A novel, long-lived, and highly engraftable immunodeficient mouse model of mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is an inherited α-L-iduronidase (IDUA, I) deficiency in which glycosaminoglycan (GAG) accumulation causes progressive multisystem organ dysfunction, neurological impairment, and death. Current MPS I mouse models, based on a NOD/SCID (NS) background, are short-lived, providing a very narrow window to assess the long-term efficacy of therapeutic interventions. They also develop thymic lymphomas, making the assessment of potential tumorigenicity of human stem cell transplantation problematic. We therefore developed a new MPS I model based on a NOD/SCID/Il2rγ (NSG) background. This model lives longer than 1 year and is tumor-free during that time. NSG MPS I (NSGI) mice exhibit the typical phenotypic features of MPS I including coarsened fur and facial features, reduced/abnormal gait, kyphosis, and corneal clouding. IDUA is undetectable in all tissues examined while GAG levels are dramatically higher in most tissues. NSGI brain shows a significant inflammatory response and prominent gliosis. Neurological MPS I manifestations are evidenced by impaired performance in behavioral tests. Human neural and hematopoietic stem cells were found to readily engraft, with human cells detectable for at least 1 year posttransplantation. This new MPS I model is thus suitable for preclinical testing of novel pluripotent stem cell-based therapy approaches.

The Autism Spectrum Disorders Stem Cell Resource at Children's Hospital of Orange County: Implications for Disease Modeling and Drug Discovery.

The autism spectrum disorders (ASDs) comprise a set of neurodevelopmental disorders that are, at best, poorly understood but are the fastest growing developmental disorders in the United States. Because animal models of polygenic disorders such as the ASDs are difficult to validate, the derivation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming offers an alternative strategy for identifying the cellular mechanisms contributing to ASDs and the development of new treatment options. Access to statistically relevant numbers of ASD patient cell lines, however, is still a limiting factor for the field. We describe a new resource with more than 200 cell lines (fibroblasts, iPSC clones, neural stem cells, glia) from unaffected volunteers and patients with a wide range of clinical ASD diagnoses, including fragile X syndrome. We have shown that both normal and ASD-specific iPSCs can be differentiated toward a neural stem cell phenotype and terminally differentiated into action-potential firing neurons and glia. The ability to evaluate and compare data from a number of different cell lines will facilitate greater insight into the cause or causes and biology of the ASDs and will be extremely useful for uncovering new therapeutic and diagnostic targets. Some drug treatments have already shown promise in reversing the neurobiological abnormalities in iPSC-based models of ASD-associated diseases. The ASD Stem Cell Resource at the Children's Hospital of Orange County will continue expanding its collection and make all lines available on request with the goal of advancing the use of ASD patient cells as disease models by the scientific community.

Identification of fragile X syndrome specific molecular markers in human fibroblasts: a useful model to test the efficacy of therapeutic drugs.

Fragile X syndrome (FXS) is the most frequent cause of inherited intellectual disability and autism. It is caused by the absence of the fragile X mental retardation 1 (FMR1) gene product, fragile X mental retardation protein (FMRP), an RNA-binding protein involved in the regulation of translation of a subset of brain mRNAs. In Fmr1 knockout mice, the absence of FMRP results in elevated protein synthesis in the brain as well as increased signaling of many translational regulators. Whether protein synthesis is also dysregulated in FXS patients is not firmly established. Here, we demonstrate that fibroblasts from FXS patients have significantly elevated rates of basal protein synthesis along with increased levels of phosphorylated mechanistic target of rapamycin (p-mTOR), phosphorylated extracellular signal regulated kinase 1/2, and phosphorylated p70 ribosomal S6 kinase 1 (p-S6K1). The treatment with small molecules that inhibit S6K1 and a known FMRP target, phosphoinositide 3-kinase (PI3K) catalytic subunit p110ß, lowered the rates of protein synthesis in both control and patient fibroblasts. Our data thus demonstrate that fibroblasts from FXS patients may be a useful in vitro model to test the efficacy and toxicity of potential therapeutics prior to clinical trials, as well as for drug screening and designing personalized treatment approaches.

Process-based expansion and neural differentiation of human pluripotent stem cells for transplantation and disease modeling.

Robust strategies for developing patient-specific, human, induced pluripotent stem cell (iPSC)-based therapies of the brain require an ability to derive large numbers of highly defined neural cells. Recent progress in iPSC culture techniques includes partial-to-complete elimination of feeder layers, use of defined media, and single-cell passaging. However, these techniques still require embryoid body formation or coculture for differentiation into neural stem cells (NSCs). In addition, none of the published methodologies has employed all of the advances in a single culture system. Here we describe a reliable method for long-term, single-cell passaging of PSCs using a feeder-free, defined culture system that produces confluent, adherent PSCs that can be differentiated into NSCs. To provide a basis for robust quality control, we have devised a system of cellular nomenclature that describes an accurate genotype and phenotype of the cells at specific stages in the process. We demonstrate that this protocol allows for the efficient, large-scale, cGMP-compliant production of transplantable NSCs from all lines tested. We also show that NSCs generated from iPSCs produced with the process described are capable of forming both glia defined by their expression of S100ß and neurons that fire repetitive action potentials.

Noninvasive diagnosis of mucopolysaccharidosis via depth-resolved optical spectroscopy of the outer ear.

Current diagnostics for lysosomal storage disorders such as mucopolysaccharidosis (MPS) rely on evaluation of ex vivo bodily fluids, which has several shortcomings. In this study, we evaluated whether Raman spectroscopy could noninvasively diagnose MPS in a murine model. Via confocal sampling of the murine outer ear, Raman spectra were obtained at multiple depths. Partial least-squares discriminant analysis of the processed Raman spectra showed a 93% sensitivity and 91% specificity for disease. The discriminant algorithm relied on several Raman bands related to glycosaminoglycans (GAGs) that typically accumulate in MPS. These findings indicate the possibility for a new, noninvasive diagnostic tool for MPS.

Derivation of induced pluripotent stem cells by lentiviral transduction.

This chapter provides a method for reprogramming human dermal fibroblasts into induced pluripotent stem cells (iPSCs) using three lentiviruses containing cDNAs for OCT4 and SOX2, KLF4 and C-MYC, and NANOG and LIN28, respectively. Lentiviral vectors are based on the human immunodeficiency virus (HIV) and provide an effective means for the delivery, integration, and expression of exogenous genes in mammalian cells. Lentiviruses are attractive gene delivery vehicles as they are able to infect both proliferating and nonproliferating cells. Lentiviruses stably integrate into the genome without incurring cellular toxicity and can maintain sustained transgene expression during prolonged host cell proliferation and differentiation. In this protocol, we describe how to prepare lentiviruses, stably transduce human fibroblasts, and identify bona fide iPSC colonies based on morphological similarity to human embryonic stem cell (ESC) colonies and live-cell immunological staining using cell-surface markers of human PSCs such as Tra-1-60 and Tra-1-81.

Traditional human embryonic stem cell culture.

Culturing human embryonic stem cells (hESCs) requires a significant commitment of time and resources. It takes weeks to establish a culture, and the cultures require daily attention. Once hESC cultures are established, they can, with skill and the methods described, be kept in continuous culture for many years. hESC lines were originally derived using very similar culture medium and conditions as those developed for the derivation and culture of mouse ESC lines. However, these methods were suboptimal for hESCs and have evolved considerably in the years since the first hESC lines were derived. Compared with mouse ESCs, hESCs are very difficult to culture - they grow slowly, and most importantly, since we have no equivalent assays for germline competence, we cannot assume that the cells that we have in our culture dishes are either stable or pluripotent. This makes it far more critical to assay the cells frequently using the characterization methods, such as karyotyping, immunocytochemistry, gene expression analysis, and flow cytometry, provided in this manual.

Immunocytochemical analysis of human pluripotent stem cells.

This chapter will describe the most common immunocytochemical method utilized in the stem cell field - using fluorescently tagged secondary antibodies to detect a primary antibody that is bound to an epitope on a molecule of interest. Secondary antibodies recognize the heavy chain of the primary antibody's isotype. Generally, these methods employ an incubation period of the sample with the primary antibody, a series of washes to remove unbound primary antibody, a secondary incubation period of the sample with the fluorescently conjugated secondary antibody, followed by washes and preparation for microscopy.

Differentiation of neural lineage cells from human pluripotent stem cells.

Human pluripotent stem cells have the unique properties of being able to proliferate indefinitely in their undifferentiated state and to differentiate into any somatic cell type. These cells are thus posited to be extremely useful for furthering our understanding of both normal and abnormal human development, providing a human cell preparation that can be used to screen for new reagents or therapeutic agents, and generating large numbers of differentiated cells that can be used for transplantation purposes. Critical among the applications for the latter are diseases and injuries of the nervous system, medical approaches to which have been, to date, primarily palliative in nature. Differentiation of human pluripotent stem cells into cells of the neural lineage, therefore, has become a central focus of a number of laboratories. This has resulted in the description in the literature of several dozen methods for neural cell differentiation from human pluripotent stem cells. Among these are methods for the generation of such divergent neural cells as dopaminergic neurons, retinal neurons, ventral motoneurons, and oligodendroglial progenitors. In this review, we attempt to fully describe most of these methods, breaking them down into five basic subdivisions: (1) starting material, (2) induction of loss of pluripotency, (3) neural induction, (4) neural maintenance and expansion, and (5) neuronal/glial differentiation. We also show data supporting the concept that undifferentiated human pluripotent stem cells appear to have an innate neural differentiation potential. In addition, we evaluate data comparing and contrasting neural stem cells differentiated from human pluripotent stem cells with those derived directly from the human brain.

Macrophages are important determinants of acute ocular HSV-1 infection in immunized mice.

PURPOSE: To determine the effect of macrophage depletion on herpes simplex virus type (HAV)-1 replication in the eye and on the establishment of latency in trigeminal ganglia (TG) of immunized and ocularly infected mice. METHODS: BALB/c mice were immunized with five HSV-1 glycoprotein DNA genes or were sham immunized. The virulent HSV-1 strain KOS was used as a positive vaccine control. Immunized mice were depleted of their macrophages by dichloromethylene diphosphonate (Cl(2)MDP) injection. After ocular infection with the HSV-1 strain McKrae, virus replication in the eye, blepharitis, corneal scarring, and dermatitis were determined. Finally, the copy numbers of latency-associated transcript (LAT) and CD4(+) and CD8(+) T-cell transcripts in the TGs of surviving mice 30 days after infection were determined by RT-PCR. RESULTS: Depletion of macrophages in immunized mice increased HSV-1 replication in the eye of infected mice between days 1 and 5 after ocular infection. Depletion of macrophages did not alter the HSV-1-induced death or corneal scarring in immunized mice. Macrophage depletion, however, resulted in increased blepharitis in immunized mice. Finally, macrophage depletion had no effect on the establishment of latency in immunized mice, as the TGs from both depleted and mock-depleted mice were negative for the presence of the LAT transcript. CONCLUSIONS: In immunized mice during primary HSV-1 ocular infection, macrophages play an important role in vaccine efficacy against HSV-1 replication in the eye and blepharitis in infected mice. During the latent stage of HSV-1 infection, however, macrophage depletion failed to have any observable effect on HSV-1 latency in the TGs of infected mice.

Reactivation phenotype in rabbits of a herpes simplex virus type 1 mutant containing an unrelated antiapoptosis gene in place of latency-associated transcript.

Latency-associated transcript (LAT) significantly enhances the spontaneous reactivation phenotype of herpes simplex virus type 1 (HSV-1). The mechanism by which LAT accomplishes this has been elusive. To determine if LAT's antiapoptosis activity is involved, the authors used a rabbit eye model to analyze the spontaneous reactivation phenotype of an HSV-1 mutant in which LAT was replaced by an unrelated antiapoptosis gene. This virus, dLAT-cpIAP, contains the open reading frame of the baculovirus inhibitor of apoptosis protein gene (cpIAP) in place of LAT, under control of the LAT promoter. The authors report here that in a rabbit ocular model of infection, dLAT-cpIAP had a spontaneous reactivation phenotype similar to wild-type virus and significantly higher than LAT(-) viruses. This was consistent with their previous findings using the mouse trigeminal ganglia explant-induced reactivation model. Whether LAT (and in the case of dLAT-cpIAP, cpIAP) enhances the spontaneous reactivation phenotype by functioning during establishment of latency, maintenance of latency, or reactivation from latency, or during two or more of these periods, remains to be determined. Regardless, the results presented in this study strongly support the hypothesis that LAT's antiapoptosis activity is the dominant function that enhances HSV-1's spontaneous reactivation phenotype.

A herpes simplex virus type 1 mutant expressing a baculovirus inhibitor of apoptosis gene in place of latency-associated transcript has a wild-type reactivation phenotype in the mouse.

The latency-associated transcript (LAT) is essential for the wild-type herpes simplex virus type 1 (HSV-1) high-reactivation phenotype since LAT- mutants have a low-reactivation phenotype. We previously reported that LAT can decrease apoptosis and proposed that this activity is involved in LAT's ability to enhance the HSV-1 reactivation phenotype. The first 20% of the primary 8.3-kb LAT transcript is sufficient for enhancing the reactivation phenotype and for decreasing apoptosis, supporting this proposal. For this study, we constructed an HSV-1 LAT- mutant that expresses the baculovirus antiapoptosis gene product cpIAP under control of the LAT promoter and in place of the LAT region mentioned above. Mice were ocularly infected with this mutant, designated dLAT-cpIAP, and the reactivation phenotype was determined using the trigeminal ganglion explant model. dLAT-cpIAP had a reactivation phenotype similar to that of wild-type virus and significantly higher than that of (i) the LAT- mutant dLAT2903; (ii) dLAT1.5, a control virus containing the same LAT deletion as dLAT-cpIAP, but with no insertion of foreign DNA, thereby controlling for potential readthrough transcription past the cpIAP insert; and (iii) dLAT-EGFP, a control virus identical to dLAT-cpIAP except that it contained the enhanced green fluorescent protein open reading frame (ORF) in place of the cpIAP ORF, thereby controlling for expression of a random foreign gene instead of the cpIAP gene. These results show that an antiapoptosis gene with no sequence similarity to LAT can efficiently substitute for the LAT function involved in enhancing the in vitro-induced HSV-1 reactivation phenotype in the mouse.

A viral model for corneal scarring and neovascularization following ocular infection of rabbits with a herpes simplex virus type 1 (HSV-1) mutant.

PURPOSE: Herpes simplex virus type 1 (HSV-1) remains a major cause of corneal scarring and visual loss. Although efforts have been made, no reproducible animal model is available to examine recurrent corneal disease. Here we propose a rabbit ocular model to study recurrent corneal disease using an HSV-1 mutant that reactivates with high efficiency. METHODS: Rabbits were ocularly infected with 2 x 10 PFU/eye of the parental McKrae, dLAT2903 (a LAT-null virus with a low-reactivation phenotype), or CJLAT (a high-reactivation virus). Acute ocular disease [days 2, 4, 7, and 10 postinfection (pi)], recurrent ocular disease, and neovascularization (days 30 to 58 pi) were monitored. RESULTS: All acute ocular disease symptoms, including conjunctivitis and corneal disease, were similar with all 3 viruses. No corneal scarring was detected in any eyes up to day 30 pi. Between days 35 and 58 pi, corneal scarring was observed in 11/14 (experiment 1) and 18/22 (experiment 2) eyes of CJLAT-infected rabbits. Significantly less corneal scarring was seen in eyes of rabbits infected with McKrae (0/18 and 0/16) or dLAT2903 (0/16 and 3/24) (P < 0.0001). Many of the eyes with corneal scarring developed obvious, measurable neovascularization. CONCLUSIONS: Rabbits infected with CJLAT developed corneal scarring and neovascularization similar to that of clinical ocular HSV-1 recurrent disease. Because this occurred well after the acute infection had resolved, the corneal scarring and neovascularization appeared to be recurrent disease. Thus, CJLAT ocular infection of rabbits may provide a good and reproducible animal model to study factors involved in corneal scarring and neovascularization from recurrent ocular HSV-1.

Methods for detecting the HSV-1 LAT anti-apoptosis activity in virus infected tissue culture cells.

Plasmids expressing the herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) reduce apoptosis in transient transfection assays in tissue culture. LAT also reduces apoptosis in the context of the virus in trigeminal ganglia of rabbits and mice at approximately 6-7 days post-infection during the switch from acute to latent HSV-1 infection, a time at which LAT is the only abundantly transcribed viral gene. Analysis of LAT's anti-apoptosis function is complicated in tissue culture by the expression of at least five additional viral gene products that can block apoptosis, and by the fact that apoptosis usually occurs in only a fraction of the cells. Here, we present two approaches for detecting LAT's anti-apoptosis activity in the context of the whole virus in tissue culture. Using a combination of serum starvation to both partially synchronize the cells and induce apoptosis, and Hoechst staining to detect chromatin condensation, we found that there was a small window of time post-infection during which Schwann cells infected with the LAT(-) mutant dLAT2903 reproducibly had more apoptotic nuclei than identically treated cells infected with the LAT(+) parental virus HSV-1 strain McKrae. Using serum starvation and/or UV treatment and a method to isolate fragmented DNA away from large chromosomal DNA, we found a similar window of time post-infection during which Neuro2A cells infected with dLAT2903 had increased DNA fragmentation (as judged by a DNA laddering assay) compared to identically treated cells infected with wild type McKrae or the LAT(+) marker rescued dLAT2903R virus. These assays should permit the use of culture assays, rather than labor intensive animal models, to examine LAT's anti-apoptosis activity in the context of the virus in a large number of existing LAT mutant viruses.

The bovine herpesvirus-1 LR ORF2 is critical for this gene's ability to restore the high wild-type reactivation phenotype to a herpes simplex virus-1 LAT null mutant.

During neuronal latency of herpes simplex virus (HSV)-1, the latency-associated transcript (LAT) is the only viral gene readily detectable. LAT is required for the high-level reactivation phenotype in animal models. LAT's anti-apoptotic activity was recently demonstrated by our group and it was proposed that LAT's anti-apoptotic function is involved in enhancing the reactivation phenotype. Recently, using chimeric virus CJLAT, it was shown that the reactivation phenotype of LAT(-) mutant dLAT2903 can be restored to wild-type levels by inserting the bovine herpes virus (BHV)-1 latency-related (LR) gene into the LAT locus of this HSV-1 LAT deletion mutant. Although transcription of the LR gene, like LAT, inhibits apoptosis, LR appears to be multifunctional. To investigate whether the LR gene's anti-apoptotic function was responsible for restoring the high-reactivation phenotype, a mutated BHV-1 LR gene was inserted into the LAT locus of HSV-1 generating the chimeric virus CJLATmut. This mutation consists of three stop codons inserted just after the ATG of the first LR open reading frame (ORF2). In plasmids and in a BHV-1 mutant, this mutation eliminated the LR gene's anti-apoptotic activity, strongly suggesting that ORF2 encodes a protein responsible for LR's anti-apoptotic activity. Reactivation of the CJLATmut virus, in both rabbits and mice, was significantly lower than in wild-type McKrae virus (P=0.0001 and P=0.0003, respectively) and CJLAT virus, containing wild-type LR in place of LAT (P<0.0001) and was similar to LAT(-) dLAT2903 (P=0.8 and P=0.7, respectively). Thus, disruption of BHV-1 LR ORF2 eliminated the high-reactivation phenotype.

Identification of herpes simplex virus type 1 latency-associated transcript sequences that both inhibit apoptosis and enhance the spontaneous reactivation phenotype.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for the high spontaneous and induced reactivation phenotype of HSV-1 in the rabbit ocular model and for the high induced reactivation phenotype in the mouse ocular model. Recently we showed that LAT has an antiapoptosis function, and we hypothesized that LAT's ability to inhibit apoptosis played an important role in LAT's ability to enhance the reactivation phenotype. Expression of just the first 1.5 kb of the 8.3-kb LAT gene is sufficient for both inhibition of apoptosis in an in vitro transient-transfection assay and the high spontaneous reactivation phenotype in vivo. Here we show the results of more complex mapping studies in which inhibition of apoptosis and the enhanced spontaneous reactivation phenotype also appear to be linked. The HSV-1 mutant virus dLAT371 has a high spontaneous reactivation phenotype in rabbits, suggesting that the LAT region deleted in this mutant (LAT nucleotides 76 to 447) is not required for this phenotype. The LAT3.3A viral mutant (which expresses LAT nucleotides 1 to 1499) also has a high spontaneous reactivation phenotype, suggesting that the region of LAT not expressed by this mutant (LAT nucleotide 1500 to the end of LAT) is also not required for this phenotype. Surprisingly, LAT2.9A, which is a combination of dLAT371 and LAT3.3A (i.e., it expresses LAT nucleotides 1 to 76 and 447 to 1499), has a low spontaneous reactivation phenotype indistinguishable from that of LAT null mutants. We report here that consistent with the low spontaneous reactivation phenotype of LAT2.9A, a plasmid expressing the identical LAT RNA did not inhibit caspase 9-induced apoptosis. In contrast, plasmids containing the same deletion but able to transcribe up to or past LAT nucleotide 2850 (rather than just up to LAT nucleotide 1499) inhibited caspase 9-induced apoptosis, consistent with the high spontaneous reactivation phenotype of dLAT371. Thus, LAT2.9A may have a low spontaneous reactivation phenotype because the LAT RNA that is made cannot block apoptosis, and dLAT371 apparently has a high spontaneous reactivation phenotype because the LAT RNA made has significant antiapoptosis activity. Furthermore, LAT appeared to have at least two regions capable of interfering with caspase 9-induced apoptosis. One region partially overlaps LAT nucleotides 76 to 447. The second region is partially (or completely) downstream of LAT nucleotide 1499.