Application of biomaterials to advance induced pluripotent stem cell research and therapy
2015; Springer Nature; Volume: 34; Issue: 8 Linguagem: Inglês
10.15252/embj.201490756
ISSN1460-2075
AutoresZhixiang Tong, Aniruddh Solanki, Allison E. Hamilos, Oren Levy, Kendall Wen, Xiaolei Yin, Jeffrey M. Karp,
Tópico(s)Tissue Engineering and Regenerative Medicine
ResumoReview12 March 2015free access Application of biomaterials to advance induced pluripotent stem cell research and therapy Zhixiang Tong Zhixiang Tong Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA Search for more papers by this author Aniruddh Solanki Aniruddh Solanki Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA Search for more papers by this author Allison Hamilos Allison Hamilos Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA Search for more papers by this author Oren Levy Oren Levy Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA Search for more papers by this author Kendall Wen Kendall Wen Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA Search for more papers by this author Xiaolei Yin Xiaolei Yin Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA Search for more papers by this author Jeffrey M Karp Corresponding Author Jeffrey M Karp Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA Search for more papers by this author Zhixiang Tong Zhixiang Tong Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA Search for more papers by this author Aniruddh Solanki Aniruddh Solanki Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA Search for more papers by this author Allison Hamilos Allison Hamilos Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA Search for more papers by this author Oren Levy Oren Levy Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA Search for more papers by this author Kendall Wen Kendall Wen Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA Search for more papers by this author Xiaolei Yin Xiaolei Yin Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA Search for more papers by this author Jeffrey M Karp Corresponding Author Jeffrey M Karp Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA Search for more papers by this author Author Information Zhixiang Tong1,2,3, Aniruddh Solanki1,2,3, Allison Hamilos1,2,3, Oren Levy1,2,3, Kendall Wen1,2,3, Xiaolei Yin1,2,3,4 and Jeffrey M Karp 1,2,3 1Division of Biomedical Engineering, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Harvard Medical School, Cambridge, MA, USA 2Harvard Stem Cell Institute, Cambridge, MA, USA 3Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA 4David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA *Corresponding author. Tel: +1 617 817 9174; E-mail: [email protected] The EMBO Journal (2015)34:987-1008https://doi.org/10.15252/embj.201490756 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Derived from any somatic cell type and possessing unlimited self-renewal and differentiation potential, induced pluripotent stem cells (iPSCs) are poised to revolutionize stem cell biology and regenerative medicine research, bringing unprecedented opportunities for treating debilitating human diseases. To overcome the limitations associated with safety, efficiency, and scalability of traditional iPSC derivation, expansion, and differentiation protocols, biomaterials have recently been considered. Beyond addressing these limitations, the integration of biomaterials with existing iPSC culture platforms could offer additional opportunities to better probe the biology and control the behavior of iPSCs or their progeny in vitro and in vivo. Herein, we discuss the impact of biomaterials on the iPSC field, from derivation to tissue regeneration and modeling. Although still exploratory, we envision the emerging combination of biomaterials and iPSCs will be critical in the successful application of iPSCs and their progeny for research and clinical translation. Introduction Induced pluripotent stem cells (iPSCs) possess a regenerative therapeutic potential comparable to embryonic stem cells (ESCs) without many of the associated ethical concerns (Bilic & Izpisua Belmonte, 2012). Nevertheless, in vitro research applications and clinical translation of iPSCs have multiple challenges: Costly and highly inefficient iPSC derivation and expansion protocols. Incomplete reprogramming of somatic cells; or genetic instability occurring during in vitro expansion and differentiation, which may result in genetic abnormalities or potential immunogenicity following iPSC transplantation (Saha & Jaenisch, 2009; Araki et al, 2013). Safety concerns, primarily attributed to the potential risk of iPSC progeny to form teratomas (due to the presence of residual undifferentiated iPSCs) or malignant transformation post-transplantation (Hong et al, 2014). Overcoming these limitations depends on designing well-defined cell culture strategies for iPSC derivation, expansion, and differentiation. Limited reprogramming efficiency may be improved by modulating the presentation and kinetics of reprogramming factors, which is challenging with traditional reprogramming methods (Hu, 2014). Once reprogrammed, the efficiency of iPSC expansion and the control over their differentiation can be improved by carefully mimicking the native microenvironment of stem cells—also known as the stem cell niche (Dellatore et al, 2008). The stem cell niche orchestrates stem cell phenotype, proliferation, and differentiation through key elements such as specific extracellular matrix (ECM) composition, 3D architecture, chemical and mechanical signals, and cell–cell interactions among resident cells (Scadden, 2006; Dingal & Discher, 2014). Thus, approaches for closely mimicking stem cell niches may substantially increase the quality and efficiency of iPSC expansion and directed lineage specification. Such advancement will further enrich our understanding of iPSC biology and facilitate the development of new therapeutics (e.g. via establishment of patient-specific disease models), as well as advance iPSC-based cell replacement therapies (Robinton & Daley, 2012). Biomaterials—materials selected or designed to interact with biological systems (Williams, 2009)—offer a unique and appealing strategy to advance iPSC research. For instance, biomaterials can be used to control the kinetics of reprogramming factors via nanoparticle- and microparticle-based systems. Biomaterials can also be used to overcome the issues associated with traditional iPSC expansion and differentiation protocols by creating stem-cell-like niches that incorporate key niche elements to enable precise regulation of stem cell fate and function (Lutolf et al, 2009; Murphy et al, 2014) (Fig 1). Beyond guiding iPSC in vitro expansion and differentiation, biomaterials may also be used to facilitate iPSC transplantation (Higuchi et al, 2011; de Peppo et al, 2013; Villa-Diaz et al, 2013; Teng et al, 2014). In this review, we discuss in detail how emerging biomaterial-based strategies may solve key iPSC safety and efficiency challenges, which may soon allow iPSCs to realize their immense potential in the study and treatment of diseases. Figure 1. Overview of biomaterial-based strategies for enhancing the safety and efficiency of existing iPSC technologies and to better probe iPSC biology and control cell fate in vitro and in vivoBiomaterials may be employed to facilitate all steps of iPSC production and consequently may help address pressing limitations of current derivation, expansion, and differentiation protocols. Traditional viral iPSC reprogramming methods, though efficient, are concerning due to insertional mutagenesis. Conversely, non-integrating iPSC reprogramming methods have lower efficiencies and require elaborate experimental procedures. To overcome the concerns associated with traditional iPSC reprogramming methods, biomaterial-based nano-/microparticles can be used to control the release kinetics of reprogramming factors to potentially avoid viral insertion, increase efficiency, and introduce simpler and less labor-intensive approaches for reprogramming. Furthermore, these nano-/microparticles can also be used to deliver soluble factors and small molecules for the expansion and differentiation of iPSCs (Corradetti et al, 2012). In parallel, biocompatible synthetic substrates can be engineered with patterned physicochemical cues and functionalized with surface-tethered factors to emulate native components of stem cell niches (Watt & Huck, 2013). Making use of biomaterial-based nano-/microparticles and biocompatible synthetic substrates can improve the scalability of traditional expansion and differentiation protocols because they can be reproducibly synthesized on large scales (as they are chemically defined) and at relatively low costs. The reduction in cost and labor will be key for the large-scale production of iPSCs and their progeny. Given the costs associated with the initial development and manufacturing of biomaterials, the cost of biomaterial-based iPSC production could be higher than that of traditional protocols at the early stage. However, in the long run, the application of biomaterials could render the iPSC production and differentiation processes more efficient (e.g. reducing the required concentrations of reprogramming factors via controlling their spatiotemporal presentation). We envision that the overall cost of biomaterial-based protocols will be significantly lower than that of traditional protocols. Download figure Download PowerPoint Advanced biomaterials for the reprogramming of iPSCs Limitations of traditional iPSC reprogramming systems Current iPSC reprogramming protocols are often associated with safety and efficiency concerns. These concerns are especially critical for therapeutic applications where a large quantity of homogenous iPSCs with clinical grade quality is required. Two general approaches exist for iPSC derivation: integrating and non-integrating approaches. The integrating approach typically employs viral vectors to deliver and integrate pluripotency genes. Unfortunately, this approach has the potential for insertional mutagenesis, or malignant transformation (Ma et al, 2013b). Hence, clinical translation of integrating methods is severely limited due to safety and efficiency concerns. Alternatively, iPSCs may be derived with safer, non-integrating approaches, in which the pluripotency factors are transiently expressed or activated without genomic integration. Emerging non-integrating methods include episomal vectors and adenovirus-/plasmid-mediated transfection as well as pluripotency induction via chemically defined molecules [proteins (Kim et al, 2009; Zhou et al, 2009), mRNAs (Warren et al, 2010), and small molecules (Hou et al, 2013)]. Nevertheless, these methods remain labor-intensive and may still induce occasional genomic integration, leading to genomic instability (Hu, 2014). Even the seemingly straightforward DNA-free non-integrating methods [e.g. small molecule cocktails (Hou et al, 2013) or proteins (Zhou et al, 2009)] remain highly inefficient, requiring intricate protocols with multiple rounds of treatment, large doses, and uncontrolled presence/kinetics of reprogramming factors. Thus, new strategies are required to overcome the limitations associated with these traditional reprogramming methods. Approaches employing biomaterials to surmount these barriers have shown promising results. Engineered biomaterials can potentially aid in iPSC derivation through controlling the kinetics of reprogramming factor delivery. Furthermore, well-defined biomaterial substrates can regulate the epigenetic state of iPSCs (Downing et al, 2013). This may work synergistically with traditional reprogramming approaches to improve reprogramming efficiency. In the following section, we highlight how these biomaterial-based strategies have been employed thus far and how these methods may impact iPSC derivation. Biomaterial-based delivery systems for iPSC reprogramming Biomaterial-based derivation of iPSCs—whether alone or in combination with existing non-integrating approaches—may improve reprogramming efficiency, safety, scalability, and reproducibility. Numerous compelling strategies involving biomaterials have emerged to deliver multiple reprogramming molecules with greater efficiency and more controlled kinetics than traditional methods (Fig 2), which we discuss herein. We recognize that while biomaterial-based strategies have great implications for iPSC reprogramming, most approaches developed are too complicated to be commercialized into research tools. Concerted efforts from biologists, materials scientists, and engineers are merited to further simplify these strategies, and in particular, efforts are needed to limit costs as well as scaled batch-to-batch variability. Figure 2. Biomaterial-based approaches for improved iPSC reprogramming(A) Well-defined, biomaterial-based micro-/nanoparticles can be formulated and engineered to load multiple reprogramming factors (e.g. Sox2, Oct4, and Klf4). The controlled distribution of different factors—on the surface of the particle (factor a) and entrapped in the particle (factor b)—can be readily achieved during the particle formulation process, and an additional factor (c), if required, can be loaded onto the particle via a stimuli-responsive linker. Given the biodegradability of chosen biomaterials, the varied distribution of multiple reprogramming factors (surface adsorption versus homogeneous encapsulation) and the degradation characteristics of the carrier particle dictate the spatiotemporally controlled release profiles of different factors such as sustained, zero-order release (curve b) and initial burst release followed by slower release or maintenance dose (curve a). Meanwhile, the surface-immobilized stimuli-responsive linker can be cleaved in response to environmental triggers, for example, pH and temperature, to release factor c on demand (e.g. pulsed release every other day, curve c). (B) Nanoparticle-based, smart, artificial transcription factor that is surface-functionalized with nuclear-targeting sequence, DNA-binding domains, and activators for the relevant transcription factors, enabling efficient nuclear localization and effective gene regulation. (C) The biomaterial substrate can be engineered with specific surface anisotropy or microgroove features, which in turn controls cell morphology and, as a result, mediates the epigenetic regulation and cellular reprogramming. (D) Reprogramming factor-laden biomaterials can be delivered into different intracellular loci via multiple engineering approaches, therefore efficiently modulating cell phenotype from inside-out. For example, high-throughput microfluidic technology can be employed to rapidly generate transient membrane disruption on the cells, enabling efficient intracellular localization of phenotype-altering agents without significantly impairing the target cells. Alternatively, this can be achieved via engineering methods such as microinjection or electroporation. Figure partially adapted with permission from Xu et al (2013b) and Patel et al (2014). Download figure Download PowerPoint 1. Biomaterials for potential spatial–temporal control of reprogramming factors Maximizing efficiency of iPSC derivation depends on controlled spatial–temporal delivery of reprogramming factors since the timing and duration of cell exposure to extracellular stimuli significantly influence cell fate (Gaeta et al, 2013; Hou et al, 2013; Liu et al, 2013a). While these kinetics are poorly controlled with traditional reprogramming approaches (e.g. via media changes), biomaterial-based nano- and microparticles (MPs) can deliver and release payloads with spatial–temporal control [e.g. via sustained or pulsatile release profiles (Ge et al, 2012)], essential for maximizing the desired biological effects (Mohamed & van der Walle, 2008). These versatile particles may effectively deliver a spectrum of small molecules and biological cargos, including, but not limited to, growth factors, nucleic acids, and plasmids, making them a customizable platform for cellular reprogramming (Panyam & Labhasetwar, 2003). Specifically, engineered degradable biomaterials can deliver factors to targeted subcellular locations with precise timing by leveraging the correlation between particle size and delivery kinetics. In general, smaller particles (typically < 1 μm, depending on target cell type and uptake mechanism) can rapidly enter cells and release encapsulated agents upon enzymolysis or hydrolysis (Woodward et al, 1985; Kou et al, 2013), whereas larger particles (typically > 1–2 μm) are not as efficiently internalized and are less accessible to degradation, permitting slower, sustained release of factors (Xu et al, 2009). This difference in release kinetics has been largely attributed to the surface area-to-volume ratios of particles with different sizes (Varde & Pack, 2007; Carpenedo et al, 2010). In addition to controlling particle size, manipulating other properties of biomaterials, including material composition, degradation rate, and inner architecture (e.g. porosity), enables tuning of delivery kinetics to produce controlled release systems (Varde & Pack, 2004; Klose et al, 2006; Giteau et al, 2008). Such tunability can be further realized by harnessing the potential responsiveness of biomaterials to environmental stimuli (Kost & Langer, 2012; Nakao, 2014; Patil & Shahiwala, 2014). Such delivery systems are capable of adjusting drug release in response to particular stimuli. In addition to microenvironmental stimuli, external stimuli can also be used to activate on-demand release and can include magnetism, electrical fields, ultrasound, and temperature changes (Mura et al, 2013). A broad set of biomaterials have been explored and designed to achieve desired temporal profiles such as daily pulsatile release and peak/plateaued release within a specific short interval. This technology could be particularly useful for controlling the temporal presentation of reprogramming factors, whether they are needed, for example, continuously for the first 5 days and then pulsatile thereafter each day every other day, or only on days 6 and 12 of reprogramming. Given the versatility of the previously mentioned release platforms, the efficiency of their intracellular delivery, essential for transcriptional regulation of pluripotency induction, relies largely on the successful transmembrane trafficking of biomaterials. This process is primarily driven by endocytosis-mediated cellular uptake; however, it is generally associated with endosomal escape or loss of delivered materials due to vesicular degradation/recycling, leading to limited delivery efficiency (Kou et al, 2013; Sahay et al, 2013). To potentially obviate this issue and maximize the reprogramming efficiency, reprogramming factor-laden biomaterials may be forced into the cytosolic space via rapid and cytocompatible mechanical deformation of somatic cells (via a microfluidic device). This deformation induces transient membrane disruption, resulting in passive diffusion of biomaterials into the cells (Sharei et al, 2013). In combination with biomaterial-based delivery platforms, this microfluidic technology could further improve the spatial presentation of reprogramming factors and maximize the reprogramming efficiency in a safe and high-throughput manner [e.g. operated at a throughput rate of 20,000 cells/second per device (Sharei et al, 2013)]. It is conceivable that such a combination could be used to localize multiple factors that are difficult to deliver, such as macromolecules with diverse and sensitive structures (Yan et al, 2010) for a broad range of iPSC applications in addition to iPSC derivation. Alternative intracellular delivery methods include microinjection, electroporation, and sonoporation which have also been explored to facilitate cellular internalization of biomolecules or nanoparticles (Miller et al, 2002; Geng & Lu, 2013). They have demonstrated excellent spatiotemporal and dose control for delivery without significantly impairing the manipulated cells (Xie et al, 2013; Boukany et al, 2014). The broad utility of the biomaterial-based controlled release strategies has been extensively demonstrated by a spectrum of materials including biodegradable polyester-based materials (Richards Grayson et al, 2003; Mohamed & van der Walle, 2008; Makadia & Siegel, 2011). For example, nano-/microparticles formulated by poly(lactic-co-glycolic acid) (PLGA) offer high stability, potential for narrow size distribution, tunability, and an excellent safety profile (FDA-approved) (Danhier et al, 2012; Ankrum et al, 2014b). It has been adopted as a gene delivery vehicle for multiple transfection applications (Seo et al, 2013; Tian et al, 2013). PLGA degrades into lactic acid and glycolic acid (natural metabolites found in the body). Its degradation rate can be tailored by balancing the ratio of lactic acid and glycolic acid and altering the molecular weight, thereby enabling controlled release of encapsulated genetic materials (Lu et al, 2000; Danhier et al, 2012). With fine-tuning of the surface chemistry, size, and drug loading, PLGA particles encapsulating phenotype-altering agents enable efficient, sustained stabilization/release of the encapsulated factors into individual cells or spheroids for prolonged control of cellular functions (Ankrum et al, 2014a,b) or morphogenesis (Carpenedo et al, 2009, 2010; Bratt-Leal et al, 2011), respectively. For instance, we have recently demonstrated loading of PLGA MPs with several agents into multiple cell types to control cell phenotype from the intercellular milieu [Fig 3A (1–3)] (Sarkar et al, 2011; Ankrum et al, 2014a,b). By selecting agents that can easily cross cell membranes (e.g. certain positively charged agents and small molecules), it is also possible to use this strategy to control the microenvironment surrounding the modified cells. Specifically, we have shown that human mesenchymal stem cells (MSCs) loaded with dexamethasone (Dex)-doped PLGA MPs release Dex to regulate the differentiation of both the modified cells and cells in the adjacent microenvironment. We have also shown that budesonide-loaded PLGA MPs enhance the MSC immunomodulatory phenotype and that iron oxide nanoparticle-loaded PLGA MPs can be used to track MSCs in vivo (Xu et al, 2012; Ankrum et al, 2014a). This platform may be used to improve reprogramming efficiency and control factor delivery because phenotype-altering agents are presented from within cells. A similar approach may also be utilized within 3D cell aggregates rather than adding soluble factors in the media (in which factors must diffuse through cellular barriers, potentially reducing efficiency and kinetic control) (Carpenedo et al, 2009; Bratt-Leal et al, 2013). For example, it has been shown that compared to conventional soluble delivery methods, bone morphogenetic protein 4 (BMP4) locally delivered via gelatin MPs inside 3D ESC spheroids led to efficient mesoderm induction, despite nearly 12-fold less total growth factors being used (Bratt-Leal et al, 2013). We envision that such biomaterial particle platforms could be adopted as stable intracellular depots of reprogramming factors, yielding direct, efficient, and prolonged pluripotency induction without frequent introduction of soluble reprogramming factors. Figure 3. Emerging applications of biomaterial-based targeted modulation of cell phenotype and gene regulation with potential application for reprogramming somatic cells(A) Confocal microscopy shows the intracellular localization of phenotype-altering agent-doped PLGA MPs in mesenchymal stem cells that can release agents for several weeks following internalization (A1) a mesenchymal stem cell, (A2) a MIN6 beta cell, and (A3) a RAW 264.7 macrophage. This robust particle platform could potentially serve as an intracellular depot for sustained presentation of reprogramming factors to achieve efficient iPSC derivation from multiple somatic cell types. Scale bars, 10 μm. Green (DiO stain), membrane; red (rhodamine 6 g), particles; blue (Hoechst), nuclei; Adapted with permission from Ankrum et al (2014b). (B) One potential biomaterial strategy for controlled regulation of gene expression is nanoparticle-based artificial transcription factors (NanoScript). This platform could be potentially adopted for the activation or expression of pluripotency-associated genes for improved iPSC derivation. B1: NanoScript is devised to emulate the structure and function of TFs by assembling the principle components, DBD, AD, and NLS, onto a single 10-nm gold nanoparticle via molecular linkers. This design enables the penetration through plasma membrane and entrance into the nuclear membrane through NLS–nuclear receptor coupling. NanoScript interacts with DNA and triggers transcriptional activity leading to desired gene regulation. B2: transmission electron microscopy (TEM) micrograph demonstrates the localization of NanoScript clusters within the nucleus (scale bar = 200 nm), with the inset showing individual nanoparticles (scale bar, 100 nm). Adapted with permission from Patel et al (2014). Download figure Download PowerPoint 2. Biomaterials for potential modulation of delivery kinetics of multiple reprogramming factors Small-molecule- or protein-based iPSC derivation protocols employ multiple cocktails to reprogram cells (Kim et al, 2009; Zhou et al, 2009; Hou et al, 2013). The sequential introduction of individual time-sensitive reprogramming factors with varied timing and duration appears to be indispensable for the activation of early biological events [e.g. epithelial-to-mesenchymal transition (EMT)], essential for the following distinct reprogramming phases (David & Polo, 2014) that are important for improved pluripotency induction (Liu et al, 2013a, 2014a). Although these state-of-the-art regimens have significantly advanced the iPSC field, further improvements for tightening control over these systems is required. Biomaterial-based methods are poised to refine, simplify, and improve the efficiency and safety of these protocols. Biomaterials may be used to control timing of factor deli
Referência(s)