Human induced pluripotent stem cells (hiPSCs) now provide unprecedented opportunities for cell replacement approaches, disease modeling and drug discovery in patient-specific manner. Particularly Lee lab is focusing on neural lineage and skeletal muscle. Recently we have developed a new approach to control protein association/aggregation with light illumination, which allows us to stimulate multiple signaling pathways in stem cells, and to model pathogenic protein aggregation. 

Skeletal muscle cells, in vitro myogenesis, and muscular dystrophies

We developed a new methodology to directly derive and prospectively isolate thousands of millions of expandable and fusion-competent myoblasts from hESCs/hiPSCs. Using multiple genetic reporter hESC lines, we are recapitulating step-wise human myogenesis, comprising pluripotent stem cells, somite cells, adipomyocytes and putative satellite stem cells. Further, hiPSCs of Duchenne muscular dystrophy and facioscapulohumeral muscular dystrophy are providing us unique opportunities to learn more about the devastating muscular dystrophies. Interestingly we found a way to direct hESCs/hiPSCs into functional satellite cells that have capabilities to engraft and repopulate in vivo niche. This project is currently funded by NIH/NIAMS, MSCRF/TEDCO, and Vita Therapeutics. 

Optical control of signaling pathways and aggregation of pathogenic proteins in human stem cells

Stem cell fate is largely determined by a complex cell signaling network. Our understanding and approaches to modulate such stem cell signaling networks is limited by the lack of precise control in single cells. In order to understand the operational principles of this network and physiological and pathological events, it is imperative to control signaling protein activities and subsequent cellular fates with great temporal and spatial precision. We have introduced light-sensing actuator modules into human pluripotent stem cells (hPSCs) to mimic fibroblast growth factor (FGF) signaling pathways via light illumination without using recombinant FGF protein. This method allowed us to maintain long-term stemness of the hPSCs. We are currently expanding this concept to other signaling pathways, to control cellular fates and promote neuronal survival of hPSC-derived neurons. Furthermore, by utilizing the protein aggregation properties of light-sensing modules, we will accelerate the aggregation of pathgoenic alpha-synuclein proteins , which will facilitate our understanding of the pathogenic mechanisms of Parkinson's disease. Using this optogenetics-assisted a-syn aggre- gation induction system (OASIS), we have develop a drug screening platform, allowing us to identify a new therapeutic small molecule with in vivo efficacy. We are currently extending this line of research for other neurogical diseases as well as a new countermeasure against galactic cosmic rays. This project is currently funded by Helis Research Foundation and TRISH/NASA. 

Neural crest and autonomic neurons 

Previously, our group studied neural crest stem cells created from fibroblasts of patients with Familial Dysautonomia (FD), also known as Riley-Day syndrome, an inherited genetic condition that affects the peripheral nervous system. Although researchers know that FD is caused by a single point mutation in the IKBKAP gene, it is not clear  how symptoms, like inability to feel pain and changes in temperature, manifest. We found that FD-specific neural crest cells expressed low levels of genes needed to make autonomous neurons—the ones needed for the “fight-or-flight” response. The FD-specific neural crest cells also moved around less than normal neural crest cells. Moving forward, as an effort to discover novel drugs to treat FD, we performed high throughput screening with a compound library using FD patient-derived neural crest stem cells to look for compounds that increased gene expression and protein levels of autonomous neuron developmental components. These studies set a paradigm of hiPSC studies, including developing differentiation protocol, disease modeling with patient hiPSCs and high throughput drug screening. Now we are advancing from neural crest to autonomic neurons and multicellular system. Our PHOX2b::GFP+ sympathetic neurons and their functional connection to target tissues (cardiac syncytia), which will lead us to investigate aberrant neuromodulation in patient-specific manner. Another research direction of this project is to study gut neuron behaviors in autoimmune disorders. We are testing the harmful effects of autoantibodies of scleroderma patients and hope to find a therapeutic target. This project is currently funded by NIH/NCATS and the Scleroderma Research Foundation. 

Nociceptive/pruriceptive neurons and congenital pain disorders

How our body can sense million of different stimuli with limited numbers of sensory neurons? How our ‘sensors’ can perceive specific stimulus?  The fate decision and physiological functions of individual sensory neurons should be choreographed by multiple molecular processes, which are closely related to the pathogenesis of many human pain disorders. Using iPSC lines of congenital sensory disorders, Congenital Insensitivity of Pain and Anhidrosis (CIPA) and Congenital Insensitivity of Pain (CIP), we are interrogating these questions with human TRPV1::GFP+, SCN9A::GFP+ and MRGPRX1::GFP+ neurons.  This project is currently funded by MSCRF/TEDCO. 

Schwann cells and Charcot Marie Tooth 1A

Charcot-Marie-Tooth 1A (CMT1A) is one of the most common genetic diseases in peripheral nervous system. We have learned lots of information from animal models, but their genetics are yet exactly same as those of CMT1A patients. Recently, in the Lee lab, the CMT1A--hiPSC-derived Schwann cells have provided us a new insight on the disease mechanism that is shared with Schwann cells derived from CMT1A-PDG-hESCs and induced neural crest of CMT1A fibroblasts. Furthermore, we now can generate myelination-competent human Schwann cells for future cell replacement therapy for many PNS diseases and related cancers. This project is currently funded by MSCRF/TEDCO and the Gilbert Family Foundation. 

Induced neural crest (iNC)

Despite of huge success of human iPSCs, there are several hurdles to overcome, such as arduously tedious time for its derivation/specification and cellular maturation issues. One potential solution is direct conversion technique. In 2014 we published a paper reporting direct conversion of human fibroblasts into induced neural crest (iNC) with single transcription factor (SOX10). As the SOX10-based iNC turns out multi-potent and behaves as their in vivo counterpart, it is a very interesting question if iNC can be ‘pattern-able’ during direct conversion process. On the other hand, we set out a chemical compound screening to replace SOX10, for generating chemically-induced neural crest (c-iNC). Our ‘genetic-factor free’ direct conversion will provide us unique opportunities to find mechanistic insight and potential malleability of c-iNC cell fates.