Construction of a human cell landscape at single-cell level.

Single-cell analysis is a valuable tool for dissecting cellular heterogeneity in complex systems1. However, a comprehensive single-cell atlas has not been achieved for humans. Here we use single-cell mRNA sequencing to determine the cell-type composition of all major human organs and construct a scheme for the human cell landscape (HCL). We have uncovered a single-cell hierarchy for many tissues that have not been well characterized. We established a 'single-cell HCL analysis' pipeline that helps to define human cell identity. Finally, we performed a single-cell comparative analysis of landscapes from human and mouse to identify conserved genetic networks. We found that stem and progenitor cells exhibit strong transcriptomic stochasticity, whereas differentiated cells are more distinct. Our results provide a useful resource for the study of human biology.

Harnessing endogenous transcription factors directly by small molecules for chemically induced pluripotency inception.

Chemistry-alone approach has recently been applied for incepting pluripotency in somatic cells, representing a breakthrough in biology. However, chemical reprogramming is hampered by low efficiency, and the underlying molecular mechanisms remain unclear. Particularly, chemical compounds do not have specific DNA-recognition domains or transcription regulatory domains, and then how do small molecules work as a driving force for reinstating pluripotency in somatic cells? Furthermore, how to efficiently clear materials and structures of an old cell to prepare the rebuilding of a new one? Here, we show that small molecule CD3254 activates endogenous existing transcription factor RXRα to significantly promote mouse chemical reprogramming. Mechanistically, CD3254-RXRα axis can directly activate all the 11 RNA exosome component genes (Exosc1-10 and Dis3) at transcriptional level. Unexpectedly, rather than degrading mRNAs as its substrates, RNA exosome mainly modulates the degradation of transposable element (TE)-associated RNAs, particularly MMVL30, which is identified as a new barrier for cell-fate determination. In turn, MMVL30-mediated inflammation (IFN-γ and TNF-α pathways) is reduced, contributing to the promotion of successful reprogramming. Collectively, our study provides conceptual advances for translating environmental cues into pluripotency inception, particularly, identifies that CD3254-RXRα-RNA exosome axis can promote chemical reprogramming, and suggests modulation of TE-mediated inflammation via CD3254-inducible RNA exosome as important opportunities for controlling cell fates and regenerative medicine.

Inhibition of Syk promotes chemical reprogramming of fibroblasts via metabolic rewiring and H2 S production.

Chemical compounds have recently been introduced as alternative and non-integrating inducers of pluripotent stem cell fate. However, chemical reprogramming is hampered by low efficiency and the molecular mechanisms remain poorly characterized. Here, we show that inhibition of spleen tyrosine kinase (Syk) by R406 significantly promotes mouse chemical reprogramming. Mechanistically, R406 alleviates Syk / calcineurin (Cn) / nuclear factor of activated T cells (NFAT) signaling-mediated suppression of glycine, serine, and threonine metabolic genes and dependent metabolites. Syk inhibition upregulates glycine level and downstream transsulfuration cysteine biosynthesis, promoting cysteine metabolism and cellular hydrogen sulfide (H2 S) production. This metabolic rewiring decreased oxidative phosphorylation and ROS levels, enhancing chemical reprogramming. In sum, our study identifies Syk-Cn-NFAT signaling axis as a new barrier of chemical reprogramming and suggests metabolic rewiring and redox homeostasis as important opportunities for controlling cell fates.

Metabolic control of TH17 and induced Treg cell balance by an epigenetic mechanism.

Metabolism has been shown to integrate with epigenetics and transcription to modulate cell fate and function. Beyond meeting the bioenergetic and biosynthetic demands of T-cell differentiation, whether metabolism might control T-cell fate by an epigenetic mechanism is unclear. Here, through the discovery and mechanistic characterization of a small molecule, (aminooxy)acetic acid, that reprograms the differentiation of T helper 17 (TH17) cells towards induced regulatory T (iTreg) cells, we show that increased transamination, mainly catalysed by GOT1, leads to increased levels of 2-hydroxyglutarate in differentiating TH17 cells. The accumulation of 2-hydroxyglutarate resulted in hypermethylation of the Foxp3 gene locus and inhibited Foxp3 transcription, which is essential for fate determination towards TH17 cells. Inhibition of the conversion of glutamate to α-ketoglutaric acid prevented the production of 2-hydroxyglutarate, reduced methylation of the Foxp3 gene locus, and increased Foxp3 expression. This consequently blocked the differentiation of TH17 cells by antagonizing the function of transcription factor RORγt and promoted polarization into iTreg cells. Selective inhibition of GOT1 with (aminooxy)acetic acid ameliorated experimental autoimmune encephalomyelitis in a therapeutic mouse model by regulating the balance between TH17 and iTreg cells. Targeting a glutamate-dependent metabolic pathway thus represents a new strategy for developing therapeutic agents against TH17-mediated autoimmune diseases.

Density- and moisture-dependent effects of arbuscular mycorrhizal fungus on drought acclimation in wheat.

Arbuscular mycorrhizal fungus (AMF) is widely viewed as an ecosystem engineer to help plants adapt to adverse environments. However, a majority of the previous studies regarding AMF's eco-physiological effects are mutually inconsistent. To clarify this fundamental issue, we conducted an experiment focused on wheat (Triticum aestivum L.) plants with or without AMF (Funneliformis mosseae) inoculation. Two water regimes (80% and 40% field water capacity, FWC80 (CK) and FWC40 (drought stress) and four planting densities (6 or 12 plants per pot as low densities, 24 or 48 plants per pot as high densities) were designed. AMF inoculation did not show significant effects on shoot biomass, grain yield, and water use efficiency (WUE) under the low densities, regardless of water regimes. However, under the high densities, AMF inoculation significantly decreased shoot biomass, grain yield and WUE in FWC80, while it significantly increased these parameters in FWC40, showing density and/or moisture-dependent effects of AMF on wheat performance. In FWC40, the relationships between reproductive biomass (y-axis) vs. vegetative biomass (x-axis) (R-V), and between grain biomass (y-axis, sink) vs. leaf biomass (x-axis, source) fell into a typical allometric pattern (α > 1, P < 0.001), and the AMF inoculation significantly increased the values of α. Yet in FWC80, they were in an isometric pattern (α ≈ 1, P < 0.001) and AMF addition had no significant effects on α. Similarly, AMF did not significantly change the isometric relationship between leaf biomass (i.e., metabolic rate) and shoot biomass (body size) in FWC80, while it significantly decreased the α of allometric relationship between both of them in FWC40 (α > 1, P < 0.001). We therefore, sketched a generalized model of R-V and sink-source relationships as affected by AMF, in which AMF inoculation might enhance the capabilities of sink acquisition and utilization under drought stress, while having no significant effect under the well watered conditions. Our findings demonstrate dual density- and moisture-dependent effects of AMF on plant development and provide new insights into current ecological applications of AMF as an ecosystem engineer.

Mouse liver repopulation with hepatocytes generated from human fibroblasts.

Human induced pluripotent stem cells (iPSCs) have the capability of revolutionizing research and therapy of liver diseases by providing a source of hepatocytes for autologous cell therapy and disease modelling. However, despite progress in advancing the differentiation of iPSCs into hepatocytes (iPSC-Heps) in vitro, cells that replicate the ability of human primary adult hepatocytes (aHeps) to proliferate extensively in vivo have not been reported. This deficiency has hampered efforts to recreate human liver diseases in mice, and has cast doubt on the potential of iPSC-Heps for liver cell therapy. The reason is that extensive post-transplant expansion is needed to establish and sustain a therapeutically effective liver cell mass in patients, a lesson learned from clinical trials of aHep transplantation. Here, as a solution to this problem, we report the generation of human fibroblast-derived hepatocytes that can repopulate mouse livers. Unlike current protocols for deriving hepatocytes from human fibroblasts, ours did not generate iPSCs but cut short reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells and subsequently hepatocytes (iMPC-Heps) could be efficiently differentiated. For this purpose we identified small molecules that aided endoderm and hepatocyte differentiation without compromising proliferation. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to those of aHeps. Unfractionated iMPC-Heps did not form tumours, most probably because they never entered a pluripotent state. Our results establish the feasibility of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy.

Recent advances and potential applications of human pluripotent stem cell-derived pancreatic ß cells.

Diabetes mellitus is characterized by chronic high blood glucose levels resulted from deficiency and/or dysfunction of insulin-producing pancreatic ß cells. Generation of large amounts of functional pancreatic ß cells is critical for the study of pancreatic biology and treatment of diabetes. Recent advances in directed differentiation of pancreatic ß-like cells from human pluripotent stem cells (hPSCs) can provide patient-specific and disease-relevant target cells. With the improved differentiation protocols, it is now possible to generate large amounts of functional human pancreatic ß-like cells that can response to high level of glucose both in vitro and in vivo. Combined with precise genomic editing, biomedical engineering, high throughput profiling, bioinformatics, and high throughput genetic and chemical screening, these hPSC-derived pancreatic ß-like cells will hold great potentials in disease modeling, drug discovery, and cell-based therapies. In this review, we summarize the recent progress in human pancreatic ß-like cells derived from hPSCs and discuss their potential applications.

Chemical strategies for pancreatic ß cell differentiation, reprogramming, and regeneration.

Generation of unlimited functional pancreatic ß cells is critical for the study of pancreatic biology and treatment of diabetes mellitus. Recent advances have suggested several promising directions, including directed differentiation of pancreatic ß cells from pluripotent stem cells, reprogramming of pancreatic ß cells from other types of somatic cells, and stimulated proliferation and enhanced functions of existing pancreatic ß cells. Small molecules are useful in generating unlimited numbers of functional pancreatic cells in vitro and could be further developed as drugs to stimulate endogenous pancreatic regeneration. Here, we provide an updated summary of recent major achievements in pancreatic ß cell differentiation, reprogramming, proliferation, and function. These studies will eventually lead to significant advances in the field of pancreatic biology and regeneration.

A field study on heavy metals phytoattenuation potential of monocropping and intercropping of maize and/or legumes in weakly alkaline soils.

The study focused on the phytoattenuation effects of monocropping and intercropping of maize (Zea mays) and/or legumes on Cu, Zn, Pb, and Cd in weakly alkaline soils. Nine growth stages of monocropping maize were chosen to study the dynamic process of extraction of heavy metals. The total content of heavy metals extracted by the aerial part of monocropped maize increased in a sigmoidal pattern over the effective accumulative temperature. The biggest biomass, highest extraction content, and lowest heavy metals bioaccumulation level occurred at physiological maturity. Among the different planting patterns, including monocropping and intercropping of maize and/or soybean (Glycine max), pea (Pisum sativum), and alfalfa (Medicago sativa), the extraction efficiency of Cu, Zn, Pb, and Cd varied greatly. Only intercropping of maize and soybean yielded relatively higher extraction efficiency for the four metals with no significant difference in the total biomass. Moreover, the heavy metals concentrations in dry biomass from all the planting patterns in the present study were within China's national legal thresholds for fodder use. Therefore, slightly polluted alkaline soils can be safely used through monocropping and intercropping of maize and/or legumes for a range of purposes. In particular, this study indicated that intercropping improves soil ecosystems polluted by heavy metals compared with monocropping.

Direct reprogramming of mouse fibroblasts to neural progenitors.

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.

N-acetyltransferase NAT10 controls cell fates via connecting mRNA cytidine acetylation to chromatin signaling.

Cell fate transition involves dynamic changes of gene regulatory network and chromatin landscape, requiring multiple levels of regulation, yet the cross-talk between epitranscriptomic modification and chromatin signaling remains largely unknown. Here, we uncover that suppression of N-acetyltransferase 10 (NAT10), the writer for mRNA N4-acetylcytidine (ac4C) modification, can notably affect human embryonic stem cell (hESC) lineage differentiation and pluripotent reprogramming. With integrative analysis, we identify that NAT10-mediated ac4C modification regulates the protein levels of a subset of its targets, which are strongly enriched for fate-instructive chromatin regulators, and among them, histone chaperone ANP32B is experimentally verified and functionally relevant. Furthermore, NAT10-ac4C-ANP32B axis can modulate the chromatin landscape of their downstream genes (e.g., key regulators of Wnt and TGFß pathways). Collectively, we show that NAT10 is an essential regulator of cellular plasticity, and its catalyzed mRNA cytidine acetylation represents a critical layer of epitranscriptomic modulation and uncover a previously unrecognized, direct cross-talk between epitranscriptomic modification and chromatin signaling during cell fate transitions.

Foliar spraying of indoleacetic acid (IAA) enhances the phytostabilization of Pb in naturally tolerant ryegrass by limiting the root-to-shoot transfer of Pb and improving plant growth.

Exogenous addition of IAA has the potential to improve the metal tolerance and phytostabilization of plants, but these effects have not been systematically investigated in naturally tolerant plants. Ryegrass (Lolium perenne L.) is a typical indigenous plant in the Lanping Pb/Zn mining area with high adaptability. This study investigated the phytostabilization ability and Pb tolerance mechanism of ryegrass in response to Pb, with or without foliar spraying of 0.1 mmol L-1 IAA. The results indicated that appropriate IAA treatment could be used to enhance the phytostabilization efficiency of naturally tolerant plants. Foliar spraying of IAA increased the aboveground and belowground biomass of ryegrass and improved root Pb phytostabilization. Compared to Pb-treated plants without exogenous IAA addition, Pb concentration in the shoots of ryegrass significantly decreased, then increased in the roots after the foliar spraying of IAA. In the 1,000 mg kg-1 Pb-treated plants, Pb concentration in the shoots decreased by 69.9% and increased by 79.1% in the roots after IAA treatment. IAA improved plant growth, especially in soils with higher Pb concentration. Foliar spraying of IAA increased shoot biomass by 35.9% and root biomass by 109.4% in 1,000 mg kg-1 Pb-treated plants, and increased shoot biomass by 196.5% and root biomass by 71.5% in 2,000 mg kg-1 Pb-treated plants. In addition, Pb stress significantly decreased the content of photosynthetic pigments and anti-oxidase activities in ryegrass, while foliar spraying of IAA remedied these negative impacts. In summary, foliar spraying of IAA could increase the biomass and improve the Pb tolerance of ryegrass.

A fast chemical reprogramming system promotes cell identity transition through a diapause-like state.

Cellular reprogramming by only small molecules holds enormous potentials for regenerative medicine. However, chemical reprogramming remains a slow process and labour intensive, hindering its broad applications and the investigation of underlying molecular mechanisms. Here, through screening of over 21,000 conditions, we develop a fast chemical reprogramming (FCR) system, which significantly improves the kinetics of cell identity rewiring. We find that FCR rapidly goes through an interesting route for pluripotent reprogramming, uniquely transitioning through a developmentally diapause-like state. Furthermore, FCR critically enables comprehensive characterizations using multi-omics technologies, and has revealed unexpected important features including key regulatory factors and epigenetic dynamics. Particularly, activation of pluripotency-related endogenous retroviruses via inhibition of heterochromatin significantly enhances reprogramming. Our studies provide critical insights into how only environmental cues are sufficient to rapidly reinstate pluripotency in somatic cells, and make notable technical and conceptual advances for solving the puzzle of regeneration.

Chemical strategies for stem cell biology and regenerative medicine.

Stem cell technology holds great promises for the cures of devastating diseases, injuries, aging, and even cancers as it is applied in regenerative medicine. Recent breakthroughs in the development of induced pluripotent stem cell techniques and efficient differentiation strategies have generated tremendous enthusiasm and efforts to explore the therapeutic potential of stem cells. Small molecules, which target specific signaling pathways and/or proteins, have been demonstrated to be particularly valuable for manipulating cell fate, state, and function. Such small molecules not only are useful in generating desired cell types in vitro for various applications but also could be further developed as conventional therapeutics to stimulate patients' endogenous cells to repair and regenerate in vivo. Here, we focus on recent progress in the use of small molecules in stem cell biology and regenerative medicine.

Brief report: combined chemical treatment enables Oct4-induced reprogramming from mouse embryonic fibroblasts.

It has been established that exogenous expression of four transcription factors (Oct4, Klf4, Sox2, and c-Myc) can reprogram mammalian somatic cells to pluripotent states. Further studies demonstrated that such induced pluripotent stem cells (iPSCs) could be generated with fewer exogenous transcription factors, facilitated by endogenous expression of reprogramming factors and/or synthetic small molecules. Here, we reported identification of a new small molecule, a protein arginine methyltransferase inhibitor AMI-5, which enabled Oct4-induced reprogramming of mouse embryonic fibroblasts in combination with transforming growth factor (TGF)-ß inhibitor A-83-01. The Oct4-induced iPSCs were shown similar to mouse embryonic stem cells with respect to typical pluripotency criteria. More importantly, they were shown to give rise to liveborn pups through tetraploid complementation assays, demonstrating the high quality of full reprogramming induced by this condition. Furthermore, this study suggests that regulation of protein arginine methylation might be involved in the reprogramming process.

The Mg-chelatase H subunit is an abscisic acid receptor.

Abscisic acid (ABA) is a vital phytohormone that regulates mainly stomatal aperture and seed development, but ABA receptors involved in these processes have yet to be determined. We previously identified from broad bean an ABA-binding protein (ABAR) potentially involved in stomatal signalling, the gene for which encodes the H subunit of Mg-chelatase (CHLH), which is a key component in both chlorophyll biosynthesis and plastid-to-nucleus signalling. Here we show that Arabidopsis ABAR/CHLH specifically binds ABA, and mediates ABA signalling as a positive regulator in seed germination, post-germination growth and stomatal movement, showing that ABAR/CHLH is an ABA receptor. We show also that ABAR/CHLH is a ubiquitous protein expressed in both green and non-green tissues, indicating that it might be able to perceive the ABA signal at the whole-plant level.

Human expandable pancreatic progenitor-derived ß cells ameliorate diabetes.

An unlimited source of human pancreatic ß cells is in high demand. Even with recent advances in pancreatic differentiation from human pluripotent stem cells, major hurdles remain in large-scale and cost-effective production of functional ß cells. Here, through chemical screening, we demonstrate that the bromodomain and extraterminal domain (BET) inhibitor I-BET151 can robustly promote the expansion of PDX1+NKX6.1+ pancreatic progenitors (PPs). These expandable PPs (ePPs) maintain pancreatic progenitor cell status in the long term and can efficiently differentiate into functional pancreatic ß (ePP-ß) cells. Notably, transplantation of ePP-ß cells rapidly ameliorated diabetes in mice, suggesting strong potential for cell replacement therapy. Mechanistically, I-BET151 activates Notch signaling and promotes the expression of key PP-associated genes, underscoring the importance of epigenetic and transcriptional modulations for lineage-specific progenitor self-renewal. In summary, our studies achieve the long-term goal of robust expansion of PPs and represent a substantial step toward unlimited supplies of functional ß cells for biomedical research and regenerative medicine.

N6-methyladenosine modification-mediated mRNA metabolism is essential for human pancreatic lineage specification and islet organogenesis.

Pancreatic differentiation from human pluripotent stem cells (hPSCs) provides promising avenues for investigating development and treating diseases. N6-methyladenosine (m6A) is the most prevalent internal messenger RNA (mRNA) modification and plays pivotal roles in regulation of mRNA metabolism, while its functions remain elusive. Here, we profile the dynamic landscapes of m6A transcriptome-wide during pancreatic differentiation. Next, we generate knockout hPSC lines of the major m6A demethylase ALKBH5, and find that ALKBH5 plays significant regulatory roles in pancreatic organogenesis. Mechanistic studies reveal that ALKBH5 deficiency reduces the mRNA stability of key pancreatic transcription factors in an m6A and YTHDF2-dependent manner. We further identify that ALKBH5 cofactor α-ketoglutarate can be applied to enhance differentiation. Collectively, our findings identify ALKBH5 as an essential regulator of pancreatic differentiation and highlight that m6A modification-mediated mRNA metabolism presents an important layer of regulation during cell-fate specification and holds great potentials for translational applications.

Conversion of mouse epiblast stem cells to an earlier pluripotency state by small molecules.

Epiblast stem cells (EpiSCs) are pluripotent cells derived from post-implantation late epiblasts in vitro. EpiSCs are incapable of contributing to chimerism, indicating that EpiSCs are less pluripotent and represent a later developmental pluripotency state compared with inner cell mass stage murine embryonic stem cells (mESCs). Using a chemical approach, we found that blockage of the TGFß pathway or inhibition of histone demethylase LSD1 with small molecule inhibitors induced dramatic morphological changes in EpiSCs toward mESC phenotypes with simultaneous activation of inner cell mass-specific gene expression. However, full conversion of EpiSCs to the mESC-like state with chimerism competence could be readily generated only with the combination of LSD1, ALK5, MEK, FGFR, and GSK3 inhibitors. Our results demonstrate that appropriate synergy of epigenetic and signaling modulations could convert cells at the later developmental pluripotency state to the earlier mESC-like pluripotency state, providing new insights into pluripotency regulation.

Identification of an important site for function of the type 2C protein phosphatase ABI2 in abscisic acid signalling in Arabidopsis.

It is known that the clade A protein phosphatase 2Cs (PP2Cs), including ABI1 and ABI2 and other PP2C members, are key players that function directly downstream of the PYR/PYL/RCAR abscisic acid (ABA) receptors. Here, identification of a crucial site for function of ABI2 protein phosphatase in ABA signalling is reported. It was observed that a calcium-dependent protein kinase (CDPK) phosphorylation site-like motif (CPL) in the ABI2 molecule is required for the interactions of ABI2 with the two members of the ABA receptors PYL5 and PYL9 and with a downstream protein kinase SnRK2.6, and for the catalytic activity of ABI2 in vitro, as well as for the response of ABI2 to the ABA receptors PYL5/PYL9 in relation to the ABA receptor-induced inhibition of the ABI2 phosphatase activity. Further, genetic evidence was provided to demonstrate that this CPL is required for the function of ABI2 to mediate ABA signalling. These data reveal that this CPL is an important site necessary for both the phosphatase activity of ABI2 and the functional interaction between ABI2 and PYL5/9 ABA receptors, providing new information to understand primary events of ABA signal transduction.