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JSRM Code: 016010300004EPA150420    
SUPPLEMENTARY INFORMATION [PDF]

Generation of transgene-free induced pluripotent stem cells from cardiac fibroblasts of goat embryos
Hanna M1,2, Sahito RGA1, Rateb M2, Kachiwal AB3, Seddiek HA2, Bhutto B4, Hescheler J1

Author Names in full: Mira Hanna1,2, Raja Ghazanfar Ali Sahito1, Moshira Rateb2, Allah Bux Kachiwal3, Hanan A. Seddiek2, Bachal Bhutto4, and Jürgen Hescheler1

1Institute of Neurophysiology, University of Cologne, Robert-Koch-Strasse 39, 50931 Cologne, Germany;2Department of physiology, Faculty of medicine (Kasr El-Aini) Cairo University, El-Maniel, Cairo 11451, Egypt; 3Department of Veterinary Physiology and Biochemistry, Sindh Agriculture University Tandojam, Pakistan. 4Department of Veterinary Parasitology, Sindh Agriculture University Tandojam, Pakistan.

Supplementary Information

Materials and Methods:

2.2. Identification of cardiac fibroblasts

a- Morphologically using light microscope

In 2D culture, fibroblasts appear as morphologically flat, spindle-shaped cells with multiple processes emerging from the main cell body.

b- Immunocytochemistry for cardiac fibroblast cell markers:

 Cells were plated on gelatin 0.2% coated autoclaved coverslips. The medium was removed and the cells were washed 1x with Tris-Wash-Buffer diluted 1:10 using MilliQ water. (99.8%) -20°C prechilled Methanol (MeOH) was added to the cultured cells on the coverslips and kept at -20°C for 20 minutes then sucked off. Cells were washed by Tris-Wash-Buffer (1:10) 3 times for 10 minutes. The samples were incubated with diluted Roti-Immuno-Block (1:10 MiliQ water) for 60 - 120 min at room temperature. Primary antibodies (Table 1) were prepared in diluted Roti–Immuno- Block (1:10) (ROTH). 200µl drop of prepared primary antibodies for each slide were placed on parafilm sitting in a plastic box then the coverslips were placed upside down and kept overnight at 4-8°C.  Coverslips were placed back into multiwell plate upside down and then washed 5 times with Tris-Wash Buffer (2 times rapid then for 5 minutes, 10 minutes and 20 minutes). The secondary antibody diluted (1:1000) using Roti-Immuno-Block and Hoechst dye (DAPI) (1µg/ml) were added and kept 90-120 minutes in darkness at room temperature and then washed 3 times with Tris- Wash- buffer. A drop of Prolong Gold 20 µl (antifade reagent) was added to the glass slide using a cut tip pipette and the coverslips were placed “upside down” on the drop.

2.3. Reprogramming (induction of pluripotency)

The process of Nucleofection of primary cardiac fibroblasts entails the following steps:

a. Cell preparation
Cardiac fibroblasts were grown to 85-90% confluency prior to nucleofection. The cells were washed by PBS then harvested by trypsinization by adding 5ml 0.05% trypsin-EDTA solution (Gibco, 25300-054) to cover  the  cell  monolayer on a  10cm  dish then incubated at 37°C for 4 minutes. Trypsin was then inactivated using culture medium containing FBS and the cells were gently resuspended and collected from the flasks by pipetting, collected by centrifugation at 1000 rpm for 4 minutes, counted and then recentrifuged and washed by PBS. Four million cells were used as control group to test for the efficiency of the method of transfection using green fluorescent protein (Using pmax GFP 2 µg) and 4 million cells were used for the DNA of interest (piggBac transposons 8 µg, HypBase (cut and paste transposase)[17] 0.5 µg, and UTF1-Neo 1.5 µg). UTF1-Neo (G418) is undifferentiated transcription factor1 driven antibiotic resistance in which G418 resistance driven by the undifferentiated transcription factor1 (UTF1) promoter plus enhancer elements[19]. The pellet was resuspended in 100 µl human MSCs nucleofector solution at room temperature, (18 µl supplement solution and 82 µl solution). The 100 μl of cell suspension of both the control and the sample of interest (test sample) were mixed with DNA (using 2 µg pmaxGFP for the control and piggyBac transposons with OSKML transcription factors 8 µg, HypBase 0.5 µg, and UTF1-Neo 1.5 µg for the other sample of interest). Then the nucleofection samples were transferred into an Amaxa certified cuvettes. Each cuvette was inserted into the cuvette holder and the program U-23 was selected and started. The cells were removed from the cuvette immediately after the program has finished to avoid cell damage. Then the cuvette was rinsed with culture medium and the cells from the cuvettes were transferred using the plastic pipettes provided in the kit to prevent damage and loss of cells to a 15ml falcon tubes. 500 μl of the pre-warmed culture medium were added to the falcon tubes then the cell mixture is transferred to 10 cm culture dish coated with gelatin 0.2% and incubated in a humidified 37°C/5% CO2 incubator. The green fluorescence of the expressed gene was detected by fluorescence microscope after 24 hours from nucleofection.

 2.4. Identification of iPSCs

a- Alkaline Phosphatase staining:

The cells were cultured for 4 days in 6 cm plate then the colonies were quickly fixed using 3 ml of 4% (wt/vol) Paraformaldehyde (PFA) for 1–2 min maximally at room temperature after removing the medium and directly adding PFA without washing. Then the PFA was aspirated and the colonies were washed with 3 ml of PBS 2 times: one time at room temperature rapidly and the other incubated for 20 min at 37 °C. This was later followed by the protocol of alkaline phosphatase staining kit (Sigma-Aldrich, Cat. No. AB0300). Once   the   colonies showed  a   clear  blue  color  (maximally  after 6 minutes), the solution mixture was aspirated then the colonies were washed 1x by 3 ml PBS then another 3 ml PBS was added and the pictures were taken.

b- ESC marker expression:

Detection of SSEA1, SSEA4 and Oct4 was achieved by staining with antibodies for Stage-specific embryonic antigen 1 and 4 (SSEA1 and SSEA4) and Oct4 (Table 2). First, murine embryonic feeders were plated on gelatine 0.2% coated autoclaved coverslips then T-GiPSCs, and GiPSCs were cultured. Medium was removed and the cells were washed once with Tris-Wash-Buffer. 4% paraformaldehyde (PFA) was added and kept at room temperature for 15 minutes. Samples were washed by Tris-Wash-Buffer (1:10) 3 times for 10 minutes. The cells were incubated with Roti-Immuno-Block for 60 - 120 min at room temperature. Primary antibodies (Table 2) were prepared in diluted Roti–Immuno- Block (1:10). 200µl drop of prepared primary antibodies were added for each slide placed on parafilm sitting in a plastic box then the coverslips were placed upside down and kept overnight at 4-8°C. Coverslips were placed back into multiwell plate upside down and then washed 5times with Tris-Wash Buffer (2 times rapidly then for 5 minutes, 10 minutes and 20 minutes). The secondary antibody (1:1000 Roti-Immuno-Block) and Hoechest dye (1:1000) were added and kept for 90 -120 minutes in darkness at room temperature and then washed 3 times with Tris- Wash- buffer. A drop of Prolong Gold 20 µl (antifade reagent) is added to the glass slide using cut tip pipette and then the coverslips were placed upside down.

c- Differentiation tests:

Trials of embryoid bodies generation:

One of the proving tests of pluripotency is the ability of the pluripotent stem cells to generate EBs and differentiate to the three germ layers (endoderm, mesoderm and ectoderm) spontaneously. There were two trials; one using clumps of cells and the other using single cells. We started by using single cells with total numbers of 100,000 per bacteriological plate but they showed cellular death and no EBs formation. Then we tried to use clumps of cells but they generated cystic type of EBs. Still in an attempt to produce compact type of EBs, we used single cells with a total number of 2 million per plate and hence, compact type of EBs were finally generated avoiding the cellular death due to trypsinization.

Cutting the colonies in to small pieces (clumps) was followed by culturing the clumps in suspension in 10 cm bacteriological dish with 20% FBS IMDM on the shaker and monitoring of embryoid body formation. The medium was changed every 2-3 days for 20 days then the EBs were cultured on 2.5µg/ml fibronectin coated 6 cm plates using 20%FBS IMDM for 7 days.

Single cell preparation was generated from a whole 6 cm plate (2 million cells) by using 0.05% trypsin for 5 minutes then the cells were filtered through 40 µm cell strainer, centrifuged 1000 rpm for 4 min and then suspended in 20% FBS IMDM in 3.5 cm  bacteriological dish. While cultured under continuous agitation on the shaker.