Travel Grant Recipients to ISSCR 2017 Announced

Do metabolites regulate stem cells in tissues? To answer this question, we developed a method to measure metabolite levels in stem cells. We found that hematopoietic stem cells (HSCs), the blood forming stem cells in the bone marrow, have high levels of ascorbate. When ascorbate is depleted from mice, HSC frequency and function is increased, and the activity of the tumor suppressor Tet2 is reduced. Tet2 is frequently deleted in human leukemia, and leukemia in mice is accelerated in the absence of ascorbate. Therefore, a circulating metabolite, ascorbate, regulates tumor suppression in stem cells.

Dr. Bundgaard’s research is focused on Osteoarthritis (OA) and lameness in horses. She is looking for cell-based therapies in the next generation treatments of OA utilizing mesenchymal stem cells (MSCs) secreted biomolecules and their suspected mechanisms of action. The research she will present at ISSCR this year describes an in vitro model that compares Secreted Protein Profiles (SPP) of MSCs after stimulation with serum amyloid A (SAA) or interleukin 1β (IL-1β). Her results indicate that the concentration of selected proteins central in the inflammatory response and breakdown of ECM are more abundant in the SPP from CH and Adipose Tissue MSC compared to the SPP from Bone Marrow MSC.

My work is to elucidate the molecular basis of insulin resistance (IR) employing human pluripotent stem cell-derived models of metabolic disease. We have generated models that replicate both impaired and constitutively activated insulin signaling through the knockout or modification of genes involved in the insulin signaling pathway. These genetic models of IR have been differentiated into several relevant metabolic cell types. The differentiated cells are currently being comprehensively analyzed via RNA-seq, metabolomics profiling, and insulin stimulation assays to generate a global picture of the effects of IR on the behavior of metabolically important cells. The ultimate goal is to illuminate novel mechanisms and identify targets in the insulin signaling pathway that would be of value in understanding and treating IR in the setting of human metabolic disease.

Articular stiffness and digital ulcers in hands affected by Systemic Sclerosis (SS) is caused by fibrosis and microangiopathy. Nowadays SS has a treatment that only allows to decrease the evolution of the illness but none of them reverse the disease. Adipose-derived Stromal Vascular Fraction (ADSVF) possesses multiple angiogenic, anti-inflammatory and regenerative properties because it contains Adipose Stromal/Stem Cells (ASC), therefore it could be a potent optional treatment,In addition, the sample collection of stem cells from adipose tissue compared to bone marrow mesenchymal stem cells (MSC) is less invasive and easier to obtain. Until now, there are studies to increase the microvascularity in human hands affected by SS, and there are studies to treat the fibrosis caused by SS only in animal models with MSC. Currently, there is research about the effect of ADSVF in SS but none of them has a control group. In our study, we apply autologous lipograft enriched with ADSVF from abdominal liposuction in 10 hands affected with articular stiffness and/or digital ulcers caused by SS and compare the evolution of the disease with 10 hands treated with conventional treatment. Medical follow-up of the patients is for six months and includes the assess of color, temperature, Raynaud Phenomenon, pain, articular hand movement, quality of life, skin thickness and digital ulcers evolution.

MitoBloCK6 (MB-6) was identified in a screen for inhibitors of the protein Augmenter of Liver Regeneration/Growth Factor Erv1-Like (ALR/GFER). ALR is a taxonomically conserved protein involved in the disulfide relay system, which facilitates import of cysteine rich proteins into the mitochondrial intermembrane space (IMS). Previous studies have shown that MB-6 causes apoptosis in human pluripotent stem cells (hPSCs) while leaving their differentiated derivatives intact. Our current work aims to determine the mechanism for MB-6 rapid cell death in hPSCs. Toward this goal, we have identified the time of differentiation, which differs based on lineage and tissue type, at which hPSC derivatives become resistant to MB-6 exposure. Antioxidants mitoTEMPO and N-acetylcysteine had little effect on hPSC death after MB-6 treatment, suggesting that reactive oxygen species (ROS) influence on the apoptotic mechanism is minimal. We hypothesize that MB-6 causes preferential death of hPSCs through differences in ALR protein import activity, and that these differences are mediated by differential protein interactions in pluripotent versus differentiated cell states. To examine this hypothesis, we are utilizing immunoprecipitation in combination with mass spectrometry (IP-MS) to identify changes in ALR interactions between undifferentiated hPSCs and their differentiated progeny. We anticipate new insights into differences in mitochondrial function in pluripotent versus differentiated cells. In addition, MB-6 could become a tool to remove pluripotent cells that fail to differentiate into the desired cell types, furthering clinical stem cell applications.

Dr. van de Leemput is an Assistant Project Scientist at the Department of Psychiatry at University of California, San Diego. In addition, she is a Collaborative Researcher with the Laboratory of Genetics at the Salk Institute for Biological Studies. She is interested in how molecular-genetic mechanisms in the earliest stages of brain development contribute to neuropsychiatric disorders. In particular, the role astrocytes play in neural network development, and how dysfunctional astrocytes might contribute to the etiology of neuropsychiatric disorders like schizophrenia. To this end, she uses human induced pluripotent stem cell (hiPSC)-derived models to study the interaction between neurons and astrocytes, comparing cells obtained from patients with those from healthy controls. Most studies to date have focused on the neurons, by investigating astrocyte’s role she hopes to uncover a crucial contributing component in the etiology of neuropsychiatric disorders, ultimately aiding the development of better treatments.

Additional Sex Combs-like 1 (ASXL1) is involved in Polycomb-dependent gene regulation via two antagonistic mechanisms: enhancing H3K27me3 deposition by recruitment of PRC2, or promoting removal of H2AK119 and subsequently H3K27me3 via activation of the BRCA1 Associated Protein 1 (BAP1). The functional importance of these mechanisms for embryonic development remains unclear. It has recently been reported that heterozygous mutations in ASXL1 lead to Bohring-Opitz-Syndrome (BOS), a developmental disorder. We found that BOS-patient-derived iPSCs, as well as hESC bearing similar ASXL1 mutations, express truncated, dominantly acting ASXL1 proteins (mutASXL1). Strikingly, differentiation to migrating neural crest-like cells was strongly impaired in mutASXL1-expressing hESCs. Transcriptional downregulation of several neural crest fate determinants, most prominently of ZIC1, was underlying the decreased developmental capacity. Interestingly, we observed a trend of increased presence of H3K27me3 at both ZIC1 and the neighboring gene ZIC4, which is also implicated in neural crest development. We believe our observations can explain the development of several neural crest- related symptoms in BOS, and are a first step towards understanding the role of (truncated) ASXL1 in Polycomb/BAP1-mediated regulation of development.

Ali received his PhD in chemical engineering (Nanotechnology) from the University of Waterloo. He worked on peptide-CNT hybrid hydrogels for tissue engineering and 3-D cancer tumor modelling. He joined Dr. Jeschke lab in Sunnybrook Research Institute (University of Toronto) in Fall 2015. His research includes developing biodegradable skin substitutes for skin regeneration in burn patients. He is uses natural and synthetic biomaterials for developing an ideal scaffold for skin regeneration. Different types of cells from dermal fibroblasts to the unique stem cells (developed in Dr. Jeschke lab) are being used to cellularize the scaffolds.

Locus-Specific Proteomics in Human Embryonic Stem Cells Identifies ZNF207 as a critical regulator of OCT4 and Self-Renewal Cell fate during development is defined by transcription factors that act as molecular switches to activate or repress specific gene expression program. The POU transcription factor OCT4 is essential for establishing and maintaining the self-renewal and pluripotent state of human embryonic stem cells (hESCs). Level of OCT4 must be precisely regulated in hESCs as a small perturbation would lead to loss of cell identity. Although genome-wide mapping of OCT4 regulatory targets and mass spectrometry analyses has identified a few OCT4-associated proteins, the complete repertoire of potential regulatory proteins for OCT4 in hESCs is not known. Here, we combine genome editing technology, chromatin immunoprecipitation (ChIP) and mass spectrometry to investigate locus-specific proteomics at OCT4 enhancer in hESCs. We identify about 150 proteins that bind to the regulatory region of OCT4, including known regulatory proteins of OCT4 (eg., OCT4, SOX2 and SALL4). We also validate the binding of a selected set of novel proteins by ChIP analysis. The identification of known regulators as well as validation of novel proteins by individual ChIP provides strong support for the reliability of our approach. More interestingly, an enrichment analysis finds the gene ontology terms involved in transcriptional regulation are overrepresented. We then perform a functional screen of transcription factors in our list by siRNAs. Strikingly, we find ZNF207, which has never been indicated a role in hESCs, an essential and indispensable regulator for maintaining the cell identity of hESCs and for cell fate change from somatic cells to stem cells during reprogramming. Knock down ZNF207 lead to differentiation of stem cells, while forced expression of ZNF207 in fibroblast significantly increase reprogramming efficiency. Furthermore, we perform genome-wide binding and transcriptional profiling of ZNF207 and integrate it to the core transcriptional network in hESCs. In summary, we provide a generalizable strategy to identify molecular components and probe dynamic regulation at a given chromosomal location in mammalian cells. Application of this approach in hESCs enable us to identify ZNF207 as a novel regulator that play essential roles in induction and maintenance of core transcriptional program of stem cells.