10. Supplementation of SMC4 to mTeSR™1 medium improves
viability of iPSCs during clonal isolation. We found that the use
of RevitaCell™ and StemFlex™ medium (Thermo Fisher Scientific) also helps increase viability and cloning efficiency for
the feeder-free culture of iPSCs.
11. To obtain a single cell per well, the number of cells seeded into
each well of 96-well plates can vary among the cell lines. The
seeding density can range from 1 to 20 cells/well. The next
day, identify the well that contains a single cell. If there are too
many empty wells, increase the number of seeded cells per well.
If there are too many wells containing multiple cells, decrease
the number of seeded cells.
Acknowledgments
M.W. is supported by Chalermphrakiat Grant, Faculty of Medicine
Siriraj Hospital, Mahidol University, Thailand.
References
1. Ruangvutilert P (2007) Thalassemia is a preventable genetic disease. Siriraj Med J
59:330–333
2. Ye L, Chang JC, Lin C, Sun X, Yu J, Kan YW
(2009) Induced pluripotent stem cells offer
new approach to therapy in thalassemia and
sickle cell anemia and option in prenatal diagnosis in genetic diseases. Proc Natl Acad Sci U
S A 106:9826–9830
3. Olivieri NF, Pakbaz Z, Vichinsky E (2011) Hb
E/beta-thalassaemia: a common & clinically
diverse disorder. Indian J Med Res
134:522–531
4. Papapetrou EP, Lee G, Malani N, Setty M,
Riviere I, Tirunagari LMS, Kadota K, Roth
SL, Giardina P, Viale A et al (2011) Genomic
safe harbors permit high β-globin transgene
expression in thalassemia induced pluripotent
stem cells. Nat Biotechnol 29:73–81
5. Tubsuwan A, Abed S, Deichmann A, Kardel
MD, Bartholoma C, Cheung A, Negre O,
Kadri Z, Fucharoen S, von Kalle C et al
(2013) Parallel assessment of globin lentiviral
transfer in induced pluripotent stem cells and
adult hematopoietic stem cells derived from the
same transplanted beta-thalassemia patient.
Stem Cells 31:1785–1794. https://doi.org/
10.1002/stem.1436
6. Liu Y, Yang Y, Kang X, Lin B, Yu Q, Song B,
Gao G, Chen Y, Sun X, Li X et al (2017)
One-step biallelic and scarless correction of a
beta-thalassemia mutation in patient-specific
iPSCs without drug selection. Mol Ther
Nucleic Acids 6:57–67. https://doi.org/10.
1016/j.omtn.2016.11.010
7. Niu X, He W, Song B, Ou Z, Fan D, Chen Y,
Fan Y, Sun X (2016) Combining single strand
oligodeoxynucleotides and CRISPR/Cas9 to
correct gene mutations in beta-thalassemiainduced pluripotent stem cells. J Biol Chem
291:16576–16585.
https://doi.org/10.
1074/jbc.M116.719237
8. Xie F, Ye L, Chang JC, Beyer AI, Wang J,
Muench MO, Kan YW (2014) Seamless gene
correction of beta-thalassemia mutations in
patient-specific iPSCs using CRISPR/Cas9
and piggyBac. Genome Res 24:1526–1533.
https://doi.org/10.1101/gr.173427.114
9. Song B, Fan Y, He W, Zhu D, Niu X, Wang D,
Ou Z, Luo M, Sun X (2015) Improved hematopoietic differentiation efficiency of genecorrected beta-thalassemia induced pluripotent
stem cells by CRISPR/Cas9 system. Stem Cells
Dev 24:1053–1065. https://doi.org/10.
1089/scd.2014.0347
10. Wattanapanitch M, Damkham N, Potirat P,
Trakarnsanga K, Janan M, Yaowalak UP,
Kheolamai P, Klincumhom N, Issaragrisil S
(2018) One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived
210
Methichit Wattanapanitch
viability of iPSCs during clonal isolation. We found that the use
of RevitaCell™ and StemFlex™ medium (Thermo Fisher Scientific) also helps increase viability and cloning efficiency for
the feeder-free culture of iPSCs.
11. To obtain a single cell per well, the number of cells seeded into
each well of 96-well plates can vary among the cell lines. The
seeding density can range from 1 to 20 cells/well. The next
day, identify the well that contains a single cell. If there are too
many empty wells, increase the number of seeded cells per well.
If there are too many wells containing multiple cells, decrease
the number of seeded cells.
Acknowledgments
M.W. is supported by Chalermphrakiat Grant, Faculty of Medicine
Siriraj Hospital, Mahidol University, Thailand.
References
1. Ruangvutilert P (2007) Thalassemia is a preventable genetic disease. Siriraj Med J
59:330–333
2. Ye L, Chang JC, Lin C, Sun X, Yu J, Kan YW
(2009) Induced pluripotent stem cells offer
new approach to therapy in thalassemia and
sickle cell anemia and option in prenatal diagnosis in genetic diseases. Proc Natl Acad Sci U
S A 106:9826–9830
3. Olivieri NF, Pakbaz Z, Vichinsky E (2011) Hb
E/beta-thalassaemia: a common & clinically
diverse disorder. Indian J Med Res
134:522–531
4. Papapetrou EP, Lee G, Malani N, Setty M,
Riviere I, Tirunagari LMS, Kadota K, Roth
SL, Giardina P, Viale A et al (2011) Genomic
safe harbors permit high β-globin transgene
expression in thalassemia induced pluripotent
stem cells. Nat Biotechnol 29:73–81
5. Tubsuwan A, Abed S, Deichmann A, Kardel
MD, Bartholoma C, Cheung A, Negre O,
Kadri Z, Fucharoen S, von Kalle C et al
(2013) Parallel assessment of globin lentiviral
transfer in induced pluripotent stem cells and
adult hematopoietic stem cells derived from the
same transplanted beta-thalassemia patient.
Stem Cells 31:1785–1794. https://doi.org/
10.1002/stem.1436
6. Liu Y, Yang Y, Kang X, Lin B, Yu Q, Song B,
Gao G, Chen Y, Sun X, Li X et al (2017)
One-step biallelic and scarless correction of a
beta-thalassemia mutation in patient-specific
iPSCs without drug selection. Mol Ther
Nucleic Acids 6:57–67. https://doi.org/10.
1016/j.omtn.2016.11.010
7. Niu X, He W, Song B, Ou Z, Fan D, Chen Y,
Fan Y, Sun X (2016) Combining single strand
oligodeoxynucleotides and CRISPR/Cas9 to
correct gene mutations in beta-thalassemiainduced pluripotent stem cells. J Biol Chem
291:16576–16585.
https://doi.org/10.
1074/jbc.M116.719237
8. Xie F, Ye L, Chang JC, Beyer AI, Wang J,
Muench MO, Kan YW (2014) Seamless gene
correction of beta-thalassemia mutations in
patient-specific iPSCs using CRISPR/Cas9
and piggyBac. Genome Res 24:1526–1533.
https://doi.org/10.1101/gr.173427.114
9. Song B, Fan Y, He W, Zhu D, Niu X, Wang D,
Ou Z, Luo M, Sun X (2015) Improved hematopoietic differentiation efficiency of genecorrected beta-thalassemia induced pluripotent
stem cells by CRISPR/Cas9 system. Stem Cells
Dev 24:1053–1065. https://doi.org/10.
1089/scd.2014.0347
10. Wattanapanitch M, Damkham N, Potirat P,
Trakarnsanga K, Janan M, Yaowalak UP,
Kheolamai P, Klincumhom N, Issaragrisil S
(2018) One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived
210
Methichit Wattanapanitch