Showing papers on "SOX2 published in 2009"
TL;DR: It is shown that the individual PB insertions can be removed from established iPS cell lines, providing an invaluable tool for discovery, and the traceless removal of reprogramming factors joined with viral 2A sequences delivered by a single transposon from murine iPS lines is demonstrated.
Abstract: Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state. The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a spectrum of adult cell types. Current reprogramming strategies involve retroviral, lentiviral, adenoviral and plasmid transfection to deliver reprogramming factor transgenes. Although the latter two methods are transient and minimize the potential for insertion mutagenesis, they are currently limited by diminished reprogramming efficiencies. piggyBac (PB) transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines. The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events. Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition. Stable iPS cells thus generated express characteristic pluripotency markers and succeed in a series of rigorous differentiation assays. By taking advantage of the natural propensity of the PB system for seamless excision, we show that the individual PB insertions can be removed from established iPS cell lines, providing an invaluable tool for discovery. In addition, we have demonstrated the traceless removal of reprogramming factors joined with viral 2A sequences delivered by a single transposon from murine iPS lines. We anticipate that the unique properties of this virus-independent simplification of iPS cell production will accelerate this field further towards full exploration of the reprogramming process and future cell-based therapies.
1,884 citations
TL;DR: It is proposed that ZEB1 links EMT-activation and stemness-maintenance by suppressingstemness-inhibiting microRNAs (miRNAs) and thereby is a promoter of mobile, migrating cancer stem cells.
Abstract: Invasion and metastasis of carcinomas is promoted by the activation of the embryonic 'epithelial to mesenchymal transition' (EMT) program, which triggers cellular mobility and subsequent dissemination of tumour cells. We recently showed that the EMT-activator ZEB1 (zinc finger E-box binding homeobox 1) is a crucial promoter of metastasis and demonstrated that ZEB1 inhibits expression of the microRNA-200 (miR-200) family, whose members are strong inducers of epithelial differentiation. Here, we report that ZEB1 not only promotes tumour cell dissemination, but is also necessary for the tumour-initiating capacity of pancreatic and colorectal cancer cells. We show that ZEB1 represses expression of stemness-inhibiting miR-203 and that candidate targets of miR-200 family members are also stem cell factors, such as Sox2 and Klf4. Moreover, miR-200c, miR-203 and miR-183 cooperate to suppress expression of stem cell factors in cancer cells and mouse embryonic stem (ES) cells, as demonstrated for the polycomb repressor Bmi1. We propose that ZEB1 links EMT-activation and stemness-maintenance by suppressing stemness-inhibiting microRNAs (miRNAs) and thereby is a promoter of mobile, migrating cancer stem cells. Thus, targeting the ZEB1-miR-200 feedback loop might form the basis of a promising treatment for fatal tumours, such as pancreatic cancer.
1,622 citations
TL;DR: It is shown that non-viral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc, Klf4, Oct4 and Sox2 linked with 2A peptides, can reprogram both mouse and human fibroblasts and can be removed once reprogramming has been achieved.
Abstract: Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to transform regenerative medicine. Most instances of direct reprogramming have been achieved by forced expression of defined factors using multiple viral vectors. However, such iPS cells contain a large number of viral vector integrations, any one of which could cause unpredictable genetic dysfunction. Whereas c-Myc is dispensable for reprogramming, complete elimination of the other exogenous factors is also desired because ectopic expression of either Oct4 (also known as Pou5f1) or Klf4 can induce dysplasia. Two transient transfection-reprogramming methods have been published to address this issue. However, the efficiency of both approaches is extremely low, and neither has been applied successfully to human cells so far. Here we show that non-viral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc, Klf4, Oct4 and Sox2 linked with 2A peptides, can reprogram both mouse and human fibroblasts. Moreover, the transgene can be removed once reprogramming has been achieved. iPS cells produced with this non-viral vector show robust expression of pluripotency markers, indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimaeric mice. When the single-vector reprogramming system was combined with a piggyBac transposon, we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This system minimizes genome modification in iPS cells and enables complete elimination of exogenous reprogramming factors, efficiently providing iPS cells that are applicable to regenerative medicine, drug screening and the establishment of disease models.
1,373 citations
TL;DR: Functional analyses of 34 p53-regulated genes demonstrate that the p53–p21 pathway serves as a barrier not only in tumorigenicity, but also in iPS cell generation in mouse and human fibroblasts.
Abstract: Induced pluripotent stem (iPS) cells can be generated from somatic cells by the introduction of Oct3/4 (also known as Pou5f1), Sox2, Klf4 and c-Myc, in mouse and in human. The efficiency of this process, however, is low. Pluripotency can be induced without c-Myc, but with even lower efficiency. A p53 (also known as TP53 in humans and Trp53 in mice) short-interfering RNA (siRNA) was recently shown to promote human iPS cell generation, but the specificity and mechanisms remain to be determined. Here we report that up to 10% of transduced mouse embryonic fibroblasts lacking p53 became iPS cells, even without the Myc retrovirus. The p53 deletion also promoted the induction of integration-free mouse iPS cells with plasmid transfection. Furthermore, in the p53-null background, iPS cells were generated from terminally differentiated T lymphocytes. The suppression of p53 also increased the efficiency of human iPS cell generation. DNA microarray analyses identified 34 p53-regulated genes that are common in mouse and human fibroblasts. Functional analyses of these genes demonstrate that the p53-p21 pathway serves as a barrier not only in tumorigenicity, but also in iPS cell generation.
1,330 citations
TL;DR: It is concluded that human iPS cells are a viable option as an autologous cell source for cardiac repair and a powerful tool for cardiovascular research.
Abstract: Human induced pluripotent stem (iPS) cells hold great promise for cardiovascular research and therapeutic applications, but the ability of human iPS cells to differentiate into functional cardiomyocytes has not yet been demonstrated. The aim of this study was to characterize the cardiac differentiation potential of human iPS cells generated using OCT4, SOX2, NANOG, and LIN28 transgenes compared to human embryonic stem (ES) cells. The iPS and ES cells were differentiated using the embryoid body (EB) method. The time course of developing contracting EBs was comparable for the iPS and ES cell lines, although the absolute percentages of contracting EBs differed. RT-PCR analyses of iPS and ES cell-derived cardiomyocytes demonstrated similar cardiac gene expression patterns. The pluripotency genes OCT4 and NANOG were downregulated with cardiac differentiation, but the downregulation was blunted in the iPS cell lines due to residual transgene expression. Proliferation of iPS and ES cell derived-cardiomyocytes based on BrdU labeling was similar, and immunocytochemistry of isolated cardiomyocytes revealed indistinguishable sarcomeric organizations. Electrophysiology studies indicated that iPS cells have a capacity like ES cells for differentiation into nodal-, atrial-, and ventricular-like phenotypes based on action potential characteristics. Both iPS and ES cell-derived cardiomyocytes exhibited responsiveness to β-adrenergic stimulation manifest by an increase in spontaneous rate and a decrease in action potential duration. We conclude that human iPS cells can differentiate into functional cardiomyocytes, and thus iPS cells are a viable option as an autologous cell source for cardiac repair and a powerful tool for cardiovascular research.
1,327 citations
TL;DR: It is reported here that expression of microRNA-145 (miR-145) is low in self-renewing human embryonic stem cells (hESCs) but highly upregulated during differentiation, and a double-negative feedback loop involving OCT4, SOX2, KLF4, and miR- 145 is uncovered.
1,133 citations
TL;DR: It is shown that reprogramming factors can activate the p53 (also known as Trp53 in mice, TP53 in humans) pathway and silencing of p53 significantly increased the reprograming efficiency of human somatic cells.
Abstract: Reprogramming somatic cells to induced pluripotent stem (iPS) cells has been accomplished by expressing pluripotency factors and oncogenes, but the low frequency and tendency to induce malignant transformation compromise the clinical utility of this powerful approach. We address both issues by investigating the mechanisms limiting reprogramming efficiency in somatic cells. Here we show that reprogramming factors can activate the p53 (also known as Trp53 in mice, TP53 in humans) pathway. Reducing signalling to p53 by expressing a mutated version of one of its negative regulators, by deleting or knocking down p53 or its target gene, p21 (also known as Cdkn1a), or by antagonizing reprogramming-induced apoptosis in mouse fibroblasts increases reprogramming efficiency. Notably, decreasing p53 protein levels enabled fibroblasts to give rise to iPS cells capable of generating germline-transmitting chimaeric mice using only Oct4 (also known as Pou5f1) and Sox2. Furthermore, silencing of p53 significantly increased the reprogramming efficiency of human somatic cells. These results provide insights into reprogramming mechanisms and suggest new routes to more efficient reprogramming while minimizing the use of oncogenes.
1,111 citations
TL;DR: Quantitative analyses define distinct cell-division-rate-dependent and -independent modes for accelerating the stochastic course of reprogramming, and suggest that the number of cell divisions is a key parameter driving epigenetic reprograming to pluripotency.
Abstract: Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc transcription factors, but only a minority of donor somatic cells can be reprogrammed to pluripotency. Here we demonstrate that reprogramming by these transcription factors is a continuous stochastic process where almost all mouse donor cells eventually give rise to iPS cells on continued growth and transcription factor expression. Additional inhibition of the p53/p21 pathway or overexpression of Lin28 increased the cell division rate and resulted in an accelerated kinetics of iPS cell formation that was directly proportional to the increase in cell proliferation. In contrast, Nanog overexpression accelerated reprogramming in a predominantly cell-division-rate-independent manner. Quantitative analyses define distinct cell-division-rate-dependent and -independent modes for accelerating the stochastic course of reprogramming, and suggest that the number of cell divisions is a key parameter driving epigenetic reprogramming to pluripotency.
1,026 citations
TL;DR: It is concluded that the silencing of Ink4/Arf locus is rate-limiting for reprogramming, and its transient inhibition may significantly improve the generation of iPS cells.
Abstract: The Ink4/Arf tumour suppressor locus encodes three potent tumour suppressors, namely p16Ink4a, p15Ink4b and p19Arf. Here Li et al. show that the locus is rate limiting for reprogramming, and its transient inhibition significantly improves the generation of iPS cells. The also show ageing upregulates the Ink4/Arf locus and, accordingly, reprogramming is less efficient in cells from old organisms. The Ink4/Arf tumour suppressor locus encodes the three potent tumour suppressors p16Ink4a, p15Ink4b and p19Arf. Here the locus is shown to be rate-limiting for reprogramming, and its transient inhibition significantly improves the generation of induced pluripotent stem cells. Furthermore, ageing is shown to upregulate the Ink4/Arf locus, with less efficient reprogramming seen in cells from old organisms. The mechanisms involved in the reprogramming of differentiated cells into induced pluripotent stem (iPS) cells by the three transcription factors Oct4 (also known as Pou5f1), Klf4 and Sox2 remain poorly understood1. The Ink4/Arf locus comprises the Cdkn2a–Cdkn2b genes encoding three potent tumour suppressors, namely p16Ink4a, p19Arf and p15Ink4b, which are basally expressed in differentiated cells and upregulated by aberrant mitogenic signals2,3,4. Here we show that the locus is completely silenced in iPS cells, as well as in embryonic stem (ES) cells, acquiring the epigenetic marks of a bivalent chromatin domain, and retaining the ability to be reactivated after differentiation. Cell culture conditions during reprogramming enhance the expression of the Ink4/Arf locus, further highlighting the importance of silencing the locus to allow proliferation and reprogramming. Indeed, the three factors together repress the Ink4/Arf locus soon after their expression and concomitant with the appearance of the first molecular markers of ‘stemness’. This downregulation also occurs in cells carrying the oncoprotein large-T, which functionally inactivates the pathways regulated by the Ink4/Arf locus, thus indicating that the silencing of the locus is intrinsic to reprogramming and not the result of a selective process. Genetic inhibition of the Ink4/Arf locus has a profound positive effect on the efficiency of iPS cell generation, increasing both the kinetics of reprogramming and the number of emerging iPS cell colonies. In murine cells, Arf, rather than Ink4a, is the main barrier to reprogramming by activation of p53 (encoded by Trp53) and p21 (encoded by Cdkn1a); whereas, in human fibroblasts, INK4a is more important than ARF. Furthermore, organismal ageing upregulates the Ink4/Arf locus2,5 and, accordingly, reprogramming is less efficient in cells from old organisms, but this defect can be rescued by inhibiting the locus with a short hairpin RNA. All together, we conclude that the silencing of Ink4/Arf locus is rate-limiting for reprogramming, and its transient inhibition may significantly improve the generation of iPS cells.
973 citations
TL;DR: It is reported that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs and is demonstrated to be similar to embryonic stem cells in vitro and in vivo.
973 citations
TL;DR: A peak of genomic amplification on chromosome 3q26.33 found in squamous cell carcinomas of the lung and esophagus contains the transcription factor gene SOX2, which is necessary for normal esophageal squamous development, promotes differentiation and proliferation of basal tracheal cells and cooperates in induction of pluripotent stem cells.
Abstract: Lineage-survival oncogenes are activated by somatic DNA alterations in cancers arising from the cell lineages in which these genes play a role in normal development. Here we show that a peak of genomic amplification on chromosome 3q26.33 found in squamous cell carcinomas (SCCs) of the lung and esophagus contains the transcription factor gene SOX2, which is mutated in hereditary human esophageal malformations, is necessary for normal esophageal squamous development, promotes differentiation and proliferation of basal tracheal cells and cooperates in induction of pluripotent stem cells. SOX2 expression is required for proliferation and anchorage-independent growth of lung and esophageal cell lines, as shown by RNA interference experiments. Furthermore, ectopic expression of SOX2 here cooperated with FOXE1 or FGFR2 to transform immortalized tracheobronchial epithelial cells. SOX2-driven tumors show expression of markers of both squamous differentiation and pluripotency. These characteristics identify SOX2 as a lineage-survival oncogene in lung and esophageal SCC.
TL;DR: Klf4 and Tbx3 are involved in mediating LIF signalling to the core circuitry but are not directly associated with the maintenance of pluripotency, because ES cells keep pluripOTency without their expression in the particular context.
Abstract: The cytokine leukaemia inhibitory factor (LIF) integrates signals into mouse embryonic stem (ES) cells to maintain pluripotency. Although the Jak-Stat3 pathway is essential and sufficient to mediate LIF signals, it is still unclear how these signals are linked to the core circuitry of pluripotency-associated transcription factors, consisting of Oct3/4 (also called Pou5f1), Sox2 and Nanog. Here we show that two LIF signalling pathways are each connected to the core circuitry via different transcription factors. In mouse ES cells, Klf4 is mainly activated by the Jak-Stat3 pathway and preferentially activates Sox2, whereas Tbx3 is preferentially regulated by the phosphatidylinositol-3-OH kinase-Akt and mitogen-activated protein kinase pathways and predominantly stimulates Nanog. In the absence of LIF, artificial expression of Klf4 or Tbx3 is sufficient to maintain pluripotency while maintaining the expression of Oct3/4. Notably, overexpression of Nanog supports LIF-independent self-renewal of mouse ES cells in the absence of Klf4 and Tbx3 activity. Therefore, Klf4 and Tbx3 are involved in mediating LIF signalling to the core circuitry but are not directly associated with the maintenance of pluripotency, because ES cells keep pluripotency without their expression in the particular context.
TL;DR: High-content chemical screening is used to identify small molecules that can replace Sox2 in reprogramming and it is found that one of these molecules functions inReprogramming by inhibiting Tgf-beta signaling in a stable and trapped intermediate cell type that forms during the process.
TL;DR: In this article, a single reprogramming factor, Klf4, was introduced into EpiSCs, which activated expression of EScell-specific transcripts including endogenous klf4, and downstream markers of lineage specification.
Abstract: Mouse embryonic stem (ES) cells derived from pluripotent early epiblast
contribute functionally differentiated progeny to all foetal lineages of
chimaeras. By contrast, epistem cell (EpiSC) lines from post-implantation
epithelialised epiblast are unable to colonise the embryo even though they
express the core pluripotency genes Oct4, Sox2 and Nanog . We
examined interconversion between these two cell types. ES cells can readily
become EpiSCs in response to growth factor cues. By contrast, EpiSCs do not
change into ES cells. We exploited PiggyBac transposition to
introduce a single reprogramming factor, Klf4, into EpiSCs. No effect was
apparent in EpiSC culture conditions, but in ground state ES cell conditions a
fraction of cells formed undifferentiated colonies. These EpiSC-derived
induced pluripotent stem (Epi-iPS) cells activated expression of ES
cell-specific transcripts including endogenous Klf4 , and
downregulated markers of lineage specification. X chromosome silencing in
female cells, a feature of the EpiSC state, was erased in Epi-iPS cells. They
produced high-contribution chimaeras that yielded germline transmission. These
properties were maintained after Cre-mediated deletion of the Klf4
transgene, formally demonstrating complete and stable reprogramming of
developmental phenotype. Thus, re-expression of Klf4 in an appropriate
environment can regenerate the naive ground state from EpiSCs.
Reprogramming is dependent on suppression of extrinsic growth factor stimuli
and proceeds to completion in less than 1% of cells. This substantiates the
argument that EpiSCs are developmentally, epigenetically and functionally
differentiated from ES cells. However, because a single transgene is the
minimum requirement to attain the ground state, EpiSCs offer an attractive
opportunity for screening for unknown components of the reprogramming
process.
TL;DR: It is demonstrated that the transcription factor OCT4 is sufficient to reprogram human neural stem cells to pluripotency and will advance the field further towards understanding reprogramming and generating patient-specific pluripotent stem cells.
Abstract: 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.
TL;DR: Using a single lentiviral vector expressing a “stem cell cassette” composed of the four transcription factors and a combination of 2A peptide and internal ribosome entry site technology achieves the most efficient reprogramming system to date and allows derivation of iPS cells with a single viral integration.
Abstract: Induced pluripotent stem (iPS) cells can be generated using retroviral vectors expressing Oct4, Klf4, Sox2, and cMyc. Most prior studies have required multiple retroviral vectors for reprogramming, resulting in high numbers of genomic integrations in iPS cells and limiting their use for therapeutic applications. Here we describe the use of a single lentiviral vector expressing a "stem cell cassette" composed of the four transcription factors and a combination of 2A peptide and internal ribosome entry site technology, generating iPS cells from postnatal fibroblasts. iPS cells generated in this manner display embryonic stem cell-like morphology, express stem cell markers, and exhibit in vivo pluripotency, as evidenced by their ability to differentiate in teratoma assays and their robust contribution to mouse chimeras. Combining all factors into a single transcript achieves the most efficient reprogramming system to date and allows derivation of iPS cells with a single viral integration. The use of a single lentiviral vector for reprogramming represents a powerful laboratory tool and a significant step toward the application of iPS technology for clinical purposes.
TL;DR: The ability to reprogram cells from human blood will allow the generation of patient-specific stem cells for diseases in which the disease-causing somatic mutations are restricted to cells of the hematopoietic lineage.
TL;DR: Genome-wide analyses of promoter binding and expression in iPS and partially reprogrammed cells reveal temporal and separable contributions of the four factors during the reprogramming process and indicate that ectopic c-Myc predominantly acts before pluripotency regulators are activated.
TL;DR: Crucially, ablation of different senescence effectors improves the efficiency of reprogramming, suggesting novel strategies for maximizing the generation of iPS cells.
Abstract: Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by overexpressing combinations of factors such as Oct4, Sox2, Klf4, and c-Myc. Reprogramming is slow and stochastic, suggesting the existence of barriers limiting its efficiency. Here we identify senescence as one such barrier. Expression of the four reprogramming factors triggers senescence by up-regulating p53, p16INK4a, and p21CIP1. Induction of DNA damage response and chromatin remodeling of the INK4a/ARF locus are two of the mechanisms behind senescence induction. Crucially, ablation of different senescence effectors improves the efficiency of reprogramming, suggesting novel strategies for maximizing the generation of iPS cells.
TL;DR: P piggyBac transposon–based reprogramming may be used to generate therapeutically applicable iPSCs and could be identified by negative selection.
Abstract: Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by transgenic expression of Oct4 (Pou5f1), Sox2, Klf4 and Myc. A major difficulty in the application of this technology for regenerative medicine, however, is the delivery of reprogramming factors. Whereas retroviral transduction increases the risk of tumorigenicity, transient expression methods have considerably lower reprogramming efficiencies. Here we describe an efficient piggyBac transposon-based approach to generate integration-free iPSCs. Transposons carrying 2A peptide-linked reprogramming factors induced reprogramming of mouse embryonic fibroblasts with equivalent efficiencies to retroviral transduction. We removed transposons from these primary iPSCs by re-expressing transposase. Transgene-free iPSCs could be identified by negative selection. piggyBac excised without a footprint, leaving the iPSC genome without any genetic alteration. iPSCs fulfilled all criteria of pluripotency, such as pluripotency gene expression, teratoma formation and contribution to chimeras. piggyBac transposon-based reprogramming may be used to generate therapeutically applicable iPSCs.
TL;DR: It is shown that induced pluripotent stem (iPS) cells can be generated from patients with T1D by reprogramming their adult fibroblasts with three transcription factors (OCT4, SOX2, KLF4).
Abstract: Type 1 diabetes (T1D) is the result of an autoimmune destruction of pancreatic β cells. The cellular and molecular defects that cause the disease remain unknown. Pluripotent cells generated from patients with T1D would be useful for disease modeling. We show here that induced pluripotent stem (iPS) cells can be generated from patients with T1D by reprogramming their adult fibroblasts with three transcription factors (OCT4, SOX2, KLF4). T1D-specific iPS cells, termed DiPS cells, have the hallmarks of pluripotency and can be differentiated into insulin-producing cells. These results are a step toward using DiPS cells in T1D disease modeling, as well as for cell replacement therapy.
TL;DR: A previously unknown Wnt-mediated regulatory mechanism that simultaneously coordinates activation of NeuroD1 and LINE-1 is described, which is important for adult neurogenesis and survival of neuronal progenitors.
Abstract: In adult hippocampus, new neurons are continuously generated from neural stem cells (NSCs), but the molecular mechanisms regulating adult neurogenesis remain elusive. We found that Wnt signaling, together with the removal of Sox2, triggered the expression of NeuroD1 in mice. This transcriptional regulatory mechanism was dependent on a DNA element containing overlapping Sox2 and T-cell factor/lymphoid enhancer factor (TCF/LEF)-binding sites (Sox/LEF) in the promoter. Notably, Sox/LEF sites were also found in long interspersed nuclear element 1 (LINE-1) elements, consistent with their critical roles in the transition of NSCs to proliferating neuronal progenitors. Our results describe a previously unknown Wnt-mediated regulatory mechanism that simultaneously coordinates activation of NeuroD1 and LINE-1, which is important for adult neurogenesis and survival of neuronal progenitors. Moreover, the discovery that LINE-1 retro-elements embedded in the mammalian genome can function as bi-directional promoters suggests that Sox/LEF regulatory sites may represent a general mechanism, at least in part, for relaying environmental signals to other nearby loci to promote adult hippocampal neurogenesis.
TL;DR: A novel approach to reduce the number of viruses necessary to reprogram somatic cells by delivering reprogramming factors in a single virus using 2A “self-cleaving” peptides, which support efficient polycistronic expression from a single promoter.
Abstract: Directed reprogramming of somatic cells by defined factors provides a novel method for the generation of patient-specific stem cells with the potential to bypass both the practical and ethical concerns associated with somatic cell nuclear transfer (SCNT) and human embryonic stem (hES) cells. Although the generation of induced pluripotent stem (iPS) cells has proven a robust technology in mouse and human, a major impediment to the use of iPS cells for therapeutic purposes has been the viral-based delivery of the reprogramming factors because multiple proviral integrations pose the danger of insertional mutagenesis. Here we report a novel approach to reduce the number of viruses necessary to reprogram somatic cells by delivering reprogramming factors in a single virus using 2A "self-cleaving" peptides, which support efficient polycistronic expression from a single promoter. We find that up to four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can be expressed from a single virus to generate iPS cells in both embryonic and adult somatic mouse cells and we show that a single proviral copy is sufficient to generate iPS cells from mouse embryonic fibroblasts. In addition we have generated human induced pluripotent stem (hiPS) cell lines from human keratinocytes, demonstrating that a single polycistronic virus can reprogram human somatic cells.
TL;DR: Silencing SOX2 in freshly derived glioblastoma tumor‐initiating cells (TICs) indicates that these cells, despite the many mutations they have accumulated, stop proliferating and lose tumorigenicity in immunodeficient mice.
Abstract: Glioblastoma, the most aggressive cerebral tumor, is invariably lethal. Glioblastoma cells express several genes typical of normal neural stem cells. One of them, SOX2, is a master gene involved in sustaining self-renewal of several stem cells, in particular neural stem cells. To investigate its role in the aberrant growth of glioblastoma, we silenced SOX2 in freshly derived glioblastoma tumor-initiating cells (TICs). Our results indicate that SOX2 silenced glioblastoma TICs, despite the many mutations they have accumulated, stop proliferating and lose tumorigenicity in immunodeficient mice. SOX2 is then also fundamental for maintenance of the self-renewal capacity of neural stem cells when they have acquired cancer properties. SOX2, or its immediate downstream effectors, would then be an ideal target for glioblastoma therapy.
TL;DR: The authors reveal that a pluripotent cell population comprises cells with temporal fluctuations in the expression of Nanog, indicating that the differentiated state of a cell population is determined simply by expression of specific marker genes.
Abstract: There is evidence that pluripotency of mouse embryonic stem (ES) cells is associated with the activity of a network of transcription factors with Sox2, Oct4, and Nanog at the core. Using fluorescent reporters for the expression of Nanog, we observed that a population of ES cells is best described by a dynamic distribution of Nanog expression characterized by two peaks defined by high (HN) and low (LN) Nanog expression. Typically, the LN state is 5%–20% of the total population, depending on the culture conditions. Modelling of the activity of Nanog reveals that a simple network of Oct4/Sox2 and Nanog activity can account for the observed distribution and its properties as long as the transcriptional activity is tuned by transcriptional noise. The model also predicts that the LN state is unstable, something that is born out experimentally. While in this state, cells can differentiate. We suggest that transcriptional fluctuations in Nanog expression are an essential element of the pluripotent state and that the function of Sox2, Oct4, and Nanog is to act as a network that promotes and maintains transcriptional noise to interfere with the differentiation signals.
TL;DR: It is concluded that adenoviral vectors can reprogram human fibroblasts to pluripotent stem cells for use in individualized cell therapy without the risk for viral or oncogene incorporation.
Abstract: Mouse and human fibroblasts have been transformed into induced pluripotent stem (iPS) cells by retroviral transduction or plasmid transfection with four genes. Unfortunately, viral and plasmid DNA incorporation into chromosomes can lead to disruption of gene transcription and malignant transformation. Tumor formation has been found in offspring of mice generated from blastocysts made mosaic with iPS cells. To proceed with iPS cells for human therapy, reprogramming should be done with transient gene expression. Recently, adenoviral vectors have been used to produce mouse iPS cells without viral integration. Here, we report the successful creation of human iPS cells from embryonic fibroblasts using adenoviral vectors expressing c-Myc, Klf4, Oct4, and Sox2. After screening 12 colonies, three stable iPS cell lines were established. Each cell line showed human embryonic stem cell morphology and surface markers. Southern blots and polymerase chain reaction demonstrated that there was no viral DNA integration into iPS cells. Fingerprinting and karyotype analysis confirmed that these iPS cell lines are derived from the parent human fibroblasts. The three human iPS cell lines can differentiate to all three germ layers in vitro, including dopaminergic neurons. After s.c. injection into nonobese diabetic-severe combined immunodeficient mice, each human iPS line produced teratomas within 5 weeks postimplantation. We conclude that adenoviral vectors can reprogram human fibroblasts to pluripotent stem cells for use in individualized cell therapy without the risk for viral or oncogene incorporation.
TL;DR: This work shows that the orphan nuclear receptor Esrrb functions in conjunction with Oct4 and Sox2 to mediate reprogramming of mouse embryonic fibroblasts (MEFs) to iPS cells, and indicates that it is possible to reprogram MEFs without exogenous Klf transcription factors and link a nuclear receptor to somatic cell reprograming.
Abstract: The dominant effect of transcription factors in imparting expanded potency is best exemplified by the reprogramming of fibroblasts to pluripotent cells using retrovirus-mediated transduction of defined transcription factors. In the murine system, Oct4, Sox2, c-Myc and Klf4 are sufficient to convert fibroblasts to induced pluripotent stem (iPS) cells that have many characteristics of embryonic stem (ES) cells. Here we show that the orphan nuclear receptor Esrrb functions in conjunction with Oct4 and Sox2 to mediate reprogramming of mouse embryonic fibroblasts (MEFs) to iPS cells. Esrrb-reprogrammed cells share similar expression and epigenetic signatures as ES cells. These cells are also pluripotent and can differentiate in vitro and in vivo into the three major embryonic cell lineages. Furthermore, these cells contribute to mouse chimaeras and are germline transmissible. In ES cells, Esrrb targets many genes involved in self-renewal and pluripotency. This suggests that Esrrb may mediate reprogramming through the upregulation of ES-cell-specific genes. Our findings also indicate that it is possible to reprogram MEFs without exogenous Klf transcription factors and link a nuclear receptor to somatic cell reprogramming.
TL;DR: It is demonstrated that autocrine TGF-beta signaling plays an essential role in retention of stemness of glioma-initiating cells (GICs) and the underlying mechanism for it is described, and an essential pathway for GICs is identified, the TGF/Sox4-Sox2 pathway, whose disruption would be a therapeutic strategy against gliomas.
TL;DR: Fibroblasts reprogrammed by human stemness factors thus acquire the potential to repair acute myocardial infarction, establishing iPS in the treatment of heart disease.
Abstract: Background—Nuclear reprogramming provides an emerging strategy to produce embryo-independent pluripotent stem cells from somatic tissue. Induced pluripotent stem cells (iPS) demonstrate aptitude for de novo cardiac differentiation, yet their potential for heart disease therapy has not been tested. Methods and Results—In this study, fibroblasts transduced with human stemness factors OCT3/4, SOX2, KLF4, and c-MYC converted into an embryonic stem cell–like phenotype and demonstrated the ability to spontaneously assimilate into preimplantation host morula via diploid aggregation, unique to bona fide pluripotent cells. In utero, iPS-derived chimera executed differentiation programs to construct normal heart parenchyma patterning. Within infarcted hearts in the adult, intramyocardial delivery of iPS yielded progeny that properly engrafted without disrupting cytoarchitecture in immunocompetent recipients. In contrast to parental nonreparative fibroblasts, iPS treatment restored postischemic contractile performance, ventricular wall thickness, and electric stability while achieving in situ regeneration of cardiac, smooth muscle, and endothelial tissue. Conclusions—Fibroblasts reprogrammed by human stemness factors thus acquire the potential to repair acute myocardial infarction, establishing iPS in the treatment of heart disease. (Circulation. 2009;120:408-416.)
TL;DR: Neural stem cells control their status, at least partly, through Sox2-dependent autocrine mechanisms, and their replication was partially rescued by the addition of SHH and was almost fully rescued by conditioned medium from normal cells.
Abstract: Neural stem cells (NSCs) are controlled by diffusible factors. The transcription factor Sox2 is expressed by NSCs and Sox2 mutations in humans cause defects in the brain and, in particular, in the hippocampus. We deleted Sox2 in the mouse embryonic brain. At birth, the mice showed minor brain defects; shortly afterwards, however, NSCs and neurogenesis were completely lost in the hippocampus, leading to dentate gyrus hypoplasia. Deletion of Sox2 in adult mice also caused hippocampal neurogenesis loss. The hippocampal developmental defect resembles that caused by late sonic hedgehog (Shh) loss. In mutant mice, Shh and Wnt3a were absent from the hippocampal primordium. A SHH pharmacological agonist partially rescued the hippocampal defect. Chromatin immunoprecipitation identified Shh as a Sox2 target. Sox2-deleted NSCs did not express Shh in vitro and were rapidly lost. Their replication was partially rescued by the addition of SHH and was almost fully rescued by conditioned medium from normal cells. Thus, NSCs control their status, at least partly, through Sox2-dependent autocrine mechanisms.