Efficient CRISPR/Cas9-based gene correction in induced pluripotent stem cells established from fibroblasts of patients with sickle cell disease

Since Takahashi and Yamanaka (1) first reported the successful establishment of induced pluripotent stem cells (iPSCs), these cells have been an important resource for regenerative medicine and gene therapy strategies (2). In particular, the use of iPSCs helps avoid the ethical concerns associated with the use of human embryonic stem cells (ESCs) established from donor oocytes, and mitigate the possibility of immune rejection by allowing the generation of “patient-specific” pluripotent stem cells from somatic cell types (3). Furthermore, the recent advent of site-specific genome-editing technologies, such as the CRISPR/Cas9 system, has enabled “gene correction” by inserting normal sequences or deleting mutated sequences from mutated sites in the genome (4,5). These techniques have opened the doors to curing genetic disorders caused by mutations in a specific gene. One promising approach is gene correction in iPSCs established from somatic cells of patients with genetic defects. The differentiated derivatives (e.g., neurons, hematopoietic cells, and cardiomyocytes) of successfully genome-edited iPSCs can be used for the replacement of damaged tissues through autologous transplantation. In fact, successful gene correction has been reported in iPSCs derived from patients with cystic fibrosis and β-thalassemia (6-10).

Generally, human ESCs derived from early embryos and iPSCs are in a primed state of pluripotency (hereafter referred to as “primed” cells) similar to mouse epiblast stem cells (11). These cells are distinct from the naïve pluripotent stem cells (hereafter referred to as “naïve” cells) of mouse ESCs and iPSCs in terms of colony morphology (flat or dome shape), single-cell passage ability [Rho-associated coiled-coil forming kinase (ROCK) inhibitor-independent or not], pluripotent gene expression profiles (similar to primed or naïve ESCs/iPSCs), signaling pathway [mitogen-activated protein kinase (MAPK) kinase (MEK)-extracellular signal-regulated kinase (ERK)-dependent or leukemia inhibitory factor (LIF)-signal transducer and activator of transcription 3 (STAT3)-dependent], and the ability to differentiate into various types of cells. The relatively low differentiation ability of primed ESCs/iPSCs is critical for their use in personalized medicine since it is often difficult to obtain the cell/tissue type that an investigator requires. Thus, several attempts have been made to convert the primed ESCs/iPSCs to naïve cells (12-15). For example, Theunissen et al. (14) incubated small colonies of iPSCs, which were transfected with vectors containing Yamanaka’s factors to induce reprogramming, in N2B27 basal medium supplemented with inhibitors of MEK, glycogen synthase kinase 3β (GSK3β), ROCK, SRC, and B-Raf (BRAF) kinases, together with LIF and activin (the so-called “5i/L/FA” medium), for about 10 days. After the incubation period, the colony morphology changed to a dome-like shape and the cells exhibited pluripotent gene expression profiles similar to those of naïve ESCs/iPSCs. Furthermore, various types of differentiated cells were generated when these iPSCs were subcutaneously transplanted into immunocompromised mice, suggesting the acquisition of multipotency.

Yang et al. (16) successfully demonstrated that it is possible to obtain naïve iPSCs directly from fibroblasts isolated from patients with β-thalassemia. β-Thalassemia, which is also called sickle cell disease (SCD), is one of the most common genetic diseases worldwide. It is an inherited blood disorder that causes severe anemia and is characterized by reduction in or absence of synthesis of hemoglobin (HB) subunit β (HB β chain). The most common molecular defects are either point mutations or small fragment deletions in the HBB gene, which affect mRNA assembly or translation. Yang et al. (16) first transfected fibroblasts carrying the β-41/42 mutation with 3 plasmids carrying Yamanaka factors (obtained from Addgene) by electroporation. The transfectants were cultured on mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeders in a conventional human ES medium for 6 days and gave rise to small iPSC colonies. Further culture of the cells for 14 to 20 days in human naïve medium (5i/L/LA medium) resulted in the generation of dome-shaped colonies. Analysis of these colonies revealed the expression of pluripotent marker genes such as NANOG, KLF4, REX1 and STELLA—a global gene expression pattern similar to that of naïve ESCs/iPSCs, which have single-cell passage ability. More interestingly, inoculation of these colonies under the skin of immunocompromised mice resulted in the generation of solid tumors containing various types of differentiated cells. Next, they attempted to correct the β-41/42 mutation in the HBB gene in the resulting naïve iPSCs. For this, they used the knock-in (KI)-based CRISPR/Cas9 genome editing system. They constructed a KI donor vector in which the normal HBB sequence was flanked by ~250-bp long 5' and 3' homologous arms of the gene. The naïve iPSCs thus obtained were subjected to electroporation in the presence of a donor vector and the pX330 vector containing both the guide RNA and Cas9. Concomitantly, primed iPSCs derived from the same patient were transfected. Seven days after transfection, 40 colonies were picked up for genomic DNA analysis. Sequencing of the PCR-amplified fragments spanning the mutated site demonstrated that 57% of clones in the naïve iPSC group were successfully corrected at one allele of the HBB gene. In contrast, in the primed iPSC group, only 32% of the clones were corrected. This result implies that naïve iPSCs are more amenable to gene correction by the CRISPR/Cas9-based genome editing system than primed iPSCs are.

The most important aspect of this study would be how to efficiently differentiate genome-modified naïve iPSCs into hematopoietic progenitor cells. Yang et al. (16) cultured gene-corrected naïve iPSCs, parental naïve iPSCs, or primed iPSCs on the OP9-GFP stromal cells, a genetically modified murine stromal cell line expressing green fluorescent protein (GFP) for 8 days. Flow cytometry revealed that the proportion of CD34⁺ cells among the corrected naïve iPSCs was approximately 4.3%, which was comparable to that of the parental naïve iPSCs (approximately 3.7%) and the primed iPSCs (approximately 4.0%). This might have been an unexpected result for the authors, because naïve iPSCs are expected to be more frequently differentiated into hematopoietic cells than primed iPSCs. In this context, a system that promotes more efficient differentiation of iPSCs into hematopoietic cells is needed.

When differentiated derivatives of iPSCs are used as therapeutic materials for treating damaged tissues, the risk of tumorigenicity arising from the residual iPSCs after autologous transplantation would be a major concern (17). Notably, Chandrakanthan et al. (18) recently demonstrated that adult human adipose cells isolated from fat tissues can be reprogrammed to tissue-regenerative multipotent stem cells (called iMS cells) by culturing them with 5-azacytidine (AZA) and platelet-derived growth factor-AB (PDGF-AB) for approximately 2 days, followed by treatment with the growth factor alone for a further 2−3 weeks. AZA is a demethylating nucleoside analog that is widely used in clinical practice. It is also known to induce cell plasticity, which is crucial for reprogramming cells. The resulting iMS cells exhibited long-term self-renewal, serial clonogenicity, and the potential to differentiate into cells of multiple germ layers. Importantly, unlike ESCs or iPSCs, iMS cells do not form teratomas. This new vector-free method of generating iMS cells from fat cells is a promising tool for safer cell-based therapy against genetic diseases.

Acknowledgements

Funding: This work was partly supported by grants (no. 24580411 for M Sato, no. 25293418 for I Saitoh, and no. 25463192 for E Inada) from The Ministry of Education, Science, Sports, and Culture, Japan.

Footnote

Provenance: This is an invited Editorial commissioned by Editor-in-Chief Zhizhuang Joe Zhao (Pathology Graduate Program, University of Oklahoma Health Sciences Center, Oklahoma City, USA).

Conflicts of Interest: The authors have no conflicts of interest to declare.

Comments on: Yang Y, Zhang X, Yi L, et al. Naïve induced pluripotent stem cells generated from β-thalassemia fibroblasts allow efficient gene correction with CRISPR/Cas9. Stem Cells Transl Med. 2016;5:8-19.

References

1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663-76. [Crossref] [PubMed]
2. Polo JM, Anderssen E, Walsh RM, et al. A molecular roadmap of reprogramming somatic cells into iPS cells. Cell 2012;151:1617-32. [Crossref] [PubMed]
3. Yamanaka S. Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell 2007;1:39-49. [Crossref] [PubMed]
4. Maeder ML, Gersbach CA. Genome-editing Technologies for Gene and Cell Therapy. Mol Ther 2016;24:430-46. [PubMed]
5. Park CY, Lee DR, Sung JJ, et al. Genome-editing technologies for gene correction of hemophilia. Hum Genet 2016;135:977-81. [Crossref] [PubMed]
6. Sebastiano V, Maeder ML, Angstman JF, et al. In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases. Stem Cells 2011;29:1717-26. [Crossref] [PubMed]
7. Zou J, Mali P, Huang X, et al. Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease. Blood 2011;118:4599-608. [Crossref] [PubMed]
8. Soldner F, Laganière J, Cheng AW, et al. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 2011;146:318-31. [Crossref] [PubMed]
9. Yusa K, Rashid ST, Strick-Marchand H, et al. Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells. Nature 2011;478:391-4. [Crossref] [PubMed]
10. Schwank G, Koo BK, Sasselli V, et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 2013;13:653-8. [Crossref] [PubMed]
11. Nichols J, Smith A. Naive and primed pluripotent states. Cell Stem Cell 2009;4:487-92. [Crossref] [PubMed]
12. Gafni O, Weinberger L, Mansour AA, et al. Derivation of novel human ground state naive pluripotent stem cells. Nature 2013;504:282-6. [Crossref] [PubMed]
13. Hanna J, Cheng AW, Saha K, et al. Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proc Natl Acad Sci U S A 2010;107:9222-7. [Crossref] [PubMed]
14. Theunissen TW, Powell BE, Wang H, et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 2014;15:471-87. [Crossref] [PubMed]
15. Ware CB, Nelson AM, Mecham B, et al. Derivation of naive human embryonic stem cells. Proc Natl Acad Sci U S A 2014;111:4484-9. [Crossref] [PubMed]
16. Yang Y, Zhang X, Yi L, et al. Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9. Stem Cells Transl Med 2016;5:8-19. [Crossref] [PubMed]
17. Ben-David U, Benvenisty N. The tumorigenicity of human embryonic and induced pluripotent stem cells. Nat Rev Cancer 2011;11:268-77. [Crossref] [PubMed]
18. Chandrakanthan V, Yeola A, Kwan JC, et al. PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells. Proc Natl Acad Sci U S A 2016;113:E2306-15. [Crossref] [PubMed]