Methods in Molecular Biology DOI 10.1007/7651_2015_202 © Springer Science+Business Media New York 2015
Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus Filipa A.C. Soares, Roger A. Pedersen, and Ludovic Vallier Abstract This protocol describes the efficient isolation of peripheral blood mononuclear cells from circulating blood via density gradient centrifugation and subsequent generation of integration-free human induced pluripotent stem cells. Peripheral blood mononuclear cells are cultured for 9 days to allow expansion of the erythroblast population. The erythroblasts are then used to derive human induced pluripotent stem cells using Sendai viral vectors, each expressing one of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc. Keywords: Reprogramming, Human induced pluripotent stem cells, Sendai virus, Peripheral blood mononuclear cells, Erythroblast
1
Introduction The discovery of human induced pluripotent stem cells (hiPSC) raised the exciting possibility of producing custom-tailored cells for disease modelling through the recapitulation of disease phenotype in differentiated hiPSCs, thereby enabling analysis of disease etiology and assessment of candidate therapeutic drugs. Furthermore, it opens the exciting possibility of personalized cell-based therapies. Since its discovery several somatic cell types and methods of reprogramming have been used to derive hiPSCs. So far fibroblasts remain the most popular donor cell type and have been used in more than 80 % of all published reprogramming experiments. However, peripheral blood is the most available adult tissue and would allow access to numerous frozen samples already stored at blood banks. Here, we describe an efficient and robust method of deriving hiPSCs from peripheral blood mononuclear cells using Sendai virus, a non-integrative method, expressing the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc. This protocol is an increment of previous published methods (1–4).
Filipa A.C. Soares et al.
2 2.1
Materials Equipment
1. Inverted phase-contrast microscope. 2. Micropipette. 3. Laminar flow cabinet. 4. Cell counter. 5. Benchtop centrifuge. 6. Incubator at 37 C, 5 % CO2.
2.2
Disposables
1. 15 and 50 mL conical tubes. 2. Plastic disposable pipettes. 3. Sterile plastic transfer pipettes. 4. 12-well plate. 5. 100 mm plate. 6. Cryovials.
2.3
Chemicals Chemical
Supplier
Catalogue number
1. Sodium citrate tubes (3.2 %)
Greiner Bio-One
455322
2. Ficoll paque premium (density 1.077 g/mL)
GE Healthcare
17-5442-02
3. D-PBS (without Ca2+ and Mg2+)
Sigma
D8537
4. Trypan blue
Lonza
17-942E
5. Knockout™ serum replacement
Invitrogen
10828028
6. DMSO
Sigma
D2650
7. StemSpan H3000
StemCell Technologies
9850
8. Pen/strep
Invitrogen
15140122
9. Recombinant human IL-3
Invitrogen
PHC0035
10. Recombinant human IGF-1
Miltenyi
130-093-885
11. Recombinant human SCF
Miltenyi
130-096-692
12. Recombinant human EPO
R&D Systems
287-TC-500
13. Dexamethasone
Sigma
D8893-1MG
14. L-ascorbic acid
Sigma
A4544-25G
15. CytoTune™-iPS Sendai Reprogramming Kit
Invitrogen
A1378001
16. Polybrene hexadimethrine bromide [10 mg/mL]
Sigma-Aldrich
107689 (continued)
Generation of Human Induced Pluripotent Stem Cells. . .
(continued)
2.4
Reagent Setup
Catalogue number
Chemical
Supplier
17. Advanced DMEM F12
Life Technologies
12634010
18. L-glutamine
Life Technologies
25030024
19. β-mercaptoethanol
Sigma
M6250
20. Recombinant Human FGF basic
R&D Systems
233-FB-01 M
21. MEF CF-1 4 M IRR
GlobalStem
GSC-6001G
22. Fetal bovine serum (FBS)
Life Technologies
10500-064
23. Gelatin
Sigma
G1890
24. Water for embryo transfer
Sigma
W1503
25. Human serum albumin (HSA)
Sigma
A1653
1. StemSpan Medium (SM). StemSpan medium (SM)
Stock concentration
Final concentration
1. StemSpan H3000 2. Pen/Strep
a
Volume 45 mL
100
1%
450 μL
a
Optional
Defrost StemSpan H3000 overnight at 4 C and aliquot 45 mL in 50 mL tubes (the medium will expand once frozen). Store aliquots at 20 C and defrost once needed. Do not freeze-thaw StemSpan H3000. Medium can be kept at 4 C for up to 2 weeks. 2. Expansion Medium (EM). Expansion medium (EM)
Stock concentration
Final concentration
1. StemSpan H3000
Volume 10 mL
2. Ascorbic acid
10 mg/mL
50 μg/mL
50 μL
3. SCF
50 μg/mL
50 ng/mL
10 μL
4. IL-3
10 μg/mL
10 ng/mL
10 μL
5. EPO
2 U/μL
2 U/mL
10 μL
100 μg/mL
40 ng/mL
4 μL
1 mM
1 μM
10 μL
6. IGF-1 7. Dexamethasone a
a
Keep dexamethasone protected from light
Filipa A.C. Soares et al.
Resuspend SCF, IL-3, EPO, IGF-1 in 0.1 % HSA (or BSA) in D-PBS to prepare stock solutions (see table for stock concentrations). Resuspend ascorbic acid in water for embryo transfer and filter to prepare 10 mg/mL stock solution. Dexamethasone is resuspended using DMSO to prepare 1 mM stock solution. Aliquot stock solutions in small volumes and store at 20 C. When defrosted keep reagents at 4 C and use within 5 days for the cytokines and 2 weeks for the ascorbic acid and dexamethasone. Also, keep dexamethasone away from light. 3. IPSC Medium. Stock Final concentration concentration Volume
IPSC medium 1. Advanced DMEM F12
400 mL
2. Knockout serum replacement
100 mL
3. L-Glutamine
5 mL
4. β-Mercaptoethanol
3.5 μL
5. Pen/strep (optional)
5 mL 4 μg/mL
6. Human bFGF
4 ng/mL
500 μL
Mix reagents 1–5 and sterilize through 0.22 μm vacuum filter. Add human bFGF and use IPSC medium within a week. Store at 4 C. Incomplete IPSC medium (reagents 1–5) can be prepared and stored up to 2 weeks at 4 C without human bFGF. 4. IPSC Medium plus Cytokines (IPSC-Cy). IPSC medium + cytokines (IPSC-Cy)
Stock concentration
Final concentration
1. IPSC medium
Volume 10 mL
2. Ascorbic acid (AA)
10 mg/mL
50 μg/mL
50 μL
3. SCF
50 μg/mL
50 ng/mL
10 μL
4. IL-3
10 μg/mL
10 ng/mL
10 μL
5. EPO
2 U/μL
2 U/mL
10 μL
100 μg/mL
40 ng/mL
4 μL
1 mM
1 μM
10 μL
6. IGF-1 7. Dexamethasone
a
a
Keep dexamethasone protected from light
Keep at 4 C and use within 5 days.
Generation of Human Induced Pluripotent Stem Cells. . .
5. MEF Medium. MEF medium
Volume
1. Advanced DMEM F12
450 mL
2. FBS
50 mL
3. L-Glutamine
5 mL
4. β-Mercaptoethanol
3.5 μL
5. Pen/strep (optional)
5 mL
Sterilize through 0.22 μm vacuum filter and keep at 4 C. Use within 2 weeks. 6. Mouse embryonic fibroblasts gelatin-coated 100 mm plates. Gelatin solution
Volume
1. Gelatin
0.5 g
2. Water for embryo transfer
500 mL
Dissolve gelatin in water for embryo transfer by heating the solution in a 57 C water bath for 30 min. Sterilize through 0.22 μm vacuum filter and keep at room temperature for up to 1 month. Coat the 100 mm plate with 6 mL of gelatin and leave at room temperature for 20 min. Aspirate excess of gelatin solution and add 6 mL of MEF medium. Defrost MEF CF-1 4 M IRR by adding the vial contents to 10 mL of MEF medium. Count and resuspend 1.2 106 mouse embryonic fibroblasts per mL. Add 1 mL to each 100 mm plate and distribute for a homogeneous distribution. Place the plate in the incubator overnight before use.
3
Methods
3.1 Isolation of Peripheral Blood Mononuclear Cells from Peripheral Blood
1. Collect 50 mL of peripheral blood into sodium citrate tubes. Invert tube 8–10 and keep upright at room temperature (10 mL is sufficient when material is limited). 2. Invert Ficoll bottle several times to mix before use. In sterile flow cabinet add 15 mL of Ficoll to empty 50 mL falcon tubes (one tube for every 12.5 mL of blood, four tubes in total). 3. Dilute blood 1:1 with D-PBS and slowly layer 25 mL on top of the Ficoll. 4. Centrifuge 30 min at 500 g at room temperature with the accelerator and BRAKE OFF (ideally within 2 h of collection). 5. Following density gradient spin, carefully collect PBMC layer using a transfer pipette (harvest about 15 mL from each tube).
Filipa A.C. Soares et al.
Use a new falcon tube for every blood tube. Top up the volume to 50 mL with D-PBS. Invert several times and spin down at room temperature for 20 min at 300 g. 6. Aspirate supernatant and resuspend pellet in 10 mL of D-PBS. 7. Count cells. 8. Expect ~60–90 106 PBMCs from 50 mL of peripheral blood (see Section 4.1). 9. Spin cells at 300 g for 5 min and freeze down 2.5 106 cells/vial (Use 90 % KOSR, 10 % DMSO) or proceed with reprogramming (see Section 3.2). 3.2 Generation of Human Induced Pluripotent Stem Cells Using Sendai Virus
1. Day
9 (Seed PBMC).
(a) Thaw one vial of frozen PBMCs (see Section 3.1, Fig. 1) into 10 mL of SM and centrifuge at 300 g for 5 min. Resuspend pellet in 2 mL of EM. For fresh isolated PBMCs add 2 106 cells to 2 mL of EM. (b) Transfer cells to 1 well of a 12-well plate and incubate at 37 C, 5 % CO2. 2. Day
6 and Day
3 (Change medium).
(a) Carefully transfer cells in suspension to 15 mL falcon tube and add 2 mL of SM. Add 1 mL of EM to well to avoid cells left in the plate drying (minimize disturbing the cells when replacing medium).
Derivation
Expansion
Day -9 2x106
Plate PBMCs in hematopoietic expansion medium
Day -6
Day -3
Day 0
Medium change
Medium change
Transduction
Fig. 1 Diagram describing the expansion of the erythroblasts population from peripheral blood mononuclear cells
Generation of Human Induced Pluripotent Stem Cells. . .
(b) Spin cells at 300 g for 5 min. (c) Resuspend cells in 1 mL of fresh EM and carefully add cells back to same well of 12-well plate (total volume of well is 2 mL). (d) Continue to culture at 37 C, 5 % CO2. 3. Day 0 (Transduction). (a) Transfer cells to sterile 15 mL conical tube and wash well 2 with 1 mL of SM to collect all cells. (b) Count cells (see Section 4.2) (c) Spin down 5 105 cells in 15 mL conical tube and add 0.3 mL of fresh EM. (d) Remove one set of CytoTune™ Sendai tubes from the 80 C storage. Thaw each tube by immersing the bottom of the tube in a 37 C water bath for 5–10 s. Remove the tube from the water bath and allow thawing at room temperature. Once thawed, briefly centrifuge the tube and place it immediately on ice. (e) Prepare 0.3 mL of fresh EM plus viruses at MOI 6 (see the CoA for the appropriate volume of virus) with 4 μg/mL of polybrene and transfer to one well of a 12-well plate (final volume/well 0.6 mL). (f) Incubate overnight at 37 C and 5 % CO2. 4. Day 1 (Wash Sendai virus). (a) Collect transduced cells into 15 mL conical tube with 2 mL SM and spin at 300 g for 5 min. Aspirate the supernatant and resupend cells in 2 mL of fresh EM. Return transduced cells to well and incubate at 37 C and 5 % CO2. 5. Day 2 (Plate Mouse Embryonic Fibroblast feeders). (a) Plate MEF feeders onto gelatin-coated 100 mm plate (see Section 2.4). 6. Day 3 (Plate transduced cells). (a) Collect transduced cells into 15 mL conical tube and spin at 300 g for 5 min. (b) Aspirate MEF medium from 100 mm plate and wash once with IPSC medium. (c) Resuspend transduced cells in 7 mL of iPSC medium plus cytokines (IPSC-Cy) as above and transfer to 100 mm plate. 7. Day 5 and Day 7 (a) Feed cells every other day with 7 mL of IPSC medium.
Filipa A.C. Soares et al.
8. Day 9–Day 12 (Small colonies emerge). (a) Feed cells daily with 8 mL of IPSC medium (see Section 4.3). (b) Add additional MEF feeders as needed. 9. ~Day 14–Day 21 (Pick colonies).
4
Notes
4.1 Isolation of Pheripheral Blood Mononuclear Cells Not Working
1. Check the expiry date of all reagents and store the components appropriately as instructed on the datasheets. 2. Limit the time between bleeding and PBMC isolation to a minimum, ideally 70 % of double positive populations of CD235a/CD45 and CD36/CD71 (Fig. 2).
4.3 Sendai Virus Reprogramming Not Working
1. Check the expiry date of the Sendai virus and store the components appropriately as instructed on the datasheet. 2. Aliquot Svirus in small aliquots to avoid repeated freeze–thaw cycles. Discard any leftover when thawed more than twice. 3. Perform reprogramming of a control line (BJ fibroblast, Stemgent catalog number 08-0027) at MOI 3 to evaluate if the Sendai virus and other reprogramming reagents are working.
Generation of Human Induced Pluripotent Stem Cells. . .
Fig. 2 Flow cytometry results after 9 days of expansion protocol (day 0). (a) CD235a APC/CD45 PE. (b) CD36 FITC/CD71 PE
Acknowledgements The authors thank Dr. Thomas Moreau for expert advice and support. The authors also acknowledge the contribution of “Fundac¸˜ao para a Cieˆncia e a Tecnologia” (SFRH/BD/69033/2010) and Cambridge Hospitals National Institute for Health Research Biomedical Research Center and ERC starting grant RelieveIMDs. References 1. Yang W, Mills JA, Sullivan S, Liu Y, French DL, Gadue P (2012) iPSC Reprogramming from human peripheral blood using sendai virus mediated gene transfer. In: StemBook (ed) The Stem Cell Research Community, StemBook 2. Chou B-K, Mali P, Huang X, Ye Z, Dowey SN, Resar LMS, Zou C, Zhang YA, Tong J, Cheng L (2011) Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures. Cell Res 21(3):518–529
3. van den Akker E, Satchwell TJ, Pellegrin S, Daniels G, Toye AM (2010) The majority of the in vitro erythroid expansion potential resides in CD34(-)cells, outweighing the contribution of CD34(+) cells and significantly increasing the erythroblast yield from peripheral blood samples. Haematologica 95(9):1594–1598 4. Leberbauer C, Boulme´ F, Unfried G, Huber J, Beug H, M€ ullner EW (2005) Different steroids co-regulate long-term expansion versus terminal differentiation in primary human erythroid progenitors. Blood 105(1):85–94
Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus Filipa A.C. Soares, Roger A. Pedersen, and Ludovic Vallier Abstract This protocol describes the efficient isolation of peripheral blood mononuclear cells from circulating blood via density gradient centrifugation and subsequent generation of integration-free human induced pluripotent stem cells. Peripheral blood mononuclear cells are cultured for 9 days to allow expansion of the erythroblast population. The erythroblasts are then used to derive human induced pluripotent stem cells using Sendai viral vectors, each expressing one of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc. Keywords: Reprogramming, Human induced pluripotent stem cells, Sendai virus, Peripheral blood mononuclear cells, Erythroblast
1
Introduction The discovery of human induced pluripotent stem cells (hiPSC) raised the exciting possibility of producing custom-tailored cells for disease modelling through the recapitulation of disease phenotype in differentiated hiPSCs, thereby enabling analysis of disease etiology and assessment of candidate therapeutic drugs. Furthermore, it opens the exciting possibility of personalized cell-based therapies. Since its discovery several somatic cell types and methods of reprogramming have been used to derive hiPSCs. So far fibroblasts remain the most popular donor cell type and have been used in more than 80 % of all published reprogramming experiments. However, peripheral blood is the most available adult tissue and would allow access to numerous frozen samples already stored at blood banks. Here, we describe an efficient and robust method of deriving hiPSCs from peripheral blood mononuclear cells using Sendai virus, a non-integrative method, expressing the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc. This protocol is an increment of previous published methods (1–4).
Filipa A.C. Soares et al.
2 2.1
Materials Equipment
1. Inverted phase-contrast microscope. 2. Micropipette. 3. Laminar flow cabinet. 4. Cell counter. 5. Benchtop centrifuge. 6. Incubator at 37 C, 5 % CO2.
2.2
Disposables
1. 15 and 50 mL conical tubes. 2. Plastic disposable pipettes. 3. Sterile plastic transfer pipettes. 4. 12-well plate. 5. 100 mm plate. 6. Cryovials.
2.3
Chemicals Chemical
Supplier
Catalogue number
1. Sodium citrate tubes (3.2 %)
Greiner Bio-One
455322
2. Ficoll paque premium (density 1.077 g/mL)
GE Healthcare
17-5442-02
3. D-PBS (without Ca2+ and Mg2+)
Sigma
D8537
4. Trypan blue
Lonza
17-942E
5. Knockout™ serum replacement
Invitrogen
10828028
6. DMSO
Sigma
D2650
7. StemSpan H3000
StemCell Technologies
9850
8. Pen/strep
Invitrogen
15140122
9. Recombinant human IL-3
Invitrogen
PHC0035
10. Recombinant human IGF-1
Miltenyi
130-093-885
11. Recombinant human SCF
Miltenyi
130-096-692
12. Recombinant human EPO
R&D Systems
287-TC-500
13. Dexamethasone
Sigma
D8893-1MG
14. L-ascorbic acid
Sigma
A4544-25G
15. CytoTune™-iPS Sendai Reprogramming Kit
Invitrogen
A1378001
16. Polybrene hexadimethrine bromide [10 mg/mL]
Sigma-Aldrich
107689 (continued)
Generation of Human Induced Pluripotent Stem Cells. . .
(continued)
2.4
Reagent Setup
Catalogue number
Chemical
Supplier
17. Advanced DMEM F12
Life Technologies
12634010
18. L-glutamine
Life Technologies
25030024
19. β-mercaptoethanol
Sigma
M6250
20. Recombinant Human FGF basic
R&D Systems
233-FB-01 M
21. MEF CF-1 4 M IRR
GlobalStem
GSC-6001G
22. Fetal bovine serum (FBS)
Life Technologies
10500-064
23. Gelatin
Sigma
G1890
24. Water for embryo transfer
Sigma
W1503
25. Human serum albumin (HSA)
Sigma
A1653
1. StemSpan Medium (SM). StemSpan medium (SM)
Stock concentration
Final concentration
1. StemSpan H3000 2. Pen/Strep
a
Volume 45 mL
100
1%
450 μL
a
Optional
Defrost StemSpan H3000 overnight at 4 C and aliquot 45 mL in 50 mL tubes (the medium will expand once frozen). Store aliquots at 20 C and defrost once needed. Do not freeze-thaw StemSpan H3000. Medium can be kept at 4 C for up to 2 weeks. 2. Expansion Medium (EM). Expansion medium (EM)
Stock concentration
Final concentration
1. StemSpan H3000
Volume 10 mL
2. Ascorbic acid
10 mg/mL
50 μg/mL
50 μL
3. SCF
50 μg/mL
50 ng/mL
10 μL
4. IL-3
10 μg/mL
10 ng/mL
10 μL
5. EPO
2 U/μL
2 U/mL
10 μL
100 μg/mL
40 ng/mL
4 μL
1 mM
1 μM
10 μL
6. IGF-1 7. Dexamethasone a
a
Keep dexamethasone protected from light
Filipa A.C. Soares et al.
Resuspend SCF, IL-3, EPO, IGF-1 in 0.1 % HSA (or BSA) in D-PBS to prepare stock solutions (see table for stock concentrations). Resuspend ascorbic acid in water for embryo transfer and filter to prepare 10 mg/mL stock solution. Dexamethasone is resuspended using DMSO to prepare 1 mM stock solution. Aliquot stock solutions in small volumes and store at 20 C. When defrosted keep reagents at 4 C and use within 5 days for the cytokines and 2 weeks for the ascorbic acid and dexamethasone. Also, keep dexamethasone away from light. 3. IPSC Medium. Stock Final concentration concentration Volume
IPSC medium 1. Advanced DMEM F12
400 mL
2. Knockout serum replacement
100 mL
3. L-Glutamine
5 mL
4. β-Mercaptoethanol
3.5 μL
5. Pen/strep (optional)
5 mL 4 μg/mL
6. Human bFGF
4 ng/mL
500 μL
Mix reagents 1–5 and sterilize through 0.22 μm vacuum filter. Add human bFGF and use IPSC medium within a week. Store at 4 C. Incomplete IPSC medium (reagents 1–5) can be prepared and stored up to 2 weeks at 4 C without human bFGF. 4. IPSC Medium plus Cytokines (IPSC-Cy). IPSC medium + cytokines (IPSC-Cy)
Stock concentration
Final concentration
1. IPSC medium
Volume 10 mL
2. Ascorbic acid (AA)
10 mg/mL
50 μg/mL
50 μL
3. SCF
50 μg/mL
50 ng/mL
10 μL
4. IL-3
10 μg/mL
10 ng/mL
10 μL
5. EPO
2 U/μL
2 U/mL
10 μL
100 μg/mL
40 ng/mL
4 μL
1 mM
1 μM
10 μL
6. IGF-1 7. Dexamethasone
a
a
Keep dexamethasone protected from light
Keep at 4 C and use within 5 days.
Generation of Human Induced Pluripotent Stem Cells. . .
5. MEF Medium. MEF medium
Volume
1. Advanced DMEM F12
450 mL
2. FBS
50 mL
3. L-Glutamine
5 mL
4. β-Mercaptoethanol
3.5 μL
5. Pen/strep (optional)
5 mL
Sterilize through 0.22 μm vacuum filter and keep at 4 C. Use within 2 weeks. 6. Mouse embryonic fibroblasts gelatin-coated 100 mm plates. Gelatin solution
Volume
1. Gelatin
0.5 g
2. Water for embryo transfer
500 mL
Dissolve gelatin in water for embryo transfer by heating the solution in a 57 C water bath for 30 min. Sterilize through 0.22 μm vacuum filter and keep at room temperature for up to 1 month. Coat the 100 mm plate with 6 mL of gelatin and leave at room temperature for 20 min. Aspirate excess of gelatin solution and add 6 mL of MEF medium. Defrost MEF CF-1 4 M IRR by adding the vial contents to 10 mL of MEF medium. Count and resuspend 1.2 106 mouse embryonic fibroblasts per mL. Add 1 mL to each 100 mm plate and distribute for a homogeneous distribution. Place the plate in the incubator overnight before use.
3
Methods
3.1 Isolation of Peripheral Blood Mononuclear Cells from Peripheral Blood
1. Collect 50 mL of peripheral blood into sodium citrate tubes. Invert tube 8–10 and keep upright at room temperature (10 mL is sufficient when material is limited). 2. Invert Ficoll bottle several times to mix before use. In sterile flow cabinet add 15 mL of Ficoll to empty 50 mL falcon tubes (one tube for every 12.5 mL of blood, four tubes in total). 3. Dilute blood 1:1 with D-PBS and slowly layer 25 mL on top of the Ficoll. 4. Centrifuge 30 min at 500 g at room temperature with the accelerator and BRAKE OFF (ideally within 2 h of collection). 5. Following density gradient spin, carefully collect PBMC layer using a transfer pipette (harvest about 15 mL from each tube).
Filipa A.C. Soares et al.
Use a new falcon tube for every blood tube. Top up the volume to 50 mL with D-PBS. Invert several times and spin down at room temperature for 20 min at 300 g. 6. Aspirate supernatant and resuspend pellet in 10 mL of D-PBS. 7. Count cells. 8. Expect ~60–90 106 PBMCs from 50 mL of peripheral blood (see Section 4.1). 9. Spin cells at 300 g for 5 min and freeze down 2.5 106 cells/vial (Use 90 % KOSR, 10 % DMSO) or proceed with reprogramming (see Section 3.2). 3.2 Generation of Human Induced Pluripotent Stem Cells Using Sendai Virus
1. Day
9 (Seed PBMC).
(a) Thaw one vial of frozen PBMCs (see Section 3.1, Fig. 1) into 10 mL of SM and centrifuge at 300 g for 5 min. Resuspend pellet in 2 mL of EM. For fresh isolated PBMCs add 2 106 cells to 2 mL of EM. (b) Transfer cells to 1 well of a 12-well plate and incubate at 37 C, 5 % CO2. 2. Day
6 and Day
3 (Change medium).
(a) Carefully transfer cells in suspension to 15 mL falcon tube and add 2 mL of SM. Add 1 mL of EM to well to avoid cells left in the plate drying (minimize disturbing the cells when replacing medium).
Derivation
Expansion
Day -9 2x106
Plate PBMCs in hematopoietic expansion medium
Day -6
Day -3
Day 0
Medium change
Medium change
Transduction
Fig. 1 Diagram describing the expansion of the erythroblasts population from peripheral blood mononuclear cells
Generation of Human Induced Pluripotent Stem Cells. . .
(b) Spin cells at 300 g for 5 min. (c) Resuspend cells in 1 mL of fresh EM and carefully add cells back to same well of 12-well plate (total volume of well is 2 mL). (d) Continue to culture at 37 C, 5 % CO2. 3. Day 0 (Transduction). (a) Transfer cells to sterile 15 mL conical tube and wash well 2 with 1 mL of SM to collect all cells. (b) Count cells (see Section 4.2) (c) Spin down 5 105 cells in 15 mL conical tube and add 0.3 mL of fresh EM. (d) Remove one set of CytoTune™ Sendai tubes from the 80 C storage. Thaw each tube by immersing the bottom of the tube in a 37 C water bath for 5–10 s. Remove the tube from the water bath and allow thawing at room temperature. Once thawed, briefly centrifuge the tube and place it immediately on ice. (e) Prepare 0.3 mL of fresh EM plus viruses at MOI 6 (see the CoA for the appropriate volume of virus) with 4 μg/mL of polybrene and transfer to one well of a 12-well plate (final volume/well 0.6 mL). (f) Incubate overnight at 37 C and 5 % CO2. 4. Day 1 (Wash Sendai virus). (a) Collect transduced cells into 15 mL conical tube with 2 mL SM and spin at 300 g for 5 min. Aspirate the supernatant and resupend cells in 2 mL of fresh EM. Return transduced cells to well and incubate at 37 C and 5 % CO2. 5. Day 2 (Plate Mouse Embryonic Fibroblast feeders). (a) Plate MEF feeders onto gelatin-coated 100 mm plate (see Section 2.4). 6. Day 3 (Plate transduced cells). (a) Collect transduced cells into 15 mL conical tube and spin at 300 g for 5 min. (b) Aspirate MEF medium from 100 mm plate and wash once with IPSC medium. (c) Resuspend transduced cells in 7 mL of iPSC medium plus cytokines (IPSC-Cy) as above and transfer to 100 mm plate. 7. Day 5 and Day 7 (a) Feed cells every other day with 7 mL of IPSC medium.
Filipa A.C. Soares et al.
8. Day 9–Day 12 (Small colonies emerge). (a) Feed cells daily with 8 mL of IPSC medium (see Section 4.3). (b) Add additional MEF feeders as needed. 9. ~Day 14–Day 21 (Pick colonies).
4
Notes
4.1 Isolation of Pheripheral Blood Mononuclear Cells Not Working
1. Check the expiry date of all reagents and store the components appropriately as instructed on the datasheets. 2. Limit the time between bleeding and PBMC isolation to a minimum, ideally 70 % of double positive populations of CD235a/CD45 and CD36/CD71 (Fig. 2).
4.3 Sendai Virus Reprogramming Not Working
1. Check the expiry date of the Sendai virus and store the components appropriately as instructed on the datasheet. 2. Aliquot Svirus in small aliquots to avoid repeated freeze–thaw cycles. Discard any leftover when thawed more than twice. 3. Perform reprogramming of a control line (BJ fibroblast, Stemgent catalog number 08-0027) at MOI 3 to evaluate if the Sendai virus and other reprogramming reagents are working.
Generation of Human Induced Pluripotent Stem Cells. . .
Fig. 2 Flow cytometry results after 9 days of expansion protocol (day 0). (a) CD235a APC/CD45 PE. (b) CD36 FITC/CD71 PE
Acknowledgements The authors thank Dr. Thomas Moreau for expert advice and support. The authors also acknowledge the contribution of “Fundac¸˜ao para a Cieˆncia e a Tecnologia” (SFRH/BD/69033/2010) and Cambridge Hospitals National Institute for Health Research Biomedical Research Center and ERC starting grant RelieveIMDs. References 1. Yang W, Mills JA, Sullivan S, Liu Y, French DL, Gadue P (2012) iPSC Reprogramming from human peripheral blood using sendai virus mediated gene transfer. In: StemBook (ed) The Stem Cell Research Community, StemBook 2. Chou B-K, Mali P, Huang X, Ye Z, Dowey SN, Resar LMS, Zou C, Zhang YA, Tong J, Cheng L (2011) Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures. Cell Res 21(3):518–529
3. van den Akker E, Satchwell TJ, Pellegrin S, Daniels G, Toye AM (2010) The majority of the in vitro erythroid expansion potential resides in CD34(-)cells, outweighing the contribution of CD34(+) cells and significantly increasing the erythroblast yield from peripheral blood samples. Haematologica 95(9):1594–1598 4. Leberbauer C, Boulme´ F, Unfried G, Huber J, Beug H, M€ ullner EW (2005) Different steroids co-regulate long-term expansion versus terminal differentiation in primary human erythroid progenitors. Blood 105(1):85–94
