Defining the Pluripotent Marker Genes for Identification of Teleost Fish Cell Pluripotency During Reprogramming.

Pluripotency is a transient state in early embryos, which is regulated by an interconnected network of pluripotency-related genes. The pluripotent state itself seems to be highly dynamic, which leads to significant differences in the description of induced pluripotent stem cells from different species at the molecular level. With the application of cell reprogramming technology in fish, the establishment of a set of molecular standards for defining pluripotency will be important for the research and potential application of induced pluripotent stem cells in fish. In this study, by BLAST search and expression pattern analysis, we screen out four pluripotent genes (Oct4, Nanog, Tdgf1, and Gdf3) in zebrafish (Danio rerio) and crucian carp (Carassius). These genes were highly expressed in the short period of early embryonic development, but significantly down-regulated after differentiation. Moreover, three genes (Oct4, Nanog and Tdgf1) have been verified that are suitable for identifying the pluripotency of induced pluripotent stem cells in zebrafish and crucian carp. Our study expands the understanding of the pluripotent markers of induced pluripotent stem cells in fish.

Genome Editing Human Pluripotent Stem Cells to Model ß-Cell Disease and Unmask Novel Genetic Modifiers.

A mechanistic understanding of the genetic basis of complex diseases such as diabetes mellitus remain elusive due in large part to the activity of genetic disease modifiers that impact the penetrance and/or presentation of disease phenotypes. In the face of such complexity, rare forms of diabetes that result from single-gene mutations (monogenic diabetes) can be used to model the contribution of individual genetic factors to pancreatic ß-cell dysfunction and the breakdown of glucose homeostasis. Here we review the contribution of protein coding and non-protein coding genetic disease modifiers to the pathogenesis of diabetes subtypes, as well as how recent technological advances in the generation, differentiation, and genome editing of human pluripotent stem cells (hPSC) enable the development of cell-based disease models. Finally, we describe a disease modifier discovery platform that utilizes these technologies to identify novel genetic modifiers using induced pluripotent stem cells (iPSC) derived from patients with monogenic diabetes caused by heterozygous mutations.

Human Organoid and Supporting Technologies for Cancer and Toxicological Research.

Recent progress in the field of organoid-based cell culture systems has enabled the use of patient-derived cells in conditions that resemble those in cancer tissue, which are better than two-dimensional (2D) cultured cell lines. In particular, organoids allow human cancer cells to be handled in conditions that resemble those in cancer tissue, resulting in more efficient establishment of cells compared with 2D cultured cell lines, thus enabling the use of multiple patient-derived cells with cells from different genetic background, in keeping with the heterogeneity of the cells. One of the most valuable points of using organoids is that human cells from either healthy or cancerous tissue can be used. Using genome editing technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein, organoid genomes can be modified to, for example, cancer-prone genomes. The normal, cancer, or genome-modified organoids can be used to evaluate whether chemicals have genotoxic or non-genotoxic carcinogenic activity by evaluating the cancer incidence, cancer progression, and cancer metastasis. In this review, the organoid technology and the accompanying technologies were summarized and the advantages of organoid-based toxicology and its application to pancreatic cancer study were discussed.

Patient-Derived Midbrain Organoids to Explore the Molecular Basis of Parkinson's Disease.

Induced pluripotent stem cell-derived organoids offer an unprecedented access to complex human tissues that recapitulate features of architecture, composition and function of in vivo organs. In the context of Parkinson's Disease (PD), human midbrain organoids (hMO) are of significant interest, as they generate dopaminergic neurons expressing markers of Substantia Nigra identity, which are the most vulnerable to degeneration. Combined with genome editing approaches, hMO may thus constitute a valuable tool to dissect the genetic makeup of PD by revealing the effects of risk variants on pathological mechanisms in a representative cellular environment. Furthermore, the flexibility of organoid co-culture approaches may also enable the study of neuroinflammatory and neurovascular processes, as well as interactions with other brain regions that are also affected over the course of the disease. We here review existing protocols to generate hMO, how they have been used so far to model PD, address challenges inherent to organoid cultures, and discuss applicable strategies to dissect the molecular pathophysiology of the disease. Taken together, the research suggests that this technology represents a promising alternative to 2D in vitro models, which could significantly improve our understanding of PD and help accelerate therapeutic developments.

Estimating the concentration of therapeutic range using disease-specific iPS cells: Low-dose rapamycin therapy for Pendred syndrome.

INTRODUCTION: Pendred syndrome is an autosomal-recessive disease characterized by congenital hearing loss and thyroid goiter. Previously, cell stress susceptibilities were shown to increase in patient-derived cells with intracellular aggregation using an in vitro acute cochlear cell model derived from patient-specific pluripotent stem (iPS) cells. Moreover, we showed that rapamycin can relieve cell death. However, studies regarding long-term cell survival without cell stressors that mimic the natural course of disease or the rational minimum concentration of rapamycin that prevents cell death are missing. METHODS: In this report, we first investigated the rational minimum concentration of rapamycin using patient-specific iPS cells derived-cochlear cells with three different conditions of acute stress. We next confirmed the effects of rapamycin in long-term cell survival and phenotypes by using cochlear cells derived from three different patient-derived iPS cells. RESULTS: We found that inner ear cells derived from Pendred syndrome patients are more vulnerable than those from healthy individuals during long-term culturing; however, this susceptibility was relieved via treatment with low-dose rapamycin. The slow progression of hearing loss in patients may be explained, in part, by the vulnerability observed in patient cells during long-term culturing. We successfully evaluated the rational minimum concentration of rapamycin for treatment of Pendred syndrome. CONCLUSION: Our results suggest that low-dose rapamycin not only decreases acute symptoms but may prevent progression of hearing loss in Pendred syndrome patients.