Reprogramming and differentiation of induced Pluripotent Stem Cells into pancreatic β-cells for diabetes research
Terézia Valkovičová 1
Ying Cai 2
Martina Škopková 1
Juraj Staník 1,3
Toshiaki Sawatani 2
Daniela Gašperíková 1
Miriam Cnop 2
1 DIABGENE, Institute of Experimental Endocrinology, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia 2 ULB Center for Diabetes Research, Universite Libre de Bruxelles, Route de Lennik 808, CP-618, 1070 Brussels, Belgium 3 Department of Paediatrics, Faculty of Medicine, Bratislava, Slovakia
|Section:||Cellular metabolism, physiology, molecular biology and genetics|
Diabetes mellitus (DM) is a chronic metabolic disease whose main characteristic is elevated levels of sugar in blood occurring due to a functional failure of β-cells in pancreas (1). There is a varied palette of genes whose single variant may be responsible for diabetes onset (monogenic diabetes) (2) including the syndromes such as MEHMO (3) and Wolfram (4). Several cell lines are used for study of pathogenic mechanisms leading to diabetes but none can precisely mimic patient´s phenotype. This problem was solved by generation of induced pluripotent stem cells (iPSC) from somatic patient´s cells and their controlled differentiation into β-like pancreatic cells. Here we describe the generation of iPSCs from blood cells of patients with MEHMO and Wolfram syndromic monogenic diabetes.
We isolated peripheral blood mononuclear cells (PBMCs) from a patient with MEHMO syndrome and a patient with Wolfram syndrome. We reprogrammed these cells into iPSCs by Sendai virus vector (5). For iPSCs colony derived from MEHMO patient, we also checked: a) iPSC potential to produce three germ layers b) Pluripotent stem cell markers of the iPSCs by real-time PCR and immunocytochemistry c) Unchanged genetic information of the cells by karyotyping and genotyping and d) Purity of culture medium e) Removal of reprogramming virus vector.
One of the selected iPSC colony from MEHMO patient passed through all of the quality control tests. a) Cells showed potential to produce three germ lines. b) Cells pluripotency was confirmed by immunostaining and real-time PCR. c) Karyotyping as well as genotyping confirmed unchanged genetic information of iPSCs. d) Cell medium was negative for mycoplasma contamination. e) Sendai virus was not present anymore in the cell culture. Vector was already removed in the 9th passage.
Reprogramming technique using Sendai virus is a powerful tool for generating unique iPSCs with patient´s genotype and phenotype. These cells have a potential to differentiate into β-like cells and may be used for study of pathogenic mechanisms leading to β-cell failure in patients with monogenic diabetes. Such studies can be focused e.g. on stress of the endoplasmic reticulum since it was shown that this leads to apoptosis of β-cells and it is involved in pathogenesis of some monogenic types of diabetes. We reprogrammed also cells from patient with Wolfram syndrome and their quality control will be commenced soon.
2. Leshinsky-Silver E, Zinger A, Bibi CN, Barash V, Sadeh M, Lev D, et al. MEHMO (Mental retardation, Epileptic seizures, Hypogenitalism, Microcephaly, Obesity): a new X-linked mitochondrial disorder. Eur J Hum Genet. 2002;10(4):226-30.
3. Leshinsky-Silver E, Zinger A, Bibi CN, Barash V, Sadeh M, Lev D, et al. MEHMO (Mental retardation, Epileptic seizures, Hypogenitalism, Microcephaly, Obesity): a new X-linked mitochondrial disorder. Eur J Hum Genet. 2002;10(4):226-30.
4. Urano F. Wolfram Syndrome: Diagnosis, Management, and Treatment. Curr Diab Rep. 2016;16(1):6.
5. Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci. 2009;85(8):348-62.