Journal of Assisted Reproduction and Genetics

, Volume 35, Issue 8, pp 1443–1455 | Cite as

Use of human-derived stem cells to create a novel, in vitro model designed to explore FMR1 CGG repeat instability amongst female premutation carriers

  • Stephanie L. F. GustinEmail author
  • Guangwen Wang
  • Valerie M. Baker
  • Gary Latham
  • Vittorio Sebastiano



Create a model, using reprogrammed cells, to provide a platform to identify the mechanisms of CGG repeat instability amongst female fragile X mental retardation 1 gene (FMR1) premutation (PM) carriers.


Female PM carriers (with and without POI) and healthy controls were enrolled from June 2013 to April 2014. Patient-derived fibroblasts (FB) were reprogrammed to induced pluripotent stem cells (iPSC) using viral vectors, encoding KLF4, OCT4, SOX2, and MYC. FMR1 CGG repeat-primed PCR was used to assess the triplet repeat structure of the FMR1 gene. FMR1 promoter methylation (%) was determined using FMR1 methylation PCR (mPCR). Quantification of FMR1 transcripts by RT-qPCR was used to evaluate the effect of reprogramming on gene transcription, as well as to correlate patient phenotype with FMR1 expression. Production of FMR1 protein (FMRP) was determined using a liquid bead array-based immunoassay.


Upon induction to pluripotency, all control clones exhibited maintenance of progenitor cell CGG repeat number, whereas 10 of 12 clones derived from PM carriers maintained their input CGG repeat number, one of which expanded and one contracted. As compared to parent FB, iPSC clones exhibited a skewed methylation pattern; however, downstream transcription and translation appeared unaffected. Further, the PM carriers, regardless of phenotype, exhibited similar FMR1 transcription and translation to the controls.


This is the first study to establish a stem cell model aimed to understand FMR1 CGG repeat instability amongst female PM carriers. Our preliminary data indicate that CGG repeat number, transcription, and translation are conserved upon induction to pluripotency.


iPSC Stem cell FMR1 Premutation Primary ovarian insufficiency CGG repeat 



The authors would like to thank Dr. Renee Reijo Pera for the initial conception of the project, and technical support in stem cell culture, and Drs. LaFauci, Dobkin and Brown, at Institute for Basic Research in Developmental Disabilities, for their FMRP analysis using the Luminex immunoassay. This work was funded by U54HD068158.

Author contributions

S.G. and V.B. conceived the project. S.G., V.S., and G.L. designed the experiments. S.G, G.W., and G.L. performed the experiments. S.G., V.B, G.L., and V.S. helped with inputs and interpretation of data. S.G. wrote the paper, and all authors read and approved the final manuscript.

Supplementary material

10815_2018_1237_MOESM1_ESM.docx (468 kb)
ESM 1 (DOCX 468 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Stephanie L. F. Gustin
    • 1
    Email author
  • Guangwen Wang
    • 2
  • Valerie M. Baker
    • 1
  • Gary Latham
    • 3
  • Vittorio Sebastiano
    • 4
  1. 1.Department Obstetrics and Gynecology, Division of Reproductive Endocrinology and InfertilityStanford University School of MedicineStanfordUSA
  2. 2.Department of GeneticsStanford UniversityStanfordUSA
  3. 3.Research and Technology DevelopmentAsuragen, Inc.AustinUSA
  4. 4.Institute for Stem Cell Biology &Regenerative Medicine, Stanford University School of MedicineStanford UniversityStanfordUSA

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