[Show abstract][Hide abstract] ABSTRACT: Stem cells self-renew and generate specialized progeny through differentiation, but vary in the range of cells and tissues they generate, a property called developmental potency. Pluripotent stem cells produce all cells of an organism, while multipotent or unipotent stem cells regenerate only specific lineages or tissues. Defining stem-cell potency relies upon functional assays and diagnostic transcriptional, epigenetic and metabolic states. Here we describe functional and molecular hallmarks of pluripotent stem cells, propose a checklist for their evaluation, and illustrate how forensic genomics can validate their provenance.
[Show abstract][Hide abstract] ABSTRACT: Somatic cell reprogramming is accompanied by changes in lipid metabolism. While attempting to dissect the molecular mechanisms of the lipid metabolic switch during reprogramming, we found that overexpression of sterol regulatory element binding protein-1 (Srebp-1), a transcriptional factor required for lipid homeostasis, enhances reprogramming efficiency, while knockdown or pharmaceutical inhibition of Srebp-1 is inhibitory. Srebp-1 overexpression blocks the formation of partially reprogrammed cells, and functions in the early phase of reprogramming. Furthermore, Srebp-1 functions in nucleus and depends on its transcriptional activity but not its ability to bind the E-box motif and regulation of canonical targets. Mechanistically, Srebp-1 interacts with c-Myc, facilitates its binding to downstream pluripotent targets, strengthens the function of c-Myc in enhancing other Yamanaka factors' binding, and thereby promotes the expression of pluripotent genes. These results elucidate a novel role for Srebp-1 in somatic cell reprogramming and provide insights into understanding the metabolic switch during reprogramming. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Hematopoiesis is a progressive process collectively controlled by an elaborate network of transcription factors (TFs). Among these TFs, GATA2 has been implicated to be critical for regulating multiple steps of hematopoiesis in mouse models. However, whether similar function of GATA2 is conserved in human hematopoiesis, especially during early embryonic development stage, is largely unknown.
To examine the role of GATA2 in human background, we generated homozygous GATA2 knockout human embryonic stem cells (GATA2 (-/-) hESCs) and analyzed their blood differentiation potential. Our results demonstrated that GATA2 (-/-) hESCs displayed attenuated generation of CD34(+)CD43(+) hematopoietic progenitor cells (HPCs), due to the impairment of endothelial to hematopoietic transition (EHT). Interestingly, GATA2 (-/-) hESCs retained the potential to generate erythroblasts and macrophages, but never granulocytes. We further identified that SPI1 downregulation was partially responsible for the defects of GATA2 (-/-) hESCs in generation of CD34(+)CD43(+) HPCs and granulocytes. Furthermore, we found that GATA2 (-/-) hESCs restored the granulocyte potential in the presence of Notch signaling.
Our findings revealed the essential roles of GATA2 in EHT and granulocyte development through regulating SPI1, and uncovered a role of Notch signaling in granulocyte generation during hematopoiesis modeled by human ESCs.
[Show abstract][Hide abstract] ABSTRACT: Oncogenic transcription factors are known to mediate the conversion of somatic cells to tumour or induced pluripotent stem cells (iPSCs). Here we report c-Jun as a barrier for iPSC formation. c-Jun is expressed by and required for the proliferation of mouse embryonic fibroblasts (MEFs), but not mouse embryonic stem cells (mESCs). Consistently, c-Jun is induced during mESC differentiation, drives mESCs towards the endoderm lineage and completely blocks the generation of iPSCs from MEFs. Mechanistically, c-Jun activates mesenchymal-related genes, broadly suppresses the pluripotent ones, and derails the obligatory mesenchymal to epithelial transition during reprogramming. Furthermore, inhibition of c-Jun by shRNA, dominant-negative c-Jun or Jdp2 enhances reprogramming and replaces Oct4 among the Yamanaka factors. Finally, Jdp2 anchors 5 non-Yamanaka factors (Id1, Jhdm1b, Lrh1, Sall4 and Glis1) to reprogram MEFs into iPSCs. Our studies reveal c-Jun as a guardian of somatic cell fate and its suppression opens the gate to pluripotency.
[Show abstract][Hide abstract] ABSTRACT: The mouse is an organism that is widely used as a mammalian model for studying human physiology or disease, and the development of immunodeficient mice has provided a valuable tool for basic and applied human disease research. Following the development of large-scale mouse knockout programs and genome-editing tools, it has become increasingly efficient to generate genetically modified mouse strains with immunodeficiency. However, due to the lack of a standardized system for evaluating the immuno-capacity that prevents tumor progression in mice, an objective choice of the appropriate immunodeficient mouse strains to be used for tumor engrafting experiments is difficult.
In this study, we developed a tumor engraftment index (TEI) to quantify the immunodeficiency response to hematologic malignant cells and solid tumor cells of six immunodeficient mouse strains and C57BL/6 wild-type mouse (WT).
Mice with a more severely impaired immune system attained a higher TEI score. We then validated that the NOD-scid-IL2Rg-/- (NSI) mice, which had the highest TEI score, were more suitable for xenograft and allograft experiments using multiple functional assays.
The TEI score was effectively able to reflect the immunodeficiency of a mouse strain.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Heteroplasmic cells, harboring both mutant and normal mitochondrial DNAs (mtDNAs), must accumulate mutations to a threshold level before respiratory activity is affected. This phenomenon has led to the hypothesis of mtDNA complementation by inter-mitochondrial content mixing. The precise mechanisms of heteroplasmic complementation are unknown, but it depends both on the mtDNA nucleoid dynamics among mitochondria as well as the mitochondrial dynamics as influenced by mtDNA. We tracked nucleoids among the mitochondria in real time to show that they are shared after complete fusion but not 'kiss-and-run'. Employing a cell hybrid model, we further show that mtDNA-less mitochondria, which have little ATP production and extensive Opa1 proteolytic cleavage, exhibit weak fusion activity among themselves, yet remain competent in fusing with healthy mitochondria in a mitofusin- and OPA1-dependent manner, resulting in restoration of metabolic function. Depletion of mtDNA by overexpression of the matrix-targeted nuclease UL12.5 resulted in heterogeneous mitochondrial membrane potential (ΔΨm) at the organelle level in mitofusin-null cells but not in wild type. In this system, overexpression of mitofusins or application of the fusion-promoting drug M1 could partially rescue the metabolic damage caused by UL12.5. Interestingly, mtDNA transcription/translation is not required for normal mitochondria to restore metabolic function to mtDNA-less mitochondria by fusion. Thus, interplay between mtDNA and fusion capacity governs a novel 'initial metabolic complementation'.
Cellular and Molecular Life Sciences CMLS 02/2015; 72(13). DOI:10.1007/s00018-015-1863-9 · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human pluripotent stem cells (hPSCs) are a promising cell source with pluripotency and capacity to differentiate into all human somatic cell types. Designing simple and safe biomaterials with an innate ability to induce osteoblastic lineage from hPSCs is desirable to realize their clinical adoption in bone regenerative medicine. To address the issue, here we developed a fully defined synthetic peptides-decorated two dimensional (2D) microenvironment assisted via polydopamine (pDA) chemistry and subsequent carboxymethyl chitosan (CMC) grafting to enhance the culture and osteogenic potential of hPSCs in vitro. The hPSCs including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were successfully cultured on the peptides-decorated surface without Matrigel- and ECM protein-coating and underwent promoted osteogenic differentiation in vitro, determined from the alkaline phosphate (ALP) activity, gene expression, and protein production as well as calcium deposit amount. It was found that directed osteogenic differentiation of hPSCs could be achieved through a peptides-decorated niche. This chemical-defined and safe 2D microenvironment which facilitates proliferation and osteo-differentiation of hPSCs, not only helps to accelerate the translational perspectives of hPSCs, but also provides tissue-specific functions such as directing stem cell differentiation commitment, having great potential in bone tissue engineering and presenting new avenues for bone regenerative medicine.
[Show abstract][Hide abstract] ABSTRACT: Successful expansion of hematopoietic stem cells (HSCs) would benefit the use of HSC transplants in the clinic. Angiopoietin-like 7 promotes the expansion of hematopoietic stem and progenitor cells (HSPC) in vitro and ex vivo. However, the impact of loss of Angptl7 on HSPCs in vivo has not been characterized. Here, we generated Angptl7-deficient mice by TALEN-mediated gene targeting and found that HSC compartments in Angptl7-null mice were compromised. In addition, wild type (WT) HSPCs failed to repopulate in the BM of Angptl7-null mice after serial transplantations while the engraftment of Angptl7-deficient HSPCs in WT mice was not impaired. These results suggest that Angptl7 is required for HSPCs repopulation in a non-cell autonomous manner.
Electronic supplementary material
The online version of this article (doi:10.1186/s13045-014-0102-4) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Valproic acid (VPA) is widely used to treat epilepsy, migraine, chronic headache, bipolar disorder, and as adjuvant chemotherapy, but potentially causes idiosyncratic liver injury. Alpers-Huttenlocher syndrome (AHS), a neurometabolic disorder caused by mutations in mitochondrial DNA polymerase gamma (POLG), is associated with an increased risk of developing fatal VPA hepatotoxicity. However, the mechanistic link of this clinical mystery remains unknown. Here, fibroblasts from 2 AHS patients were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to hepatocyte-like cells (AHS iPSCs-Hep). Both AHS iPSCs-Hep are more sensitive to VPA-induced mitochondrial-dependent apoptosis than controls, showing more activated caspase-9 and cytochrome c release. Strikingly, levels of both soluble and oligomeric optic atrophy 1, which together keep cristae junctions tight, are reduced in AHS iPSCs-Hep. Furthermore, POLG mutation cells show reduced POLG expression, mitochondrial DNA (mtDNA) amount, mitochondrial adenosine triphosphate production, as well as abnormal mitochondrial ultrastructure after differentiation to hepatocyte-like cells. Superoxide flashes, spontaneous bursts of superoxide generation, caused by opening of the mitochondrial permeability transition pore (mPTP), occur more frequently in AHS iPSCs-Hep. Moreover, the mPTP inhibitor, cyclosporine A, rescues VPA-induced apoptotic sensitivity in AHS iPSCs-Hep. This result suggests that targeting mPTP opening could be an effective method to prevent hepatotoxicity by VPA in AHS patients. In addition, carnitine or N-acetylcysteine, which has been used in the treatment of VPA-induced hepatotoxicity, is able to rescue VPA-induced apoptotic sensitivity in AHS iPSCs-Hep.
AHS iPSCs-Hep are more sensitive to the VPA-induced mitochondrial-dependent apoptotic pathway, and this effect is mediated by mPTP opening. Toxicity models in genetic diseases using iPSCs enable the evaluation of drugs for therapeutic targets.
[Show abstract][Hide abstract] ABSTRACT: Recent studies have boosted our understanding of long noncoding RNAs (lncRNAs) in numerous biological processes, but few have examined their roles in somatic cell reprogramming. Through expression profiling and functional screening, we have identified that the large intergenic noncoding RNA p21 (lincRNA-p21) impairs reprogramming. Notably, lincRNA-p21 is induced by p53 but does not promote apoptosis or cell senescence in reprogramming. Instead, lincRNA-p21 associates with the H3K9 methyltransferase SETDB1 and the maintenance DNA methyltransferase DNMT1, which is facilitated by the RNA-binding protein HNRNPK. Consequently, lincRNA-p21 prevents reprogramming by sustaining H3K9me3 and/or CpG methylation at pluripotency gene promoters. Our results provide insight into the role of lncRNAs in reprogramming and establish a novel link between p53 and heterochromatin regulation.Cell Research advance online publication 16 December 2014; doi:10.1038/cr.2014.165.
Cell Research 12/2014; 25(1). DOI:10.1038/cr.2014.165 · 12.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reactivation of the pluripotency network during somatic cell reprogramming by exogenous transcription factors involves chromatin remodeling and the recruitment of RNA polymerase II (Pol II) to target loci. Here, we report that Pol II is engaged at pluripotency promoters in reprogramming but remains paused and inefficiently released. We also show that bromodomain-containing protein 4 (BRD4) stimulates productive transcriptional elongation of pluripotency genes by dissociating the pause release factor P-TEFb from an inactive complex containing HEXIM1. Consequently, BRD4 overexpression enhances reprogramming efficiency and HEXIM1 suppresses it, whereas Brd4 and Hexim1 knockdown do the opposite. We further demonstrate that the reprogramming factor KLF4 helps recruit P-TEFb to pluripotency promoters. Our work thus provides a mechanism for explaining the reactivation of pluripotency genes in reprogramming and unveils an unanticipated role for KLF4 in transcriptional pause release.
[Show abstract][Hide abstract] ABSTRACT: The process that converts somatic cells to pluripotent ones has enormous potential not only as a tool to generate cells for disease therapy and modeling, but also as an experimental system to investigate fundamental biological questions. The discovery of mesenchymal-to-epithelial transitions at the initial phase of reprogramming provides a conceptual framework to understand reprogramming in a cellular context and it helps to resolve the mechanistic roles of the original Yamanaka factors as well as newly identified modulators of reprogramming. Emerging concept such as sequential EMT-MET in reprogramming further suggests the value of this model to the understanding of cell fate conversions. We highlight recent advances about the function and regulation of MET in reprogramming and discuss their potential implications here.
Current Opinion in Genetics & Development 08/2014; 28C:32-37. DOI:10.1016/j.gde.2014.08.005 · 7.57 Impact Factor