As introduced above, while the hiPSC approach to study pathogenesis has been attempted for a few monogenic diseases, disease modeling strategies to study more complex diseases face even greater challenges. A major concern to the study of human diseases in the cell culture dish is that it may be difficult or not practical to in vitro
model diseases with a long-latency such as Alzheimer’s or Parkinson’s. In such cases, the dynamics of disease progression in the patient is likely to be vastly different from any phenotype developing in vitro
in cells differentiated from patient-specific hiPSCs (). One possibility to overcome this challenge would be the attempt to accelerate the appearance of pathological phenotypes in the cell culture dish by exposing the cells to environmental effects that may contribute to the disease, such as oxidative stress. Issues such as the kinetics of disease pathology have yet to be addressed with the strategies that have been published to date. A second major concern for the study of disease pathogenesis is that it may be difficult or impractical to model diseases in vitro
with a single purified lineage-committed cell type. In current hiPSC modeling approaches possible interactions of the cell type that is affected in the patient with other cell types within a tissue or within the diseased patient’s body have yet to be systematically reconstructed (), and in some cases differentiation protocols required to generate cell types of interest from pluripotent populations have not been established. Lastly, diseases with significant epigenetic components may be difficult to study in iPSCs, as the reprogramming process is expected to remove any epigenetic alterations associated with disease phenotypes. Hence, epigenetic alterations will not persist in the pluripotent iPSCs, an issue particularly relevant to sporadic and multifactorial disorders caused by a combination of genetic and environmental factors. Environmental factors, such as toxic metals and pesticides, general lifestyle and dietary habits have been associated with increased risk in some diseases and may affect the epigenome (Jaenisch and Bird, 2003
). Thus, iPSCs from patients with sporadic diseases, which are caused predominantly by epigenetic alterations, may be of little value for mechanistic studies unless the epigenetic alterations also associate with unidentified genetic alterations.
Technical challenges in generating cellular models of disease
Defining a disease-relevant phenotype will critically depend on the choice of ‘healthy wild-type’ control cells. A wide range of control cell lines could be used for comparison with a given patient-specific hiPSC line, including established hESC lines, or established hiPSC lines from healthy donors. To establish a general model of disease, a panel of lines derived from the same patient, as well as additional, unrelated patients suffering from the same disease should be compared to ensure that any observations are not specific to a given cell line, or a particular patient. For example, given the genetic background diversity that exists between unrelated individuals, the use of control cells lines derived from healthy siblings may be less likely to result in background-specific confounding results during experimental comparisons. In single gene diseases genetically-rescued hiPSC lines could represent an ideal isogenic control. In diseases with somatically acquired mutations, hiPSC lines isolated from unaffected cell types could be used as controls. For example in myeloid proliferative disorders that affect the hematopoietic system, hiPSC lines derived from the skin of the patient would serve as control lines for studies involving hiPSC lines derived from the diseased blood.
In the following we will highlight four technical challenges: 1) creation of reprogramming factor-free hiPSCs to minimize or eliminate genetic alterations in the derived iPSC lines; 2) gene targeting strategies to generate markers for differentiation and gene corrections; 3) establishing disease-relevant phenotypes in vitro; and 4) establishing disease-relevant phenotypes in vivo. In each of these areas, new tools are emerging that address these challenges and will make modeling with hiPSCs more tractable for complex diseases (). These challenges and emerging solutions are described below in the chronological order likely encountered by researchers in this field. First, only virus mediated reprogramming has been used thus far to generate hiPSCs that display some disease specific phenotypes from patients, and because the reprogramming vectors remained integrated in these disease-specific hiPSCs, it cannot be excluded that residual vector expression contributed to the observed phenotype.
Emerging tools that may be used in disease modeling efforts
Recently, novel derivation strategies have been devised to create ‘reprogramming factor-free’ hiPSCs. Second, gene targeting interventions aimed to disrupt, repair or overexpress genes in hiPSCs are integral in current strategies to mark and purify the differentiation stage of hiPSC derivatives and for genetic rescue of diseased cellular phenotypes. Current prospects for perturbing gene function in hiPSCs are described. Finally, traditional cell culture techniques with single differentiated hiPSC types may provide inadequate stresses or microenvironments to truly model the onset and/or progression of disease processes. Both biomaterials and animal-human chimeras are tools that overcome some of the limitations of traditional cell culture.
Strategies of deriving reprogramming factor-free hiPSCs
Residual expression of integrated copies of reprogramming factors in hiPSCs can affect the gene expression and potentially biological properties of the resulting iPSC derivatives. In the most salient example, use of c-Myc as a reprogramming factor led to high incidence of tumors in chimeras generated with mouse iPSCs and it is expected that this oncogene would function similarly in hiPSCs (Nakagawa et al., 2008
). In a more systematic study through use of Cre-recombinase excisable viruses, hiPSCs were first derived through viral vector mediated transduction of reprogramming factors and subsequently followed by Cre-mediated excision of the vectors (Soldner et al., 2009
). Such factor-free hiPSCs displayed a global gene expression profile that was more closely related to hESCs than to hiPSCs carrying the transgenes, consistent with the possibility that basal vector expression may affect the phenotype of the hiPSCs.
Various new methods have been developed to improve reprogramming technology to generate genetically unmodified or reprogramming “factor-free” hiPSCs (reviewed in (O’Malley et al., 2009
)). At present, there is no clear optimal method, as each approach has strengths and disadvantages (). As noted above, the Cre-recombinase method efficiently reprograms cells, however viral elements flanking the loxP sites still remain after excision. Like the Cre-loxP recombination strategy, piggyBac transposition has achieved removal of exogenous reprogramming factors from genomic integration sites in iPSCs (Kaji et al., 2009
; Woltjen et al., 2009
). The piggyBac transposon/transposase system requires the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events. However, the identification of hiPSCs with minimal-copy vector insertions, integration site mapping, excision of the reprogramming cassettes and validation of factor-free clones can be a laborious process. Non-integrating strategies using episomes (Yu et al., 2009
), adenoviral transfection (Stadtfeld et al., 2008
), RNA viruses (Fusaki et al., 2009
), or plasmid transfection (Gonzalez et al., 2009
; Okita et al., 2008
) are extremely inefficient. Though these approaches circumvent a few of these obstacles, it is difficult to completely exclude the possibility that vector subfragments integrated in the resulting iPSCs. Lastly, protein transfection can generate genetically-unmodified iPSCs, but at exceedingly low efficiencies (Kim et al., 2009
; Zhou et al., 2009
). A variety of small molecules could singly replace reprogramming factors (Huangfu et al., 2008
; Ichida et al., 2009
; Lyssiotis et al., 2009
; Shi et al., 2008
; Xu et al., 2008
), but there has yet to be a demonstration of using only small molecules to reprogram somatic cells.
Strategies for deriving “reprogramming factor-free” hiPSCs
Genetic modification of hESCs and hiPSCs
Tracking, accentuating, or accelerating pathological phenotypes in the lab could greatly benefit from cell type–specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes. First, cell type–specific lineage reporters would aid in the enrichment for specific cell types during in vitro differentiation, as differentiation techniques to generate specific somatic cell types affected by disease typically also produce progenitors and mixed cell cultures, which may interfere with in vitro assays of disease. Indeed, for many cell types of interest, efficient in vitro generation techniques have yet to be determined, and thus lineage-tracking tools will likely be required to achieve this important early step in the effort to model some human diseases. In addition, such reporters may facilitate the tracking of diseased cells in co-cultures and in chimeric animals after grafting or transplantation. Further, tools to disrupt, repair or overexpress genes could help isolate individual genetic components in complex disease models. Building up to a complex disease phenotype from combinations of single genetic modifications as well as rescuing phenotypes through gene modification would also be of interest. Lastly, overexpression of genes that stress or age cells might help to accentuate phenotypesand/or mimic the induction of disease onset in the laboratory context. hiPSCs provide an attractive pool of cells to modify since they indefinitely self-renew, although most methods could also be applied to hiPSC derivatives such as differentiated progenitors that can be easily expanded and banked.
Tools to achieve expression of transgenes in hESCs or hiPSCs by random integration of vectors include retroviruses, lentiviruses, bacterial artificial chromosomes, synthetic gene delivery reagents, and a transposon/transposase system (Giudice and Trounson, 2008
; Placantonakis et al., 2009
). Viral gene transfer into hESCs can be inefficient, as adeno-associated virus and adenovirus vectors have been shown to transduce only 0.01–11% of undifferentiated hESCs (Smith-Arica et al., 2003
). Lentiviral vectors are typically used instead for transgene expression, as these approaches achieve <40% transduction efficiency in hESCs (Xia et al., 2007
). Recently, synthetic gene delivery approaches have been developed to rival viral delivery, as engineered polymers and cationic reagents have the ability to condense DNA into particles that facilitate cellular uptake and endosomal escape (Green et al., 2008
). Finally, piggyBac transposition is host-factor independent and has recently been demonstrated to be functional in various hiPSCs (Kaji et al., 2009
; Lacoste et al., 2009
; Woltjen et al., 2009
). Copy number and integration patterns of the transgenes are not easily controlled in these strategies.
Targeting specific endogenous genetic loci is a key technology to study gene function, as this strategy preserves the flanking genomic context of the target including important regulatory elements. Since the derivation of the first hESCs, only a few reports have described successful gene targeting by homologous recombination in hESCs (Costa et al., 2007
; Davis et al., 2008
; Irion et al., 2007
; Zwaka and Thomson, 2003
). These studies used both nonisogenic and isogenic constructs encoding a drug selectable cassette introduced into hESCs by electroporation or transfection with a cationic reagent. Isolation of correctly targeted clones involved drug selection and screening of clones through PCR or Southern analysis to check for proper integration of the vectors into the human genome. Recently, a technique, called ‘genome editing’, based on the introduction of DNA double-strand breaks by site-specific zinc-finger nucleases (ZFNs) to facilitate homologous recombination (Lombardo et al., 2007
) has been used to target endogenous genes in hESCs and hiPSCs (Hockemeyer et al., 2009
; Zou et al., 2009
). A ZFN is generated by fusing the FokI nuclease domain to a DNA recognition domain composed of engineered zinc-finger motifs that specify the genomic DNA binding site for the chimeric protein. Upon binding of two such fusion proteins at adjacent genomic sites, the nuclease domains dimerize, become active and cut the genomic DNA. When a donor DNA that is homologous to the target on both sides of the double-strand break is provided, the genomic site can be repaired by homology-directed repair, allowing the incorporation of exogenous sequences placed between the homologous regions. To ensure the uniqueness of intended targets within the human genome, ZFNs containing multiple zinc fingers need to recognize composite sites of 20–50 bp. ZFNs were used to engineer several loci in hiPSCs: the disease-related pig-a
locus (Zhou et al, 2009
), the pitx3
locus, which is not expressed in hESCs, the oct4
locus to report on cell fate, and the aavs1
locus to be a ‘safe harbor’ for an inducible transgene (Hockemeyer et al., 2009
). Though vector insertions into these four loci has been efficient, it is not clear as yet what fraction of genes can be targeted by this approach.
Towards tissue engineering with hiPSC derivatives to generate disease-relevant phenotypes
Cellular functions are influenced not only by cell-autonomous programs but also by microenvironmental stimuli, which include neighboring cells, extracellular matrix, soluble factors and physical forces. Engineered biomaterials and co-cultures may provide a powerful way to provide a richer context for studying disease relevant cell–cell interactions (Guilak et al., 2009
). These contextual cues are particularly important for modeling non-cell autonomous pathology. In amyotrophic lateral sclerosis (ALS) for example, co-cultures of wild-type, hESC-derived motor neurons with mutant ALS astrocytes induced motor neuron death (Di Giorgio et al., 2008
; Marchetto et al., 2008
While full recapitulation of tissue architecture remains an elusive goal of tissue engineering, smaller functional units (10–100 μm) have been developed to study cellular responses to distinct local stimuli. Bhatia and colleagues used ‘soft lithography’ techniques to create micropatterned cell clusters, in which 500–μm-islands of human hepatic cells are surrounded by fibroblasts. These micropatterned cell cultures were then assessed for liver function through gene expression profiles, metabolism, secretion of liver-specific products and susceptibility to hepatotoxins (Khetani and Bhatia, 2008
). Patterning approaches can also be applied in three-dimensional (3D) scaffolds, which have been generated from purified molecules such as collagen I, synthetic biomaterials, and from native extracellular matrices from which living cells were previously extracted (Yamada and Cukierman, 2007
). Using hiPSC derivatives in combination with these and other advances in biomaterials such as microscale cell patterning and 3D tissue scaffolds could bridge the gap between traditional cell culture and animal models.
Generating disease-relevant phenotypes with human-animal chimeras
It may not be practical to in vitro
model diseases with long latencies of onset and/or with complex pathophysiology. Thus, for some types of disease modeling in vivo
approaches may be required. Chimeras provide long-term access to complex and changing environmental context for hiPSCs and are being currently being experimentally explored and optimized. A chimera is an organism in which tissues of genetically different constitution co-exist as a result of grafting, mutation, or some other process. Human-animal interspecific chimeras can be generated by grafting hiPSC-derived cells into embryos, fetuses, or adult animals (Behringer, 2007
; Shultz et al., 2007
). In several instances, xenografts created by transplantation of human cells into immune-privileged sites (e.g., anterior chamber of the eye or cheek pouch) has been used, however, the most widespread approaches utilize immunodeficient mice, such as the nude mouse, severe combined immunodeficiency (SCID) mouse, and NOG mouse. In this way animal chimeras engrafted with human tissues at orthotopic sites have been produced in efforts to generate ‘humanized’ animals (Friese et al., 2006
Humanized mouse systems have recently had the most notable progress with hematopoietic, nervous system, and hepatic reconstitution with human adult stem cells or hESC derivatives (Behringer, 2007
; Shultz et al., 2007
). Adult human hematopoietic stem cells have been injected intravenously into irradiated adult or newborn recipients with significant engraftment (Ishikawa et al., 2005
; Shultz et al., 2005
). When undifferentiated hESCs were injected directly into the brain ventricles of fetal mice, human neurons and glia formed (Muotri et al., 2005
), although it is not clear how differentiated cells were generated after injection and became incorporated into the brains of the host animal and why no hESC-derived teratomas formed. Human adult neural stem cells survive, migrate, and express differentiation markers for neurons and oligodendrocytes after long-term engraftment in spinal cord-injured NOD-SCID mice, and in the neonatal, the adult, or the injured rodent brain (Cummings et al., 2005
; Guzman et al., 2007
). Lastly, a hepatocyte-humanized mouse has been generated to exhibit human-type responses in a series of in vivo
drug processing experiments and in the infection and propagation of hepatic viruses (Kneteman and Mercer, 2005
). Currently, there have been no reports of using hiPSCs or their derivatives with such animal models, and these protocols will likely need to be refined to enable more robust engraftment and functionality of the transplanted human cells.