Induction of myelinating oligodendrocytes in human cortical spheroids.

Cerebral organoids provide an accessible system for investigations of cellular composition, interactions, and organization but have lacked oligodendrocytes, the myelinating glia of the central nervous system. Here we reproducibly generated oligodendrocytes and myelin in 'oligocortical spheroids' derived from human pluripotent stem cells. Molecular features consistent with those of maturing oligodendrocytes and early myelin appeared by week 20 in culture, with further maturation and myelin compaction evident by week 30. Promyelinating drugs enhanced the rate and extent of oligodendrocyte generation and myelination, and spheroids generated from human subjects with a genetic myelin disorder recapitulated human disease phenotypes. Oligocortical spheroids provide a versatile platform for studies of myelination of the developing central nervous system and offer new opportunities for disease modeling and therapeutic development.

Modeling the Mutational and Phenotypic Landscapes of Pelizaeus-Merzbacher Disease with Human iPSC-Derived Oligodendrocytes.

Pelizaeus-Merzbacher disease (PMD) is a pediatric disease of myelin in the central nervous system and manifests with a wide spectrum of clinical severities. Although PMD is a rare monogenic disease, hundreds of mutations in the X-linked myelin gene proteolipid protein 1 (PLP1) have been identified in humans. Attempts to identify a common pathogenic process underlying PMD have been complicated by an incomplete understanding of PLP1 dysfunction and limited access to primary human oligodendrocytes. To address this, we generated panels of human induced pluripotent stem cells (hiPSCs) and hiPSC-derived oligodendrocytes from 12 individuals with mutations spanning the genetic and clinical diversity of PMD-including point mutations and duplication, triplication, and deletion of PLP1-and developed an in vitro platform for molecular and cellular characterization of all 12 mutations simultaneously. We identified individual and shared defects in PLP1 mRNA expression and splicing, oligodendrocyte progenitor development, and oligodendrocyte morphology and capacity for myelination. These observations enabled classification of PMD subgroups by cell-intrinsic phenotypes and identified a subset of mutations for targeted testing of small-molecule modulators of the endoplasmic reticulum stress response, which improved both morphologic and myelination defects. Collectively, these data provide insights into the pathogeneses of a variety of PLP1 mutations and suggest that disparate etiologies of PMD could require specific treatment approaches for subsets of individuals. More broadly, this study demonstrates the versatility of a hiPSC-based panel spanning the mutational heterogeneity within a single disease and establishes a widely applicable platform for genotype-phenotype correlation and drug screening in any human myelin disorder.

Molecular-based diagnosis of multiple sclerosis and its progressive stage.

OBJECTIVE: Biomarkers aid diagnosis, allow inexpensive screening of therapies, and guide selection of patient-specific therapeutic regimens in most internal medicine disciplines. In contrast, neurology lacks validated measurements of the physiological status, or dysfunction(s) of cells of the central nervous system (CNS). Accordingly, patients with chronic neurological diseases are often treated with a single disease-modifying therapy without understanding patient-specific drivers of disability. Therefore, using multiple sclerosis (MS) as an example of a complex polygenic neurological disease, we sought to determine whether cerebrospinal fluid (CSF) biomarkers are intraindividually stable, cell type-, disease- and/or process-specific, and responsive to therapeutic intervention. METHODS: We used statistical learning in a modeling cohort (n = 225) to develop diagnostic classifiers from DNA-aptamer-based measurements of 1,128 CSF proteins. An independent validation cohort (n = 85) assessed the reliability of derived classifiers. The biological interpretation resulted from in vitro modeling of primary or stem cell-derived human CNS cells and cell lines. RESULTS: The classifier that differentiates MS from CNS diseases that mimic MS clinically, pathophysiologically, and on imaging achieved a validated area under the receiver operating characteristic curve (AUROC) of 0.98, whereas the classifier that differentiates relapsing-remitting from progressive MS achieved a validated AUROC of 0.91. No classifiers could differentiate primary progressive from secondary progressive MS better than random guessing. Treatment-induced changes in biomarkers greatly exceeded intraindividual and technical variabilities of the assay. INTERPRETATION: CNS biological processes reflected by CSF biomarkers are robust, stable, disease specific, or even disease stage specific. This opens opportunities for broad utilization of CSF biomarkers in drug development and precision medicine for CNS disorders. Ann Neurol 2017;82:795-812.

Abnormal corneal epithelial maintenance in mice heterozygous for the micropinna microphthalmia mutation Mp.

We investigated the corneal morphology of adult Mp/+ mice, which are heterozygous for the micropinna microphthalmia mutation, and identified several abnormalities, which implied that corneal epithelial maintenance was abnormal. The Mp/+ corneal epithelium was thin, loosely packed and contained goblet cells in older mice. Evidence also suggested that the barrier function was compromised. However, there was no major effect on corneal epithelial cell turnover and mosaic patterns of radial stripes indicated that radial cell movement was normal. Limbal blood vessels formed an abnormally wide limbal vasculature ring, K19-positive cells were distributed more widely than normal and K12 was weakly expressed in the peripheral cornea. This raises the possibilities that the limbal-corneal boundary was poorly defined or the limbus was wider than normal. BrdU label-retaining cell numbers and quantitative clonal analysis suggested that limbal epithelial stem cell numbers were not depleted and might be higher than normal. However, as corneal epithelial homeostasis was abnormal, it is possible that Mp/+ stem cell function was impaired. It has been shown recently that the Mp mutation involves a chromosome 18 inversion that disrupts the Fbn2 and Isoc1 genes and produces an abnormal, truncated fibrillin-2(MP) protein. This abnormal protein accumulates in the endoplasmic reticulum (ER) of cells that normally express Fbn2 and causes ER stress. It was also shown that Fbn2 is expressed in the corneal stroma but not the corneal epithelium, suggesting that the presence of truncated fibrillin-2(MP) protein in the corneal stroma disrupts corneal epithelial homeostasis in Mp/+ mice.

Epigenetic Modulation of Human Induced Pluripotent Stem Cell Differentiation to Oligodendrocytes.

Pluripotent stem cells provide an invaluable tool for generating human, disease-relevant cells. Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system, characterized by myelin damage. Oligodendrocytes are the myelinating cells of the central nervous system (CNS); they differentiate from progenitor cells, and their membranes ensheath axons, providing trophic support and allowing fast conduction velocity. The current understanding of oligodendrocyte biology was founded by rodent studies, where the establishment of repressive epigenetic marks on histone proteins, followed by activation of myelin genes, leads to lineage progression. To assess whether this epigenetic regulation is conserved across species, we differentiated human embryonic and induced pluripotent stem cells to oligodendrocytes and asked whether similar histone marks and relative enzymatic activities could be detected. The transcriptional levels of enzymes responsible for methylation and acetylation of histone marks were analyzed during oligodendrocyte differentiation, and the post-translational modifications on histones were detected using immunofluorescence. These studies showed that also in human cells, differentiation along the oligodendrocyte lineage is characterized by the acquisition of multiple repressive histone marks, including deacetylation of lysine residues on histone H3 and trimethylation of residues K9 and K27. These data suggest that the epigenetic modulation of oligodendrocyte identity is highly conserved across species.

Expression of podocalyxin separates the hematopoietic and vascular potentials of mouse embryonic stem cell-derived mesoderm.

In the mouse embryo and differentiating embryonic stem cells, the hematopoietic, endothelial, and cardiomyocyte lineages are derived from Flk1+ mesodermal progenitors. Here, we report that surface expression of Podocalyxin (Podxl), a member of the CD34 family of sialomucins, can be used to subdivide the Flk1+ cells in differentiating embryoid bodies at day 4.75 into populations that develop into distinct mesodermal lineages. Definitive hematopoietic potential was restricted to the Flk1+Podxl+ population, while the Flk1-negative Podxl+ population displayed only primitive erythroid potential. The Flk1+Podxl-negative population contained endothelial cells and cardiomyocyte potential. Podxl expression distinguishes Flk1+ mesoderm populations in mouse embryos at days 7.5, 8.5, and 9.5 and is a marker of progenitor stage primitive erythroblasts. These findings identify Podxl as a useful tool for separating distinct mesodermal lineages.

BMP4 signaling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm identity.

The visceral endoderm (VE) is an epithelial tissue in the early postimplantation mouse embryo that encapsulates the pluripotent epiblast distally and the extraembryonic ectoderm proximally. In addition to facilitating nutrient exchange before the establishment of a circulation, the VE is critical for patterning the epiblast. Since VE is derived from the primitive endoderm (PrE) of the blastocyst, and PrE-derived eXtraembryonic ENdoderm (XEN) cells can be propagated in vitro, XEN cells should provide an important tool for identifying factors that direct VE differentiation. In this study, we demonstrated that BMP4 signaling induces the formation of a polarized epithelium in XEN cells. This morphological transition was reversible, and was associated with the acquisition of a molecular signature comparable to extraembryonic (ex) VE. Resembling exVE which will form the endoderm of the visceral yolk sac, BMP4-treated XEN cells regulated hematopoiesis by stimulating the expansion of primitive erythroid progenitors. We also observed that LIF exerted an antagonistic effect on BMP4-induced XEN cell differentiation, thereby impacting the extrinsic conditions used for the isolation and maintenance of XEN cells in an undifferentiated state. Taken together, our data suggest that XEN cells can be differentiated towards an exVE identity upon BMP4 stimulation and therefore represent a valuable tool for investigating PrE lineage differentiation.

Association of VEGF gene polymorphisms with the development of heart failure in patients after myocardial infarction.

OBJECTIVES: Vascular endothelial growth factor (VEGF) is upregulated in vivoin the ischemic human myocardium. Since several polymorphisms have been shown to influence VEGF expression, we evaluated the contribution of such polymorphisms to the clinical outcome of patients after an acute myocardial infarction (AMI). METHODS: PCR and restriction fragment length polymorphism analysis was performed to genotype 10 VEGF polymorphisms in 102 patients who had suffered an AMI and in 98 age- and sex-matched healthy individuals. Distribution of these polymorphisms was assessed by logistic regression analysis. RESULTS: No significant differences were found between patients and normal individuals. However, when patients were subdivided into 2 groups based on the development of heart failure after their AMI judged by heart ultrasound measurements (ejection fraction <40%), the distribution of the -634 polymorphism differed significantly (p = 0.016). Specifically, patients with a CC genotype had 7 times higher risk of developing heart failure. Additionally, the co-inheritance of -634 with other VEGF polymorphisms was found to be significant for the development of heart failure between these 2 groups. CONCLUSIONS: Our data indicate that the -634 polymorphism and its co-inheritance with genotypes of other VEGF polymorphisms might be considered as risk factors playing a role in the clinical outcome of AMI patients.

Oligodendrocyte differentiation of induced pluripotent stem cells derived from subjects with schizophrenias implicate abnormalities in development.

Abnormalities of brain connectivity and signal transduction are consistently observed in individuals with schizophrenias (SZ). Underlying these anomalies, convergent in vivo, post mortem, and genomic evidence suggest abnormal oligodendrocyte (OL) development and function and lower myelination in SZ. Our primary hypothesis was that there would be abnormalities in the number of induced pluripotent stem (iPS) cell-derived OLs from subjects with SZ. Our secondary hypothesis was that these in vitro abnormalities would correlate with measures of white matter (WM) integrity and myelination in the same subjects in vivo, estimated from magnetic resonance imaging. Six healthy control (HC) and six SZ iPS cell lines, derived from skin fibroblasts from well-characterized subjects, were differentiated into OLs. FACS analysis of the oligodendrocyte-specific surface, glycoprotein O4, was performed at three time points of development (days 65, 75, and 85) to quantify the number of late oligodendrocyte progenitor cells (OPCs) and OLs in each line. Significantly fewer O4-positive cells developed from SZ versus HC lines (95% CI 1.0: 8.6, F1,10 = 8.06, p = 0.02). The difference was greater when corrected for age (95% CI 5.4:10.4, F1,8 = 53.6, p < 0.001). A correlation between myelin content in WM in vivo, estimated by magnetization transfer ratio (MTR) and number of O4-positive cells in vitro was also observed across all time points (F1,9 = 4.3, p = 0.07), reaching significance for mature OLs at day 85 in culture (r = 0.70, p < 0.02). Low production of OPCs may be a contributing mechanism underlying WM reduction in SZ.

GFAP Mutations in Astrocytes Impair Oligodendrocyte Progenitor Proliferation and Myelination in an hiPSC Model of Alexander Disease.

Alexander disease (AxD) is a leukodystrophy that primarily affects astrocytes and is caused by mutations in the astrocytic filament gene GFAP. While astrocytes are thought to have important roles in controlling myelination, AxD animal models do not recapitulate critical myelination phenotypes and it is therefore not clear how AxD astrocytes contribute to leukodystrophy. Here, we show that AxD patient iPSC-derived astrocytes recapitulate key features of AxD pathology such as GFAP aggregation. Moreover, AxD astrocytes inhibit proliferation of human iPSC-derived oligodendrocyte progenitor cells (OPCs) in co-culture and reduce their myelination potential. CRISPR/Cas9-based correction of GFAP mutations reversed these phenotypes. Transcriptomic analyses of AxD astrocytes and postmortem brains identified CHI3L1 as a key mediator of AxD astrocyte-induced inhibition of OPC activity. Thus, this iPSC-based model of AxD not only recapitulates patient phenotypes not observed in animal models, but also reveals mechanisms underlying disease pathology and provides a platform for assessing therapeutic interventions.

Directed Differentiation of Human Pluripotent Stem Cells to Microglia.

Microglia, the immune cells of the brain, are crucial to proper development and maintenance of the CNS, and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology, we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes, expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca2+ transients, whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines.

Generation and isolation of oligodendrocyte progenitor cells from human pluripotent stem cells.

In the CNS, oligodendrocytes act as the myelinating cells. Oligodendrocytes have been identified to be key players in several neurodegenerative disorders. This protocol describes a robust, fast and reproducible differentiation protocol to generate human oligodendrocytes from pluripotent stem cells (PSCs) using a chemically defined, growth factor-rich medium. Within 8 d, PSCs differentiate into paired box 6-positive (PAX6(+)) neural stem cells, which give rise to OLIG2(+) progenitors by day 12. Oligodendrocyte lineage transcription factor 2-positive (OLIG2(+)) cells begin to express the transcription factor NKX2.2 around day 18, followed by SRY-box 10 (SOX10) around day 40. Oligodendrocyte progenitor cells (OPCs) that are positive for the cell surface antigen recognized by the O4 antibody (O4(+)) appear around day 50 and reach, on average, 43% of the cell population after 75 d of differentiation. O4(+) OPCs can be isolated by cell sorting for myelination studies, or they can be terminally differentiated to myelin basic protein-positive (MBP(+)) oligodendrocytes. This protocol also describes an alternative strategy for markedly reducing the length and the costs of the differentiation and generating ∼30% O4(+) cells after only 55 d of culture.

Characterization of molecular and cellular phenotypes associated with a heterozygous CNTNAP2 deletion using patient-derived hiPSC neural cells.

Neurodevelopmental disorders, such as autism spectrum disorders (ASD) and schizophrenia (SZ), are complex disorders with a high degree of heritability. Genetic studies have identified several candidate genes associated with these disorders, including contactin-associated protein-like 2 (CNTNAP2). Traditionally, in animal models or in vitro, the function of CNTNAP2 has been studied by genetic deletion or transcriptional knockdown, which reduce the expression of the entire gene; however, it remains unclear whether the mutations identified in clinical settings are sufficient to alter CNTNAP2 expression in human neurons. Here, using human induced pluripotent stem cells (hiPSCs) derived from two individuals with a large (289kb) and heterozygous deletion in CNTNAP2 (affecting exons 14-15) and discordant clinical outcomes, we have characterized CNTNAP2 expression patterns in hiPSC neural progenitor cells (NPCs), two independent populations of hiPSC-derived neurons and hiPSC-derived oligodendrocyte precursor cells (OPCs). First, we observed exon-specific changes in CNTNAP2 expression in both carriers; although the expression of exons 14-15 is significantly decreased, the expression of other exons is upregulated. Second, we observed significant differences in patterns of allele-specific expression in CNTNAP2 carriers that were consistent with clinical outcome. Third, we observed a robust neural migration phenotype that correlated with diagnosis and exon- and allele-specific CNTNAP2 expression patterns, but not with genotype. In all, our data highlight the importance of considering the nature, location and regulation of mutated alleles when attempting to connect GWAS studies to gene function.

Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells.

Multiple sclerosis (MS) is a chronic demyelinating disease of unknown etiology that affects the CNS. While current therapies are primarily directed against the immune system, the new challenge is to address progressive MS with remyelinating and neuroprotective strategies. Here, we develop a highly reproducible protocol to efficiently derive oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes from induced pluripotent stem cells (iPSCs). Key elements of our protocol include adherent cultures, dual SMAD inhibition, and addition of retinoids from the beginning of differentiation, which lead to increased yields of OLIG2 progenitors and high numbers of OPCs within 75 days. Furthermore, we show the generation of viral and integration-free iPSCs from primary progressive MS (PPMS) patients and their efficient differentiation to oligodendrocytes. PPMS OPCs are functional, as demonstrated by in vivo myelination in the shiverer mouse. These results provide encouraging advances toward the development of autologous cell therapies using iPSCs.

Increased corneal epithelial turnover contributes to abnormal homeostasis in the Pax6(+/-) mouse model of aniridia.

We aimed to test previous predictions that limbal epithelial stem cells (LESCs) are quantitatively deficient or qualitatively defective in Pax6(+/-) mice and decline with age in wild-type (WT) mice. Consistent with previous studies, corneal epithelial stripe patterns coarsened with age in WT mosaics. Mosaic patterns were also coarser in Pax6(+/-) mosaics than WT at 15 weeks but not at 3 weeks, which excludes a developmental explanation and strengthens the prediction that Pax6(+/-) mice have a LESC-deficiency. To investigate how Pax6 genotype and age affected corneal homeostasis, we compared corneal epithelial cell turnover and label-retaining cells (LRCs; putative LESCs) in Pax6(+/-) and WT mice at 15 and 30 weeks. Limbal BrdU-LRC numbers were not reduced in the older WT mice, so this analysis failed to support the predicted age-related decline in slow-cycling LESC numbers in WT corneas. Similarly, limbal BrdU-LRC numbers were not reduced in Pax6(+/-) heterozygotes but BrdU-LRCs were also present in Pax6(+/-) corneas. It seems likely that Pax6(+/-) LRCs are not exclusively stem cells and some may be terminally differentiated CD31-positive blood vessel cells, which invade the Pax6(+/-) cornea. It was not, therefore, possible to use this approach to test the prediction that Pax6(+/-) corneas had fewer LESCs than WT. However, short-term BrdU labelling showed that basal to suprabasal movement (leading to cell loss) occurred more rapidly in Pax6(+/-) than WT mice. This implies that epithelial cell loss is higher in Pax6(+/-) mice. If increased corneal epithelial cell loss exceeds the cell production capacity it could cause corneal homeostasis to become unstable, resulting in progressive corneal deterioration. Although it remains unclear whether Pax6(+/-) mice have LESC-deficiency, we suggest that features of corneal deterioration, that are often taken as evidence of LESC-deficiency, might occur in the absence of stem cell deficiency if corneal homeostasis is destabilised by excessive cell loss.

Normal X-inactivation mosaicism in corneas of heterozygous FlnaDilp2/+ female mice--a model of human filamin A (FLNA) diseases.

BACKGROUND: Some abnormalities of mouse corneal epithelial maintenance can be identified by the atypical mosaic patterns they produce in X-chromosome inactivation mosaics and chimeras. Human FLNA/+ females, heterozygous for X-linked, filamin A gene (FLNA) mutations, display a range of disorders and X-inactivation mosaicism is sometimes quantitatively unbalanced. FlnaDilp2/+ mice, heterozygous for an X-linked filamin A (Flna) nonsense mutation have variable eye, skeletal and other abnormalities, but X-inactivation mosaicism has not been investigated. The aim of this study was to determine whether X-inactivation mosaicism in the corneal epithelia of FlnaDilp2/+ mice was affected in any way that might predict abnormal corneal epithelial maintenance. RESULTS: X-chromosome inactivation mosaicism was studied in the corneal epithelium and a control tissue (liver) of FlnaDilp2/+ and wild-type (WT) female X-inactivation mosaics, hemizygous for the X-linked, LacZ reporter H253 transgene, using ß-galactosidase histochemical staining. The corneal epithelia of FlnaDilp2/+ and WT X-inactivation mosaics showed similar radial, striped patterns, implying epithelial cell movement was not disrupted in FlnaDilp2/+ corneas. Corrected stripe numbers declined with age overall (but not significantly for either genotype individually), consistent with previous reports suggesting an age-related reduction in stem cell function. Corrected stripe numbers were not reduced in FlnaDilp2/+ compared with WT X-inactivation mosaics and mosaicism was not significantly more unbalanced in the corneal epithelia or livers of FlnaDilp2/+ than wild-type Flna+/+ X-inactivation mosaics. CONCLUSIONS: Mosaic analysis identified no major effect of the mouse FlnaDilp2 mutation on corneal epithelial maintenance or the balance of X-inactivation mosaicism in the corneal epithelium or liver.

Stem cells and corneal epithelial maintenance: insights from the mouse and other animal models.

Maintenance of the corneal epithelium is essential for vision and is a dynamic process incorporating constant cell production, movement and loss. Although cell-based therapies involving the transplantation of putative stem cells are well advanced for the treatment of human corneal defects, the scientific understanding of these interventions is poor. No definitive marker that discriminates stem cells that maintain the corneal epithelium from the surrounding tissue has been discovered and the identity of these elusive cells is, therefore, hotly debated. The key elements of corneal epithelial maintenance have long been recognised but it is still not known how this dynamic balance is co-ordinated during normal homeostasis to ensure the corneal epithelium is maintained at a uniform thickness. Most indirect experimental evidence supports the limbal epithelial stem cell (LESC) hypothesis, which proposes that the adult corneal epithelium is maintained by stem cells located in the limbus at the corneal periphery. However, this has been challenged recently by the corneal epithelial stem cell (CESC) hypothesis, which proposes that during normal homeostasis the mouse corneal epithelium is maintained by stem cells located throughout the basal corneal epithelium with LESCs only contributing during wound healing. In this chapter we review experimental studies, mostly based on animal work, that provide insights into how stem cells maintain the normal corneal epithelium and consider the merits of the alternative LESC and CESC hypotheses. Finally, we highlight some recent research on other stem cell systems and consider how this could influence future research directions for identifying the stem cells that maintain the corneal epithelium.

Rare corneal clones in mice suggest an age-related decrease of stem cell activity and support the limbal epithelial stem cell hypothesis.

The anterior ocular surface comprises the cornea, conjunctiva and a narrow intermediate region called the limbus. It is widely accepted that the corneal epithelium is maintained by stem cells but different hypotheses propose that the stem cells that maintain the mouse corneal epithelium during normal homeostasis are located either in the basal limbal epithelium or throughout the basal corneal epithelium. There are no specific markers to help test these alternatives and new methods are required to distinguish between them. We observed that KRT5(LacZ/-) transgenic mice produced rare ß-galactosidase (ß-gal)-positive radial stripes in the corneal epithelium. These stripes are likely to be clonal lineages of cells derived from stem cells, so they provide a lineage marker for actively proliferating stem cells. The distributions of the ß-gal-positive radial stripes suggested they extended centripetally from the limbus, supporting the limbal epithelial stem cell (LESC) hypothesis. Stripe frequency declined between 15 and 30 weeks, which predicts a reduction in stem cell function with age. Pax6(+/-), KRT5(LacZ/-) corneas had small patches rather than stripes, which confirms that corneal maintenance is abnormal in Pax6(+/-) mice.

The sixth annual translational stem cell research conference of the New York Stem Cell Foundation.

The New York Stem Cell Foundation's "Sixth Annual Translational Stem Cell Research Conference" convened on October 11-12, 2011 at the Rockefeller University in New York City. Over 450 scientists, patient advocates, and stem cell research supporters from 14 countries registered for the conference. In addition to poster and platform presentations, the conference featured panels entitled "Road to the Clinic" and "The Future of Regenerative Medicine."

Effects of aberrant Pax6 gene dosage on mouse corneal pathophysiology and corneal epithelial homeostasis.

BACKGROUND: Altered dosage of the transcription factor PAX6 causes multiple human eye pathophysiologies. PAX6⁺/⁻ heterozygotes suffer from aniridia and aniridia-related keratopathy (ARK), a corneal deterioration that probably involves a limbal epithelial stem cell (LESC) deficiency. Heterozygous Pax6(+/Sey-Neu) (Pax6⁺/⁻) mice recapitulate the human disease and are a good model of ARK. Corneal pathologies also occur in other mouse Pax6 mutants and in PAX77(Tg/-) transgenics, which over-express Pax6 and model human PAX6 duplication. METHODOLOGY/PRINCIPAL FINDINGS: We used electron microscopy to investigate ocular defects in Pax6⁺/⁻ heterozygotes (low Pax6 levels) and PAX77(Tg/-) transgenics (high Pax6 levels). As well as the well-documented epithelial defects, aberrant Pax6 dosage had profound effects on the corneal stroma and endothelium in both genotypes, including cellular vacuolation, similar to that reported for human macular corneal dystrophy. We used mosaic expression of an X-linked LacZ transgene in X-inactivation mosaic female (XLacZ(Tg/-)) mice to investigate corneal epithelial maintenance by LESC clones in Pax6⁺/⁻ and PAX77(Tg/-) mosaic mice. PAX77(Tg/-) mosaics, over-expressing Pax6, produced normal corneal epithelial radial striped patterns (despite other corneal defects), suggesting that centripetal cell movement was unaffected. Moderately disrupted patterns in Pax6⁺/⁻ mosaics were corrected by introducing the PAX77 transgene (in Pax6⁺/⁻, PAX77(Tg/-) mosaics). Pax6(Leca4/+), XLacZ(Tg/-) mosaic mice (heterozygous for the Pax6(Leca4) missense mutation) showed more severely disrupted mosaic patterns. Corrected corneal epithelial stripe numbers (an indirect estimate of active LESC clone numbers) declined with age (between 15 and 30 weeks) in wild-type XLacZ(Tg/-) mosaics. In contrast, corrected stripe numbers were already low at 15 weeks in Pax6⁺/⁻ and PAX77(Tg/-) mosaic corneas, suggesting Pax6 under- and over-expression both affect LESC clones. CONCLUSIONS/SIGNIFICANCE: Pax6⁺/⁻ and PAX77(Tg/-) genotypes have only relatively minor effects on LESC clone numbers but cause more severe corneal endothelial and stromal defects. This should prompt further investigations of the pathophysiology underlying human aniridia and ARK.