Urine of the patient was collected

Urine of the patient was collected for tubulin isolation and expansion. The urine cells were tested for free of mycoplasma. For reprogramming, the urine cells were transfected with episomal plasmids expressing Oct4, Sox2, Klf4 and miR-302-367 cluster. About 3weeks after transfection, many colonies with human embryonic stem cell-like morphology come out and 3 clones were picked for expansion and frozen. One of them was selected for further characterization. The iPS cell has embryonic stem cell-like morphology with a high nuclear to cytoplasmic ratio (Fig. 1.A). Integration of the exogenous transgenes in the genome of iPS cells was excluded by PCR (Fig. 1.B). Mutation analysis demonstrated the iPSC line has a point mutation in exon 9 that is the exact mutation among the urine cells, iPSC and the blood cell DNA (Fig. 1.C). It is a heterozygous mutation in exon-9 (c.1288 G>T, Glu430, encoding a stop codon) resulting in aberrant splicing of a defected RNA. The pluripotency markers were measured by immune-staining for Oct4, Nanog, Tra-1-60 and alkaline phosphatase (AP) (Fig. 1.D, E, F), and their RNA expressions were at a comparable level with human embryonic stem cell H1 (Fig. 1.G). This cell line displayed normal karyotype (Fig. 1.H). Furthermore, this cell retained the potential to differentiate into three germ layers upon embryo body formation in vitro, as showed by RNA expression analysis for key genes representing different germ layers (Fig. 1.I, J). Taken together, we obtained iPSC lines from a MEN1 patient who sustains a point mutant in exon-9 of Men1 gene. It is a unique cell line resource for the molecular pathology study as well as drug development of MEN1.
Materials and methods
Conflict of interest
Acknowledgements We thank all the other members in the lab of Prof. Yin-xiong Li. This work was financially supported by Haizhu District Science and Technology Plan (2011-ZD-02, 2014-CG-05), Ministry of Science and Technology 973 Program (2015CB964700) and Guangdong Province Science and Technology Plan (2016B030301007).
Resource table. Resource details Multiple endocrine neoplasia type 1 (MEN1) (also termed Wermer syndrome) is an autosomal dominant hereditary and associated with tumorigenesis of parathyroid glands, pancreatic islets, anterior pituitary gland. MEN1 was caused by mutations in the tumor suppressor gene MEN1 (Thakker, 2014). Previously we reported two patients in one family with MEN1 (Li et al., 2013). Both of the two patients carry a heterozygous mutation (c.1288 G>T, Glu430 encoding a stop codon) on exon-9 of MEN1 gene and suffered with pancreatic bate cell tumors, but one was hypoglycemia, and the other was not. In details, the CRISPR/Cas9 plasmid and ssODN were transfected into UC-015 cells. After G418 treatment and single cell screening we got 4 clones with specific mutation site and one clone was chosen for further characterization. The mutation iPS cells have the same mutation site with the two MEN1 patients (Fig. 1.B). These iPS cells expressed pluripotent marker Oct4 and Nanog as demonstrated by immune-staining (Fig. 1.C) and their karyotype analysis were displayed normal chromosome appearances and numbers (Fig. 1.D). Furthermore, this cell line retained the potential to differentiate into three germ layers upon embryo body formation in vitro, as showed by RNA expression analysis for key genes representing different germ layers (Fig. 1.E). More importantly no mutation was detected at potential off-target sites (POTs) (Supplementary Fig. 1). Taken together, we established iPSC lines carrying MEN1 patient specific mutation in wild type iPS cells. It is a unique cell line for MEN1 pathology research. In particular, it helps to understand the genotype and phenotype relationship on hypoglycemia in MEN1 syndrome.
Materials and methods
Conflict of interest
Acknowledgements We thank all the other members in the lab of Prof. Yin-xiong Li. This work was financially supported by Haizhu District Science and Technology grant (2011-ZD-02, 2014-CG-05), Ministry of Science and Technology 973 Program (2015CB964700) and Guangdong Province Science and Technology Plan (2016B030301007).