Hans R. Schöler

Formation of Pluripotent Stem Cells in the Mammalian Embryo Depends on the POU Transcription Factor Oct4

Oct4 is a mammalian POU transcription factor expressed by early embryo cells and germ cells. We report that the activity of Oct4 is essential for the identity of the pluripotential founder cell population in the mammalian embryo. Oct4-deficient embryos develop to the blastocyst stage, but the inner cell mass cells are not pluripotent. Instead, they are restricted to differentiation along the extraembryonic trophoblast lineage. Furthermore, in the absence of a true inner cell mass, trophoblast proliferation is not maintained in Oct4-/- embryos. Expansion of trophoblast precursors is restored, however, by an Oct4 target gene product, fibroblast growth factor-4. Therefore, Oct4 also determines paracrine growth factor signaling from stem cells to the trophectoderm.

Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors

Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility of generating patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4 (also known as Pou5f1), Sox2, c-Myc and Klf4 (refs 1–11). Considering that ectopic expression of c-Myc causes tumorigenicity in offspring and that retroviruses themselves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here we show that adult mouse neural stem cells express higher endogenous levels of Sox2 and c-Myc than embryonic stem cells, and that exogenous Oct4 together with either Klf4 or c-Myc is sufficient to generate iPS cells from neural stem cells. These two-factor iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germ line, and form chimaeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors.

OCT4-INDUCED PLURIPOTENCY IN ADULT NEURAL STEM CELLS

The four transcription factors Oct4, Sox2, Klf4, and c-Myc can induce pluripotency in mouse and human fibroblasts. We previously described direct reprogramming of adult mouse neural stem cells (NSCs) by Oct4 and either Klf4 or c-Myc. NSCs endogenously express Sox2, c-Myc, and Klf4 as well as several intermediate reprogramming markers. Here we report that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs. These one-factor induced pluripotent stem cells (1F iPS) are similar to embryonic stem cells in vitro and in vivo. Not only can these cells can be efficiently differentiated into NSCs, cardiomyocytes, and germ cells in vitro, but they are also capable of teratoma formation and germline transmission in vivo. Our results demonstrate that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency.

Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds.

Somatic cells can be induced into pluripotent stem cells (iPSCs) with a combination of four transcription factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtain patient-specific cells for various therapeutic and research applications. However, several problems remain for this approach to be therapeutically relevant due to drawbacks associated with efficiency and viral genome integration. Recently, it was shown that neural progenitor cells (NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs. However, NPCs express Sox2 endogenously, possibly facilitating reprogramming in the absence of exogenous Sox2. In this study, we identified a small-molecule combination, BIX-01294 and BayK8644, that enables reprogramming of Oct4/Klf4-transduced mouse embryonic fibroblasts, which do not endogenously express the factors essential for reprogramming. This study demonstrates that small molecules identified through a phenotypic screen can compensate for viral transduction of critical factors, such as Sox2, and improve reprogramming efficiency.

OCT-4: GATEKEEPER IN THE BEGINNINGS OF MAMMALIAN DEVELOPMENT

The Oct‐4 POU transcription factor is expressed in mouse totipotent embryonic stem and germ cells. Differentiation of totipotent cells to somatic lineages occurs at the blastocyst stage and during gastrulation, simultaneously with Oct‐4 downregulation. Stem cell lines derived from the inner cell mass and the epiblast of the mouse embryo express Oct‐4 only if undifferentiated. When embryonic stem cells are triggered to differentiate, Oct‐4 is downregulated thus providing a model for the early events linked to somatic differentiation in the developing embryo. In vivo mutagenesis has shown that loss of Oct‐4 at the blastocyst stage causes the cells of the inner cell mass to differentiate into trophectoderm cells. Recent experiments indicate that an Oct‐4 expression level of roughly 50%‐150% of the endogenous amount in embryonic stem cells is permissive for self‐renewal and maintenance of totipotency. However, upregulation above these levels causes stem cells to express genes involved in the lineage differentiation of primitive endoderm. These novel advances along with latest findings on Oct‐4‐associated factors, target genes, and dimerization ability, provide new insights into the understanding of the early steps regulating mammalian embryogenesis.

Direct reprogramming of human neural stem cells by OCT4

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of four transcription factors (OCT4 (also called POU5F1), SOX2, c-Myc and KLF4). We previously reported that Oct4 alone is sufficient to reprogram directly adult mouse neural stem cells to iPS cells. Here we report the generation of one-factor human iPS cells from human fetal neural stem cells (one-factor (1F) human NiPS cells) by ectopic expression of OCT4 alone. One-factor human NiPS cells resemble human embryonic stem cells in global gene expression profiles, epigenetic status, as well as pluripotency in vitro and in vivo. These findings demonstrate that the transcription factor OCT4 is sufficient to reprogram human neural stem cells to pluripotency. One-factor iPS cell generation will advance the field further towards understanding reprogramming and generating patient-specific pluripotent stem cells.

OCT-4 - A GERMLINE-SPECIFIC TRANSCRIPTION FACTOR MAPPING TO THE MOUSE T-COMPLEX

Oct‐4 is a maternally expressed octamer‐binding protein encoded by the murine Oct‐4 gene. It is present in unfertilized oocytes, but also in the inner cell mass and in primordial germ cells. Here we show that the ectopic expression of Oct‐4 in HeLa cells is sufficient for transcriptional activation from the octamer motif, indicating that Oct‐4 is a transcription factor. Therefore, Oct‐4 is the first transcription factor described that is specific for the early stages of mouse development. The spatial and temporal expression patterns were further determined using in situ hybridization. With this technique Oct‐4 expression is detected in the oocyte, in the blastocyst and before gastrulation in the embryonic ectoderm. After day 8 Oct‐4 expression decreases and is restricted to primordial germ cells from about day 8.5 onwards. Therefore Oct‐4 is a transcription factor that is specifically expressed in cells participating in the generation of the germline lineage. Linkage analysis using B X D recombinant inbred mouse strains demonstrates that Oct‐4 maps to chromosome 17 in or near the major histocompatibility complex. Several mouse mutants in the distal region of the mouse t‐complex affecting blastocyst and embryonic ectoderm formation also map to this region.

A family of octamer-specific proteins present during mouse embryogenesis: Evidence for germline-specific expression of an Oct factor.

We have analysed various adult organs and different developmental stages of mouse embryos for the presence of octamer‐binding proteins. A variety of new octamer‐binding proteins were identified in addition to the previously described Oct1 and Oct2. Oct1 is ubiquitously present in murine tissues, in agreement with cell culture data. Although Oct2 has been described as a B‐cell‐specific protein, similar complexes were also found with extracts from brain, kidney, embryo and sperm. In embryo and brain at least two other proteins, Oct3 and Oct7, are present. A new microextraction procedure allowed the detection of two maternally expressed octamer‐binding proteins, Oct4 and Oct5. Both proteins are present in unfertilized oocytes and embryonic stem cells, the latter containing an additional protein, Oct6. Whereas Oct4 was not found in sperm or testis, it is expressed in male and female primordial germ cells. Therefore Oct4 expression is specific for the female germline at later stages of germ cell development. Our results indicate that a family of octamer‐binding proteins is present during mouse development and is differentially expressed during early embryogenesis. Protease clipping experiments of Oct4 and Oct1 suggest that both proteins contain similar DNA‐binding domains.

Regulatory networks in embryo-derived pluripotent stem cells.

Mammalian development requires the specification of over 200 cell types from a single totipotent cell. Investigation of the regulatory networks that are responsible for pluripotency in embryo-derived stem cells is fundamental to understanding mammalian development and realizing therapeutic potential. Extracellular signals and second messengers modulate cell-autonomous regulators such as OCT4, SOX2 and Nanog in a combinatorial complexity. Knowledge of this circuitry might reveal how to achieve phenotypic changes without the genetic manipulation of Oct4, Nanog and other toti/pluripotency-associated genes.

Differential expression of the Oct-4 transcription factor during mouse germ cell differentiation.

The POU transcription factor Oct-4 is expressed in early mouse embryogenesis and in pluripotent ES and EC stem cell lines. After gastrulation in the embryo, Oct-4 expression is confined to the germline. The present study provides evidence that Oct-4 undergoes downregulation during oogenesis and spermatogenesis, coincident with entry into meiosis. Furthermore, analysis of maturation stages of oocytes showed that Oct-4 is upregulated de novo in the final stages of meiotic prophase I in female germ cells. These data suggest that Oct-4 downregulation in germ cells in both sexes might represent one of the molecular triggers involved in the commitment to meiosis. The upregulation of Oct-4 in oocytes at the completion of the prophase I of meiotic division further suggests a specific involvement of this transcription factor in oocyte growth or the acquisition of meiotic competence.