Gabsang Lee, D.V.M., Ph.D. - US grants

? DESCRIPTION (provided by applicant): The prevalent genetic defects and acquired injuries of the peripheral nervous system (PNS) cause a significant socioeconomic issue on our healthcare system. However, there are relatively few detailed studies of human PNS tissues, due to the difficulty to obtaining patient samples. We have shown that peripheral neurons and Schwann cells can be derived from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs); however, the process is arduously long (at least over 5 months). Recently we developed an alternative, a new method that directly converts human fibroblasts into induced neural crest (iNC) in only two weeks. Our iNC population exhibits neural crest- specific cellular and molecular characteristics. Although the iNC population shows multipotency in a single cell level, they are more prone to a sensory neuron fate, rather than autonomic neuron lineages. During development, cell fates are determined by cell extrinsic factors, such as growth factors and morphogens. If such cell extrinsic factors also govern cell fates during generation of iNC, we may be able to change the differentiation potential of the iNC. This hypothesis incites a question, whether modulation of cell extrinsic factors (such as ventralization and/or caudalization cues) can influence the cellular fate during direct conversion, in the same way that this mechanism operates in developmental cell determination. Currently direct conversion, exemplified by our iNC, is dependent on transcription factors delivered by oncogenic viral transduction. Our second question is whether and how `non-genetic' small molecules can achieve sufficient direct conversion of human fibroblasts to induced neural crest? Our current iNC induction system is suitable for investigating these fundamental questions about cell fate plasticity, because it employs only a single transcription factor, along with highly quantitative detection method (SOX10::GFP detection by FACS). Our proposed experiments are expected to: (i) expand and strengthen our current conception of general `genetic factor-dependent' direct conversion, and (ii) accelerate a wide range of research on PNS disorders, e.g. by providing disease-specific Schwann cells or sympathetic neurons directly from fibroblasts of Charcot-Marie- Tooth1A (CMT1A) or familial dysautonomia patients.

PI: Lee, Gabsang
Proposal Number: 1547515

Recent advances in stem cell biology hold great potential in regenerative medicine. To realize the therapeutic promise of stem cells, approach to realize high-quality and high-precision culture system is essential. The investigator proposes an innovative approach of optical control of stem cell behaviors through blue light illumination. If successful the approach will be expanded to other stem cell applications by controlling multiple signaling pathways.

Current stem cell culture systems include different growth factors, but their uneven distribution in the media (lack of spatial control), and gradually decreased activity due to temporal-instability, often cause significant problems in a large-scale stem cell biomanufacturing. To address this problem, the investigator proposes photo-inducible modulation of fibroblast growth factor (FGF) signaling in human pluripotent stem cells and muscle stem cells, using an innovative optically controlled fibrobast growth factor receptor (OptoFGFR) system allowing precise control of FGF signaling. The proposed investigation will accelerate the in vitro and in vivo spatio-temporal modulation of stem cells, and will be the first example of the 'opto-signaling' application in stem cell biology with potential translational characteristics. More importantly, the OptoFGFR system will be expanded to other signaling pathways such as epidermal growth factor (EGF), vascular endothelial growth factor VEGF, and nerve growth factor (NGF), and if successful the approach could be useful for multiple human diseases.

  Muscle wasting, caused by aging, genetic mutations, cancan-associated cachexia, or traumatic injury, can result in significant functional impairment, and is a challenging clinical problem with a significant socioeconomic burden on our healthcare system. We have shown that functional myoblasts are readily derived from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), allowing us to begin to study Duchenne muscular dystrophy (DMD), the most common genetic disorder of muscle. However, we know little about i) how early myogenic events are genetically controlled during development, ii) whether embryonic PAX7 expressing myogenic stem/progenitor cells adopt postnatal `satellite-like' fate, and iii) how DMD is occurred in skeletal muscle stem/progenitor cell stage as well as their relevance for cell replacement therapy. First, using multiple genetic reporter lines to recapitulate human myogenic events, we will depict a time- course analysis of transcriptional landscape followed by `loss of function' analysis to address essential questions regarding which critical cell intrinsic/extrinsic component(s) govern the skeletal muscle specification process and stem cell maintenance. Secondly, by performing serial transplantation of human PAX7::GFP+ putative skeletal muscle stem/progenitor cells in mouse model, we will interrogate how the embryonic cells become to postnatal satellite-like cell fate. Thirdly, based on our observation on DYSTROPHIN expression in human skeletal muscle stem/progenitor cells, we will investigate stage-specific role(s) of DYSTROPHIN and its long intergenic non- coding RNAs (LincRNAs), in healthy and DMD condition. In addition, we will interrogate in vivo regeneration ability of patient-specific PAX7::GFP+ cells of genetically corrected DMD-hiPSC lines. Our proposed experiments are expected to expand and strengthen our current conception of myogenic specification events, and to accelerate a wide range of research on skeletal muscle disorders, e.g. traumatic muscle damages, genetic muscular dystrophies, neuromuscular diseases, type II diabetes and cancer-induced cachexia. 

PROJECT SUMMARY Alzheimer's disease (AD) is the most common neurodegenerative disease and its pathology is characterized by deposition of ?-amyloid and tau accumulations, leading to extensive neuron loss. The converging evidence from in vivo and in vitro studies support that aberrant behaviors of tau protein and their aggregation play a central role in the development of Alzheimer's disease pathology, but there is no experimental approach to control tau aggregation with great temporal and spatial precision. To address this fundamental issue, we have developed a new synthetic technique to optically control aggregation of pathogenic proteins upon light illumination. We propose to use this approach to control tau aggregation in order to model AD with human pluripotent stem cells (hiPSCs)-derived neurons. In future, our proposed studies will be complementary to existing animal models, and providing a novel platform for drug screening/validation. The optical control of tau aggregation will transform the current concepts of disease modeling and drug screening toward innovative translational approaches.

Skeletal muscle makes up almost half of the human lean body mass and approximately 40% of all traumatic injuries involve skeletal muscle damage. This results in a global economic burden of roughly $6 billion. While skeletal muscle possesses an intrinsic self-regeneration capacity, in clinical scenarios of volumetric muscle loss (VML) where the muscle's natural repair mechanisms are overwhelmed, regeneration fails. Tissue engineering strategies using human skeletal muscle stem or progenitor cells combined with novel biomaterials have unprecedented potential to provide effective therapies. In this study, we propose to harness the myogenic potential and regenerative capacity of sorted skeletal muscle stem/progenitor reporter cells (PAX7::GFP+) derived from human pluripotent stem cells (hPSCs). Specifically, we hypothesize that PAX7::GFP+ myogenic progenitors grown on electrospun fibrin microfiber bundles will proliferate, upregulate their expression of myogenic genes and form aligned, multi-nucleated myotubes assembled into 3D muscle grafts. These engineered grafts will be used to regenerate skeletal muscle tissue and restore normal function following VML. We further hypothesize that the use of agrin in combination with insulin-like growth factor-1 (IGF-1) will promote the formation of more densely packed PAX7::GFP+ derived myotubes in the engineered muscle grafts and enable the formation of mature neuromuscular junctions (NMJs) in the regenerating skeletal muscle. We will test these hypotheses in three Specific Aims. In Sp. Aim 1, we will engineer uniform, densely seeded skeletal muscle grafts by (i) electrospinning PAX7::GFP+ cell aggregates into the fibrin microfiber bundles and (ii) coating the microfiber bundles with PAX7::GFP+ cell-seeded bulk fibrin. We will stimulate their maturation into contractile 3D skeletal muscle tissues using biophysical stimulation. We will quantitatively evaluate cell morphology, proliferation, multi-nucleation, and myogenic differentiation and utilize single-cell RNA-sequencing to compare the cellular heterogeneity and myogenic gene expression profiles with that of native muscle cells. In Sp. Aim 2, we will evaluate the potential of soluble and tethered agrin/IGF-1 individually and in combination to enhance the proliferation and myogenesis of PAX7::GFP+ cells. We will also characterize the effects of tethering these molecules on the physicochemical and pro-myogenic properties of the modified scaffolds. In Sp. Aim 3, we will implant PAX7::GFP+ derived muscle grafts engineered with and without soluble or tethered agrin/IGF-1 into small incisions into the tibialis anterior (TA) muscle of immunodeficient mice to assess cell survival, integration, and regenerative potential. We will use these data to optimize the engineered skeletal muscle grafts that we will implant into VML defects to quantitatively assess muscle regeneration, vascular and neural infiltration, the formation of mature neuromuscular junctions, and functional recovery at 1 and 3 months post-transplantation. To successfully accomplish these aims, we combine complementary expertise in tissue engineering, stem cell biology, biomaterials, murine models of VML, and skeletal muscle physiology.