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Stem cells reside in a niche, a local environment whose cellular and molecular complexity is still being elucidated. In ovaries, germline stem cells depend on cap cells for self-renewing signals and physical attachment. Germline stem cells also contact the anterior escort cells, and here we report that anterior escort cells are absolutely required for germline stem cell maintenance. When escort cells die from impaired Wnt signaling or expression, the loss of anterior escort cells causes loss of germline stem cells. Anterior escort cells function as an integral niche component by promoting DE-cadherin anchorage and by transiently expressing the Dpp ligand to promote full-strength BMP signaling in germline stem cells. Anterior escort cells are maintained by Wnt6 ligands produced by cap cells; without Wnt6 signaling, anterior escort cells die leaving vacancies in the niche, leading to loss of germline stem cells. Our data identify anterior escort cells as constituents of the germline stem cell niche, maintained by a cap cell-produced Wnt6 survival signal.
© 2018. Published by The Company of Biologists Ltd.
Purpose - Mammalian central nervous system axons fail to regenerate after injury. Contributing factors include limited intrinsic growth capacity and an inhibitory glial environment. Inflammation-induced optic nerve regeneration (IIR) is thought to boost retinal ganglion cell (RGC) intrinsic growth capacity through progrowth gene expression, but effects on the inhibitory glial environment of the optic nerve are unexplored. To investigate progrowth molecular changes associated with reactive gliosis during IIR, we developed an imaging mass spectrometry (IMS)-based approach that identifies discriminant molecular signals in and around optic nerve crush (ONC) sites.
Methods - ONC was performed in rats, and IIR was established by intravitreal injection of a yeast cell wall preparation. Optic nerves were collected at various postcrush intervals, and longitudinal sections were analyzed with matrix-assisted laser desorption/ionization (MALDI) IMS and data mining. Immunohistochemistry and confocal microscopy were used to compare discriminant molecular features with cellular features of reactive gliosis.
Results - IIR increased the area of the crush site that was occupied by a dense cellular infiltrate and mass spectral features consistent with lysosome-specific lipids. IIR also increased immunohistochemical labeling for microglia and macrophages. IIR enhanced clearance of lipid sulfatide myelin-associated inhibitors of axon growth and accumulation of simple GM3 gangliosides in a spatial distribution consistent with degradation of plasma membrane from degenerated axons.
Conclusions - IIR promotes a robust phagocytic response that improves clearance of myelin and axon debris. This growth-permissive molecular remodeling of the crush injury site extends our current understanding of IIR to include mechanisms extrinsic to the RGC.
Cell therapies suffer from poor survival post-transplant due to placement into hostile implant sites characterized by host immune response and innate production of high levels of reactive oxygen species (ROS). We hypothesized that cellular encapsulation within an injectable, antioxidant hydrogel would improve viability of cells exposed to high oxidative stress. To test this hypothesis, we applied a dual thermo- and ROS-responsive hydrogel comprising the ABC triblock polymer poly[(propylene sulfide)-block-(N,N-dimethyl acrylamide)-block-(N-isopropylacrylamide)] (PPS-b-PDMA-b-PNIPAAM, PDN). The PPS chemistry reacts irreversibly with ROS such as hydrogen peroxide (HO), imparting inherent antioxidant properties to the system. Here, PDN hydrogels were successfully integrated with type 1 collagen to form ROS-protective, composite hydrogels amenable to spreading and growth of adherent cell types such as mesenchymal stem cells (MSCs). It was also shown that, using a control hydrogel substituting nonreactive polycaprolactone in place of PPS, the ROS-reactive PPS chemistry is directly responsible for PDN hydrogel cytoprotection of both MSCs and insulin-producing β-cell pseudo-islets against HO toxicity. In sum, these results establish the potential of cytoprotective, thermogelling PDN biomaterials for injectable delivery of cell therapies.
Paneth cells (PCs), a secretory population located at the base of the intestinal crypt, support the intestinal stem cells (ISC) with growth factors and participate in innate immunity by releasing antimicrobial peptides, including lysozyme and defensins. PC dysfunction is associated with disorders such as Crohn's disease and necrotizing enterocolitis, but the specific pathways regulating PC development and function are not fully understood. Here we tested the role of the neuregulin receptor ErbB3 in control of PC differentiation and the ISC niche. Intestinal epithelial ErbB3 knockout caused precocious appearance of PCs as early as postnatal day 7, and substantially increased the number of mature PCs in adult mouse ileum. ErbB3 loss had no effect on other secretory lineages, but increased expression of the ISC marker Lgr5. ErbB3-null intestines had elevated levels of the Atoh1 transcription factor, which is required for secretory fate determination, while Atoh1 cells had reduced ErbB3, suggesting reciprocal negative regulation. ErbB3-null intestinal progenitor cells showed reduced activation of the PI3K-Akt and ERK MAPK pathways. Inhibiting these pathways in HT29 cells increased levels of ATOH1 and the PC marker LYZ. Conversely, ErbB3 activation suppressed LYZ and ATOH1 in a PI3K-dependent manner. Expansion of the PC compartment in ErbB3-null intestines was accompanied with elevated ER stress and inflammation markers, raising the possibility that negative regulation of PCs by ErbB3 is necessary to maintain homeostasis. Taken together, our data suggest that ErbB3 restricts PC numbers through PI3K-mediated suppression of Atoh1 levels leading to inhibition of PC differentiation, with important implications for regulation of the ISC niche.
Although intestinal homeostasis is maintained by intestinal stem cells (ISCs), regeneration is impaired upon aging. Here, we first uncover changes in intestinal architecture, cell number, and cell composition upon aging. Second, we identify a decline in the regenerative capacity of ISCs upon aging because of a decline in canonical Wnt signaling in ISCs. Changes in expression of Wnts are found in stem cells themselves and in their niche, including Paneth cells and mesenchyme. Third, reactivating canonical Wnt signaling enhances the function of both murine and human ISCs and, thus, ameliorates aging-associated phenotypes of ISCs in an organoid assay. Our data demonstrate a role for impaired Wnt signaling in physiological aging of ISCs and further identify potential therapeutic avenues to improve ISC regenerative potential upon aging.
Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Multiple myeloma (MM) patients frequently develop tumor-induced bone destruction, yet no therapy completely eliminates the tumor or fully reverses bone loss. Transforming growth factor-β (TGF-β) activity often contributes to tumor-induced bone disease, and pre-clinical studies have indicated that TGF-β inhibition improves bone volume and reduces tumor growth in bone metastatic breast cancer. We hypothesized that inhibition of TGF-β signaling also reduces tumor growth, increases bone volume, and improves vertebral body strength in MM-bearing mice. We treated myeloma tumor-bearing (immunocompetent KaLwRij and immunocompromised Rag2-/-) mice with a TGF-β inhibitory (1D11) or control (13C4) antibody, with or without the anti-myeloma drug bortezomib, for 4weeks after inoculation of murine 5TGM1 MM cells. TGF-β inhibition increased trabecular bone volume, improved trabecular architecture, increased tissue mineral density of the trabeculae as assessed by ex vivo micro-computed tomography, and was associated with significantly greater vertebral body strength in biomechanical compression tests. Serum monoclonal paraprotein titers and spleen weights showed that 1D11 monotherapy did not reduce overall MM tumor burden. Combination therapy with 1D11 and bortezomib increased vertebral body strength, reduced tumor burden, and reduced cortical lesions in the femoral metaphysis, although it did not significantly improve cortical bone strength in three-point bending tests of the mid-shaft femur. Overall, our data provides rationale for evaluating inhibition of TGF-β signaling in combination with existing anti-myeloma agents as a potential therapeutic strategy to improve outcomes in patients with myeloma bone disease.
Published by Elsevier Inc.
White blood cells play diverse roles in innate and adaptive immunity. Genetic association analyses of phenotypic variation in circulating white blood cell (WBC) counts from large samples of otherwise healthy individuals can provide insights into genes and biologic pathways involved in production, differentiation, or clearance of particular WBC lineages (myeloid, lymphoid) and also potentially inform the genetic basis of autoimmune, allergic, and blood diseases. We performed an exome array-based meta-analysis of total WBC and subtype counts (neutrophils, monocytes, lymphocytes, basophils, and eosinophils) in a multi-ancestry discovery and replication sample of ∼157,622 individuals from 25 studies. We identified 16 common variants (8 of which were coding variants) associated with one or more WBC traits, the majority of which are pleiotropically associated with autoimmune diseases. Based on functional annotation, these loci included genes encoding surface markers of myeloid, lymphoid, or hematopoietic stem cell differentiation (CD69, CD33, CD87), transcription factors regulating lineage specification during hematopoiesis (ASXL1, IRF8, IKZF1, JMJD1C, ETS2-PSMG1), and molecules involved in neutrophil clearance/apoptosis (C10orf54, LTA), adhesion (TNXB), or centrosome and microtubule structure/function (KIF9, TUBD1). Together with recent reports of somatic ASXL1 mutations among individuals with idiopathic cytopenias or clonal hematopoiesis of undetermined significance, the identification of a common regulatory 3' UTR variant of ASXL1 suggests that both germline and somatic ASXL1 mutations contribute to lower blood counts in otherwise asymptomatic individuals. These association results shed light on genetic mechanisms that regulate circulating WBC counts and suggest a prominent shared genetic architecture with inflammatory and autoimmune diseases.
Copyright © 2016 American Society of Human Genetics. All rights reserved.
The zebrafish pineal complex consists of four cell types (rod and cone photoreceptors, projection neurons and parapineal neurons) that are derived from a single pineal complex anlage. After specification, parapineal neurons migrate unilaterally away from the rest of the pineal complex whereas rods, cones and projection neurons are non-migratory. The transcription factor Tbx2b is important for both the correct number and migration of parapineal neurons. We find that two additional transcription factors, Flh and Nr2e3, negatively regulate parapineal formation. Flh induces non-migratory neuron fates and limits the extent of parapineal specification, in part by activation of Nr2e3 expression. Tbx2b is positively regulated by Flh, but opposes Flh action during specification of parapineal neurons. Loss of parapineal neuron specification in Tbx2b-deficient embryos can be partially rescued by loss of Nr2e3 or Flh function; however, parapineal migration absolutely requires Tbx2b activity. We conclude that cell specification and migration in the pineal complex are regulated by a network of at least three transcription factors.
© 2016. Published by The Company of Biologists Ltd.
Pdx1 and Oc1 are co-expressed in multipotent pancreatic progenitors and regulate the pro-endocrine gene Neurog3. Their expression diverges in later organogenesis, with Oc1 absent from hormone+ cells and Pdx1 maintained in mature β cells. In a classical genetic test for cooperative functional interactions, we derived mice with combined Pdx1 and Oc1 heterozygosity. Endocrine development in double-heterozygous pancreata was normal at embryonic day (E)13.5, but defects in specification and differentiation were apparent at E15.5, the height of the second wave of differentiation. Pancreata from double heterozygotes showed alterations in the expression of genes crucial for β-cell development and function, decreased numbers and altered allocation of Neurog3-expressing endocrine progenitors, and defective endocrine differentiation. Defects in islet gene expression and β-cell function persisted in double heterozygous neonates. These results suggest that Oc1 and Pdx1 cooperate prior to their divergence, in pancreatic progenitors, to allow for proper differentiation and functional maturation of β cells.
Published by Elsevier Inc.
Biomaterial substrates composed of semi-aligned electrospun fibers are attractive supports for the regeneration of connective tissues because the fibers are durable under cyclic tensile loads and can guide cell adhesion, orientation, and gene expression. Previous studies on supported electrospun substrates have shown that both fiber diameter and mechanical deformation can independently influence cell morphology and gene expression. However, no studies have examined the effect of mechanical deformation and fiber diameter on unsupported meshes. Semi-aligned large (1.75 μm) and small (0.60 μm) diameter fiber meshes were prepared from degradable elastomeric poly(esterurethane urea) (PEUUR) meshes and characterized by tensile testing and scanning electron microscopy (SEM). Next, unsupported meshes were aligned between custom grips (with the stretch axis oriented parallel to axis of fiber alignment), seeded with C3H10T1/2 cells, and subjected to a static load (50 mN, adjusted daily), a cyclic load (4% strain at 0.25 Hz for 30 min, followed by a static tensile loading of 50 mN, daily), or no load. After 3 days of mechanical stimulation, confocal imaging was used to characterize cell shape, while measurements of deoxyribonucleic acid (DNA) content and messenger ribonucleic acid (mRNA) expression were used to characterize cell retention on unsupported meshes and expression of the connective tissue phenotype. Mechanical testing confirmed that these materials deform elastically to at least 10%. Cells adhered to unsupported meshes under all conditions and aligned with the direction of fiber orientation. Application of static and cyclic loads increased cell alignment. Cell density and mRNA expression of connective tissue proteins were not statistically different between experimental groups. However, on large diameter fiber meshes, static loading slightly elevated tenomodulin expression relative to the no load group, and tenascin-C and tenomodulin expression relative to the cyclic load group. These results demonstrate the feasibility of maintaining cell adhesion and alignment on semi-aligned fibrous elastomeric substrates under different mechanical conditions. The study confirms that cell morphology is sensitive to the mechanical environment and suggests that expression of select connective tissue genes may be enhanced on large diameter fiber meshes under static tensile loads.