Sungtae Kim is a scientific investigator and has his expertise in stem cells, CRISPR/Cas9 genome editing, genomics and disease modeling. He has been implementing cutting-edge technologies and platforms to establish cellular disease models and for target validation studies. He has lead genome engineering tasks at GSK in multiple cell lines and primary cells by introducing novel strategies/technologies to the group and created new technological platforms for drug discovery in higher throughput manners. He is the first line leader in genome editing who cross-trains scientists in several discovery performance units, enjoys disseminating updates in science, organizes meetings for scientific discussion and established himself as a go-to person in this area. He is actively involved in several target identification studies and attempts to bridge genome editing strategies with advanced cellular modeling for better assessment of targets.

Statement of the Problem: Repertoire of genomic alterations via CRISPR/Cas9 genome editing depends on the efficiency of dsDNA breaks and the subsequent DNA repair events, which consists of two distinct pathways: Non-Homologous End Joining (NHEJ) and Homology-Directed Repair (HDR). HDR permits precise genome modifications which can be used to mimic disease relevant genotypes or to integrate functional domains of interest into the genome. However, the efficiency of HDR via genome editing remains quite low, mostly ranging from 0.1~10%. Although several strategies have been claimed to enhance HDR efficiency, there are still concerns about variability and reproducibility. Strategies: Relevant literature reporting HDR enhancement has been extensively surveyed. We hypothesized 1) high NHEJ event without a donor DNA also increase the chance of HDR in the presence of a donor. 2)HDR may increases by antagonizing NHEJ since NHEJ and HDR compete for the repair of limited number of dsDNA breaks. Combinations of modifications of gRNA and donor DNA, use of chemical inhibitors and targeted knock down have been examined. Findings: Temporal delivery of ribonucleoprotein (RNP) and chemical modifications of donor DNA increased HDR efficiency. Optimized conditions with KU0060648 or NU7441, DNA-PK inhibitors, reproducibly increase the HDR efficiency up to 50% in several different cell lines. Conclusion & Significance: Enhancement of HDR up to 50% has been achieved using combinations of different strategies. Our findings provide useful guideline to improve the efficiency of precise genome modifications in human cells.

Natella Enukashvily has completed her PhD at the age of 27 years from St. Petersburg State Universiy and postdoctoral studies from Institute of Cytology. She is a Senior Researcher and a Group Leader in the Institute of Cytology (St. Petersburg, Russia) and the Head of RnD Department in Pokrovsky Stem Cell Bank, Russia. She has published 14 papers in reputed journals.

Preclinical development and clinical trials require reliable methods of cell labeling for biomedical cell products kinetics study. MRI is considered to be a perspective method of transplantat visualization in a human body. Use of MRI demands the development of intravital cell labeling with contrast agents. SuperParamagnetic Iron Oxide Nanoparticles (SPIONp) of Fe3O4 (magnetite) are considered as an appropriate material. Aim: To establish a protocol of in vitro labeling of cells by SPIONp suitable for visualisation at 1.5 and 1T MRI scanners and verify viability, immunophenotype and trilineage differentiation of labelled cells. Materials and methods: Mesenchymal stromal cells (MSC) were obtained from human umbilical cord (UC) vein and adipose tissue (AT) after obtaning an informed consent from a donor (AT) or his/her parents (UC). UC and AT MSC were incubated with sonicated uncoated SPIONp (5-20 nm) (PNT, Russia). In 24 hours, an uptake of SPIONp was estimated by light microscopy. Cells immunophenotype, trilineage differentiation, proliferation and the possibility of cryoconservation were checked. Results: Cells with SPIONp maintained viability (95%). The optimal concentration for good uptake was 200-400 ng/ml. The SPIONp containing cells maintained MSC immunophenotype and the differentiation and proliferation potentials. SPIONp labelled cells could be frozen and succefully hawed. SPIONp cells could be visualized in animal tissues at 1/5 and 1T MRI Scanners. Conclusion: The protocol established can be used for labelling cells with SPIONp and future visualisation at clinical MRI scanners.

Michal Izrael has a broad background in developmental neuroscience and pluripotent stem cells biology. During her Ph.D at Prof. Michel Revel laboratory, she developed the protocols for derivation of human oligodendrocytes and astrocytes from human Pluripotent Stem Cells (iPSC and hESC) for drug screening applications and for regenerative medicine. These technologies are protected by several patent families. Dr. Izrael Joined Kadimastem on 2009, she served as the head of the drug screening team, then she was appointed as a VP for R&D in ALS and neurodegenerative diseases and developed Kadimastem’s cell product for ALS (AstroRx®). She holds a PhD from the molecular genetics department at the Weizmann Institute of Science and M.Sc. in Neurobiology from the Hebrew University in Jerusalem. Dr. Izrael has 16 scientific papers and patents in the field of Neurobiology and stem cell-based technologies for the treatment of CNS system disease and drug screening.

ALS is a Motor Neuron (MN) disease that is characterized by the loss of MNs in the central nervous systems. As motor neurons die, patients progressively lose their ability to control voluntary movements, become paralyzed and eventually die from respiratory/deglutition related failure. Despite the selective MN cell death in ALS, there is growing evidence that astrocyte dysfunction play a crucial role in ALS disease progression, and that transplantation of functional astrocytes may have therapeutic effects. Here we describe the derivation of Astrocyte Progenitor Cells (APC) from human Embryonic Stem Cells (hESC). These APC can be expanded in large quantities and stored frozen as cell banks. By a short further differentiation of the APC one obtains an enriched population of astrocytes (>90% GFAP+ cells). In vitro, these cells express the specific markers and possess the activities of functional healthy astrocytes, including uptake of glutamate, promotion of neurite and axon outgrowth and protection of motor neurons from oxidative stress. A secretome analysis shows that these hESC-derived astrocytes (AstroRx) abundantly secrete several inhibitors of metalloproteases as well as a variety of astrocytic neuroprotective factors (e.g. TIMP-1&2, OPN, MIF, Midkine) different from the classical BDNF and GDNF. Intrathecal transplantation of hESC derived astrocytes to the CerebroSpinal Fluid (CSF) of NSG mice shows that the cells distribute all along the neural axis (Brain and Spinal cord) and attach to CNS meninges, mainly to Pia matter. In animal models of ALS, human SOD1G93A high-copy number transgenic mice and rats, intrathecal injections of the AstroRx significantly delayed disease onset and improved motor performance compared to sham-injected animals. These findings indicate the feasibility and potential efficacy of intrathecal astrocyte injections for treatment of ALS.