Summary of pluripotent stem cells and gene editing tools to generate human disease models.
Human pluripotent stem cells (PSCs) in the form of human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) are capable of growing indefinitely in vitro, maintaining their capacity to differentiate into the three primary germ layers: mesoderm, endoderm and ectoderm. Different protocols have been developed to differentiate PSCs into almost any cellular type with different degree of success. This technology has allowed scientists to use patient‐derived iPSCs to study the physiopathology of the disease by analyzing the phenotype of the cells derived from these iPSCs. However, control iPSCs obtained from healthy individuals will always have different genomic environment than patient's iPSCs, making it difficult the interpretation of the cells phenotype. The recent appearance of specific nucleases [zinc‐finger nucleases (ZFNs), the transcription activator‐like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)] has made it possible to edit the genome of PSCs. We can now generate syngeneic hESCs or iPSCs harboring the desired mutation and comparing the emerging cells with those derived from genetically identical PSCs that will differ only in the mutated gene. In this chapter, we summarize the progress made in this field and discuss the different approaches that have been used recently for the generation of syngeneic human pluripotent cellular models for different pathologies.
- human pluripotent stem cells
- disease models
Disease models are an essential tool for elucidating the molecular basis of several pathologies, allowing the development of novel therapies. Historically, and taking into account the scarcity of the patient cells, the use of model organism made possible the clarification of cellular mechanisms underlying human disease.
2. Disease modeling with human stem cells derived from patients
The advantage of using human stem cells derived from patients is that they can be isolated at different stages of disease severity. For example, by isolating stem cells from patients with end‐stage disease, we could study those phenotypes resulting from a combination of insults in the patient that take the patient to that particular stage. These particular phenotypes are practically impossible to mimic by other means such as genetic or epigenetic manipulations.
2.1. Human adult stem cells
Adult stem cells are multipotent cells found in all adult tissues, and they participate in the physiological regeneration of the tissues where they belong. The adult stem cell better characterized and with better perspectives for use as human models of disease are the neural progenitor cells (NPCs), mesenchymal stromal cells (MSCs) and the HSCs:
2.1.1. Neural progenitor cells
NPCs comprise relatively undifferentiated cell population in the central nervous systems (CNSs) that give rise to a broad array of specialized cells, including neurons and glial cells. These cells can be isolated and cultured
2.1.2. Mesenchymal stromal cells
MSCs represent another interesting alternative for disease modeling. The International Society for Cellular Therapy (ISCT) established that MSCs must be purified from stromal populations based on plastic adherence and must be positive for CD105, CD90 and CD73, negative for MHC‐II, CD11b, CD14, CD34, CD45 and CD31 and express low levels of MHC‐I. In addition, MSCs must differentiate
MSCs are a very attractive source for disease modeling because they are able to give rise to different tissue types. Therefore, MSCs derived from patients could be used to model diseases affecting tissues to which MSCs can be derived to. Contrary to induced pluripotent stem cells (iPSCs), MSCs are not induced by the expression of gene involved in oncogenic cells transformation (discussed later). Due to their relative abundance within the body, the MSCs could be used as novel human
2.1.3. Hematopoietic stem cells
HSCs have the potential to give rise to all hematopoietic cells
2.2. Human embryonic stem cells from embryos with genetic diseases
Human embryonic stem cells (hESCs), derived from pre‐implantation embryos, were the first human pluripotent stem cells (PSCs) to be isolated. Thanks to the pre‐implantation genetic diagnosis (PGD), it is now possible to generate hESCs from monogenetic diseases [15–17]. Using these approaches, hESCs derived from embryos with FXS, Huntington's disease (HD) and familial dysautonomia (FD) have been generated [18–21]. The authors observed that
2.3. Induced pluripotent stem cells
The development of the iPSCs technologies  bypassed these limitations since we can now generate hESC‐like cells from patients. The ability of iPSCs to differentiate into virtually any tissue or cell type makes these cells an excellent tool for modeling human disease. These cells allow the generation of patient‐specific disease‐relevant cells in virtually a continuous manner. This is imperative in the case where the disease is a result of an interplay genetics risk, instead of a punctual mutation. This is the case of the vast majority of the neurodegenerative disorders where only the 5–10% are Mendelian disorders caused by a punctual mutation, the rest are a result of multifactorial genetic association. Several iPSCs models have been developed for neurodegenerative diseases including HD [24–29], Alzheimer's disease  and spinal muscular atrophy [31–36]. Several inherited bone marrow failure (BMF) syndromes have also been modeled
3. Disease modeling with gene editing tools and stem cells
iPSCs and hESCs from embryos with genetic diseases (hESCs‐GD) provide powerful tools for modeling human diseases, although the different genetic background of the control cells versus the patient‐specific cells makes the interpretation of the results difficult . A solution to this problem is the use of genome editing (GE) technologies to generate the desired mutations into iPSCs or hESCs . This approach allows the direct comparison of PSCs harboring the desired mutation with isogenic control cells lines. However, until recently, this approach presents low efficiency and specificity. This situation changed considerably with the appearance of specific nucleases (SNs). The introduction of double‐strand breaks (DSBs) in the targeted DNA sequence has dramatically improved the homologous recombination up to 10,000. Several SNs have been described such as the meganuclease I‐SceI, the zinc‐finger nucleases (ZFNs), the transcription activator‐like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)‐associated system 9 (CRISPR/Cas9). Meganucleases, also called homing endonucleases, are a class of highly sequence‐specific enzymes that recognize a relative large long DNA sequence ranging from 12 to 30 bp. The recognition site of MNs can be engineered in order to target specific sites within the genome. One of the major advantages of the meganucleases is their small size making them amenable to be packaged into single viral vector and allowing efficient delivery. However, this technology requires good knowledge of protein engineering and has been limited to some DNA targets . A major breakthrough arises with the discovery of ZFNs [46–48], the first SNs capable to target almost any DNA sequence in the human genome. ZFNs are chimeric proteins that combine a nuclease domain (FokI) and a zinc‐finger domain (ZFD) that recognizes the targeted DNA sequence. The specificity is therefore determined by the ZFD that harbors four‐six zinc fingers of 30 amino acids. ZFNs are easier to construct in comparison with MNs, but still required intensive labor to obtain site‐specific ZFNs. Soon after the appearance of ZFNs, a new protein‐based SNs, TALENs, were described [49–51]. TALENs are a combination of the catalytic domain of an endonuclease (FokI) fused with a DNA‐binding domain derived from transcription activator‐like effectors from plant pathogen
All these GE technologies have opened up the possibility to obtain almost any mutation in any cell type. Therefore, different groups have already applied these technologies to model human disease by generating disease‐causing mutations in primary stem cells:
3.1. Editing hHSCs for disease modeling
Gene therapy targeting HSCs is a real option now for several genetic diseases, including severe combined immunodeficiency (SCID) and other severe non‐SCID PID. The PID patient has been treated classically by allogeneic HSCs transplantation or by the correction of the patient's own HSCs by the insertion of a functional copy of the affected gene by a viral vector [52, 53]. However, to further improve HSCs‐based gene therapy, HSCs cellular models capable to recapitulate the disease phenotype are needed. The low frequency of these disorders and the difficulties to obtain HSCs from the patients preclude the use of patient HSCs as standard tools for disease modeling and preclinical testing. As an alternative, GE tools can be used to generate the disease‐causing mutation in HSCs obtained from umbilical cord. For example, a X‐linked severe combined immunodeficiency (SCID‐X1) induced by a mutation in the IL2RG gene using specific ZFNs has been recently modeled
3.2. Editing hESCs for disease modeling
As we mentioned before, hESCs‐GD can be generated and could in theory be an excellent tool for any disease. We also mentioned the problems this strategy face if we want to extend its use to model any disease . GE technologies have allowed the development of a very potent alternative: the generation of gene‐edited hESCs harboring the disease‐causing mutation. This strategy has the additional advantage of count with isogenic hESCs that only differed in the mutation and are otherwise genetically identical.
Then, we will describe some examples of how different groups have used GE to model different diseases:
Several other monogenetic diseases have been modeled by GE of hESCs (see Table 1). Martinez
|Disease||Gene||Gene editing tools||Cell type||References|
|Hemophilia A||Coagulation factor VIII|
|AIDS||Chemokine (C‐C motif)|
receptor 5 (CCR5)
|Imaging purpose||AAVS1||ZFN||iPSC, hESCs|||
|Fragile X syndrome (FXS)||FMR1||CRISPR/Cas9||iPSCs|||
|Ewing sarcoma and|
anaplastic large cell
|TALEN and ZFNs||hESCs|||
|Coronary artery disease||SORT1||TALEN||hESCs|||
|X‐linked severe combined|
subtype of partial
3.3. Editing iPSCs for disease modeling
As mentioned before, iPSCs can be obtained from any patient representing very potent tools for disease modeling. However, several considerations should be taken into account when interpreting the phenotypes of iPSCs from patients and controls since they have different genetic backgrounds. Indeed, a major concern to interpret the data coming out from a disease‐associated iPSCs is the choice of appropriate controls. In addition, several studies have shown that the generation and expansion of different iPSCs from some patients’ lines shows some genetic aberration ranging from single nucleotide variant, chromosomal deletion and differences in the methylation pattern of their genomes. An interesting solution to this problem is the GE of iPSCs from healthy individuals, as we have described previously for hESCs, in order to generate “home‐made” disease‐specific iPSCs Figure 1. This approach allows the direct comparison of “gene‐edited” iPSCs cell line with its isogenic control. Then, we will describe some examples of modeling human disease using this approach:
Parkinson's disease (PD) iPSCs were generated by mutating the LRRK2 gene in iPSCs lines from a healthy control using ZFNs. Neurons derived from gene‐edited PD iPSCs exhibit reduced neurite outgrowth and increased apoptosis in response to oxidative stress when compared with neuron derived from isogenic control iPSCs [60, 82]. Park
This work has been financed by Fondo de Investigaciones Sanitarias ISCIII (Spain) and Fondo Europeo de Desarrollo Regional (FEDER) from the European Union through the research grants PI12/01097 (F.M.) and by the CICE and CS de la Junta de Andalucía FEDER/Fondo de Cohesion Europeo (FSE) de Andalucía 2007–2013 through research grants P09‐CTS‐04532, PI‐57069, PI‐0001/2009 and PAIDI‐Bio‐326 (F.M.); PI‐0160/2012 (K.B.).
Casci, I. and U.B. Pandey, A fruitful endeavor: modeling ALS in the fruit fly.Brain Res, 2014. 1607: p. 47–74.
Lepesant, J.A., The promises of neurodegenerative disease modeling.C R Biol, 2015. 338(8–9): p. 584–92.
Jucker, M., The benefits and limitations of animal models for translational research in neurodegenerative diseases.Nat Med, 2010. 16(11): p. 1210–4.
Legrand, N., K. Weijer, and H. Spits, Experimental models to study development and function of the human immune system in vivo.J Immunol, 2006. 176(4): p. 2053–8.
Shultz, L.D., F. Ishikawa, and D.L. Greiner, Humanized mice in translational biomedical research.Nat Rev Immunol, 2007. 7(2): p. 118–30.
Zhang, B., Z. Duan, and Y. Zhao, Mouse models with human immunity and their application in biomedical research.J Cell Mol Med, 2009. 13(6): p. 1043–58.
Haidet‐Phillips, A.M., et al., Astrocytes from familial and sporadic ALS patients are toxic to motor neurons.Nat Biotechnol, 2011. 29(9): p. 824–8.
Schwartz, P.H., et al., Neural progenitor cells from an adult patient with fragile X syndrome.BMC Med Genet, 2005. 6: p. 2.
Dossena, M., et al., Human adipose‐derived mesenchymal stem cells as a new model of spinal and bulbar muscular atrophy.PLoS One, 2014. 9(11): p. e112746.
Orr, H.T., Cell biology of spinocerebellar ataxia.J Cell Biol, 2012. 197(2): p. 167–77.
Joseph, A., et al., Inhibition of in vivo HIV infection in humanized mice by gene therapy of human hematopoietic stem cells with a lentiviral vector encoding a broadly neutralizing anti‐HIV antibody.J Virol, 2010. 84(13): p. 6645–53.
Ludtke, A., et al., Ebola virus disease in mice with transplanted human hematopoietic stem cells.J Virol, 2015. 89(8): p. 4700–4.
Scaramuzza, S., et al., Preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vector for the treatment of Wiskott‐Aldrich syndrome.Mol Ther, 2013. 21(1): p. 175–84.
Levasseur, D.N., et al., Correction of a mouse model of sickle cell disease: lentiviral/antisickling beta‐globin gene transduction of unmobilized, purified hematopoietic stem cells.Blood, 2003. 102(13): p. 4312–9.
Mateizel, I., et al., Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders.Hum Reprod, 2006. 21(2): p. 503–11.
Ben‐Yosef, D., M. Malcov, and R. Eiges, PGD‐derived human embryonic stem cell lines as a powerful tool for the study of human genetic disorders.Mol Cell Endocrinol, 2008. 282(1–2): p. 153–8.
Verlinsky, Y., et al., Human embryonic stem cell lines with genetic disorders.Reprod Biomed Online, 2005. 10(1): p. 105–10.
Niclis, J., et al., Human embryonic stem cell models of Huntington disease.Reprod Biomed Online, 2009. 19(1): p. 106–13.
Eiges, R., et al., Developmental study of fragile X syndrome using human embryonic stem cells derived from preimplantation genetically diagnosed embryos.Cell Stem Cell, 2007. 1(5): p. 568–77.
Niclis, J.C., et al., Characterization of forebrain neurons derived from late‐onset Huntington's disease human embryonic stem cell lines.Front Cell Neurosci, 2013. 7: p. 37.
Lefler, S., et al., Familial dysautonomia (FD) Human embryonic stem cell derived PNS neurons reveal that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation.PLoS One, 2015. 10(10): p. e0138807.
Feyeux, M., et al., Early transcriptional changes linked to naturally occurring Huntington's disease mutations in neural derivatives of human embryonic stem cells.Hum Mol Genet, 2012. 21(17): p. 3883–95.
Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell, 2006. 126(4): p. 663–76.
Arber, C., et al., Activin A directs striatal projection neuron differentiation of human pluripotent stem cells.Development, 2015. 142(7): p. 1375–86.
Juopperi, T.A., et al., Astrocytes generated from patient induced pluripotent stem cells recapitulate features of Huntington's disease patient cells.Mol Brain, 2012. 5: p. 17.
Mattis, V.B., et al., HD iPSC‐derived neural progenitors accumulate in culture and are susceptible to BDNF withdrawal due to glutamate toxicity.Hum Mol Genet, 2015. 24(11): p. 3257–71.
Mattis, V.B. and C.N. Svendsen, Modeling Huntington's disease with patient‐derived neurons.Brain Res, 2015. doi: 10.1016/j.brainres.2015.10.001 [Epub ahead of print].
Russo, F.B., et al., Induced pluripotent stem cells for modeling neurological disorders.World J Transplant, 2016. 5(4): p. 209–21.
Zhang, N., et al., iPSC‐based drug screening for Huntington's disease.Brain Res, 2015. doi: 10.1016/j.brainres.2015.09.020 [Epub].
Sullivan, S.E. and T.L. Young‐Pearse, Induced pluripotent stem cells as a discovery tool for Alzheimer's disease.Brain Res, 2015. doi: 10.1016/j.brainres.2015.10.005 [Epub ahead of print].
Frattini, E., et al., Pluripotent stem cell‐based models of spinal muscular atrophy.Mol Cell Neurosci, 2014. 64: p. 44–50.
Liu, H., et al., Spinal muscular atrophy patient‐derived motor neurons exhibit hyperexcitability.Sci Rep, 2015. 5: p. 12189.
Ohuchi, K., et al., Established stem cell model of spinal muscular atrophy is applicable in the evaluation of the efficacy of thyrotropin‐releasing hormone analog.Stem Cells Transl Med, 2015. 5(2): p. 152–63.
Valetdinova, K.R., S.P. Medvedev, and S.M. Zakian, Model systems of motor neuron diseases as a platform for studying pathogenic mechanisms and searching for therapeutic agents.Acta Naturae, 2015. 7(1): p. 19–36.
Yoshida, M., et al., Modeling the early phenotype at the neuromuscular junction of spinal muscular atrophy using patient‐derived iPSCs.Stem Cell Reports, 2015. 4(4): p. 561–8.
Fuller, H.R., et al., Spinal muscular atrophy patient iPSC‐derived motor neurons have reduced expression of proteins important in neuronal development.Front Cell Neurosci, 2016. 9: p. 506.
Winkler, T., et al., Defective telomere elongation and hematopoiesis from telomerase‐mutant aplastic anemia iPSCs.J Clin Invest, 2013. 123(5): p. 1952–63.
Raya, A., et al., A protocol describing the genetic correction of somatic human cells and subsequent generation of iPS cells.Nat Protoc, 2010. 5(4): p. 647–60.
Liu, G.H., et al., Modelling Fanconi anemia pathogenesis and therapeutics using integration‐free patient‐derived iPSCs.Nat Commun, 2014. 5: p. 4330.
Suzuki, N.M., et al., Pluripotent cell models of fanconi anemia identify the early pathological defect in human hemoangiogenic progenitors.Stem Cells Transl Med, 2015. 4(4): p. 333–8.
Hirata, S., et al., Congenital amegakaryocytic thrombocytopenia iPS cells exhibit defective MPL‐mediated signaling.J Clin Invest, 2013. 123(9): p. 3802–14.
Wang, G., et al., Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart‐on‐chip technologies.Nat Med, 2014. 20(6): p. 616–23.
Saha, K. and R. Jaenisch, Technical challenges in using human induced pluripotent stem cells to model disease.Cell Stem Cell, 2009. 5(6): p. 584–95.
Musunuru, K., Genome editing of human pluripotent stem cells to generate human cellular disease models.Dis Model Mech, 2013. 6(4): p. 896–904.
Arnould, S., et al., The I‐CreI meganuclease and its engineered derivatives: applications from cell modification to gene therapy.Protein Eng Des Sel, 2011. 24(1–2): p. 27–31.
Bibikova, M., et al., Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc‐finger nucleases.Genetics, 2002. 161(3): p. 1169–75.
Bibikova, M., et al., Enhancing gene targeting with designed zinc finger nucleases.Science, 2003. 300(5620): p. 764.
Urnov, F.D., et al., Highly efficient endogenous human gene correction using designed zinc‐finger nucleases.Nature, 2005. 435(7042): p. 646–51.
Wood, A.J., et al., Targeted genome editing across species using ZFNs and TALENs.Science, 2011. 333(6040): p. 307.
Boch, J., et al., Breaking the code of DNA binding specificity of TAL‐type III effectors.Science, 2009. 326(5959): p. 1509–12.
Moscou, M.J. and A.J. Bogdanove, A simple cipher governs DNA recognition by TAL effectors.Science, 2009. 326(5959): p. 1501.
Fischer, A., et al., Gene therapy for primary immunodeficiencies.Clin Genet, 2015. 88(6): p. 507–15.
Ott de Bruin, L.M., S. Volpi, and K. Musunuru, Novel genome‐editing tools to model and correct primary immunodeficiencies.Front Immunol, 2015. 6: p. 250.
Genovese, P., et al., Targeted genome editing in human repopulating haematopoietic stem cells.Nature, 2014. 510(7504): p. 235–40.
Urbinati, F., et al., Potentially therapeutic levels of anti‐sickling globin gene expression following lentivirus‐mediated gene transfer in sickle cell disease bone marrow CD34+ cells.Exp Hematol, 2015. 43(5): p. 346–51.
Hoban, M.D., et al., Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells.Blood, 2015. 125(17): p. 2597–604.
Mandal, P.K., et al., Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9.Cell Stem Cell, 2014. 15(5): p. 643–52.
Tebas, P., et al., Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.N Engl J Med, 2014. 370(10): p. 901–10.
Piganeau, M., et al., Cancer translocations in human cells induced by zinc finger and TALE nucleases.Genome Res, 2013. 23(7): p. 1182–93.
Reinhardt, P., et al., Genetic correction of a LRRK2 mutation in human iPSCs links Parkinsonian neurodegeneration to ERK‐dependent changes in gene expression.Cell Stem Cell, 2013. 12(3): p. 354–67.
Li, Y., et al., Global transcriptional and translational repression in human‐embryonic‐stem‐cell‐derived Rett syndrome neurons.Cell Stem Cell, 2014. 13(4): p. 446–58.
Toscano, M.G., et al., Absence of WASp enhances hematopoietic and megakaryocytic differentiation in a human embryonic stem cell model.Mol Ther, 2015. 24(2): p. 342–53.
Ingrungruanglert, P., et al., Wiskott‐Aldrich syndrome iPS cells produce megakaryocytes with defects in cytoskeletal rearrangement and proplatelet formation.Thromb Haemost, 2015. 113(4): p. 792–805.
Hockemeyer, D., et al., Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc‐finger nucleases.Nat Biotechnol, 2009. 27(9): p. 851–7.
Brunet, E., et al., Chromosomal translocations induced at specified loci in human stem cells.Proc Natl Acad Sci U S A, 2009. 106(26): p. 10620–5.
Park, C.Y., et al., Targeted inversion and reversion of the blood coagulation factor 8 gene in human iPS cells using TALENs.Proc Natl Acad Sci U S A, 2014. 111(25): p. 9253–8.
Lee, H.J., et al., Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases.Genome Res, 2012. 22(3): p. 539–48.
Kang, H., et al., CCR5 disruption in induced pluripotent stem cells using CRISPR/Cas9 provides selective resistance of immune cells to CCR5‐tropic HIV‐1 virus.Mol Ther Nucleic Acids, 2015. 4: p. e268.
Ramalingam, S., et al., Generation and genetic engineering of human induced pluripotent stem cells using designed zinc finger nucleases.Stem Cells Dev, 2012. 22(4): p. 595–610.
Ru, R., et al., Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs.Cell Regen (Lond), 2013. 2(1): p. 5.
Lei, Y., et al., Gene editing of human embryonic stem cells via an engineered baculoviral vector carrying zinc‐finger nucleases.Mol Ther, 2011. 19(5): p. 942–50.
Wang, Y., et al., Genome editing of human embryonic stem cells and induced pluripotent stem cells with zinc finger nucleases for cellular imaging.Circ Res, 2012. 111(12): p. 1494–503.
Zhang, N., et al., CRISPR/Cas9‐mediated conversion of human platelet alloantigen allotypes.Blood, 2015. doi: 10.1182/blood‐2015‐10‐675751 [Epub].
Wang, P., et al., CRISPR/Cas9‐mediated heterozygous knockout of the autism gene CHD8 and characterization of its transcriptional networks in neurodevelopment.Mol Autism, 2015. 6: p. 55.
Park, C.Y., et al., Reversion of FMR1 methylation and silencing by editing the triplet repeats in fragile X iPSC‐derived neurons.Cell Rep, 2015. 13(2): p. 234–41.
Liu, J., et al., CRISPR/Cas9 facilitates investigation of neural circuit disease using human iPSCs: mechanism of epilepsy caused by an SCN1A loss‐of‐function mutation.Transl Psychiatry, 2016. 6: p. e703.
Zou, J., et al., Gene targeting of a disease‐related gene in human induced pluripotent stem and embryonic stem cells.Cell Stem Cell, 2009. 5(1): p. 97–110.
Flaherty, E.K. and K.J. Brennand, Using hiPSCs to model neuropsychiatric copy number variations (CNVs) has potential to reveal underlying disease mechanisms.Brain Res. 2015 Nov 12. pii: S0006-8993(15)00852‐5. doi: 10.1016/j.brainres.2015.11.009. [Epub ahead of print].
Vanuytsel, K., et al., FANCA knockout in human embryonic stem cells causes a severe growth disadvantage.Stem Cell Res, 2014. 13(2): p. 240–50.
Martinez, R.A., et al., Genome engineering of isogenic human ES cells to model autism disorders.Nucleic Acids Res, 2015. 43(10): p. e65.
Ding, Q., et al., A TALEN genome‐editing system for generating human stem cell‐based disease models.Cell Stem Cell, 2012. 12(2): p. 238–51.
Soldner, F., et al., Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations.Cell, 2011. 146(2): p. 318–31.
Yao, Y., et al., Generation of CD34+ cells from CCR5‐disrupted human embryonic and induced pluripotent stem cells.Hum Gene Ther, 2011. 23(2): p. 238–42.
Wang, Y., et al., Genome editing of isogenic human induced pluripotent stem cells recapitulates long QT phenotype for drug testing.J Am Coll Cardiol, 2014. 64(5): p. 451–9.
Liu, Y. and W. Deng, Reverse engineering human neurodegenerative disease using pluripotent stem cell technology.Brain Res. 2016 May 1; 1638(Pt A):30‐41. doi: 10.1016/j.brainres.2015.09.023. Epub 2015 Sep 28.