Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

Podsumowanie

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Streszczenie

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.

Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Wprowadzenie

Human embryonic stem cells (hESCs) have a capacity to self-renew in vitro and have pluripotency, which could be potentially useful for disease modeling, for drug screening, and to develop cell-based therapies to treat disease and tissue injuries. However, hESCs have a limitation for cell replacement therapy because of immunological, oncological and ethical barriers, and to study disease related genes, disease-specific hESCs could be isolated through pre-implantation genetic diagnosis (PGD) approaches, but it is still technically challenging and the embryo donations are pretty rare. These issues are related to the progress in stem cell biology, which has led to the development of induced pluripotent stem cells (hiPSCs).

Human iPSCs are genetically reprogrammed from human adult somatic cells and harbor pluripotent stem cell-like features similar to hESCs, which makes them a useful source for regenerative medicine such as drug discovery, disease modeling and cell therapy in patient-specific manner^1,2 .

Till now, there are several methods to generate human iPSCs, including virus-mediated (retrovirus and adenovirus)³, non-virus mediated (BAC system and vectors transfection)⁴ gene transductions, and protein delivery system^5-7.

Although a delivery of virus-mediated genes can ensure a certain level of efficiency, viral vectors could leave genetic footprint, because they integrate into host chromosomes to express reprogramming genes in an uncontrolled manner. Even when viral integration of transcription factors may activate or inactivate host genes⁸, it can cause an unexpected genetic aberration and the risk of tumorigenesis^5,9. On the other hand, the direct introduction of proteins or RNA into somatic cells were reported, but have some disadvantages such as labor-intensive, repeated transfection, and low level of reprogramming^7,10. Even episomal and non-integrating adenovirus, adeno-associated virus, and plasmid vectors are still relatively less efficient¹¹. For these reasons, it is plausible to choose non-integration reprogramming methods with high efficacy of iPSC generation and fewer genetic abnormalities. In this study, we use a Sendai-virus based reprogramming. This method is known to be non-integrated into the host genome and consistently produces human iPSCs without transgene integrations.

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Protokół

1. Preparation of Cell and Media (Day 1)

1. Culture and expand human fibroblast with DMEM media containing 10% FBS.
2. Plate human fibroblasts (Figure 1) onto a 24-well plate at the appropriate density per well on the day before transduction.
   NOTE: The following serial dilutions are recommended (200K, 100K, 50K, 25K, 12.5K and 6.25K) because different cell types have different attachment ability.
3. Incubate the cells for one more day in a 37 °C, 5% CO₂ incubator, ensuring the cells have fully adhered and extended.

2. Perform Transduction (Day 2)

1. On the day of transduction, check the cell density and chose the most efficient density wells (Figure 2). It is better to pick three different densities: high (80~90%), middle (50~70%) and low (20~40%).
2. At least 1 hr before transduction, aspirate the fibroblast media from the cells and change new 300 μl of fibroblast medium.
3. Remove one set of 4 different Sendai virus tubes from the -80 °C storage. Thaw each tube at the same time in a 37 °C water bath for few seconds, and then take the tubes out from the water bath. After that, thaw them to room temperature; centrifuge tubes at 6,000 x g for 10 sec and place them on ice till use. Do not re-freeze and thaw the virus since the titers will not maintain.
4. Add the indicated volumes of each of the four Sendai virus tubes (Oct-4, Klf-4, c- Myc and Sox-2) to micro-centrifuge tube. Make sure that the solution is mixed well by pipetting gently.
   For example, if 50K cells/one well of 24-well plate lookwell for transduction:
   (Titer based on the Certificate of Analysis from Life Technologies, can be different from batch to batch)
   Tube hOct-4 = 6.0 x 10⁷ CIU,3 MOI = 5 μl
   Tube hSox-2 = 6.5 x 10⁷ CIU, 3 MOI = 4.6 μl
   Tube hKlf-4 = 6.3 x 10⁷ CIU, 3 MOI = 4.8 μl
   Tube hc-Myc = 7.8 x 10⁷ CIU, 3 MOI = 3.8 μl
   Total = 18.2 μl of four virus factors mixture/one well (50K cells) of 24-well plate
5. Depending on cell densities; add 2X, 1X, and 0.5X volume of the virus mixture into the well. Gently shake the plate front to back, left and right (2X: 36.4 μl of virus mixture, 1X: 18.2 μl and 0.5X: 9.1 μl).
6. Place the cells in a 37 °C, 5% CO₂ incubator and incubate overnight.

3. Replacement of Culture Medium (Day 3 & Day 5)

1. 24 hr after transduction, replace the medium with 500 μl fresh fibroblast medium.
2. On Day 5, change Medium with fresh fibroblast media.

4. Prepare MEF Dishes for the Co-culture (Day 8)

1. Prepare MEF cells in 60 mm culture dishes with 10% FBS contained DMEM medium one day before passaging the transduced fibroblast onto MEF feeder-cells.
   Note: We are using 5 x 10⁵ of MEF in each 60 mm dish.

5. Start Co-culture Transduced Cells with MEF Feeder Cells (Day 9)

1. Before plating transduced cells, aspirate 10% FBS contained DMEM media from MEF feeder dishes and add fresh 10% FBS contained DMEM medium.
2. Remove the medium from the transduced fibroblasts that are in the 24-well plate and wash cells once with D-PBS. Add 200 μl of 0.25% Trypsin/EDTA into the fibroblast cells inthe 24-well plate. After less than 5 min incubation with Trypsin/EDTA, when the cells start to detach from the plate, collect the cells with growth medium in 15 ml conical tube. Then centrifuge them in 6,000 x g for 4 min.
   NOTE: Pooling 2X, 1X, and 0.5X virus transduced fibroblast is recommended.
3. Remove the supernatant medium and wash to re-suspend the cell pellet with 4-5 ml of fresh fibroblast medium for neutralization from trypsin. Then centrifuge them at 135 x g for 4 min.
4. Plate cells as serial dilution density and start to co-culture with fibroblast media onto MEF: such as 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, and 1/128 per 60 mm dish. Depending on cell death rate during the first week of transduction, one may not need to dilute to 1/2 or 1/128 density. Put aside remaining cells to extract total RNA as a positive control for Transgene RT-PCR.
5. Place 60 mm dishes in 37 °C, 5% CO₂ incubator overnight.

6. Feed Human Embryonic Stem Cell Medium and Monitor the Cells (Day 10)

1. The next day, change fibroblast media to human ES media with 10 μM Y-27632. After that, change medium daily with human ES medium: 800 ml DMEM/F12 + 200 ml Knockout Serum Replacement + 10 ml of L-Glutamine + 10 ml of 10 mM MEM minimum non-essential amino acids + 1 ml of 2-mercaptoethanol + 10 ng/ml of basic Fibroblast Growth Factor.
2. Do a daily media change with fresh human ES media. After a week, check the dishes once per 2~3 days under a microscope for the formation of ES colony -like cell clumps. Depending on the cell type, the culture will take more or less time before colonies are found.

7. Picking the Induced Pluripotent Stem Cell Colonies and Expand the Cells (Day 20~)

1. Three weeks after transduction, colonies should appear grown properly for picking.
2. The day before picking the colonies, prepare MEF feeder in 24-well plate.
   NOTE: We are using 12.5 x 10⁵ of MEF in one 24-well plate.
3. The next day, change medium of MEF dishes from fibroblast media to human ES media with 10 μM Y-27632. Manually pick one colony at each time under the microscope in the picking-hood and make smaller clumps by pipetting and transfer them onto newly prepared MEF dishes.Try to pick several clones.
4. Passage and expand the each well from a 24-well plate to a 6 well plate initially. Then from a 6-well plate to a 60 mm dishes before further propagation.

8. Characterization of Human iPSCs (after 10 Passages)

1. Immunofluorescence
   1. Plate human iPSCs in 24-well plate and culture during 4-5 days with daily media change.
   2. For starting Immunofluorescence assay, wash the plate with D-PBS once. Then fix the cells immediately in 0.4% paraformaldehyde for 30 min. After that wash three times in D-PBS for 5 min.
   3. Treat fixed cells with 0.1% Triton X-100 solution in PBS for 15 min at room temperature.
   4. Aspirate the permeablization solution (0.1% triton X-100 solution), then add 0.5% BSA blocking solution for 1 hr at room temperature.
   5. Perform a detection of Nanog, Oct-4, SSEA (stage-specific embryonic antigen) and TRA-1-81 (tumor rejection antigen) using an anti-human antibody in 0.5% BSA solution (check the material table for antibody dilution rate). Incubate Primary Antibody for 2 hr at room temperature. Then wash three times in D-PBS for 5 min each.
   6. Check using fluorescence microscopy after 1 hr of incubation with Alexa Fluor 488 Goat anti-mouse IgG antibodies as 2^nd antibody, which are diluted 1:2,000 in 0.5% BSA solution at room temperature. Stain all the nuclei of each MEF feeder cells and hiPSCs with DAPI.
2. RT-PCR of Transgene Confirmation
   1. Extract total mRNA from cell pellet by using RNA extraction reagents.
   2. Do a reverse transcription on aliquots (1 μg) of total RNA and the resultant cDNA for PCR amplification by Reverse Transcriptase.
   3. Prepare the PCR mixtures to contain 2 μl of cDNA template, 10 μl of 2X PCR master mix, 2 μl of 2 pmol of primers (forward: 1 μl and reverse: 1 μl) and 6 μl of DW. To detect the gene expression, do the RT-PCR with the primers listed in Table 1.
   4. Perform the RT-PCRs with GAPDH gene as a control for efficiency of the amplification reactions. Visualize and analyze PCR product by 1% agarose-gel electrophoresis.

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Wyniki

Usually infected fibroblasts do not show any morphological changes in several days after Sendai virus transduction, but five days later, they start to have different shapes (Figure 1). As described in a right panel of Figure 1, cells do not have typical fibroblast morphology any more. They have a round shape and bigger nucleus than cytoplasm. Even when transduction is performed in 80% cellular confluency, it looks like they are less confluent in the well after they start to reprogram. If...

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Dyskusje

Reprogramming human somatic cells to hiPSCs holds unprecedented promises in basic biology, personal medicine, and transplantation¹². Previously, human iPSC generations required DNA virus that has integration risk into the host genome, which can create undesirable genetic mutations that limit further clinical applications such as drug development and transplantation therapies¹³. With this reason, many studies have been reported to generate vector- and transgene-free system human iPSCs by several alte...

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Materiały

Name	Company	Catalog Number	Comments
CytoTune-iPS Reprogramming Kit	Invitrogen	A1378002
CF-6,MEFs, neomycin-resistant, mitomycin C treated	Global stem	GSC-6105M	5 x 105/6 cm or 12.5 x 105/24-well plate
Trypsin EDTA 0.25% Trypsin with EDTA 4Na 1X	Invitrogen	25200114
DMEM/F-12 medium	Invitrogen	11330-032
24-well Cell Culture Plate, flat-bottom with lid	BD	353935
Y-27632	TOCRIS	1254	10 μM (Stock: 10 mM)
basic fibroblast growth factor	LIFE TECHNOLOGIES	PHG0263	10 ng (Stock : 100 ug)
Knock-out serum replacement	Gibco	10828028
Dulbecco's Modified Eagle Medium (D-MEM, DMEM) (1X), liquid (high glucose)	Invitrogen	11965118
Fetal bovine serum	Thermo Scientific Fermentas	SH30071.03
L-Glutamine-200 mM (100X), liquid	GIBCO	25030-081	1/100
MEM Non-Essential Amino Acids Solution, 100X	LIFE TECHNOLOGIES	11140050	1/100
2-Mercaptoethanol (1,000X), liquid	GIBCO	21985023	1/1,000
Hausser Phase Contrast Hemacytometers	Hausser Scientific	02-671-54
EmbryoMax 0.1% Gelatin Solution	Millipore	ES-006-B
SSEA-4	DSHB	MC-813-70	1/200
anti-Tra-1-81	Cell Signaling	4745S	1/200
mouse monoclonal Oct4 antibody	Santa Cruz	SC-5279	1/1,000
Nanog	R&D	AF1997	1/1,000
Alexa Flouor 488 goat anti-mouse	Invitrogen	948492	1/2,000
DPBS (Dulbecco's Phosphate-Buffered Saline), 1X without calcium & magnesium	Cellgro	21-031-CV
QuantiTect Reverse Transcription Kit	QIAGEN	205313
PCR Master Mix [2X]	Thermo Scientific Fermentas	K0171
Trizol	Invitrogen	15596018
picking hood	NuAire	NU-301
dissecting scope	Nikon	SMZ745