Pluripotency without Proliferation.

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.

The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity.

The RNA modification N6-methyladenosine (m6A) has critical roles in many biological processes1,2. However, the function of m6A in the early phase of mammalian development remains poorly understood. Here we show that the m6A reader YT521-B homology-domain-containing protein 1 (YTHDC1) is required for the maintenance of mouse embryonic stem (ES) cells in an m6A-dependent manner, and that its deletion initiates cellular reprogramming to a 2C-like state. Mechanistically, YTHDC1 binds to the transcripts of retrotransposons (such as intracisternal A particles, ERVK and LINE1) in mouse ES cells and its depletion results in the reactivation of these silenced retrotransposons, accompanied by a global decrease in SETDB1-mediated trimethylation at lysine 9 of histone H3 (H3K9me3). We further demonstrate that YTHDC1 and its target m6A RNAs act upstream of SETDB1 to repress retrotransposons and Dux, the master inducer of the two-cell stage (2C)-like program. This study reveals an essential role for m6A RNA and YTHDC1 in chromatin modification and retrotransposon repression.

Resolving Cell Fate Decisions during Somatic Cell Reprogramming by Single-Cell RNA-Seq.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs), which is a highly heterogeneous process. Here we report the cell fate continuum during somatic cell reprogramming at single-cell resolution. We first develop SOT to analyze cell fate continuum from Oct4/Sox2/Klf4- or OSK-mediated reprogramming and show that cells bifurcate into two categories, reprogramming potential (RP) or non-reprogramming (NR). We further show that Klf4 contributes to Cd34+/Fxyd5+/Psca+ keratinocyte-like NR fate and that IFN-γ impedes the final transition to chimera-competent pluripotency along the RP cells. We analyze more than 150,000 single cells from both OSK and chemical reprograming and identify additional NR/RP bifurcation points. Our work reveals a generic bifurcation model for cell fate decisions during somatic cell reprogramming that may be applicable to other systems and inspire further improvements for reprogramming.

Regeneration of the human segmentation clock in somitoids in vitro.

Each vertebrate species appears to have a unique timing mechanism for forming somites along the vertebral column, and the process in human remains poorly understood at the molecular level due to technical and ethical limitations. Here, we report the reconstitution of human segmentation clock by direct reprogramming. We first reprogrammed human urine epithelial cells to a presomitic mesoderm (PSM) state capable of long-term self-renewal and formation of somitoids with an anterior-to-posterior axis. By inserting the RNA reporter Pepper into HES7 and MESP2 loci of these iPSM cells, we show that both transcripts oscillate in the resulting somitoids at ~5 h/cycle. GFP-tagged endogenous HES7 protein moves along the anterior-to-posterior axis during somitoid formation. The geo-sequencing analysis further confirmed anterior-to-posterior polarity and revealed the localized expression of WNT, BMP, FGF, and RA signaling molecules and HOXA-D family members. Our study demonstrates the direct reconstitution of human segmentation clock from somatic cells, which may allow future dissection of the mechanism and components of such a clock and aid regenerative medicine.

A Novel CCK Receptor GPR173 Mediates Potentiation of GABAergic Inhibition.

Cholecystokinin (CCK) enables excitatory circuit long-term potentiation (LTP). Here, we investigated its involvement in the enhancement of inhibitory synapses. Activation of GABA neurons suppressed neuronal responses in the neocortex to a forthcoming auditory stimulus in mice of both sexes. High-frequency laser stimulation (HFLS) of GABAergic neurons potentiated this suppression. HFLS of CCK interneurons could induce the LTP of their inhibition toward pyramidal neurons. This potentiation was abolished in CCK knock-out mice but intact in mice with both CCK1R and 2R knockout of both sexes. Next, we combined bioinformatics analysis, multiple unbiased cell-based assays, and histology examinations to identify a novel CCK receptor, GPR173. We propose GPR173 as CCK3R, which mediates the relationship between cortical CCK interneuron signaling and inhibitory LTP in the mice of either sex. Thus, GPR173 might represent a promising therapeutic target for brain disorders related to excitation and inhibition imbalance in the cortex.SIGNIFICANCE STATEMENT CCK, the most abundant and widely distributed neuropeptide in the CNS, colocalizes with many neurotransmitters and modulators. GABA is one of the important inhibitory neurotransmitters, and much evidence shows that CCK may be involved in modulating GABA signaling in many brain areas. However, the role of CCK-GABA neurons in the cortical microcircuits is still unclear. We identified a novel CCK receptor, GPR173, localized in the CCK-GABA synapses and mediated the enhancement of the GABA inhibition effect, which might represent a promising therapeutic target for brain disorders related to excitation and inhibition imbalance in the cortex.

Exclusion of HDAC1/2 complexes by oncogenic nuclear condensates.

Nuclear condensates have been shown to regulate cell fate control, but its role in oncogenic transformation remains largely unknown. Here we show acquisition of oncogenic potential by nuclear condensate remodeling. The proto-oncogene SS18 and its oncogenic fusion SS18-SSX1 can both form condensates, but with drastically different properties and impact on 3D genome architecture. The oncogenic condensates, not wild type ones, readily exclude HDAC1 and 2 complexes, thus, allowing aberrant accumulation of H3K27ac on chromatin loci, leading to oncogenic expression of key target genes. These results provide the first case for condensate remodeling as a transforming event to generate oncogene and such condensates can be targeted for therapy. One sentence summary: Expulsion of HDACs complexes leads to oncogenic transformation.

Metabolic switch and epithelial-mesenchymal transition cooperate to regulate pluripotency.

Both metabolic switch from oxidative phosphorylation to glycolysis (OGS) and epithelial-mesenchymal transition (EMT) promote cellular reprogramming at early stages. However, their connections have not been elucidated. Here, when a chemically defined medium was used to induce early EMT during mouse reprogramming, a facilitated OGS was also observed at the same time. Additional investigations suggested that the two events formed a positive feedback loop via transcriptional activation, cooperated to upregulate epigenetic factors such as Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5, and accelerated pluripotency induction at the early stage. However, at late stages, by over-inducing glycolysis and preventing the necessary mesenchymal-epithelial transition, the two events trapped the cells at a new pluripotency state between naïve and primed states and inhibited further reprogramming toward the naïve state. In addition, the pluripotent stem cells at the new state have high similarity to epiblasts from E4.5 and E5.5 embryos, and have distinct characteristics from the previously reported epiblast-like or formative states. Therefore, the time-dependent cooperation between OGS and EMT in regulating pluripotency should extend our understanding of related fields.

Heterochromatin loosening by the Oct4 linker region facilitates Klf4 binding and iPSC reprogramming.

The success of Yamanaka factor reprogramming of somatic cells into induced pluripotent stem cells suggests that some factor(s) must remodel the nuclei from a condensed state to a relaxed state. How factor-dependent chromatin opening occurs remains unclear. Using FRAP and ATAC-seq, we found that Oct4 acts as a pioneer factor that loosens heterochromatin and facilitates the binding of Klf4 and the expression of epithelial genes in early reprogramming, leading to enhanced mesenchymal-to-epithelial transition. A mutation in the Oct4 linker, L80A, which shows impaired interaction with the BAF complex component Brg1, is inactive in heterochromatin loosening. Oct4-L80A also blocks the binding of Klf4 and retards MET. Finally, vitamin C or Gadd45a could rescue the reprogramming deficiency of Oct4-L80A by enhancing chromatin opening and Klf4 binding. These studies reveal a cooperation between Oct4 and Klf4 at the chromatin level that facilitates MET at the cellular level and shed light into the research of multiple factors in cell fate determination.

iPS cells: mapping the policy issues.

Given the explosion of research on induced pluripotent stem (iPS) cells, it is timely to consider the various ethical, legal, and social issues engaged by this fast-moving field. Here, we review issues associated with the procurement, basic research, and clinical translation of iPS cells.

UBE2O and USP7 co-regulate RECQL4 ubiquitinylation and homologous recombination-mediated DNA repair.

The human RecQ DNA helicase, RECQL4, plays a pivotal role in maintaining genomic stability by regulating the DNA double-strand breaks (DSBs) repair pathway, and is, thus, involved in the regulation of aging and cancer onset. However, the regulatory mechanisms of RECQL4, especially its post-translational modifications, have not been fully illustrated. Here, we report that the E2/E3 hybrid ubiquitin-conjugating enzyme, UBE2O, physically interacts with RECQL4, and mediates the multi-monoubiquitinylation of RECQL4, subsequently leading to its proteasomal degradation. Functionally, we showed that UBE2O inhibits homologous recombination (HR)-mediated DSBs repair, and this inhibition depends on its E2 catalytic activity and RECQL4 expression. Mechanistically, we showed that UBE2O attenuates the interaction of RECQL4 and DNA damage repair proteins, the MRE11-RAD50-NBS1 complex and CtIP. Furthermore, we show that deubiquitinylase USP7 interacts with both UBE2O and RECQL4, and in that it antagonizes UBE2O-mediated regulation of RECQL4 stability and function. Collectively, we found a novel regulatory mechanism of ubiquitin-mediated regulation of RECQL4 in HR-mediated DSBs repair process.

Chromatin accessibility dynamics during cell fate reprogramming.

Genome architecture and chromatin dynamics govern the fate and identify of a cell. Recent advances in mapping chromatin landscapes offer valuable tools for the acquisition of accurate information regarding chromatin dynamics. Here we discuss recent findings linking chromatin dynamics to cell fate control. Specifically, chromatin undergoes a binary off/on switch during iPSC reprogramming, closing and opening loci occupied by somatic and pluripotency transcription factors, respectively. This logic of a binary off/on switch may also be operational in cell fate control during normal development and implies that further approaches could potentially be developed to direct cell fate changes both in vitro and in vivo.

The nuclear factor CECR2 promotes somatic cell reprogramming by reorganizing the chromatin structure.

Somatic cells can be reprogrammed into pluripotent stem cells with a minimal set of defined factors, Oct3/4, Sox2, Klf4, and c-Myc, also known as OKSM, although this reprogramming is somewhat inefficient. Recent work has identified other nuclear factors, including SALL4, that can synergize with the OSK factors to improve reprogramming dynamics, but the specific role of each of these factors remains poorly understood. In this study, we sought to learn more about the role of SALL4. We observed that SALL4 was the most significant factor in promoting OKS-induced reprogramming. To look for molecules downstream of SALL4, we screened a set of putative targets to determine whether they could promote OKS-induced reprogramming. We identified CECR2, a multidomain nuclear factor and histone acetyl-lysine reader, as a SALL4 effector. Mechanistically, we determined that SALL4 activates Cecr2 expression by directly binding to its promotor region. CECR2 in turn promotes reprogramming by forming a chromatin remodeling complex; this complex contained the SWI/SNF family member SMARCA1 and was dependent on CECR2's DTT domain. In combination, our findings suggest that CECR2 is a novel reprogramming factor and works through a protein network to overcome epigenetic barriers during reprogramming.

AP-1 activity is a major barrier of human somatic cell reprogramming.

Human induced pluripotent stem cells (iPSCs) technology has been widely applied to cell regeneration and disease modeling. However, most mechanism of somatic reprogramming is studied on mouse system, which is not always generic in human. Consequently, the generation of human iPSCs remains inefficient. Here, we map the chromatin accessibility dynamics during the induction of human iPSCs from urine cells. Comparing to the mouse system, we found that the closing of somatic loci is much slower in human. Moreover, a conserved AP-1 motif is highly enriched among the closed loci. The introduction of AP-1 repressor, JDP2, enhances human reprogramming and facilitates the reactivation of pluripotent genes. However, ESRRB, KDM2B and SALL4, several known pluripotent factors promoting mouse somatic reprogramming fail to enhance human iPSC generation. Mechanistically, we reveal that JDP2 promotes the closing of somatic loci enriching AP-1 motifs to enhance human reprogramming. Furthermore, JDP2 can rescue reprogramming deficiency without MYC or KLF4. These results indicate AP-1 activity is a major barrier to prevent chromatin remodeling during somatic cell reprogramming.

Beyond Boolean: Ternary networks and dynamics.

Boolean networks introduced by Kauffman, originally intended as a prototypical model for gaining insights into gene regulatory dynamics, have become a paradigm for understanding a variety of complex systems described by binary state variables. However, there are situations, e.g., in biology, where a binary state description of the underlying dynamical system is inadequate. We propose random ternary networks and investigate the general dynamical properties associated with the ternary discretization of the variables. We find that the ternary dynamics can be either ordered or disordered with a positive Lyapunov exponent, and the boundary between them in the parameter space can be determined analytically. A dynamical event that is key to determining the boundary is the emergence of an additional fixed point for which we provide numerical verification. We also find that the nodes playing a pivotal role in shaping the system dynamics have characteristically distinct behaviors in different regions of the parameter space, and, remarkably, the boundary between these regions coincides with that separating the ordered and disordered dynamics. Overall, our framework of ternary networks significantly broadens the classical Boolean paradigm by enabling a quantitative description of richer and more complex dynamical behaviors.

[Culture of pluripotent stem cell-derived hepatocytes and organoids].

Liver is one of the most important organs in the human body. Liver diseases are also a major threat to human health and longevity. Hepatic decompensation treatment is quite difficult due to multiple reasons. Extracorporeal liver support devices are unable to solve this problem, and there is a severe shortage of orthotopic liver transplant donors. Study of pluripotent stem cell-derived hepatocytes and organoids can determine not only hepatocyte fate, but also liver development, regeneration mechanisms, and pathophysiology. Furthermore, it can be used for drug screening in order to provide a stable source of functional hepatocytes for future transplantation therapy. Culture of pluripotent stem cell-derived hepatocytes and organoids has a self-organizing process similar to liver development, i.e., starting with changes in several key factors, and eventually forming functionally complex cells/organs. This paper introduces the main methods and progress of pluripotent stem cell-derived hepatocytes and organoids, with hope to provide clues for future research.

Naloxone regulates the differentiation of neural stem cells via a receptor-independent pathway.

The abilities of opioids to activate downstream signaling pathways normally depend on the binding between opioids and their receptors. However, opioids may also function in a receptor-independent manner, especially in neural stem cells (NSCs) in which the expression of opioid receptors and endogenous opioid agonists is low. When two opioids, morphine and naloxone, were used during the early stage of NSC differentiation, increased neurogenesis was observed. However, naloxone methiodide, a membrane impenetrable analog of naloxone, did not affect the NSC differentiation. The abilities of morphine and naloxone to facilitate neurogenesis were also observed in opioid receptor-knockout NSCs. Therefore, morphine and naloxone promote neurogenesis in a receptor-independent manner at least during the early stage. In addition, the receptor-independent functions of opioids were not observed in methylcytosine dioxygenase ten-eleven translocation 1 (Tet1) knockout NSCs. When the expression of opioid receptors increased and the expression of Tet1 decreased during the late stage of NSC differentiation, morphine, but not naloxone, inhibited neurogenesis via traditional receptor-dependent and miR181a-Prox1-Notch-related pathway. In summary, the current results demonstrated the time-dependent effects of opioids during the differentiation of NSCs and provided additional insight on the complex functions of opioids.

Hallmarks of pluripotency.

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.

Vitamin C-dependent lysine demethylase 6 (KDM6)-mediated demethylation promotes a chromatin state that supports the endothelial-to-hematopoietic transition.

Hematopoietic stem cells (HSCs)/progenitor cells (HPCs) are generated from hemogenic endothelial cells (HECs) during the endothelial-to-hematopoietic transition (EHT); however, the underlying mechanism remains poorly understood. Here, using an array of approaches, including CRSPR/Cas9 gene knockouts, RNA-Seq, ChIP-Seq, ATAC-Seq etc., we report that vitamin C (Vc) is essential in HPC generation during human pluripotent stem cell (hPSC) differentiation in defined culture conditions. Mechanistically, we found that the endothelial cells generated in the absence of Vc fail to undergo the EHT because of an apparent failure in opening up genomic loci essential for hematopoiesis. Under Vc deficiency, these loci exhibited abnormal accumulation of histone H3 trimethylation at Lys-27 (H3K27me3), a repressive histone modification that arose because of lower activities of demethylases that target H3K27me3. Consistently, deletion of the two H3K27me3 demethylases, Jumonji domain-containing 3 (JMJD3 or KDM6B) and histone demethylase UTX (UTX or KDM6A), impaired HPC generation even in the presence of Vc. Furthermore, we noted that Vc and jmjd3 are also important for HSC generation during zebrafish development. Together, our findings reveal an essential role for Vc in the EHT for hematopoiesis, and identify KDM6-mediated chromatin demethylation as an important regulatory mechanism in hematopoietic cell differentiation.

The homeobox transcription factor MSX2 partially mediates the effects of bone morphogenetic protein 4 (BMP4) on somatic cell reprogramming.

Bone morphogenetic proteins (BMPs) induce mesenchymal-epithelial transition (MET) and enhance the generation of induced pluripotent stem cells (iPSCs). However, BMPs are also signaling molecules critical for arresting reprogramming in the pre-iPSC state. In this study, using mouse embryonic fibroblasts, we found that the time- and concentration-dependent effects of BMPs on reprogramming are mediated by Msh homeobox 2 (MSX2), a homeobox-containing transcription factor. BMPs up-regulated Msx2 by activating SMAD1/5, and MSX2 then directly bound to the promoters and up-regulated the expression of the cadherin 1 (Cdh1, also known as E-cadherin), GATA-binding protein 3 (Gata3), and Nanog genes. Cdh1 contributed to BMP4- and MSX2-induced MET and subsequently promoted reprogramming. On the other hand, GATA3 promoted reprogramming, possibly by up-regulating Spalt-like transcription factor 4 (SALL4) expression. As key transcriptional factors in maintaining pluripotency, up-regulation of SALL4 and NANOG enhanced reprogramming. Moreover, the ability of MSX2 to up-regulate Cdh1, Gata3, Nanog, and Sall4 was further potentiated in the presence of Krüppel-like factor 4 (KLF4). However, MSX2 did not mediate the effects of BMP4 signaling on activation of the microRNAs miR-205 and miR-200 or the inhibitory effects that arrested reprogramming in the pre-iPSC state. In conclusion, MSX2 partially mediates the effects of BMP4 signaling during reprogramming, improving our understanding of the role of BMP signaling in MET and of the connection between cell lineage specifiers such as MSX2 and GATA3 and pluripotency.