No abstract

Fine-scale genetic structure in Eucalyptus globulus ssp. globulus native forest was detected using 69 randomly amplified polymorphic DNA (RAPD) markers. The association between genetic similarity and geographic distance was studied among 51 trees from the Tinderbox locality in Tasmania (distance ranging from 2 m to 4 km apart) and compared to 18 trees from localities up to 100 km away. Twenty pedigreed F1s were used as controls to scale the RAPD similarity among individuals to pedigree similarity. The association between genetic similarity and geographic distance was weak, yet at Tinderbox, highly related trees were shown to occur within 25 m of one another. There is an abrupt drop in average similarity after about 25 m, with no significant change with distances up to 14 km. Nevertheless, Tinderbox trees outside the 25 m genetic patches are still more similar to each other than they are to trees from the Mayfield Bay locality 100 km away. These results suggest that E. globulus native forests have a family group structure, superimposed on a noisy, background level of lower relatedness which extends over a wider geographical range. This study is unique in demonstrating the congruence between fine-scale genetic structure as revealed by molecular data and previous quantitative genetic data.

Needle-free multijet electrospinning can be used to mass produce artificial ECMs with intrinsic biocompatibility and desirable integration of stem cells for large-scale applications.

Two isolates of a novel babuvirus causing "bunchy top" symptoms were characterised, one from abacá (Musa textilis) from the Philippines and one from banana (Musa sp.) from Sarawak (Malaysia). The name abacá bunchy top virus (ABTV) is proposed. Both isolates have a genome of six circular DNA components, each ca. 1.0-1.1 kb, analogous to those of isolates of Banana bunchy top virus (BBTV). However, unlike BBTV, both ABTV isolates lack an internal ORF in DNA-R, and the ORF in DNA-U3 found in some BBTV isolates is also absent. In all phylogenetic analyses of nanovirid isolates, ABTV and BBTV fall in the same clade, but on separate branches. However, ABTV and BBTV isolates shared only 79-81% amino acid sequence identity for the putative coat protein and 54-76% overall nucleotide sequence identity across all components. Stem-loop and major common regions were present in ABTV, but there was less than 60% identity with the major common region of BBTV. ABTV and BBTV were also shown to be serologically distinct, with only two out of ten BBTV-specific monoclonal antibodies reacting with ABTV. The two ABTV isolates may represent distinct strains of the species as they are less closely related to each other than are isolates of the two geographic subgroups (Asian and South Pacific) of BBTV.

Novel clinical grade electrospinning methods could provide three-dimensional (3D) nanostructured biomaterials comprising of synthetic or natural biopolymer nanofibers. Such advanced materials could potentially mimic the natural extracellular matrix (ECM) accurately and may provide superior niche-like spaces on the subcellular scale for optimal stem-cell attachment and individual cell homing in regenerative therapies. The goal of this study was to design several novel "nanofibrous extracellular matrices" (NF-ECMs) with a natural mesh-like 3D architecture through a unique needle-free multi-jet electrospinning method in highly controlled manner to comply with good manufacturing practices (GMP) for the production of advanced healthcare materials for regenerative medicine, and to test cellular behavior of human mesenchymal stem cells (HMSCs) on these. Biopolymers manufactured as 3D NF-ECM meshes under clinical grade GMP-like conditions show higher intrinsic cytobiocompatibility with superior cell integration and proliferation if compared to their 2D counterparts or a clinically-approved collagen membrane.

Age related macular degeneration (AMD) is a leading cause of vision loss in the developed world, with an increasing number of people suffering from blindness or severe visual impairment. Transplantation of retinal pigment epithelium (RPE) cells supported on a synthetic, biomimetic-like Bruch's membrane (BM) is considered a promising treatment. However, the synthetic scaffolds used do not mimic the microenvironment of the RPE cell supporting layers, required for the development of a functional RPE monolayer. This study indicated that porous, laminin coated, 70nm PLLA ENMs supported functional RPE monolayers, exhibiting 3D polygonal-cobblestone morphology, apical microvilli, basal infoldings, high transepithelial resistance (TER), phagocytic activity and expression of signature RPE markers. These findings indicate the potential clinical use of porous, laminin coated, 70nm PLLA ENMs in fabricating retinal constructs aimed at treating dry AMD.

No abstract

Age-related macular degeneration (AMD) is a highly prevalent form of blindness caused by loss death of cells of the retinal pigment epithelium (RPE). Transplantation of pluripotent stem cell (PSC)-derived RPE cells is considered a promising therapy to regenerate cell function and vision. Objective The objective of this study is to develop a rapid directed differentiation method for production of RPE cells from PSC which is rapid, efficient, and fully defined and produces cells suitable for clinical use. Design A protocol for cell growth and differentiation from hESCs was developed to induce differentiation through screening small molecules which regulated a primary stage of differentiation to the eyefield progenitor, and then, a subsequent set of molecules to drive differentiation to RPE cells. Methods for cell plating and maintenance have been optimized to give a homogeneous population of cells in a short 14-day period, followed by a procedure to support maturation of cell function. Results We show here the efficient production of RPE cells from human embryonic stem cells (hESCs) using small molecules in a feeder-free system using xeno-free/defined medium. Flow cytometry at day 14 showed ~ 90% of cells expressed the RPE markers MITF and PMEL17. Temporal gene analysis confirmed differentiation through defined cell intermediates. Mature hESC-RPE cell monolayers exhibited key morphological, molecular, and functional characteristics of the endogenous RPE. Conclusion This study identifies a novel cell differentiation process for rapid and efficient production of retinal RPE cells directly from hESCs. The described protocol has utility for clinical-grade cell production for human therapy to treat AMD.