Lens Progenitor Cells Research Articles

Generation of Lens Progenitor Cells and Lentoid Bodies from Pluripotent Stem Cells: Novel Tools for Human Lens Development and Ocular Disease Etiology.

In vitro differentiation of human pluripotent stem cells (hPSCs) into specialized tissues and organs represents a powerful approach to gain insight into those cellular and molecular mechanisms regulating human development. Although normal embryonic eye development is a complex process, generation of ocular organoids and specific ocular tissues from pluripotent stem cells has provided invaluable insights into the formation of lineage-committed progenitor cell populations, signal transduction pathways, and self-organization principles. This review provides a comprehensive summary of recent advances in generation of adenohypophyseal, olfactory, and lens placodes, lens progenitor cells and three-dimensional (3D) primitive lenses, "lentoid bodies", and "micro-lenses". These cells are produced alone or "community-grown" with other ocular tissues. Lentoid bodies/micro-lenses generated from human patients carrying mutations in crystallin genes demonstrate proof-of-principle that these cells are suitable for mechanistic studies of cataractogenesis. Taken together, current and emerging advanced in vitro differentiation methods pave the road to understand molecular mechanisms of cataract formation caused by the entire spectrum of mutations in DNA-binding regulatory genes, such as PAX6, SOX2, FOXE3, MAF, PITX3, and HSF4, individual crystallins, and other genes such as BFSP1, BFSP2, EPHA2, GJA3, GJA8, LIM2, MIP, and TDRD7 represented in human cataract patients.

ATRIP protects progenitor cells against DNA damage in vivo

The maintenance of genomic stability during the cell cycle of progenitor cells is essential for the faithful transmission of genetic information. Mutations in genes that ensure genome stability lead to human developmental syndromes. Mutations in Ataxia Telangiectasia and Rad3-related (ATR) or in ATR-interacting protein (ATRIP) lead to Seckel syndrome, which is characterized by developmental malformations and short life expectancy. While the roles of ATR in replicative stress response and chromosomal segregation are well established, it is unknown how ATRIP contributes to maintaining genomic stability in progenitor cells in vivo. Here, we generated the first mouse model to investigate ATRIP function. Conditional inactivation of Atrip in progenitor cells of the CNS and eye led to microcephaly, microphthalmia and postnatal lethality. To understand the mechanisms underlying these malformations, we used lens progenitor cells as a model and found that ATRIP loss promotes replicative stress and TP53-dependent cell death. Trp53 inactivation in Atrip-deficient progenitor cells rescued apoptosis, but increased mitotic DNA damage and mitotic defects. Our findings demonstrate an essential role of ATRIP in preventing DNA damage accumulation during unchallenged replication.

Jagged1 protein processing in the developing mammalian lens.

ABSTRACTNotch signaling regulates a multitude of cellular processes. During ocular lens development this pathway is required for lens progenitor growth, differentiation and maintenance of the transition zone. After ligand-receptor binding, the receptor proteins are processed, first by ADAM proteases, then by γ-secretase cleavage. This results in the release of a Notch intracellular domain (N-ICD), which is recruited into a nuclear transcription factor complex that activates Notch target genes. Previous in vitro studies showed that the Delta-like and Jagged ligand proteins can also be cleaved by the γ-secretase complex, but it remains unknown whether such processing occurs during in vivo vertebrate development. Here we show that mouse and human lens progenitor cells endogenously express multiple Jagged1 protein isoforms, including a Jagged1 intracellular domain. We also found that pharmacologic blockage of γ-secretase activity in vitro resulted in an accumulation of Jagged1 polypeptide intermediates. Finally, overexpression of an epitope-tagged Jagged1 intracellular domain displayed nuclear localization and induced the upregulation of endogenous JAG1 mRNA expression. These findings support the idea that along with its classical role as a Notch pathway ligand, Jagged1 is regulated post-translationally, to produce multiple active protein isoforms.

Separate and coincident expression of Hes1 and Hes5 in the developing mouse eye.

Notch signaling is broadly required during embryogenesis, frequently activating the transcription of two basic helix-loop-helix transcription factors, Hes1 and Hes5. But, it remains unresolved when and where Hes1 and Hes5 act alone or together during development. Here, we analyzed a Hes5-green fluorescent protein (GFP) bacterial artificial chromosome (BAC) transgenic mouse, as a proxy for endogenous Hes5. We directly compared transgenic GFP expression with Hes1, and particular markers of embryonic lens and retina development. Hes5-GFP is dynamic within subsets of retinal and lens progenitor cells, and differentiating retinal ganglion neurons, in contrast to Hes1 found in all progenitor cells. In the adult retina, only Müller glia express Hes5-GFP. Finally, Hes5-GFP is up-regulated in Hes1 germline mutants, consistent with previous demonstration that Hes1 suppresses Hes5 transcription. Hes5-GFP BAC transgenic mice are useful for identifying Hes5-expressing cells. Although Hes5-GFP and Hes1 are coexpressed in particular developmental contexts, we also noted cohorts of lens or retinal cells expressing just one factor. The dynamic Hes5-GFP expression pattern, coupled with its derepressed expression in Hes1 mutants, suggests that this transgene contains the relevant cis-regulatory elements that regulate endogenous Hes5 in the mouse lens and retina. Developmental Dynamics 247:212-221, 2018. © 2017 Wiley Periodicals, Inc.

Generation of Functional Lentoid Bodies From Human Induced Pluripotent Stem Cells Derived From Urinary Cells

The pathological mechanisms underlying cataract formation remain largely unknown on account of the lack of appropriate in vitro cellular models. The aim of this study is to develop a stable in vitro system for human lens regeneration using pluripotent stem cells. Isolated human urinary cells were infected with four Yamanaka factors to generate urinary human induced pluripotent stem cells (UiPSCs), which were induced to differentiate into lens progenitor cells and lentoid bodies (LBs). The expression of lens-specific markers was examined by real-time PCR, immunostaining, and Western blotting. The structure and magnifying ability of LBs were investigated using transmission electron microscopy and observing the magnification of the letter "X," respectively. We developed a "fried egg" differentiation method to generate functional LBs from UiPSCs. The UiPSC-derived LBs exhibited crystalline lens-like morphology and a transparent structure and expressed lens-specific markers αA-, αB-, β-, and γ-crystallin and MIP. During LB differentiation, the placodal markers SIX1, EYA1, DLX3, PAX6, and the specific early lens markers SOX1, PROX1, FOXE3, αA-, and αB-crystallin were observed at certain time points. Microscopic examination revealed the presence of lens epithelial cells adjacent to the lens capsule as well as both immature and mature fiber-like cells. Optical analysis further demonstrated the magnifying ability (1.7×) of the LBs generated from UiPSCs. Our study provides the first evidence toward generating functional LBs from UiPSCs, thereby establishing an in vitro system that can be used to study human lens development and cataractogenesis and perhaps even be useful for drug screening.

The cellular and molecular mechanisms of vertebrate lens development.

The ocular lens is a model system for understanding important aspects of embryonic development, such as cell specification and the spatiotemporally controlled formation of a three-dimensional structure. The lens, which is characterized by transparency, refraction and elasticity, is composed of a bulk mass of fiber cells attached to a sheet of lens epithelium. Although lens induction has been studied for over 100 years, recent findings have revealed a myriad of extracellular signaling pathways and gene regulatory networks, integrated and executed by the transcription factor Pax6, that are required for lens formation in vertebrates. This Review summarizes recent progress in the field, emphasizing the interplay between the diverse regulatory mechanisms employed to form lens progenitor and precursor cells and highlighting novel opportunities to fill gaps in our understanding of lens tissue morphogenesis.

C-myc Regulates Cell Proliferation during Lens Development

Myc protooncogenes play important roles in the regulation of cell proliferation, growth, differentiation and survival during development. In various developing organs, c-myc has been shown to control the expression of cell cycle regulators and its misregulated expression is detected in many human tumors. Here, we show that c-myc gene (Myc) is highly expressed in developing mouse lens. Targeted deletion of c-myc gene from head surface ectoderm dramatically impaired ocular organogenesis, resulting in severe microphtalmia, defective anterior segment development, formation of a lens stalk and/or aphakia. In particular, lenses lacking c-myc presented thinner epithelial cell layer and growth impairment that was detectable soon after its inactivation. Defective development of c-myc-null lens was not caused by increased cell death of lens progenitor cells. Instead, c-myc loss reduced cell proliferation, what was associated with an ectopic expression of Prox1 and p27Kip1 proteins within epithelial cells. Interestingly, a sharp decrease in the expression of the forkhead box transcription factor Foxe3 was also observed following c-myc inactivation. These data represent the first description of the physiological roles played by a Myc family member in mouse lens development. Our findings support the conclusion that c-myc regulates the proliferation of lens epithelial cells in vivo and may, directly or indirectly, modulate the expression of classical cell cycle regulators in developing mouse lens.

Dual function of Yap in the regulation of lens progenitor cells and cellular polarity

Hippo-Yap signaling has been implicated in organ size determination via its regulation of cell proliferation, growth and apoptosis (Pan, 2007). The vertebrate lens comprises only two major cell types, lens progenitors and differentiated fiber cells, thereby providing a relatively simple system for studying size-controlling mechanisms. In order to investigate the role of Hippo-Yap signaling in lens size regulation, we conditionally ablated Yap in the developing mouse lens. Lens progenitor-specific deletion of Yap led to near obliteration of the lens primarily due to hypocellularity in the lens epithelium (LE) and accompanying lens fiber (LF) defects. A significantly reduced LE progenitor pool resulted mainly from failed self-renewal and increased apoptosis. Additionally, Yap-deficient lens progenitor cells precociously exited the cell cycle and expressed the LF marker, β-Crystallin. The mutant progenitor cells also exhibited multiple cellular and subcellular alterations including cell and nuclear shape change, organellar polarity disruption, and disorganized apical polarity complex and junction proteins such as Crumbs, Pals1, Par3 and ZO-1. Yap-deficient LF cells failed to anchor to the overlying LE layer, impairing their normal elongation and packaging. Furthermore, our localization study results suggest that, in the developing LE, Yap participates in the cell context-dependent transition from the proliferative to differentiation-competent state by integrating cell density information. Taken together, our results shed new light on Yap's indispensable and novel organizing role in mammalian organ size control by coordinating multiple events including cell proliferation, differentiation, and polarity.

Efficient Generation of Lens Progenitor Cells from Cataract Patient–Specific Induced Pluripotent Stem Cells

The development of a technique to induce the transformation of somatic cells to a pluripotent state via the ectopic expression of defined transcription factors was a transformational event in the field of regenerative medicine. The development of this technique also impacted ophthalmology, as patient-specific induced pluripotent stemcells (iPSCs) may be useful resources for some ophthalmological diseases. The lens is a key refractive element in the eye that focuses images of the visual world onto the retina. To establish a new model for drug screening to treat lens diseases and investigating lens aging and development, we examined whether human lens epithelial cells (HLECs) could be induced into iPSCs and if lens-specific differentiation of these cells could be achieved under defined chemical conditions. We first efficiently reprogrammed HLECs from age-related cataract patients to iPSCs with OCT-4, SOX-2, and KLF-4. The resulting HLEC-derived iPS (HLE-iPS) colonies were indistinguishable from human ES cells with respect to morphology, gene expression, pluripotent marker expression and their ability to generate all embryonic germ-cell layers. Next, we performed a 3-step induction procedure: HLE-iPS cells were differentiated into large numbers of lens progenitor-like cells with defined factors (Noggin, BMP and FGF2), and we determined that these cells expressed lens-specific markers (PAX6, SOX2, SIX3, CRYAB, CRYAA, BFSP1, and MIP). In addition, HLE-iPS-derived lens cells exhibited reduced expression of epithelial mesenchymal transition (EMT) markers compared with human embryonic stem cells (hESCs) and fibroblast-derived iPSCs. Our study describes a highly efficient procedure for generating lens progenitor cells from cataract patient HLEC-derived iPSCs. These patient-derived pluripotent cells provide a valuable model for studying the developmental and molecular biological mechanisms that underlie cell determination in lens development and cataract pathophysiology.

Efficient generation of lens progenitor cells and lentoid bodies from human embryonic stem cells in chemically defined conditions

The eye lens is an encapsulated avascular organ whose function is to focus light on the retina. Lens comprises a single progenitor cell lineage in multiple states of differentiation. Disruption of lens function leading to protein aggregation and opacity results in age-onset cataract. Cataract is a complex disease involving genetic and environmental factors. Here, we report the development of a new 3-stage system that differentiates human embryonic stem cells (hESCs) into large quantities of lens progenitor-like cells and differentiated 3-dimensional lentoid bodies. Inhibition of BMP signaling by noggin triggered differentiation of hESCs toward neuroectoderm. Subsequent reactivation of BMP and activation of FGF signaling stimulated formation of lens progenitor cells marked by the expression of PAX6 and alpha-crystallins. The formation of lentoid bodies was most efficient in the presence of FGF2 and Wnt-3a, yielding approximately 1000 lentoid bodies/30-mm well. Lentoid bodies expressed and accumulated lens-specific markers including alphaA-, alphaB-, beta-, and gamma-crystallins, filensin, CP49, and MIP/aquaporin 0. Collectively, these studies identify a novel procedure to generate lens cells from hESCs that can be applied for studies of lens differentiation and cataractogenesis using induced pluripotent stem (iPS) cells derived from various cataract patients.

Genetic and epigenetic mechanisms of gene regulation during lens development

Recent studies demonstrated a number of links between chromatin structure, gene expression, extracellular signaling and cellular differentiation during lens development. Lens progenitor cells originate from a pool of common progenitor cells, the pre-placodal region (PPR) which is formed from a combination of extracellular signaling between the neural plate, naïve ectoderm and mesendoderm. A specific commitment to the lens program over alternate choices such as the formation of olfactory epithelium or the anterior pituitary is manifested by the formation of a thickened surface ectoderm, the lens placode. Mouse lens progenitor cells are characterized by the expression of a complement of lens lineage-specific transcription factors including Pax6, Six3 and Sox2, controlled by FGF and BMP signaling, followed later by c-Maf, Mab21like1, Prox1 and FoxE3. Proliferation of lens progenitors together with their morphogenetic movements results in the formation of the lens vesicle. This transient structure, comprised of lens precursor cells, is polarized with its anterior cells retaining their epithelial morphology and proliferative capacity, whereas the posterior lens precursor cells initiate terminal differentiation forming the primary lens fibers. Lens differentiation is marked by expression and accumulation of crystallins and other structural proteins. The transcriptional control of crystallin genes is characterized by the reiterative use of transcription factors required for the establishment of lens precursors in combination with more ubiquitously expressed factors (e.g. AP-1, AP-2alpha, CREB and USF) and recruitment of histone acetyltransferases (HATs) CBP and p300, and chromatin remodeling complexes SWI/SNF and ISWI. These studies have poised the study of lens development at the forefront of efforts to understand the connections between development, cell signaling, gene transcription and chromatin remodeling.

Tissue-specific Regulation of the Mouse αA-crystallin Gene in Lens via Recruitment of Pax6 and c-Maf to its Promoter

Pax6 is a lineage-restricted DNA-binding transcription factor regulating the formation of mammalian organs including brain, eye and pancreas. Pax6 plays key roles during the initial formation of lens lineage, proliferation of lens progenitor and precursor cells and their terminal differentiation. In addition to Pax6, lens fiber cell differentiation is regulated by c-Maf, Prox1 and Sox1. Crystallins are essential lens structural proteins required for light refraction and transparency. Mouse alphaA-crystallin represents about 17% of all crystallins at the protein level and ranks as one of the most abundant tissue-specific proteins. Lens-specific expression of this gene is regulated at the level of transcription. A promoter fragment of -88 to +46 is capable of driving lens-specific expression in transgenic mouse. Here we provide data suggesting that this lens-specific promoter fragment is comprised of multiple Pax6 and Maf-binding sites. Site-directed mutagenesis of regions within these sites resulted in partially or completely reduced promoter activities in lens cells. Co-transfections using Pax6 and c-Maf alone revealed moderate and strong activations of this promoter, respectively. In contrast to synergistic activation of alphaB-crystallin by Pax6 and c-Maf, Pax6 has a neutral effect on c-Maf-mediated alphaA-crystallin promoter activation. Chromatin immunoprecipitations established in vivo interactions of Pax6 and c-Maf with the alphaA-crystallin promoter in lens cells. Collectively, the present data support a molecular model in which tissue-specific expression of alphaA-crystallin is regulated by recruitment of Pax6 and c-Maf, two proteins regulating multiple processes of lens differentiation, to its promoter. In addition, the data suggest a molecular model of temporal and spatial regulation of alphaB, alphaA and gamma-crystallin genes in mouse embryonic lens by using variants of the Pax6/Maf regulatory module.

Regulation of gene expression by Pax6 in ocular cells: a case of tissue-preferred expression of crystallins in lens

Lens development is an excellent model for genetic and biochemical studies of embryonic induction, cell cycle regulation, cellular differentiation and signal transduction. Differentiation of lens is characterized by lens-preferred expression and accumulation of water-soluble proteins, crystallins. Crystallins are required for light transparency, refraction and maintenance of lens integrity. Here, we review mechanisms of lens-preferred expression of crystallin genes by employing synergism between developmentally regulated DNA-binding transcription factors: Pax6, c-Maf, MafA/L-Maf, MafB, NRL, Sox2, Sox1, RARbeta/RXRbeta, RORalpha, Prox1, Six3, gammaFBP-B and HSF2. These factors are differentially expressed in lens precursor cells, lens epithelium and primary and secondary lens fibers. They exert their function in combination with ubiquitously expressed factors (e.g. AP-1, CREB, pRb, TFIID and USF) and co-activators/chromatin remodeling proteins (e.g. ASC-2 and CBP/p300). A special function belongs to Pax6, a paired domain and homeodomain-containing protein, which is essential for lens formation. Pax6 is expressed in lens progenitor cells before the onset of crystallin expression and it serves as an important regulatory factor required for expression of c-Maf, MafA/L-Maf, Six3, Prox1 and retinoic acid signaling both in lens precursor cells and the developing lens. The roles of these factors are illustrated by promoter studies of mouse alphaA-, alphaB-, gammaF- and guinea pig zeta-crystallins. Pax6 forms functional complexes with a number of transcription factors including the retinoblastoma protein, pRb, MafA, Mitf and Sox2. We present novel data showing that pRb antagonizes Pax6-mediated activation of the alphaA-crystallin promoter likely by inhibiting binding of Pax6 to DNA.