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1.  Efficient derivation of cortical glutamatergic neurons from human pluripotent stem cells: A model system to study neurotoxicity in Alzheimer's disease 
Neurobiology of disease  2013;62:62-72.
Alzheimer's disease (AD) is among the most prevalent forms of dementia affecting the aging population, and pharmacological therapies to date have not been successful in preventing disease progression. Future therapeutic efforts may benefit from the development of models that enable basic investigation of early disease pathology. In particular, disease-relevant models based on human pluripotent stem cells (hPSCs) may be promising approaches to assess the impact of neurotoxic agents in AD on specific neuronal populations and thereby facilitate the development of novel interventions to avert early disease mechanisms. We implemented an efficient paradigm to convert hPSCs into enriched populations of cortical glutamatergic neurons emerging from dorsal forebrain neural progenitors, aided by modulating Sonic hedgehog (Shh) signaling. Since AD is generally known to be toxic to glutamatergic circuits, we exposed glutamatergic neurons derived from hESCs to an oligomeric pre-fibrillar forms of Aβ known as “globulomers”, which have shown strong correlation with the level of cognitive deficits in AD. Administration of such Aβ oligomers yielded signs of the disease, including cell culture age-dependent binding of Aβ and cell death in the glutamatergic populations. Furthermore, consistent with previous findings in postmortem human AD brain Aβ-induced toxicity was selective for glutamatergic rather than GABAeric neurons present in our cultures. This in vitro model of cortical glutamatergic neurons thus offers a system for future mechanistic investigation and therapeutic development for AD pathology using human cell types specifically affected by this disease.
doi:10.1016/j.nbd.2013.09.005
PMCID: PMC4122237  PMID: 24055772
2.  Single-cell western blotting 
Nature methods  2014;11(7):749-755.
To measure cell-to-cell variation in protein-mediated functions — a hallmark of biological processes — we developed an approach to conduct ~103 concurrent single-cell western blots (scWesterns) in ~4 hours. A microscope slide supporting a 30 µm-thick photoactive polyacrylamide gel enables western blotting comprised of: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins, and antibody probing. We apply this scWestern to monitor single rat neural stem cell differentiation and responses to mitogen stimulation. The scWestern quantifies target proteins even with off-target antibody binding, multiplexes to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supports analyses of low starting cell numbers (~200) when integrated with fluorescence activated cell sorting. The scWestern thus overcomes limitations in single-cell protein analysis (i.e., antibody fidelity, sensitivity, and starting cell number) and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.
doi:10.1038/nmeth.2992
PMCID: PMC4077215  PMID: 24880876
3.  Advances in AAV Vector Development for Gene Therapy in the Retina 
Adeno-associated virus (AAV) is a small, non-pathogenic dependovirus that has shown great potential for safe and long-term expression of a genetic pay-load in the retina. AAV has been used to treat a growing number of animal models of inherited retinal degeneration, though drawbacks—including a limited carrying capacity, slow onset of expression, and a limited ability to transduce some retinal cell types from the vitreous—restrict the utility of AAV for treating some forms of inherited eye disease. Next generation AAV vectors are being created to address these needs, through rational design efforts such as the creation of self-complementary AAV vectors for faster onset of expression and specific mutations of surface-exposed residues to increase transduction of viral particles. Furthermore, directed evolution has been used to create, through an iterative process of selection, novel variants of AAV with newly acquired, advantageous characteristics. These novel AAV variants have been shown to improve the therapeutic potential of AAV vectors, and further improvements may be achieved through rational design, directed evolution, or a combination of these approaches, leading to broader applicability of AAV and improved treatments for inherited retinal degeneration.
doi:10.1007/978-1-4614-3209-8_86
PMCID: PMC4267879  PMID: 24664759
Adeno-associated virus; Gene therapy; Mutagenesis; Directed evolution; Retinal degeneration
4.  Retinoschisin gene therapy in photoreceptors, Müller glia, or all retinal cells in the Rs1h−/− mouse 
Gene therapy  2014;21(6):585-592.
X-linked retinoschisis, a disease characterized by splitting of the retina, is caused by mutations in the retinoschisin gene, which encodes a secreted cell adhesion protein. Currently, there is no effective treatment for retinoschisis, though viral vector-mediated gene replacement therapies offer promise. We used intravitreal delivery of three different AAV vectors to target delivery of the RS1 gene to Müller glia, photoreceptors, or multiple cell types throughout the retina. Müller glia radially span the entire retina, are accessible from the vitreous, and remain intact throughout progression of the disease. However, photoreceptors, not glia, normally secrete retinoschisin. We compared the efficacy of rescue mediated by retinoschisin secretion from these specific subtypes of retinal cells in the Rs1h−/− mouse model of retinoschisis. Our results indicate that all three vectors deliver the RS1 gene, and that several cell types can secrete retinoschisin, leading to transport of the protein across the retina. The greatest long-term rescue was observed when photoreceptors produce retinoschisin. Similar rescue was observed with photoreceptor-specific or generalized expression, though photoreceptor secretion may contribute to rescue in the latter case. These results collectively point to the importance of cell targeting and appropriate vector choice in the success of retinal gene therapies.
doi:10.1038/gt.2014.31
PMCID: PMC4047144  PMID: 24694538
Gene therapy; X-linked retinoschisis; AAV vectors; photoreceptors; Müller glia; cell targeting
5.  Imaging Light Responses of Foveal Ganglion Cells in the Living Macaque Eye 
The Journal of Neuroscience  2014;34(19):6596-6605.
The fovea dominates primate vision, and its anatomy and perceptual abilities are well studied, but its physiology has been little explored because of limitations of current physiological methods. In this study, we adapted a novel in vivo imaging method, originally developed in mouse retina, to explore foveal physiology in the macaque, which permits the repeated imaging of the functional response of many retinal ganglion cells (RGCs) simultaneously. A genetically encoded calcium indicator, G-CaMP5, was inserted into foveal RGCs, followed by calcium imaging of the displacement of foveal RGCs from their receptive fields, and their intensity-response functions. The spatial offset of foveal RGCs from their cone inputs makes this method especially appropriate for fovea by permitting imaging of RGC responses without excessive light adaptation of cones. This new method will permit the tracking of visual development, progression of retinal disease, or therapeutic interventions, such as insertion of visual prostheses.
doi:10.1523/JNEUROSCI.4438-13.2014
PMCID: PMC4012315  PMID: 24806684
calcium imaging; in vivo adaptive optics imaging; intrinsic signal imaging; primate fovea; retinal ganglion cells
6.  Biogenic Gas Nanostructures as Ultrasonic Molecular Reporters 
Nature nanotechnology  2014;9(4):311-316.
Ultrasound is among the most widely used non-invasive imaging modalities in biomedicine1, but plays a surprisingly small role in molecular imaging due to a lack of suitable molecular reporters on the nanoscale. Here we introduce a new class of reporters for ultrasound based on genetically encoded gas nanostructures from microorganisms, including bacteria and archaea. Gas vesicles are gas-filled protein-shelled compartments with typical widths of 45–250 nm and lengths of 100–600 nm that exclude water and are permeable to gas2, 3. We show that gas vesicles produce stable ultrasound contrast that is readily detected in vitro and in vivo, that their genetically encoded physical properties enable multiple modes of imaging, and that contrast enhancement through aggregation permits their use as molecular biosensors.
doi:10.1038/nnano.2014.32
PMCID: PMC4023545  PMID: 24633522
7.  Mechanisms of action of hESC-secreted proteins that enhance human and mouse myogenesis 
Aging (Albany NY)  2014;6(8):602-620.
Adult stem cells grow poorly in vitro compared to embryonic stem cells, and in vivo stem cell maintenance and proliferation by tissue niches progressively deteriorates with age. We previously reported that factors produced by human embryonic stem cells (hESCs) support a robust regenerative capacity for adult and old mouse muscle stem/progenitor cells. Here we extend these findings to human muscle progenitors and investigate underlying molecular mechanisms. Our results demonstrate that hESC-conditioned medium enhanced the proliferation of mouse and human muscle progenitors. Furthermore, hESC-produced factors activated MAPK and Notch signaling in human myogenic progenitors, and Delta/Notch-1 activation was dependent on MAPK/pERK. The Wnt, TGF-β and BMP/pSmad1,5,8 pathways were unresponsive to hESC-produced factors, but BMP signaling was dependent on intact MAPK/pERK. c-Myc, p57, and p18 were key effectors of the enhanced myogenesis promoted by the hECS factors. To define some of the active ingredients of the hESC-secretome which may have therapeutic potential, a comparative proteomic antibody array analysis was performed and identified several putative proteins, including FGF2, 6 and 19 which as ligands for MAPK signaling, were investigated in more detail. These studies emphasize that a “youthful” signaling of multiple signaling pathways is responsible for the pro-regenerative activity of the hESC factors.
PMCID: PMC4169857  PMID: 25109702
hESC; muscle; myogenesis; Notch; MAPK; aging; rejuvenation; stem cell
8.  Soft microenvironments promote the early neurogenic differentiation but not self-renewal of human pluripotent stem cells 
Human pluripotent stem cells (hPSCs) are of great interest in biology and medicine due to their ability to self-renew and differentiate into any adult or fetal cell type. Important efforts have identified biochemical factors, signaling pathways, and transcriptional networks that regulate hPSC biology. However, recent work investigating the effect of biophysical cues on mammalian cells and adult stem cells suggests that the mechanical properties of the microenvironment, such as stiffness, may also regulate hPSC behavior. While several studies have explored this mechanoregulation in mouse embryonic stem cells (mESCs), it has been challenging to extrapolate these findings and thereby explore their biomedical implications in hPSCs. For example, it remains unclear whether hPSCs can be driven down a given tissue lineage by providing tissue-mimetic stiffness cues. Here we address this open question by investigating the regulation of hPSC neurogenesis by microenvironmental stiffness. We find that increasing extracellular matrix (ECM) stiffness in vitro increases hPSC cell and colony spread area but does not alter self-renewal, in contrast to past studies with mESCs. However, softer ECMs with stiffnesses similar to that of neural tissue promote the generation of early neural ectoderm. This mechanosensitive increase in neural ectoderm requires only a short 5-day soft stiffness “pulse,” which translates into downstream increases in both total neurons as well as therapeutically relevant dopaminergic neurons. These findings further highlight important differences between mESCs and hPSCs and have implications for both the design of future biomaterials as well as our understanding of early embryonic development.
doi:10.1039/c2ib20083j
PMCID: PMC3459311  PMID: 22854634
9.  Multivalent ligands to control stem cell behaviour in vitro and in vivo 
Nature nanotechnology  2013;8(11):10.1038/nnano.2013.205.
There is broad interest in designing nanostructured materials that can interact with cells and regulate key downstream functions1–7. In particular, materials with nanoscale features may enable control over multivalent interactions, which involve the simultaneous binding of multiple ligands on one entity to multiple receptors on another and are ubiquitous throughout biology8–10. Cellular signal transduction of growth factor and morphogen cues that play critical roles in regulating cell function and fate often begins with such multivalent binding of ligands, either secreted or cell-surface tethered, to target cell receptors, leading to receptor clustering11–18. Cellular mechanisms that orchestrate ligand-receptor oligomerisation are complex, however, and the capacity to control multivalent interactions and thereby modulate key signaling events within living systems is therefore currently very limited. Here we demonstrate the design of potent multivalent conjugates that can organise stem cell receptors into nanoscale clusters and control stem cell behaviour in vitro and in vivo. The ectodomain of ephrin-B2, normally an integral membrane protein ligand, was conjugated to a soluble biopolymer to yield multivalent nanoscale conjugates that potently induced signaling in neural stem cells and promoted their neuronal differentiation both in culture and within the brain. Super-resolution microscopy analysis yielded insights into the organisation of receptor-ligand clusters at the nanoscale. We also found that synthetic multivalent conjugates of ephrin-B1 strongly enhanced human embryonic and induced pluripotent stem cell differentiation into functional dopaminergic neurons. Multivalent bioconjugates thus represent powerful tools and potential nanoscale therapeutics for controlling the behaviour of target stem cells in vitro and in vivo.
doi:10.1038/nnano.2013.205
PMCID: PMC3830932  PMID: 24141540
10.  Engineering a serum-resistant and thermostable vesicular stomatitis virus G glycoprotein for pseudotyping retroviral and lentiviral vectors 
Gene therapy  2013;20(8):807-815.
Vesicular stomatitis virus G glycoprotein (VSV-G) is the most widely used envelope protein for retroviral and lentiviral vector pseudotyping; however, serum inactivation of VSV-G pseudotyped vectors is a significant challenge for in vivo gene delivery. To address this problem, we conducted directed evolution of VSV-G to increase its resistance to human serum neutralization. After six selection cycles, numerous common mutations were present. Based on their location within VSV-G, we analyzed whether substitutions in several surface exposed residues could endow viral vectors with higher resistance to serum. S162T, T230N, and T368A mutations enhanced serum resistance, and additionally K66T, T368A, and E380K substitutions increased the thermostability of VSV-G pseudotyped retroviral vectors, an advantageous byproduct of the selection strategy. Analysis of a number of combined mutants revealed that VSV-G harboring T230N + T368A or K66T + S162T + T230N + T368A mutations exhibited both higher in vitro resistance to human serum and higher thermostability, as well as enhanced resistance to rabbit and mouse serum. Finally, lentiviral vectors pseudotyped with these variants were more resistant to human serum in a murine model. These serum-resistant and thermostable VSV-G variants may aid the application of retroviral and lentiviral vectors to gene therapy.
doi:10.1038/gt.2013.1
PMCID: PMC3735647  PMID: 23364315
serum-resistant; thermostable; VSV-G; directed evolution; pseudotyping
11.  Molecular characterization of multivalent bioconjugates by size-exclusion chromatography (SEC) with multi-angle laser light scattering (MALS) 
Bioconjugate chemistry  2012;23(9):1794-1801.
The degree of substitution and valency of bioconjugate reaction products are often poorly judged or require multiple time- and product- consuming chemical characterization methods. These aspects become critical when analyzing and optimizing the potency of costly polyvalent bioactive conjugates. In this study, size-exclusion chromatography with multi-angle laser light scattering was paired with refractive index detection and ultraviolet spectroscopy (SEC-MALS-RI-UV) to characterize the reaction efficiency, degree of substitution, and valency of the products of conjugation of either peptides or proteins to a biopolymer scaffold, i.e., hyaluronic acid (HyA). Molecular characterization was more complete compared to estimates from a protein quantification assay, and exploitation of this method led to more accurate deduction of the molecular structures of polymer bioconjugates. Information obtained using this technique can improve macromolecular engineering design principles and better understand multivalent macromolecular interactions in biological systems.
doi:10.1021/bc3000595
PMCID: PMC3900495  PMID: 22794081
12.  Akt Increases Sox2 Expression in Adult Hippocampal Neural Progenitor Cells, but Increased Sox2 Does Not Promote Proliferation 
Stem Cells and Development  2010;20(7):1153-1161.
Multiple extracellular factors have been shown to modulate adult hippocampal neural progenitor cell (NPC) proliferation and self-renewal, and we have previously shown that Akt is an important mediator of the effects of these extracellular factors on NPC proliferation and differentiation. However, very little work has investigated how and whether Akt is involved in maintaining the multipotency of these cells. Here we demonstrate that Akt promotes expression of Sox2, a core transcription factor important for the self-renewal of NPCs. Retroviral-mediated overexpression of wild-type Akt increased Sox2 protein expression, particularly under conditions that promote cell differentiation, whereas Akt inhibition decreased Sox2. Similarly, quantitative reverse transcription (RT)–PCR in differentiating cultures indicated that Akt rescued Sox2 mRNA to levels present under conditions that promote cell proliferation. Additionally, pharmacological inhibition of Akt did not affect Sox2 protein levels in cells constitutively expressing Sox2 from a retroviral vector, indicating that Akt does not affect Sox2 protein stability. Further, in contrast to Akt overexpression, Sox2 overexpression does not increase NPC viable cell number or proliferation yet does inhibit differentiation. Collectively, these results indicate that Akt promotes cell proliferation and maintenance of a multipotent state via two downstream paths.
doi:10.1089/scd.2010.0130
PMCID: PMC3121932  PMID: 21028992
13.  Biophysical regulation of stem cell behavior within the niche 
Stem cells reside within most tissues throughout the lifetimes of mammalian organisms. To maintain their capacities for division and differentiation and thereby build, maintain, and regenerate organ structure and function, these cells require extensive and precise regulation, and a critical facet of this control is the local environment or niche surrounding the cell. It is well known that soluble biochemical signals play important roles within such niches, and a number of biophysical aspects of the microenvironment, including mechanical cues and spatiotemporally varying biochemical signals, have also been increasingly recognized to contribute to the repertoire of stimuli that regulate various stem cells in various tissues of both vertebrates and invertebrates. For example, biochemical factors immobilized to the extracellular matrix or the surface of neighboring cells can be spatially organized in their placement. Furthermore, the extracellular matrix provides mechanical support and regulatory information, such as its elastic modulus and interfacial topography, which modulate key aspects of stem cell behavior. Numerous examples of each of these modes of regulation indicate that biophysical aspects of the niche must be appreciated and studied in conjunction with its biochemical properties.
doi:10.1186/scrt141
PMCID: PMC3580480  PMID: 23241436
14.  AAV-Mediated, Optogenetic Ablation of Müller Glia Leads to Structural and Functional Changes in the Mouse Retina 
PLoS ONE  2013;8(9):e76075.
Müller glia, the primary glial cell in the retina, provide structural and metabolic support for neurons and are essential for retinal integrity. Müller cells are closely involved in many retinal degenerative diseases, including macular telangiectasia type 2, in which impairment of central vision may be linked to a primary defect in Müller glia. Here, we used an engineered, Müller-specific variant of AAV, called ShH10, to deliver a photo-inducibly toxic protein, KillerRed, to Müller cells in the mouse retina. We characterized the results of specific ablation of these cells on visual function and retinal structure. ShH10-KillerRed expression was obtained following intravitreal injection and eyes were then irradiated with green light to induce toxicity. Induction of KillerRed led to loss of Müller cells and a concomitant decrease of Müller cell markers glutamine synthetase and cellular retinaldehyde-binding protein, reduction of rhodopsin and cone opsin, and upregulation of glial fibrillary acidic protein. Loss of Müller cells also resulted in retinal disorganization, including thinning of the outer nuclear layer and the photoreceptor inner and outer segments. High resolution imaging of thin sections revealed displacement of photoreceptors from the ONL, formation of rosette-like structures and the presence of phagocytic cells. Furthermore, Müller cell ablation resulted in increased area and volume of retinal blood vessels, as well as the formation of tortuous blood vessels and vascular leakage. Electrophysiologic measures demonstrated reduced retinal function, evident in decreased photopic and scotopic electroretinogram amplitudes. These results show that loss of Müller cells can cause progressive retinal degenerative disease, and suggest that AAV delivery of an inducibly toxic protein in Müller cells may be useful to create large animal models of retinal dystrophies.
doi:10.1371/journal.pone.0076075
PMCID: PMC3785414  PMID: 24086689
15.  Surface immobilization of hexa-histidine-tagged adeno-associated viral vectors for localized gene delivery 
Gene therapy  2010;17(11):1384-1389.
Adeno-associated viral vectors, which are undergoing broad exploration in clinical trials, have significant promise for therapeutic gene delivery due to their safety and delivery efficiency. Gene delivery technologies capable of mediating localized gene expression may further enhance AAV’s potential in a variety of therapeutic applications by reducing spread outside of a target region, which may thereby reduce off-target side effects. We have genetically engineered an AAV variant capable of binding to surfaces with high affinity via a hexahistidine-metal binding interaction. This immobilized AAV vector system mediates high efficiency delivery to cells that contact the surface and thus may have promise for localized gene delivery, which may aid numerous applications of AAV delivery to gene therapy.
doi:10.1038/gt.2010.81
PMCID: PMC2932747  PMID: 20508598
adeno-associated virus; localized gene delivery; substrate-mediated gene delivery; hexa-histidine
16.  Genetic Selection for Context-Dependent Stochastic Phenotypes: Sp1 and TATA Mutations Increase Phenotypic Noise in HIV-1 Gene Expression 
PLoS Computational Biology  2013;9(7):e1003135.
The sequence of a promoter within a genome does not uniquely determine gene expression levels and their variability; rather, promoter sequence can additionally interact with its location in the genome, or genomic context, to shape eukaryotic gene expression. Retroviruses, such as human immunodeficiency virus-1 (HIV), integrate their genomes into those of their host and thereby provide a biomedically-relevant model system to quantitatively explore the relationship between promoter sequence, genomic context, and noise-driven variability on viral gene expression. Using an in vitro model of the HIV Tat-mediated positive-feedback loop, we previously demonstrated that fluctuations in viral Tat-transactivating protein levels generate integration-site-dependent, stochastically-driven phenotypes, in which infected cells randomly ‘switch’ between high and low expressing states in a manner that may be related to viral latency. Here we extended this model and designed a forward genetic screen to systematically identify genetic elements in the HIV LTR promoter that modulate the fraction of genomic integrations that specify ‘Switching’ phenotypes. Our screen identified mutations in core promoter regions, including Sp1 and TATA transcription factor binding sites, which increased the Switching fraction several fold. By integrating single-cell experiments with computational modeling, we further investigated the mechanism of Switching-fraction enhancement for a selected Sp1 mutation. Our experimental observations demonstrated that the Sp1 mutation both impaired Tat-transactivated expression and also altered basal expression in the absence of Tat. Computational analysis demonstrated that the observed change in basal expression could contribute significantly to the observed increase in viral integrations that specify a Switching phenotype, provided that the selected mutation affected Tat-mediated noise amplification differentially across genomic contexts. Our study thus demonstrates a methodology to identify and characterize promoter elements that affect the distribution of stochastic phenotypes over genomic contexts, and advances our understanding of how promoter mutations may control the frequency of latent HIV infection.
Author Summary
The sequence of a gene within a cellular genome does not uniquely determine its expression level, even for a single type of cell under fixed conditions. Numerous other factors, including gene location on the chromosome and random gene-expression “noise,” can alter expression patterns and cause differences between otherwise identical cells. This poses new challenges for characterizing the genotype–phenotype relationship. Infection by the human immunodeficiency virus-1 (HIV-1) provides a biomedically important example in which transcriptional noise and viral genomic location impact the decision between viral replication and latency, a quiescent but reversible state that cannot be eliminated by anti-viral therapies. Here, we designed a forward genetic screen to systematically identify mutations in the HIV promoter that alter the fraction of genomic integrations that specify noisy/reactivating expression phenotypes. The mechanisms by which the selected mutations specify the observed phenotypic enrichments are investigated through a combination of single-cell experiments and computational modeling. Our study provides a framework for identifying genetic sequences that alter the distribution of stochastic expression phenotypes over genomic locations and for characterizing their mechanisms of regulation. Our results also may yield further insights into the mechanisms by which HIV sequence evolution can alter the propensity for latent infections.
doi:10.1371/journal.pcbi.1003135
PMCID: PMC3708878  PMID: 23874178
17.  Chromatin accessibility at the HIV LTR promoter sets a threshold for NF-κB mediated viral gene expression 
Integrative Biology  2012;4(6):661-671.
Higher order chromatin structure in eukaryotes can lead to differential gene expression in response to the same transcription factor; however, how transcription factor inputs integrate with quantitative features of the chromatin environment to regulate gene expression is not clear. In vitro models of HIV gene regulation, in which repressive mechanisms acting locally at an integration site keep proviruses transcriptionally silent until appropriately stimulated, provide a powerful system to study gene expression regulation in different chromatin environments. Here we quantified HIV expression as a function of activating transcription factor nuclear factor-κB RelA/p65 (RelA) levels and chromatin features at a panel of viral integration sites. Variable RelA overexpression demonstrated that the viral genomic location sets a threshold RelA level necessary to induce gene expression. However, once the induction threshold is reached, gene expression increases similarly for all integration sites. Furthermore, we found that higher induction thresholds are associated with repressive histone marks and a decreased sensitivity to nuclease digestion at the LTR promoter. Increasing chromatin accessibility via inhibition of histone deacetylation or DNA methylation lowered the induction threshold, demonstrating that chromatin accessibility sets the level of RelA required to activate gene expression. Finally, a functional relationship between gene expression, RelA level, and chromatin accessibility accurately predicted synergistic HIV activation in response to combinatorial pharmacological perturbations. Different genomic environments thus set a threshold for transcription factor activation of a key viral promoter, which may point toward biological principles that underlie selective gene expression and inform strategies for combinatorial therapies to combat latent HIV.
doi:10.1039/c2ib20009k
PMCID: PMC3362694  PMID: 22555315
18.  Pan-neuronal maturation but not neuronal subtype differentiation of adult neural stem cells is mechanosensitive 
Scientific Reports  2013;3:1817.
Most past studies of the biophysical regulation of stem cell differentiation have focused on initial lineage commitment or proximal differentiation events. It would be valuable to understand whether biophysical inputs also influence distal endpoints more closely associated with physiological function, such as subtype specification in neuronal differentiation. To explore this question, we cultured adult neural stem cells (NSCs) on variable stiffness ECMs under conditions that promote neuronal fate commitment for extended time periods to allow neuronal subtype differentiation. We find that ECM stiffness does not modulate the expression of NeuroD1 and TrkA/B/C or the percentages of pan-neuronal, GABAergic, or glutamatergic neuronal subtypes. Interestingly, however, an ECM stiffness of 700 Pa maximizes expression of pan-neuronal markers. These results suggest that a wide range of stiffnesses fully permit pan-neuronal NSC differentiation, that an intermediate stiffness optimizes expression of pan-neuronal genes, and that stiffness does not impact commitment to particular neuronal subtypes.
doi:10.1038/srep01817
PMCID: PMC3650663  PMID: 23660869
19.  hESC-secreted proteins can be enriched for multiple regenerative therapies by heparin-binding 
Aging (Albany NY)  2013;5(5):357-372.
This work builds upon our findings that proteins secreted by hESCs exhibit pro-regenerative activity, and determines that hESC-conditioned medium robustly enhances the proliferation of both muscle and neural progenitor cells. Importantly, this work establishes that it is the proteins that bind heparin which are responsible for the pro-myogenic effects of hESC-conditioned medium, and indicates that this strategy is suitable for enriching the potentially therapeutic factors. Additionally, this work shows that hESC-secreted proteins act independently of the mitogen FGF-2, and suggests that FGF-2 is unlikely to be a pro-aging molecule in the physiological decline of old muscle repair. Moreover, hESC-secreted factors improve the viability of human cortical neurons in an Alzheimer's disease (AD) model, suggesting that these factors can enhance the maintenance and regeneration of multiple tissues in the aging body.
PMCID: PMC3701111  PMID: 23793469
rejuvenation; embryonic stem cell; myoblast; satellite cell
20.  Astrocytes regulate adult hippocampal neurogenesis through ephrin-B signaling 
Nature neuroscience  2012;15(10):1399-1406.
Neurogenesis in the adult hippocampus involves activation of quiescent neural stem cells (NSCs) to yield transiently amplifying NSCs and progenitors, and ultimately neurons that affect learning and memory. This process is tightly controlled by microenvironmental cues, though few endogenous factors are known to regulate neuronal differentiation. While astrocytes have been implicated, their role in juxtacrine (i.e. cell-cell contact-dependent) signaling within NSC niches has not been investigated. We show that ephrin-B2 presented from rodent hippocampal astrocytes regulates neurogenesis in vivo. Furthermore, clonal analysis in NSC fate-mapping studies reveals a novel role for ephrin-B2 in instructing neuronal differentiation. Additionally, ephrin-B2 signaling, transduced by EphB4 receptors on NSCs, activates β-catenin in vitro and in vivo independent of Wnt signaling and upregulates proneural transcription factors. Ephrin-B2+ astrocytes thus promote neuronal differentiation of adult NSCs through juxtacrine signaling, findings that advance our understanding of adult neurogenesis and may have future regenerative medicine implications.
doi:10.1038/nn.3212
PMCID: PMC3458152  PMID: 22983209
21.  DNA Shuffling of Adeno-associated Virus Yields Functionally Diverse Viral Progeny 
Adeno-associated virus (AAV) vectors are extremely effective gene-delivery vehicles for a broad range of applications. However, the therapeutic efficacy of these and other vectors is currently limited by barriers to safe, efficient gene delivery, including pre-existing antiviral immunity, and infection of off-target cells. Recently, we have implemented directed evolution of AAV, involving the generation of randomly mutagenized viral libraries based on serotype 2 and high-throughput selection, to engineer enhanced viral vectors. Here, we significantly extend this capability by performing high-efficiency in vitro recombination to create a large (107), diverse library of random chimeras of numerous parent AAV serotypes (AAV1, 2, 4–6, 8, and 9). In order to analyze the extent to which such highly chimeric viruses can be viable, we selected the library for efficient viral packaging and infection, and successfully recovered numerous novel chimeras. These new viruses exhibited a broad range of cell tropism both in vitro and in vivo and enhanced resistance to human intravenous immunoglobulin (IVIG), highlighting numerous functional differences between these chimeras and their parent serotypes. Thus, directed evolution can potentially yield unlimited numbers of new AAV variants with novel gene-delivery properties, and subsequent analysis of these variants can further extend basic knowledge of AAV biology.
doi:10.1038/mt.2008.167
PMCID: PMC2683895  PMID: 18728640
23.  Enhanced Sialic Acid-Dependent Endocytosis Explains the Increased Efficiency of Infection of Airway Epithelia by a Novel Adeno-Associated Virus▿ 
Journal of Virology  2011;85(17):9023-9030.
We previously used directed evolution in human airway epithelia to create adeno-associated virus 2.5T (AAV2.5T), a highly infectious chimera of AAV2 and AAV5 with one point mutation (A581T). We hypothesized that the mechanism for its increased infection may be a higher binding affinity to the surface of airway epithelia than its parent AAV5. Here, we show that, like AAV5, AAV2.5T, uses 2,3N-linked sialic acid as its primary receptor; however, AAV2.5T binds to the apical surface of human airway epithelia at higher levels and has more receptors than AAV5. Furthermore, its binding affinity is similar to that of AAV5. An alternative hypothesis is that AAV2.5T interaction with 2,3N-linked sialic acid may instead be required for cellular internalization. Consistent with this, AAV2.5T binds but fails to be internalized by CHO cells that lack surface expression of sialic acid. Moreover, whereas AAV2.5T binds similarly to human (rich in 2,3N-linked sialic acid) and pig airway epithelia (2,6N-linked sialic acid), significantly more virus was internalized by human airway. Subsequent transduction correlated with the level of internalized rather than surface-bound virus. We also found that human airway epithelia internalized significantly more AAV2.5T than AAV5. These data suggest that AAV2.5T has evolved to utilize specific 2,3N-linked sialic acid residues on the surface of airway epithelia that mediate rapid internalization and subsequent infection. Thus, sialic acid serves as not just an attachment factor but is also required for AAV2.5T internalization, possibly representing an important rate-limiting step for other viruses that use sialic acids.
doi:10.1128/JVI.05154-11
PMCID: PMC3165813  PMID: 21697483
24.  Colloids as Mobile Substrates for the Implantation and Integration of Differentiated Neurons into the Mammalian Brain 
PLoS ONE  2012;7(1):e30293.
Neuronal degeneration and the deterioration of neuronal communication lie at the origin of many neuronal disorders, and there have been major efforts to develop cell replacement therapies for treating such diseases. One challenge, however, is that differentiated cells are challenging to transplant due to their sensitivity both to being uprooted from their cell culture growth support and to shear forces inherent in the implantation process. Here, we describe an approach to address these problems. We demonstrate that rat hippocampal neurons can be grown on colloidal particles or beads, matured and even transfected in vitro, and subsequently transplanted while adhered to the beads into the young adult rat hippocampus. The transplanted cells have a 76% cell survival rate one week post-surgery. At this time, most transplanted neurons have left their beads and elaborated long processes, similar to the host neurons. Additionally, the transplanted cells distribute uniformly across the host hippocampus. Expression of a fluorescent protein and the light-gated glutamate receptor in the transplanted neurons enabled them to be driven to fire by remote optical control. At 1-2 weeks after transplantation, calcium imaging of host brain slice shows that optical excitation of the transplanted neurons elicits activity in nearby host neurons, indicating the formation of functional transplant-host synaptic connections. After 6 months, the transplanted cell survival and overall cell distribution remained unchanged, suggesting that cells are functionally integrated. This approach, which could be extended to other cell classes such as neural stem cells and other regions of the brain, offers promising prospects for neuronal circuit repair via transplantation of in vitro differentiated, genetically engineered neurons.
doi:10.1371/journal.pone.0030293
PMCID: PMC3266246  PMID: 22295079
25.  Neural stem cell adhesion and proliferation on phospholipid bilayers functionalized with RGD peptides 
Biomaterials  2010;31(33):8706-8715.
Peptide-functionalized materials show promise in controlling stem cell behavior by mimicking cell-matrix interactions. Supported lipid bilayers are an excellent platform for displaying peptides due to their ease of fabrication and low non-specific interactions with cells. In this paper, we report on the behavior of adult hippocampal neural stem cells (NSCs) on phospholipid bilayers functionalized with different RGD-containing peptides: either GGGNGEPRGDTYRAY (‘bsp-RGD(15)’) or GRGDSP. Fluid supported bilayers were prepared on glass surfaces by adsorption and fusion of small lipid vesicles incorporating synthetic peptide amphiphiles. NSCs adhered to bilayers with either GRGDSP or bsp-RGD(15) peptide. After 5 days in culture, NSCs formed neurosphere-like aggregates on GRGDSP bilayers, whereas on bsp-RGD(15) bilayers a large fraction of single adhered cells were observed, comparable to monolayer growth seen on laminin controls. NSCs retained their ability to differentiate into neurons and astrocytes on both peptide surfaces. This work illustrates the utility of supported bilayers in displaying peptide ligands and demonstrates that RGD peptides may be useful in synthetic culture systems for stem cells.
doi:10.1016/j.biomaterials.2010.07.104
PMCID: PMC2949453  PMID: 20728935

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