Differential Transgene Silencing of Myeloid-Specific Promoters in the AAVS1 Safe Harbor Locus of Induced Pluripotent Stem Cell-Derived Myeloid Cells

Targeted integration into a genomic safe harbor, such as the AAVS1 locus on chromosome 19, promises predictable transgene expression and reduces the risk of insertional mutagenesis in the host genome. The application of gamma-retroviral LTR-driven vectors, which semi-randomly integrate into the genome, has previously caused severe adverse events in some clinical studies due to transactivation of neighboring proto-oncogenes. Consequently, the site-specific integration of a therapeutic transgene into a genomic safe harbor locus would allow stable genetic correction with a reduced risk of insertional mutagenesis. However, recent studies revealed that transgene silencing, especially in case of weaker cell type-specific promoters, can occur in the AAVS1 locus of human pluripotent stem cells (PSC) and can impede transgene expression during differentiation. In this study, we aimed to correct p47phox-deficiency, which is the second most common cause of chronic granulomatous disease, by insertion of a therapeutic p47phox transgene into the AAVS1 locus of human induced PSC (iPSC) using CRISPR-Cas9. We analyzed transgene expression and functional correction from three different myeloid-specific promoters (miR223, CatG/cFes and MRP8). Upon myeloid differentiation of corrected iPSC clones, we observed that the miR223 and CatG/cFes promoter achieved therapeutic-relevant levels of p47phox expression and NADPH oxidase activity, whereas the MRP8 promoter was less efficient. Analysis of the different promoters revealed high CpG methylation of the MRP8 promoter in differentiated cells, which correlated with the transgene expression data. In summary, we identified the miR223 and CatG/cFes promoters as cell type-specific promoters that allow stable transgene expression in the AAVS1 locus of iPSC-derived myeloid cells. Our findings further indicate that promoter silencing can occur in the AAVS1 safe harbor locus in differentiated hematopoietic cells and that a comparison of different promoters is necessary to achieve optimal transgene expression for therapeutic application of iPSC-derived cells

Human Amniocytes Are Receptive to Chemically Induced Reprogramming to Pluripotency

Restoring pluripotency using chemical compounds alone would be a major step forward in developing clinical-grade pluripotent stem cells, but this has not yet been reported in human cells. We previously demonstrated that VPA_ AFS cells, human amniocytes cultivated with valproic acid (VPA) acquired functional pluripotency while remaining distinct from human embryonic stem cells (hESCs), questioning the relationship between the modulation of cell fate and molecular regulation of the pluripotency network. Here, we used single-cell analysis and functional assays to reveal that VPA treatment resulted in a homogeneous population of self-renewing non-transformed cells that fulfill the hallmarks of pluripotency, i.e., a short G1 phase, a dependence on glycolytic metabolism, expression of epigenetic modifications on histones 3 and 4, and reactivation of endogenous OCT4 and downstream targets at a lower level than that observed in hESCs. Mechanistic insights into the process of VPA-induced reprogramming revealed that it was dependent on OCT4 promoter activation, which was achieved independently of the PI3K (phosphatidylinositol 3-kinase)/ AKT/ mTOR (mammalian target of rapamycin) pathway or GSK3 beta inhibition but was concomitant with the presence of acetylated histones H3K9 and H3K56, which promote pluripotency. Our data identify, for the first time, the pluripotent transcriptional and molecular signature and metabolic status of human chemically induced pluripotent stem cells

Cellular Hydraulics Suggests a Poroelastic Cytoplasm Rheology

The cytoplasm represents the largest part of the cell by volume and hence its rheology sets the rate at which cellular shape change can occur. Recent experimental evidence suggests that cytoplasmic rheology can be described using a poroelastic formulation in which the cytoplasm is considered a biphasic material constituted of a porous elastic solid meshwork (cytoskeleton, organelles, macromolecules) bathed in an interstitial fluid (cytosol). In this picture, the rate of cellular deformation is limited by the rate at which intracellular water can redistribute within the cytoplasm. Though this is a conceptually attractive model, direct supporting evidence has been lacking. Here we present such evidence and directly validate this concept to explain cellular rheology at physiologically relevant time-scales using microindentation tests in conjunction with mechanical, chemical and genetic treatments. Our results show that water redistribution through the solid phase of cytoplasm (cytoskeleton and crowders) plays a fundamental role in setting cellular rheology.Engineering and Applied Science

A Framework for Understanding Modifications to Measures for Diverse Populations

Research on health disparities and determinants of health disparities among ethnic minorities and vulnerable older populations necessitates use of self-report measures. Most established instruments were developed on mainstream populations and may need adaptation for research with diverse populations. Although information is increasingly available on various problems using these measures in diverse groups, there is little guidance on how to modify the measures. We provide a framework of issues to consider when modifying measures for diverse populations

A Process and Outcome Evaluation of a Shelter for Homeless Young Women

To evaluate the processes and outcomes of a short-term shelter, both quantitative and qualitative data were gathered via participant observation, focus group interviews with shelter staff and residents, and individual interviews with a sample of 40 young women who had been homeless prior to using the shelter. The process evaluation showed that the shelter staff strived to utilize an empowerment philosophy in their relationships with residents, but that there were many challenges to implementing this philosophy. The outcome evaluation showed that, at a 3-month follow-up, the participants reported significant improvements in housing, income, independence, and life satisfaction, but most continued to experience poverty and a number of other difficulties. The results were discussed in terms of the implications for future research and the value and limitations of shelters for dealing with homeless youth. The need for more sustained and comprehensive program interventions and supportive social policies was underscored

Correction of both immunodeficiency and hypoparathyroidism by thymus transplantation in complete DiGeorge Syndrome

Combined immune deficiency due to athymia in patients with complete DiGeorge syndrome can be corrected by allogeneic thymus transplantation. Hypoparathyroidism is a frequent concomitant clinical problem in these patients, which persists after thymus transplantation. Cotransplantation of allogeneic thymus and parental parathyroid tissue has been attempted but does not achieve durable correction of the patients' hypoparathyroidism due to parathyroid graft rejection. Surprisingly, we observed correction of hypoparathyroidism in one patient after thymus transplantation. Immunohistochemical analysis and fluorescence in situ hybridization confirmed the presence of allogeneic parathyroid tissue in the patient's thymus transplant biopsy. Despite a lack of HLA‐matching between thymus donor and recipient, the reconstituted immune system displays tolerance toward the thymus donor. Therefore we expect this patient's hypoparathyroidism to be permanently cured. It is recognised that ectopic parathyroid tissue is not infrequently found in the thymus. If such thymuses could be identified, we propose that their use would offer a compelling approach to achieving lasting correction of both immunodeficiency and hypoparathyroidism