The enteric neural crest progressively loses capacity to form enteric nervous system

Cells of the vagal neural crest (NC) form most of the enteric nervous system (ENS) by a colonising wave in the embryonic gut, with high cell proliferation and differentiation. Enteric neuropathies have an ENS deficit and cell replacement has been suggested as therapy. This would be performed post-natally, which raises the question of whether the ENS cell population retains its initial ENS-forming potential with age. We tested this on the avian model in organ culture in vitro (3 days) using recipient aneural chick midgut/hindgut combined with ENS- donor quail midgut or hindgut of ages QE5 to QE10. ENS cells from young donor tissues (<= QE6) avidly colonised the aneural recipient, but this capacity dropped rapidly 2-3 days after the transit of the ENS cell wavefront. This loss in capability was autonomous to the ENS population since a similar decline was observed in ENS cells isolated by HNK1 FACS. Using QE5, 6, 8 and 10 midgut donors and extending the time of assay to 8 days in chorio-allantoic membrane grafts did not produce 'catch up' colonisation. NC-derived cells were counted in dissociated quail embryo gut and in transverse sections of chick embryo gut using NC, neuron and glial marker antibodies. This showed that the decline in ENS-forming ability correlated with a decrease in proportion of ENS cells lacking both neuronal and glial differentiation markers, but there were still large numbers of such cells even at stages with low colonisation ability. Moreover, ENS cells in small numbers from young donors were far superior in colonisation ability to larger numbers of apparently undifferentiated cells from older donors. This suggests that the decline of ENS-forming ability has both quantitative and qualitative aspects. In this case, ENS cells for cell therapies should aim to replicate the embryonic ENS stage rather than using post-natal ENS stem/progenitor cells

Enteric Neural Cells From Hirschsprung Disease Patients Form Ganglia in Autologous Aneuronal Colon

Background & Aims: Hirschsprung disease (HSCR) is caused by failure of cells derived from the neural crest (NC) to colonize the distal bowel in early embryogenesis, resulting in absence of the enteric nervous system (ENS) and failure of intestinal transit postnatally. Treatment is by distal bowel resection, but neural cell replacement may be an alternative. We tested whether aneuronal (aganglionic) colon tissue from patients may be colonized by autologous ENS-derived cells. Methods: Cells were obtained and cryopreserved from 31 HSCR patients from the proximal resection margin of colon, and ENS cells were isolated using flow cytometry for the NC marker p75 (nine patients). Aneuronal colon tissue was obtained from the distal resection margin (23 patients). ENS cells were assessed for NC markers immunohistologically and by quantitative reverse-transcription polymerase chain reaction, and mitosis was detected by ethynyl-2\u27-deoxyuridine labeling. The ability of human HSCR postnatal ENS-derived cells to colonize the embryonic intestine was demonstrated by organ coculture with avian embryo gut, and the ability of human postnatal HSCR aneuronal colon muscle to support ENS formation was tested by organ coculture with embryonic mouse ENS cells. Finally, the ability of HSCR patient ENS cells to colonize autologous aneuronal colon muscle tissue was assessed. Results: ENS-derived p75-sorted cells from patients expressed multiple NC progenitor and differentiation markers and proliferated in culture under conditions simulating Wnt signaling. In organ culture, patient ENS cells migrated appropriately in aneural quail embryo gut, and mouse embryo ENS cells rapidly spread, differentiated, and extended axons in patient aneuronal colon muscle tissue. Postnatal ENS cells derived from HSCR patients colonized autologous aneuronal colon tissue in cocultures, proliferating and differentiating as neurons and glia. Conclusions: NC-lineage cells can be obtained from HSCR patient colon and can form ENS-like structures in aneuronal colonic muscle from the same patient

Yield and critical levels of P in ‘Cabernet Sauvignon’ vineyards grown in subtropical soils

Tropical and subtropical soils in vineyards show high phospho­rus (P) adsorption ability and, consequently, low P availability to plants. However, P critical levels (CL) and sufficiency ranges (SR) in soils and leaves in subtropical regions for yield and the must variables remain unknown. This study aimed to assess the yield response of ‘Cabernet Sauvignon’ grown in acidic soils with a high organic matter content in subtropical regions and to suggest CL and SR of P in the soil and leaves using bayesian change point quantile regression models. The study was carried out in the 2013/14, 2014/15, 2015/16, 2016/17, and 2017/18 crop sea­sons in a vineyard located in southern Brazil, treated with 0, 13, 26, 40, and 52 kg Pha-1 year-1, which were assessed. The P avail­ability in the soil was determined by Mehlich-1. The P in leaves (veraison), yield parameters, and must composition were eval­uated. Phosphate fertilizer applications to ‘Cabernet Sauvignon’ increased soil P, mainly on the soil surface. However, it did not increase the P concentration in leaves or influence yield or alter must pH and composition, such as pH, total soluble solids (TSS), total titratable acidity (TTA), and total polyphenols (TP). While P treatments had no effect on yield and fruit quality it was possible to utilize natural variation and Bayesian change point quantile regression models to determine CL’s for various attrib­utes. The CL of P in the 0-20 cm soil layer was 13.2 mg dm-3. The CL of P in leaves, regarding yield, TSS, and TTA, was 0.61, 0.88, and 0.52%, respectively

Morphological Analysis of Nanostructured PVDF β-Phase Films Obtained by Solution Blow Spinning

This work aimed to compare the characteristics of micro and nanofibers of the poly (vinylidene fluoride) - PVDF polymer obtained by the Solution Blow Spinning (SBS) method, and films with those produced by other techniques, such as: casting and hot pressing. Thermogravimetric analysis (TGA) revealed that the films are thermally stable up to a temperature of 420 ° C. X-ray diffractometry (XRD) indicated the presence of the crystalline phases α and β, the β phase being more evident for nanofibers and PVDF casting. The film obtained by SBS showed a greater contact angle, showing to be more hydrophobic. Strain tests showed that the nanofiber films showed a 72% rupture to rupture, about 1.7 and 3.1 times greater than those obtained by casting and hot pressing, respectively. There was also a decrease in the modulus of elasticity and the limit of tensile strength of nanofibers, compared to other films.</jats:p

Non-linear elasticity of core/shell spun PGS/PLLA fibres and their effect on cell proliferation

AbstractAn efficient delivery system is critical for the success of cell therapy. To deliver cells to a dynamic organ, the biomaterial vehicle should mechanically match with the non-linearly elastic host tissue. In this study, non-linearly elastic biomaterials have been fabricated from a chemically crosslinked elastomeric poly(glycerol sebacate) (PGS) and thermoplastic poly(l-lactic acid) (PLLA) using the core/shell electrospinning technique. The spun fibrous materials containing a PGS core and PLLA shell demonstrate J-shaped stress–strain curves, having ultimate tensile strength (UTS), rupture elongation and stiffness constants of 1 ± 0.2 MPa, 25 ± 3% and 12 ± 2, respectively, which are comparable to skin tissue properties reported previously. Our ex vivo and in vivo trials have shown that the elastomeric mesh supports and fosters the growth of enteric neural crest (ENC) progenitor cells, and that the cell-seeded elastomeric fibrous sheet physically remains in intimate contact with guts after grafting, providing the effective delivery of the progenitor cells to an embryonic and post-natal gut environment