A number of potential improvements of the differentiation procedure described above should be considered and empirically tested. In principle, the system can be improved through testing of different 3-D gels and extracellular matrix proteins that are found in the lens capsule, growth of lentoid bodies on lens capsule, specific activators and inhibitors of differentiation, chemical libraries, 3-D scaffolds to generate a gradient of growth factor(s), and any combination of these procedures. In addition, genetically engineered human and mouse ES cells that carry fluorescent reporter genes, under the control of lens regulatory elements, can be used to aid in the analysis of the differentiation process.

There are at least three commercially available 3-D systems: ExtraCel hydrogel (Glycosan Biosystems), HyStem-C Cell Culture Scaffold kit (Sigma), and Cultrex 3-D Culture Matrix Extract (R&D Systems). Each system allows for the incorporation of variable amounts/ratios of laminin, collagen IV, entactin/nidogen, perlecan, fibronectin, collagen XVIII and sparc/osteonectin, extracellular matrix (ECM) proteins found in the lens [21, 117].

A number of drugs have been shown to promote cellular differentiation with some of the tested in lens cell cultures. These include specific inhibitors of DNA methylation such as 5-azacytidine and 5-deazacytidine [12, 49, 94], inhibitors of histone methyltransferases (cytarabine and decitabine [84]), inhibitors of histone deacetylases (valproic acid and sodium butyrate [22, 24, 34, 74, 78]), and inhibitors of cyclin-dependent kinases (olomoucine and roscovitine [70, 73, 89, 115]). Of particular interest are the rho-kinase (ROCK) inhibitors, Y27632 and PP-1, as the PP-1 drug has been successfully used to promote cell cycle withdrawal and commitment of lens cells to differentiate [113, 114].

Considering the specific roles of Notch and Wnt signaling pathways for lens fiber cell differentiation, and the role of Wnt signaling in the differentiation of lens epithelial cells described above (“Mammalian Lens Development and Lessons for a Rational Design of ES Cell-Based Differentiation Systems”), stimulation of ES cell differentiation may be considered. Recombinant Notch ligands, Jagged 1 and 2, can be added transiently during the thirds stage of the differentiation procedure. Concerning Wnt signaling, the situation is more complex as multiple Wnts and their receptors, the frizzled proteins, can regulate lens development both in the epithelial and fiber cell compartments. Nevertheless, inclusion of Wnt3a improved the quantitative parameters of the current procedure of lentoid body formation [122].

Ongoing experiments in the laboratory are aimed to improve differentiation of lentoid bodies using a combinatorial approach as outlined above. The procedure can be improved via genetically engineered ES cells [5] that carry fluorescent markers under the control of lens regulatory regions from genes known to control different stages of the lens lineage formation, cell cycle exit, and terminal differentiation. For this purpose, the EGFP, or enhanced green fluorescent protein marker can be inserted into a specific BAC clone with PAX6 (early marker), HSF4 (late marker), β-/γ-crystallins, DNase IIβ, MIP/aquaporin 0, paralemmin, and other genes expressed in terminally differentiated lens fiber cells as established for similar differentiation systems [83, 110].

For the first time in human lens research, we are about to establish a general strategy to model human lens development and diseases with an unlimited supply of lens cells that originate from genetically and phenotypically defined human source(s). In addition, these materials can be shared between multiple laboratories to accelerate research. The pioneering work of S. Yamanaka at the Institute for Frontier Medical Sciences, Kyoto University, Japan, to establish the reprogramming procedure using skin fibroblasts provided proof-of-principle that the iPS cell can be established from somatic terminally differentiated cells, and these iPS cells behaved like authentic ES cells in a series of functional tests [108, 109]. A large follow-up effort in a number of laboratories worldwide resulted in expansion of the reprogramming procedures and cell types suitable for these manipulations. The majority of currently existing procedures are summarized in Table 4.1. It has been shown recently that iPS cells can be produced from a cataract patient using lens epithelial cells as the starting material [85]. Most importantly, these iPS cells were differentiated into lentoid bodies using the procedure described here (see Fig. 4.1) [85]. Nevertheless, whether iPS cells, generated through other reprogramming protocols and cell types, are capable of producing lentoid bodies similar to those generated from human ES cells, remains to be formally proven.

One of the most pressing objectives of medical research today is to develop novel approaches to model formation of human organs, tissues and diseases. Use of human ES and iPS cells differentiated into individual tissues provides the highest possible promise to achieve this objective as it is now possible to understand the contribution of genetic and environmental factors in various diseases including those related to aging such as age-onset cataract.

Thus, the present cell culture system can be used to modulate these common signaling pathways during lens formation [62] via siRNA technology and through the use of small drug molecules, inhibitors of FGF and BMP signaling (e.g., SB431542—an inhibitor of the Alk1 receptor, SU5402—an inhibitor of FGFR and U0126—an inhibitor of MEK) to study formation of lens lineage and formation of alternate cell fates that originate from the common pre-placodal region [105].

It is now possible to produce iPS cells from human patients that carry heterozygous mutations in regulatory genes such as PAX6, FOXE3, MAF, HSF4, PITX3, and others and to identify those genes that are not properly regulated during early stages of lens development. In contrast, studies of cataractogenesis using the system of ES/iPS cells seems be premature until procedures to generate enucleated lentoid bodies with distinct epithelium/fiber cell compartments are established. The long-term benefits of the research to model human cataract using iPS cells should stimulate our efforts to achieve this challenging goal.