic body shape compared with an embryonic body differentiated from iPSC derived from fibroblasts isolated from a normal patient without CFC syndrome and which do not encode a BRAF protein mutation; b) unexpression of one or more neuroectoderm marker genes selected from the group consisting of Paired box 6 (PAX6), Orthodenticle homeobox 2 (OTX2), and Neural cadherin (NCAD) compared with the embryonic body differentiated from iPSC derived from fibroblasts isolated from a normal patient without CFC syndrome and without a BRAF protein mutation; and c) increase of phosphorylation levels of Phosphorylated Mothers against decapentaplegic homolog 1 (p-SMAD1), Phosphorylated Mothers against decapentaplegic homolog 2 (p-SMAD2), and Phosphorylated extracellular signal-regulated kinase (p-ERK), compared with the embryonic body differentiated from the iPSC derived from fibroblasts isolated from a normal patient without CFC syndrome and which do not encode the BRAF protein mutation, iii) selecting iPSC generated in step i), which produce embryonic bodies having broken embryonic shape, unexpression of the one or more neuroectoderm marker genes, and increased phosphorylation levels of p-SMAD1, p-SMAD2, and p-ERK, as CFC syndrome model cells.

2. The method of claim 1 wherein the broken embryonic body shape is a broken sphere shape.

3. A method of using induced pluripotent stem cells (iPSC) as a cardiofaciocutaneous syndrome (CFC syndrome) model comprising the following steps: i) inducing dedifferentiation of fibroblasts separated from a CFC syndrome patient into induced pluripotent stem cells (iPSC) in vitro, dedifferentiating the iPSC into embryonic bodies, and inducing differentiation of neurons from the embryonic bodies, wherein the fibroblasts comprise a BRAF gene which encodes a Gln257Arg BRAF protein mutation; ii) analyzing expression of neural differentiation markers of a) and b) below in the neurons differentiated in step i): a) down-regulation of one or more neural differentiation marker proteins selected from the group consisting of PAX6, OTX2, and NCAD, compared with neurons differentiated from iPSC derived from fibroblasts isolated from a normal patient without CFC syndrome and which do not encode a BRAF protein mutation; and b) decrease of neural rosette formation compared with neurons differentiated from the iPSC derived from fibroblasts isolated from a normal patient without CFC syndrome and which do not encode a BRAF protein mutation, iii) selecting neurons which exhibit down-regulation of the one or more neural differentiation marker proteins and decrease of neural rosette formation as CFC syndrome model cells.

4. A method for screening agents potentially useful for treating cardiofaciocutaneous syndrome (CFC syndrome), comprising the following steps: i) inducing dedifferentiation of fibroblasts separated from a CFC syndrome patient into induced pluripotent stem cells (iPSC) in vitro, and dedifferentiating the iPSC into an embryonic body, wherein the fibroblasts comprise a BRAF gene which encodes a Gln257Arg BRAF protein mutation; ii) treating the embryonic body of step i) with a test agent capable of inhibiting p-SMAD1 iii) analyzing characteristics of the treated embryonic body of step ii), wherein said characteristics comprise a)˜c) below; and iv) comparing results of step iii) with results from a non-treated control, wherein production of the characteristics of the following a)˜c) in the treated embryonic body by the agent indicates that the agent is potentially a treating agent for CFC syndrome; a) normal embryonic body shape; b) expression of one or more neuroectoderm marker genes selected from the group consisting of PAX6, OTX2, and NCAD; and c) decrease of phosphorylation level of p-SMAD1, p-ERK1, or p-ERK2, compared with those of the non-treated control.

5. A method for screening agents potentially useful for treating cardiofaciocutaneous syndrome (CFC syndrome), comprising the following steps: i) inducing dedifferentiation of fibroblasts separated from a CFC syndrome patient into induced pluripotent stem cells (iPSC) in vitro, dedifferentiating the iPSC into embryonic bodies, and inducing differentiation of neurons from the embryonic bodies, wherein the fibroblasts comprise a BRAF gene which encodes a Gln257Arg BRAF protein mutation; ii) treating the neurons of step i) with a test agent capable of inhibiting p-SMAD1; iii) analyzing characteristics of the treated neurons of step ii), wherein said characteristics comprise a) ˜c) below; and iv) comparing results of step iii) with results from a non-treated control, wherein production of the characteristics of the following a)˜c) in the treated neurons by the agent indicates that the agent is potentially a treating agent for CFC syndrome; a) expression of one or more neuroectoderm marker proteins selected from the group consisting of PAX6, OTX2, and NCAD; b) decrease of phosphorylation level of p-SMAD1, p-ERK1, or p-ERK2, compared with those of the non-treated control; and c) neural rosette formation.

2011/0217774 September 2011 Kim et al.
10-2012-0118619 October 2012
10-2013-0133598 December 2013
2011-130217 October 2011
WO 2011/130217 October 2011

Koch et al A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration PNAS_Mar. 3, 2009_vol. 106_No. 9_3225-3230. cited by examiner
Han et al Enhanced SMAD1 Signaling Contributes to Impairments of Early Development in CFC-iPSCs SMAD1 Signaling in Early Development of CFCiPSCs Stem cells (Dayton, Ohio) / Stem Cells (Durham, NC, U. S.)vol. and Issue No. vol. 33 Issue: 5 pp. 1447-1455 Publication Date: May 2015. cited by examiner
Chambers et al Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling Nature Biotechnology vol. 27 No. 3 Mar. 2009 pp. 275-280. cited by examiner
Carvajal-Vergara et al., Patient-specific induced pluripotent stem cell derived models of LEOPARD syndrome Nature. Jun. 10, 2010; 465(7299): 808-812. cited by examiner
Lowry et al ., Generation of human induced pluripotent stem cells from dermal fibroblasts PNAS Feb. 26, 2008 vol. 105 No. 8 2883-2888. cited by examiner
Carvajal-Vergara et al. (2010) “Patient-Specific Induced Pluripotent Stem-Cell-Derived Models of LEOPARD Syndrome,” Nature 465:808-812. cited by applicant
International Search Report for PCT/KR2013/011484, dated Aug. 28, 2014. cited by applicant
Bentires-Alj et al., (2006) Nature Medicine, 12(3), 283-285. cited by applicant
Lee et. al, (2009) Journal of Genetic Medicine, 6, 87-90. cited by applicant
Myers et. al, (2014) Am J Med Genet A, 146(11), 2814-2821. cited by applicant
Robinton et. al, (2012) Nature, 481, 295-305. cited by applicant
Rodriguez-Viciana et al., (2006) Science, 311, 1287. cited by applicant
Schubbert et. al, (2007) Nature Reviews Cancer, 7(4), 295-308. cited by applicant
Tidyman et al., (2008) Expert reviews in molecular medicine 10: e37. cited by applicant

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