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Read the Shioda Lab 2017-2018 Annual Report
Toshihiro Shioda, MD, PhDAssociate Professor of MedicineHarvard Medical School
DirectorMolecular Profiling Laboratory
The Shioda laboratory is interested in biology and diseases of human germline, which is the specialized population of cells destined to generate sperm or eggs. The germline is solely responsible for conveying the entire genetic information to the next generation. Thus, all heritable, disease-causing genetic mutations occur only in the germline. The first germline cells, which are known as primordial germ cells (PGCs), are observed in human embryos during the third week of gestation as a cluster of only 40 cells, and this is the only single opportunity to generate the germline in each lifespan. Because of the extreme difficulty to obtain human PGCs for research, scientific knowledge of normal biology and mechanisms of genetic damages in human PGCs is very limited. To overcome this restriction, our laboratory has been generating PGC-like cell culture models from human induced pluripotent stem cells. Using these models, we attempt to examine how drugs or environmental factors can introduce disease-causing damages into the genome of human germline.
Toshihiro Shioda, MD, PhDPrincipal Investigator Group Members
In vitro generation of mammalian germ cells from pluripotent stem cells
The germline is the specialized population of cells destined for gametogenesis and solely responsible for conveying genetic and epigenetic information to the subsequent generation. During the third week of gestation, human primordial germ cells (PGCs) are observed in the wall of yolk sac as a cluster of only 40 cells. While rapidly proliferating, PGCs migrate towards genital ridges, supported by the SCF/KIT survival signal and guided by the SDF1/CXCR4 chemotaxis signal. PGCs colonized in the genital ridges differentiate into sex-specific germline cells upon gonadal sex differentiation.
All heritable genetic aberrations, including cancer predisposition mutations, occur exclusively in the germline. There is also evidence that shows that in utero exposure of mammalian embryos and/or fetuses to various types of environmental stresses such as therapeutic drugs, toxic industrial chemicals, or malnutrition may cause trans-generationally heritable epigenetic changes that could convey predisposition to various adult-onset diseases, including obesity and malignancies. However, epigenetic marks are subjected to global and robust erasure in PGCs and then re-established during later stages of germline differentiation. How the transgenerational epimutations can escape this reprogramming process is unknown.
One of the major hurdles of studying mammalian germline is the difficulty to obtain sufficient amounts of early-stage germline cells from embryos or fetuses. Moreover, animal experiments using rodent models often result in total absence of germline or embryonic lethality after exposure to stresses or genetic manipulations. To overcome these problems, we produce and characterize PGC-like cell culture models (PGC-LCs for PGC-Like Cells) from human and mouse pluripotent stem cells to establish their usefulness in studying mechanisms, effects, and prevention of germline mutations or heritable epigenetic aberrations. Last year we published a study demonstrating very robust and global DNA demethylation in the genome of mouse PGC-LCs, resembling the epigenetic erasure process in embryonic PGCs. Several types of repetitive elements, such as the IAP class endogenous retroviruses, escaped the global epigenetic erasure in mouse PGC-LCs as well as embryonic PGCs. Mouse PGC-LCs were also able to erase iPSC-derived, aberrant DNA hypermethylation at the Dlk1-Dio3 imprinting control region, and this is the first experimental demonstration that a specific epimutation is erased during the epigenetic reprogramming in the germline. To distinguish the epigenetic characteristics between paternally and maternally derived alleles, we have established mouse iPSC clones whose paternal and maternal chromosomes are derived from Mus spretus and Mus musculus, respectively. Taking advantage of their rich and evenly distributed SNPs, we are presently developing a computational data analysis pipeline for sensitive and quantitative determination of allele-biased gene expression and epigenetic marks in these iPS cells and their PGC-LC products.
Extending our PGC-LC studies, we have established a novel protocol for robust and highly reproducible production of human PGC-LCs from iPSCs. We have demonstrated a well-conserved transcriptomal signature of human PGC-LCs produced in multiple independent laboratories including ours, thus contributing to establish a foundation of practical applications of this novel cell culture model. Our ongoing study has been accumulating evidence that human PGC-LCs resemble embryonic PGCs in the early migrating stage, during which PGCs receive the SCF/KIT survival signal but not the SDF1/CXCR4 chemotactic signal yet. Because this stage of PGCs have been presumed as a precursor of the extra-gonadal germ cell tumors, we are trying to reconstitute the oncogenic process of germ cell tumors by introducing known cancer predisposition mutations into human PGC-LCs. Attempts are also being made to study mechanisms through which therapeutic drugs or toxic chemicals introduce genetic mutations and/or epigenetic aberrations in the genomes of human and mouse PGC-LCs.
The Molecular Profiling Laboratory (MPL) is one of the Cancer Center research centers that provide technological support to Cancer Center investigators.
Technologies available through MPL include Affymetrix DNA microarray (mRNA expression, tiling, resequencing, promoter, and SNP), RTQ-PCR (TaqMan assay), nucleic acid characterization (microscale UV spec and Agilent Bioanalyzer microcapillary electrophoresis), basic bioinformatics (array data analysis and computational data profiling), and RNAi knockdown of human and mouse genes using the shRNA lentivirus library clones generated by The RNAi Consortium (TRC) of the Broad Institute.
MPL is presently developing a laboratory automation system for RNAi knockdown screening using this resource.
Toshihiro Shioda, MD, PhD
View a list of publications by researchers at the Shioda Laboratory
Shoucri BM, Matinez ES, Abreo TJ, Hung VT, Moosova Z, Shioda T, and Blumberg B. Retinoid X receptor activation alters the chromatin landscape to commit mesenchymal stem cells to the adipose lineage. Endocrinology 2017 Oct 1;158(10):3109-3125.
Miyoshi N, Stel JM, Shioda K, Qu N, Odajima J, Mitsunaga S, Zhang X, Nagano M, Hochedlinger K, Isselbacher KJ, and Shioda T. Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells. Proc Natl Acad Sci U S A. 113(34):9545-50 (2016).
Miyoshi N, Wittner BS, Shioda K, Hirota T, Ito T, Ramaswamy S., Isselbacher KJ, Sgroi DC, and Shioda T. Nodes-and-connections RNAi knockdown screening: Identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis. Oncogenesis 4:e172 (2015).
National Research Council Committee (Berg AO, Bailor III JC, Gandolfi AJ, Kriebel D, Morris JB, Pinkerton KE, Rusyn I, Shioda T, Smith TJ, Wetzler M, ZeiseL, and Zweidler-McKay P). Review of the Formaldehyde Assessment in the National Toxicology Program 12th Report on Carcinogens. The National Academies Press (2014).
Janesick AS, Shioda T, Blumberg B. Transgenerational inheritance of prenatal obesogen exposure. Molecular and Cellular Endocrinology 398:31-35 (2014).
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