The generation of beta-thalassemia (β-Thal) patient-specific induced pluripotent stem cells (iPSCs), subsequent homologous recombination-based gene correction of disease-causing mutations/deletions in the β-globin gene (HBB), and their derived hematopoietic stem cell (HSC) transplantation offers an ideal therapeutic solution for treating this disease. However, the hematopoietic differentiation efficiency of gene-corrected β-Thal iPSCs has not been well evaluated in the previous studies. In this study, we used the latest gene-editing tool, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), to correct β-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and full pluripotency as human embryonic stem cells (hESCs) showed no off-targeting effects. Then, we evaluated the differentiation efficiency of the gene-corrected β-Thal iPSCs. We found that during hematopoietic differentiation, gene-corrected β-Thal iPSCs showed an increased embryoid body ratio and various hematopoietic progenitor cell percentages. More importantly, the gene-corrected β-Thal iPSC lines restored HBB expression and reduced reactive oxygen species production compared with the uncorrected group. Our study suggested that hematopoietic differentiation efficiency of β-Thal iPSCs was greatly improved once corrected by the CRISPR/Cas9 system, and the information gained from our study would greatly promote the clinical application of β-Thal iPSC-derived HSCs in transplantation.
Get full access to this article
View all access options for this article.
References
1.
GalanelloR and OrigaR. (2010). Beta-thalassemia. Orphanet J Rare Dis, 5:11.
2.
RivellaS and RachmilewitzE. (2009). Future alternative therapies for β-thalassemia. Expert Rev Hematol, 2:685.
3.
FanY, LuoY, ChenX, LiQ and SunX. (2012). Generation of human β-thalassemia induced pluripotent stem cells from amniotic fluid cells using a single excisable lentiviral stem cell cassette. J Reprod Dev, 58:404–409.
4.
MarzialiM, IsgròA, GazievJ and LucarelliG. (2009). Hematopoietic stem cell transplantation in thalassemia and sickle cell disease. Unicenter experience in a multi-ethnic population. Mediterr J Hematol Infect Dis, 1:e2009027.
5.
CooleyTB and LeeP. (1925). A series of cases of splenomegaly in children with anemia and peculiar bone changes. Trans Am Pediatr Soc, 37:29–30.
6.
WeatherallDJ. (2001). Phenotype-genotype relationships in monogenic disease: lessons from the thalassaemias. Nat Rev Genet, 2:245–255.
7.
TheinSL. (2005). Pathophysiology of {beta} thalassemia: a guide to molecular therapies. Hematology Am Soc Hematol Educ Program, 1:31–37.
8.
LibaniIV, GuyEC, MelchioriL, SchiroR, RamosP, BredaL, ScholzenT, ChadburnA, LiuY, et al. (2008). Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in beta-thalassemia. Blood, 112:875–885.
9.
TuzmenS and SchechterAN. (2001). Genetic diseases of hemoglobin: diagnostic methods for elucidating beta-thalassemia mutations. Blood Rev, 15:19–29.
10.
RivellaS. (2009). Ineffective erythropoiesis and thalassemias. Curr Opin Hematol, 16:187–194.
11.
RibeilJA, ArletJB, DussiotM, MouraIC, CourtoisG and HermineO. (2013). Ineffective erythropoiesis in β-thalassemia. ScientificWorldJournal, 2013:394295.
12.
WeatherallDJ. (2010). Thalassemia as a global health problem: recent progress toward its control in the developing countries. Ann N Y Acad Sci, 1202:17–23.
13.
IsgròA, GazievJ, SodaniP and LucarelliG. (2010). Progress in hematopoietic stem cell transplantation as allogeneic cellular gene therapy in thalassemia. Ann N Y Acad Sci, 1202:149–154.
14.
BouladF, RivièreI and SadelainM. (2009). Gene therapy for homozygous beta-thalassemia. Is it a reality?. Hemoglobin, 33: S188–S196.
15.
HannaJ, WernigM, MarkoulakiS, SunCW, MeissnerA, CassadyJP, BeardC, BrambrinkT, WuLC, TownesTM and JaenischR. (2007). Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science, 318:1920–1923.
16.
WangY, JiangY, LiuS, SunX and GaoS. (2009). Generation of induced pluripotent stem cells from human beta-thalassemia fibroblast cells. Cell Res, 19:1120–1123.
17.
YeL, ChangJC, LinC, SunX, YuJ and KanYW. (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.
WangY, ZhengCG, JiangY, ZhangJ, ChenJ, YaoC, ZhaoQ, LiuS, ChenK, et al. (2012). Genetic correction of β-thalassemia patient-specific iPS cells and its use in improving hemoglobin production in irradiated SCID mice. Cell Res, 22:637–648.
MaliP, YangL, EsveltKM, AachJ, GuellM, DiCarloJE, NorvilleJE and ChurchGM. (2013). RNA-guided human genome engineering via Cas9. Science, 339:823–826.
22.
SchwankG, KooBK, SasselliV, DekkersJF, HeoI, DemircanT, SasakiN, BoymansS, CuppenE, et al. (2013). Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell, 13:653–658.
23.
XieF, YeL, ChangJC, BeyerAI, WangJ, MuenchMO and KanYW. (2014). Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Res, 24:1526–1533.
24.
YeL, MuenchMO, FusakiN, BeyerAI, WangJ, QiZ, YuJ and KanYW. (2013). Blood cell-derived induced pluripotent stem cells free of reprogramming factors generated by Sendai viral vectors. Stem Cells Transl Med, 2:558–566.
25.
FanY, LuoY, ChenX and SunX. (2010). A modified culture medium increases blastocyst formation and the efficiency of human embryonic stem cell derivation from poor-quality embryos. J Reprod Dev, 56:533–539.
26.
CaoA and KanYW. (2013). The prevention of thalassemia. Cold Spring Harb Perspect Med, 3: a011775.
27.
ChangKH, NelsonAM, CaoH, WangL, NakamotoB, WareCB and PapayannopoulouT. (2006). Definitive-like erythroid from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin. Blood, 108:1515–1523.
28.
ChangKH, HuangA, HirataRK, WangPR, RussellDW and PapayannopoulouT. (2010). Globin phenotype of erythroid cells derived from human induced pluripotent stem cells. Blood, 115:2553–2554.
29.
WangL, LiL, ShojaeiF, LevacK, CerdanC, MenendezP, MartinT, RouleauA and BhatiaM. (2004). Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties. Immunity, 21:31–41.
30.
ChoiKD, YuJ, Smuga-OttoK, SalvagiottoG, RehrauerW, VodyanikM, ThomsonJ and SlukvinI. (2009). Hematopoietic and endothelial differentiation of human induced pluripotent stem cells. Stem Cells, 27:559–567.
31.
Inoue-YokooT, TaniK and SugiyamaD. (2013). Mesodermal and hematopoietic differentiation from ES and iPS cells. Stem Cell Rev, 9:422–434.
32.
KabrunN, BühringHJ, ChoiK, UllrichA, RisauW and KellerG. (1997). Flk-1 expression defines a population of early embryonic hematopoietic precursors. Development, 124:2039–2048.
33.
HuberTL, KouskoffV, FehlingHJ, PalisJ and KellerG. (2004). Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature, 432:625–630.
34.
FresonK, MatthijsG, ThysC, MariënP, HoylaertsMF, VermylenJ and Van GeetC. (2002). Different substitutions at residue D218 of the X-linked transcription factor GATA1 lead to altered clinical severity of macrothrombocytopenia and anemia and are associated with variable skewed X inactivation. Hum Mol Genet, 11:147–152.
35.
VakocCR, LettingDL, GheldofN, SawadoT, BenderMA, GroudineM, WeissMJ, DekkerJ and BlobelGA. (2005). Proximity among distant regulatory elements at the beta-globin locus requires GATA-1 and FOG-1. Mol Cell, 17:453–462.
36.
HasegawaA, ShimizuR, MohandasN and YamamotoM. (2012). Mature erythrocyte membrane homeostasis is compromised by loss of the GATA1-FOG1 interaction. Blood, 119:2615–2623.
37.
SattaS, PerseuL, MaccioniL, GiaguN and GalanelloR. (2012). Delayed fetal hemoglobin switching in subjects with KLF1 gene mutation. Blood Cells Mol Dis, 48:22–24.
38.
LiakhovitskaiaA, RybtsovS, SmithT, BatsivariA, RybtsovaN, RodeC, de BruijnM, BuchholzF, Gordon-KeylockS, ZhaoS and MedvinskyA. (2014). Runx1 is required for progression of CD41+embryonic precursors into HSCs but not prior to this. Development, 141:3319–3323.
39.
DumonS, WaltonDS, VolpeG, WilsonN, DasséE, Del PozzoW, LandryJR, TurnerB, O'NeillLP, GöttgensB and FramptonJ. (2012). Itga2b regulation at the onset of definitive hematopoiesis and commitment to differentiation. PLoS One, 7: e43300.
40.
MathiasLA, FisherTC, ZengL, MeiselmanHJ, WeinbergKI, HitiAL and MalikP. (2000). Ineffective erythropoiesis in beta-thalassemia major is due to apoptosis at the polychromatophilic normoblast stage. Exp Hematol, 28:1343–1353.
41.
De FranceschiL, BertoldiM, De FalcoL, Santos FrancoS, RonzoniL, TurriniF, ColanceccoA, CamaschellaC, CappelliniMD and IolasconA. (2011). Oxidative stress modulates heme synthesis and induces peroxiredoxin-2 as a novel cytoprotective response in β-thalassemic erythropoiesis. Haematologica, 96:1595–1604.
42.
CentisF, TabelliniL, LucarelliG, BuffiO, TonucciP, PersiniB, AnnibaliM, EmilianiR, IliescuA, et al. (2000). The importance of erythroid expansion in determining the extent of apoptosis in erythroid precursors in patients with beta-thalassemia major. Blood, 96:3624–3629.
43.
PalHC, SharmaS, StricklandLR, AgarwalJ, AtharM, ElmetsCA and AfaqF. (2013). Delphinidin reduces cell proliferation and induces apoptosis of non-small-cell lung cancer cells by targeting EGFR/VEGFR2 signaling pathways. PLoS One, 8:e77270.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
