title: Direct Reprogramming of Human Primordial Germ Cells into Induced Pluripotent Stem Cells: Efficient Generation of Genetically Engineered Germ Cells.
abstract: Primordial germ cells (PGCs) share many properties with embryonic stem cells (ESCs) and innately express several key pluripotency-controlling factors, including OCT4, NANOG, and LIN28. Therefore, PGCs may provide a simple and efficient model for studying somatic cell reprogramming to induced pluripotent stem cells (iPSCs), especially in determining the regulatory mechanisms that fundamentally define pluripotency. Here, we report a novel model of PGC reprogramming to generate iPSCs via transfection with SOX2 and OCT4 using integrative lentiviral. We also show the feasibility of using nonintegrative approaches for generating iPSC from PGCs using only these two factors. We show that human PGCs express endogenous levels of KLF4 and C-MYC protein at levels similar to embryonic germ cells (EGCs) but lower levels of SOX2 and OCT4. Transfection with both SOX2 and OCT4 together was required to induce PGCs to a pluripotent state at an efficiency of 1.71%, and the further addition of C-MYC increased the efficiency to 2.33%. Immunohistochemical analyses of the SO-derived PGC-iPSCs revealed that these cells were more similar to ESCs than EGCs regarding both colony morphology and molecular characterization. Although leukemia inhibitory factor (LIF) was not required for the generation of PGC-iPSCs like EGCs, the presence of LIF combined with ectopic exposure to C-MYC yielded higher efficiencies. Additionally, the SO-derived PGC-iPSCs exhibited differentiation into representative cell types from all three germ layers in vitro and successfully formed teratomas in vivo. Several lines were generated that were karyotypically stable for up to 24 subcultures. Their derivation efficiency and survival in culture significantly supersedes that of EGCs, demonstrating their utility as a powerful model for studying factors regulating pluripotency in future studies.
authors.affiliation: 1 Department of Biomedical Engineering, The Johns Hopkins University School of Medicine , Baltimore, Maryland.
authors.forename: Faith A
authors.initials: FA
authors.lastname: Bazley
authors.affiliation: 2 Department of Genecology and Obstetrics, The Johns Hopkins University School of Medicine , Baltimore, Maryland.|3 Institute for Cell Engineering, The Johns Hopkins University School of Medicine , Baltimore, Maryland.
authors.forename: Cyndi F
authors.initials: CF
authors.lastname: Liu
authors.affiliation: 4 Department of Medicine, Division of Hematology, The Johns Hopkins University School of Medicine , Baltimore, Maryland.
authors.forename: Xuan
authors.initials: X
authors.lastname: Yuan
authors.affiliation: 5 JHMI Deep Sequencing and Microarray Core, High Throughput Biology Center, Johns Hopkins University , Baltimore, Maryland.
authors.forename: Haiping
authors.initials: H
authors.lastname: Hao
authors.affiliation: 1 Department of Biomedical Engineering, The Johns Hopkins University School of Medicine , Baltimore, Maryland.
authors.forename: Angelo H
authors.initials: AH
authors.lastname: All
authors.affiliation: 6 Stem Cell Transplantation Program, Division of Pediatric Hematology Oncology, Children's Hospital Boston , Massachusetts.|7 Department of Biological Chemistry and Molecular Pharmacology, Dana-Farber Cancer Institute , Harvard Medical School, Boston, Massachusetts.|8 Harvard Stem Cell Institute , Cambridge, Massachusetts.
authors.forename: Alejandro
authors.initials: A
authors.lastname: De Los Angeles
authors.affiliation: 3 Institute for Cell Engineering, The Johns Hopkins University School of Medicine , Baltimore, Maryland.|9 Division of Pediatric Oncology at the Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine , Baltimore, Maryland.
authors.forename: Elias T
authors.initials: ET
authors.lastname: Zambidis
authors.affiliation: 10 Department of Cell and Developmental Biology, School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania.|11 Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania , Philadelphia, Pennsylvania.
authors.forename: John D
authors.initials: JD
authors.lastname: Gearhart
authors.affiliation: 2 Department of Genecology and Obstetrics, The Johns Hopkins University School of Medicine , Baltimore, Maryland.|12 Department of Biochemistry and Molecular Biology, University of Maryland , Baltimore, Maryland.
authors.forename: Candace L
authors.initials: CL
authors.lastname: Kerr
mesh_terms: D002478:Cells, Cultured; D065150:Cellular Reprogramming; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005854:Germ Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors