- Email: [email protected]
Reprogramming of somatic cells
CHAPTER
Reprogramming of somatic cells: iPS and iN cells
2 Vania Broccoli1
San Raffaele Scientific Institute, Milan, Italy CNR-Institute of Neuroscience, Milan, Italy 1 Corresponding author: Tel.: +39-226434616; Fax: +39-226436164, e-mail address: [email protected]
Abstract Limited access to human neurons has posed a significant barrier to progress in biological and preclinical studies of the human nervous system. The advent of cell reprogramming technologies has widely disclosed unprecedented opportunities to generate renewable sources of human neural cells for disease modeling, drug discovery, and cell therapeutics. Both somatic reprogramming into induced pluripotent stem cells (iPSCs) and directly induced Neurons (iNeurons) rely on transcription factor-based cellular conversion processes. Nevertheless, they rely on very distinct mechanisms, biological barriers, technical limitations, different levels of efficiency, and generate neural cells with distinctive properties. Human iPSCs represent a long-term renewable source of neural cells, but over time genomic aberrations might erode the quality of the cultures and the in vitro differentiation process requires extensive time. Conversely, direct neuronal reprogramming ensures a fast and straightforward generation of iNeurons endowed with functional properties. However, in this last case, conversion efficiency is reduced when starting from adult human cells, and the molecular and functional fidelity of iNeurons with respect to their corresponding native neuronal subtype is yet to be fully ascertained in many cases. For any biomedical research application, it should be carefully pondered the reprogramming method to use for generating reprogrammed human neuronal subtypes that best fit with the following analysis considering the existing limitations and gap of knowledge still present in this young field of investigation.
Keywords iNeurons, iNeuronal cells, iPSCs, Pluripotent stem cells, Direct cell reprogramming, Cell therapy, Disease modeling, Pharmacological reprogramming, CRISPR/Cas9, Genome editing
Progress in Brain Research, Volume 230, ISSN 0079-6123, http://dx.doi.org/10.1016/bs.pbr.2016.12.009 © 2017 Elsevier B.V. All rights reserved.
53
54
CHAPTER 2 iPS and iN cells
1 OPTIMIZED STRATEGIES FOR iPS CELL REPROGRAMMING AND HIGH-QUALITY VALIDATION Human-induced pluripotent stem cells (hiPSCs) have provided an unprecedented experimental setting for disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics (Takahashi and Yamanaka, 2015; Tapia and Sch€ oler, 2016). To reach these applications in clinics, however, reprogramming of hiPSCs and their differentiation procedures should be in compliance with a high safety profile without inherent risks of tumorigenesis (Kimmelman et al., 2016). Importantly, new “scarless” technologies have been developed to generate adult somatic cells into hiPSCs without leaving genetic traces of the reprogramming event. In fact, nonintegrating technologies based on episomal vectors, synthetic mRNAs, and Sendai viruses have proven efficient to reprogram high-quality hiPSCs from various somatic cell types and can be employed in GMP settings to generate hiPSC adequate for future applications in clinics (Schlaeger et al., 2015). A further obstacle to carefully consider is the tendency of hiPSCs to accumulate genomic alterations during both the reprogramming process and the extensive periods of in vitro culture (Ben-David et al., 2011; Weissbein et al., 2014). In fact, beyond structural and numerical chromosomal aberrations, hiPSCs can suffer from small copy number variations on the kilobase scale, which are not present in the somatic cells of origin (Gore et al., 2011). Although one study was able to identify a recurrent set of point mutations in the mouse iPSC clones tested (Young et al., 2012), none of the studies could detect any recurrent single-nucleotide variations in hiPSCs, indicating that no single significant mutation tends to arise during successful reprogramming nor provides a substantial growth advantage in culture (Gore et al., 2011; Ruiz et al., 2013). Thus, genomic content of hiPSCs should be carefully evaluated after reprogramming and during culture expansion and use of cells at late passages should be avoided when possible. For a sufficiently in-depth genomic analysis, standard G-band karyotyping should be associated with higher-resolution methods like CGH (comparative genomic hybridization) or SNP (single-nucleotide polymorphism) genomic arrays for detection of small genetic changes (Mayshar et al., 2010). An alternative to DNA-based methods is the e-karyotyping assay, which predicts chromosomal aberrations from gene-expression biases, and, for instance, a chromosomal gain can be identified by consistent overexpression of genes throughout the aberrant region (Ben-David et al., 2013). Hence, this last approach provides an accurate estimation of chromosomal integrity as well as the full gene-expression profiling of the hiPSCs at the same time. A remaining bottleneck in hiPSC reprogramming is the overall low efficiency of the reprogramming event and the extended time necessary to obtain the reprogrammed primary clones. Intriguingly, a recent report has shown that efficiency in iPSC reprogramming can be dramatically improved in a microfluidic environment. In fact, microliter-volume confinement resulted in a 50-fold increase in
2 Generation of iNeurons by direct cell conversion
efficiency over traditional reprogramming by delivery of synthetic mRNAs encoding transcription factors (TFs) (Luni et al., 2016). High quality and pure hiPSCs were obtained in 2 weeks from initial reprogramming and subsequently differentiated into functional hepatocyte- and cardiomyocyte-like cells in the same platform without additional expansion (Luni et al., 2016). Thus, the microfluidic platform promotes a highly efficient and fast iPSC reprogramming while consistently reducing the required culture media and reagents, ultimately strongly lowering the overall costs. Thus, this technology can provide the opportunity for the generation of high numbers of hiPSCs from a large cohort of patients in an accelerated time frame and with affordable costs. iPSCs are long-term and homogenous self-renewal stem cells which are perfectly suited for genetic engineering. The advent of the CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas)) gene-editing technology has provided an easy and straightforward tool for targeted gene modifications in hiPSCs (Hockemeyer and Jaenisch, 2016). In fact, differently from previous gene-editing technologies like zinc-finger nucleases and TALENs, the CRISPR/Cas9 system can be easily set up in any regular laboratory, providing a powerful and flexible approach for any type of genetic modifications (Doudna and Charpentier, 2014; Hsu et al., 2015; Mali et al., 2013). Gene-editing technologies for gene manipulation in hiPSCs have already proven successful for: (i) correcting disease-causing gene mutations in patient hiPSCs (Smith et al., 2014), (ii) inserting disease-causing gene mutations in control hiPSCs (Liu et al., 2016a), (iii) performing straight and conditional gene mutagenesis (Chen et al., 2015; Rubio et al., 2016), and (iv) inducing targeted genomic alterations (Park et al., 2016). In all these applications, the CRISPR/Cas9 machinery beyond targeting the correct sequence might be engaged with similar other sequences in the genome, leading to off-target effects. Thus, it is mandatory to undertake a close analysis of the putative off-target sequences, as predicted in silico by on-line computational tools or by an unbiased full sequencing analysis (Tycko et al., 2016). Off-target mutations can be mitigated by taking advantage of new Cas9 engineered variants which present a substantially higher fidelity profile while maintaining comparable efficiency for the on-target site (Kleinstiver et al., 2016; Slaymaker et al., 2016). Thus, CRISPR/Cas9 gene editing offers a new level of precision in genome manipulation of hiPSCs, providing an efficient and safe technological platform for correcting disease-causing gene mutations or defining the molecular contributors to the pathogenesis of numerous human diseases.
2 GENERATION OF iNEURONS BY DIRECT CELL CONVERSION The seminal discovery of iPSCs in 2006 has provided the conceptual evidence that supraphysiological misexpression of developmental TFs is sufficient to promote conversion between two cell types even with very different embryonic origin,
55
56
CHAPTER 2 iPS and iN cells
developmental potential, and functional state (Takahashi and Yamanaka, 2006). iPSC generation appears not to recapitulate the developmental milestones occurring during physiological differentiation of pluripotent stem cells. Conversely, reprogramming factors stimulate the pluripotency gene network directly, thereby promoting the emergence of the pluripotent stem cell identity in the host cell followed by the dismantlement of the original cell identity. In this perspective, any cell type can in principle be reprogrammed into a new fate whenever the correct transcriptional program is initiated and self-maintained over time. Based on this framework, the field of cell reprogramming has recently exploded, providing numerous examples of striking transitions from one cell type into another promoted by a minimal combination of developmental TFs (Graf and Enver, 2009; Masserdotti et al., 2016; Xu et al., 2015). Among them, the direct conversion of fibroblasts into neurons has provided the first example of direct reprogramming between two cell types developed from different embryonic germinal layers (Vierbuchen et al., 2010). Forced expression of the three neurodevelopmental TFs Ascl1, Brn2, and Myt1l (ABM) is sufficient to convert mouse fibroblasts into induced neuronal (iN) cells that—beyond developing long and polarized neurites—acquired sophisticated functional properties, such as membrane excitability and synapse activity. Considering that the reprogrammed cells closely resemble primary neurons in their morphology, transcriptome, and electrophysiological properties, I favor referring to them simply as induced neurons (iNeurons), much in line with the nomenclature for other reprogrammed cell types, e.g., induced hepatocytes (Lim et al., 2016) and cardiomyocytes (Qian et al., 2012). Importantly, expression of the reprogramming factors can be silenced in both mouse and human iNeurons without altering their acquired morphological and functional neuronal properties. Interestingly, the stable maintenance of the iNeuronal identity is achieved when the corresponding endogenous genes of the reprogramming factors are stably activated, and their expression is maintained even after silencing of the exogenous transgenes (Pang et al., 2011; Vierbuchen et al., 2010). Importantly, after transplantation into the brains of adult rats, human iNeurons survive with high efficiency in the host neural parenchyma preserving and further developing their neuronal morphology (Pereira et al., 2014).
2.1 MOLECULAR MECHANISMS OF DIRECT NEURONAL REPROGRAMMING Mechanistically, during the fibroblast-to-neuron conversion, Ascl1 acts as a powerful pioneer TF by accessing its authentic neuronal target genes in the repressive and silent chromatin state of the fibroblasts. Subsequently, Brn2 and Myt1l are recruited to the chromatin by Ascl1, while they alone lack the ability to access the fibroblast chromatin (Wapinski et al., 2013). In fact, Ascl1 is sufficient by itself to generate functional iNeurons from mouse and human fibroblasts, indicating that Ascl1 is the key driver in reprogramming (Chanda et al., 2014). However, Myt1l and Brn2 play a critical role in enhancing the neuronal maturation process and to prevent Ascl1
2 Generation of iNeurons by direct cell conversion
from activating myogenic target genes caused by its close homology with the myogenic inducer MyoD (Treutlein et al., 2016; Wapinski et al., 2013). Another powerful combination to generate iNeurons with cholinergic identity is the enforced expression of Neurogenin-2 (Neurog2) with the two small molecules forskolin and dorsomorphin (Liu et al., 2013). Interestingly, similar to Ascl1, Neurog2 acts as a pioneer TF accessing closed chromatin, but small molecules synergize with Neurog2 to enhance chromatin accessibility and H3K27 acetylation (Smith et al., 2016). Mechanistically, forskolin is an activator of cAMP synthesis and through PRKACA (protein kinase cAMP-activated catalytic subunit A) kinase activity phosphorylates, CREB1 which binds and costimulates a subset of the Neurog2 downstream genes. Thus, small molecules might have a strong impact in regulating and promoting the cell conversion efficiency of the reprogramming factors.
2.2 STRATEGIES TO IMPROVE EFFICIENCY AND MATURITY OF iNEURONS Despite that the ABM cocktail is very efficient in reprogramming mouse primary fibroblasts, the conversion rate is strongly reduced when starting from human cells, and the functional maturation of human iNeurons is generally limited (Pang et al., 2011). Notably, recent findings have identified additional factors, epigenetic regulators, microRNAs (miRNAs), and small molecules that favor neuronal cell lineage reprogramming. In particular, expressing ABM in human fibroblasts with either Neurog2 or NeuroD1, two neurodevelopmental helix-loop-helix factors, provided a significant increase in the iNeuron yield (Ladewig et al., 2012; Pang et al., 2011). Interestingly, both TFs have a prominent role in promoting the glutamatergic neuronal cell fate during cerebral cortex development and, therefore, have a synergic action on the same neuronal lineage promoted by the ABM combination. Four-factor reprogrammed human iNeurons displayed an increased neuronal maturation both in morphology and in functional properties although they appeared relatively immature as indicated by their slightly depolarized membrane potentials and the relatively low-amplitude synaptic responses (Pang et al., 2011). While additional reprogramming factors might enhance the neuronal conversion, increasing numbers of factors pose substantial practical hurdles for their efficient delivery, as for instance the increasing number of vectors that need to be employed independently and the optimal relative expression levels among the different factors. miRNAs are small noncoding elements with an emerging role in regulating cell-fate genes and their associated chromatin state. Notably, the overexpression of the neuronal-specific miR-9/9* and miR-124 alone in human fibroblasts can induce MAP-2-expressing iNeurons, although the addition of TFs is required for developing iNeurons with functional activity (Yoo et al., 2011). In another study, the overexpression of miR-124 also promoted human iNeuronal induction mediated via Brn2 and Myt1l overexpression (Ambasudhan et al., 2011). Interestingly, miR-9/9* and miR-124 regulate the composition of the Brg1-associated factors (BAF) complex during neural development promoting the formation of neuron-specific nBAF
57
58
CHAPTER 2 iPS and iN cells
complexes essential for postmitotic neurons (Yoo et al., 2009). The significant role of miRNAs in cell reprogramming has been further revealed by the surprising finding that the inhibition of the miRNA regulator PTB alone can generate functional murine iNeurons (Xue et al., 2013). PTB blocks miRNA-mediated activity of the REST complex; thus, PTB inhibition leads to derepression of multiple miRNA-regulated neuronal genes stimulating neuronal cell reprogramming. Interestingly, REST is also a negative regulator of the astrocyte-to-neuronal conversion (Masserdotti et al., 2015). In this system, REST was shown to directly repress critical downstream targets of the neuronal reprogramming factors, thus, limiting their action in stimulating the neuronal cell identity. Expression of miRNAs on cells is technically handy since due to their small size their insertion in expression vectors is easy and flexible. However, expression of either reprogramming genes or miRNAs is generally achieved by using lentiviral technology, which raises safety concerns for its clinical applications caused by their random integration in the host cell genome. A promising solution for this drawback is the induction of the cell-fate conversion by small molecules. In fact, chemical inhibitors of the SMAD signaling and GSK3beta facilitated neuronal reprogramming in combination with TFs (Ladewig et al., 2012). SMADs and GSK3beta have a significant role also in repressing neural fate during early embryogenesis, thus suggesting evident similarities between the processes that regulate normal neuronal differentiation during development and direct neuronal reprogramming. Importantly, two recent studies completed fibroblast neuronal reprogramming using only a defined set of small molecules (Hu et al., 2015; Li et al., 2015). In particular, Li et al. (2015) carried out neuronal conversion by using a combination of four chemicals, which included three inducers of neuronal fate (ISX9, GSK3beta inhibitor, and Forskolin) and I-BET151, a suppressor of the host cell molecular program. I-BET151 was reported to competitively bind the BRD domain of BET family proteins (Seal et al., 2012). Interestingly, BET family proteins were described to specifically associate with the activated chromatin domains and maintain the cell-fate-specific gene-expression pattern (Wu et al., 2015). In particular, inhibition of BRD4 can disrupt cell-fate maintenance and alter the gene-expression pattern controlling cell-type identity (Di Micco et al., 2014). Thus, these results support the view that BRD chemical inhibition concurs in disrupting the fibroblast-specific gene-expression program in early-stage reprogramming facilitating neuronal conversion. In the second study, Hu et al. (2015) selected a cocktail of seven small molecules including inhibitors for the JNK, PKC, and ROCK signaling that was sufficient to generate human iNeurons with functional activity. However, the exact contribution of these pathways in promoting the neuronal cell fate and their downstream molecular mechanisms is yet to be discerned. Another intriguing question is how small molecules could replace the function of exogenous TFs during the chemical conversion process. Although the difference of global gene expression between different cell types may involve thousands of genes, the core gene regulatory network that determines one specific cell type may only be comprised of several master genes. For example, a recent study indicated that the
2 Generation of iNeurons by direct cell conversion
essential TF program of naı¨ve mouse pluripotent stem cells involves 16 interactions, 12 components, and 3 inputs (Dunn et al., 2014). Thus, it is conceivable that small molecules can initially activate sufficient levels of one or only a few reprogramming factors sufficient to initiate the cell-fate switch. However, the exact mechanism connecting the small molecule-dependent external stimuli with the activation of the endogenous reprogramming factors is yet to be elucidated.
2.3 APPROACHES FOR THE GENERATION OF iNEURONAL SUBTYPES iNeurons generated with the original BAM reprogramming cocktail exhibit functional properties of excitatory glutamatergic neurons lacking a defined regional identity (Vierbuchen et al., 2010). However, a large body of recent work has identified new sets of neurodevelopmental TFs able to reprogram somatic cells into a variety of neuronal subtypes that can be potentially useful for cell-based therapies as well as for disease modeling. Altogether, these findings suggest that neuronal subtype specification can be achieved in vitro by expressing lineage-specific TFs without recapitulating the entire developmental program occurring during brain development. A growing number of minimal combinations of TFs with various technical platforms have recently been reported, and some of them will be described to shed light on the basic principles controlling neuronal subtype reprogramming.
2.3.1 Forebrain-specific neuronal subtypes Glutamatergic iNeurons can be generated from fibroblasts using the ABM cocktail or from astrocytes by enforced expression of Neurog2, thus, indicating that the starting cell type strongly determines the reprogramming conditions and factors required to trigger successful reprogramming (Heinrich et al., 2010; Masserdotti et al., 2015; Vierbuchen et al., 2010). This implies that iNeurons obtained from the two systems, despite having a common glutamatergic phenotype, must rely on different reprogramming mechanisms, distinguished by specific features and properties. A direct comparison of the molecular mechanisms triggered by the two reprogramming methods will reveal the cell type specificity and their constraints in neuronal cell switch. However, the next crucial step is to generate distinct neuronal glutamatergic subtypes with the characteristics of those located in different layers of the cerebral cortex with their specific afferent projections and efferent connections (Lodato and Arlotta, 2015). Notably, forced expression of the single TF Fezf2 triggers the callosal projection neurons of cortical layer II/III into layer V/VI subcortical projection neurons (Lodato and Arlotta, 2015). This neuronal fate switch occurred also when Fezf2 was expressed in postmitotic neuroblasts, indicating that this process is at least in part independent by both the cell cycle and the more intrinsic plastic state of the proliferating neuronal progenitors. Several other developmental TFs have been described to have a prominent role in determining the identity of cortical layer-specific neurons, thus, suggesting that combining these factors together might trigger these particular fates starting from nonneuronal cells.
59
60
CHAPTER 2 iPS and iN cells
Cerebral cortex is a complex ensemble of excitatory and inhibitory neurons, and their interactions regulate the net balance of excitation and inhibition that underlies normal physiological processing. Inhibitory GABAergic neurons are local-projecting neurons within the cortex and are classified in several groups depending on their embryonic origin, connections, and molecular repertoire. Dysfunctions or loss of these neurons are the initial cause or a strong contributor for several brain disorders, including various forms of epilepsies and cognitive disorders. Interestingly, iGABA interneurons (iGABA-iNs) were recently generated by the direct conversion of murine and human fibroblasts using five developmental TFs (Colasante et al., 2015). This is the first time that the expression of the reprogramming transgenes has been modulated in different time windows; in order to enable an efficient functional maturation of the iGABA-iNs, two of the five factors were expressed for 12–14 days only and then silenced through an inducible promoter system (Colasante et al., 2015). Indeed, some of the reprogramming factors, although indispensable for the initial cell lineage conversion, might direct the process to a progenitor cell stage blocking the progression toward a mature functional state of the induced cell type. This phenomenon might occur as well in other cases of direct cell conversion. The capacity to modulate the expression of each factor independently by the others is thereby a relevant technical advance which increases the flexibility of these approaches. In a separate study, three of the same factors were shown to promote differentiation of hiPSCs into GABAergic neurons in a single-step process (Sun et al., 2016). This is a convenient approach since the same differentiation process with small molecules is a labor-intensive procedure that requires more than 3 months to obtain functional GABAergic neurons with several intermediate steps of cell manipulation (Maroof et al., 2013; Nicholas et al., 2013).
2.3.2 Dopaminergic neurons Midbrain dopaminergic neurons are of great interest since their loss is the leading cause of Parkinson’s disease. During development, they are generated in the floor plate of the mesencephalon and several developmental TFs have been implicated in their generation and specification. These include Otx2, which is involved in early patterning; FoxA1/2, which instructs the commitment of the progenitor cells; Lmx1a/b, Ascl1, and Ngn2 that are important for progenitor cell differentiation; and Pitx3 and Nurr1 (Nr4a2), which are involved in the maturation and long-term survival of midbrain dopaminergic neurons (Arenas et al., 2015). Accordingly, many of these TFs have been successfully used to generate induced dopaminergic (iDA) neurons from fibroblasts or astrocytes (Caiazzo et al., 2011; Kim et al., 2011; Pfisterer et al., 2011; Torper et al., 2013). Ascl1, Nurr1, and Lmx1a (ANL) are the minimal combination able to generate iDA neurons endowed with distinguished subtype functional features as dopamine production, functional D2 autoreceptors, and pacemaker-like firing of action potentials (Caiazzo et al., 2011). Importantly, transplanted ANL-induced iDA neurons matured and connected within the host neuronal tissue and could rescue in large part the behavioral deficits caused by the six-OHDA-mediated acute loss of endogenous DA neurons (Dell’Anno et al., 2014).
3 Direct reprogramming of glial subtypes
Functional replacement in vivo obtained with iNeurons of the corresponding endogenous neuronal subtype is the most rigorous and convincing assay to demonstrate the equivalence of the two neuronal cell types. Thus, in cases where it is possible to perform cell replacement with clear functional and behavioral readouts in vivo, this should represent a mandatory requirement before claiming a functional correspondence between induced and somatic neuronal subtypes. However, none of these studies has yet been able to specify iNeurons with a frank VTA (A10) or substantia nigra (A9)-specific identity (Hegarty et al., 2013). This, indeed, remains a challenging task until the molecular determinants discriminating between these two DA neuronal populations have been identified.
2.3.3 Spinal motor neurons Direct reprogramming to spinal cord motor neurons was recently obtaining by combining common neurogenic factors such as Ascl1, Neurog2, Myt1l, and Brn2 (Pou3f2) with TFs specific to spinal cord motor neuron development, such as Lhx3, Isl1, and Hb9 (Lee et al., 2009; Son et al., 2011). The combination of these seven factors generated functional induced motor neurons (iMNs) from mouse embryonic fibroblasts that were able to establish functional neuromuscular junctions with cocultured myotubes, and which could survive after transplantation in vivo (Son et al., 2011). With a similar rationale, Liu et al. combined two panneurogenic TFs, Neurog2 and Sox11, and two neuronal subtype-specific TFs, Isl1 and Sox11, together with forskolin, dorsomorphin, and Fgf2 treatment, converting human fibroblasts into HB9 and ChAT-positive human iMNs with an extremely high efficiency (>80%) (Liu et al., 2016b). These data therefore further support the key role of region-specific TFs for the specification of distinct neuronal subtypes.
3 DIRECT REPROGRAMMING OF GLIAL SUBTYPES Recent findings have demonstrated that TF-mediated reprogramming can be applied to generate induced oligodendrocytes, Schwann cells (SCs), and astrocytes. Two independent studies identified a three TF combination which share the factors Sox10 and Olig2 capable of converting mouse fibroblasts into oligodendrocyte precursor cells (iOPCs) that express appropriate OPC markers, produce myelin sheaths in vitro, and sustain myelin regeneration in mouse brains with genetic dysmyelination (Najm et al., 2013; Yang et al., 2013). Sox10 fulfills widespread and essential functions in myelinating glia of the central as well as peripheral nervous system at multiple stages of development such as glial specification, survival, and terminal differentiation. Interestingly, iOPCs were shown to lack myelin protein zero protein, a specific SC marker, and myelinated multiple axons confirming their central glial cell identity. Conversely, Sox10 only, combined with secreted (BMP4 and WNT) and epigenetic factors (VPA and 5-Aza), was sufficient to reprogram fibroblasts into induced neural crest cells (iNCCs) (Kim et al., 2014). Despite the observation that iNCCs could differentiate into both peripheral neurons and glial cells, they remained
61
62
CHAPTER 2 iPS and iN cells
at a very immature stage lacking an evident functional state. Intriguingly, when Sox10 was combined with Egr2 (known also as Krox-20), a developmental factor necessary for SC myelination, fibroblasts were directly converted into induced SCs with peripheral identity able to wrap axons in vitro for generating compact myelin sheaths with regular nodal structures (Mazzara et al., 2017). Thus, Sox10 is a crucial factor for reprogramming myelinating glia together with selective intrinsic or extrinsic factors which further specify the central or peripheral identity of the induced cells. Astrocytes are the most representative glial component in the brain with trophic and regulatory functions on neurons. Notably, direct reprogramming of murine and human fibroblasts into induced astrocytes (iAstrocytes) was recently obtained using the minimal combination of the three glial molecular determinants NFIA, NFIB, and Sox9. For gene-expression profiling, electrophysiological properties, glutamate uptake, and inflammatory response iAstrocytes had a comparable behavior compared to native brain astrocytes (Caiazzo et al., 2015). Altogether, these results demonstrate that genetic methods of direct cell reprogramming are available to convert adult somatic cells into all three main glial cell types populating the central and peripheral neural tissue.
4 iNEURONS AND GLIA CELLS FOR DISEASE IN VITRO CELL MODELING iNeurons and iGlia are generated with a fast and straightforward process, which make these cells particularly convenient for applications in cell replacement therapies and in vitro disease modeling. Currently, however, a strong hurdle for in vivo applications of these cells is the general use of genome integrating technologies, such as lentiviruses, for transgene expression, which raises severe concerns of genotoxicity for the integration of the viral transgenes in the host genome. Thus, alternative methods are needed to achieve cell reprogramming, which ensure safety of the reprogramming process. Notably, neuronal reprogramming was recently obtained with nonintegrating lentiviruses or AAV, suggesting that these modifications in principle are suitable for clinical use (Lau et al., 2014). A major issue, however, is to maintain high levels of cell reprogramming efficiency, even using clinical-grade methods in order to ensure sufficient numbers of cells for the subsequent clinical applications. Methodologies of direct neuronal reprogramming can be also exploited for establishing models of human diseases in vitro. In fact, this technical platform has important advantages in this application, strongly complementary to those associated with iPSC reprogramming. Importantly, while iPSCs and derived neurons do not retain the aging phenotype of the starting cells, iNeurons maintain the age-related gene expression and phenotypic features according to the age of the reprogrammed cells (Mertens et al., 2015; Miller et al., 2013). This is a crucial aspect which makes direct neuronal reprogramming a convenient system to model age-related disorders, as the case for most of the human neurodegenerative diseases. However, iNeuronal
References
reprogramming generally skips out any intermediate proliferating cell stage; and therefore, it is always necessary to start back from the patient cells for each reprogramming experiment. This can result in a strong drawback when patient cells are difficult to obtain, and their prolonged passaging in vitro might reduce reprogramming efficiency. In such case, a potential solution would be the direct generation of proliferating neural progenitors as a self-expanding intermediate step for subsequent generation of postmitotic neurons. In fact, recent studies have identified a minimal combination of TFs sufficient to convert mouse and human fibroblasts into induced neural progenitor cells (iNPCs) able to largely expand in vitro maintaining the trilineage differentiation potential to generate neurons, astrocytes, and oligodendrocytes (Han et al., 2012; Lujan et al., 2012; Their et al., 2012). Intriguingly, Zhang et al. (2016) have recently devised a pharmacological method based on nine small molecules to reprogram murine fibroblasts into iNPCs. A future development of this method for generating iNPCs from human fibroblasts will open exciting opportunities for in vitro modeling of human disorders. Nevertheless, recent studies have confirmed the value of human iNeurons to dissect the pathological deficits associated to human disorders. In fact, iMNs generated from fibroblasts of amyotrophic lateral sclerosis (ALS) patients recapitulated some crucial aspects of ALS pathology, like the mislocalization of the ribonucleoprotein FUS, smaller soma, lower firing frequency, and higher susceptibility to cell death over time (Liu et al.,2016b). In addition, this approach has been extended to generate iNeurons from familial Alzheimer’s disease patients, showing a pathological increase in both Ab42/Ab40 ratio and release of the amyloid Ab42 fragment (Hu et al., 2015). These results stand as a proof of concept that iNeurons can be informative for disease mechanisms only if their subtype specification, close resemblance to the native neuronal counterpart, and functional maturation are granted by the neuronal reprogramming process. In such case, neuronal reprogramming can offer the unique advantage to be fast and easy to replicate in large numbers in order to obtain iNeurons from multiple patients in a short time. Thus, these reprogramming technologies might have invaluable applications for the future modeling of idiopathic and sporadic disorders where many patients need to be sampled and their cells analyzed in comparative studies.
ACKNOWLEDGMENTS I apologize to all those scientists whose outstanding work could not be cited due to space limitations. I thank all members of my laboratory for their comments on this manuscript.
REFERENCES Ambasudhan, R., Talantova, M., Coleman, R., Yuan, X., Zhu, S., Lipton, S.A., et al., 2011. Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions. Cell Stem Cell 9, 113–118.
63
64
CHAPTER 2 iPS and iN cells
Arenas, E., Denham, M., Villaescusa, J.C., 2015. How to make a midbrain dopaminergic neuron. Development 142, 1918–1936. Ben-David, U., Mayshar, Y., Benvenisty, N., 2011. Large-scale analysis reveals acquisition of lineage-specific chromosomal aberrations in human adult stem cells. Cell Stem Cell 9, 97–102. Ben-David, U., Mayshar, Y., Benvenisty, N., 2013. Virtual karyotyping of pluripotent stem cells on the basis of their global gene expression profiles. Nat. Protoc. 8 (5), 989–997. Caiazzo, M., Dell’Anno, M.T., Dvoretskova, E., Lazarevic, D., Taverna, S., Leo, D., et al., 2011. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476, 224–227. Caiazzo, M., Giannelli, S., Valente, P., Lignani, G., Carissimo, A., Sessa, A., et al., 2015. Direct conversion of fibroblasts into functional astrocytes by defined transcription factors. Stem Cell Rep. 4, 25–36. Chanda, S., Ang, C.E., Davila, J., Pak, C., Mall, M., Lee, Q.Y., et al., 2014. Generation of induced neuronal cells by the single reprogramming factor ASCL1. Stem Cell Rep. 3, 282–296. Chen, Y., Cao, J., Xiong, M., Petersen, A.J., Dong, Y., Tao, Y., et al., 2015. Engineering human stem cell lines with inducible gene knockout using CRISPR/Cas9. Cell Stem Cell 17, 233–244. Colasante, G., Lignani, G., Rubio, A., Medrihan, L., Yekhlef, L., Sessa, A., et al., 2015. Rapid conversion of fibroblasts into functional forebrain GABAergic interneurons by direct genetic reprogramming. Cell Stem Cell 17, 719–734. Dell’Anno, M.T., Caiazzo, M., Leo, D., Dvoretskova, E., Medrihan, L., Colasante, G., et al., 2014. Remote control of induced dopaminergic neurons in Parkinsonian rats. J. Clin. Invest. 124, 3215–3229. Di Micco, R., Fontanals-Cirera, B., Low, V., Ntziachristos, P., Yuen, S.K., Lovell, C.D., et al., 2014. Control of embryonic stem cell identity by BRD4-dependent transcriptional elongation of super-enhancer-associated pluripotency genes. Cell Rep. 9, 234–247. Doudna, J.A., Charpentier, E., 2014. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096. Dunn, S.J., Martello, G., Yordanov, B., Emmott, S., Smith, A.G., 2014. Defining an essential transcription factor program for naı¨ve pluripotency. Science 344, 1156–1160. Gore, A., Li, Z., Fung, H.L., Young, J.E., Agarwal, S., Antosiewicz-Bourget, J., et al., 2011. Somatic coding mutations in human induced pluripotent stem cells. Nature 471, 63–67. Graf, T., Enver, T., 2009. Forcing cells to change lineages. Nature 462, 587–594. Han, D.W., Tapia, N., Hermann, A., Hemmer, K., H€ oing, S., Arau´zo-Bravo, M.J., et al., 2012. Direct reprogramming of fibroblasts into neural stem cells by defined factors. Cell Stem Cell 10, 465–472. Hegarty, S.V., Sullivan, A.M., O’Keeffe, G.W., 2013. Midbrain dopaminergic neurons: a review of the molecular circuitry that regulates their development. Dev. Biol. 379, 123–138. Heinrich, C., Blum, R., Gasco´n, S., Masserdotti, G., Tripathi, P., Sa´nchez, R., et al., 2010. Directing astroglia from the cerebral cortex into subtype specific functional neurons. PLoS Biol. 8, e1000373. Hockemeyer, D., Jaenisch, R., 2016. Induced pluripotent stem cells meet genome editing. Cell Stem Cell 18, 573–586. Hsu, P.D., Lander, E.S., Zhang, F., 2015. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278. Hu, W., Qiu, B., Guan, W., Wang, Q., Wang, M., Li, W., et al., 2015. Direct conversion of normal and Alzheimer’s disease human fibroblasts into neuronal cells by small molecules. Cell Stem Cell 17, 204–212.
References
Kim, J., Su, S.C., Wang, H., Cheng, A.W., Cassady, J.P., Lodato, M.A., et al., 2011. Functional integration of dopaminergic neurons directly converted from mouse fibroblasts. Cell Stem Cell 9 (5), 413–419. Kim, Y.J., Lim, H., Li, Z., Oh, Y., Kovlyagina, I., Choi, I.Y., et al., 2014. Generation of multipotent induced neural crest by direct reprogramming of human postnatal fibroblasts with a single transcription factor. Cell Stem Cell 15, 497–506. Kimmelman, J., Heslop, H.E., Sugarman, J., Studer, L., Benvenisty, N., Caulfield, T., et al., 2016. New ISSCR guidelines: clinical translation of stem cell research. Lancet 387, 1979–1981. Kleinstiver, B.P., Pattanayak, V., Prew, M.S., Tsai, S.Q., Nguyen, N.T., Zheng, Z., et al., 2016. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495. Ladewig, J., Mertens, J., Kesavan, J., Doerr, J., Poppe, D., Glaue, F., et al., 2012. Small molecules enable highly efficient neuronal conversion of human fibroblasts. Nat. Methods 9, 575–578. Lau, S., Rylander Ottosson, D., Jakobsson, J., Parmar, M., 2014. Direct neural conversion from human fibroblasts using self-regulating and nonintegrating viral vectors. Cell Rep. 9, 1673–1680. Lee, S., Lee, B., Lee, J.W., Lee, S.K., 2009. Retinoid signaling and neurogenin2 function are coupled for the specification of spinal motor neurons through a chromatin modifier CBP. Neuron 62, 641–654. Li, X., Zuo, X., Jing, J., Ma, Y., Wang, J., Liu, D., et al., 2015. Small-molecule-driven direct reprogramming of mouse fibroblasts into functional neurons. Cell Stem Cell 17, 195–203. Lim, K.T., Lee, S.C., Gao, Y., Kim, K.P., Song, G., An, S.Y., et al., 2016. Small molecules facilitate single factor-mediated hepatic reprogramming. Cell Rep. 16, 30360–30366. S2211–1247. Liu, M.L., Zang, T., Zou, Y., Chang, J.C., Gibson, J.R., Huber, K.M., et al., 2013. Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons. Nat. Commun. 4, 2183. Liu, J., Gao, C., Chen, W., Ma, W., Li, X., Shi, Y., et al., 2016a. CRISPR/Cas9 facilitates investigation of neural circuit disease using human iPSCs: mechanism of epilepsy caused by an SCN1A loss-of-function mutation. Transl. Psychiatry 6, e703. Liu, M.L., Zang, T., Zhang, C.L., 2016b. Direct lineage reprogramming reveals disease-specific phenotypes of motor neurons from human ALS patients. Cell Rep. 14, 115–128. Lodato, S., Arlotta, P., 2015. Generating neuronal diversity in the mammalian cerebral cortex. Annu. Rev. Cell Dev. Biol. 31, 699–720. Lujan, E., Chanda, S., Ahlenius, H., S€udhof, T.C., Wernig, M., 2012. Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells. Proc. Natl. Acad. Sci. U.S.A. 109, 2527–2532. Luni, C., Giulitti, S., Serena, E., Ferrari, L., Zambon, A., Gagliano, O., et al., 2016. High-efficiency cellular reprogramming with microfluidics. Nat. Methods. 13 (5), 446–452. Mali, P., Esvelt, K.M., Church, G.M., 2013. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963. Maroof, A.M., Keros, S., Tyson, J.A., Ying, S.W., Ganat, Y.M., Merkle, F.T., et al., 2013. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell 12, 559–572.
65
66
CHAPTER 2 iPS and iN cells
Masserdotti, G., Gillotin, S., Sutor, B., Drechsel, D., Irmler, M., Jørgensen, H.F., et al., 2015. Transcriptional mechanisms of proneural factors and REST in regulating neuronal reprogramming of astrocytes. Cell Stem Cell 17, 74–88. Masserdotti, G., Gasco´n, S., G€otz, M., 2016. Direct neuronal reprogramming: learning from and for development. Development 143, 2494–2510. Mayshar, Y., Ben-David, U., Lavon, N., Biancotti, J.C., Yakir, B., Clark, A.T., et al., 2010. Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell 7, 521–531. Mazzara, P.G., Massimino, L., Pellegatta, M., Ronchi, G., Ricca, A., Iannielli, A., et al., 2017. Two factor based reprogramming of rodent and human fibroblasts into myelinogenic Schwann cells. Nat. Commun. In press. Mertens, J., Paquola, A.C., Ku, M., Hatch, E., B€ ohnke, L., Ladjevardi, S., et al., 2015. Directly reprogrammed human neurons retain aging-associated transcriptomic signatures and reveal age-related nucleocytoplasmic defects. Cell Stem Cell 17, 705–718. Miller, J.D., Ganat, Y.M., Kishinevsky, S., Bowman, R.L., Liu, B., Tu, E.Y., et al., 2013. Human iPSC-based modeling of late-onset disease via progerin-induced aging. Cell Stem Cell 13, 691–705. Najm, F.J., Lager, A.M., Zaremba, A., Wyatt, K., Caprariello, A.V., Factor, D.C., et al., 2013. Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells. Nat. Biotechnol. 31, 426–433. Nicholas, C.R., Chen, J., Tang, Y., Southwell, D.G., Chalmers, N., Vogt, D., et al., 2013. Functional maturation of hPSC-derived forebrain interneurons requires an extended timeline and mimics human neural development. Cell Stem Cell 12, 573–586. Pang, Z.P., Yang, N., Vierbuchen, T., Ostermeier, A., Fuentes, D.R., Yang, T.Q., et al., 2011. Induction of human neuronal cells by defined transcription factors. Nature 476, 220–223. Park, C.Y., Sung, J.J., Choi, S.H., Lee, D.R., Park, I.H., Kim, D.W., 2016. Modeling and correction of structural variations in patient-derived iPSCs using CRISPR/Cas9. Nat. Protoc. 11, 2154–2169. Pereira, M., Pfisterer, U., Rylander, D., Torper, O., Lau, S., Lundblad, M., et al., 2014. Highly efficient generation of induced neurons from human fibroblasts that survive transplantation into the adult rat brain. Sci. Rep. 4, 6330. Pfisterer, U., Kirkeby, A., Torper, O., Wood, J., Nelander, J., Dufour, A., et al., 2011. Direct conversion of human fibroblasts to dopaminergic neurons. Proc. Natl. Acad. Sci. U.S.A. 108, 10343–10348. Qian, L., Huang, Y., Spencer, C.I., Foley, A., Vedantham, V., Liu, L., et al., 2012. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 485, 593–598. Rubio, A., Luoni, M., Giannelli, S.G., Radice, I., Iannielli, A., Cancellieri, C., et al., 2016. Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming. Sci. Rep. 6, 37540. Ruiz, S., Gore, A., Li, Z., Panopoulos, A.D., Montserrat, N., Fung, H.L., et al., 2013. Analysis of protein-coding mutations in hiPSCs and their possible role during somatic cell reprogramming. Nat. Commun. 4, 1382. Schlaeger, T.M., Daheron, L., Brickler, T.R., Entwisle, S., Chan, K., Cianci, A., et al., 2015. A comparison of non-integrating reprogramming methods. Nat. Biotechnol. 33, 58–63.
References
Seal, J., Lamotte, Y., Donche, F., Bouillot, A., Mirguet, O., Gellibert, F., et al., 2012. Identification of a novel series of BET family bromodomain inhibitors: binding mode and profile of I-BET151 (GSK1210151A). Bioorg. Med. Chem. Lett. 22, 2968–2972. Slaymaker, I.M., Gao, L., Zetsche, B., Scott, D.A., Yan, W.X., Zhang, F., 2016. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88. Smith, C., Gore, A., Yan, W., Abalde-Atristain, L., Li, Z., He, C., et al., 2014. Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. Cell Stem Cell 15, 12–13. Smith, D.K., Yang, J., Liu, M.L., Zhang, C.L., 2016. Small molecules modulate chromatin accessibility to promote NEUROG2-mediated fibroblast-to-neuron reprogramming. Stem Cell Rep. 7, 955–969. Son, E.Y., Ichida, J.K., Wainger, B.J., Toma, J.S., Rafuse, V.F., Woolf, C.J., et al., 2011. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell 9, 205–218. Sun, A.X., Yuan, Q., Tan, S., Xiao, Y., Wang, D., Khoo, A.T., et al., 2016. Direct induction and functional maturation of forebrain GABAergic neurons from human pluripotent stem cells. Cell Rep. 16, 1942–1953. Takahashi, K., Yamanaka, S., 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676. Takahashi, K., Yamanaka, S., 2015. A developmental framework for induced pluripotency. Development 142, 3274–3285. Tapia, N., Sch€oler, H.R., 2016. Molecular obstacles to clinical translation of iPSCs. Cell Stem Cell 19, 298–309. Their, M., W€orsd€orfer, P., Lakes, Y.B., Gorris, R., Herms, S., Opitz, T., et al., 2012. Direct conversion of fibroblasts into stably expandable neural stem cells. Cell Stem Cell 10, 473–479. Torper, O., Pfisterer, U., Wolf, D.A., Pereira, M., Lau, S., Jakobsson, J., et al., 2013. Generation of induced neurons via direct conversion in vivo. Proc. Natl. Acad. Sci. U.S.A. 110, 7038–7043. Treutlein, B., Lee, Q.Y., Camp, J.G., Mall, M., Koh, W., Shariati, S.A., et al., 2016. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq. Nature 534, 391–395. Tycko, J., Myer, V.E., Hsu, P.D., 2016. Methods for optimizing CRISPR-Cas9 genome editing specificity. Mol. Cell 63, 355–370. Vierbuchen, T., Ostermeier, A., Pang, Z.P., Kokubu, Y., S€ udhof, T.C., Wernig, M., 2010. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041. Wapinski, O.L., Vierbuchen, T., Qu, K., Lee, Q.Y., Chanda, S., Fuentes, D.R., et al., 2013. Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons. Cell 155, 621–635. Weissbein, U., Benvenisty, N., Ben-David, U., 2014. Quality control: genome maintenance in pluripotent stem cells. J. Cell Biol. 204, 153–163. Wu, T., Pinto, H.B., Kamikawa, Y.F., Donohoe, M.E., 2015. The BET family member BRD4 interacts with OCT4 and regulates pluripotency gene expression. Stem Cell Rep. 4, 390–403. Xu, J., Du, Y., Deng, H., 2015. Direct lineage reprogramming: strategies, mechanisms, and applications. Cell Stem Cell 16, 119–134.
67
68
CHAPTER 2 iPS and iN cells
Xue, Y., Ouyang, K., Huang, J., Zhou, Y., Ouyang, H., Li, H., et al., 2013. Direct conversion of fibroblasts to neurons by reprogramming PTB-regulated microRNA circuits. Cell 152, 82–96. Yang, N., Zuchero, J.B., Ahlenius, H., Marro, S., Ng, Y.H., Vierbuchen, T., et al., 2013. Generation of oligodendroglial cells by direct lineage conversion. Nat. Biotechnol. 31, 434–439. Yoo, A.S., Staahl, B.T., Chen, L., Crabtree, GR., 2009. MicroRNA-mediated switching of chromatin-remodelling complexes in neural development. Nature 460 (7255), 642–646. Yoo, A.S., Sun, A.X., Li, L., Shcheglovitov, A., Portmann, T., Li, Y., et al., 2011. MicroRNA-mediated conversion of human fibroblasts to neurons. Nature 476, 228–231. Young, M.A., Larson, D.E., Sun, C.W., George, D.R., Ding, L., Miller, C.A., et al., 2012. Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells. Cell Stem Cell 10, 570–582. Zhang, M., Lin, Y.H., Sun, Y.J., Zhu, S., Zheng, J., Liu, K., et al., 2016. Pharmacological reprogramming of fibroblasts into neural stem cells by signaling-directed transcriptional activation. Cell Stem Cell 18, 653–667.
Reprogramming of somatic cells: iPS and iN cells
2 Vania Broccoli1
San Raffaele Scientific Institute, Milan, Italy CNR-Institute of Neuroscience, Milan, Italy 1 Corresponding author: Tel.: +39-226434616; Fax: +39-226436164, e-mail address: [email protected]
Abstract Limited access to human neurons has posed a significant barrier to progress in biological and preclinical studies of the human nervous system. The advent of cell reprogramming technologies has widely disclosed unprecedented opportunities to generate renewable sources of human neural cells for disease modeling, drug discovery, and cell therapeutics. Both somatic reprogramming into induced pluripotent stem cells (iPSCs) and directly induced Neurons (iNeurons) rely on transcription factor-based cellular conversion processes. Nevertheless, they rely on very distinct mechanisms, biological barriers, technical limitations, different levels of efficiency, and generate neural cells with distinctive properties. Human iPSCs represent a long-term renewable source of neural cells, but over time genomic aberrations might erode the quality of the cultures and the in vitro differentiation process requires extensive time. Conversely, direct neuronal reprogramming ensures a fast and straightforward generation of iNeurons endowed with functional properties. However, in this last case, conversion efficiency is reduced when starting from adult human cells, and the molecular and functional fidelity of iNeurons with respect to their corresponding native neuronal subtype is yet to be fully ascertained in many cases. For any biomedical research application, it should be carefully pondered the reprogramming method to use for generating reprogrammed human neuronal subtypes that best fit with the following analysis considering the existing limitations and gap of knowledge still present in this young field of investigation.
Keywords iNeurons, iNeuronal cells, iPSCs, Pluripotent stem cells, Direct cell reprogramming, Cell therapy, Disease modeling, Pharmacological reprogramming, CRISPR/Cas9, Genome editing
Progress in Brain Research, Volume 230, ISSN 0079-6123, http://dx.doi.org/10.1016/bs.pbr.2016.12.009 © 2017 Elsevier B.V. All rights reserved.
53
54
CHAPTER 2 iPS and iN cells
1 OPTIMIZED STRATEGIES FOR iPS CELL REPROGRAMMING AND HIGH-QUALITY VALIDATION Human-induced pluripotent stem cells (hiPSCs) have provided an unprecedented experimental setting for disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics (Takahashi and Yamanaka, 2015; Tapia and Sch€ oler, 2016). To reach these applications in clinics, however, reprogramming of hiPSCs and their differentiation procedures should be in compliance with a high safety profile without inherent risks of tumorigenesis (Kimmelman et al., 2016). Importantly, new “scarless” technologies have been developed to generate adult somatic cells into hiPSCs without leaving genetic traces of the reprogramming event. In fact, nonintegrating technologies based on episomal vectors, synthetic mRNAs, and Sendai viruses have proven efficient to reprogram high-quality hiPSCs from various somatic cell types and can be employed in GMP settings to generate hiPSC adequate for future applications in clinics (Schlaeger et al., 2015). A further obstacle to carefully consider is the tendency of hiPSCs to accumulate genomic alterations during both the reprogramming process and the extensive periods of in vitro culture (Ben-David et al., 2011; Weissbein et al., 2014). In fact, beyond structural and numerical chromosomal aberrations, hiPSCs can suffer from small copy number variations on the kilobase scale, which are not present in the somatic cells of origin (Gore et al., 2011). Although one study was able to identify a recurrent set of point mutations in the mouse iPSC clones tested (Young et al., 2012), none of the studies could detect any recurrent single-nucleotide variations in hiPSCs, indicating that no single significant mutation tends to arise during successful reprogramming nor provides a substantial growth advantage in culture (Gore et al., 2011; Ruiz et al., 2013). Thus, genomic content of hiPSCs should be carefully evaluated after reprogramming and during culture expansion and use of cells at late passages should be avoided when possible. For a sufficiently in-depth genomic analysis, standard G-band karyotyping should be associated with higher-resolution methods like CGH (comparative genomic hybridization) or SNP (single-nucleotide polymorphism) genomic arrays for detection of small genetic changes (Mayshar et al., 2010). An alternative to DNA-based methods is the e-karyotyping assay, which predicts chromosomal aberrations from gene-expression biases, and, for instance, a chromosomal gain can be identified by consistent overexpression of genes throughout the aberrant region (Ben-David et al., 2013). Hence, this last approach provides an accurate estimation of chromosomal integrity as well as the full gene-expression profiling of the hiPSCs at the same time. A remaining bottleneck in hiPSC reprogramming is the overall low efficiency of the reprogramming event and the extended time necessary to obtain the reprogrammed primary clones. Intriguingly, a recent report has shown that efficiency in iPSC reprogramming can be dramatically improved in a microfluidic environment. In fact, microliter-volume confinement resulted in a 50-fold increase in
2 Generation of iNeurons by direct cell conversion
efficiency over traditional reprogramming by delivery of synthetic mRNAs encoding transcription factors (TFs) (Luni et al., 2016). High quality and pure hiPSCs were obtained in 2 weeks from initial reprogramming and subsequently differentiated into functional hepatocyte- and cardiomyocyte-like cells in the same platform without additional expansion (Luni et al., 2016). Thus, the microfluidic platform promotes a highly efficient and fast iPSC reprogramming while consistently reducing the required culture media and reagents, ultimately strongly lowering the overall costs. Thus, this technology can provide the opportunity for the generation of high numbers of hiPSCs from a large cohort of patients in an accelerated time frame and with affordable costs. iPSCs are long-term and homogenous self-renewal stem cells which are perfectly suited for genetic engineering. The advent of the CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas)) gene-editing technology has provided an easy and straightforward tool for targeted gene modifications in hiPSCs (Hockemeyer and Jaenisch, 2016). In fact, differently from previous gene-editing technologies like zinc-finger nucleases and TALENs, the CRISPR/Cas9 system can be easily set up in any regular laboratory, providing a powerful and flexible approach for any type of genetic modifications (Doudna and Charpentier, 2014; Hsu et al., 2015; Mali et al., 2013). Gene-editing technologies for gene manipulation in hiPSCs have already proven successful for: (i) correcting disease-causing gene mutations in patient hiPSCs (Smith et al., 2014), (ii) inserting disease-causing gene mutations in control hiPSCs (Liu et al., 2016a), (iii) performing straight and conditional gene mutagenesis (Chen et al., 2015; Rubio et al., 2016), and (iv) inducing targeted genomic alterations (Park et al., 2016). In all these applications, the CRISPR/Cas9 machinery beyond targeting the correct sequence might be engaged with similar other sequences in the genome, leading to off-target effects. Thus, it is mandatory to undertake a close analysis of the putative off-target sequences, as predicted in silico by on-line computational tools or by an unbiased full sequencing analysis (Tycko et al., 2016). Off-target mutations can be mitigated by taking advantage of new Cas9 engineered variants which present a substantially higher fidelity profile while maintaining comparable efficiency for the on-target site (Kleinstiver et al., 2016; Slaymaker et al., 2016). Thus, CRISPR/Cas9 gene editing offers a new level of precision in genome manipulation of hiPSCs, providing an efficient and safe technological platform for correcting disease-causing gene mutations or defining the molecular contributors to the pathogenesis of numerous human diseases.
2 GENERATION OF iNEURONS BY DIRECT CELL CONVERSION The seminal discovery of iPSCs in 2006 has provided the conceptual evidence that supraphysiological misexpression of developmental TFs is sufficient to promote conversion between two cell types even with very different embryonic origin,
55
56
CHAPTER 2 iPS and iN cells
developmental potential, and functional state (Takahashi and Yamanaka, 2006). iPSC generation appears not to recapitulate the developmental milestones occurring during physiological differentiation of pluripotent stem cells. Conversely, reprogramming factors stimulate the pluripotency gene network directly, thereby promoting the emergence of the pluripotent stem cell identity in the host cell followed by the dismantlement of the original cell identity. In this perspective, any cell type can in principle be reprogrammed into a new fate whenever the correct transcriptional program is initiated and self-maintained over time. Based on this framework, the field of cell reprogramming has recently exploded, providing numerous examples of striking transitions from one cell type into another promoted by a minimal combination of developmental TFs (Graf and Enver, 2009; Masserdotti et al., 2016; Xu et al., 2015). Among them, the direct conversion of fibroblasts into neurons has provided the first example of direct reprogramming between two cell types developed from different embryonic germinal layers (Vierbuchen et al., 2010). Forced expression of the three neurodevelopmental TFs Ascl1, Brn2, and Myt1l (ABM) is sufficient to convert mouse fibroblasts into induced neuronal (iN) cells that—beyond developing long and polarized neurites—acquired sophisticated functional properties, such as membrane excitability and synapse activity. Considering that the reprogrammed cells closely resemble primary neurons in their morphology, transcriptome, and electrophysiological properties, I favor referring to them simply as induced neurons (iNeurons), much in line with the nomenclature for other reprogrammed cell types, e.g., induced hepatocytes (Lim et al., 2016) and cardiomyocytes (Qian et al., 2012). Importantly, expression of the reprogramming factors can be silenced in both mouse and human iNeurons without altering their acquired morphological and functional neuronal properties. Interestingly, the stable maintenance of the iNeuronal identity is achieved when the corresponding endogenous genes of the reprogramming factors are stably activated, and their expression is maintained even after silencing of the exogenous transgenes (Pang et al., 2011; Vierbuchen et al., 2010). Importantly, after transplantation into the brains of adult rats, human iNeurons survive with high efficiency in the host neural parenchyma preserving and further developing their neuronal morphology (Pereira et al., 2014).
2.1 MOLECULAR MECHANISMS OF DIRECT NEURONAL REPROGRAMMING Mechanistically, during the fibroblast-to-neuron conversion, Ascl1 acts as a powerful pioneer TF by accessing its authentic neuronal target genes in the repressive and silent chromatin state of the fibroblasts. Subsequently, Brn2 and Myt1l are recruited to the chromatin by Ascl1, while they alone lack the ability to access the fibroblast chromatin (Wapinski et al., 2013). In fact, Ascl1 is sufficient by itself to generate functional iNeurons from mouse and human fibroblasts, indicating that Ascl1 is the key driver in reprogramming (Chanda et al., 2014). However, Myt1l and Brn2 play a critical role in enhancing the neuronal maturation process and to prevent Ascl1
2 Generation of iNeurons by direct cell conversion
from activating myogenic target genes caused by its close homology with the myogenic inducer MyoD (Treutlein et al., 2016; Wapinski et al., 2013). Another powerful combination to generate iNeurons with cholinergic identity is the enforced expression of Neurogenin-2 (Neurog2) with the two small molecules forskolin and dorsomorphin (Liu et al., 2013). Interestingly, similar to Ascl1, Neurog2 acts as a pioneer TF accessing closed chromatin, but small molecules synergize with Neurog2 to enhance chromatin accessibility and H3K27 acetylation (Smith et al., 2016). Mechanistically, forskolin is an activator of cAMP synthesis and through PRKACA (protein kinase cAMP-activated catalytic subunit A) kinase activity phosphorylates, CREB1 which binds and costimulates a subset of the Neurog2 downstream genes. Thus, small molecules might have a strong impact in regulating and promoting the cell conversion efficiency of the reprogramming factors.
2.2 STRATEGIES TO IMPROVE EFFICIENCY AND MATURITY OF iNEURONS Despite that the ABM cocktail is very efficient in reprogramming mouse primary fibroblasts, the conversion rate is strongly reduced when starting from human cells, and the functional maturation of human iNeurons is generally limited (Pang et al., 2011). Notably, recent findings have identified additional factors, epigenetic regulators, microRNAs (miRNAs), and small molecules that favor neuronal cell lineage reprogramming. In particular, expressing ABM in human fibroblasts with either Neurog2 or NeuroD1, two neurodevelopmental helix-loop-helix factors, provided a significant increase in the iNeuron yield (Ladewig et al., 2012; Pang et al., 2011). Interestingly, both TFs have a prominent role in promoting the glutamatergic neuronal cell fate during cerebral cortex development and, therefore, have a synergic action on the same neuronal lineage promoted by the ABM combination. Four-factor reprogrammed human iNeurons displayed an increased neuronal maturation both in morphology and in functional properties although they appeared relatively immature as indicated by their slightly depolarized membrane potentials and the relatively low-amplitude synaptic responses (Pang et al., 2011). While additional reprogramming factors might enhance the neuronal conversion, increasing numbers of factors pose substantial practical hurdles for their efficient delivery, as for instance the increasing number of vectors that need to be employed independently and the optimal relative expression levels among the different factors. miRNAs are small noncoding elements with an emerging role in regulating cell-fate genes and their associated chromatin state. Notably, the overexpression of the neuronal-specific miR-9/9* and miR-124 alone in human fibroblasts can induce MAP-2-expressing iNeurons, although the addition of TFs is required for developing iNeurons with functional activity (Yoo et al., 2011). In another study, the overexpression of miR-124 also promoted human iNeuronal induction mediated via Brn2 and Myt1l overexpression (Ambasudhan et al., 2011). Interestingly, miR-9/9* and miR-124 regulate the composition of the Brg1-associated factors (BAF) complex during neural development promoting the formation of neuron-specific nBAF
57
58
CHAPTER 2 iPS and iN cells
complexes essential for postmitotic neurons (Yoo et al., 2009). The significant role of miRNAs in cell reprogramming has been further revealed by the surprising finding that the inhibition of the miRNA regulator PTB alone can generate functional murine iNeurons (Xue et al., 2013). PTB blocks miRNA-mediated activity of the REST complex; thus, PTB inhibition leads to derepression of multiple miRNA-regulated neuronal genes stimulating neuronal cell reprogramming. Interestingly, REST is also a negative regulator of the astrocyte-to-neuronal conversion (Masserdotti et al., 2015). In this system, REST was shown to directly repress critical downstream targets of the neuronal reprogramming factors, thus, limiting their action in stimulating the neuronal cell identity. Expression of miRNAs on cells is technically handy since due to their small size their insertion in expression vectors is easy and flexible. However, expression of either reprogramming genes or miRNAs is generally achieved by using lentiviral technology, which raises safety concerns for its clinical applications caused by their random integration in the host cell genome. A promising solution for this drawback is the induction of the cell-fate conversion by small molecules. In fact, chemical inhibitors of the SMAD signaling and GSK3beta facilitated neuronal reprogramming in combination with TFs (Ladewig et al., 2012). SMADs and GSK3beta have a significant role also in repressing neural fate during early embryogenesis, thus suggesting evident similarities between the processes that regulate normal neuronal differentiation during development and direct neuronal reprogramming. Importantly, two recent studies completed fibroblast neuronal reprogramming using only a defined set of small molecules (Hu et al., 2015; Li et al., 2015). In particular, Li et al. (2015) carried out neuronal conversion by using a combination of four chemicals, which included three inducers of neuronal fate (ISX9, GSK3beta inhibitor, and Forskolin) and I-BET151, a suppressor of the host cell molecular program. I-BET151 was reported to competitively bind the BRD domain of BET family proteins (Seal et al., 2012). Interestingly, BET family proteins were described to specifically associate with the activated chromatin domains and maintain the cell-fate-specific gene-expression pattern (Wu et al., 2015). In particular, inhibition of BRD4 can disrupt cell-fate maintenance and alter the gene-expression pattern controlling cell-type identity (Di Micco et al., 2014). Thus, these results support the view that BRD chemical inhibition concurs in disrupting the fibroblast-specific gene-expression program in early-stage reprogramming facilitating neuronal conversion. In the second study, Hu et al. (2015) selected a cocktail of seven small molecules including inhibitors for the JNK, PKC, and ROCK signaling that was sufficient to generate human iNeurons with functional activity. However, the exact contribution of these pathways in promoting the neuronal cell fate and their downstream molecular mechanisms is yet to be discerned. Another intriguing question is how small molecules could replace the function of exogenous TFs during the chemical conversion process. Although the difference of global gene expression between different cell types may involve thousands of genes, the core gene regulatory network that determines one specific cell type may only be comprised of several master genes. For example, a recent study indicated that the
2 Generation of iNeurons by direct cell conversion
essential TF program of naı¨ve mouse pluripotent stem cells involves 16 interactions, 12 components, and 3 inputs (Dunn et al., 2014). Thus, it is conceivable that small molecules can initially activate sufficient levels of one or only a few reprogramming factors sufficient to initiate the cell-fate switch. However, the exact mechanism connecting the small molecule-dependent external stimuli with the activation of the endogenous reprogramming factors is yet to be elucidated.
2.3 APPROACHES FOR THE GENERATION OF iNEURONAL SUBTYPES iNeurons generated with the original BAM reprogramming cocktail exhibit functional properties of excitatory glutamatergic neurons lacking a defined regional identity (Vierbuchen et al., 2010). However, a large body of recent work has identified new sets of neurodevelopmental TFs able to reprogram somatic cells into a variety of neuronal subtypes that can be potentially useful for cell-based therapies as well as for disease modeling. Altogether, these findings suggest that neuronal subtype specification can be achieved in vitro by expressing lineage-specific TFs without recapitulating the entire developmental program occurring during brain development. A growing number of minimal combinations of TFs with various technical platforms have recently been reported, and some of them will be described to shed light on the basic principles controlling neuronal subtype reprogramming.
2.3.1 Forebrain-specific neuronal subtypes Glutamatergic iNeurons can be generated from fibroblasts using the ABM cocktail or from astrocytes by enforced expression of Neurog2, thus, indicating that the starting cell type strongly determines the reprogramming conditions and factors required to trigger successful reprogramming (Heinrich et al., 2010; Masserdotti et al., 2015; Vierbuchen et al., 2010). This implies that iNeurons obtained from the two systems, despite having a common glutamatergic phenotype, must rely on different reprogramming mechanisms, distinguished by specific features and properties. A direct comparison of the molecular mechanisms triggered by the two reprogramming methods will reveal the cell type specificity and their constraints in neuronal cell switch. However, the next crucial step is to generate distinct neuronal glutamatergic subtypes with the characteristics of those located in different layers of the cerebral cortex with their specific afferent projections and efferent connections (Lodato and Arlotta, 2015). Notably, forced expression of the single TF Fezf2 triggers the callosal projection neurons of cortical layer II/III into layer V/VI subcortical projection neurons (Lodato and Arlotta, 2015). This neuronal fate switch occurred also when Fezf2 was expressed in postmitotic neuroblasts, indicating that this process is at least in part independent by both the cell cycle and the more intrinsic plastic state of the proliferating neuronal progenitors. Several other developmental TFs have been described to have a prominent role in determining the identity of cortical layer-specific neurons, thus, suggesting that combining these factors together might trigger these particular fates starting from nonneuronal cells.
59
60
CHAPTER 2 iPS and iN cells
Cerebral cortex is a complex ensemble of excitatory and inhibitory neurons, and their interactions regulate the net balance of excitation and inhibition that underlies normal physiological processing. Inhibitory GABAergic neurons are local-projecting neurons within the cortex and are classified in several groups depending on their embryonic origin, connections, and molecular repertoire. Dysfunctions or loss of these neurons are the initial cause or a strong contributor for several brain disorders, including various forms of epilepsies and cognitive disorders. Interestingly, iGABA interneurons (iGABA-iNs) were recently generated by the direct conversion of murine and human fibroblasts using five developmental TFs (Colasante et al., 2015). This is the first time that the expression of the reprogramming transgenes has been modulated in different time windows; in order to enable an efficient functional maturation of the iGABA-iNs, two of the five factors were expressed for 12–14 days only and then silenced through an inducible promoter system (Colasante et al., 2015). Indeed, some of the reprogramming factors, although indispensable for the initial cell lineage conversion, might direct the process to a progenitor cell stage blocking the progression toward a mature functional state of the induced cell type. This phenomenon might occur as well in other cases of direct cell conversion. The capacity to modulate the expression of each factor independently by the others is thereby a relevant technical advance which increases the flexibility of these approaches. In a separate study, three of the same factors were shown to promote differentiation of hiPSCs into GABAergic neurons in a single-step process (Sun et al., 2016). This is a convenient approach since the same differentiation process with small molecules is a labor-intensive procedure that requires more than 3 months to obtain functional GABAergic neurons with several intermediate steps of cell manipulation (Maroof et al., 2013; Nicholas et al., 2013).
2.3.2 Dopaminergic neurons Midbrain dopaminergic neurons are of great interest since their loss is the leading cause of Parkinson’s disease. During development, they are generated in the floor plate of the mesencephalon and several developmental TFs have been implicated in their generation and specification. These include Otx2, which is involved in early patterning; FoxA1/2, which instructs the commitment of the progenitor cells; Lmx1a/b, Ascl1, and Ngn2 that are important for progenitor cell differentiation; and Pitx3 and Nurr1 (Nr4a2), which are involved in the maturation and long-term survival of midbrain dopaminergic neurons (Arenas et al., 2015). Accordingly, many of these TFs have been successfully used to generate induced dopaminergic (iDA) neurons from fibroblasts or astrocytes (Caiazzo et al., 2011; Kim et al., 2011; Pfisterer et al., 2011; Torper et al., 2013). Ascl1, Nurr1, and Lmx1a (ANL) are the minimal combination able to generate iDA neurons endowed with distinguished subtype functional features as dopamine production, functional D2 autoreceptors, and pacemaker-like firing of action potentials (Caiazzo et al., 2011). Importantly, transplanted ANL-induced iDA neurons matured and connected within the host neuronal tissue and could rescue in large part the behavioral deficits caused by the six-OHDA-mediated acute loss of endogenous DA neurons (Dell’Anno et al., 2014).
3 Direct reprogramming of glial subtypes
Functional replacement in vivo obtained with iNeurons of the corresponding endogenous neuronal subtype is the most rigorous and convincing assay to demonstrate the equivalence of the two neuronal cell types. Thus, in cases where it is possible to perform cell replacement with clear functional and behavioral readouts in vivo, this should represent a mandatory requirement before claiming a functional correspondence between induced and somatic neuronal subtypes. However, none of these studies has yet been able to specify iNeurons with a frank VTA (A10) or substantia nigra (A9)-specific identity (Hegarty et al., 2013). This, indeed, remains a challenging task until the molecular determinants discriminating between these two DA neuronal populations have been identified.
2.3.3 Spinal motor neurons Direct reprogramming to spinal cord motor neurons was recently obtaining by combining common neurogenic factors such as Ascl1, Neurog2, Myt1l, and Brn2 (Pou3f2) with TFs specific to spinal cord motor neuron development, such as Lhx3, Isl1, and Hb9 (Lee et al., 2009; Son et al., 2011). The combination of these seven factors generated functional induced motor neurons (iMNs) from mouse embryonic fibroblasts that were able to establish functional neuromuscular junctions with cocultured myotubes, and which could survive after transplantation in vivo (Son et al., 2011). With a similar rationale, Liu et al. combined two panneurogenic TFs, Neurog2 and Sox11, and two neuronal subtype-specific TFs, Isl1 and Sox11, together with forskolin, dorsomorphin, and Fgf2 treatment, converting human fibroblasts into HB9 and ChAT-positive human iMNs with an extremely high efficiency (>80%) (Liu et al., 2016b). These data therefore further support the key role of region-specific TFs for the specification of distinct neuronal subtypes.
3 DIRECT REPROGRAMMING OF GLIAL SUBTYPES Recent findings have demonstrated that TF-mediated reprogramming can be applied to generate induced oligodendrocytes, Schwann cells (SCs), and astrocytes. Two independent studies identified a three TF combination which share the factors Sox10 and Olig2 capable of converting mouse fibroblasts into oligodendrocyte precursor cells (iOPCs) that express appropriate OPC markers, produce myelin sheaths in vitro, and sustain myelin regeneration in mouse brains with genetic dysmyelination (Najm et al., 2013; Yang et al., 2013). Sox10 fulfills widespread and essential functions in myelinating glia of the central as well as peripheral nervous system at multiple stages of development such as glial specification, survival, and terminal differentiation. Interestingly, iOPCs were shown to lack myelin protein zero protein, a specific SC marker, and myelinated multiple axons confirming their central glial cell identity. Conversely, Sox10 only, combined with secreted (BMP4 and WNT) and epigenetic factors (VPA and 5-Aza), was sufficient to reprogram fibroblasts into induced neural crest cells (iNCCs) (Kim et al., 2014). Despite the observation that iNCCs could differentiate into both peripheral neurons and glial cells, they remained
61
62
CHAPTER 2 iPS and iN cells
at a very immature stage lacking an evident functional state. Intriguingly, when Sox10 was combined with Egr2 (known also as Krox-20), a developmental factor necessary for SC myelination, fibroblasts were directly converted into induced SCs with peripheral identity able to wrap axons in vitro for generating compact myelin sheaths with regular nodal structures (Mazzara et al., 2017). Thus, Sox10 is a crucial factor for reprogramming myelinating glia together with selective intrinsic or extrinsic factors which further specify the central or peripheral identity of the induced cells. Astrocytes are the most representative glial component in the brain with trophic and regulatory functions on neurons. Notably, direct reprogramming of murine and human fibroblasts into induced astrocytes (iAstrocytes) was recently obtained using the minimal combination of the three glial molecular determinants NFIA, NFIB, and Sox9. For gene-expression profiling, electrophysiological properties, glutamate uptake, and inflammatory response iAstrocytes had a comparable behavior compared to native brain astrocytes (Caiazzo et al., 2015). Altogether, these results demonstrate that genetic methods of direct cell reprogramming are available to convert adult somatic cells into all three main glial cell types populating the central and peripheral neural tissue.
4 iNEURONS AND GLIA CELLS FOR DISEASE IN VITRO CELL MODELING iNeurons and iGlia are generated with a fast and straightforward process, which make these cells particularly convenient for applications in cell replacement therapies and in vitro disease modeling. Currently, however, a strong hurdle for in vivo applications of these cells is the general use of genome integrating technologies, such as lentiviruses, for transgene expression, which raises severe concerns of genotoxicity for the integration of the viral transgenes in the host genome. Thus, alternative methods are needed to achieve cell reprogramming, which ensure safety of the reprogramming process. Notably, neuronal reprogramming was recently obtained with nonintegrating lentiviruses or AAV, suggesting that these modifications in principle are suitable for clinical use (Lau et al., 2014). A major issue, however, is to maintain high levels of cell reprogramming efficiency, even using clinical-grade methods in order to ensure sufficient numbers of cells for the subsequent clinical applications. Methodologies of direct neuronal reprogramming can be also exploited for establishing models of human diseases in vitro. In fact, this technical platform has important advantages in this application, strongly complementary to those associated with iPSC reprogramming. Importantly, while iPSCs and derived neurons do not retain the aging phenotype of the starting cells, iNeurons maintain the age-related gene expression and phenotypic features according to the age of the reprogrammed cells (Mertens et al., 2015; Miller et al., 2013). This is a crucial aspect which makes direct neuronal reprogramming a convenient system to model age-related disorders, as the case for most of the human neurodegenerative diseases. However, iNeuronal
References
reprogramming generally skips out any intermediate proliferating cell stage; and therefore, it is always necessary to start back from the patient cells for each reprogramming experiment. This can result in a strong drawback when patient cells are difficult to obtain, and their prolonged passaging in vitro might reduce reprogramming efficiency. In such case, a potential solution would be the direct generation of proliferating neural progenitors as a self-expanding intermediate step for subsequent generation of postmitotic neurons. In fact, recent studies have identified a minimal combination of TFs sufficient to convert mouse and human fibroblasts into induced neural progenitor cells (iNPCs) able to largely expand in vitro maintaining the trilineage differentiation potential to generate neurons, astrocytes, and oligodendrocytes (Han et al., 2012; Lujan et al., 2012; Their et al., 2012). Intriguingly, Zhang et al. (2016) have recently devised a pharmacological method based on nine small molecules to reprogram murine fibroblasts into iNPCs. A future development of this method for generating iNPCs from human fibroblasts will open exciting opportunities for in vitro modeling of human disorders. Nevertheless, recent studies have confirmed the value of human iNeurons to dissect the pathological deficits associated to human disorders. In fact, iMNs generated from fibroblasts of amyotrophic lateral sclerosis (ALS) patients recapitulated some crucial aspects of ALS pathology, like the mislocalization of the ribonucleoprotein FUS, smaller soma, lower firing frequency, and higher susceptibility to cell death over time (Liu et al.,2016b). In addition, this approach has been extended to generate iNeurons from familial Alzheimer’s disease patients, showing a pathological increase in both Ab42/Ab40 ratio and release of the amyloid Ab42 fragment (Hu et al., 2015). These results stand as a proof of concept that iNeurons can be informative for disease mechanisms only if their subtype specification, close resemblance to the native neuronal counterpart, and functional maturation are granted by the neuronal reprogramming process. In such case, neuronal reprogramming can offer the unique advantage to be fast and easy to replicate in large numbers in order to obtain iNeurons from multiple patients in a short time. Thus, these reprogramming technologies might have invaluable applications for the future modeling of idiopathic and sporadic disorders where many patients need to be sampled and their cells analyzed in comparative studies.
ACKNOWLEDGMENTS I apologize to all those scientists whose outstanding work could not be cited due to space limitations. I thank all members of my laboratory for their comments on this manuscript.
REFERENCES Ambasudhan, R., Talantova, M., Coleman, R., Yuan, X., Zhu, S., Lipton, S.A., et al., 2011. Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions. Cell Stem Cell 9, 113–118.
63
64
CHAPTER 2 iPS and iN cells
Arenas, E., Denham, M., Villaescusa, J.C., 2015. How to make a midbrain dopaminergic neuron. Development 142, 1918–1936. Ben-David, U., Mayshar, Y., Benvenisty, N., 2011. Large-scale analysis reveals acquisition of lineage-specific chromosomal aberrations in human adult stem cells. Cell Stem Cell 9, 97–102. Ben-David, U., Mayshar, Y., Benvenisty, N., 2013. Virtual karyotyping of pluripotent stem cells on the basis of their global gene expression profiles. Nat. Protoc. 8 (5), 989–997. Caiazzo, M., Dell’Anno, M.T., Dvoretskova, E., Lazarevic, D., Taverna, S., Leo, D., et al., 2011. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476, 224–227. Caiazzo, M., Giannelli, S., Valente, P., Lignani, G., Carissimo, A., Sessa, A., et al., 2015. Direct conversion of fibroblasts into functional astrocytes by defined transcription factors. Stem Cell Rep. 4, 25–36. Chanda, S., Ang, C.E., Davila, J., Pak, C., Mall, M., Lee, Q.Y., et al., 2014. Generation of induced neuronal cells by the single reprogramming factor ASCL1. Stem Cell Rep. 3, 282–296. Chen, Y., Cao, J., Xiong, M., Petersen, A.J., Dong, Y., Tao, Y., et al., 2015. Engineering human stem cell lines with inducible gene knockout using CRISPR/Cas9. Cell Stem Cell 17, 233–244. Colasante, G., Lignani, G., Rubio, A., Medrihan, L., Yekhlef, L., Sessa, A., et al., 2015. Rapid conversion of fibroblasts into functional forebrain GABAergic interneurons by direct genetic reprogramming. Cell Stem Cell 17, 719–734. Dell’Anno, M.T., Caiazzo, M., Leo, D., Dvoretskova, E., Medrihan, L., Colasante, G., et al., 2014. Remote control of induced dopaminergic neurons in Parkinsonian rats. J. Clin. Invest. 124, 3215–3229. Di Micco, R., Fontanals-Cirera, B., Low, V., Ntziachristos, P., Yuen, S.K., Lovell, C.D., et al., 2014. Control of embryonic stem cell identity by BRD4-dependent transcriptional elongation of super-enhancer-associated pluripotency genes. Cell Rep. 9, 234–247. Doudna, J.A., Charpentier, E., 2014. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096. Dunn, S.J., Martello, G., Yordanov, B., Emmott, S., Smith, A.G., 2014. Defining an essential transcription factor program for naı¨ve pluripotency. Science 344, 1156–1160. Gore, A., Li, Z., Fung, H.L., Young, J.E., Agarwal, S., Antosiewicz-Bourget, J., et al., 2011. Somatic coding mutations in human induced pluripotent stem cells. Nature 471, 63–67. Graf, T., Enver, T., 2009. Forcing cells to change lineages. Nature 462, 587–594. Han, D.W., Tapia, N., Hermann, A., Hemmer, K., H€ oing, S., Arau´zo-Bravo, M.J., et al., 2012. Direct reprogramming of fibroblasts into neural stem cells by defined factors. Cell Stem Cell 10, 465–472. Hegarty, S.V., Sullivan, A.M., O’Keeffe, G.W., 2013. Midbrain dopaminergic neurons: a review of the molecular circuitry that regulates their development. Dev. Biol. 379, 123–138. Heinrich, C., Blum, R., Gasco´n, S., Masserdotti, G., Tripathi, P., Sa´nchez, R., et al., 2010. Directing astroglia from the cerebral cortex into subtype specific functional neurons. PLoS Biol. 8, e1000373. Hockemeyer, D., Jaenisch, R., 2016. Induced pluripotent stem cells meet genome editing. Cell Stem Cell 18, 573–586. Hsu, P.D., Lander, E.S., Zhang, F., 2015. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278. Hu, W., Qiu, B., Guan, W., Wang, Q., Wang, M., Li, W., et al., 2015. Direct conversion of normal and Alzheimer’s disease human fibroblasts into neuronal cells by small molecules. Cell Stem Cell 17, 204–212.
References
Kim, J., Su, S.C., Wang, H., Cheng, A.W., Cassady, J.P., Lodato, M.A., et al., 2011. Functional integration of dopaminergic neurons directly converted from mouse fibroblasts. Cell Stem Cell 9 (5), 413–419. Kim, Y.J., Lim, H., Li, Z., Oh, Y., Kovlyagina, I., Choi, I.Y., et al., 2014. Generation of multipotent induced neural crest by direct reprogramming of human postnatal fibroblasts with a single transcription factor. Cell Stem Cell 15, 497–506. Kimmelman, J., Heslop, H.E., Sugarman, J., Studer, L., Benvenisty, N., Caulfield, T., et al., 2016. New ISSCR guidelines: clinical translation of stem cell research. Lancet 387, 1979–1981. Kleinstiver, B.P., Pattanayak, V., Prew, M.S., Tsai, S.Q., Nguyen, N.T., Zheng, Z., et al., 2016. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495. Ladewig, J., Mertens, J., Kesavan, J., Doerr, J., Poppe, D., Glaue, F., et al., 2012. Small molecules enable highly efficient neuronal conversion of human fibroblasts. Nat. Methods 9, 575–578. Lau, S., Rylander Ottosson, D., Jakobsson, J., Parmar, M., 2014. Direct neural conversion from human fibroblasts using self-regulating and nonintegrating viral vectors. Cell Rep. 9, 1673–1680. Lee, S., Lee, B., Lee, J.W., Lee, S.K., 2009. Retinoid signaling and neurogenin2 function are coupled for the specification of spinal motor neurons through a chromatin modifier CBP. Neuron 62, 641–654. Li, X., Zuo, X., Jing, J., Ma, Y., Wang, J., Liu, D., et al., 2015. Small-molecule-driven direct reprogramming of mouse fibroblasts into functional neurons. Cell Stem Cell 17, 195–203. Lim, K.T., Lee, S.C., Gao, Y., Kim, K.P., Song, G., An, S.Y., et al., 2016. Small molecules facilitate single factor-mediated hepatic reprogramming. Cell Rep. 16, 30360–30366. S2211–1247. Liu, M.L., Zang, T., Zou, Y., Chang, J.C., Gibson, J.R., Huber, K.M., et al., 2013. Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons. Nat. Commun. 4, 2183. Liu, J., Gao, C., Chen, W., Ma, W., Li, X., Shi, Y., et al., 2016a. CRISPR/Cas9 facilitates investigation of neural circuit disease using human iPSCs: mechanism of epilepsy caused by an SCN1A loss-of-function mutation. Transl. Psychiatry 6, e703. Liu, M.L., Zang, T., Zhang, C.L., 2016b. Direct lineage reprogramming reveals disease-specific phenotypes of motor neurons from human ALS patients. Cell Rep. 14, 115–128. Lodato, S., Arlotta, P., 2015. Generating neuronal diversity in the mammalian cerebral cortex. Annu. Rev. Cell Dev. Biol. 31, 699–720. Lujan, E., Chanda, S., Ahlenius, H., S€udhof, T.C., Wernig, M., 2012. Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells. Proc. Natl. Acad. Sci. U.S.A. 109, 2527–2532. Luni, C., Giulitti, S., Serena, E., Ferrari, L., Zambon, A., Gagliano, O., et al., 2016. High-efficiency cellular reprogramming with microfluidics. Nat. Methods. 13 (5), 446–452. Mali, P., Esvelt, K.M., Church, G.M., 2013. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963. Maroof, A.M., Keros, S., Tyson, J.A., Ying, S.W., Ganat, Y.M., Merkle, F.T., et al., 2013. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell 12, 559–572.
65
66
CHAPTER 2 iPS and iN cells
Masserdotti, G., Gillotin, S., Sutor, B., Drechsel, D., Irmler, M., Jørgensen, H.F., et al., 2015. Transcriptional mechanisms of proneural factors and REST in regulating neuronal reprogramming of astrocytes. Cell Stem Cell 17, 74–88. Masserdotti, G., Gasco´n, S., G€otz, M., 2016. Direct neuronal reprogramming: learning from and for development. Development 143, 2494–2510. Mayshar, Y., Ben-David, U., Lavon, N., Biancotti, J.C., Yakir, B., Clark, A.T., et al., 2010. Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell 7, 521–531. Mazzara, P.G., Massimino, L., Pellegatta, M., Ronchi, G., Ricca, A., Iannielli, A., et al., 2017. Two factor based reprogramming of rodent and human fibroblasts into myelinogenic Schwann cells. Nat. Commun. In press. Mertens, J., Paquola, A.C., Ku, M., Hatch, E., B€ ohnke, L., Ladjevardi, S., et al., 2015. Directly reprogrammed human neurons retain aging-associated transcriptomic signatures and reveal age-related nucleocytoplasmic defects. Cell Stem Cell 17, 705–718. Miller, J.D., Ganat, Y.M., Kishinevsky, S., Bowman, R.L., Liu, B., Tu, E.Y., et al., 2013. Human iPSC-based modeling of late-onset disease via progerin-induced aging. Cell Stem Cell 13, 691–705. Najm, F.J., Lager, A.M., Zaremba, A., Wyatt, K., Caprariello, A.V., Factor, D.C., et al., 2013. Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells. Nat. Biotechnol. 31, 426–433. Nicholas, C.R., Chen, J., Tang, Y., Southwell, D.G., Chalmers, N., Vogt, D., et al., 2013. Functional maturation of hPSC-derived forebrain interneurons requires an extended timeline and mimics human neural development. Cell Stem Cell 12, 573–586. Pang, Z.P., Yang, N., Vierbuchen, T., Ostermeier, A., Fuentes, D.R., Yang, T.Q., et al., 2011. Induction of human neuronal cells by defined transcription factors. Nature 476, 220–223. Park, C.Y., Sung, J.J., Choi, S.H., Lee, D.R., Park, I.H., Kim, D.W., 2016. Modeling and correction of structural variations in patient-derived iPSCs using CRISPR/Cas9. Nat. Protoc. 11, 2154–2169. Pereira, M., Pfisterer, U., Rylander, D., Torper, O., Lau, S., Lundblad, M., et al., 2014. Highly efficient generation of induced neurons from human fibroblasts that survive transplantation into the adult rat brain. Sci. Rep. 4, 6330. Pfisterer, U., Kirkeby, A., Torper, O., Wood, J., Nelander, J., Dufour, A., et al., 2011. Direct conversion of human fibroblasts to dopaminergic neurons. Proc. Natl. Acad. Sci. U.S.A. 108, 10343–10348. Qian, L., Huang, Y., Spencer, C.I., Foley, A., Vedantham, V., Liu, L., et al., 2012. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 485, 593–598. Rubio, A., Luoni, M., Giannelli, S.G., Radice, I., Iannielli, A., Cancellieri, C., et al., 2016. Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming. Sci. Rep. 6, 37540. Ruiz, S., Gore, A., Li, Z., Panopoulos, A.D., Montserrat, N., Fung, H.L., et al., 2013. Analysis of protein-coding mutations in hiPSCs and their possible role during somatic cell reprogramming. Nat. Commun. 4, 1382. Schlaeger, T.M., Daheron, L., Brickler, T.R., Entwisle, S., Chan, K., Cianci, A., et al., 2015. A comparison of non-integrating reprogramming methods. Nat. Biotechnol. 33, 58–63.
References
Seal, J., Lamotte, Y., Donche, F., Bouillot, A., Mirguet, O., Gellibert, F., et al., 2012. Identification of a novel series of BET family bromodomain inhibitors: binding mode and profile of I-BET151 (GSK1210151A). Bioorg. Med. Chem. Lett. 22, 2968–2972. Slaymaker, I.M., Gao, L., Zetsche, B., Scott, D.A., Yan, W.X., Zhang, F., 2016. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88. Smith, C., Gore, A., Yan, W., Abalde-Atristain, L., Li, Z., He, C., et al., 2014. Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. Cell Stem Cell 15, 12–13. Smith, D.K., Yang, J., Liu, M.L., Zhang, C.L., 2016. Small molecules modulate chromatin accessibility to promote NEUROG2-mediated fibroblast-to-neuron reprogramming. Stem Cell Rep. 7, 955–969. Son, E.Y., Ichida, J.K., Wainger, B.J., Toma, J.S., Rafuse, V.F., Woolf, C.J., et al., 2011. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell 9, 205–218. Sun, A.X., Yuan, Q., Tan, S., Xiao, Y., Wang, D., Khoo, A.T., et al., 2016. Direct induction and functional maturation of forebrain GABAergic neurons from human pluripotent stem cells. Cell Rep. 16, 1942–1953. Takahashi, K., Yamanaka, S., 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676. Takahashi, K., Yamanaka, S., 2015. A developmental framework for induced pluripotency. Development 142, 3274–3285. Tapia, N., Sch€oler, H.R., 2016. Molecular obstacles to clinical translation of iPSCs. Cell Stem Cell 19, 298–309. Their, M., W€orsd€orfer, P., Lakes, Y.B., Gorris, R., Herms, S., Opitz, T., et al., 2012. Direct conversion of fibroblasts into stably expandable neural stem cells. Cell Stem Cell 10, 473–479. Torper, O., Pfisterer, U., Wolf, D.A., Pereira, M., Lau, S., Jakobsson, J., et al., 2013. Generation of induced neurons via direct conversion in vivo. Proc. Natl. Acad. Sci. U.S.A. 110, 7038–7043. Treutlein, B., Lee, Q.Y., Camp, J.G., Mall, M., Koh, W., Shariati, S.A., et al., 2016. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq. Nature 534, 391–395. Tycko, J., Myer, V.E., Hsu, P.D., 2016. Methods for optimizing CRISPR-Cas9 genome editing specificity. Mol. Cell 63, 355–370. Vierbuchen, T., Ostermeier, A., Pang, Z.P., Kokubu, Y., S€ udhof, T.C., Wernig, M., 2010. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041. Wapinski, O.L., Vierbuchen, T., Qu, K., Lee, Q.Y., Chanda, S., Fuentes, D.R., et al., 2013. Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons. Cell 155, 621–635. Weissbein, U., Benvenisty, N., Ben-David, U., 2014. Quality control: genome maintenance in pluripotent stem cells. J. Cell Biol. 204, 153–163. Wu, T., Pinto, H.B., Kamikawa, Y.F., Donohoe, M.E., 2015. The BET family member BRD4 interacts with OCT4 and regulates pluripotency gene expression. Stem Cell Rep. 4, 390–403. Xu, J., Du, Y., Deng, H., 2015. Direct lineage reprogramming: strategies, mechanisms, and applications. Cell Stem Cell 16, 119–134.
67
68
CHAPTER 2 iPS and iN cells
Xue, Y., Ouyang, K., Huang, J., Zhou, Y., Ouyang, H., Li, H., et al., 2013. Direct conversion of fibroblasts to neurons by reprogramming PTB-regulated microRNA circuits. Cell 152, 82–96. Yang, N., Zuchero, J.B., Ahlenius, H., Marro, S., Ng, Y.H., Vierbuchen, T., et al., 2013. Generation of oligodendroglial cells by direct lineage conversion. Nat. Biotechnol. 31, 434–439. Yoo, A.S., Staahl, B.T., Chen, L., Crabtree, GR., 2009. MicroRNA-mediated switching of chromatin-remodelling complexes in neural development. Nature 460 (7255), 642–646. Yoo, A.S., Sun, A.X., Li, L., Shcheglovitov, A., Portmann, T., Li, Y., et al., 2011. MicroRNA-mediated conversion of human fibroblasts to neurons. Nature 476, 228–231. Young, M.A., Larson, D.E., Sun, C.W., George, D.R., Ding, L., Miller, C.A., et al., 2012. Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells. Cell Stem Cell 10, 570–582. Zhang, M., Lin, Y.H., Sun, Y.J., Zhu, S., Zheng, J., Liu, K., et al., 2016. Pharmacological reprogramming of fibroblasts into neural stem cells by signaling-directed transcriptional activation. Cell Stem Cell 18, 653–667.