Systems biology discoveries using non-human primate pluripotent stem and germ cells: novel gene and genomic imprinting interactions as well as unique expression patterns

The study of pluripotent stem cells has generated much interest in both biology and medicine. Understanding the fundamentals of biological decisions, including what permits a cell to maintain pluripotency, that is, its ability to self-renew and thereby remain immortal, or to differentiate into multiple types of cells, is of profound importance. For clinical applications, pluripotent cells, including both embryonic stem cells and adult stem cells, have been proposed for cell replacement therapy for a number of human diseases and disorders, including Alzheimer's, Parkinson's, spinal cord injury and diabetes. One challenge in their usage for such therapies is understanding the mechanisms that allow the maintenance of pluripotency and controlling the specific differentiation into required functional target cells. Because of regulatory restrictions and biological feasibilities, there are many crucial investigations that are just impossible to perform using pluripotent stem cells (PSCs) from humans (for example, direct comparisons among panels of inbred embryonic stem cells from prime embryos obtained from pedigreed and fertile donors; genomic analysis of parent versus progeny PSCs and their identical differentiated tissues; intraspecific chimera analyses for pluripotency testing; and so on). However, PSCs from nonhuman primates are being investigated to bridge these knowledge gaps between discoveries in mice and vital information necessary for appropriate clinical evaluations. In this review, we consider the mRNAs and novel genes with unique expression and imprinting patterns that were discovered using systems biology approaches with primate pluripotent stem and germ cells

Embryonic and induced pluripotent stem cells: understanding, creating, and exploiting the nano-niche for regenerative medicine.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the capacity to differentiate into any specialized cell type of the human body, and therefore, ESC/iPSC-derived cell types offer great potential for regenerative medicine. However, key to realizing this potential requires a strong understanding of stem cell biology, techniques to maintain stem cells, and strategies to manipulate cells to efficiently direct cell differentiation toward a desired cell type. As nanoscale science and engineering continues to produce novel nanotechnology platforms, which inform, infiltrate, and impinge on many aspects of everyday life, it is no surprise that stem cell research is turning toward developments in nanotechnology to answer research questions and to overcome obstacles in regenerative medicine. Here we discuss recent advances in ESC and iPSC manipulation using nanomaterials and highlight future challenges within this area of research

Rv2074 is a novel F420H2-dependent biliverdin reductase in Mycobacterium tuberculosis

Bilirubin is a potent antioxidant that is produced from the reduction of the heme degradation product biliverdin. In mammalian cells and Cyanobacteria, NADH/NADPH-dependent biliverdin reductases (BVRs) of the Rossmann-fold have been shown to catalyze this reaction. Here, we describe the characterization of Rv2074 from Mycobacterium tuberculosis, which belongs to a structurally and mechanistically distinct family of F420H2-dependent BVRs (F-BVRs) that are exclusively found in Actinobacteria. We have solved the crystal structure of Rv2074 bound to its cofactor, F-420, and used this alongside molecular dynamics simulations, site-directed mutagenesis and NMR spectroscopy to elucidate its catalytic mechanism. The production of bilirubin by Rv2074 could exploit the anti-oxidative properties of bilirubin and contribute to the range of immuno-evasive mechanisms that have evolved in M. tuberculosis to allow persistent infection

Genome-Wide Analysis Reveals the Unique Stem Cell Identity of Human Amniocytes

<div><p>Human amniotic fluid contains cells that potentially have important stem cell characteristics, yet the programs controlling their developmental potency are unclear. Here, we provide evidence that amniocytes derived from multiple patients are marked by heterogeneity and variability in expression levels of pluripotency markers. Clonal analysis from multiple patients indicates that amniocytes have large pools of self-renewing cells that have an inherent property to give rise to a distinct amniocyte phenotype with a heterogeneity of pluripotent markers. Significant to their therapeutic potential, genome-wide profiles are distinct at different gestational ages and times in culture, but do not differ between genders. Based on hierarchical clustering and differential expression analyses of the entire transcriptome, amniocytes express canonical regulators associated with pluripotency and stem cell repression. Their profiles are distinct from human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), and newborn foreskin fibroblasts. Amniocytes have a complex molecular signature, coexpressing trophoblastic, ectodermal, mesodermal, and endodermal cell-type-specific regulators. In contrast to the current view of the ground state of stem cells, ESCs and iPSCs also express high levels of a wide range of cell-type-specific regulators. The coexpression of multilineage differentiation markers combined with the strong expression of a subset of ES cell repressors in amniocytes suggests that these cells have a distinct phenotype that is unlike any other known cell-type or lineage.</p> </div

Core stem cell markers are variably expressed, depending on GA and time in culture.

<p>(A–B) Dot plots of (A) RNA-seq and (B) qPCR results reveal significant variability in transcript levels for key genes known to be required for establishment and maintenance of pluripotency. (A) RNA-seq measurements for 37 datasets are presented as variance-stabilized read counts. The string of horizontal dots at the lower detection limit for genes Oct4, Sox2 and cKit indicates samples that had no reads in those genes. (B) qPCR units for 17 datasets are presented as normalized Cp values (Cp value of target gene minus Cp value of reference gene Gapdh). (C–E) Hierarchical clustering of C) qPCR results for eight genes; (D) qPCR results for 17 patients; and (E) RNA-seq results for 37 datasets using measurements of 250 stem cell markers. Clustering similarities in transcript levels were calculated by Pearson's r² correlation coefficient as a measure of dendrogramatic distance and bootstrapping values were calculated from 10,000 random replications. (E) Culture time point T1 was taken on average from 1.3 days (0–8 range), T2 was taken on average from 15.2 days (13–22 range), and T3 was taken on average from 28.0 days (24–36 range).</p

Amniocyte genome-wide transcriptional profile varies depending on GA and time in culture.

<p>(A) Hierarchical clustering of 11 independent amniocyte isolates at different times in culture (37 RNA-seq datasets; 24,609 ensemble genes per dataset). Four major clusters correlate with gestational age and culture time since amniocentesis. (B–D) Volcano plots display the results of differential expression analyses using these three variables (n = 49,235 Ensembl genes). Genes plotted above the red line have adjusted <i>p</i>-values<0.05, and genes plotted outside of the green lines are >2-fold differentially expressed. Comparing (B) early vs. late gestational age revealed the most differentially expressed genes (n = 2,197), followed by (C) time in culture (n = 1039) and (D) gender (n = 208). (A, C) Culture time point T1 was taken on average from 1.3 days (0–8 range), T2 was taken on average from 15.2 days (13–22 range), and T3 was taken on average from 28.0 days (24–36 range).</p