Embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocyst [12
], and because they are pluripotent can give rise to all three germ layers (Box 1
). Due to the pluripotent and self-renewing capacities, ESCs are considered cellular resources for modeling human diseases as well as treating damaged human organs and tissues [14
]. iPSCs have been created by reprogramming differentiated cells by the ectopic expression of the four transcription factors Oct4
, and Klf-4
]. iPSCs and ESCs share characteristics such as self-renewing capacity, morphology, expression of surface markers, and pluripotent differentiation potential. Because iPSCs retain the genetic composition of the parental somatic cells, the phenotypes of cells differentiated from iPSCs presumably represent those manifested by parental cells [20
]. Thus, iPSCs from patients enable the development of disease-specific cellular models, platforms for drug screening, and autologous sources for cell replacement therapies. Furthermore, iPSCs bypass the ethical issues associated with destroying embryos to obtain human ESCs and immunological barriers that prevent the use of heterologous cells [26
]. depicts a scheme in studying ASD using genomics, animal models and iPSCs. When a person is diagnosed by ASD, genomics method can identify the culprit candidate genes. The physiological functions of the genes can be examined by transgenic murine models [9
]. Meanwhile, iPSCs are readily available by reprogramming somatic cells from ASD patients. Following neuronal differentiation, functional analysis can be performed to examine in vitro
phenotypes manifested by ASD iPSCs. Quantifiable measures, such as neuronal connectivity, synaptic activity and neuronal migration that are associated with ASDs, can be used to test the efficacy of chemicals as a screening platform to ameliorate ASD.
Box 1. Definition of potency in stem cells
The ability of self-renewal and expanded differentiation potential define stem cells. Self-renewal is a form of proliferation without differentiation. Differentiation potential is classified into subtypes according to the Potency of stem cells. Totipotency is the ability of a single cell to produce all the types of cells necessary to give rise to an organism, including extraembryonic tissues. A zygote is a typical example of cells with totipotency. Pluripotency is the potential to differentiate into three germ layers: the endoderm, mesoderm, and ectoderm. Pluripotent stem cells, however, cannot generate extraembryonic tissue or an entire organism. Embryonic stem cells (ESCs) are pluripotent stem cells. Multipotency is the ability to differentiate into multiple cell types, but only a limited number of closely related lineages. An example of multipotent stem cells is hematopoietic stem cells (HSCs). HSCs can give rise to all the cells in blood lineage, but not into other lineages, such as neurons or bone. Oligopotency is the potential to differentiate into a few cell types. Lymphoid and myeloid stem cells are typical examples of oligopotent cells. Unipotency is the limited ability to differentiate into only one cell type. Spermatogonial stem cells (SSCs) can self-renew but only differentiate into sperm.
When using iPSCs as ASD cellular model, a proper selection of standard control determines the interpretation of the in vitro
phenotypes. Thus far, when iPSCs are used in cellular models, iPSCs from healthy persons have been used as controls for most disease models [21
]. Because individual genetic variations have a large influence on cellular physiology, use of iPSCs derived from closely genetically related persons, such as siblings or parent, can reduce the compounding genetic effect. Nevertheless, isogenic iPSC lines are the ideal control. One method for generating isogenic lines is to take advantage of the fact that when a disease is X-linked and prominent in females, reprogramming produces unique sets of isogenic female iPSCs by retaining the active/repressed X chromosome status of fibroblasts [27
]. Thus, iPSCs are produced with either the wild type or mutant allele on the active X chromosome from female Rett syndrome patients by taking advantage of this feature (see below and Box 2
]. However, recent study showed that inactive X chromosome in female iPSCs undergo erosion of X chromosome inactivation over time in culture, raising a concern in modeling X linked disorders using iPSCs [32
]. As gene editing technologies such as zinc finger nuclease (ZFN) or Transcription activator-like effector nuclease (TALEN) are advanced, manipulating single gene within iPSCs has become possible (Box 3
]. Gene-editing technology will become an attractive approach for generating isogenic controls or correcting mutated genes, when modeling ASDs.
Box 2. X chromosome status in human iPSCs and its relevance in ASD study
Female has two X chromosomes, one of which undergoes random inactivation during early embryonic development. Female somatic cells are mosaic in the expression of genes on X chromosome. Pluripotent ICM cells and thus the in vitro
counterpart ESCs have cellular machinery to maintain two active X chromosomes. Murine iPSCs have two active X chromosomes as ESCs. However, X chromosome status of human ESCs is not as definite as murine ESCs. Human ESCs are considered derived from epiblast cells that have undergone random X chromosome inactivation, and have one active X chromosome. Despite a success in isolating human ESCs with two active X chromosomes, X chromosomes in human ESCs is not stable and subject to random inactivation by physiological stress [93
]. Thus, most female human ESCs have one active X chromosome. Reprogramming of human female somatic cells results in iPSCs that have one active X chromosome [27
]. While most groups report the retention of parental X chromosome status [27
], some reports showed the isolation of iPSCs with two active X chromosomes [50
]. Whether the erosion of inactive X chromosome of iPSC during long-term culture is solely responsible [32
], or reprogramming results in X reactivation should be determined by further investigation. Nonetheless, the generation of mono-allelic iPSC line having one active X chromosome offers a unique opportunity to obtain isogenic controls for X-linked diseases. A handful of genes on X chromosomes were shown linked to ASDs, including NLGN4X, NLGN3, and RPL10 in addition to RTT and FX [95
]. Reprogramming fibroblasts of unaffected carrier females that have cells expressing either wild type or mutant form of these genes will result in pure wild type or mutant iPSCs for direct comparative analysis as cellular model.
Box 3. Gene editing by ZFN and TALEN
Zinc finger nucleases (ZFNs) are modular enzymes generated by linking DNA cleavage domain to specific DNA binding domain to induce double strand break (DSB) in specific DNA sequence [97
]. FokI endonuclease is used as DNA cleavage domain. Dimerization of FokI is required for the nuclease activity, and thus a pair of ZFN is designed to cleave target DNA. A zinc finger used for ZFN is comprised of a combination of cysteine and histidine residues, which recognize 3 bp of DNA sequence. DNA binding domain of ZFN is designed to target specific DNA sequence. The number of zinc finger repeats gives the specificity of ZFN. Once double strand break in target DNA sequence is generated, it is repaired by intrinsic DNA repair mechanisms such as non-homologous end joining (NHEJ) or homology directed repair (HDR). Insertions or mutations at the cleavage site accompany the NHEJ repair pathway, which disrupt the locus of interest during repairing process. Meanwhile, targeted insertion can be introduced into DSB sites by HDR, when provided with a homologous donor DNA. Targeted insertion allows for gene correction by homology-based approach. Although the ZFNs have been successfully introduced to genome editing in various organisms including human pluripotent stem cell (hPSC) [33
], nuclease specificity should be improved to prevent off-target cleavage that may cause toxicity in target cells. In addition, repairing DSB can result in unintended mutations and abnormal integration of donor DNA.
Transcription activator like effector nucleases (TALENs) provides an alternative approach for gene editing of specific locus. TALENs are another restriction enzymes generated by combining the TAL effector DNA binding domain with DNA cleavage domain [99
]. In general, FokI endonuclease is used to make DSB in target site like in ZFNs. TAL effector proteins consist of repetitive 33–35 amino acid sequences that are highly conserved except for two amino acids of 12th
. Those two key amino acids determine the specificity of TALEN binding to target DNA sequence [100
]. TALENs showed much less off target effects and similar gene targeting efficiency, compared with ZFNs [101
]. Improving tools to deliver TALENs into target and to select the modified cells is needed in applying TALEN in iPSCs.
A robust neuronal differentiation method is critical in studying ASDs in vitro
. Particularly, success in generating cortical neurons is essential, because potential mechanisms underlying autism include the defect in cortical connectivity and neural migration to cerebral cortex [34
]. There are two major types of neurons in cerebral cortex; excitatory glutamatergic projection neurons, and inhibitory GABAergic interneurons. Excitatory neurons are produced within cortical epithelium, while inhibitory interneurons are generated in the subpallium and migrate into dorsal telencephalon [36
]. There have been much efforts to produce cortical excitatory neurons from pluripotent stem cells (). Broadly, neuronal differentiation is initiated either by forming embryoid body (EB) aggregates or by plating cells as adherent layer. Using SFEBq (serum-free EB-like quick aggregate) method, Sasai and colleagues have succeeded in generating cortical progenitors with regional specification by modulating Wnt3a and BMP pathways [37
]. In adherent culture, Studer and colleagues expedited the neuronal differentiation and obtained Pax6+ neuronal progenitors within a week by inhibiting BMP and Nodal signaling [38
]. Although Studer and colleagues did not extend the efforts to isolate forebrain neurons, Livesey and colleagues further refined the approach to enrich the cortical excitatory neurons by using retinoids in addition to BMP and Nodal inhibitors [39
]. The method developed by the Livesey group has not been used for any ASD study yet, but will be important in delineating the phenotypes critical in cortical neurons for ASD cellular models.
Strategies for differentiating human pluripotent stem cells into cortical neurons