Poster Presentation 6th Australian Health and Medical Research Congress 2012

mRNA-mediated efficient non-integrated reprogramming of T1D patient fibroblasts to pluripotent stem cells (#396)

Jun Liu 1 , Huseyin Sumer 1 , Helena Teede 2 3 , Paul J. Verma 1 4
  1. Monash Institute of Medical Research, Monash University, Clayton, VIC, Australia
  2. School of Public Health and Preventive Medicine, Monash University, Clayton, VIC, Australia
  3. Diabetes Unit, Southern Health , Clayton, VIC, Australia
  4. Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, SA, Australia

Type 1 diabetes (T1D) occurs as consequence of autoimmune destruction of the isulin-producing ß-cells in the islet of the pancreas. T1D patient-specific induced pluripotent stem cells (iPSC) are preferable to ESC to study the disease, as they represent the disease genotype. Delivery of reprogramming factors by viral vectors results in integration of exogenous DNA into the genome, which presents a formidable hurdle to therapeutic application of iPSCs1,2 .  The objective of the study was to derive non-integration iPSCs from a T1D patient using synthetic mRNAs and demonstrated complete pluripotency of the iPSCs.

Skin fibroblasts (designated as MMCF1) from an adult male T1D patient were derived from a dermal biopsy. Commercial BJ fibroblasts were included in the study as a non-disease control line. MMCF1 and BJ cells were seeded in 6-well culture plates at density of 2.5×104 and 1×104 cells/well, respectively. mRNAs encoding the reprogramming factors OCT4, SOX2, KLF4, cMYC, LIN28 along with eGFP as a transfection indicator were transfected into the cells for 18 days. The cells were monitored for morphology changes and GFP expression. The generated iPSCs were characterized for gene expression by RT-PCR and immunocytochemistry, whole-genome expression analysis by DNA microarray, DNA finger printing, karyotyping, methylation analysis of OCT4 and NANOG promoter regions, and spontaneous differentiations by embryoid body formation in vitro and teratoma formation in vivo.

GFP positive cells could be observed 15 h after the first transfection and more cells became green following repeat transfections up to day 5 in both cell lines. Human iPSC colonies were recognizable at day 11 in BJ cell cultures, and at day 18 in MMCF1 cell cultures. A total of 56 and 11 iPSC colonies were picked from transfected BJ and MMCF1 cells, respectively. The efficiency of iPSC derivation was 0.56% and 0.044% in BJ and MMCF1 fibroblasts, respectively. Two BJ iPSC and 3 MMCF1 iPSC cell lines were characterized and showed complete pluripotency with normal karyotypes.

  1. Takahashi K, et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861-872.
  2. Yu J, et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318(5858):1917-1920.