Figure 2 – Timeline showing points at which Simplicon Reprogramming steps were performed.


were selected based on colony morphology and expanded for future experiments. Figure 2 provides a timeline showing when each repro- gramming step was performed.


Results


Transfection effi ciency HFFs were transfected with either a control RNA replicon encoding green fl uorescent protein (GFP) alone (Figure 3a) or with the Simplicon Reprogramming RNA (Figure 3b). The cells were analyzed the following day to determine trans- fection effi ciency. A high percentage of cells in Figure 3a showed GFP signal; transfection of HFFs using Simplicon Reprogramming RNA re- sulted in roughly 5–10% Oct-4 expression one day after transfection (green, Figure 3b).


Reprogramming kinetics To determine the kinetics of reprogramming, Simplicon transfected HFFs were analyzed at four timepoints by brightfi eld microscopy (Figure 4). Colonies of human iPSCs emerged between days 17 and 21 after a single transfec- tion at day 1.


Characterization of human iPSCs Simplicon reprogrammed fi broblasts were assayed for alkaline phosphatase activity, a pluripotency marker, 28 days after transfection (Figure 5). Both HFF and BJ fi broblasts showed high expression levels of alkaline phosphatase activity, with the HFF cells showing the most intense signal.


Lot-to-lot reproducibility To compare the performance of diff erent manufacturing lots of the synthetic Simplicon


AMERICAN LABORATORY • 43 • MARCH 2015


RNA strands, four diff erent lots were utilized to transfect HFFs, using 10,000 cells per trans- fection. The number of hiPSC colonies that appeared 17–21 days later was then counted. By calculating the reprogramming effi ciency as the number of reprogrammed colonies divided by the number of original cells, it was determined that all the manufacturing lots


Figure 3 – Fluorescent micrographs of HFFs transfected with a GFP RNA replicon control (a) or Simplicon Reprogramming RNA (b) and ana- lyzed the following day to estimate transfec- tion effi ciency. The green signal in (b) refl ects OCT-4 expression.


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