Maf and Mafb control mouse pallial interneuron fate and maturation through neuropsychiatric disease gene regulation.

​Maf (c-Maf) and Mafb transcription factors (TFs) have compensatory roles in repressing somatostatin (SST+) interneuron (IN) production in medial ganglionic eminence (MGE) secondary progenitors in mice. Maf and Mafb conditional deletion (cDKO) decreases the survival of MGE-derived cortical interneurons (CINs) and changes their physiological properties. Herein, we show that (1) Mef2c and Snap25 are positively regulated by Maf and Mafb to drive IN morphological maturation; (2) Maf and Mafb promote Mef2c expression which specifies parvalbumin (PV+) INs; (3) Elmo1, Igfbp4 and Mef2c are candidate markers of immature PV+ hippocampal INs (HIN). Furthermore, Maf/Mafb neonatal cDKOs have decreased CINs and increased HINs, that express Pnoc, an HIN specific marker. Our findings not only elucidate key gene targets of Maf and Mafb that control IN development, but also identify for the first time TFs that differentially regulate CIN vs. HIN production.

Matrix stiffness modulates the differentiation of neural crest stem cells in vivo.

Stem cells are often transplanted with scaffolds for tissue regeneration; however, how the mechanical property of a scaffold modulates stem cell fate in vivo is not well understood. Here we investigated how matrix stiffness modulates stem cell differentiation in a model of vascular graft transplantation. Multipotent neural crest stem cells (NCSCs) were differentiated from induced pluripotent stem cells, embedded in the hydrogel on the outer surface of nanofibrous polymer grafts, and implanted into rat carotid arteries by anastomosis. After 3 months, NCSCs differentiated into smooth muscle cells (SMCs) near the outer surface of the polymer grafts; in contrast, NCSCs differentiated into glial cells in the most part of the hydrogel. Atomic force microscopy demonstrated a stiffer matrix near the polymer surface but much lower stiffness away from the polymer graft. Consistently, in vitro studies confirmed that stiff surface induced SMC genes whereas soft surface induced glial genes. These results suggest that the scaffold's mechanical properties play an important role in directing stem cell differentiation in vivo, which has important implications in biomaterials design for stem cell delivery and tissue engineering.

Author Correction: Discovery of stimulation-responsive immune enhancers with CRISPR activation.

In this Letter, analysis of steady-state regulatory T (Treg) cell percentages from Il2ra enhancer deletion (EDEL) and wild-type (WT) mice revealed no differences between them (Extended Data Fig. 9d). This analysis included two mice whose genotypes were incorrectly assigned. Even after correction of the genotypes, no significant differences in Treg cell percentages were seen when data across experimental cohorts were averaged (as was done in Extended Data Fig. 9d). However, if we normalize the corrected data to account for variation among experimental cohorts, a subtle decrease in EDEL Treg cell percentages is revealed and, using the corrected and normalized data, we have redrawn Extended Data Fig. 9d in Supplementary Fig. 1. The Supplementary Information to this Amendment contains the corrected and reanalysed Extended Data Fig. 9d. The sentence "This enhancer deletion (EDEL) strain also had no obvious T cell phenotypes at steady state (Extended Data Fig. 9)." should read: "This enhancer deletion (EDEL) strain had a small decrease in the percentage of Treg cells (Extended Data Fig. 9).". This error does not affect any of the main figures in the Letter or the data from mice with the human autoimmune-associated single nucleotide polymorphism (SNP) knocked in or with a 12-base-pair deletion at the site (12DEL). In addition, we stated in the Methods that we observed consistent immunophenotypes of EDEL mice across three founders, but in fact, we observed consistent phenotypes in mice from two founders. This does not change any of our conclusions and the original Letter has not been corrected.

Discovery of stimulation-responsive immune enhancers with CRISPR activation.

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

Heritability of cardiovascular risk factors in a Chinese population--Taichung Community Health Study and Family Cohort.

BACKGROUND AND AIM: Previous studies reporting on estimates of heritability of cardiovascular risk factors in Chinese are limited. This study aims to estimate the heritability of cardiovascular risk factors in relatives of residents who participated in the Taichung Community Health Study (TCHS) and Family Cohort (TCHS-FC) while controlling as many potential confounders as possible. METHODS: A total of 1564 study subjects from 494 families with members aged 12-91 years were enrolled from a random sample of participants of TCHS and their family members (TCHS-FC) from 2009 to 2012. Anthropometric measurement, body composition, blood pressure, plasma lipids, fasting glucose, insulin, highly sensitive C-reactive protein (hs-CRP), brachial-ankle pulse wave velocity (baPWV), and the ankle-brachial index (ABI), as well as a questionnaire interview, were obtained from each participant. RESULTS: Cardiovascular risk factors with estimates of heritability greater than 30% after multivariate adjustment were triglyceride (h(2) = 0.41), HDL-C (h(2) = 0.49), LDL-C (h(2) = 0.47), total cholesterol (h(2) = 0.46), hip circumference (h(2) = 0.44), weight (h(2) = 0.42), insulin (h(2) = 0.39), hs-CRP (h(2) = 0.38), BMI (h(2) = 0.38), and percent body fat mass (h(2) = 0.35). Correlation coefficients for significant sibling varied from 0.10 for weight to 0.47 for LDL-C whereas those for significant parent-offspring varied from 0.09 for fasting plasma glucose to 0.43 for baPWV. CONCLUSIONS: This study demonstrated significant heritability and familial aggregation of cardiovascular risk factors in a random sample of ethnic Chinese population.

Synovial stem cells and their responses to the porosity of microfibrous scaffold.

Tissue-specific stem cells can be coaxed or harvested for tissue regeneration. In this study, we identified and characterized a new type of stem cells from the synovial membrane of knee joint, named neural crest cell-like synovial stem cells (NCCL-SSCs). NCCL-SSCs showed the characteristics of neural crest stem cells: they expressed markers such as Sox10, Sox17 and S100ß, were clonable, and could differentiate into neural lineages as well as mesenchymal lineages, although NCCL-SSCs were not derived from neural crest during the development. When treated with transforming growth factor ß1 (TGF-ß1), NCCL-SSCs differentiated into mesenchymal stem cells (MSCs), lost the expression of Sox17 and the differentiation potential into neural lineages, but retained the potential of differentiating into mesenchymal lineages. To determine the responses of NCCL-SSCs to microfibrous scaffolds for tissue engineering, electrospun composite scaffolds with various porosities were fabricated by co-electrospinning of structural and sacrificial microfibers. The increase in the porosity in microfibrous scaffolds enhanced cell infiltration in vitro and in vivo, but did not affect the morphology and the proliferation of NCCL-SSCs. Interestingly, microfibrous scaffolds with higher porosity increased the expression of chondrogenic and osteogenic genes but suppressed smooth muscle and adipogenic genes. These results suggest that the differentiation of NCCL-SSCs can be controlled by both soluble chemical factors and biophysical factors such as the porosity of the scaffold. Engineering both NCCL-SSCs and scaffolds will have tremendous potential for tissue regeneration.

Differentiation of multipotent vascular stem cells contributes to vascular diseases.

It is generally accepted that the de-differentiation of smooth muscle cells, from the contractile to the proliferative/synthetic phenotype, has an important role during vascular remodelling and diseases. Here we provide evidence that challenges this theory. We identify a new type of stem cell in the blood vessel wall, named multipotent vascular stem cells. Multipotent vascular stem cells express markers, including Sox17, Sox10 and S100ß, are cloneable, have telomerase activity, and can differentiate into neural cells and mesenchymal stem cell-like cells that subsequently differentiate into smooth muscle cells. On the other hand, we perform lineage tracing with smooth muscle myosin heavy chain as a marker and find that multipotent vascular stem cells and proliferative or synthetic smooth muscle cells do not arise from the de-differentiation of mature smooth muscle cells. In response to vascular injuries, multipotent vascular stem cells, instead of smooth muscle cells, become proliferative, and differentiate into smooth muscle cells and chondrogenic cells, thus contributing to vascular remodelling and neointimal hyperplasia. These findings support a new hypothesis that the differentiation of multipotent vascular stem cells, rather than the de-differentiation of smooth muscle cells, contributes to vascular remodelling and diseases.

Anisotropic effects of mechanical strain on neural crest stem cells.

Neural crest stem cells (NCSCs) are multipotent and play an important role during the development and tissue regeneration. However, the anisotropic effects of mechanical strain on NCSCs are not known. To investigate the anisotropic mechanosensing by NCSCs, NCSCs derived from induced pluripotent stem cells were cultured on micropatterned membranes, and subjected to cyclic uniaxial strain in the direction parallel or perpendicular to the microgrooves. Cell and nuclear shape were both regulated by micropatterning and mechanical strain. Among the unpatterned, parallel-patterned and perpendicular-patterned groups, mechanical strain caused an increase in histone deacetylase activity in the parallel-patterned group, accompanied by the increase of cell proliferation. In addition, mechanical strain increased the expression of contractile marker calponin-1 but not other differentiation markers in the unpatterned and parallel-patterned groups. These results demonstrated that NCSCs responded differently to the anisotropic mechanical environment. Understanding the mechanical regulation of NCSCs will reveal the role of mechanical factors in NCSC differentiation during development, and provide a basis for using NCSCs for tissue engineering.

Shared genetic architecture in the relationship between adult stature and subclinical coronary artery atherosclerosis.

BACKGROUND: Short stature is associated with increased risk of coronary heart disease (CHD); although the mechanisms for this relationship are unknown, shared genetic factors have been proposed. Subclinical atherosclerosis, measured by coronary artery calcification (CAC), is associated with CHD events and represents part of the biological continuum to overt CHD. Many molecular mechanisms of CAC development are shared with bone growth. Thus, we examined whether there was evidence of shared genes (pleiotropy) between adult stature and CAC. METHODS: 877 Asymptomatic white adults (46% men) from 625 families in a community-based sample had computed tomography measures of CAC. Pleiotropy between height and CAC was determined using maximum-likelihood estimation implemented in SOLAR. RESULTS: Adult height was significantly and inversely associated with CAC score (P = 0.01). After adjusting for age, sex and CHD risk factors, the estimated genetic correlation between height and CAC score was -0.37 and was significantly different than 0 (P = 0.001) and -1 (P < 0.001). The environmental correlation between height and CAC score was 0.60 and was significantly different than 0 (P = 0.024). CONCLUSIONS: Further studies of shared genetic factors between height and CAC may provide important insight into the complex genetic architecture of CHD, in part through increased understanding of the molecular pathways underlying the process of both normal growth and disease development. Bivariate genetic linkage analysis may provide a powerful mechanism for identifying specific genomic regions associated with both height and CAC.

Biophysical regulation of histone acetylation in mesenchymal stem cells.

Histone deacetylation and acetylation are catalyzed by histone deacetylase (HDAC) and histone acetyltransferase, respectively, which play important roles in the regulation of chromatin remodeling, gene expression, and cell functions. However, whether and how biophysical cues modulate HDAC activity and histone acetylation is not well understood. Here, we tested the hypothesis that microtopographic patterning and mechanical strain on the substrate regulate nuclear shape, HDAC activity, and histone acetylation. Bone marrow mesenchymal stem cells (MSCs) were cultured on elastic membranes patterned with parallel microgrooves 10 µm wide that kept MSCs aligned along the axis of the grooves. Compared with MSCs on an unpatterned substrate, MSCs on microgrooves had elongated nuclear shape, a decrease in HDAC activity, and an increase of histone acetylation. To investigate anisotropic mechanical sensing by MSCs, cells on the elastic micropatterned membranes were subjected to static uniaxial mechanical compression or stretch in the direction parallel or perpendicular to the microgrooves. Among the four types of loads, compression or stretch perpendicular to the microgrooves caused a decrease in HDAC activity, accompanied by the increase in histone acetylation and slight changes of nuclear shape. Knocking down nuclear matrix protein lamin A/C abolished mechanical strain-induced changes in HDAC activity. These results demonstrate that micropattern and mechanical strain on the substrate can modulate nuclear shape, HDAC activity, and histone acetylation in an anisotropic manner and that nuclear matrix mediates mechanotransduction. These findings reveal a new mechanism, to our knowledge, by which extracellular biophysical signals are translated into biochemical signaling events in the nucleus, and they will have significant impact in the area of mechanobiology and mechanotransduction.