Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency.

Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. The level of octamer-binding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC.

Genetic modification of airway progenitors after lentiviral gene delivery to the amniotic fluid of murine fetuses.

Lentiviral vectors with the firefly luciferase or enhanced green fluorescent protein (EGFP) transgenes were delivered to the amniotic fluid of murine fetuses at Embryonic Day (E) 14.5 or E16.5. Whole-body imaging of luciferase recipients after birth demonstrated transgene expression in the peritoneal and thoracic regions. Organ imaging showed luciferase expression in lung, skin, stomach, and/or intestine. Histological immunofluorescence analysis of EGFP recipients demonstrated that small clusters (≤ three cells) of EGFP-positive epithelial cells were present in the large and small airways of recipients at up to 7 months (n = 11). There was no difference in the frequency of transgene expression in mice injected at E14.5 or E16.5 in respiratory or nonrespiratory organs. Analysis of the bronchoalveolar duct junctions on tissue sections of recipient mice identified multiple EGFP-positive epithelial cells. Cells coexpressing EGFP, Clara cell 10-kd protein, and surfactant protein C (SPC) were also found in lungs, consistent with the transduction of bronchoalveolar stem cells. Next, naphthalene lung injury in both luciferase and EGFP recipients was performed to determine whether transduced cells could contribute to tissue repair. In luciferase recipients, the whole-body luciferase signal increased 2- to 20-fold at 2 weeks after naphthalene treatment. Remarkably, immunohistological analysis of the lungs of EGFP recipients after lung injury repair demonstrated repopulation of airways with long stretches of EGFP-positive epithelial cells (n = 4). Collectively, these data demonstrate that lentiviral gene delivery to the amniotic fluid of murine fetuses genetically modifies long-lived epithelial progenitors capable of contributing to lung injury repair.