Understanding the nature of stem cell interaction with microenvironment
during tissue regeneration and in cancers
Recent findings, including our studies of the bladder, and a survey of various epithelial tissues that we carried out suggest that, in many endoderm-derived epithelial tissues, Hedgehog (Hh) signaling acts in a non-cell autonomous way in which Hh response occurs only stromal cells to mediate epithelial and stromal interactions. The role of Hh signaling in various diseases of these tissues in the context of stem cell regulation, however, remains unclear. One
focus of our current research is to understand how Hh signaling operates in regeneration and carcinogenesis of various endoderm-derived epithelial tissues in which Hh signaling operates in non-cell autonomous manner; Hh ligand is expressed in epithelial cells and Hh response is restricted to the stromal compartment.
Development of miniature organs for disease modelling
Development of bladder organoid. With the advance of stem cell technologies, the efforts have been made to develop in vitro three-dimensional organ-like tissues called organoids that mimic in vivo tissues, providing reliable tools to study various human diseases. Recently, we successfully cultured bladder organoids in vitro and have investigated the role of multiple key signaling pathways associated with various bladder diseases including bladder cancer. This provides strong conceptual and experimental foundationfor our
research, in which we generate more consistent 3D multicellular bladder tissues, to establish an in vitro model that recapitulates in vivo bladder diseases. One focus of our research is to develop 3D bladder organoid that can mimic normal bladder and various bladder diseases and that can be maintained for long period of time without losing its ability to generate new organdies. To this end, we use various stem cells such as tissue/adult stem cells, embryonic stem cells, and induced pluripotent stem cells and innovative 3D bioprinting technology.
Establishment of cortex specific mini brain. The biggest challenge to study human brain is the lack of experimental systems that can precisely represent the complexity of human brain. For many decades, in vitro two-dimensional cell cultures have been utilized to understand physiological function of neurons, but these approaches are limited to the study of physiological properties of individual neurons, which makes them
inadequate model for the development of human brain and neural connections. To compensate for the limitation of in vitro cell culture-based approaches, researchers have traditionally used animals to model the human brain development in vivo. Although contributing significantly to our current understanding of brain development and neurological diseases, animal modes cannot faithfully recapitulate various features of human brain because of the structural and functional differences among species. Thus, to understand the function of neurodevelopment as well as the neural circuits of human brain, it is necessary to develop experimental models of neural tissue development. In our research group, we develop several new strategies to generate consistent nd mature cerebral cortex specific organoids that mimics human brain development and that can be used to study various brain disorders involving cortical connectivity and neurodevelopment.
Development of individualized cancer therapy using 3-dimensional in vitro organoid cancer model
Recent advances in early detection technologies and cancer genomics demand the development of personalized therapeutic interventions. Designing precise therapeutic strategies for individual patients with genetic variability at different cancer stages is required to prevent the potentially harmful effects of drugs developed from population-based research. To achieve this goal, it is imperative to develop in vitro cancer model systems that accurately recapitulate in vivo cancer progression, and that integrate the genomic profiles of individual cancer patients. These in vitro cancer model systems are also necessary to better understand the molecular and cellular basis of individual patients’ cancer pathogenesis. Although cell culture approaches have been utilized in pharmacological studies, cancer cell lines significantly underrepresent the genetic lesions of individual cancer patients due to a limited number of available cell lines. More importantly, cell culture approaches have been limited in their applications because they do not represent the full spectrum of in vivo disease progression. This research projects is designed to develop three-dimensional (3D) in vitro organoid systems that accurately model in vivo bladder cancer progression and to further establish human bladder tumor organoid lines that represent the pathology and molecular diversity of original tumors from individual patients.
Establishment of patient-derived organoid lines from human cancer patients. In vitro tumor models that integrate individual genomic profiles will be necessary to customize new drugs for the specific genetic contexts of individual patients. Although widely used in pharmacological studies, a 2D culture system
with a limited number of available cell lines significantly underrepresents the genetic lesions of human bladder cancers. The goals of this project are to generate a large repertoire of patient-derived bladder cancer organoid lines, and to establish a human cancer model that recapitulates patient-specific cancer patients
We hypothesize that patient-derived bladder cancer organoids will accurately represent the pathology and the molecular diversity of individual patient tumors. Recent studies have shown that prostate cancer cells from biopsy specimens can be cultured and maintained using a 3D organoid system. We plan to generate a
large repertoire of bladder tumor organoid lines from primary and metastatic bladder cancer cells using our recently developed tumor organoid culture methodology.
Establishment of an in vitro cancer model system that recapitulates in vivo cancer progression. We are currently establishing an in vitro bladder cancer model by coupling a 3D bladder organoid culture system to an advanced genome engineering technology based on the CRISPRCas9 system, followed by validation of that model. Previous studies have shown that combined inactivation of p53 and PTEN in the bladder epithelium is sufficient to induce invasive bladder cancer in vivo. Our preliminary data showed that urothelial stem cells with reduced expression of p53 and PTEN developed neoplastic organoids. By combining our bladder organoid culture with the CRISPR-Cas9 system, we are developing a 3D organoid culture system that mimics in vivo bladder cancer progression.
In vivo reprogramming coupled with genome editing technology to develop novel strategy for regenerative medicine
The major goal of this research project is to develop an innovative strategy to overcome three major obstacles to current cell replacement therapies for a variety of disorders: 1) the shortage of specific cell types available from donors; 2) host immune rejection of cells from exogenous donors; and 3) safety concerns stemming from the in vitro culture of exogenous cells. We plan to genetically reprogram somatic cells directly into different lineages within endogenous tissues to restore the function of other damaged tissues.
For degenerative diseases caused by genetic mutations, it is important to correct the genetic defects prior to generation of new tissues by reprogramming because newly generated cells otherwise would be subject to degeneration due to the same genetic defects. Recent progress in genome engineering technology, such as the CRISPR-Cas9 system, allows for precise and efficient genome editing, suggesting the use of this approach as a therapeutic intervention for treating genetic disorders. By coupling this technology with in vivo reprogramming, the defective genes of precursor cells may be corrected prior to their reprogramming to generate functional cells. In this research project, we envision a new paradigm for regenerative medicine in which functional cells with corrected genetic defects are generated in the bladder to restore healthy and functional tissues.