Abstract
Induced pluripotent stem (iPS) cells have attracted enormous attention due to their vast potential in regenerative medicine, pharmaceutical screening and basic research. Most prior established iPS cell lines were derived and maintained on mouse embryonic fibroblast (MEF) cells supplemented with exogenous leukemia inhibitory factor (LIF). Drawbacks of MEF cells impede optimization as well as dissection of reprogramming events and limit the usage of iPS cell derivatives in therapeutic applications. In this study, we develop a reproducible protocol for efficient reprogramming mouse neural progenitor cells (NPCs) on human foreskin fibroblast (HFF) cells via retroviral transfer of human transcriptional factors OCT4/SOX2/KLF4/C-MYC. Two independent iPS cell lines are derived without exogenous LIF. They display typical undifferentiated morphology and express pluripotency markers Oct4 and Sox2. Transgenes are inactivated and the endogenous Oct4 promoter is completely demethylated in the established iPS cell lines, indicating a fully reprogrammed state. Moreover, the iPS cells can spontaneously differentiate or be induced into various cell types of three embryonic germ layers in vitro and in vivo when they are injected into immunodeficient mice for teratoma formation. Importantly, iPS cells extensively integrate with various host tissues and contribute to the germline when injected into the blastocysts. Interestingly, these two iPS cell lines, while both pluripotent, exhibit distinctive differentiation tendencies towards different lineages. Taken together, the data describe the first genuine mouse iPS cell lines generated on human feeder cells without exogenous LIF, providing a reliable tool for understanding the molecular mechanisms of nuclear reprogramming.
Highlights
The generation of induced pluripotent stem cells from mouse embryonic and adult fibroblasts by retroviral introduction of transcription factors (Oct4, Sox2, Klf4 and c-Myc) was first reported by Yamanaka and his colleagues in 2006 [1]
Primary culture of mouse neural progenitor cells (NPCs) Based upon reports that mouse NPCs could be converted to the pluripotent state with higher efficiency than fully differentiated somatic cells [19] or with fewer reprogramming factors [13,14,15], we utilized NPCs as recipient cells to test the feasibility of using human foreskin fibroblast (HFF) cells in derivation of induced pluripotent stem (iPS)
There were Oct4-positive spermatogonial stem cells and Oct4-negative spermatocytes, providing evidence of the contribution of injected iPS cells to the germline. This is the first report of generation and maintenance of mouse iPS cells on human feeder cells without exogenous leukemia inhibitory factor (LIF)
Summary
The generation of induced pluripotent stem (iPS) cells from mouse embryonic and adult fibroblasts by retroviral introduction of transcription factors (Oct, Sox, Klf and c-Myc) was first reported by Yamanaka and his colleagues in 2006 [1]. A drug-inducible reprogramming system was developed for multiple mouse cells [6]. Among the cell types tested, NPCs are interesting. They can be reprogrammed by ectopic expression of two factors (Oct and Klf4) or even one factor (Oct4) alone [14,15], providing a unique tool to analyze reprogramming events at a molecular level. Using NPCs, Silva et al delineated two phases in the reprogramming process (pre-pluripotency and ground state pluripotency) and demonstrated that dual inhibition (2i) of mitogen-activated protein kinase (MAKP) signaling and glycogen synthase kinase-3
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