[show abstract][hide abstract] ABSTRACT: Tet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion.
[show abstract][hide abstract] ABSTRACT: Background: Oct4 is a transcription factor that plays a major role for the preservation of the pluripotent state in embryonic stem cells as well as for efficient reprogramming of somatic cells to induced pluripotent stem cells (iPSC) or other progenitors. Protein-based reprogramming methods mainly rely on the addition of a fused cell penetrating peptide. This study describes that Oct4 inherently carries a protein transduction domain, which can translocate into human and mouse cells. Results: A 16 amino acid peptide representing the third helix of the human Oct4 homeodomain, referred to as Oct4 protein transduction domain (Oct4-PTD), can internalize in mammalian cells upon conjugation to a fluorescence moiety thereby acting as a cell penetrating peptide (CPP). The cellular distribution of Oct4-PTD shows diffuse cytosolic and nuclear staining, whereas penetratin is strictly localized to a punctuate pattern in the cytoplasm. By using a Cre/loxP-based reporter system, we show that this peptide also drives translocation of a functionally active Oct4-PTD-Cre-fusion protein. We further provide evidence for translocation of full length Oct4 into human and mouse cell lines without the addition of any kind of cationic fusion tag. Finally, physico-chemical properties of the novel CPP are characterized, showing that in contrast to penetratin a helical structure of Oct4-PTD is only observed if the FITC label is present on the N-terminus of the peptide. Conclusions: Oct4 is a key transcription factor in stem cell research and cellular reprogramming. Since it has been shown that recombinant Oct4 fused to a cationic fusion tag can drive generation of iPSCs, our finding might contribute to further development of protein-based methods to generate iPSCs. Moreover, our data support the idea that transcription factors might be part of an alternative paracrine signalling pathway, where the proteins are transferred to neighbouring cells thereby actively changing the behaviour of the recipient cell.
[show abstract][hide abstract] ABSTRACT: Induced pluripotent stem cells (iPSCs) derived from somatic cells have enormous potential for clinical applications. Notably, it was recently reported that reprogramming from somatic cells to iPSCs can induce genomic copy number variation (CNV), which is one of the major genetic causes of human diseases. However it was unclear if this genome instability is dependent on reprogramming methods and/or the genetic background of donor cells. Furthermore, genome-wide CNV analysis is technically challenging and CNV data need to be interpreted with care.
In order to carefully investigate the possible CNV instability during somatic reprogramming, we performed genome-wide CNV analyses with 41 mouse iPSC lines generated from the same parental donor; therefore, the donor's genetic background can be controlled. Different reprogramming factor combinations and dosages were used for investigating potential method-dependent effects on genome integrity. We detected 63 iPSC CNVs using high-resolution comparative genomic hybridization. Intriguingly, CNV rates were negatively associated with the dosages of classic factor(s). Furthermore, the use of high-performance engineered factors led to less CNVs than the classic factor(s) of the same dosage.
Our observations suggest that sufficient reprogramming force can protect the genome from CNV instability during the reprogramming process.
[show abstract][hide abstract] ABSTRACT: The breakthrough development of induced pluripotent stem cells (iPSCs) raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells. However, whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear. In this study, we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with autogenous peripheral blood mononuclear cells (PBMCs), we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation. However, a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs. Furthermore, no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells (CD3(+)CD8(-) T cells, CD3(+)CD8(+) T cells or CD3(-)CD56(+) NK cells) by NPCs in both PBMC and T cell co-culture systems. These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants, and thus set a base for further preclinical evaluation of human iPSCs.
Science China. Life sciences 01/2014; · 2.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: Zfyve9 is a FYVE domain protein first identified as a binding partner for SMAD2/3. In vitro studies indicate that it can function either positively or negatively in the TGF-beta signaling pathway depending on the cell lines used. However, the in vivo function of this protein remains to be investigated. We first analyzed the tissue distribution of zebrafish zfyve9a by in situ hybridization. To investigate the in vivo function of this gene, we performed morpholino mediated loss-of-function assays. We analyzed the expression patterns of liver (cp and fabp10a), pancreas (trypsin and insulin) or gut (fabp2) specific markers to determine whether the formation of these organs is affected by zfyve9a knockdown. We determined the specification of hepatoblast in the zfyve9a morphants (prox1a) and investigated the proliferation and survival of hepatic cells in the morphants by P-H3 staining and TUNEL assay respectively. We report here that zfyve9a is enriched in the zebrafish embryonic liver and required for hepatogenesis. Morpholino mediated knockdown of zfyve9a inhibits the formation of liver by day 4 while the other endoderm-derived organs appear unaffected. We demonstrated that the specification of hepatoblasts is normal in the zfyve9a morphants; however, the proliferation rate of these cells is reduced. Thus, our results reveal the liver-specific function of zfyve9a during early embryogenesis and indicate that the zfyve9a mediated signal is essential for the proliferation of hepatic cells during the expansion of liver bud.
The International journal of developmental biology 11/2013; · 2.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Vitamin C, a micronutrient known for its anti-scurvy activity in humans, promotes the generation of induced pluripotent stem cells (iPSCs) through the activity of histone demethylating dioxygenases. TET hydroxylases are also dioxygenases implicated in active DNA demethylation. Here we report that TET1 either positively or negatively regulates somatic cell reprogramming depending on the absence or presence of vitamin C. TET1 deficiency enhances reprogramming, and its overexpression impairs reprogramming in the context of vitamin C by modulating the obligatory mesenchymal-to-epithelial transition (MET). In the absence of vitamin C, TET1 promotes somatic cell reprogramming independent of MET. Consistently, TET1 regulates 5-hydroxymethylcytosine (5hmC) formation at loci critical for MET in a vitamin C-dependent fashion. Our findings suggest that vitamin C has a vital role in determining the biological outcome of TET1 function at the cellular level. Given its benefit to human health, vitamin C should be investigated further for its role in epigenetic regulation.
[show abstract][hide abstract] ABSTRACT: β-thalassemia (β-Thal) is a group of life-threatening blood disorders caused by either point mutations or deletions of nucleotides in β-globin gene (HBB). It is estimated that 4.5% of the population in the world carry β-Thal mutants (1), posing a persistent threat to public health. The generation of patient specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations offer an ideal therapeutic solution to this problem. However, homologous recombination-based gene correction in human iPS cells remains largely inefficient. Here, we describe a robust process combining efficient generation of integration-free β-Thal iPSCs from patients' cells and TALEN-based universal correction of HBB mutations in situ. We generated integration-free and gene-corrected iPSC lines from two patients carrying different types of homozygous mutations and showed that these iPSCs are pluripotent and have normal karyotype. We showed that the correction process did not generate TALEN induced off-targeting mutations by sequencing. More importantly, the gene-corrected β-Thal iPS cell lines from each patient can be induced to differentiate into hematopoietic progenitor cells (HPCs) and then further to erythroblasts expressing normal β-globin. Our studies provide an efficient and universal strategy to correct different types of β-globin mutations in β-Thal iPSCs for disease modeling and applications.
Journal of Biological Chemistry 10/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Realization of the full potential of human induced pluripotent stem cells (hiPSCs) in clinical applications requires the development of well-defined culture conditions for their long-term growth and directed differentiation. Here, we describe a novel fully defined synthetic peptide-decorated substrate that supports self-renewal of hiPSCs in commercially available xeno-free, chemically defined medium. The Au surface was deposited by a poly(OEGMA-co-HEMA) film using surface-initiated polymerization method (SIP) with the further step of carboxylation. The hiPSCs generated from umbilical cord mesenchymal cells were successfully cultured for 10 passages on the peptide-tethered poly(OEGMA-co-HEMA) brushes for the first time. Cells maintained their characteristic morphology, proliferation and expressed high levels of markers of pluripotency, similar to the cells cultured on Matrigel(TM). Moreover, the cell adhesion could be tuned by the pattern and peptide concentration on the substrate. Such well-defined, xeno-free and safe substrate that supports long-term proliferation and self-renewal of hiPSCs will not only help to accelerate the translational perspectives of hiPSCs, but also provide a platform to elucidate the underlying molecular mechanisms that regulate stem cell proliferation and differentiation via SIP technology.
[show abstract][hide abstract] ABSTRACT: CTCF is a ubiquitously expressed master weaver and plays multiple functions in the genome, including transcriptional activation/repression, chromatin insulation, imprinting, X chromosome inactivation and high order chromatin organization. It has been shown that CTCF facilitates the recruitment of UBF onto ribosomal DNA (rDNA) and regulates the local epigenetic state of rDNA repeats. However, the mechanism by which CTCF modulates rRNA gene transcription has not been well understood. Here we found that wild-type CTCF augmented pre-rRNA level, cell size and cell growth in cervical cancer cells. In contrast, RNA interference-mediated knockdown of CTCF reduced pre-rRNA transcription. CTCF positively regulates rRNA gene transcription in a RNA polymerase I dependent manner. We identified a RRGR motif as a putative Nucleolar Localization Sequence (NoLS) in C-terminal region of CTCF which is required for activating rRNA gene transcription. Using mass spectrometry, we identified SMC2 and SMC4, two subunits of condensin complexes which interact with CTCF. Condensin negatively regulates CTCF-mediated rRNA gene transcription. Knockdown of SMC2 expression significantly facilitates the loading of CTCF and UBF onto the rDNA locus, increases histone acetylation across the rDNA locus. Taken together, our study suggests that condensin competes with CTCF in binding to specific rDNA locus and negatively regulates CTCF-mediated rRNA gene transcription.
Journal of Biological Chemistry 07/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Catching the mutants: Pyrimido[4,5-d]pyrimidin-4(1H)-one derivatives (see example) were identified as specific inhibitors of EGFR(T790M) mutants. The compounds bound with T790M or L858R/T790M mutants with significantly lower Kd values than that with EGFR(WT) . They also selectively inhibited EGFR signal transduction and proliferation of NSCLC cells harboring EGFR(L858R/T790M) mutation, but were significantly less potent to cells with EGFR(WT) .
Angewandte Chemie International Edition 06/2013; · 13.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Present practices for reprogramming somatic cells to induced pluripotent stem cells involve simultaneous introduction of reprogramming factors. Here we report that a sequential introduction protocol (Oct4-Klf4 first, then c-Myc and finally Sox2) outperforms the simultaneous one. Surprisingly, the sequential protocol activates an early epithelial-to-mesenchymal transition (EMT) as indicated by the upregulation of Slug and N-cadherin followed by a delayed mesenchymal-to-epithelial transition (MET). An early EMT induced by 1.5-day TGF-β treatment enhances reprogramming with the simultaneous protocol, whereas 12-day treatment blocks reprogramming. Consistent results were obtained when the TGF-β antagonist Repsox was applied in the sequential protocol. These results reveal a time-sensitive role of individual factors for optimal reprogramming and a sequential EMT-MET mechanism at the start of reprogramming. Our studies provide a rationale for further optimizing reprogramming, and introduce the concept of a sequential EMT-MET mechanism for cell fate decision that should be investigated further in other systems, both in vitro and in vivo.
[show abstract][hide abstract] ABSTRACT: Sorting nexins (SNXs) are PX (phox-homology) domain-containing proteins involved in diverse intracellular endosomal trafficking pathways. The PX domain binds to certain phosphatidylinositols and is recruited to vesicles rich in those lipids. The structure of the PX domain is highly conserved, containing a three-stranded β-sheet followed by three α -helices. Here, we report the crystal structures of truncated human sorting nexin 11 (SNX11). The structures reveal that SNX11 contains a novel PX domain, hereby named extended PX (PXe) domain, with two additional α-helices at the C-terminal. We demonstrated that these α-helices were indispensible for the in vitro functions of SNX11. We propose that this PXe domain is present in sorting nexin 10 (SNX10) and is responsible for the vacuolation activity of SNX10. Thus, this novel PXe domain constitutes a structurally and functionally important subfamily of the PX domain.
Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Class IIa histone deacetylases (HDACs) and myocyte enhancer factor-2 (MEF2) proteins compose a signaling module that orchestrates lineage specification during embryogenesis. We show here that this module also regulates the generation of mouse induced pluripotent stem cells (iPSCs) by defined transcription factors. Class IIa HDACs and MEF2 proteins rise steadily during fibroblast reprogramming to iPSCs. MEF2 proteins tend to block the process by inducing the expression of Tgfβ cytokines, which impairs the necessary phase of mesenchymal-to-epithelial transition (MET). Conversely, class IIa HDACs endeavor to suppress the activity of MEF2 proteins, thus enhancing the MET and colony formation efficiency. Our work highlights an unexpected role for a developmental axis in somatic cell reprogramming and provides new insight into how the MET is regulated in this context.
Journal of Biological Chemistry 03/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Induced pluripotent stem cells (iPSCs) hold promise for the treatment of motoneuron diseases because of their distinct features including pluripotency, self-derivation and potential ability to differentiate into motoneurons. However, it is still unknown whether human iPSC-derived motoneurons can functionally innervate target muscles in vivo, which is the definitive sign of successful cell therapy for motoneuron diseases. In the present study, we demonstrated that human iPSCs derived from mesenchymal cells of the umbilical cord possessed a high yield in neural differentiation. Using a chemically-defined in vitro system, human iPSCs efficiently differentiated into motoneurons which displayed typical morphology, expressed specific molecules, and generated repetitive trains of action potentials. When transplanted into the injured musculocutaneous nerve of rats, they survived robustly, extended axons along the nerve, and formed functional connections with the target muscle (biceps brachii), thereby protecting the muscle from atrophy. Our study provides evidence for the first time that human iPSC-derived motoneurons are truly functional not only in vitro but also in vivo, and they have potential for stem cell-based therapies for motoneuron diseases.
Stem cell research 03/2013; 11(1):529-539. · 3.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: The generation of induced pluripotent stem (iPS) cells by exogenous transcription factors involves a comprehensive rearrangement of cellular functions, including the microRNA profile. The resulting cell lines are similar to embryonic stem (ES) cells and have therefore raised much interest for in vitro studies and the perspective of clinical application. Yet, microRNAs are not mere listeners of the reprogramming orchestra but play an active role in the process. In consequence, overexpression or suppression of individual microRNAs has profound effects in colony formation efficiency, and in combination they can produce iPS cells without added transcription factors. Moreover, variations in microRNA expression of iPS/ES cells can predict their differentiation potential and may have consequences at other levels. Altogether, these findings highlight the relevance of pursuing further these studies.
Current opinion in cell biology 01/2013; · 14.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Induced pluripotent stem cells (iPSCs) exhibit reduced efficiency and higher variability in neural differentiation compared to embryonic stem cells (ESCs). In this study, we showed that mouse iPSCs failed to efficiently give rise to neuronal cells using conventional methods previously established for driving mouse ESC differentiation. We reported a novel approach which remarkably increases neural differentiation of mouse iPSCs. This novel approach initiated embryoid body (EB) formation directly from the whole cell clones isolated from the top of feeder cells. Compared to conventional neural induction methods such as single cell suspension or monolayer culture, the cell clone-derived EB method led to a pronounced increase in directed generation of various types of neural cells including neural stem cells, motoneurons and dopaminergic neurons in response to different inducers. Through gene expression microarray analysis, we identified 14 genes that were highly expressed in the cell clone-derived EBs. Among them, we found that Cdh2, also known as N-cadherin, played important roles in controlling the neural differentiation efficiency of mouse iPSCs. Forced expression of Cdh2 in iPSCs substantially enhanced the differentiation efficiency while knocking-down of Cdh2 by shRNA blocked the neural differentiation. Our results revealed a critical role of Cdh2 in the process of efficient neural differentiation of mouse iPS cells.
Stem cell research 01/2013; 10(3):338-348. · 3.39 Impact Factor