Instruct: Given a scientific claim, retrieve relevant documents that support or refute the claim.
Query: Ectopic expression of Sall4, Nanog, Esrrb, and Lin28 generates induced pluripotent stem cells from mouse embryonic fibroblasts more efficiently than other factor combinations.
Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.
During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry.
Ectopic expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the fibroblast genome, generating induced pluripotent stem (iPS) cells. It remains unknown if nuclear reprogramming induced by these four factors globally resets epigenetic differences between differentiated and pluripotent cells. Here, using novel selection approaches, we have generated iPS cells from fibroblasts to characterize their epigenetic state. Female iPS cells showed reactivation of a somatically silenced X chromosome and underwent random X inactivation upon differentiation. Genome-wide analysis of two key histone modifications indicated that iPS cells are highly similar to ES cells. Consistent with these observations, iPS cells gave rise to viable high-degree chimeras with contribution to the germline. These data show that transcription factor-induced reprogramming leads to the global reversion of the somatic epigenome into an ES-like state. Our results provide a paradigm for studying the epigenetic modifications that accompany nuclear reprogramming and suggest that abnormal epigenetic reprogramming does not pose a problem for the potential therapeutic applications of iPS cells.
Since the initial discovery that OCT4, SOX2, KLF4, and c-MYC overexpression sufficed for the induction of pluripotency in somatic cells, methodologies replacing the original factors have enhanced our understanding of the reprogramming process. However, unlike in mouse, OCT4 has not been replaced successfully during reprogramming of human cells. Here we report on a strategy to accomplish this replacement. Through a combination of transcriptome and bioinformatic analysis we have identified factors previously characterized as being lineage specifiers that are able to replace OCT4 and SOX2 in the reprogramming of human fibroblasts. Our results show that it is possible to replace OCT4 and SOX2 simultaneously with alternative lineage specifiers in the reprogramming of human cells. At a broader level, they also support a model in which counteracting lineage specification networks underlies the induction of pluripotency.
We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.
The simple yet powerful technique of induced pluripotency may eventually supply a wide range of differentiated cells for cell therapy and drug development. However, making the appropriate cells via induced pluripotent stem cells (iPSCs) requires reprogramming of somatic cells and subsequent redifferentiation. Given how arduous and lengthy this process can be, we sought to determine whether it might be possible to convert somatic cells into lineage-specific stem/progenitor cells of another germ layer in one step, bypassing the intermediate pluripotent stage. Here we show that transient induction of the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can efficiently transdifferentiate fibroblasts into functional neural stem/progenitor cells (NPCs) with appropriate signaling inputs. Compared with induced neurons (or iN cells, which are directly converted from fibroblasts), transdifferentiated NPCs have the distinct advantage of being expandable in vitro and retaining the ability to give rise to multiple neuronal subtypes and glial cells. Our results provide a unique paradigm for iPSC-factor-based reprogramming by demonstrating that it can be readily modified to serve as a general platform for transdifferentiation.
The generation of induced pluripotent stem cells (iPSCs) provides the opportunity to use patient-specific somatic cells, which are a valuable source for disease modeling and drug discovery. To promote research involving these cells, it is important to make iPSCs from easily accessible and less invasive tissues, like blood. We have recently reported the efficient generation of human iPSCs from adult fibroblasts using a combination of plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA for TP53. We herein report a modified protocol enabling efficient iPSC induction from CD34+ cord blood cells and from peripheral blood isolated from healthy donors using these plasmid vectors. The original plasmid mixture could induce iPSCs; however, the efficiency was low. The addition of EBNA1, an essential factor for episomal amplification of the vectors, by an extra plasmid greatly increased the efficiency of iPSC induction, especially when the induction was performed from αβT cells. This improvement enabled the establishment of blood-derived iPSCs from seven healthy donors ranging in age from their 20s to their 60s. This induction method will be useful for the derivation of patient-specific integration-free iPSCs and would also be applicable to the generation of clinical-grade iPSCs in the future.
Human embryonic stem (ES) cell lines that have the ability to self-renew and differentiate into specific cell types have been established. The molecular mechanisms for self-renewal and differentiation, however, are poorly understood. We determined the transcriptome profiles for two proprietary human ES cell lines (HES3 and HES4, ES Cell International), and compared them with murine ES cells and other human tissues. Human and mouse ES cells appear to share a number of expressed gene products although there are numerous notable differences, including an inactive leukemia inhibitory factor pathway and the high preponderance of several important genes like POU5F1 and SOX2 in human ES cells. We have established a list of genes comprised of known ES-specific genes and new candidates that can serve as markers for human ES cells and may also contribute to the "stemness" phenotype. Of particular interest was the downregulation of DNMT3B and LIN28 mRNAs during ES cell differentiation. The overlapping similarities and differences in gene expression profiles of human and mouse ES cells provide a foundation for a detailed and concerted dissection of the molecular and cellular mechanisms governing their pluripotency, directed differentiation into specific cell types, and extended ability for self-renewal.
We compared two genetically highly defined transgenic systems to identify parameters affecting reprogramming of somatic cells to a pluripotent state. Our results demonstrate that the level and stoichiometry of reprogramming factors during the reprogramming process strongly influence the resulting pluripotency of iPS cells. High expression of Oct4 and Klf4 combined with lower expression of c-Myc and Sox2 produced iPS cells that efficiently generated "all-iPSC mice" by tetraploid (4n) complementation, maintained normal imprinting at the Dlk1-Dio3 locus, and did not create mice with tumors. Loss of imprinting (LOI) at the Dlk1-Dio3 locus did not strictly correlate with reduced pluripotency though the efficiency of generating "all-iPSC mice" was diminished. Our data indicate that stoichiometry of reprogramming factors can influence epigenetic and biological properties of iPS cells. This concept complicates efforts to define a "generic" epigenetic state of iPSCs and ESCs and should be considered when comparing different iPS and ES cell lines.
Transcription factors, such as Oct4, are critical for establishing and maintaining pluripotent cell identity. Whereas the genomic locations of several pluripotency transcription factors have been reported, the spectrum of their interaction partners is underexplored. Here, we use an improved affinity protocol to purify Oct4-interacting proteins from mouse embryonic stem cells (ESCs). Subsequent purification of Oct4 partners Sall4, Tcfcp2l1, Dax1, and Esrrb resulted in an Oct4 interactome of 166 proteins, including transcription factors and chromatin-modifying complexes with documented roles in self-renewal, but also many factors not previously associated with the ESC network. We find that Esrrb associated with the basal transcription machinery and also detect interactions between transcription factors and components of the TGF-beta, Notch, and Wnt signaling pathways. Acute depletion of Oct4 reduced binding of Tcfcp2l1, Dax1, and Esrrb to several target genes. In conclusion, our purification protocol allowed us to bring greater definition to the circuitry controlling pluripotent cell identity.
The RNA-binding proteins LIN28A and LIN28B play critical roles in embryonic development, tumorigenesis, and pluripotency, but their exact functions are poorly understood. Here, we show that, like LIN28A, LIN28B can function effectively with NANOG, OCT4, and SOX2 in reprogramming to pluripotency and that reactivation of both endogenous LIN28A and LIN28B loci are required for maximal reprogramming efficiency. In human fibroblasts, LIN28B is activated early during reprogramming, while LIN28A is activated later during the transition to bona fide induced pluripotent stem cells (iPSCs). In murine cells, LIN28A and LIN28B facilitate conversion from naive to primed pluripotency. Proteomic and metabolomic analysis highlighted roles for LIN28 in maintaining the low mitochondrial function associated with primed pluripotency and in regulating one-carbon metabolism, nucleotide metabolism, and histone methylation. LIN28 binds to mRNAs of proteins important for oxidative phosphorylation and modulates protein abundance. Thus, LIN28A and LIN28B play cooperative roles in regulating reprogramming, naive/primed pluripotency, and stem cell metabolism.
Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.
The interconversion between naive and primed pluripotent states is accompanied by drastic epigenetic rearrangements. However, it is unclear whether intrinsic epigenetic events can drive reprogramming to naive pluripotency or if distinct chromatin states are instead simply a reflection of discrete pluripotent states. Here, we show that blocking histone H3K4 methyltransferase MLL1 activity with the small-molecule inhibitor MM-401 reprograms mouse epiblast stem cells (EpiSCs) to naive pluripotency. This reversion is highly efficient and synchronized, with more than 50% of treated EpiSCs exhibiting features of naive embryonic stem cells (ESCs) within 3 days. Reverted ESCs reactivate the silenced X chromosome and contribute to embryos following blastocyst injection, generating germline-competent chimeras. Importantly, blocking MLL1 leads to global redistribution of H3K4me1 at enhancers and represses lineage determinant factors and EpiSC markers, which indirectly regulate ESC transcription circuitry. These findings show that discrete perturbation of H3K4 methylation is sufficient to drive reprogramming to naive pluripotency.
As is the case for embryo-derived stem cells, application of reprogrammed human induced pluripotent stem cells is limited by our understanding of lineage specification. Here we demonstrate the ability to generate progenitors and mature cells of the haematopoietic fate directly from human dermal fibroblasts without establishing pluripotency. Ectopic expression of OCT4 (also called POU5F1)-activated haematopoietic transcription factors, together with specific cytokine treatment, allowed generation of cells expressing the pan-leukocyte marker CD45. These unique fibroblast-derived cells gave rise to granulocytic, monocytic, megakaryocytic and erythroid lineages, and demonstrated in vivo engraftment capacity. We note that adult haematopoietic programs are activated, consistent with bypassing the pluripotent state to generate blood fate: this is distinct from haematopoiesis involving pluripotent stem cells, where embryonic programs are activated. These findings demonstrate restoration of multipotency from human fibroblasts, and suggest an alternative approach to cellular reprogramming for autologous cell-replacement therapies that avoids complications associated with the use of human pluripotent stem cells.
Transcription factor-based cellular reprogramming has opened the way to converting somatic cells to a pluripotent state, but has faced limitations resulting from the requirement for transcription factors and the relative inefficiency of the process. We show here that expression of the miR302/367 cluster rapidly and efficiently reprograms mouse and human somatic cells to an iPSC state without a requirement for exogenous transcription factors. This miRNA-based reprogramming approach is two orders of magnitude more efficient than standard Oct4/Sox2/Klf4/Myc-mediated methods. Mouse and human miR302/367 iPSCs display similar characteristics to Oct4/Sox2/Klf4/Myc-iPSCs, including pluripotency marker expression, teratoma formation, and, for mouse cells, chimera contribution and germline contribution. We found that miR367 expression is required for miR302/367-mediated reprogramming and activates Oct4 gene expression, and that suppression of Hdac2 is also required. Thus, our data show that miRNA and Hdac-mediated pathways can cooperate in a powerful way to reprogram somatic cells to pluripotency.
Deciphering the molecular basis of stem cell pluripotency is fundamental to the understanding of stem cell biology, early embryonic development, and to the clinical application of regenerative medicine. We report here that the molecular chaperone heat shock protein 90 (Hsp90) is essential for mouse embryonic stem cell (ESC) pluripotency through regulating multiple pluripotency factors, including Oct4, Nanog, and signal transducer and activator of transcription 3. Inhibition of Hsp90 by either 17-N-Allylamino-17-demethoxygeldanamycin or miRNA led to ESC differentiation. Overexpression of Hsp90β partially rescued the phenotype; in particular, the levels of Oct4 and Nanog were restored. Notably, Hsp90 associated with Oct4 and Nanog in the same cellular complex and protected them from degradation by the ubiquitin proteasome pathway, suggesting that Oct4 and Nanog are potential novel Hsp90 client proteins. In addition, Hsp90 inhibition reduced the mRNA level of Oct4, but not that of Nanog, indicating that Hsp90 participates in Oct4 mRNA processing or maturation. Hsp90 inhibition also increased expression of some protein markers for mesodermal lineages, implying that Hsp90 suppresses mesodermal differentiation from ESCs. These findings support a new role for Hsp90 in maintaining ESC pluripotency by sustaining the level of multiple pluripotency factors, particularly Oct4 and Nanog.
Polycomb repressive complex two (PRC2) has been implicated in embryonic stem (ES) cell pluripotency; however, the mechanistic roles of this complex are unclear. It was assumed that ES cells contain PRC2 with the same subunit composition as that identified in HeLa cells and Drosophila embryos. Here, we report that PRC2 in mouse ES cells contains at least three additional subunits: JARID2, MTF2, and a novel protein denoted esPRC2p48. JARID2, MTF2, and esPRC2p48 are highly expressed in mouse ES cells compared to differentiated cells. Importantly, knockdowns of JARID2, MTF2, or esPRC2p48 alter the level of PRC2-mediated H3K27 methylation and result in the expression of differentiation-associated genes in ES cells. Interestingly, expression of JARID2, MTF2, and esPRC2p48 together, but not individually, enhances Oct4/Sox2/Klf4-mediated reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells, whereas knockdown or knockout of JARID2, MTF2, or esPRC2p48 significantly inhibits reprogramming. JARID2, MTF2, and esPRC2p48 modulate H3K27 methylation and facilitate repression of lineage-associated gene expression when transduced into MEFs, and synergistically stimulate the histone methyltransferase activity of PRC2 in vitro. Therefore, these studies identify JARID2, MTF2, and esPRC2p48 as important regulatory subunits of PRC2 in ES cells and reveal critical functions of these subunits in modulating PRC2's activity and gene expression both in ES cells and during somatic cell reprogramming.
Pluripotential cells are present in a mouse embryo until at least an early post-implantation stage, as shown by their ability to take part hi the formation of chimaeric animals1 and to form teratocarcinomas2. Until now it has not been possible to establish progressively growing cultures of these cells in vitro, and cell lines have only been obtained after teratocarcinoma formation in vivo. We report here the establishment in tissue culture of pluripotent cell lines which have been isolated directly from in vitro cultures of mouse blastocysts. These cells are able to differentiate either in vitro or after innoculation into a mouse as a tumour in vivo. They have a normal karyotype.
We describe robust induction of autophagy during the reprogramming of mouse fibroblasts to induced pluripotent stem cells by four reprogramming factors (Sox2, Oct4, Klf4 and c-Myc), henceforth 4F. This process occurs independently of p53 activation, and is mediated by the synergistic downregulation of mechanistic target of rapamycin complex 1 (mTORC1) and the induction of autophagy-related genes. The 4F coordinately repress mTORC1, but bifurcate in their regulation of autophagy-related genes, with Klf4 and c-Myc inducing them but Sox2 and Oct4 inhibiting them. On one hand, inhibition of mTORC1 facilitates reprogramming by promoting cell reshaping (mitochondrial remodelling and cell size reduction). On the other hand, mTORC1 paradoxically impairs reprogramming by triggering autophagy. Autophagy does not participate in cell reshaping in reprogramming but instead degrades p62, whose accumulation in autophagy-deficient cells facilitates reprogramming. Our results thus reveal a complex signalling network involving mTORC1 inhibition and autophagy induction in the early phase of reprogramming, whose delicate balance ultimately determines reprogramming efficiency.
Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine.
Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) and the epiblast, and have been suggested to be a homogeneous population with characteristics intermediate between them. These cells express Oct3/4 and Rex1 genes, which have been used as markers to indicate the undifferentiated state of ES cells. Whereas Oct3/4 is expressed in totipotent and pluripotent cells in the mouse life cycle, Rex1 expression is restricted to the ICM, and is downregulated in pluripotent cell populations in the later stages, i.e. the epiblast and primitive ectoderm (PrE). To address whether ES cells comprise a homogeneous population equivalent to a certain developmental stage of pluripotent cells or a heterogeneous population composed of cells corresponding to various stages of differentiation, we established knock-in ES cell lines in which genes for fluorescent proteins were inserted into the Rex1 and Oct3/4 gene loci to visualize the expression of these genes. We found that undifferentiated ES cells included at least two different populations, Rex1(+)/Oct3/4(+) cells and Rex1(-)/Oct3/4(+) cells. The Rex1(-)/Oct3/4(+) and Rex1(+)/Oct3/4(+) populations could convert into each other in the presence of LIF. In accordance with our assumption that Rex1(+)/Oct3/4(+) cells and Rex1(-)/Oct3/4(+) cells have characteristics similar to those of ICM and early-PrE cells, Rex1(+)/Oct3/4(+) cells predominantly differentiated into primitive ectoderm and contributed to chimera formation, whereas Rex1(-)/Oct3/4(+) cells differentiated into cells of the somatic lineage more efficiently than non-fractionated ES cells in vitro and showed poor ability to contribute to chimera formation. These results confirmed that undifferentiated ES cell culture contains subpopulations corresponding to ICM, epiblast and PrE.
Embryonic stem (ES) cells homozygous for a Shp-2 mutation (Shp-2(Delta46-110)) demonstrate leukemia inhibitory factor (LIF) hypersensitivity and increased LIF-stimulated phosphorylation of signal transducer and activator of transcription (STAT3). We hypothesized that LIF-responsive genes in Shp-2(Delta46-110) cells would represent potential candidates for molecules vital for ES cell self-renewal. Using microarray analysis, we detected 41 genes whose expression was modified by LIF in Shp-2(Delta46-110) ES cells. Induction of 2 significantly up-regulated genes, suppressor of cytokine signaling-3 (SOCS-3) and Kruppel-like factor 4 (Klf4), was verified using Northern blotting. ES cells overexpressing SOCS-3 had an increased capacity to differentiate to hematopoietic progenitors, rather than to self-renew. In contrast, ES cells overexpressing Klf4 had a greater capacity to self-renew based on secondary embryoid body (EB) formation. Klf4-transduced d6 EBs expressed higher levels of Oct-4, consistent with the notion that Klf4 promotes ES cell self-renewal. These findings verify the negative role of SOCS-3 on LIF signaling and provide a novel role for Klf4 in ES cell function.
Pluripotency is the remarkable capacity of a single cell to engender all the specialized cell types of an adult organism. This property can be captured indefinitely through derivation of self-renewing embryonic stem cells (ESCs), which represent an invaluable platform to investigate cell fate decisions and disease. Recent advances have revealed that manipulation of distinct signaling cues can render ESCs in a uniform "ground state" of pluripotency, which more closely recapitulates the pluripotent naive epiblast. Here we discuss the extrinsic and intrinsic regulatory principles that underpin the nature of pluripotency and consider the emerging spectrum of pluripotent states.
Somatic cell nuclear transfer (SCNT) technology has recently been used to generate animals with a common genetic composition. In this study, we report the derivation of a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned human blastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surface markers and were capable of differentiating into embryoid bodies in vitro and of forming teratomas in vivo containing cell derivatives from all three embryonic germ layers in severe combined immunodeficient mice. After continuous proliferation for more than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and were genetically identical to the somatic nuclear donor cells. Although we cannot completely exclude the possibility that the cells had a parthenogenetic origin, imprinting analyses support a SCNT origin of the derived human ES cells.
Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iPSC, and imaged the conversion of fibroblasts to iPSC. We combined fluorescence microscopy with long-term single cell tracking, and used live-cell imaging to analyze the emergence and composition of early iPSC clusters. Applying our engineered lentiviral vectors, we demonstrate that vector silencing typically occurs prior to or simultaneously with the induction of an Oct4-EGFP pluripotency marker. Around 7 days post-transduction (pt), a subfraction of cells in clonal colonies expressed Oct4-EGFP and rapidly expanded. Cell tracking of single cell-derived iPSC colonies supported the concept that stochastic epigenetic changes are necessary for reprogramming. We also found that iPSC colonies may emerge as a genetic mosaic originating from different clusters. Improved vector design with continuous cell tracking thus creates a powerful system to explore the subtle dynamics of biological processes such as early reprogramming events.