Janine Davis

Efficient and Rapid Derivation of Primitive Neural Stem Cells and Generation of Brain Subtype Neurons From Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are unique cell sources for disease modeling, drug discovery screens, and cell therapy applications. The first step in producing neural lineages from hPSCs is the generation of neural stem cells (NSCs). Current methods of NSC derivation involve the time‐consuming, labor‐intensive steps of an embryoid body generation or coculture with stromal cell lines that result in low‐efficiency derivation of NSCs. In this study, we report a highly efficient serum‐free pluripotent stem cell neural induction medium that can induce hPSCs into primitive NSCs (pNSCs) in 7 days, obviating the need for time‐consuming, laborious embryoid body generation or rosette picking. The pNSCs expressed the neural stem cell markers Pax6, Sox1, Sox2, and Nestin; were negative for Oct4; could be expanded for multiple passages; and could be differentiated into neurons, astrocytes, and oligodendrocytes, in addition to the brain region‐specific neuronal subtypes GABAergic, dopaminergic, and motor neurons. Global gene expression of the transcripts of pNSCs was comparable to that of rosette‐derived and human fetal‐derived NSCs. This work demonstrates an efficient method to generate expandable pNSCs, which can be further differentiated into central nervous system neurons and glia with temporal, spatial, and positional cues of brain regional heterogeneity. This method of pNSC derivation sets the stage for the scalable production of clinically relevant neural cells for cell therapy applications in good manufacturing practice conditions.

Evidence for gelsolin as a corneal crystallin in zebrafish.

We have shown that gelsolin is one of the most prevalent water-soluble proteins in the transparent cornea of zebrafish. There are also significant amounts of actin. In contrast to actin, gelsolin is barely detectable in other eye tissues (iris, lens, and remaining eye) of the zebrafish. Gelsolin cDNA hybridized intensely in Northern blots to RNA from the cornea but not from the lens, brain, or headless body. The deduced zebrafish gelsolin is ∼60% identical to mammalian cytosolic gelsolin and has the characteristic six segmental repeats as well as the binding sites for actin, calcium, and phosphatidylinositides. In situhybridization tests showed that gelsolin mRNA is concentrated in the zebrafish corneal epithelium. The zebrafish corneal epithelium stains very weakly with rhodamine-phalloidin, indicating little F-actin in the cytoplasm. In contrast, the mouse corneal epithelium contains relatively little gelsolin and stains intensely with rhodamine-phalloidin, as does the zebrafish extraocular muscle. We propose, by analogy with the diverse crystallins of the eye lens and with the putative enzyme-crystallins (aldehyde dehydrogenase class 3 and other enzymes) of the mammalian cornea, that gelsolin and actin-gelsolin complexes act as water-soluble crystallins in the zebrafish cornea and contribute to its optical properties.

Identification of Candidate Klf4 Target Genes Reveals the Molecular Basis of the Diverse Regulatory Roles of Klf4 in the Mouse Cornea

PURPOSE Krüppel-like factor 4 (Klf4) plays a crucial role in the development and maintenance of the mouse cornea. In the current study, wild-type (WT) and Klf4-conditional null (Klf4CN) corneal gene expression patterns were examined, to gain understanding of the molecular basis of the Klf4CN corneal phenotype. METHODS Expression of more than 22,000 genes in 10 WT and Klf4CN corneas was compared by microarrays, analyzed using BRB ArrayTools (National Cancer Institute, Bethesda, MD) and validated by Q-RT-PCR. Transient cotransfections were used to test whether Klf4 activates the aquaporin-3, Aldh3a1, and TKT promoters. RESULTS Scatterplot analysis identified 740 and 529 genes up- and downregulated by more than twofold, respectively, in the Klf4CN corneas. Cell cycle activators were upregulated, whereas the inhibitors were downregulated, consistent with the increased Klf4CN corneal epithelial cell proliferation. Desmosomal components were downregulated, consistent with the Klf4CN corneal epithelial fragility. Downregulation of aquaporin-3, detected by microarray, was confirmed by immunoblot and immunohistochemistry. Aquaporin-3 promoter activity was stimulated 7- to 10-fold by cotransfection with pCI-Klf4. The corneal crystallins Aldh3A1 and TKT were downregulated in the Klf4CN cornea, and their respective promoter activities were upregulated 16- and 9-fold by pCI-Klf4 in cotransfections. The expression of epidermal keratinocyte differentiation markers was affected in the Klf4CN cornea. Although the cornea-specific keratin-12 was downregulated, most other keratins were upregulated, suggesting hyperkeratosis. CONCLUSIONS Functionally diverse candidate Klf4 target genes were identified, revealing the molecular basis of the diverse aspects of the Klf4CN corneal phenotype. These results establish Klf4 as an important node in the genetic network of transcription factors regulating the corneal homeostasis.

A Multiplex High-Throughput Gene Expression Assay to Simultaneously Detect Disease and Functional Markers in Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium

There is continuing interest in the development of lineage‐specific cells from induced pluripotent stem (iPS) cells for use in cell therapies and drug discovery. Although in most cases differentiated cells show features of the desired lineage, they retain fetal gene expression and do not fully mature into “adult‐like” cells. Such cells may not serve as an effective therapy because, once implanted, immature cells pose the risk of uncontrolled growth. Therefore, there is a need to optimize lineage‐specific stem cell differentiation protocols to produce cells that no longer express fetal genes and have attained “adult‐like” phenotypes. Toward that goal, it is critical to develop assays that simultaneously measure cell function and disease markers in high‐throughput format. Here, we use a multiplex high‐throughput gene expression assay that simultaneously detects endogenous expression of multiple developmental, functional, and disease markers in iPS cell‐derived retinal pigment epithelium (RPE). We optimized protocols to differentiate iPS cell‐derived RPE that was then grown in 96‐ and 384‐well plates. As a proof of principle, we demonstrate differential expression of eight genes in iPS cells, iPS cell‐derived RPE at two different differentiation stages, and primary human RPE using this multiplex assay. The data obtained from the multiplex gene expression assay are significantly correlated with standard quantitative reverse transcription‐polymerase chain reaction‐based measurements, confirming the ability of this high‐throughput assay to measure relevant gene expression changes. This assay provides the basis to screen for compounds that improve RPE function and maturation and target disease pathways, thus providing the basis for effective treatments of several retinal degenerative diseases.

Small-molecule–directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells

Significance Cell-based approaches utilizing retinal pigment epithelial (RPE)-like cells derived from human pluripotent stem cells (hPSCs) are being developed for the treatment of retinal degeneration. In most research published to date, the choice of the factors used to induce RPE differentiation is based on data from developmental studies. Here, we developed an unbiased approach directed at identifying novel RPE differentiation-promoting factors using a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. We identified chetomin, a dimeric epidithiodiketopiperazine, as a strong inducer of RPE; combination with nicotinamide resulted in efficient RPE differentiation. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE. Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule–only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.

Adaptive evolution of small heat shock protein/ αB-crystallin promoter activity of the blind subterranean mole rat, Spalax ehrenbergi

Blind mole rats have degenerated subcutaneous eyes that are visually nonfunctional. In this investigation, we have compared the tissue specificity of the small heat shock protein (shsp)/αB-crystallin promoter of the mole rat superspecies, Spalax ehrenbergi, with that of the mouse. Earlier experiments showed that mouse shsp/αB-crystallin promoter/enhancer activity is high in the lens and moderate in the heart and skeletal muscle of transgenic mice. Here, we show in transgenic mouse experiments using the firefly luciferase reporter gene that, despite relatively few changes in sequence, the mole rat shsp/αB-crystallin promoter/enhancer has selectively lost lens activity after 13.5 days of embryogenesis (E13.5). The ratios of mole rat/mouse promoter activity were 0.01 for lens, 1.7 for heart, and 13.6 for skeletal muscle in 8-wk-old transgenic mice. Our data indicate that the shsp/αB-crystallin promoter/enhancer has undergone adaptive changes corresponding to the subterranean evolution of the blind mole rat. We speculate that selective pressures on metabolic economy may have contributed to these tissue-specific modifications of promoter/enhancer function during adaptation to life underground.

Human Adult Retinal Pigment Epithelial Stem Cell-Derived RPE Monolayers Exhibit Key Physiological Characteristics of Native Tissue.

PURPOSE We tested what native features have been preserved with a new culture protocol for adult human RPE. METHODS We cultured RPE from adult human eyes. Standard protocols for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and ELISA were used. RESULTS Confluent monolayers of adult human RPE cultures exhibit characteristics of native RPE. Immunohistochemistry demonstrated polarized expression of RPE markers. Electron microscopy illustrated characteristics of native RPE. The mean transepithelial potential (TEP) was 1.19 ± 0.24 mV (mean ± SEM, n = 31), apical positive, and the mean transepithelial resistance (RT) was 178.7 ± 9.9 Ω·cm2 (mean ± SEM, n = 31). Application of 100 μM adenosine triphosphate (ATP) apically increased net fluid absorption (Jv) by 6.11 ± 0.53 μL·cm2·h-1 (mean ± SEM, n = 6) and TEP by 0.33 ± 0.048 mV (mean ± SEM, n = 25). Gene expression of cultured RPE was comparable to native adult RPE (n = 5); however, native RPE RNA was harvested between 24 and 40 hours after death and, therefore, may not accurately reflect healthy native RPE. Vascular endothelial growth factor secreted preferentially basally 2582 ± 146 pg/mL/d, compared to an apical secretion of 1548 ± 162 pg/mL/d (n = 14, P < 0.01), while PEDF preferentially secreted apically 1487 ± 280 ng/mL/d compared to a basolateral secretion of 864 ± 132 ng/mL/d (n = 14, P < 0.01). CONCLUSIONS The new culture model preserves native RPE morphology, electrophysiology, and gene and protein expression patterns, and may be a useful model to study RPE physiology, disease, and transplantation.

Overexpression of Pax6 in Mouse Cornea Directly Alters Corneal Epithelial Cells: Changes in Immune Function, Vascularization, and Differentiation

PURPOSE To assess whether Pax6 functions directly in the cornea, a corneal-preferred promoter was used to overexpress Pax6 specifically in the cornea. METHODS Transgenic mice harboring a construct containing mouse Pax6 coding sequences fused downstream of the aldehyde dehydrogenase 3a1 (Aldh3a1) promoter were generated (Pax6 Tg). Pax6 expression was analyzed by Western blot and immunohistochemistry. Eye sections were stained with hematoxylin and eosin, Schiff reagent, and fluorescein, to assess morphologic changes, the presence of goblet cells, and barrier integrity, respectively. Gene expression changes in mildly affected Pax6 Tg corneas were compared to age-matched, wild-type (WT) corneas by microarray analysis and quantitative PCR. Promoter regulation of several differentially expressed genes was examined by monitoring luciferase activity of reporter constructs after cotransfection with Pax6 in COS7 cells. RESULTS Corneal overexpression of Pax6 produces an abnormal cornea with altered epithelial cell morphology, neovascularization, immune cell invasion, and a compromised barrier; the lens appeared normal. Major changes in expression of genes involved in immune function, vascularization, and epithelial differentiation occurred in corneas from Pax6 Tg versus WT mice. The keratin (K) profile was dramatically altered in the Pax6 Tg corneas, as were several components of the Wnt signaling pathway. In severely affected Pax6 Tg corneas, K12 was reduced, and Pax6 was redistributed into the cytoplasm. Promoters from the chitinase 3-like 3, Wnt inhibitory factor 1, and fms-related tyrosine kinase 1/soluble VEGF receptor genes were upregulated five-, seven-, and threefold, respectively, by Pax6 in transfected COS7 cells. CONCLUSIONS Pax6 functions directly to maintain normal, corneal epithelial cells.