First Report of Colletotrichum cordylinicola Causing Anthracnose on Cordyline fruticosa in China.

Cordyline fruticosa is a shrub plant, commonly used in landscape, and distributed in the tropical regions of southern China. In September 2022, anthracnose symptoms were found on this species in Nanning, Guangxi, China. The disease incidence was between 30% to 80% and disease severity was 10% to 30% in five surveyed planting areas. The symptoms initially appeared as small, round, brown spots on leaves. As the disease developed, the lesions turned gray-white with brown borders and yellow halos. Some spots coalesced into larger irregular shapes and even leading to leaf blight. Small segments of the diseased tissues (3×3 mm) were cut from the leaves, surface-sterilized by dipping in a 1% sodium hypochlorite solution for 1 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar (PDA). These plates were incubated at 28°C in the dark for 5 days. Ten fungal isolates with similar morphology were consistently isolated from these diseased tissues. The colonies on PDA were initially white with sparse aerial mycelia and turned pale orange with abundant orange conidial masses on the center after 8 days of culture. The reverse color was pale orange. No sclerotia or setae were found in culture. Conidia were single-celled, hyaline, straight, cylindrical with round ends, and 12.2 to 17.8 µm long (mean 14.9 µm) and 3.9 to 7.3 µm wide (mean 4.8 µm, n=50). The morphological characteristics of these isolates were similar to the Colletotrichum cordylinicola (Sharma et al., 2014). Genomic DNA of two isolates Z3 and Z4 generated from monospore culture was extracted using a fungal DNA extraction kit (Solarbio, Beijing, China). Partial sequences of internal transcribed spacer (ITS), partial actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2) were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GDF1/GDR1, and BT2A/BT2B (Lin et al., 2022), respectively. All the sequences (GenBank accession nos. OQ509909, OQ509910, OQ658690, OQ658691, and OK649310 to OK649314) showed 99% to 100% identity with those of C. cordylinicola in GenBank database. A phylogenetic tree based on concatenated sequences of ITS, ACT, CHS-1, TUB, and GAPDH using maximum likelihood analysis by MEGA X software revealed that Z3 and Z4 clade with reference strains of C. cordylinicola (OJX010226 and MK935473). Based on morphological observation and multi-gene sequence analysis, the isolates were identified as C. cordylinicola (Phoulivong et al., 2010). To assess their pathogenicity, conidial suspensions (106 conidia/ml) of C. cordylinicola were inoculated onto 10 healthy living leaves wounded by slight puncturing (10 µl/wounded spot). Control leaves were treated with sterile water. All inoculated and control plants were maintained under high relative humidity (~90%) and 28℃ in a climate chamber. After 8 days, all the inoculated leaves showed brown lesions resembling natural symptoms, whereas the control group remained symptom-free. The same fungus was re-isolated from the symptomatic leaves, thus completing Koch's postulates. C. cordylinicola is a species of the C. gloeosporioides complex (Weir et al., 2012). It has been reported to cause anthracnose on C. fruticosa in USA and Thailand (Phoulivong et al., 2010; Sharma et al., 2014). To our knowledge, this is the first report of C. cordylinicola causing anthracnose on C. fruticosa in China. Knowing the causal agent is essential to control the serious disease effectively.

Fungus-derived protein particles as cell-adhesive matrices for cell-cultivated food.

Cell-adhesive factors mediate adhesion of cells to substrates via peptide motifs such as the Arg-Gly-Asp (RGD) sequence. With the onset of sustainability issues, there is a pressing need to find alternatives to animal-derived cell-adhesive factors, especially for cell-cultivated food applications. In this paper, we show how data mining can be a powerful approach toward identifying fungal-derived cell-adhesive proteins and present a method to isolate and utilize these proteins as extracellular matrices (ECM) to support cell adhesion and culture in 3D. Screening of a protein database for fungal and plant proteins uncovered that ~5.5% of the unique reported proteins contain RGD sequences. A plot of fungi species vs RGD percentage revealed that 98% of the species exhibited an RGD percentage > = 1%. We observed the formation of protein particles in crude extracts isolated from basidiomycete fungi, which could be correlated to their stability towards particle aggregation at different temperatures. These protein particles were incorporated in 3D fiber matrices encapsulating mouse myoblast cells, showing a positive effect on cell alignment. We demonstrated a cell traction stress on the protein particles (from Flammulina velutipes) that was comparable to cells on fibronectin. A snapshot of the RGD-containing proteins in the fungal extracts was obtained by combining SDS-PAGE and mass spectrometry of the peptide fragments obtained by enzymatic cleavage. Therefore, a sustainable source of cell-adhesive proteins is widely available in the fungi kingdom. A method has been developed to identify candidate species and produce cell-adhesive matrices, applicable to the cell-cultivated food and healthcare industries.

First report of Fusarium falciforme causing root rot of pomegranate (Punica granatum L.) in China.

Pomegranate (Punica granatum L.) is a deciduous shrub or small tree that is native to Iran and Afghanistan. It is also a commercially important fruit tree in China and worldwide. In the summer of 2022, a serious root rot disease occurred in some pomegranate orchards in Xichuan County(32º42´ N, 111º48´ E), Henan Province, China, with an incidence of ~30%. Symptoms included leaf yellowing and wilting, root browning and rotting, and stem-base cracking, eventually leading to defoliation and death. To isolate the causal agent, small pieces (5×5 mm) of diseased root from six trees were surface-sterilized by dipping in 2% NaClO for 8 min followed by 70% ethanol for 15 s, rinsed five times with sterile water, and plated on potato dextrose agar (PDA), then incubated at 28°C in the dark for 5 days. Fifteen pure fungal isolates with the same morphological characteristics were obtained from 24 pieces of roots. All isolates produced white fluffy mycelia. Microconidia were hyaline, oval or reniform, with zero to one septa and dimensions of 7.1 to 19.9 (average 14.5 )× 3.8 to 8.0 (average 5.6) µm (n = 100). Macroconidia were sickle-shaped, one to four septate, and 20.1 to 40.8 (average 26.5) × 4.8 to 8.6 (average 6.5) µm (n = 100). Chlamydospores were spherical, single, in pairs or chains, and 5.6 to 9.8 (average 6.8) µm in diameter (n = 100). Based on the above characteristics, the pathogens were identified as Fusarium sp. (Leslie and Summerell 2006). Genomic DNA was extracted from mycelia of two representative isolates Fs1 and Fs3. The internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and RNA polymerase II second largest subunit (RPB2) sequences were PCR amplified using primer pairs of ITS1/ITS4, EF1/EF2, and RPB2-5f2/RPB2-7cr, RPB2-7cf/RPB2-11ar (O'Donnell et al., 2022), respectively. BLAST analysis showed that the ITS, TEF-1α and RPB2 sequences of isolates Fs1(GenBank accession nos. OK001765, OQ921726 and OQ928396) and Fs3 (GenBank accession nos. OK001771, OQ921727 and OQ928397) showed 99%-100% identity with multiple GenBank sequences of Fusarium falciforme (KY617066, MN064683, KF255514, OQ933361, KY556711 and ON331935). A phylogenetic tree based on concatenated sequences of ITS, TEF-1α and RPB2 using maximum-likelihood analysis revealed that both isolates Fs1 and Fs3 were in the same clade with F. falciforme strains. Based on the morphological and molecular characteristics, the isolates were identified as members of F. falciforme. For pathogenicity testing, conidial suspensions (1×108 spores /mL) of isolates Fs1 and Fs3 were poured onto the roots of healthy pomegranate that had been planted in pots two months previously. Ten plants were inoculated for each isolate. Control plants were drenched with sterile water. After 3 months, inoculated plants developed leaf yellowing and wilting accompanied by root browning and rotting, much like symptoms observed in field plants. The same fungi re-isolated from the experimental plants were confirmed to be F. falciforme by morphology and sequence analysis. This is the first report of F. falciforme causing root rot on pomegranate. F. falciforme is a ubiquitous soil-borne pathogen that causes root rot on multiple plants around the world (Xu F., et al. 2022; Qiu R., et al. 2023). The results of pathogen identification are essential precursors to development of effective control of the disease.

First Report of Leaf Black Spot Caused by Alternaria alternata on Rosemary (Rosmarinus officinalis L.) in China.

Rosemary (Rosmarinus officinalis L.) is an aromatic, evergreen, medicinally important shrub and widely used for cooking, tea, cosmetics as well as medicinal materials. It is grown in many countries including China that had more than 9300 hm2 of commercial cultivation area in 2021. In March 2020, a leaf spot disease sporadic occurred in field rosemarry plants in Nanyang City (32º51´ N, 111º36´ E), Henan Province, China. The disease outbreaked in September with a disease incidence of 57-83%. Symptoms initially appeared as small brown leaf spots that gradually expanded into dark blackbrown irregular lesions. Most of the spots started from the leaf tip or leaf margin, and gradually spread to the leaf base, resulting in heavy defoliation especially on rainy days. Diseased leaf segments (1×3 mm) were surface-sterilized by dipping in 1% sodium hypochlorite for 1 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar, then incubated at 28°C in the dark for 5 days. Twelve fungal isolates with the same morphological characteristics were obtained from nine affected leaves. The fungal colonies were initially white and turned gray brown with flocculent aerial mycelia and a whorled back. Conidia were frequently born in a long chain, with a short beak, brown or light-brown, 13.2 to 48. 7 (average 26.1) × 4.0 to 13.1 (average 8.0) µm in size (n=148) with 0 to 8 transverse and 0 to 3 longitudinal/oblique septa. Phenotypic features of the isolates agreed with those of Alternaria alternata (Simmons et al. 2007). Two isolates Aa1 and Aa2 were randomly selected for molecular and pathogenicity tests. DNA was extracted from mycelia. Partial sequences of internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF1-α) were amplified using the primer pairs ITS1/ITS4 and EFI-728F/EFI-986R (Wei et al. 2022), respectively. The GenBank accession nos. were OK036714 and OK036715 for ITS, and ON951980 and ON951981 for TEF1-α of Aa1 and Aa2, respectively, with a maximal identity of greater than 99% to multiple A. alternata strains. In the neighbour joining phylogenetic tree of the amplified ITS and TEF1-α sequences both Aa1 and Aa2 clustered with A. alternata strains, clearly separating them from other Alternaria spp. For pathogenicity test, conidial suspensions (1×106 spores /mL) of Aa1 and Aa2 were separately sprayed on healthy one-year-old rosemary plants (n=3) with their leaves slightly wounded with a sterilized needle. Control plants (n=3) were sprayed with sterile water. Both inoculated and control plants were incubated at 90% RH, 28 °C. After 14 days, all the inoculated leaves showed black brown lesions similar to those on naturally affected field plants, whereas controls remained symptomless. Fungal cultures with the same phenotypic features as the inocula were constantly re-isolated from the infected leaves. A. alternata was reported as pathogen causing foliar necrosis on rosemary in Italy (Perello et al.1995) and leaf spot (or leaf blight) on multiple plant species such as Actaea dahurica (Hai et al. 2022), and Ligustrum japonicum (Wei et al. 2022) in China. This is the first report of A. alternata causing leaf black spot on rosemary in China.

Influence of hydroquinone content on thermotropic liquid crystalline copolymers and nanocomposites: thermo-mechanical properties and morphology.

Thermotropic liquid crystalline copolyesters (Co-TLCPs) were synthesized by varying the hydroquinone (HQ) molar ratio from 1-5 with respect to the 2,5-diethoxyterephthalic acid (ETA) monomer. The thermal properties and liquid crystalline mesophases of the synthesized Co-TLCP were investigated. All of the Co-TLCPs synthesized using a HQ molar ratio of 1-5 showed a nematic liquid crystalline phase. Among the Co-TLCPs obtained using HQ in various molar ratios, the most stable physical properties and a clear liquid crystalline phase were obtained when HQ was 4 mol. Among the various Co-TLCPs synthesized, hybrids were prepared using Co-TLCP synthesized with a 1 : 4 = ETA : HQ ratio and organoclay. A 1-10% loading of the organoclay Cloisite 93A was employed per weight of TLCP, and the clay was dispersed using the melt intercalation method. Among the Co-TLCP hybrids, the morphology and thermal properties of the hybrids were investigated according to the changes in the Cloisite 93A in the 1-10 wt% range. In general, the thermal properties were superior when the organoclay loading was 3 wt% and were inferior when the organoclay amount was 5 wt% or more. This result was confirmed by the dispersibility of the clay through transmission electron microscopy.

Modular access to 1,2-allenyl ketones based on a photoredox-catalysed radical-polar crossover process.

Herein, a new protocol dealing with the preparation of 1,2-allenyl ketones has been successfully developed via the reactions of enynes with radicals enabled by dual photoredox/copper catalysis. Based on the results of a deuteration experiment and the competition reaction between cyclopropanation and allenation, the mechanism based on a photoredox-neutral-catalysed radical-polar crossover process has been proposed. Synthetic applications of allenes have also been demonstrated.

First Report of Neofusicoccum parvum Causing Leaf Spot on Geodorum eulophioides in China.

Geodorum eulophioides Schltr. is a critically endangered orchid listed in the International Union for Conservation of Nature (IUCN) Red List of threatened species. At present, only two natural populations were found in China. It has important scientific and ornamental values because of its uniqueness. During the summer of 2019, a black leaf spot disease occurred on G. eulophioides, in Yachang Orchid National Nature Reserve (E106°13'32″，N24°44'19″) in Guangxi province, China. More than 60% of leaves of these plants were infected. The disease symptoms initially appeared as small yellow circular spots, which enlarged into irregular brown spots (6 to 9 cm length and 3 to 5 cm width). In later stages of the disease development, the center of the spots became dark brown with a clear edge and surrounded by a yellow halo. In severe infections, the spots coalesced covering the entire leaf. Six symptomatic leaves were collected from three infected plants, surface sterilized in 75% ethanol for 15 s and 0.1% HgCl2 for 4 min, and subsequently washed three times with sterile water, then plated onto potato dextrose agar (PDA), and incubated at 28℃ for three days. Eighteen fungal cultures with similar morphological characteristics were obtained from the infected tissues. Colonies were initially white, then turned dark grey after nine days. To induce sporulation, isolates were grown on 2% water agar and incubated under UVA light at 28℃ for nine days. Three isolates were selected for morphological characterization. Conidia were hyaline, unicellular, nonseptate, ellipsoidal to fusiform, externally smooth, thin-walled, and ranged from 10.7 to 16.6 µm (avg. 13.8 µm) × 4.1 to 6.7 µm (avg. 5.1 µm) (n=50). The isolate DBL-1 was selected as a representative for molecular identification. Genomic DNA was extracted and used for PCR to amplify the rDNA internal transcribed spacer region (ITS), translation elongation factor 1-alpha gene (EF1-α), and beta-tubulin gene (TUB2), using the primer pairs ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R（Alves et al. 2008；Carbone & Kohn, 1999）, and T1/T2 (O'Donnell et al., 1997), respectively. The obtained ITS sequence (GenBank Accession No. MN918440), EF1-α sequence (MN963815), and TUB2 sequence (MN963816) showed >99% homology with several GenBank sequences of Neofusicoccum parvum (JX513636, KU997497 for ITS, KU997261, MH252401 for EF1-α, and KJ841779, MK412882 for TUB2, respectively). Based on morphological characteristics of the asexual morph and maximum likelihood analyses of a combined rDNA-ITS, EF1-α and TUB2 gene sequences, was identified as N. parvum. Pathogenicity test was performed using isolate DBL-1 by inoculating 3 leaves of G. eulophioides plants. The test was repeated three times. Each leaf was wounded using a sterile needle, and a mycelial plug (6 mm diameter) harvested from the periphery of a 3-day-old colony grown on PDA was placed on each wound. Plants were then covered with plastic bags to maintain high relative humidity of 90% and kept at 28℃ in a greenhouse under natural daylight conditions. An equal number of leaves on the same plant were inoculated using sterile PDA plugs and served as mock inoculated controls. After three days, all the inoculated leaves showed black spot symptoms resembling those observed in the field, whereas controls remained symptomless. The fungus was re-isolated from the symptomatic leaves, thus completing Koch's postulates. N. parvum has been reported to cause leaf spot disease on Myristica fragrans (Jayakumar, et al., 2011), Ginkgo biloba (Mirhosseini, et al., 2014), Vitis heyneana (Wu, et al., 2015), and Hevea brasiliensis (Liu et al., 2017), respectively. To the best of our knowledge, this is the first report of N. parvum causing leaf spot disease on G. eulophioides in China. The disease control measures and in-situ conservation method need to be strengthened to protect this rare species.

Synthetic Poly(Vinylalcohol)-Based Membranes for Cartilage Surgery and Repair.

Cell-based therapies for cartilage repair are continually being developed to treat osteoarthritis. The cells are either introduced directly by intra-articular injection or via a cell-seeded matrix scaffold. Here, poly(vinylalcohol)-based membranes are developed to be used for mesenchymal stem cell implantation in cartilage repair procedures, having controllable physicochemical properties such as porosity, mechanical strength, and permeability, and a unique self-sealing property. The membranes possess a bilayer structure with a less porous layer providing mechanical strength and selective permeability, exhibit an elastic modulus of between 0.3 and 0.9 MPa, and are permeable to molecules <40 kDa, which is in the range of cartilage permeability. Three different peptide ligands with the sequences Ac-GCGYGRGDSPG, Ac-GCG(OPG)4REGOFG(OPG)4, and Ac-GCG(OPG)7, respectively, are conjugated to the membranes and subject to in vitro cell adhesion and differentiation assays. Col I/Col II gene expression ratios indicated that the collagen-mimetic peptide, Ac-GCG(OPG)7, best supported mesenchymal stem cell differentiation into the chondrogenic lineage. Although low retention of the membrane is observed in vivo in a rabbit knee model, results suggest that the membrane was able to facilitate mesenchymal stem cell implantation and differentiation to chondrocytes. These PVA-based membranes provide a feasible, synthetic, off-the-shelf material for the delivery of stem cells, and can be modified for other surgical applications.

Engineering a functional three-dimensional human cardiac tissue model for drug toxicity screening.

Cardiotoxicity is one of the major reasons for clinical drug attrition. In vitro tissue models that can provide efficient and accurate drug toxicity screening are highly desired for preclinical drug development and personalized therapy. Here, we report the fabrication and characterization of a human cardiac tissue model for high throughput drug toxicity studies. Cardiac tissues were fabricated via cellular self-assembly of human transgene-free induced pluripotent stem cells-derived cardiomyocytes in pre-fabricated polydimethylsiloxane molds. The formed tissue constructs expressed cardiomyocyte-specific proteins, exhibited robust production of extracellular matrix components such as laminin, collagen and fibronectin, aligned sarcomeric organization, and stable spontaneous contractions for up to 2 months. Functional characterization revealed that the cardiac cells cultured in 3D tissues exhibited higher contraction speed and rate, and displayed a significantly different drug response compared to cells cultured in age-matched 2D monolayer. A panel of clinically relevant compounds including antibiotic, antidiabetic and anticancer drugs were tested in this study. Compared to conventional viability assays, our functional contractility-based assays were more sensitive in predicting drug-induced cardiotoxic effects, demonstrating good concordance with clinical observations. Thus, our 3D cardiac tissue model shows great potential to be used for early safety evaluation in drug development and drug efficiency testing for personalized therapy.

Electrospun polystyrene scaffolds as a synthetic substrate for xeno-free expansion and differentiation of human induced pluripotent stem cells.

The use of human induced pluripotent stem cells (hiPSCs) for clinical tissue engineering applications requires expansion and differentiation of the cells using defined, xeno-free substrates. The screening and selection of suitable synthetic substrates however, is tedious, as their performance relies on the inherent material properties. In the present work, we demonstrate an alternative concept for xeno-free expansion and differentiation of hiPSCs using synthetic substrates, which hinges on the structure-function relationship between electrospun polystyrene scaffolds (ESPS) and pluripotent stem cell growth. ESPS of differential porosity was obtained by fusing the fibers at different temperatures. The more porous, loosely fused scaffolds were found to efficiently trap the cells, leading to a large number of three-dimensional (3D) aggregates which were shown to be pluripotent colonies. Immunostaining, PCR analyses, in vitro differentiation and in vivo teratoma formation studies demonstrated that these hiPSC aggregates could be cultured for up to 10 consecutive passages (P10) with maintenance of pluripotency. Flow cytometry showed that more than 80% of the cell population stained positive for the pluripotent marker OCT4 at P1, P5 and P10. P10 cells could be differentiated to neuronal-like cells and cultured within the ESPS for up to 18months. Our results suggest the usefulness of a generic class of synthetic substrates, exemplified by ESPS, for 'trapped aggregate culture' of hiPSCs. STATEMENT OF SIGNIFICANCE: To realize the potential of human induced pluripotent stem cells (hiPSCs) in clinical medicine, robust, xeno-free substrates for expansion and differentiation of iPSCs are required. In the existing literature, synthetic materials have been reported that meet the requirement for non-xenogeneic substrates. However, the self-renewal and differentiation characteristics of hiPSCs are affected differently by the biocompatibility and physico-chemical properties of individual substrates. Although some rules based on chemical structure and substrate rigidity have been developed, most of these efforts are still empirical, and most synthetic substrates must still be rigorously screened for suitability. In this paper, we demonstrate an alternative concept for xeno-free expansion and differentiation of hiPSCs using synthetic substrates, which hinges on the structure-function relationship between electrospun polystyrene scaffolds (ESPS) and pluripotent stem cell growth. ESPS of differential porosity was obtained by fusing the fibers at different temperatures. The more porous, loosely fused scaffold was found to efficiently trap the cells, leading to a large number of three-dimensional (3D) aggregates. In the form of these trapped aggregates, we showed that hiPSCs could be cultured for up to 10 consecutive passages (P10) with maintenance of pluripotency, following which they could be differentiated to a chosen lineage. We believe that this novel, generic class of synthetic substrates that employs 'trapped aggregate culture' for expansion and differentiation of hiPSCs is an important conceptual advance, and would be of high interest to the readership of Acta Biomaterialia.

Functional Kidney Bioengineering with Pluripotent Stem-Cell-Derived Renal Progenitor Cells and Decellularized Kidney Scaffolds.

Recent advances in developmental biology and stem cell technology have led to the engineering of functional organs in a dish. However, the limited size of these organoids and absence of a large circulatory system poses limits to its clinical translation. To overcome these issues, decellularized whole kidney scaffolds with native microstructure and extracellular matrix (ECM) are employed for kidney bioengineering, using human-induced pluripotent-stem-cell-derived renal progenitor cells and endothelial cells. To demonstrate ECM-guided cellular assembly, the present work is focused on generating the functional unit of the kidney, the glomerulus. In the repopulated organ, the presence of endothelial cells broadly upregulates the expression level of genes related to renal development. When the cellularized native scaffolds are implanted in SCID mice, glomeruli assembly can be achieved by co-culture of the renal progenitors and endothelial cells. These individual glomerular units are shown to be functional in the context of the whole organ using a simulated bio-reactor set-up with urea and creatinine excretion and albumin reabsorption. Our results indicate that the repopulation of decellularized native kidney using clinically relevant, expandable patient-specific renal progenitors and endothelial cells may be a viable approach for the generation of a functional whole kidney.

Induced pluripotent stem cell-derived hepatocytes and endothelial cells in multi-component hydrogel fibers for liver tissue engineering.

Liver tissue engineering requires a suitable cell source, methodologies to assemble the cells within their niche microenvironments in a spatially defined manner, and vascularization of the construct in vivo for maintenance of hepatocyte viability and function. Recently, we have developed methods of encapsulating cells within separate domains in multi-component hydrogel fibers and methods of assembling fibers to form 3D-patterned tissue constructs. In the present work, we have combined these approaches to encapsulate hepatocytes and endothelial cells within their specific niches, and to assemble them into endothelialized liver tissue constructs. The hepatocytes and endothelial cells were obtained in parallel by differentiating human recombinant protein-induced human pluripotent stem cells, resulting in a construct which contained genetically identical endothelial and parenchymal elements. We were able to demonstrate that the presence of endothelial cells in the scaffold significantly improved hepatocyte function in vitro and facilitated vascularization of the scaffold when implanted in a mouse partial hepatectomy model. The in vivo studies further asserted that integration of the scaffold with host vasculature had occurred, as demonstrated by the presence of human albumin in the mouse serum.

Polysulfone membranes coated with polymerized 3,4-dihydroxy-l-phenylalanine are a versatile and cost-effective synthetic substrate for defined long-term cultures of human pluripotent stem cells.

Clinical and industrial applications of human pluripotent stem cells (hPSC) require large amounts of cells that have been expanded under defined conditions. Labor-intensive techniques and ill-defined or expensive compounds and substrates are not applicable. Here we describe a chemically defined synthetic substrate consisting of polysulfone (PSF) membranes coated with polymerized 3,4-dihydroxy-l-phenylalanine (DOPA). DOPA/PSF is inexpensive and can be easily produced at various shapes and sizes. DOPA/PSF supports long-term self-renewal of undifferentiated human embryonic (hESC) and human induced pluripotent stem cells (hiPSC) under defined conditions. Pluripotency is maintained for at least 10 passages. Adhesion of hPSC to DOPA/PSF is mainly mediated by a specific integrin heterodimer. Proliferation and gene expression patterns on DOPA/PSF and control substrates are comparable. Labor-intensive cultivation methods and use of serum or coating with proteins are not required. Together, these features make DOPA/PSF attractive for applications where large-scale expansion of human pluripotent stem cells under defined conditions is essential.

Enzymatic conjugation of a bioactive peptide into an injectable hyaluronic acid-tyramine hydrogel system to promote the formation of functional vasculature.

In this study, one-step enzyme-mediated preparation of a multi-functional injectable hyaluronic-acid-based hydrogel system is reported. Hydrogel was formed through the in situ coupling of phenol moieties by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2), and bioactive peptides were simultaneously conjugated into the hydrogel during the gel formation process. The preparation of this multi-functional hydrogel was made possible by synthesizing peptides containing phenols which could couple with the phenol moieties of hyaluronic-acid-tyramine (HA-Tyr) during the HRP-mediated crosslinking reaction. Preliminary studies demonstrated that two phenol moieties per molecule resulted in a consistently high degree of conjugation into the HA-Tyr hydrogel network, unlike the one modified with one phenol moiety per molecule. Therefore, an Arg-Gly-Asp (RGD) peptide bearing two phenol moieties (phenol2-poly(ethylene glycol)-RGD) was designed for conjugation to endow the HA-Tyr hydrogel with adhesion signals and enhance its bioactivities. Human umbilical vein endothelial cells (HUVECs) cultured on or within the RGD-modified hydrogels showed significantly different adhesion behavior, from non-adherence on the HA-Tyr hydrogel to strong adhesion on hydrogels modified with phenol2-poly(ethylene glycol)-RGD. This altered cell adhesion behavior led to improved cell proliferation, migration and formation of capillary-like network in the hydrogel in vitro. More importantly, when HUVECs and human fibroblasts (HFF1) were encapsulated together in the RGD-modified HA-Tyr hydrogel, functional vasculature was observed inside the cell-laden gel after 2weeks in the subcutaneous tissue. Taken together, the in situ conjugation of phenol2-poly(ethylene glycol)-RGD into HA-Tyr hydrogel system, coupled with the ease of incorporating cells, offers a simple and effective means to introduce biological signals for preparation of multi-functional injectable hydrogels for tissue engineering application.

Modulation of chondrocyte functions and stiffness-dependent cartilage repair using an injectable enzymatically crosslinked hydrogel with tunable mechanical properties.

We developed an injectable hydrogel system to evaluate the effect of hydrogel stiffness on chondrocyte cellular functions in a three-dimensional (3D) environment and its subsequent influence on ectopic cartilage formation and early-stage osteochondral defect repair in a rabbit model. The hydrogels, composed of gelatin-hydroxyphenylpropionic acid (Gtn-HPA) conjugate, were formed using oxidative coupling of HPA moieties catalyzed by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). The storage modulus (G') of the hydrogels, which was tunable by changing the H2O2 and Gtn-HPA concentrations, ranged from 570 Pa to 2750 Pa. It was found that the cellular functions of chondrocytes encapsulated in hydrogels, including cell proliferation, biosynthesis of collagen and sulfated glycosaminoglycans (sGAG), as well as gene expression of type I (Col-I) and type II collagen (Col-II), were strongly affected by the stiffness of the hydrogels. Of note, chondrocytes cultured within the Gtn-HPA hydrogel of medium stiffness (G' = 1000 Pa) produced highest level of sGAG production, as well as highest ratio of Col-II to Col-I gene expression among the Gtn-HPA hydrogels of different stiffness. Consistent with the results from in vitro and in vivo ectopic cartilage formation, osteochondral defect repair in a rabbit model showed stiffness-dependent tissue repair, with defects implanted with chondrocytes in hydrogels of medium stiffness having markedly more hyaline cartilage formation, smoother surface and better integration with adjacent cartilage, compared to defects treated with hydrogels of low or high stiffness. These results suggest that the tunable stiffness of Gtn-HPA hydrogels modulates chondrocyte cellular functions, and has a dramatic impact on cartilage tissue histogenesis and repair.

Multi-channel hyperspectral fluorescence detection excited by coupled plasmon-waveguide resonance.

We propose in this paper a biosensor scheme based on coupled plasmon-waveguide resonance (CPWR) excited fluorescence spectroscopy. A symmetrical structure that offers higher surface electric field strengths, longer surface propagation lengths and depths is developed to support guided waveguide modes for the efficient excitation of fluorescence. The optimal parameters for the sensor films are theoretically and experimentally investigated, leading to a detection limit of 0.1 nM (for a Cy5 solution). Multiplex analysis possible with the fluorescence detection is further advanced by employing the hyperspectral fluorescence technique to record the full spectra for every pixel on the sample plane. We demonstrate experimentally that highly overlapping fluorescence (Cy5 and Dylight680) can be distinguished and ratios of different emission sources can be determined accurately. This biosensor shows great potential for multiplex detections of fluorescence analytes.

Patterned prevascularised tissue constructs by assembly of polyelectrolyte hydrogel fibres.

The in vivo efficacy of engineered tissue constructs depends largely on their integration with the host vasculature. Prevascularisation has been noted to facilitate integration of the constructs via anastomosis of preformed microvascular networks. Here we report a technique to fabricate aligned, spatially defined prevascularised tissue constructs with endothelial vessels by assembling individually tailored cell-laden polyelectrolyte hydrogel fibres. Stable, aligned endothelial vessels form in vitro within these constructs in 24 h, and these vessels anastomose with the host circulation in a mouse subcutaneous model. We create vascularised adipose and hepatic tissues by co-patterning the respective cell types with the preformed endothelial vessels. Our study indicates that the formation of aligned endothelial vessels in a hydrogel is an efficient prevascularisation approach in the engineering of tissue constructs.

Follicular dermal papilla structures by organization of epithelial and mesenchymal cells in interfacial polyelectrolyte complex fibers.

The hair follicle is a regenerating organ that produces a new hair shaft during each growth cycle. Development and cycling of the hair follicle is governed by interactions between the epithelial and mesenchymal components. Therefore, development of an engineered 3D hair follicle would be useful for studying these interactions to identify strategies for treatment of hair loss. We have developed a technique suitable for assembly of different cell types in close proximity in fibrous hydrogel scaffolds with resolutions of ∼50 µm. By assembly of dermal papilla (DP) and keratinocytes, structures similar to the native hair bulb arrangement are formed. Gene expression of these constructs showed up-regulation of molecules involved in epithelial-mesenchymal interactions of the hair follicle. Implantation of the follicular structures in SCID mice led to the formation of hair follicle-like structures, thus demonstrating their hair inductive ability. The transparency of the fiber matrix and the small dimensions of the follicular structures allowed the direct quantitation of DP cell proliferation by confocal microscopy, clearly illustrating the promoting or inhibitory effects of hair growth regulating agents. Collectively, our results suggested a promising application of these 3D engineered follicular structures for in vitro screening and testing of drugs for hair growth therapy.

Delivery of reprogramming factors into fibroblasts for generation of non-genetic induced pluripotent stem cells using a cationic bolaamphiphile as a non-viral vector.

Protein delivery allows a clinical effect to be directly realized without genetic modification of the host cells. We have developed a cationic bolaamphiphile as a non-viral vector for protein delivery application. The relatively low toxicity and efficient protein delivery by the cationic bolaamphiphile prompted us to test the system for the generation of induced pluripotent stem cells (iPSCs) as an alternative to the conventional vector-based genetic approach. Studies on the kinetics and cytotoxicity of the protein delivery system led us to use an optimized cationic bolaamphiphile-protein complex ratio of 7:1 (wt/wt) and a 3 h period of incubation with human fibroblasts, to ensure complete and non-toxic protein delivery of the reprogramming proteins. The reprogrammed cells were shown to exhibit the characteristics of embryonic stem cells, including expression of pluripotent markers, teratoma formation in SCID mice, and ability to be differentiated into a specific lineage, as exemplified by neuronal differentiation.