Highly efficient induced pluripotent stem cell reprogramming of cryopreserved lymphoblastoid cell lines

Abstract

Tissue culture based in-vitro experimental modeling of human inherited disorders provides insight into the cellular and molecular mechanisms involved and the underlying genetic component influencing the disease phenotype. The breakthrough development of induced pluripotent stem cell (iPSC) technology represents a quantum leap in experimental modeling of human diseases, providing investigators with a self-renewing and thus unlimited source of pluripotent cells for targeted differentiation into functionally relevant disease specific tissue/cell types. The existing rich bio-resource of Epstein-Barr virus (EBV) immortalized lymphoblastoid cell line (LCL) repositories generated from a wide array of patients in genetic and epidemiological studies worldwide, many of them with extensive genotypic, genomic and phenotypic data already existing, provides a great opportunity to reprogram iPSCs from any of these LCL donors in the context of their own genetic identity for disease modeling and disease gene identification. However, due to the low reprogramming efficiency and poor success rate of LCL to iPSC reprogramming, these LCL resources remain severely underused for this purpose. Here, we detailed step-by-step instructions to perform our highly efficient LCL-to-iPSC reprogramming protocol using EBNA1/OriP episomal plasmids encoding pluripotency transcription factors (i.e., OCT3/4, SOX2, KLF4, L-MYC, and LIN28), mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate (> 200 reprogrammed iPSC lines) using this protocol.

Keywords: lymphoblastoid cell line, iPSC reprogramming, human, disease modeling, disease genetics

BACKGROUND

MATERIALS

Reagents, media and solutions

• RPMI 1640 Medium (Cat. # 11875-085, Gibco, Thermo Fisher Scientific, USA)
• Fetal Bovine Serum (Cat. # 10082-147, Gibco, Thermo Fisher Scientific, USA)
• MEM Non-Essential Amino Acids (Cat. # 11140-050, Gibco, Thermo Fisher Scientific, USA)
• Sodium Pyruvate (Cat. # 11360-070, Gibco, Thermo Fisher Scientific, USA)
• HEPES (Cat. # 15630-080, Gibco, Thermo Fisher Scientific, USA)
• Antibiotic-Antimycotic (Cat. # 15240-062, Gibco, Thermo Fisher Scientific, USA)
• Phosphate buffered saline (PBS) without CaCl₂ and MgCl₂ (Cat. # 14190-144, Gibco, Thermo Fisher Scientific, USA)
• Myco Alert Detection Kit (Optional) (Cat. # LT07-318, Lonza, USA)
• Cell Culture Treated T-25 Flasks (Cat. # 430639, Corning, USA)
• Cell Culture Treated 35 mm dish (Cat. # 150318, Nunc, Thermo Fisher Scientific, USA)
• SE Cell Line 4D-Nucleofector X Kit S (32 RCT) (Cat. # V4XC-1032, Lonza, USA)
• Episomal plasmid pCE-hOCT3/4 (Cat. # 41813, Addgene, USA)
• Episomal plasmid pCE-hSK (Cat. # 41814, Addgene, USA)
• Episomal plasmid pCE-hUL (Cat. # 41855, Addgene, USA)
• Episomal plasmid pCE-mp53DD (Cat. # 41856, Addgene, USA)
• Countess Cell Counting Chamber Slides (Cat. # C10228, Invitrogen, Thermo Fisher Scientific, USA)
• Cell Culture Treated 6 well plate (Cat. # 353934, Corning, USA)
• DMEM/F-12 Medium (Cat. # 10565018, Gibco, Thermo Fisher Scientific, USA)
• Matrigel hESC Qualified Matrix (Cat. # 354277, Corning, USA)
• TeSR-E7 Medium (Cat. # 5910, Stem Cell Technologies, Canada)
• mTeSR-1 Medium (Cat. # 5850, Stem Cell Technologies, Canada)
• ROCK inhibitor Y-27632 (Cat. # 04-0012-02, Stemgent, USA)
• Stain Alive TRA-1-81 Antibody (Optional) (Cat. # 09-0069, Stemgent, USA)
• Alkaline Phosphatase Live Stain (Optional) (Cat. # A14353, Invitrogen, Thermo Fisher Scientific, USA)
• Cell Scraper (Cat. # 08-100-241, Fisher Scientific, USA)
• Dispase (Cat. # 7923, Stem Cell Technologies, Canada)
• ReLeSR dissociation reagent (Cat. # 5872, Stem Cell Technologies, Canada)
• mFreSR (Cat. # 05854, Stem Cell Technologies, Canada)
• CryoStem Freezing Medium (Cat. # 01-0013-50, Stemgent, USA)
• Tissue Culture Treated 4 well plate (Cat. # 229103, CELLTREAT Scientific Products, USA)
• Paraformaldehyde (Cat. # 158127-100G, Sigma Aldrich, USA)
• 1.0 N NaOH solution (Cat. # S2770-100ML, Sigma Aldrich, USA)
• Triton™ X-100 (Cat. # T8787-100ML, Sigma Aldrich, USA)
• Bovine Serum Albumin (Cat. # A7906-100 G, Sigma Aldrich, USA)
• Donkey serum (Cat. # D9663-10ML, Sigma Aldrich, USA)
• TRA-1-60 Antibody Mouse (Cat. # 09-0010, Stemgent, USA)
• TRA-1-81 Antibody Mouse (Cat. # 09-0011, Stemgent, USA)
• Oct4 Antibody Rabbit (Cat. # 09-0023, Stemgent, USA)
• SSEA-4 Antibody Mouse (Cat. # 09-0006, Stemgent, USA)
• Donkey anti-Mouse Secondary Antibody, Alexa Fluor 488 (Cat. # A21202, Invitrogen, Thermo Fisher Scientific, USA)
• Donkey anti-Rabbit Secondary Antibody, Alexa Fluor 594 (Cat. # A21207, Invitrogen, Thermo Fisher Scientific, USA)
• 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Cat. # D9542-1MG, Sigma Aldrich, USA)
• Human Pluripotent Stem Cell Functional Identification Kit (Cat. # SC027B, R&D Systems, USA)
• Anti-Goat Secondary Antibodies, CF™ 488A antibody (Cat. # SAB4600032-50UL, Sigma Aldrich, USA)

Recipes

• RPMI complete medium: RPMI 1640 medium containing 15% heat inactivated fetal bovine serum, 1% MEM nonessential amino acids, 1 mM sodium pyruvate, and 10 mM HEPES buffer.
• Paraformaldehyde (PFA) 4% solution: 4 % w/v paraformaldehyde in PBS. Heat to 55°C–60°C and add 1.0 N NaOH dropwise to dissolve PFA.
• Permeabilization buffer: 0.2% v/v Triton X-100, 1% w/v Bovine serum albumin (BSA) in PBS.
• Blocking buffer: 2% w/v BSA, 5% v/v secondary antibodies host species serum or equivalent in PBS.

Equipment

• Countess Automated Cell Counter (Thermo Fisher Scientific)
• 4D-Nucleofector System (Lonza)
• Plate Vortexer (Electron Microscopy Sciences)
• Mr. Frosty Cryo 1°C freezing container (Nalgene, Thermo Fisher Scientific, USA)
• Inverted Microscope (Zeiss or any equivalent)
• Centrifuge (Eppendorf or any equivalent)
• CO₂ Cell culture Incubator (Panasonic or any equivalent)
• Magnetic hot plate stirrer (Any make)
• Pipet-Aid Pipette Controller (Drummond or any equivalent)
• Variable volume pipette set 0.5 µl to 1000 µl (Eppendorf or any equivalent)

PROCEDURE

Lymphoblastoid cell culture

1.Culturing cryopreserved lymphoblastoid cell line(s)

1.1.Warm phosphate buffered saline (PBS) without CaCl₂ and MgCl₂ and RPMI complete medium to 37°C before starting the protocol to ensure that the thawing procedure is done as quickly as possible.

1.2.Label a T-25 cell culture flask, add 5 ml of RPMI complete medium and incubate in a CO₂ incubator at 37°C, 5% CO₂ and atmospheric O₂.

1.3.Quickly thaw a vial of cryopreserved cells in a 37°C water bath by gently shaking the cryovial continuously until only a small frozen cell pellet remains.

1.4.Remove the cryovial from the water bath and wipe it with 70% ethanol or isopropanol.

1.5.Transfer cells (~1 ml) from the cryovial to a pre-labeled 15 ml tube and add 9 ml of pre-warmed (37°C) PBS without CaCl₂ and MgCl₂ with gentle mixing.

1.6.Centrifuge cells at 500 × g for 5 min at room temperature.

1.7.Aspirate supernatant, leaving the cell pellet intact. Gently re-suspend the cell pellet in 2 ml of pre-warmed RPMI complete medium by pipetting 2–3 times using a 5 ml pipette.

1.8.Transfer the cells to the T-25 cell culture flask prepared in step 1.2 and incubate in a CO₂ incubator at 37°C, 5% CO₂ and atmospheric O₂.

1.9.Culture cells for 5–7 d or until the desired number of cells are achieved, changing medium every 2–3 d.

iPSC reprogramming

2.Pre-nucleofection

2.1.Inspect cell culture microscopically for any sign of infection. A mycoplasma test is optional but recommended.

2.2.To keep the cell in logarithmic growth phase, passage LCL culture 24 h before nucleofection at a density of about 0.2 to 0.5 × 10⁶ cells/ml.

3.Nucleofection

3.1.Label a 35 mm cell culture dish and add 1.4 ml RPMI complete medium. Incubate the dish in a CO₂ incubator at 37°C, 5% CO₂ and atmospheric O₂.

3.2.Warm PBS without CaCl₂ and MgCl₂ and RPMI complete medium to 37°C.

3.3.Start 4D-Nucleofector system and create or upload the file with following experimental parameters: SE Cell Line solution and 4D-Nucleofector DN-100 program.

3.4.Add 3.6 μl of nucleofector supplement to 16.4 μl of nucleofector solution per sample nucleofected. Alternatively, add entire supplement supplied in the kit to the nucleofector solution, and use 20 μl of this mix per sample nucleofected. When stored at 4°C, the nucleofector solution and supplement mix is stable for 3 months.

3.5.Thaw plasmid DNA on ice and prepare plasmid mix by adding equal amounts of pCEh-OCT3/4, pCE-hSK, pCE-hUL, and pCE-mp53DD plasmid DNA (250 ng each/sample) in a 0.5 ml tube. Keep the tube on ice.

3.6.To prepare LCLs for nucleofection, gently pipette LCL culture 3–5 times to make single cell suspension, then collect 5 ml of cell suspension in a 15 ml tube and centrifuge at 500 × g for 5 min at room temperature.

3.7.Aspirate the supernatant and resuspend cells in 3–5 ml of pre-warmed PBS without CaCl₂ and MgCl₂.

3.8.Determine the cell density using preferred cell-counting method and aliquot 4 × 10⁵ cells in a 1.5 ml microcentrifuge tube.

3.9.Centrifuge the LCL aliquot at 500 × g for 5 min at room temperature. Remove supernatant completely.

3.10.Add 1 μg of the plasmid DNA mix prepared in step 3.5 and resuspend the cell pellet gently in 20 μl of room temperature nucleofector solution mix prepared in step 3.4.

3.11.Transfer 20 μl of the cells and plasmid DNA mix into a well of 16-well nucleocuvette strip (supplied with nucleofection kit).

3.12.Gently tap the nucleocuvette strip to make sure the sample covers the bottom of the cuvette.

3.13.Place nucleocuvette strip into the retainer of the 4D-Nucleofector X Unit and start nucleofection.

3.14.After run completion, remove the nucleocuvette strip from the retainer, add 80 μl of pre-warmed (37°C) RPMI complete medium, and incubate for 5 min at room temperature.

3.15.Mix cells by gently pipetting 2–3 times and transfer to the 35 mm cell culture dish prepared in step 3.1.

3.16.Transfer the culture dish to a CO₂ incubator at 37°C, 5% CO₂ and atmospheric O₂ and allow the cells to recover overnight (16–18 h).

4.Maintenance of iPSC reprogramming culture

4.1.Day 1, after overnight recovery, plate the nucleofected cells into two wells of a Matrigel coated six well plate (~750 µl per well) and add 750 µl of iPSC reprogramming medium (TeSR-E7) in each well. Place the plate in a CO₂ incubator at 37°C, 5% CO₂ and atmospheric O₂.

4.2.Day 3 and 5, add 0.5 ml pre-warmed (37°C) TeSR-E7 medium to each well.

4.3.Day 7 and 9, replace 1 ml of spent medium with pre-warmed (37°C) TeSR-E7 medium.

4.4.On day 11, replace spent medium with 2 ml of pre-warmed (37°C) TeSR-E7 medium.

4.5.Day 13 to 14 when iPSC like cell colonies start to appear (Fig. 1), transition the culture to human pluripotent stem cell maintenance medium (mTeSR-1) by replacing spent medium with fresh 1 ml mTeSR-1 medium.

4.6.From day 15 onwards, change spent medium with 2 ml of pre-warmed (37°C) mTeSR-1 medium daily.

4.7.On days 18–21, 10 to 15 colonies morphologically similar to human ESCs (Fig. 1 and Fig. 2A) are manually picked for further expansion. Refer to following section for detailed protocol.

Figure 1. Schematic diagram of LCL to iPSC reprogramming methodology.

5.iPSC colony selection and expansion

5.1.At least 1 h before picking the iPSC colonies from the reprogramming plate, coat two wells of a six well plate with Matrigel hESC qualified matrix. Follow the manufacturer protocol.

5.2.Warm a 4 ml aliquot of mTeSR-1 medium to room temperature (15°C–25°C) and add 10 μM ROCK inhibitor (Y-27632).

5.3.Aliquot 1 ml of mTeSR-1 medium supplemented with ROCK inhibitor in a 15 ml tube and 1.5 ml in a Matrigel coated well for each iPSC clone to be established. Place the plate in CO₂ incubator at 37°C, 5% CO₂ and atmospheric O₂.

5.4.Place the reprogramming plate on an inverted microscope kept in a laminar flow hood and pick 10–15 iPSC colonies that are morphologically similar to human ESCs (Fig. 1 and Fig. 2A) using a stretched glass Pasteur pipette or 200 μl pipette tip. Collect the picked colonies in the 15 ml tube(s) prepared in step 5.3. Alternatively, colonies may be live stained for TRA-1-81 or alkaline phosphatase for easy identification.

5.5.After picking the desired number of colonies, gently pipette the medium containing iPSC colonies 2–3 times with a 1 ml pipette to achieve desired iPSC aggregate size.

5.6.Plate the iPSC aggregates (0.5 ml in each well) in two wells of the six well plate prepared in step 5.3.

5.7.Place the plate in a CO₂ incubator at 37°C, 5% CO₂ and atmospheric O₂. Move the plate in several quick, short, back-and-forth and side-to-side motions to evenly distribute the cell aggregates. Do not disturb the plate for next 24 h.

5.8.Change medium (mTeSR-1 without ROCK inhibitor) daily and visually assess cultures to monitor growth.

5.9.On day 3 or 4 after plating, when iPSC colonies are easily distinguishable from differentiated cells. Remove the differentiated areas using a stretched glass Pasteur pipette or 200 μl pipette tip.

5.10.On day 5 to 7 when iPSC colonies are ready for passaging, coat appropriate number of six well plate(s) with Matrigel hESC qualified matrix.

5.11.Warm a 12 ml aliquot/six well plate of mTeSR-1 medium to room temperature (15°C–25°C) and add 10 μM ROCK inhibitor (Y-27632).

5.12. Add 1.5 ml per well to the Matrigel coated six well plate. Place the plate in CO₂ incubator at 37°C, 5% CO₂ and atmospheric O₂.

5.13.Warm sufficient volumes of DMEM/F12 and Dispase to 37°C in a water bath.

5.14.Aspirate spent medium from the iPSC well and wash with 2 ml of pre-warmed (37°C) DMEM/F12.

5.15.Add 1 ml of Dispase (1 U/ml) solution and incubate for 6–8 min in a CO₂ incubator at 37°C, 5% CO₂ and atmospheric O₂.

5.16.Aspirate Dispase and wash each well 3 times with 2 ml of pre-warmed (37°C) DMEM/F12.

5.17.Add 1 ml of pre-warmed (37°C) DMEM/F12 to each well and detach iPSC colonies by gentle scrapping with a cell scrapper.

5.18.Collect the scrapped iPSC colonies in a 15 ml tube and wash each well with an additional 1 ml of pre-warmed (37°C) DMEM/F12 and combine with the iPSC colonies collected in the 15 ml tube.

5.19.Centrifuge the 15 ml tube containing cell aggregates at 90 × g for 5 min at room temperature.

5.20.Remove supernatant and resuspend cell aggregates in 3 ml of mTeSR-1 medium supplemented with 10 μM ROCK inhibitor (Y-27632) prepared in step 5.11.

5.21.Plate the iPSC aggregates at a density of 1:6 to 1:10 depending upon the confluency of the iPSC wells, into the six well plate prepared in step 5.12.

5.22.Place the plate in a CO₂ incubator at 37°C, 5% CO₂ and atmospheric O₂. Move the plate in several quick, short, back-and-forth and side-to-side motions to evenly distribute iPSC aggregates. Do not disturb the plate for 24 h.

5.23.Change medium (mTeSR-1 without ROCK inhibitor) daily and visually assess cultures to monitor growth.

5.24.The cultures can be split every 4–7 d upon maturity of the iPSC colonies. The iPSCs should be passaged at least 3–5 times before characterization, functional validation, and experimental use.

Figure 2. Characterization of LCL generated iPSCs. A. Morphology of a reprogrammed iPSC colony at 50×, 100×, and 200× original magnifications, respectively. B. Immunocytochemistry analysis of reprogrammed iPSC colonies showing expression of pluripotency markers TRA-1-81, TRA-1-60, Oct3/4 and SSEA4.

Cryopreservation

6.Cryopreserving generated iPSC clones/lines

6.1.Warm sufficient volumes of DMEM/F12 and Dispase to 37°C in a water bath.

6.2.Using an inverted microscope identify and remove any differentiated areas by scrapping with a stretched glass Pasteur pipette or 200 μl pipette tip.

6.3.Follow steps 5.14 through 5.19.

6.4.Remove supernatant and resuspend cell aggregates in an appropriate volume (1 ml/harvested well) of iPSC freezing media (CryoStem or mFreSR freezing media).

6.5.Immediately aliquot resuspended cell aggregates into pre-labelled cryovial(s) (1 ml/cryovial).

6.6.Transfer the cryovials into Mr. Frosty Cryo 1°C freezing container and store at –80°C for 4–6 h, followed by long-term storage in liquid nitrogen.

iPSC characterization/validation

7.Immunocytochemistry analysis of pluripotency markers (TRA-1-60, TRA-1-81, Oct 3/4, SSEA4)

7.1.Using the methodology described above grow iPSCs on a Matrigel coated four well plate.

7.2.On day 3–4 after plating iPSCs on Matrigel coated four well plate or when iPSC colonies acquire characteristic morphology.

7.3.Wash the cells with 0.5 ml ice-cold PBS and then fix with 0.5 ml 4% PFA, for 15 min at room temperature.

7.4.Briefly wash with 0.5 ml of ice-cold PBS 2 times. If not immediately proceeding to immunostaining, add fresh 0.5 ml PBS and seal the culture plate with parafilm. The fixed cells can be stored at 4°C for a week.

7.5.Permeabilize cells with 0.5 ml of permeabilization buffer for 12 min at room temperature.

7.6.Aspirate permeabilization buffer and add 0.5 ml of blocking buffer. Incubate at room temperature for 1 h.

7.7.During the incubation period prepare 1× working solution of each primary antibodies (i.e., anti TRA-1-60, anti TRA-1-81, anti Oct 3/4 and anti SSEA4) in blocking buffer and keep on ice.

7.8.After an hour aspirate blocking buffer and add 250 μl of respective primary antibodies to each well.

7.9.Incubate at 4°C overnight in a humidifying chamber.

7.10.Next day prepare respective fluorochrome-conjugated secondary antibodies 1× working solution(s). Keep the solution on ice protected from light.

7.11.Wash each well 3 times with 0.5 ml of blocking buffer for 5 min each.

7.12.Add 250 μl of respective fluorochrome-conjugated secondary antibodies to each well and incubate for 1 h at room temperature protected from light.

7.13.Dilute DAPI stock solution (5 mg/ml) 1:10 in H₂O and store protected from light.

7.14.Wash each well with 0.5 ml of PBS for 5 min.

7.15.Add 5 ul of DAPI working solution in 0.5 ml of PBS to each well, incubate 2 min at room temperature.

7.16.Wash each well for 5 min twice with 0.5 ml PBS. Add 0.5 ml of fresh PBS and seal the plate with parafilm.

7.17.Image stained iPSC colonies immediately or it can also be stored at 4°C in the dark for 1–2 d.

Functional validation of reprogrammed iPSCs

8.Culturing iPSCs for differentiation assays

8.1.Coat 2 wells in each of three 4 well plates with Cultrex PathClear BME supplied with the kit.

8.2.Warm a 3 ml aliquot of mTeSR-1 medium supplemented with 10 μM ROCK inhibitor (Y-27632) to room temperature (15°C–25°C).

8.3.Warm an aliquot of PBS and DMEM/F12 medium to 37°C in the water bath.

8.4.Wash iPSC culture plate(s) with pre-warmed (37°C) PBS.

8.5.Add appropriate amount of pre-warmed (37°C) Accutase to iPSC well(s) and incubate at 37°C for 5–7 min.

8.6.Collect the dissociated cells in a 15 ml tube and dilute 1:4 with pre-warmed DMEM/F12 medium.

8.7.Centrifuge the cells at 200 × g for 4 min at room temperature. Aspirate the supernatant and resuspend cell pellet in 5 ml pre-warmed DMEM/F12 medium.

8.8.Determine cell density using preferred cell-counting method. Aliquot 1.2 × 10⁶ cells in a fresh 15 ml tube.

8.9.Centrifuge at 200 × g for 4 min at room temperature. Aspirate the supernatant and resuspend cell pellet in 3 ml of mTeSR-1 medium supplemented with ROCK inhibitor.

8.10.Plate 0.5 ml cell suspension (1.1 × 10⁵ cells/cm²) in each Cultrex PathClear BME coated wells of the 4 well plates prepared in step 8.1.

8.11.Place the plate in a CO₂ incubator at 37°C, 5% CO₂ and atmospheric O₂. The next day cells should be approximately 50% confluent. If cells are not 50% confluent, replace medium with fresh mTeSR-1 medium and continue iPSC culture until 50% confluency is achieved.

8.12.Label the plates as “endoderm”, “mesoderm” and “ectoderm” and proceed for respective differentiation.

9.Endoderm differentiation

9.1.Prepare 2 ml of endoderm differentiation medium-I, as described in the kit’s manual.

9.2.Replace mTeSR-1 with endoderm differentiation medium-I (1 ml/well) in the plate labeled as “endoderm”. Return the plate to CO₂ incubator.

9.3.On day 2 about 16–24 h after replacing endoderm differentiation medium-I, prepare endoderm differentiation medium-II.

9.4.Replace endoderm differentiation medium-I with endoderm differentiation medium-II. Return the plate to CO₂ incubator.

9.5.Replace spent medium with fresh endoderm differentiation medium-II on day 3.

9.6.On day 4, cells are ready of immunocytochemistry analysis of endoderm markers. Proceed to immunocytochemistry analysis of germ layer markers.

10.Mesoderm differentiation

10.1.Prepare 2 ml of mesoderm differentiation medium, as described in the kit’s manual.

10.2.Replace mTeSR-1 with mesoderm differentiation medium (1 ml/well) in the plate labeled as “mesoderm”. Return the plate to CO₂ incubator.

10.3.After 12 to 16 h, replace spent medium with fresh mesoderm differentiation medium.

10.4.Cells are ready for immunocytochemistry analysis of mesoderm markers within 24–36 h of differentiation. Proceed to immunocytochemistry analysis of germ layer markers.

11.Ectoderm differentiation

11.1.Prepare 2 ml of ectoderm differentiation medium, as described in the kit’s manual.

11.2.Replace mTeSR-1 with ectoderm differentiation medium (1 ml/well) in the plate labeled as “ectoderm”. Return the plate to CO₂ incubator.

11.3.Repeat medium change with fresh ectoderm differentiation medium on day 2 and 3.

11.4.On day 4 cells are ready of immunocytochemistry analysis of ectoderm markers. Proceed to immunocytochemistry analysis of germ layer markers.

12.Immunocytochemistry analysis of germ layer markers

12.1.Follow steps 7.3 to 7.5 to fix and permeabilize cells.

12.2.Aspirate permeabilization buffer and add 0.5 ml of blocking buffer and incubate at room temperature for 1 h.

12.3.During incubation period, dilute the following primary antibodies supplied with the kit in blocking buffer to a final concentration of 10 µg/ml and keep on ice: Goat anti-human SOX17 (Endoderm marker); Goat anti-human Brachyury (Mesoderm marker); Goat anti-human Otx2 (Ectoderm marker)

12.4.After 1 h incubation, aspirate blocking buffer and add 300 μl of respective primary antibodies to 1 well of the endoderm, mesoderm and ectoderm plates. Use the second well as a negative control by adding 300 μl of blocking buffer.

12.5.Incubate at 4°C overnight in a humidifying chamber.

12.6.Next day prepare 1× working solution of fluorochrome-conjugated Donkey Anti-Goat secondary antibodies. Keep the solution on ice protected from light.

12.7.Repeat steps 7.11 to 7.16.

12.8.Image stained cells immediately or it can also be stored at 4°C in the dark for 1–2 d.

Figure 3. Differential gene expression analysis of pluripotency and LCL specific markers in LCL and their reprogrammed iPSCs. A-C. Graphs showing gene expression of core pluripotency markers (POU5F1, NANOG, SOX2) in LCLs and their reprogrammed iPSCs. D. The differential gene expression graph showing significant down-regulation of LCL specific markers (CD23, CD70, CD30, CD39, CD11a, CD58 and CD54).

Genomic integrity of reprogrammed iPSCs

ANTICIPATED RESULTS

Figure 4. Functional validation of LCL generated iPSCs. Immunocytochemistry analysis of the cells of three embryonic germ layers differentiated from reprogrammed iPSCs using monolayer differentiation assay.

Figure 5. Generation of terminally differentiated cells of all the three germ layers from LCL reprogrammed iPSCs. Immunocytochemistry analysis of neurons (ectoderm), cardiomyocytes (mesoderm) and hepatocytes (endoderm) differentiated from LCL reprogrammed iPSCs.

TROUBLESHOOTING

Acknowledgments

References