[Show abstract][Hide abstract]ABSTRACT: During embryonic organogenesis, the odontogenic potential resides in dental mesenchyme from the bud stage until birth. Mouse dental mesenchymal cells (mDMCs) isolated from the inductive dental mesenchyme of developing molars are frequently used in the context of tooth development and regeneration. We wondered if and how the odontogenic potential could be retained when mDMCs were cultured in vitro. In the present study, we undertook to test the odontogenic potential of cultured mDMCs and attempted to maintain the potential during culturing. We found that cultured mDMCs could retain the odontogenic potential for 24 h with a ratio of 60% for tooth formation, but mDMCs were incapable of supporting tooth formation after more than 24 h in culture. This loss of odontogenic potential was accompanied by widespread transcriptomic alteration and, specifically, the downregulation of some dental mesenchyme-specific genes, such as Pax9, Msx1, and Pdgfrα. To prolong the odontogenic potential of mDMCs in vitro, we then cultured mDMCs in a serum-free medium with Knockout Serum Replacement (KSR) and growth factors (fibroblastic growth factor 2 and epidermal growth factor). In this new micromilieu, mDMCs could maintain the odontogenic potential for 48 h with tooth formation ratio of 50%. Moreover, mDMCs cultured in KSR-supplemented medium gave rise to tooth-like structures when recombined with non-dental second-arch epithelium. Among the supplements, KSR is essential for the survival and adhesion of mDMCs, and both Egf and Fgf2 induced the expression of certain dental mesenchyme-related genes. Taken together, our results demonstrated that the transcriptomic changes responded to the alteration of odontogenic potential in cultured mDMCs and a new micromilieu partly retained this potential in vitro, providing insight into the long-term maintenance of odontogenic potential in mDMCs.
[Show abstract][Hide abstract]ABSTRACT: Human urine cells (HUCs) can be reprogrammed into neural progenitor cells (NPCs) or induced pluripotent stem cells (iPSCs) with defined factors and a small molecule cocktail, but the underlying fate choice remains unresolved. Here, through sequential removal of individual compound from small molecule cocktail, we showed that A8301, a TGFβ signaling inhibitor, is sufficient to switch the cell fate from iPSCs into NPCs in OSKM-mediated HUCs reprogramming. However, TGFβ exposure at early stage inhibits HUCs reprogramming by promoting EMT. Base on these data, we developed an optimized approach for generation of NPCs or iPSCs from HUCs with significantly improved efficiency by regulating TGFβ activity at different reprogramming stages. This approach provides a simplified and improved way for HUCs reprogramming, thus would be valuable for banking human iPSCs or NPCs from people with different genetic background.
[Show abstract][Hide abstract]ABSTRACT: Mouse embryonic stem cells (mESCs) are capable of unlimited proliferation without losing pluripotency. Scognamiglio et al. now reveal that Myc depletion shifts mESCs into a dormant state reminiscent of embryonic diapause in which pluripotency remains fully preserved, thus decoupling pluripotency from proliferative programs.
[Show abstract][Hide abstract]ABSTRACT: Human pluripotent stem cells (hPSCs) possess great value in the aspect of cellular therapies due to its self-renewal and potential to differentiate into all somatic cell types. A few defined synthetic surfaces such as polymers and adhesive biological materials conjugated substrata were established for the self-renewal of hPSCs. However, none of them was effective in the generation of human induced pluripotent stem cells (hiPSCs) and long-term maintenance of multiple hPSCs, and most of them required complicated manufacturing processes. Polydopamine has good biocompatiblity, is able to form a stable film on nearly all solid substrates surface, and can immobilize adhesive biomolecules. In this manuscript, a polydopamine-mediated surface was developed, which not only supported the reprogramming of human somatic cells into hiPSCs under defined conditions, but also sustained the growth of hiPSCs on diverse substrates. Moreover, the proliferation and pluripotency of hPSCs cultured on the surface were comparable to Matrigel for more than 20 passages. Besides, hPSCs were able to differentiate to cardiomyocytes and neural cells on the surface. This polydopamine-based synthetic surface represents a chemically-defined surface extensively applicable both forfundamental research and cell therapies of hPSCs.
[Show abstract][Hide abstract]ABSTRACT: The trafficking of ion channels to/from the plasma membrane is considered an important mechanism for cellular activity and an interesting approach for disease therapies. The transient receptor potential vanilloid 3 (TRPV3) ion channel is widely expressed in skin keratinocytes, and its trafficking mechanism to/from the plasma membrane is unknown. Here, we report that the vesicular trafficking protein sorting nexin 11 (SNX11) downregulates the level of the TRPV3 plasma membrane protein. Overexpression of SNX11 causes a decrease in the level of TRPV3 current and TRPV3 plasma membrane protein in TRPV3-transfected HEK293T cells. Subcellular localizations and Western blots indicate that SNX11 interacts with TRPV3 and targets it to lysosomes for degradation, which is blocked by the lysosomal inhibitors chloroquine and leupeptin. Both TRPV3 and SNX11 are highly expressed in HaCaT cells. We show that TRPV3 agonists-activated Ca(2+) influxes and the level of native TRPV3 total protein in HaCaT cells are decreased by overexpression of SNX11 and increased by knockdown of SNX11. Our findings reveal that SNX11 promotes the trafficking of TRPV3 from the plasma membrane to lysosomes for degradation via protein-protein interactions, which demonstrates a previously unknown function of SNX11 as a regulator of TRPV3 trafficking from the plasma membrane to lysosomes.
[Show abstract][Hide abstract]ABSTRACT: Background:
Currently, direct conversion from somatic cells to neurons requires virus-mediated delivery of at least one transcriptional factor or a combination of several small-molecule compounds. Delivery of transcriptional factors may affect genome stability, while small-molecule compounds may require more evaluations when applied in vivo. Thus, a defined medium with only conventional growth factors or additives for cell culture is desirable for inducing neuronal trans-differentiation.
Here, we report that a defined medium (5C) consisting of basic fibroblast growth factor (bFGF), N2 supplement, leukemia inhibitory factor, vitamin C (Vc), and β-mercaptoethanol (βMe) induces the direct conversion of somatic cells to cells with neuronal characteristics. Application of 5C medium converted mouse embryonic fibroblasts (MEFs) into TuJ+ neuronal-like cells, which were capable of survival after being transplanted into the mouse brain. The same 5C medium could convert primary rat astrocytes into neuronal-like cells with mature electrophysiology characteristics in vitro and facilitated the recovery of brain injury, possibly by inducing similar conversions, when infused into the mouse brain in vivo. Crucially, 5C medium could also induce neuronal characteristics in several human cell types.
In summary, this 5C medium not only provides a means to derive cells with neuronal characteristics without viral transfection in vitro but might also be useful to produce neurons in vivo for neurodegenerative disease treatment.
[Show abstract][Hide abstract]ABSTRACT: Nucleosome positioning and histone modification play a critical role in gene regulation, but their role during reprogramming has not been fully elucidated. Here, we determined the genome-wide nucleosome coverage and histone methylation occupancy in mouse embryonic fibroblasts (MEFs), induced pluripotent stem cells (iPSCs) and pre-iPSCs. We found that nucleosome occupancy increases in promoter regions and decreases in intergenic regions in pre-iPSCs, then recovers to an intermediate level in iPSCs. We also found that nucleosomes in pre-iPSCs are much more phased than those in MEFs and iPSCs. During reprogramming, nucleosome reorganization and histone methylation around transcription start sites (TSSs) are highly coordinated with distinctively transcriptional activities. Bivalent promoters gradually increase, while repressive promoters gradually decrease. High CpG (HCG) promoters of active genes are characterized by nucleosome depletion at TSSs, while low CpG (LCG) promoters exhibit the opposite characteristics. In addition, we show that vitamin C (VC) promotes reorganizations of canonical, H3K4me3- and H3K27me3-modified nucleosomes on specific genes during transition from pre-iPSCs to iPSCs. These data demonstrate that pre-iPSCs have a more open and phased chromatin architecture than that of MEFs and iPSCs. Finally, this study reveals the dynamics and critical roles of nucleosome positioning and chromatin organization in gene regulation during reprogramming.
Full-text · Article · Dec 2015 · Scientific Reports
[Show abstract][Hide abstract]ABSTRACT: The mechanisms of somatic cell reprogramming have been revealed at multiple levels. However, the lack of tools to monitor different reactive oxygen species (ROS) has left their distinct signals and roles in reprogramming unknown. We hypothesized that mitochondrial flashes (mitoflashes), recently identified spontaneous bursts of mitochondrial superoxide signaling, play a role in reprogramming. Here we show that the frequency of mitoflashes transiently increases, accompanied by flash amplitude reduction, during the early stages of reprogramming. This transient activation of mitoflashes at the early stage enhances reprogramming, whereas sustained activation impairs reprogramming. The reprogramming-promoting function of mitoflashes occurs via the upregulation of Nanog expression that is associated with decreases in the methylation status of the Nanog promoter through Tet2 occupancy. Together our findings provide a previously unknown role for superoxide signaling mediated epigenetic regulation in cell fate determination.
[Show abstract][Hide abstract]ABSTRACT: Fibrodysplasia ossificans progressiva (FOP) is a rare disease characterized by progressive ossification of soft tissues, for which there is no effective treatment. Mutations in the bone morphogenetic protein (BMP) type I receptor activin receptor-like kinase 2 (ACVR1/ALK2) are the main cause of FOP. We generated human induced pluripotent stem cells (hiPSCs) from FOP patients with the ALK2 R206H mutation. The mutant ALK2 gene changed differentiation efficiencies of hiPSCs into FOP bone-forming progenitors: endothelial cells (ECs) and pericytes. ECs from FOP hiPSCs showed reduced expression of vascular endothelial growth factor receptor 2 and could transform into mesenchymal cells through endothelial-mesenchymal transition. Increased mineralization of pericytes from FOP hiPSCs could be partly inhibited by the ALK2 kinase inhibitor LDN-212854. Thus, differentiated FOP hiPSCs recapitulate some aspects of the disease phenotype in vitro, and they could be instrumental in further elucidating underlying mechanisms of FOP and development of therapeutic drug candidates.
[Show abstract][Hide abstract]ABSTRACT: Aging is considered an irreversible biological process and also a major risk factor for a spectrum of geriatric diseases. Advanced age-related decline in physiological functions, such as neurodegeneration, development of cardiovascular disease, endocrine and metabolic dysfunction, and neoplastic transformation, has become the focus in aging research. Natural aging is not regarded as a programmed process. However, accelerated aging due to inherited genetic defects in patients of progeria is programmed and resembles many aspects of natural aging. Among several premature aging syndromes, Werner syndrome (WS) and Hutchinson-Gilford progeria syndrome (HGPS) are two broadly investigated diseases. In this review, we discuss how stem cell aging in WS helps us understand the biology of aging. We also discuss briefly how the altered epigenetic landscape in aged cells can be reversed to a "juvenile" state. Lastly, we explore the potential application of the latest genomic editing technique for stem cell-based therapy and regenerative medicine in the context of aging.
[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.
Full-text · Article · Jun 2015 · Nature Cell Biology