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 SOderived 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.

Porcine pluripotent stem cells: progress, challenges and prospects

“…In human PGCs, the expression of endogenous KLF4 and C-MYC is similar to EGCs, but the expression levels of SOX2 and OCT4 are lower than EGCs. Thus, the reprogramming of PGCs into iPSCs can occur only by employing two transcription factors, SOX2 and OCT4 [55] . Only one study has found that a porcine iPSC line could be established by transfecting six human reprogramming factors (OCT4, SOX2, NANOG, KLF4, LIN28 and C-MYC) which possessed the ability to produce chimeric offspring [42] (Table 1).…”

Frequency and Markers of Precursor Lesions and Implications for the Pathogenesis of Testicular Germ Cell Tumors

“…A panel of OCT4, homeobox transcription factor NANOG (NANOG), Krüppel-like factor 4 (KLF4) and the oncogene avian myelocytomatosis viral oncogene homolog, MYC (C-MYC) might induce reprogramming of testicular germ cells to a stem cell pattern. 32 Correspondingly, malignant germ cells of GCNIS have an especially high expression of OCT4, NANOG, C. elegans homolog of LIN28 (LIN28), SRYbox 17 (SOX17), and KLF4. 33,34 These genetic similarities might be significant for the pathogenesis of TGCT.…”

“…Transfection with OCT4 and SRY-box 2 (SOX2) might make primordial germ cells undergo reprogramming to an induced pluripotent stem cell pattern, like that of EC. 32 Also in human fibroblasts, transduction of OCT4, SOX2, NANOG, and LIN28 can induce reprogramming to a pluripotent stem cell pattern. 35 Also, a combination of the transcription factors OCT4, NANOG, and KFL4 and the oncogene MYC might induce the stem cell pattern.…”

Environmental challenges may impact on somatic repair

“…In addition to this, protein homoeostasis in somatic cells is well-maintained when germ cells are damaged and it is significantly downgraded when germ cell function increases [28]. There exist mechanisms in germ cells which may induce somatic cell reprogramming and somatic stem cell pluripotency [29,30]. Kim et al [31] have shown that when male sticklebacks experience a benign and safe environment:…”