NCRM supported 11 applications this year from multiple institutes and centers including: NHRGI, NIA, NHLBI, NINDS, NIDCD, NIDCR, NEI, NCI and NIMH. These projects included development of iPSC-Based Disease Model of Niemann-Pick Disease Type C, which proposed to generate iPSCs from NPC patient skin fibroblasts, differentiate them into neuronal cells, and utilize them in high-throughput screening (HTS) of large drug collections to identify probes to study NPC pathophysiology, and drugs that may be repurposed for the treatment of NPC patients. Another project investigated the cardiovascular applications of iPSCs. The project involves generation human iPSCs from individuals with the newly described LACA (Localized Arterial Calcifications in the Adult) syndrome, as well as from patients affected with Arrhythmogenic Right Ventricular Cardiomyopathy. These iPSCs will be used for cell-based models of these conditions in the hopes of discerning novel mechanistic and therapeutic insights. In addition, it is proposed to develop the technology to generate porcine iPSCs and to use their capacity for targeted myocardial cell delivery to test the therapeutic value of cardiac progenitor cells in a pre-clinical large animal model of advanced heart failure. A third project was the generation of iPSCs for mechanistic and therapeutic studies of motor neuron disease including familial amyotrophic lateral sclerosis (ALS) and spinal bulbar muscular atrophy (SBMA). It was proposed to generate neurons and other cells that can then be manipulated and studied in vivo to better understand the disease mechanisms. Specifically the investigators will study the cellular phenotype by assessing proteasome activity, cell survival in co-cultures, axonal transport, RNA localization and transport, mitochondrial function, and gene expression. A fourth funded project is the induction of hair cell regeneration using iPSCs. The investigators will determine whether induction of iPSC identity within adult vestibular epithelia is sufficient to induce hair cell regeneration. Initially, in vitro assays will be used to determine conditions that will optimize the generation of new hair cells following hair cell injury. Based on these results, the functional consequences of hair cell regeneration in vivo will be assessed using a combination of behavioral and electrophysiological testing. Finally, in order to confirm that new hair cells are in fact arising from iPSCs, specific transgenic reporter mice will be used to mark newly generated hair cells. A fifth project analyzing Smads2/3 dependent epigenetic landscape in iPSCs -They are now carrying out a genome wide analysis to determine the full extent of the contribution of Polycomb repressive histone methylation mechanism to ESC pluripotency and in differentiated derivatives. By carrying out similar analyses on iPSCs they can potentially gain important insight into additional epigenetic differences present in iPSCs that impact their potential. Other recent work in the iPSC field has shown that a pulse of Smads2/3 signaling inhibitors during the late phase of reprogramming of fibroblasts to iPSC can improve the efficiency and kinetics of their derivation. A critical issue for iPSC research is the generation of standards. As a result, another appropriate project was the development of a Reference Set of Comprehensively Characterized iPSC lines. The investigators propsed developing high throughput technologies to comprehensively characterize several existing iPSC lines from healthy individuals at multiple levels including: whole genome sequencing, whole genome analysis of DNA methylation, whole genome analysis of histone modifications and whole genome transcript analysis. They will generate a reference set of iPSC lines from healthy individuals enrolled in the ClinSeq Project, who have been extensively phenotyped and consented to genome sequencing. In addition, the investigators will engage begin screens to identify small molecules that will inhibit the spontaneous differentiatiion of iPSCs. As a first step toward using patient-specific induced iPSCs for creating functional retinal pigment epithelium (RPE) for transplantation in the diseased eye, a seventh funded project proposed to provide a rigorous molecular and physiological characterization of RPE derived from iPSC lines from six healthy donors. As a control, they will carry out the same experiments using RPE derived from two ES cell lines. The eighth project approved for funding was to reprogram tumor-specific T cells using iPSCs for cancer immunotherapy. The investigators propose generating iPSC-derived, tumor-specific lymphocytes, which will be analzyed in terms of telomere length and are unencumbered by the epigenetic changes associated with terminal differentiation, exhaustion and senescence. Another initiative is devoted to differentiating human iPSCs into bone and cartilage. Specifically, the investigators are assessing conditions htat favor osteogenic differentiation using HSF-6 (UC06) hES cell line. HSF-6 cells were cultured in a number of conditions, including with KO-DMEM-based media with FBS, dexamethasone, and ascorbic acid on a carpet of hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic particles. They propose to apply these culture conditions to human iPSCs, and to further improve upon them to develop more consistent bone formation. In addition to forming bone, they have also been devising techniques for the development of cartilage by human bone marrow stromal cells (BMSCs, also known as mesenchymal stem cells). In pellet cultures with chondrogenic factors, BMSCs are able to form cartilage, however, these pellets undergo hypertrophy. The tenth project involved the validation of human GABA inter-neurons derived from IPSCs by comparing them to GABA neurons directly isolated from the brains . Reprogramming approaches will generate three biological replicate iPSC lines from each genome. Successful reprogramming will be assessed by comparison with an existing comprehensive database of human ES and IPSCs. Cell lines with a fully reprogrammed status will be differentiated to ventral forebrain neural precursors and inhibitory GABA-ergic inter-neurons will be derived. Finally, the eleventh project will be to define approaches for optimal isolation of homogeneous human iPSC populations. To begin the process of identifying lineage markers and marker panels, the investigators intend to identify cell surface glycoproteins using state of the art biochemical methods and mass spectrometry. Analyses of the cell surface proteome will include at least two human embryonic stem cell (ESC, H1 and H9) lines, which will serve as standards for pluripotency, and at least 3 to 5 human iPSC (skin fibroblast- and fat-derived) lines. Data will be compared to a proprietary Cell Surface Protein Atlas to identify proteins whose expression is restricted to pluripotent cells.
