Scientists have developed a durable stand-in for human germ cell precursors that can be used to ethically study human development and reproductive tract cancers.
Explore the Shioda Lab
The Shioda laboratory is interested in human primordial germ cells (PGCs), the most upstream precursors of gametes (eggs and sperm). Due to the technical and ethical barriers to obtain PGCs from human embryos, a cell culture model of human PGC-like cells (hPGC-LCs) has been generated from pluripotent stem cells in several laboratories, including ours. Overcoming the well-known difficulties in maintaining hPGC-LC cultures over three weeks, our lab has recently accomplished long-term culture of actively proliferating hPGC-LCs that can be maintained for at least three months without losing their PGC state. This novel resource provides unprecedented opportunities to study development, epigenetics, and diseases of human PGCs. For example, as transformed male PGCs cause testicular cancer (the most common malignancy in juvenile and young-adult men), we took advantage of our long-term hPGC-LC culture and genetic engineering techniques and have successfully generated hPGC-LCs harboring various genetic mutations linked to testicular cancers. Attempts are currently ongoing in our lab to establish the first synthetic tumor model of human testicular cancer with defined genetic mutations.
Long-term maintenance of hPGC-LCs
Several laboratories, including ours, have developed various protocols of hPGC-LC production from human ESCs or iPSCs. Our previous study demonstrated strong transcriptomal similarities among hPGC-LCs generated using various protocols, supporting the notion that these hPGC-LCs are reflecting the same type of cells that exist in vivo – namely, embryonic PGCs. We also reported that our iPSC-derived hPGC-LCs reflect a very early stage of human embryonic PGCs during their passive midline movement before the CXCL12/CXCR4-guided, active lateral migration towards gonadal anlagen. The major technical barrier that prevents immediate application of hPGC-LCs to various biological studies such as chemical or CRISPR-knockout screening has been their limited lifespan in cell culture conditions and relatively low yields achievable by each experiment performed in the standard lab settings. The latest published study claimed only three weeks of hPGC-LC maintenance in cell culture before cells were lost or nonspecifically differentiated.
To overcome this hurdle, our laboratory has performed a systemic evaluation of cell culture conditions and successfully established a protocol that permits active proliferation of hPGC-LCs over at least three months without losing their PGC-like state. We have developed two variations of the long-term hPGC-LC culture protocols that reproducibly support stable maintenance of hPGC-LCs derived from various human iPSCs, males or females. The amplified hPGC-LCs are readily stored frozen in the conventional freezing media without losing viability or the PGC-like state. This novel resource will provide the relevant fields of research with unique and important opportunities to use practically unlimited amounts of hPGC-LCs without generating relatively small numbers of them from the precursor pluripotent stem cells each time through laborious protocols.
Genetic modeling of human testicular cancers
Testicular cancer is the most common malignancy that hits juvenile and young-adult males of 15-35 years old. The vast majority of various subtypes of testicular cancer is the Type II germ cell tumor, which derives from PGCs. Most pathologically diagnosed invasive testicular cancers are associated with adjacent Germ Cell Neoplasia In Situ (GCNIS), which consist of morphologically atypical PGC-like cells harboring chromosome 12p amplification in practically all cases and are believed to be precursor lesions of the invasive carcinoma. Testicular cancers uniquely lack genetic mutations commonly found in many other types of adult cancers, and instead they often harbor a specific set of mutations such as heterozygous gain-of-function. c-KIT, and genome-wide association studies have repeatedly suggested the involvement of the pro-apoptotic gene BAK1 in testicular carcinogenesis. Genes locating in chromosome 12p, such as the pluripotency gene NANOG, are also candidates of the driver genes.
However, due to the unexplained difficulties in modeling human testicular cancers in rodent models harboring genetic mutations (except for embryonic carcinoma, a subtype of testicular cancer), experimental demonstrations of the importance of these driver gene candidates in human testicular carcinogenesis are still awaited. Taking advantage of our long-term hPGC-LC culture technique, our lab has generated a panel of hPGC-LCs harboring various driver mutation candidates – for example, gain-of-function c-KIT knock-in, BAK1/BAX single or double mutations that remarkably affected cellular sensitivities to apoptotic stimuli, and/or overexpression of NANOG. Attempts are being made to establish hPGC-LC cell cultures that show reproducible evidence of partial or full malignant transformation caused by defined genetic changes.
On the other hand, in collaboration with Dr. Chin-Lee We of MGH Urological Pathology, we have established novel human testicular cancer cell lines with accompanying normal testicular somatic cell cultures. Attempts are being made in our lab to reprogram these cancerous and normal human testicular cells harboring the same genetic background into iPSCs and then hPGC-LCs. This compensating approach will determine whether genetic mutations alone can readily cause human testicular cancers.
Molecular Profiling Lab
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.
Diaz-Castillo C, Chamarro-Garcia R, Shioda T, and Blumberg B. Transgenerational self-reconstruction of disrupted chromatin organization after exposure to an environmental stressor in mice. Scientific Reports. 2019;9(1):13057.
Mitsunaga S, Shioda K, Owa C, Isselbacher KJ, Hanna JH, and Shioda T. Generation of human primordial germ cell-like cells at the surface of embryoid bodies from primed-pluripotency induced pluripotent stem cells. J Vis Exp. 2019; 11(143).
Taylor JA, Shioda K, Mitsunaga S, Yawata S, Angle BM, Nagel SC, vom Saal FS and Shioda T. Prenatal Exposure to Bisphenol A Disrupts Naturally Occurring Bimodal DNA Methylation at Proximal Promoter of fggy, an Obesity-Relevant Gene Encoding a Carbohydrate Kinase, in Gonadal White Adipose Tissues of CD-1 Mice. Endocrinology. 2018;159(2):779-794.
Mitsunaga S, Odajima J, Yawata S, Shioda K, Owa C, Isselbacher KJ, Hanna JH, and Shioda T. Relevance of iPSC-derived human PGC-like cells at the surface of embryoid bodies to prechemotaxis migrating PGCs. Proc Natl Acad Sci U S A. 2017; 114(46):E9913-E9922.
Chamorro-Garcia R, Diaz-Castillo C, Shoucri BM, Kach H, Leavitt R, Shioda T, and Blumberg B. Ancestral perinatal obesogen exposure results in a transgenerational thrifty phenotype in mice. Nature Communications. 2017; 8(1):2012.
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).
Emergence of human PGC-LCs on the surface of embryoid bodies. Human PGC-LCs are visualized by anti-OCT4 immunohistochemistry of FFPE slides.
Most PGC-LCs are localized in the outermost surface layer of embryoid bodies (left). PGC-LCs often form dense clusters (arrows; right), which may mimic the embryonic niche involved in germline commitment of precursor cells.
Toshihiro Shioda, MD, PhDPrincipal Investigator
- Hikari Hagihara, BS
- Ayako Nakashoji, MD, PhD
- Junko Odajima, PhD
- Keiko Shioda, RN, BS