BACKGROUND

The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1.

RESULTS

In the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death.

CONCLUSIONS

Appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death.

Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments using stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with induced pluripotent stem cells (iPSCs) of swine. Here, we subjected iPSCs of swine to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real-time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1, NANOG, and SOX2 and induction of rod photoreceptor genes RCVRN, NRL, RHO, and ROM1. While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of rhodopsin (RHO) and rod outer segment-specific membrane protein 1 in outer segment-like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO+ cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that iPSCs of swine can differentiate into photoreceptors in culture, and these cells can integrate into the damaged swine neural retina, thus, laying a foundation for future studies using the pig as a model for retinal stem cell transplantation.

BACKGROUND

Given the significant drawbacks of using human embryonic stem (hES) cells for regenerative medicine, the search for alternative sources of multipotent cells is ongoing. Studies in mice have shown that multipotent ES-like cells can be derived from neonatal and adult testis. Here we report the derivation of ES-like cells from adult human testis.

METHODS

Testis material was donated for research by four men undergoing bilateral castration as part of prostate cancer treatment. Testicular cells were cultured using StemPro medium. Colonies that appeared sharp edged and compact were collected and subcultured under hES-specific conditions. Molecular characterization of these colonies was performed using RT-PCR and immunohistochemistry. (Epi)genetic stability was tested using bisulphite sequencing and karyotype analysis. Directed differentiation protocols in vitro were performed to investigate the potency of these cells and the cells were injected into immunocompromised mice to investigate their tumorigenicity.

RESULTS

In testicular cell cultures from all four men, sharp-edged and compact colonies appeared between 3 and 8 weeks. Subcultured cells from these colonies showed alkaline phosphatase activity and expressed hES cell-specific genes (Pou5f1, Sox2, Cripto1, Dnmt3b), proteins and carbohydrate antigens (POU5F1, NANOG, SOX2 and TRA-1-60, TRA-1-81, SSEA4). These ES-like cells were able to differentiate in vitro into derivatives of all three germ layers including neural, epithelial, osteogenic, myogenic, adipocyte and pancreatic lineages. The pancreatic beta cells were able to produce insulin in response to glucose and osteogenic-differentiated cells showed deposition of phosphate and calcium, demonstrating their functional capacity. Although we observed small areas with differentiated cell types of human origin, we never observed extensive teratomas upon injection of testis-derived ES-like cells into immunocompromised mice.

CONCLUSIONS

Multipotent cells can be established from adult human testis. Their easy accessibility and ethical acceptability as well as their non-tumorigenic and autogenic nature make these cells an attractive alternative to human ES cells for future stem cell therapies.

BACKGROUND

To investigate how long fetal germ cells retain pluripotency, which may be linked to their ability to transform into histologically variable tumours, we examined the expression of OCT-3/4 (POU5F1), a transcription factor essential for the maintenance of totipotency in embryonic stem cells.

METHODS

The ontogeny of expression of OCT-3/4 was studied in 74 specimens of normal human gonads during development and in 58 samples of gonads from cases with testicular dysgenesis syndrome (TDS), including disorders of sex differentiation and malignant changes.

RESULTS

OCT-3/4 expression was found in primordial germ cells during migration to the gonadal ridges and in the indifferent gonad. The expression in testes gradually decreased until approximately 20 weeks of gestation, and thereafter it was more rapidly down-regulated, but persisted in a few cells until 3-4 months of postnatal age, which coincides with the final differentiation of gonocytes into infantile spermatogonia. Subsequently, OCT-3/4 was not detected in normal testes. In the ovaries, OCT-3/4 was expressed in primordial oogonia, but was down-regulated in oocytes that formed primary follicles. The pattern of expression was heterogeneous in dysgenetic and intersex cases, with OCT-3/4-positive gonocytes detected in this series until 14 months of age. Visibly neoplastic gonadoblastoma and carcinoma in situ (CIS) expressed abundant OCT-3/4 regardless of the age.

CONCLUSIONS

In the human ovary, OCT-3/4 is silenced at the onset of the first meiotic prophase, whereas in the testis, down-regulation of OCT-3/4 is a gradual process associated with differentiation of gonocytes. This normal pattern of expression is disturbed in dysgenetic gonads, especially in rare intersex cases, thus increasing the risk of malignant transformation. The high abundance of OCT-3/4 in CIS cells is consistent with their early fetal origin and pluripotency.

BACKGROUND

Dedifferentiation occurs naturally in mature cell types during epimorphic regeneration in fish and some amphibians. Dedifferentiation also occurs in the induction of pluripotent stem cells when a set of transcription factors (Oct4, Sox2, Klf4 and c-Myc) is over expressed in mature cell types.

RESULTS

We hypothesised that there are parallels between dedifferentiation or reprogramming of somatic cells to induced pluripotent stem cells and the natural process of dedifferentiation during epimorphic regeneration. We analysed expression levels of the most commonly used pluripotency associated factors in regenerating and non-regenerating tissue and compared them with levels in a pluripotent reference cell. We found that some of the pluripotency associated factors (oct4/pou5f1, sox2, c-myc, klf4, tert, sall4, zic3, dppa2/4 and fut1, a homologue of ssea1) were expressed before and during regeneration and that at least two of these factors (oct4, sox2) were also required for normal fin regeneration in the zebrafish. However these factors were not upregulated during regeneration as would be expected if blastema cells acquired pluripotency.

CONCLUSIONS

By comparing cells from the regeneration blastema with embryonic pluripotent reference cells we found that induced pluripotent stem and blastema cells do not share pluripotency. However, during blastema formation some of the key reprogramming factors are both expressed and are also required for regeneration to take place. We therefore propose a link between partially reprogrammed induced pluripotent stem cells and the half way state of blastema cells and suggest that a common mechanism might be regulating these two processes.

Cancer stem cells (CSC) play a significant role in tumor progression, disease recurrence, and treatment failure. Here, we show that the endogenously expressed ETS transcription factor ESE3/EHF controls prostate epithelial cell differentiation and stem-like potential. We found that loss of ESE3/EHF induced epithelial-to-mesenchymal transition (EMT), stem-like features, and tumor-initiating and metastatic properties in prostate epithelial cells, and reexpression of ESE3/EHF inhibited the stem-like properties and tumorigenic potential of prostate cancer cells. Mechanistically, ESE3/EHF repressed the expression of key EMT and CSC genes, including TWIST1, ZEB2, BMI1, and POU5F1. Analysis of human tissue microarrays showed that reduced ESE3/EHF expression is an early event in tumorigenesis, frequently occurring independently of other ETS gene alterations. Additional analyses linked loss of ESE3/EHF expression to a distinct group of prostate tumors with distinctive molecular and biologic characteristics, including increased expression of EMT and CSC genes. Low ESE3/EHF expression was also associated with increased biochemical recurrence of prostate cancer and reduced overall survival after prostatectomy. Collectively, our findings define a key role for ESE3/EHF in the development of a subset of prostate tumors and highlight the clinical importance of identifying molecularly defined tumor subgroups.

We previously reported that Zscan4 showed heterogeneous expression patterns in mouse embryonic stem (ES) cells. To identify genes that show similar expression patterns, we carried out high-throughput in situ hybridization assays on ES cell cultures for 244 genes. Most of the genes are involved in transcriptional regulation, and were selected using microarray-based comparisons of gene expression profiles in ES and embryonal carcinoma (EC) cells versus differentiated cell types. Pou5f1 (Oct4, Oct3/4) and Krt8 (EndoA) were used as controls. Hybridization signals were detected on ES cell colonies for 147 genes (60%). The majority (136 genes) of them showed relatively homogeneous expression in ES cell colonies. However, we found that two genes unequivocally showed Zscan4-like spotted expression pattern (spot-in-colony pattern; Whsc2 and Rhox9). We also found that nine genes showed relatively heterogeneous expression pattern (mosaic-in-colony pattern: Zfp42/Rex1, Rest, Atf4, Pa2g4, E2f2, Nanog, Dppa3/Pgc7/Stella, Esrrb, and Fscn1). Among these genes, Zfp42/Rex1 showed unequivocally heterogeneous expression in individual ES cells prepared by the CytoSpin. These results show the presence of different types or states of cells within ES cell cultures otherwise thought to be undifferentiated and homogeneous, suggesting a previously unappreciated complexity in ES cell cultures.

The B1 SOX transcription factors SOX1/2/3/19 have been implicated in various processes of early embryogenesis. However, their regulatory functions in stages from the blastula to early neurula remain largely unknown, primarily because loss-of-function studies have not been informative to date. In our present study, we systematically knocked down the B1 sox genes in zebrafish. Only the quadruple knockdown of the four B1 sox genes sox2/3/19a/19b resulted in very severe developmental abnormalities, confirming that the B1 sox genes are functionally redundant. We characterized the sox2/3/19a/19b quadruple knockdown embryos in detail by examining the changes in gene expression through in situ hybridization, RT-PCR, and microarray analyses. Importantly, these phenotypic analyses revealed that the B1 SOX proteins regulate the following distinct processes: (1) early dorsoventral patterning by controlling bmp2b/7; (2) gastrulation movements via the regulation of pcdh18a/18b and wnt11, a non-canonical Wnt ligand gene; (3) neural differentiation by regulating the Hes-class bHLH gene her3 and the proneural-class bHLH genes neurog1 (positively) and ascl1a (negatively), and regional transcription factor genes, e.g., hesx1, zic1, and rx3; and (4) neural patterning by regulating signaling pathway genes, cyp26a1 in RA signaling, oep in Nodal signaling, shh, and mdkb. Chromatin immunoprecipitation analysis of the her3, hesx1, neurog1, pcdh18a, and cyp26a1 genes further suggests a direct regulation of these genes by B1 SOX. We also found an interesting overlap between the early phenotypes of the B1 sox quadruple knockdown embryos and the maternal-zygotic spg embryos that are devoid of pou5f1 activity. These findings indicate that the B1 SOX proteins control a wide range of developmental regulators in the early embryo through partnering in part with Pou5f1 and possibly with other factors, and suggest that the B1 sox functions are central to coordinating cell fate specification with patterning and morphogenetic processes occurring in the early embryo.

BACKGROUND

The post-translational addition of the monosaccharide O-linked beta-N-acetylglucosamine (O-GlcNAc) regulates the activity of a wide variety of nuclear and cytoplasmic proteins. The enzymes O-GlcNAc Transferase (Ogt) and O-GlcNAcase (Oga) catalyze, respectively, the attachment and removal of O-GlcNAc to target proteins. In adult mice, Ogt and Oga attenuate the response to insulin by modifying several components of the signal transduction pathway. Complete loss of ogt function, however, is lethal to mouse embryonic stem cells, suggesting that the enzyme has additional, unstudied roles in development. We have utilized zebrafish as a model to determine role of O-GlcNAc modifications in development. Zebrafish has two ogt genes, encoding six different enzymatic isoforms that are expressed maternally and zygotically.

RESULTS

We manipulated O-GlcNAc levels in zebrafish embryos by overexpressing zebrafish ogt, human oga or by injecting morpholinos against ogt transcripts. Each of these treatments results in embryos with shortened body axes and reduced brains at 24 hpf. The embryos had 23% fewer cells than controls, and displayed increased rates of cell death as early as the mid-gastrula stages. An extensive marker analysis indicates that derivatives of three germ layers are reduced to variable extents, and the embryos are severely disorganized after gastrulation. Overexpression of Ogt and Oga delayed epiboly and caused a severe disorganization of the microtubule and actin based cytoskeleton in the extra-embryonic yolk syncytial layer (YSL). The cytoskeletal defects resemble those previously reported for embryos lacking function of the Pou5f1/Oct4 transcription factor spiel ohne grenzen. Consistent with this, Pou5f1/Oct4 is modified by O-GlcNAc in human embryonic stem cells.

CONCLUSIONS

We conclude that O-GlcNAc modifications control the activity of proteins that regulate apoptosis and epiboly movements, but do not seem to regulate germ layer specification. O-GlcNAc modifies the transcription factor Spiel ohne grenzen/Pou5f1 and may regulate its activity.

The Oct4 protein, encoded by the Pou5f1 gene was the very first master gene, discovered 25 years ago, to be absolutely required for the stemness properties of murine and primate embryonic stem cells. This transcription factor, which has also been shown to be essential for somatic cell reprogrammation, displays various functions depending upon its level of expression and has been quoted as a "rheostat" gene. Oct4 protein is in complexes with many different partners and its activity depends upon fine post-translational modifications. This review aims at revisiting some properties of this protein, which has not yet delivered all its potentialities.

Cancer stem-like cells (CSLC) are crucial in tumor initiation and progression; however, the underlying mechanism for the self-renewal of cancer cells remains undefined. In the study, immunohistochemical analysis of specimens freshly excised from patients with lung adenocarcinoma showed that high expression of insulin-like growth factor I receptor (IGF-IR) in lung adenocarcinoma cells was positively correlated with the expressions of cancer stem cell markers CD133 and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). IGF-IR activation enhanced POU class 5 homeobox 1 (POU5F1) expression on human lung adenocarcinoma stem-like cells (LACSLC) through PI3K/AKT/GSK3β/β-catenin cascade. POU5F1 could form a novel complex with β-catenin and SOX2 to bind Nanog promoter for transcription to maintain self-renewal of LACSLCs, which was dependent on the functional IGF-IR. Genetic and pharmacologic inhibition of IGF-IR abrogated LACSLC capabilities for self-renewal and tumorigenicity in vitro. In an in vivo xenograft tumor model, knockdown of either IGF-IR or POU5F1 impeded tumorigenic potentials of LACSLCs. By analyzing pathologic specimens excised from 200 patients with lung adenocarcinoma, we found that colocalization of highly expressed IGF-IR with β-catenin and POU5F1 predicted poor prognosis. Taken together, we show that IGF-IR-mediated POU5F1 expression to form a complex with β-catenin and SOX2 is crucial for the self-renewal and oncogenic potentials of LACSLCs, and the integrative clinical detection of the expressions of IGF-IR, β-catenin, and POU5F1 is indicatory for predicting prognosis in the patients of lung adenocarcinoma.

Histone H3 methylation at R17 and R26 recently emerged as a novel epigenetic mechanism regulating pluripotency in mouse embryos. Blastomeres of four-cell embryos with high H3 methylation at these sites show unrestricted potential, whereas those with lower levels cannot support development when aggregated in chimeras of like cells. Increasing histone H3 methylation, through expression of coactivator-associated-protein-arginine-methyltransferase 1 (CARM1) in embryos, elevates expression of key pluripotency genes and directs cells to the pluripotent inner cell mass. We demonstrate CARM1 is also required for the self-renewal and pluripotency of embryonic stem (ES) cells. In ES cells, CARM1 depletion downregulates pluripotency genes leading to their differentiation. CARM1 associates with Oct4/Pou5f1 and Sox2 promoters that display detectable levels of R17/26 histone H3 methylation. In CARM1 overexpressing ES cells, histone H3 arginine methylation is also at the Nanog promoter to which CARM1 now associates. Such cells express Nanog at elevated levels and delay their response to differentiation signals. Thus, like in four-cell embryo blastomeres, histone H3 arginine methylation by CARM1 in ES cells allows epigenetic modulation of pluripotency.

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

The modular DNA recognition code of the transcription-activator-like effectors (TALEs) from plant pathogenic bacterial genus Xanthomonas provides a powerful genetic tool to create designer transcription factors (dTFs) targeting specific DNA sequences for manipulating gene expression. Previous studies have suggested critical roles of enhancers in gene regulation and reprogramming. Here, we report dTF activator targeting the distal enhancer of the Pou5f1 (Oct4) locus induces epigenetic changes, reactivates its expression, and substitutes exogenous OCT4 in reprogramming mouse embryonic fibroblast cells (MEFs) to induced pluripotent stem cells (iPSCs). Similarly, dTF activator targeting a Nanog enhancer activates Nanog expression and reprograms epiblast stem cells (EpiSCs) to iPSCs. Conversely, dTF repressors targeting the same genetic elements inhibit expression of these loci, and effectively block reprogramming. This study indicates that dTFs targeting specific enhancers can be used to study other biological processes such as transdifferentiation or directed differentiation of stem cells.

Lhx8 is a member of the LIM-homeobox transcription factor family and preferentially expressed in oocytes and germ cells within the mouse ovary. We discovered that Lhx8 knockout females lose oocytes within 7 days after birth. At the time of birth, histological examination shows that Lhx8-deficient (Lhx8-/-) ovaries are grossly similar to the newborn wild-type ovaries. Lhx8-/- ovaries fail to maintain the primordial follicles, and the transition from primordial to growing follicles does not occur. Lhx8-/- ovaries misexpress oocyte-specific genes, such as Gdf9, Pou5f1, and Nobox. Very rapid loss of oocytes may partly be due to the drastic downregulation of Kit and Kitl in Lhx8-/- ovaries. We compared Lhx8-/- and wild-type ovaries using an Affymetrix 430 2.0 microarray platform. A total of 80 (44%) of 180 of the genes downregulated more than 5-fold in Lhx8-/- ovaries were preferentially expressed in oocytes, whereas only 3 (2%) of 146 genes upregulated more than 5-fold in the absence of Lhx8 were preferentially expressed in oocytes. In addition, the comparison of genes regulated in Lhx8-/- and Nobox-/- newborn ovaries discovered a common set of 34 genes whose expression level was affected in both Lhx8- and Nobox-deficient mice. Our findings show that Lhx8 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis, and it acts in part by downregulating the Nobox pathway.

OCT4 (POU5F1) is a transcription factor expressed in embryonic stem and germ cells and is involved in the regulation and maintenance of pluripotency. It has been detected in primary testicular germ cell tumors with pluripotent potential, seminoma, and embryonal carcinoma. We undertook immunohistochemical staining of OCT4 in a wide variety of primary testicular neoplasms (germ cell tumors and other tumors) to assess the specificity and usefulness of this marker as a diagnostic tool. We examined histologic sections from 91 primary testicular neoplasms, including 64 cases of mixed germ cell tumors containing embryonal carcinoma (54), seminoma (51), yolk sac tumor (38), mature teratoma (31), immature teratoma (20), and choriocarcinoma (15). In addition, we examined sections from spermatocytic seminomas (5), Leydig cell tumors (8), Sertoli cell tumors (6), unclassified sex-cord stromal tumors (4), adenomatoid tumors (2), testicular tumor of adrenogenital syndrome (1), and granulosa cell tumor (1). Each tumor was examined with hematoxylin and eosin staining and with antibodies to OCT4. In all cases of mixed germ cell tumor with components of embryonal carcinoma (54) and seminoma (51), there was greater than 90% nuclear staining of the embryonal carcinoma and seminoma tumor cells with little to no background staining. In all but 1 of these cases (embryonal carcinoma), there was strong (3+) staining intensity. The other germ cell tumor components (yolk sac tumor, mature teratoma, immature teratoma, and choriocarcinoma) showed no staining. Syncytiotrophoblast cells, which were present in 15 of the cases, were also completely negative, as were all 5 of the spermatocytic seminomas. The 22 cases of non-germ cell tumors were all immunohistochemically negative for OCT4. Fifteen of the 54 germ cell tumors containing embryonal carcinoma were also examined with antibodies to CD30. These embryonal carcinoma components were all positive for CD30 with staining of greater than 90% of the tumor cells but with variable staining intensity. We conclude that immunostaining with antibodies to OCT4 is a useful diagnostic tool in the identification of primary testicular embryonal carcinomas and "usual," but not spermatocytic, seminomas. OCT4 immunostaining has comparable sensitivity but greater consistency compared with CD30 in the diagnosis of embryonal carcinoma.

pou5f1, also known as Oct4, is required to establish the pluripotent cell population necessary for embryogenesis in mouse. Additional roles during development, including endoderm formation, have been proposed. In zebrafish, the zygotic pou5f1/pou2 mutant spiel ohne grenzen (spg) shows neural plate patterning defects and reduced endoderm at the tailbud stage. To investigate the function of maternal and early zygotic pou5f1 expression, we rescued zygotic spg(m793) mutants by injecting pou5f1 mRNA at the one-cell stage and raised them into fertile homozygous spg(m793) adults that mate to produce maternal-zygotic spg (MZspg) mutant embryos. Although neurectoderm, mesoderm, and germ cells develop in MZspg mutants, gastrulation is delayed and proceeds abnormally. Further, MZspg mutants do not maintain expression of sox32/casanova, express little or no sox17, and fail to develop endodermal tissue. Constitutively active Nodal receptor TARAM-A or sox32 overexpression induces ubiquitous sox17 expression in wild-type embryos, but not in MZspg mutants. Overexpression of a Pou5f1-VP16 activator fusion protein can rescue gastrulation and endodermal tissues in MZspg mutants. We propose that pou5f1 plays an activating role in zebrafish endodermal development, where it maintains sox32 expression during gastrulation and acts with sox32 to induce sox17 expression in endodermal precursor cells.

Reprogramming somatic cells to become induced pluripotent stem cells (iPSCs) by using defined factors represents an important breakthrough in biology and medicine, yet remains inefficient and poorly understood. We therefore devised synthetic factors by fusing the VP16 transactivation domain to OCT4 (also known as Pou5f1), NANOG and SOX2, respectively. These synthetic factors could reprogramme both mouse and human fibroblasts with enhanced efficiency and accelerated kinetics. Remarkably, Oct4-VP16 alone could efficiently reprogramme mouse embryonic fibroblasts (MEFs) into germline-competent iPSCs. Furthermore, episomally delivered synthetic factors could reproducibly generate integration-free iPSCs from MEFs with enhanced efficiency. Our results not only demonstrate the feasibility of engineering more potent reprogramming factors, but also suggest that transcriptional reactivation of OCT4 target genes might be a rate-limiting step in the conversion of somatic cells to pluripotent cells. Synthetic factor-based reprogramming might lead to a paradigm shift in reprogramming research.

Stem cells are characterized by two defining features, the ability to self-renew and to differentiate into highly specialized cell types. The POU homeodomain transcription factor Oct4 (Pou5f1) is an essential mediator of the embryonic stem cell state and has been implicated in lineage specific differentiation, adult stem cell identity, and cancer. Recent description of the regulatory networks which maintain 'ES' have highlighted a dual role for Oct4 in the transcriptional activation of genes required to maintain self-renewal and pluripotency while concomitantly repressing genes which facilitate lineage specific differentiation. However, the molecular mechanism by which Oct4 mediates differential activation or repression at these loci to either maintain stem cell identity or facilitate the emergence of alternate transcriptional programs required for the realization of lineage remains to be elucidated. To further investigate Oct4 function, we employed gene expression profiling together with a robust statistical analysis to identify genes highly correlated to Oct4. Gene Ontology analysis to categorize overrepresented genes has led to the identification of themes which may prove essential to stem cell identity, including chromatin structure, nuclear architecture, cell cycle control, DNA repair, and apoptosis. Our experiments have identified previously unappreciated roles for Oct4 for firstly, regulating chromatin structure in a state consistent with self-renewal and pluripotency, and secondly, facilitating the expression of genes that keeps the cell poised to respond to cues that lead to differentiation. Together, these data define the mechanism by which Oct4 orchestrates cellular regulatory pathways to enforce the stem cell state and provides important insight into stem cell function and cancer.

Considerable levels of testosterone and dihydrotestosterone (DHT) are found in prostate cancer (PCa) tissue after androgen deprivation therapy. Treatment of surviving cancer-initiating cells and the ability to metabolize steroids from precursors may be the keystones for the appearance of recurrent tumors. To study this hypothesis, we assessed the expression of several steroidogenic enzymes and stem cell markers in clinical PCa samples and cell cultures during androgen depletion. Gene expression profiles were determined by microarray or qRT-PCR. In addition, we measured cell viability and analyzed stem cell marker expression in DuCaP cells by immunocytochemistry. Seventy patient samples from different stages of PCa, and the PCa cell line DuCaP were included in this study. The androgen receptor (AR) and enzymes (AKR1C3, HSD17B2, HSD17B3, UGT2B15 and UGT2B17 ) that are involved in the metabolism of adrenal steroids were upregulated in castration resistant prostate cancer (CRPC). In vitro, some DuCaP cells survived androgen depletion, and eventually gave rise to a culture adapted to these conditions. During and after this transition, most of the steroidogenic enzymes were upregulated. These cells also are enriched with stem/progenitor cell markers cytokeratin 5 (CK5) and ATP-binding cassette sub-family G member 2 (ABCG2). Similarly, putative stem/progenitor cell markers CK5, c-Kit, nestin, CD44, c-met, ALDH1A1, α2-integrin, CD133, ABCG2, CXCR4 and POU5F1 were upregulated in clinical CRPC. The upregulation of steroidogenic enzymes and stem cell markers in recurrent tumors suggests that cancer initiating cells can expand by adaptation to their T/DHT deprived environment. Therapies targeting the metabolism of adrenal steroids by the tumor may prove effective in preventing tumor regrowth.

Although genome-wide hypomethylation is a hallmark of many cancers, roles for active DNA demethylation during tumorigenesis are unknown. Here, loss of the APC tumor suppressor gene causes upregulation of a DNA demethylase system and the concomitant hypomethylation of key intestinal cell fating genes. Notably, this hypomethylation maintained zebrafish intestinal cells in an undifferentiated state that was released upon knockdown of demethylase components. Mechanistically, the demethylase genes are directly activated by Pou5f1 and Cebpβ and are indirectly repressed by retinoic acid, which antagonizes Pou5f1 and Cebpβ. Apc mutants lack retinoic acid as a result of the transcriptional repression of retinol dehydrogenase l1 via a complex that includes Lef1, Groucho2, Ctbp1, Lsd1, and Corest. Our findings imply a model wherein APC controls intestinal cell fating through a switch in DNA methylation dynamics. Wild-type APC and retinoic acid downregulate demethylase components, thereby promoting DNA methylation of key genes and helping progenitors commit to differentiation.

Despite new therapies, breast cancer continues to be the second leading cause of cancer mortality in women, a consequence of recurrence and metastasis. In recent years, a population of cancer cells has been identified, called cancer stem cells (CSCs) with self-renewal capacity, proposed to underlie tumor recurrence and metastasis. We previously showed that the adipose tissue cytokine LEPTIN, increased in obesity, promotes the survival of CSCs in vivo. Here, we tested the hypothesis that the leptin receptor (LEPR), expressed in mammary cancer cells, is necessary for maintaining CSC-like and metastatic properties. We silenced LEPR via shRNA lentivirus transduction and determined that the expression of stem cell self-renewal transcription factors NANOG, SOX2, and OCT4 (POU5F1) is inhibited. LEPR-NANOG signaling pathway is conserved between species because we can rescue NANOG expression in human LEPR-silenced cells with the mouse LepR. Using a NANOG promoter GFP reporter, we showed that LEPR is enriched in NANOG promoter active (GFP+) cells. In lineage tracing studies, we showed that the GFP+ cells divide in a symmetric and asymmetric manner. LEPR-silenced MDA-MB-231 cells exhibit a mesenchymal to epithelial transition morphologically, increased E-CADHERIN and decreased VIMENTIN expression compared with control cells. Finally, LEPR-silenced cells exhibit reduced cell proliferation, self-renewal in tumor sphere assays, and tumor outgrowth in xenotransplant studies. Given the emergence of NANOG as a pro-carcinogenic protein in multiple cancers, these studies suggest that inhibition of LEPR may be a promising therapeutic approach to inhibit NANOG and thereby neutralize CSC functions.

Canonical Wnt signaling supports the pluripotency of embryonic stem cells (ESCs) but also promotes differentiation of early mammalian cell lineages. To explain these paradoxical observations, we explored the gene regulatory networks at play. Canonical Wnt signaling is intertwined with the pluripotency network comprising Nanog, Oct4, and Sox2 in mouse ESCs. In defined media supporting the derivation and propagation of ESCs, Tcf3 and β-catenin interact with Oct4; Tcf3 binds to Sox motif within Oct-Sox composite motifs that are also bound by Oct4-Sox2 complexes. Furthermore, canonical Wnt signaling upregulates the activity of the Pou5f1 distal enhancer via the Sox motif in ESCs. When viewed in the context of published studies on Tcf3 and β-catenin mutants, our findings suggest Tcf3 counters pluripotency by competition with Sox2 at these sites, and Tcf3 inhibition is blocked by β-catenin entry into this complex. Wnt pathway stimulation also triggers β-catenin association at regulatory elements with classic Lef/Tcf motifs associated with differentiation programs. The failure to activate these targets in the presence of a mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor essential for ESC culture suggests MEK/ERK signaling and canonical Wnt signaling combine to promote ESC differentiation.

The fields of both developmental and stem cell biology explore how functionally distinct cell types arise from a self-renewing founder population. Multipotent, proliferative human neural crest cells (hNCC) develop toward the end of the first month of pregnancy. It is assumed that most differentiate after migrating throughout the organism, although in animal models neural crest stem cells reportedly persist in postnatal tissues. Molecular pathways leading over time from an invasive mesenchyme to differentiated progeny such as the dorsal root ganglion, the maxillary bone or the adrenal medulla are altered in many congenital diseases. To identify additional components of such pathways, we derived and maintained self-renewing hNCC lines from pharyngulas. We show that, unlike their animal counterparts, hNCC are able to self-renew ex vivo under feeder-free conditions. While cross species comparisons showed extensive overlap between human, mouse and avian NCC transcriptomes, some molecular cascades are only active in the human cells, correlating with phenotypic differences. Furthermore, we found that the global hNCC molecular profile is highly similar to that of pluripotent embryonic stem cells when compared with other stem cell populations or hNCC derivatives. The pluripotency markers NANOG, POU5F1 and SOX2 are also expressed by hNCC, and a small subset of transcripts can unambiguously identify hNCC among other cell types. The hNCC molecular profile is thus both unique and globally characteristic of uncommitted stem cells.