Investigating the functionality of an OCT4-short response element in human induced pluripotent stem cells.

Pluripotent stem cells offer great therapeutic promise for personalized treatment platforms for numerous injuries, disorders, and diseases. Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene maintaining pluripotency and self-renewal of mammalian cells. With site-specific integration for gene correction in cellular therapeutics, use of the OCT4 promoter may have advantages when expressing a suicide gene if pluripotency remains. However, the human OCT4 promoter region is 4 kb in size, limiting the capacity of therapeutic genes and other regulatory components for viral vectors, and decreasing the efficiency of homologous recombination. The purpose of this investigation was to characterize the functionality of a novel 967bp OCT4-short response element during pluripotency and to examine the OCT4 titer-dependent response during differentiation to human derivatives not expressing OCT4. Our findings demonstrate that the OCT4-short response element is active in pluripotency and this activity is in high correlation with transgene expression in vitro, and the OCT4-short response element is inactivated when pluripotent cells differentiate. These studies demonstrate that this shortened OCT4 regulatory element is functional and may be useful as part of an optimized safety component in a site-specific gene transferring system that could be used as an efficient and clinically applicable safety platform for gene transfer in cellular therapeutics

Modulation of Cell Adhesion and Migration by the Histone Methyltransferase Subunit mDpy-30 and Its Interacting Proteins

We have previously shown that a subset of mDpy-30, an accessory subunit of the nuclear histone H3 lysine 4 methyltransferase (H3K4MT) complex, also localizes at the trans-Golgi network (TGN), where its recruitment is mediated by the TGN-localized ARF guanine nucleotide exchange factor (ArfGEF) BIG1. Depletion of mDpy-30 inhibits the endosome-to-TGN transport of internalized CIMPR receptors and concurrently promotes their accumulation at the cell protrusion. These observations suggest mDpy-30 may play a novel role at the crossroads of endosomal trafficking, nuclear transcription and adhesion/migration. Here we provide novel mechanistic and functional insight into this association. First, we demonstrate a direct interaction between mDpy-30 and BIG1 and locate the binding region in the N-terminus of BIG1. Second, we provide evidence that the depletion or overexpression of mDpy-30 enhances or inhibits cellular adhesion/migration of glioma cells in vitro, respectively. A similar increase in cell adhesion/migration is observed in cells with reduced levels of BIG1 or other H3K4MT subunits. Third, knockdown of mDpy-30, BIG1, or the RbBP5 H3K4MT subunit increases the targeting of β1 integrin to cell protrusions, and suppression of H3K4MT activity by depleting mDpy-30 or RbBP5 leads to increased protein and mRNA levels of β1 integrin. Moreover, stimulation of cell adhesion/migration via mDpy-30 knockdown is abolished after treating cells with a function-blocking antibody to β1 integrin. Taken together, these data indicate that mDpy-30 and its interacting proteins function as a novel class of cellular adhesion/migration modulators partially by affecting the subcellular distribution of endosomal compartments as well as the expression of key adhesion/migration proteins such as β1 integrin

Ancient Lowland Maya neighborhoods: Average Nearest Neighbor analysis and kernel density models, environments, and urban scale

Many humans live in large, complex political centers, composed of multi-scalar communities including neighborhoods and districts. Both today and in the past, neighborhoods form a fundamental part of cities and are defined by their spatial, architectural, and material elements. Neighborhoods existed in ancient centers of various scales, and multiple methods have been employed to identify ancient neighborhoods in archaeological contexts. However, the use of different methods for neighborhood identification within the same spatiotemporal setting results in challenges for comparisons within and between ancient societies. Here, we focus on using a single method—combining Average Nearest Neighbor (ANN) and Kernel Density (KD) analyses of household groups—to identify potential neighborhoods based on clusters of households at 23 ancient centers across the Maya Lowlands. While a one-size-fits all model does not work for neighborhood identification everywhere, the ANN/KD method provides quantifiable data on the clustering of ancient households, which can be linked to environmental zones and urban scale. We found that centers in river valleys exhibited greater household clustering compared to centers in upland and escarpment environments. Settlement patterns on flat plains were more dispersed, with little discrete spatial clustering of households. Furthermore, we categorized the ancient Maya centers into discrete urban scales, finding that larger centers had greater variation in household spacing compared to medium-sized and smaller centers. Many larger political centers possess heterogeneity in household clustering between their civic-ceremonial cores, immediate hinterlands, and far peripheries. Smaller centers exhibit greater household clustering compared to larger ones. This paper quantitatively assesses household clustering among nearly two dozen centers across the Maya Lowlands, linking environment and urban scale to settlement patterns. The findings are applicable to ancient societies and modern cities alike; understanding how humans form multi-scalar social groupings, such as neighborhoods, is fundamental to human experience and social organization

BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells

Abstract Introduction The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. Methods We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. Results We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. Conclusions For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics

Putative Immunogenicity Expression Profiling Using Human Pluripotent Stem Cells and Derivatives

Autologous human induced pluripotent stem cells (hiPSCs) should allow cellular therapeutics without an associated immune response. This concept has been controversial since the original report that syngeneic mouse iPSCs elicited an immune response after transplantation. However, an investigative analysis of any potential acute immune responses in hiPSCs and their derivatives has yet to be conducted. In the present study, we used correlative gene expression analysis of two putative mouse "immunogenicity" genes, ZG16 and HORMAD1, to assay their human homologous expression levels in human pluripotent stem cells and their derivatives. We found that ZG16 expression is heterogeneous across multiple human embryonic stem cell and hiPSC-derived cell types. Additionally, ectopic expression of ZG16 in antigen-presenting cells is insufficient to trigger a detectable response in a peripheral blood mononuclear cell coculture assay. Neither of the previous immunogenicity-associated genes in the mouse currently appears to be relevant in a human context

Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status

Abstract Introduction The reprogramming of a patient’s somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known. Methods In this study, we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions. Results We found that a STEMCCA-derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status. Conclusion For the first time, these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics