PhD Scientific Days 2021

MO_V_L: Molecular Sciences V. Lectures

Generation of iPSC lines from identical diabetic twins

Text of the abstract

Aims : The main goal of our work is to generate induced pluripotent stem cells (iPSc) from monozygotic twins diagnosed with type 2 diabetes. With this model system we may able to investigate DM2 related cellular phenotypes to improve therapeutic options.
Methods: We generated induced pluripotent stem cell lines from peripheral blood samples of monoclonal twin sisters (66 years old) with DM2. We established two clonal lines from each patient. The samples were reprogramed via Sendai viral transduction of the four Yamanaka factors (Oct3/4, Sox2, klf4, cMyc).
The cell lines' identity was validated by STR analysis and the genetic integrity was validated by karyotyping. Pluripotency was confirmed by flow cytometry analysis of surface marker Stage Specific Antigen-4, as well as assessment of morphology immunofluorescent staining of endogenous pluripotency transcription factors Oct4 and Nanog. In vitro spontaneous differentiation potential was examined via embryoid body formation. Expression of alpha-fetoprotein (AFP) as endoderm, smooth muscle actin as mesoderm and beta-III tubulin as ectoderm markers were detected by confocal microscopy. Besides RNA expression levels of pluripotency marker Nanog, endoderm marker AFP, mesoderm marker Brachyury and ectoderm marker Paired Box 6 were determined and compared to a positive control by RT-PCR.
Results: Two stabilized, validated IPS cell lines were generated from both of the twins.
Future plans: Generation of neuronal cell lines from stabilized DM2 iPSCs, and comparative studies with healthy, sex- and age-matched controls to characterize the effects of DM2 on neural cultures.
Funding:
This study was funded by the National Brain Research Program (NAP) of Hungary (grant numbers: 2017-1.2.1-NKP-2017-00002), National Research, Development and Innovation Office (OTKA- K128369) and PRIME Consortium (H2020 Programme): Prevention and Remediation of Insulin Multimorbidity in Europe. Grant agreement ID: 847879.

University and Doctoral School

Semmelweis University, Doctoral School of Mental Health Sciences