Non cell-autonomous reprogramming of adult ocular progenitors: generation of pluripotent stem cells without exogenous transcription factors.

Direct reprogramming of differentiated cells to induced pluripotent stem (iPS) cells by ectopic expression of defined transcription factors (TFs) represents a significant breakthrough towards the use of stem cells in regenerative medicine (Takahashi and Yamanaka Cell 2006;126:663-676). However, the virus-mediated expression of exogenous transcription factors could be potentially harmful and, therefore, represents a barrier to the clinical use of iPS cells. Several approaches, ranging from plasmid-mediated TF expression to introduction of recombinant TFs (Yamanaka Cell 2009;137:13-17; Zhou, Wu, Joo et al. Cell Stem Cell 2009;4:381-384), have been reported to address the risk associated with viral integration. We describe an alternative strategy of reprogramming somatic progenitors entirely through the recruitment of endogenous genes without the introduction of genetic materials or exogenous factors. To this end, we reprogrammed accessible and renewable progenitors from the limbal epithelium of adult rat eye by microenvironment-based induction of endogenous iPS cell genes. Non cell-autonomous reprogramming generates cells that are pluripotent and capable of differentiating into functional neurons, cardiomyocytes, and hepatocytes, which may facilitate autologous cell therapy to treat degenerative diseases.

Ciliary neurotrophic factor-mediated signaling regulates neuronal versus glial differentiation of retinal stem cells/progenitors by concentration-dependent recruitment of mitogen-activated protein kinase and Janus kinase-signal transducer and activator of transcription pathways in conjunction with Notch signaling.

In the retina, as elsewhere in the central nervous system, neurogenesis precedes gliogenesis; that is, the only glia in the retina, Müller cells, are born when the majority of neurons have already been generated. However, our understanding of how the multipotent retinal stem cells/progenitors choose to differentiate along neuronal and glial lineages is unclear. This information is important in promoting directed differentiation of retinal stem cells/progenitors in an ex vivo or in vivo stem cell approach to treating degenerative retinal diseases. Here, using the neurosphere assay, we demonstrate that ciliary neurotrophic factor (CNTF), acting in a concentration-dependent manner, influences the simultaneous differentiation of retinal stem cells/progenitors into neurons or glia. At low CNTF concentrations differentiation of bipolar cells is promoted, whereas high CNTF concentrations facilitate Müller cell differentiation. The two concentrations of CNTF lead to differential activation of mitogen-activated protein kinase and Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathways, with recruitment of the former and the latter for the differentiation of bipolar and Müller cells, respectively. The concentration-dependent recruitment of two disparate pathways toward neurogenesis and gliogenesis occurs in concert with Notch signaling. Furthermore, we demonstrate that the attenuation of Jak-STAT signaling along with Notch signaling facilitates the differentiation of retinal stem cells/progenitors along the rod photoreceptor lineage in vivo. Our observations posit CNTF-mediated signaling as a molecular switch for neuronal versus glial differentiation of retinal stem cells/progenitors and a molecular target for directed neuronal differentiation of retinal stem cells/progenitors as an approach to addressing degenerative changes in the retina. Disclosure of potential conflicts of interest is found at the end of this article.

Derivation of neurons with functional properties from adult limbal epithelium: implications in autologous cell therapy for photoreceptor degeneration.

The limbal epithelium (LE), a circular and narrow epithelium that separates cornea from conjunctiva, harbors stem cells/progenitors in its basal layer that regenerate cornea. We have previously demonstrated that cells in the basal LE, when removed from their niche and cultured in reduced bond morphogenetic protein signaling, acquire properties of neural progenitors. Here, we demonstrate that LE-derived neural progenitors generate neurons with functional properties and can be directly differentiated along rod photoreceptor lineage in vitro and in vivo. These observations posit the LE as a potential source of neural progenitors for autologous cell therapy to treat photoreceptor degeneration in age-related macular degeneration and retinitis pigmentosa.

The canonical Wnt pathway regulates retinal stem cells/progenitors in concert with Notch signaling.

The canonical Wnt pathway is known to influence multiple developmental events such as patterning, cell proliferation and cell specification. Recent studies have provided evidence of the involvement of the canonical Wnt pathway in the emergence and development of the optic neuroepithelium and its derivatives, particularly the retina. However, the mechanism of its action during retinal development remains rather obscure. Here, we demonstrate that (in agreement with observations in the blood, intestine, and skin) the canonical Wnt pathway influences retinal development by maintaining stem cells/progenitors. For example, the activation of this pathway keeps the early retinal stem cells/progenitors proliferating and uncommitted, while its attenuation facilitates their differentiation into retinal ganglion cells in vitro and in vivo. In addition, we demonstrate that Wnt signaling acts in concert with Notch signaling during retinal histogenesis, where the latter calibrates the influence of the former on the differentiation status of retinal stem cells/progenitors by regulating Lef1 and sFRP2.

Membrane properties of retinal stem cells/progenitors.

The membrane properties of cells help integrate extrinsic information relayed through growth factors, chemokines, extracellular matrix, gap junctions and neurotransmitters towards modulating cell-intrinsic properties, which in turn determine whether cells remain quiescent, proliferate, differentiate, establish contact with other cells or remove themselves by activating programmed cell death. This review highlights some of the membrane properties of early and late retinal stem cells/progenitors, which are likely to be helpful in the identification and enrichment of these cells and in understanding mechanisms underlying their maintenance and differentiation. Understanding of membrane properties of retinal stem cells/progenitors is essential for the successful formulation of approaches to treat retinal degeneration and diseases by cell therapy.

In vitro generation of early-born neurons from late retinal progenitors.

Evidence suggests that, as development ensues, the competence of neural progenitors is progressively altered, such that they become fated to give rise to neurons of a particular stage. Here, we demonstrate that late retinal progenitors can give rise to retinal ganglion cells (RGCs), an example of an early-born cell type in the retina. A subset of late retinal progenitors in vitro responds to cues that favor RGC differentiation by displaying markers characteristic of RGCs. In addition, mechanisms used during normal RGC differentiation are recruited by these cells toward their differentiation along RGC lineage. Our observations suggest that late neural progenitors may not be irreversibly fated but may appear as such under the constraints dictated by epigenetic cues.

Retinal properties and potential of the adult mammalian ciliary epithelium stem cells.

The ciliary epithelium (CE) in the adult mammalian eye harbors a mitotic quiescent population of neural stem cells. Here we have compared the cellular and molecular properties of CE stem cells and populations of retinal progenitors that define the early and late stages of histogenesis. The CE stem cells and retinal progenitors proliferate in the presence of mitogens and share the expression of universal neural and retinal progenitor markers. However, the expression of the majority of retinal progenitor markers (e.g., Chx10) is transient in the former when compared to the latter, in vitro. They are similar to early than late retinal progenitors in their proliferative response to FGF2 and/or EGF. Analysis of the differentiation potential of CE stem cells shows that they are capable of generating both early (e.g., retinal ganglion cells) and late (e.g., rod photoreceptors) born retinal neurons. However, under identical differentiation conditions, i.e., in the presence of 1% FBS, they generate more early-born retinal neurons than late-born retinal neurons showing a preference for generating early retinal neurons. Transcription profiling of these cells and retinal progenitors demonstrate that they share approximately 80% of the expressed genes. The CE stem cells have more unique genes in common with early retinal progenitors than late retinal progenitors. Both proliferative/differential potential and transcription profiles suggest that CE stem cells may be a residual population of stem cells of optic neuroepithelium, representing a stage antecedent to retinal progenitors.

Direct identification and enrichment of retinal stem cells/progenitors by Hoechst dye efflux assay.

PURPOSE: The present study describes a method for isolating neural stem cells/progenitors directly from the freshly dissociated embryonic retina (prospective identification) and compares their characteristics with those enriched from mitogen-exposed embryonic retinal cell culture. METHODS: Cell dissociates from embryonic rat retina and mitogen-exposed embryonic retinal cultures were stained with Hoechst 33342 fluorescent dye. The emission patterns of cells were analyzed in both blue and red wavelength using flow cytometry to enrich cells that retained or excluded the dye. The phenotype characteristics and differentiation potential of enriched cells were analyzed by immunocytochemical, RT-PCR, and electrophysiological analyses. RESULTS: The Hoechst dye efflux assay identified a minor population of cells, called side population (SP) cells, in fresh retinal dissociates. These cells that preferentially excluded the Hoechst 33342 fluorescent dye were proliferative and expressed both neural progenitor and retinal progenitor markers. The retinal SP cells generated functional neurons and glia and possessed the ability to differentiate along lineages of different late-born retinal cell types. Cells of similar phenotypes and potential were observed in the SP obtained from mitogen-exposed retinal culture. CONCLUSIONS: The Hoechst dye efflux assay represents an effective method for direct identification of retinal stem cells/progenitors. These results demonstrate that the prospectively isolated retinal stem cells/progenitors and those enriched as SP cells from mitogen-exposed retinal cell culture may be similar in their properties and potential.

Transplantation of ocular stem cells: the role of injury in incorporation and differentiation of grafted cells in the retina.

The incorporation of transplanted cells into the host retina is one of the prerequisites for successful cell replacement therapy to treat retinal degeneration. To test the hypothesis that injury promotes cell incorporation, stem cells/progenitors were isolated from the retina, ciliary epithelium or limbal epithelium and transplanted into the eyes of rats with retinal injury. Different stem cell/progenitor populations incorporated into traumatized or diseased retina but not into the normal retina. The proportion of cells incorporated into the inner retina was consistently higher than in the outer retina. The transplanted cells expressed markers specific to cells of the lamina into which they were incorporated suggesting that cues for specific differentiation are localized within the inner and outer retina. These findings demonstrate that injury-induced cues play a significant role in promoting the incorporation of ocular stem cells/progenitors regardless of their origin or their differentiation along specific retinal sublineage.

Maintenance of retinal stem cells by Abcg2 is regulated by notch signaling.

ABCG2 belongs to the ATP-binding cassette superfamily of transmembrane proteins and is ubiquitously expressed in stem cells including those in the developing nervous system. The ability of ABCG2 to preferentially exclude DNA-intercalating dyes is regarded to be the basis for the enrichment of stem cells or progenitors as dye(low) side population (SP) cells. However, the role of ABCG2 in neural stem cells remains speculative and poorly understood. Here, we demonstrate using retinal stem cells, that ABCG2 is the molecular determinant of SP cell phenotype of neural stem cells and plays an important role in their maintenance. Overexpression of ABCG2 prevents the SP cell phenotype and adversely affects the lineage commitment of retinal stem cells. By contrast, targeted attenuation of ABCG2 depletes retinal SP cells and promotes their differentiation along pan neural and retinal lineages. In addition, we demonstrate for the first time that ABCG2 is a target of Notch signaling, and as such, constitutes one of the genes in the regulatory network of Notch signaling, involved in the maintenance of stem cells.

Characterization of early retinal progenitor microenvironment: presence of activities selective for the differentiation of retinal ganglion cells and maintenance of progenitors.

The maintenance and differentiation of retinal progenitors take place in the context of the microenvironment in which they reside at a given time during retinal histogenesis. To understand the nature of the microenvironment in the developing retina, we have examined the influence of activities present during the early stage of retinal histogenesis on enriched retinal progenitors, using the neurosphere model. Early and late retinal progenitors, enriched as neurospheres from embryonic day 14 (E14) and E18 rat retina, respectively, were cultured in embryonic day 3 (E3) chick retinal conditioned medium, simulating the microenvironment present during early retinal histogenesis. Examination of the differentiation and proliferation of retinal progenitors revealed that the early microenvironment contains at least three regulatory activities, which are partitioned in different size fractions of the conditioned medium with different heat sensitivity. First, it is characterized by activities, present in heat stable <30 kDa fraction, that promote the differentiation of retinal ganglion cells (RGCs), the early born neurons. Second, it contains activities, present in heat-sensitive >30 kDa fraction, that regulate the number of early born neurons and maintain the pool of retinal progenitors. Third, it possesses activities, present in heat-sensitive <30 kDa fraction, that prevent the premature differentiation of early retinal progenitors into the late born neurons. Thus, our observations demonstrate the regulatory influence of microenvironment on the maintenance and differentiation of retinal progenitors and establish neurospheres as a viable model system for the examination of such influences.

SWI/SNF chromatin remodeling ATPase Brm regulates the differentiation of early retinal stem cells/progenitors by influencing Brn3b expression and Notch signaling.

Based on a variety of approaches, evidence suggests that different cell types in the vertebrate retina are generated by multipotential progenitors in response to interactions between cell intrinsic and cell extrinsic factors. The identity of some of the cellular determinants that mediate such interactions has emerged, shedding light on mechanisms underlying cell differentiation. For example, we know now that Notch signaling mediates the influence of the microenvironment on states of commitment of the progenitors by activating transcriptional repressors. Cell intrinsic factors such as the proneural basic helix-loop-helix and homeodomain transcription factors regulate a network of genes necessary for cell differentiation and maturation. What is missing from this picture is the role of developmental chromatin remodeling in coordinating the expression of disparate classes of genes for the differentiation of retinal progenitors. Here we describe the role of Brm, an ATPase in the SWI/SNF chromatin remodeling complex, in the differentiation of retinal progenitors into retinal ganglion cells. Using the perturbation of expression and function analyses, we demonstrate that Brm promotes retinal ganglion cell differentiation by facilitating the expression and function of a key regulator of retinal ganglion cells, Brn3b, and the inhibition of Notch signaling. In addition, we demonstrate that Brm promotes cell cycle exit during retinal ganglion cell differentiation. Together, our results suggest that Brm represents one of the nexus where diverse information of cell differentiation is integrated during cell differentiation.

Neural stem cell properties of Müller glia in the mammalian retina: regulation by Notch and Wnt signaling.

The retina in adult mammals, unlike those in lower vertebrates such as fish and amphibians, is not known to support neurogenesis. However, when injured, the adult mammalian retina displays neurogenic changes, raising the possibility that neurogenic potential may be evolutionarily conserved and could be exploited for regenerative therapy. Here, we show that Müller cells, when retrospectively enriched from the normal retina, like their radial glial counterparts in the central nervous system (CNS), display cardinal features of neural stem cells (NSCs), i.e., they self-renew and generate all three basic cell types of the CNS. In addition, they possess the potential to generate retinal neurons, both in vitro and in vivo. We also provide direct evidence, by transplanting prospectively enriched injury-activated Müller cells into normal eye, that Müller cells have neurogenic potential and can generate retinal neurons, confirming a hypothesis, first proposed in lower vertebrates. This potential is likely due to the NSC nature of Müller cells that remains dormant under the constraint of non-neurogenic environment of the adult normal retina. Additionally, we demonstrate that the mechanism of activating the dormant stem cell properties in Müller cells involves Wnt and Notch pathways. Together, these results identify Müller cells as latent NSCs in the mammalian retina and hence, may serve as a potential target for cellular manipulation for treating retinal degeneration.

Neural stem cells in the mammalian eye: types and regulation.

Neural stem cells/progenitors that give rise to neurons and glia have been identified in different regions of the brain, including the embryonic retina. Recently, such cells have been reported to be present, in a mitotically quiescent state, in the ciliary epithelium of the adult mammalian eye. The retinal and ciliary epithelium stem cells/progenitors appear to share similar signaling pathways that are emerging as important regulators of stem cells in general. Yet, they are different in certain respects, such as in the potential to self-renew. These two neural stem cell/progenitor populations not only will serve as models for investigating stem cell biology but also will help explain the relationships between embryonic and adult neural stem cells/progenitors.

Involvement of Ath3 in CNTF-mediated differentiation of the late retinal progenitors.

The cellular diversity of the mammalian retina is underpinned by multipotential neural progenitors that generate retinal neurons and glia with temporal and spatial specificity. It is thought, based on studies using a variety of approaches, that the fate of retinal progenitors is determined through interactions between temporally and spatially arrayed epigenetic cues with intrinsic factors that regulate the competence of cells to respond to such cues. Here, we demonstrate interactions between an intrinsic factor Ath3, a neural bHLH protein, and an extrinsic factor CNTF during the differentiation of the late retinal progenitors along the bipolar cell lineage. Expression of Ath3 is predominantly associated with the late stage of retinal histogenesis when bipolar cells are specified, and in adult it is detected in cells expressing bipolar cell-specific markers. We demonstrate that CNTF-induced bipolar cell differentiation is accompanied by an increase in levels of Ath3 transcripts and compromised when Ath3 expression is attenuated. Our study suggests that the influence of CNTF on the differentiation of late retinal progenitors is mediated through Ath3.

Extraction and purification of depurinated benzo[a]pyrene-adducted DNA bases from human urine by immunoaffinity chromatography coupled with HPLC and analysis by LC/quadrupole ion-trap MS.

In this paper, we describe implementation and testing of an immunoaffinity (IA) column for rapid and selective extraction of 7-(benzo[a]pyren-6-yl)adenine (BP-6-N7Ade) and 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) from urine, where BP is benzo[a]pyrene. The BP radical cation is a carcinogenic metabolite that reacts with double-stranded DNA, producing depurinated BP-adducted DNA bases excreted in urine. The expected modified nucleobases are BP-6-N7Gua, BP-6-N7Ade, and 8-(benzo[a]pyren-6-yl)guanine (BP-6-C8Gua), and they may serve as important biomarkers for DNA damage by PAHs. IA extracts of urine from a cigarette smoker and a nonsmoker contained less than 5% of contaminants present in Sep-Pak extracts and, unlike the latter, were suitable for analytical HPLC. IA extraction achieved 75-95% recovery of BP-6-N7Gua (10 fmol/mL) and BP-6-N7Ade (1 fmol/mL) added to urine samples. Tandem mass spectrometry of IA/HPLC fractions of urine from two coal smoke-exposed women at high risk for lung cancer demonstrated the presence of 20 and 50 fmol BP-6-N7Gua per mL of urine. Unexposed controls were negative. With proposed modifications, the IA-based protocol can achieve a detection limit of 0.1 fmol/mL urine, which is sufficient for routine quantification of BP-adducted bases in urine of cigarette smokers. This procedure may allow screening of persons at risk for lung cancer associated with exposure to PAH in cigarette and other forms of smoke.