Training the next generation of pluripotent stem cell researchers

Human pluripotent stem cells (PSCs) have the unique properties of being able to proliferate indefinitely in their undifferentiated state and of being able to differentiate into any somatic cell type. These cells are thus posited to be extremely useful for furthering our understanding of both normal and abnormal human development, providing a human cell preparation that can be used to screen for new reagents or therapeutic agents, and generating large numbers of differentiated cells that can be used for transplantation purposes. PSCs in culture have a specific morphology and they express characteristic surface antigens and nuclear transcription factors; thus, PSC culture is very specific and requires a core skill set for successful propagation of these unique cells. Specialized PSC training courses have been extremely valuable in seeding the scientific community with researchers that possess this skill set

Ataxin-1 Fusion Partners Alter PolyQ Lethality and Aggregation

Intranuclear inclusion bodies (IBs) are the histopathologic markers of multiple protein folding diseases. IB formation has been extensively studied using fluorescent fusion products of pathogenic polyglutamine (polyQ) expressing proteins. These studies have been informative in determining the cellular targets of expanded polyQ protein as well as the methods by which cells rid themselves of IBs. The experimental thrust has been to intervene in the process of polyQ aggregation in an attempt to alleviate cytotoxicity. However new data argues against the notion that polyQ aggregation and cytotoxicity are inextricably linked processes. We reasoned that changing the protein context of a disease causing polyQ protein could accelerate its precipitation as an IB, potentially reducing its cytotoxicity. Our experimental strategy simply exploited the fact that conjoined proteins influence each others folding and aggregation properties. We fused a full-length pathogenic ataxin-1 construct to fluorescent tags (GFP and DsRed1-E5) that exist at different oligomeric states. The spectral properties of the DsRed1-E5-ataxin-1 transfectants had the additional advantage of allowing us to correlate fluorochrome maturation with cytotoxicity. Each fusion protein expressed a distinct cytotoxicity and IB morphology. Flow cytometric analyses of transfectants expressing the greatest fluorescent signals revealed that the DsRed1-E5-ataxin-1 fusion was more toxic than GFP fused ataxin-1 (31.8±4.5% cell death versus 12.85±3%), although co-transfection with the GFP fusion inhibited maturation of the DsRed1-E5 fluorochrome and diminished the toxicity of the DsRed1-E5-ataxin-1 fusion. These data show that polyQ driven aggregation can be influenced by fusion partners to generate species with different toxic properties and provide new opportunities to study IB aggregation, maturation and lethality

Single domain antibody multimers confer protection against rabies infection

Post-exposure prophylactic (PEP) neutralizing antibodies against Rabies are the most effective way to prevent infection-related fatality. The outer envelope glycoprotein of the Rabies virus (RABV) is the most significant surface antigen for generating virus-neutralizing antibodies. The small size and uncompromised functional specificity of single domain antibodies (sdAbs) can be exploited in the fields of experimental therapeutic applications for infectious diseases through formatting flexibilities to increase their avidity towards target antigens. In this study, we used phage display technique to select and identify sdAbs that were specific for the RABV glycoprotein from a naïve llama-derived antibody library. To increase their neutralizing potencies, the sdAbs were fused with a coiled-coil peptide derived from the human cartilage oligomeric matrix protein (COMP48) to form homogenous pentavalent multimers, known as combodies. Compared to monovalent sdAbs, the combodies, namely 26424 and 26434, exhibited high avidity and were able to neutralize 85-fold higher input of RABV (CVS-11 strain) pseudotypes in vitro, as a result of multimerization, while retaining their specificities for target antigen. 26424 and 26434 were capable of neutralizing CVS-11 pseudotypes in vitro by 90–95% as compared to human rabies immunoglobulin (HRIG), currently used for PEP in Rabies. The multimeric sdAbs were also demonstrated to be partially protective for mice that were infected with lethal doses of rabies virus in vivo. The results demonstrate that the combodies could be valuable tools in understanding viral mechanisms, diagnosis and possible anti-viral candidate for RABV infection

Genomic and Proteomic Analysis of the Impact of Mitotic Quiescence on the Engraftment of Human CD34+ Cells

It is well established that in adults, long-term repopulating hematopoietic stem cells (HSC) are mitotically quiescent cells that reside in specialized bone marrow (BM) niches that maintain the dormancy of HSC. Our laboratory demonstrated that the engraftment potential of human HSC (CD34+ cells) from BM and mobilized peripheral blood (MPB) is restricted to cells in the G0 phase of cell cycle but that in the case of umbilical cord blood (UCB) -derived CD34+ cells, cell cycle status is not a determining factor in the ability of these cells to engraft and sustain hematopoiesis. We used this distinct in vivo behavior of CD34+ cells from these tissues to identify genes associated with the engraftment potential of human HSC. CD34+ cells from BM, MPB, and UCB were fractionated into G0 and G1 phases of cell cycle and subjected in parallel to microarray and proteomic analyses. A total of 484 target genes were identified to be associated with engraftment potential of HSC. System biology modeling indicated that the top four signaling pathways associated with these genes are Integrin signaling, p53 signaling, cytotoxic T lymphocyte-mediated apoptosis, and Myc mediated apoptosis signaling. Our data suggest that a continuum of functions of hematopoietic cells directly associated with cell cycle progression may play a major role in governing the engraftment potential of stem cells. While proteomic analysis identified a total of 646 proteins in analyzed samples, a very limited overlap between genomic and proteomic data was observed. These data provide a new insight into the genetic control of engraftment of human HSC from distinct tissues and suggest that mitotic quiescence may not be the requisite characteristic of engrafting stem cells, but instead may be the physiologic status conducive to the expression of genetic elements favoring engraftment

Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection

The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse.Deutscher Akademischer Austausch Dienst (DAAD); International Graduate School in Development Health and Disease (IGS-DHD); Deutsche For-schungsgemeinschaft (SFBs 635, 670, 680); Max-Planck-Gesellschaft (Max Planck Fellowship)

Inactivation of a Single Copy of Crebbp Selectively Alters Pre-mRNA Processing in Mouse Hematopoietic Stem Cells

Global expression analysis of fetal liver hematopoietic stem cells (FL HSCs) revealed the presence of unspliced pre-mRNA for a number of genes in normal FL HSCs. In a subset of these genes, Crebbp+/− FL HSCs had less unprocessed pre-mRNA without a corresponding reduction in total mRNA levels. Among the genes thus identified were the key regulators of HSC function Itga4, Msi2 and Tcf4. A similar but much weaker effect was apparent in Ep300+/− FL HSCs, indicating that, in this context as in others, the two paralogs are not interchangeable. As a group, the down-regulated intronic probe sets could discriminate adult HSCs from more mature cell types, suggesting that the underlying mechanism is regulated with differentiation stage and is active in both fetal and adult hematopoiesis. Consistent with increased myelopoiesis in Crebbp hemizygous mice, targeted reduction of CREBBP abundance by shRNA in the multipotent EML cell line triggered spontaneous myeloid differentiation in the absence of the normally required inductive signals. In addition, differences in protein levels between phenotypically distinct EML subpopulations were better predicted by taking into account not only the total mRNA signal but also the amount of unspliced message present. CREBBP thus appears to selectively influence the timing and degree of pre-mRNA processing of genes essential for HSC regulation and thereby has the potential to alter subsequent cell fate decisions in HSCs