Induced pluripotent stem (iPS) cells are generated by reprogramming a differentiated somatic cell into a pluripotent stem cell. This source of pluripotent cells enables drug discovery and disease modeling, and ultimately gives us a chance to find cures for today’s most devastating diseases. With iPS cells, researchers can generate patient- or disease-specific cells, enabling new frontiers in disease modeling (Meissner, Wernig, and Jaenisch 2007).

Although there are several methods for reprogramming cells, the Sendai virus method was shown to be highly reliable and efficient in comparison to other non-integrating methods (Schlaeger et al. 2015). The most commonly used reprogramming factors are the Yamanaka factors, Oct3/4, Sox2, c-Myc, and Klf4, which are suitable for efficient reprogramming (Takahashi et al. 2007).

The Cellartis DEF-CS 500 Culture System promotes reliable growth of hiPS cells in a defined, feeder-free environment. Cells are grown as a homogeneous monolayer and are enzymatically passaged as single cells that maintain pluripotency with a stable karyotype for more than 20 passages (Asplund et al. 2016). Here we show that the Cellartis DEF-CS 500 Culture System efficiently and robustly reprograms human fibroblasts using Sendai virus, in a 21-day protocol (Figure 1).

Figure 1. Reprogramming timeline with the Cellartis DEF-CS 500 Culture System.

We have reprogrammed cells from four different human fibroblast cultures using Sendai virus and the Cellartis DEF-CS 500 Culture System (Figure 1). First, human BJ fibroblasts were reprogrammed to evaluate the method, then fibroblasts from three different donors (A, B, and C) were reprogrammed. Most of the data shown are from Donors A, B, and C, which were analyzed in greater detail.

All tested fibroblast lines generated many iPS cell colonies on the first transduction attempt. Only a selection of the generated colonies was picked. We transduced on the order of 10⁶ cells, which generated large numbers of colonies, and picked 37–49 colonies from each of the donors for further culturing.

The survival rate of picked colonies from Donors A, B, and C varied between 33–65%, depending on the donor, as shown in Table I. Here, the survival rate reflects the number of healthy, pluripotent colonies with robust growth.

The morphology of the cells was monitored regularly. Figure 2 shows the morphological changes in a colony from the day of picking until the clone is ready to be frozen as a seed bank (Day 22 post-picking, Passage 6, Day 2).

Figure 2. The progress from a colony at the day of picking to a clone ready to be frozen as a seed bank.

Nine of the generated iPS cell lines, three from each donor, were further passaged to study the loss of the Sendai virus. The amount of Sendai virus RNA present in the cells was measured using quantitative real-time PCR. Ct values ≥36 were considered negative. At Passages 7–8, two-thirds of the iPS cell lines were cleared of Sendai virus RNA. All tested iPS cell lines were cleared of Sendai virus RNA by Passage 11 (see Table II). Ct values ≥36 were considered negative.

At Passage 11, the iPS cell lines were analyzed for the stem cell markers Oct4, SSEA-4, and TRA-1-81 using immunocytochemistry (ICC). A vast majority of the cells were positive for all three markers (see Figure 3), whereas no expression of SSEA-1 was observed. 

Figure 3. Using immunocytochemical analysis to detect the stem cell markers Oct4, SSEA-4, and TRA-1-81 in one of the iPS cell lines at Passage 11. SSEA-1 was used as a negative marker. DAPI was used for nuclear staining.

Using the Cellartis DEF-CS 500 Culture System to reprogram human fibroblasts with Sendai virus allowed for generation of multiple iPS cell lines from each donor with only one transduction step. The Cellartis DEF-CS 500 Culture System promoted high rates of survival in the emerging iPS cell colonies and robust proliferation into iPS cell lines. The reprogrammed iPS cell lines showed high expression levels of the stem cell markers Oct4, SSEA-4, and TRA-1-81, and the typical morphology of undifferentiated stem cells. The highly efficient and reliable performance of the Cellartis DEF-CS 500 Culture System in these studies demonstrates the time- and cost-effectiveness of this method.

Culture of fibroblasts

Human BJ fibroblasts were thawed and seeded in fibroblast medium. They were maintained in fibroblast medium for about two weeks prior to the start of the Sendai reprogramming.

Sendai reprogramming and transfer to the Cellartis DEF-CS 500 Culture System

The CytoTune-iPS 2.0 Sendai Reprogramming Kit (Fisher Scientific) was used to reprogram the fibroblasts. The reprogramming vectors included the four Yamanaka factors, Oct3/4, Sox2, c-Myc, and Klf4. The major reprogramming steps are shown in Figure 1. Plating of fibroblasts, transduction, and maintenance of transduced cells until Day 6 were performed according to the kit user guide. At Day 7 post-transduction, the transduced cells were plated in COAT-1-coated culture dishes in fibroblast medium. At Day 8, the medium was changed to complete DEF-CS Medium. This medium was replaced daily until the colonies were ready to be picked.

Picking and expansion of colonies

Three to four weeks post-transduction, the colonies were ready to be manually cut and transferred to fresh COAT-1-coated culture dishes. Thereafter, the clones were cultured according to the Cellartis DEF-CS 500 Culture System User Manual.

Quantitative real-time PCR

Gene expression was analyzed using the TaqMan Gene Expression Assay SEV (Sendai virus), and the amplification reactions were carried out in a real-time PCR system. The samples were normalized to the expression of CREBBP (CREB binding protein).

Immunocytochemistry

The iPS cell lines were stained with Oct4, SSEA-4, TRA-1-81, and SSEA-1 antibodies, and further stained with secondary antibodies conjugated with Alexa Fluor 488 or 594. DAPI was used for cell nuclear staining. Stainings were examined using a fluorescence microscope.

Asplund, A. et al. One Standardized Differentiation Procedure Robustly Generates Homogenous Hepatocyte Cultures Displaying Metabolic Diversity from a Large Panel of Human Pluripotent Stem Cells. Stem Cell Rev. Reports 12, 90–104 (2016).    

Meissner, A., Wernig, M. & Jaenisch, R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat. Biotechnol. 25, 1177–81 (2007).  

Schlaeger, T. M. et al. A comparison of non-integrating reprogramming methods. Nat. Biotechnol. 33, 58–63 (2015). 

Takahashi, K. et al. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell 131, 861–872 (2007).