Background: Nanog is a core factor that is required for the maintenance of embryonic stem (ES) cell pluripotency and self-renewal. Results: Alternative splicing results in Nanog proteins with different capacities for maintaining the undifferentiated ES cell state. Conclusion:The Nanog N-terminal domain is regulated by post-transcriptional modification. Significance: Nanog protein variants either fully support the maintenance of pluripotency or facilitate differentiation.

Gap junctions are intercellular aqueous channels composed of transmembrane proteins called connexins (1). The gap junction-dependent or independent functions of connexins are important in the regulation of several cellular processes, including growth, proliferation, differentiation, protection, and cell death (2, 3). Distinct expression patterns and highly dynamic turnover rates are the key components that regulate tissue-specific activity of different connexin molecules. The expression and turnover of connexins are fine-tuned balances of several processes such as gene expression, mRNA stability, protein synthesis and transport, and degradation (4, 5). Connexin turnover and function is also modulated by several intrinsic and extrinsic factors, including intra-and extracellular pH, various phosphorylation events, cellular status, and chemical reagents such as the tumor-promoting phorbol ester, TPA 3 (6 -10). One tissue that relies on the gap junction-mediated communication for normal function and growth is the vertebrate lens. The lens is naturally avascular and, therefore, gap junctionmediated functions play a major role in maintaining proper homeostasis and transparency of the lens. The vertebrate lens endogenously expresses three connexin proteins required for proper lens development and function, connexin43 (Cx43), connexin46 (Cx46), and connexin50 (Cx50) (11)(12)(13)(14)(15)(16)(17). These connexins show differential spatial distributions that are related to their specific functions at different regions of the lens.

Retinal cells which become ischemic will pass apoptotic signal to adjacent cells, resulting in the spread of damage. This occurs through open gap junctions. A class of novel drugs, based on primaquine (PQ), was tested for binding to connexin 43 using simulated docking studies. A novel drug has been synthesized and tested for inhibition of gap junction activity using R28 neuro-retinal cells in culture. Four drugs were initially compared to mefloquine, a known gap junction inhibitor. The drug with optimal inhibitory activity, PQ1, was tested for inhibition and was found to inhibit dye transfer by 70% at 10 μM. Retinal ischemia was produced in R28 cells using cobalt chloride as a chemical agent. This resulted in activation of caspase-3 which was prevented by the PQ1 gap junction inhibitor. Results demonstrate that novel gap junction inhibitors may provide a means to prevent retinal damage during ischemia.

PKCγ has a major role in the regulation of gap junction expression and coupling in the normal lens.

The development of a hematopoietic reporter is crucial for determining the fate of lineages derived from cell-based therapies. A marking system will enable safer embryonic stem (ES) and induced pluripotent stem (iPS) cell-based derivation of blood lineages, and facilitate the development of efficient cellular reprogramming strategies based on direct fibroblast conversion. Here we report that the protein tyrosine phosphatase CD45 is an ideal candidate gene on which to base a hematopoietic reporter. CD45 regulatory elements were discovered by analyzing transcription factor chromatin occupancy (ChIP-seq) and promoter nuclease sensitivity (DNase-seq) to identify minimally sufficient sequences required for expression. After cloning the CD45 regulatory elements into an attenuated lentiviral backbone, we found that two transcriptional initiation regions were essential for high-level expression. Expressing CD45 promoters containing these regions and tethered to GFP in a primary B cell differentiation assay and a transplantation model resulted in high levels of GFP in lymphoid, myeloid and nucleated erythroid cells in mouse and human blood cell lineages. Moreover, high GFP levels remained five months following secondary transplantation, indicating persistence of the reporter. No CD45 driven GFP expression is observed following fibroblast or ES cell transduction. The GFP reporter is seen only after ES cells differentiate into hematopoietic cell progenitors and lineages, suggesting that this hematopoietic reporter system could be useful in validating potential autologous blood cell therapies.