The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle

One of the obstacles to the potential clinical utility of bioengineered skeletal muscle is its limited force generation capacity. Since engineered muscle, unlike most native muscle tissue, is composed of relatively short myofibers, we hypothesized that its force production and transmission would be profoundly influenced by cell-matrix interactions. To test this hypothesis, we systematically varied the matrix protein type (collagen I/fibrin/Matrigel) and concentration in engineered, hydrogel-based neonatal rat skeletal muscle bundles and assessed the resulting tissue structure, generation of contractile force, and intracellular Ca2+ handling. After two weeks of culture, the muscle bundles consisted of highly aligned and cross-striated myofibers and exhibited standard force-length and force-frequency relationships achieving tetanus at 40 Hz. The use of 2 mg/ml fibrin (control) yielded isometric tetanus amplitude of 1.4±0.3 mN as compared to 0.9±0.4 mN measured in collagen I-based bundles. Higher fibrin and Matrigel concentrations synergistically yielded further increase in active force generation to 2.8±0.5 mN without significantly affecting passive mechanical properties, tetanus-to-twitch ratio, and twitch kinetics. Optimized matrix composition yielded significant cellular hypertrophy (protein/DNA ratio=11.4±4.1 vs. 6.5±1.9 μg/μg in control) and a prolonged Ca2+ transient half-width (Ca50=232.8±33.3 vs. 101.7±19.8 ms). The use of growth-factor-reduced Matrigel instead of standard Matrigel did not alter the obtained results suggesting enhanced cell-matrix interactions rather than growth factor supplementation as an underlying cause for the measured increase in contractile force. In summary, biomaterial-based manipulation of cell-matrix interactions represents an important target for improving contractile force generation in engineered skeletal muscle

Implantation of Mouse Embryonic Stem Cell-Derived Cardiac Progenitor Cells Preserves Function of Infarcted Murine Hearts

Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart

Tissue Engineering of a Differentiated Skeletal Muscle Construct with Controllable Structure and Function

<p>Transplantation of a functional engineered skeletal muscle substitute is a promising therapeutic option to repair irreversible muscle damage, and, on the other hand, functional muscle tissue constructs can serve as in vitro 3D tissue models that complement the conventional 2D cell cultures and animal models to advance our limited understanding of intrinsic myogenesis and muscle regeneration process. However, the engineering of skeletal muscle constructs with comparable contractile function to the native muscle is hampered by the lack of 1) effective and reproducible methods to form relatively large muscle constructs composed of viable, dense, aligned and matured myofibers, and 2) beneficial microenvironmental cues as well as physiological stimulations that favor the growth, differentiation and maturation of myogenic cells. Thus, in this thesis, I have developed a mesoscopic hydrogel molding approach to fabricate relatively large engineered muscle tissue networks with controllable thickness, pore dimensions, overall myofiber alignment and regional myofiber orientation. I then investigated the effect of variation in pore length on the force generation and passive properties of engineered muscle networks and the potential to improve the contractile function of engineered muscle networks with the treatment of a soluble neurotrophic factor, agrin.</p><p>Specifically, high aspect-ratio soft lithography was utilized to precisely fabricate elastomeric molds containing an array of staggered hexagonal posts which created elliptical pores in muscle tissue sheets made from a mixture of primary skeletal myoblasts, fibrin and Matrigel. The improved oxygen and nutrient access through the pores increased the viability of the embedded muscle cells and prevented the formation of an acellular core. The differentiated myofibers were locally aligned in tissue bundles surrounding the elliptical pores. The length and direction of the microfabricate posts arbitrarily determined the length of elliptical pores and the mean orientation of myofibers formed around the pores, which enables the control of pore dimensions and regional myofiber orientation. Contractile force analysis revealed that engineered muscle networks with more elongated pores generated larger contractile force due to the increased myonuclear density and degree of overall myofiber alignment, despite the larger porosity and reduced tissue volume. Furthermore, the introduction of elliptical pores resulted in distinct deformational changes in tissue bundles and node regions that connect the ends of bundles with the applied unaxial macroscopic stretch, but such spatial alteration of local strain field resulted in no significant change in macroscopic length- tension relationships among engineered muscle networks with different pore length. </p><p>In addition, supplementing culture medium with soluble recombinant agrin significantly increased contractile force production of engineered muscle networks in the absence of nerve-muscle interaction, primarily or partially due to the agrin-induced upregulation of dystrophin. As expected, alteration in the levels endogenous ACh or ACh-like compound affected the agrin-induced AChR aggregation. Furthermore, increased autocrine AChR stimulation, a novel mechanism underlying survival and maturation of aneural myotubes, attenuated the agrin-induced force increase, while suppressed autocrine AChR stimulation severely comproised the overall force production of engineered muscle networks, of which the underlying mechanisms remains to be elucidated in the future studies. </p><p>In summary, a novel tissue engineering methodology that enables the fabrication of relative large muscle tissue constructs with controllable structure and function has been developed in this thesis. Future studies, such as optimizing cell-matrix interaction, incorporating beneficial regulatory proteins in the fibrin-based matrix, and applying specific patterns of electro-mechanical stimulations are expected to further augment the contractile function of engineered muscle networks. The potential application of this versatile tissue fabrication approach to engineer different types of soft tissue might further advance the development of tissue regeneration therapies as well as deepen our understanding of intrinsic tissue morphogenesis and regeneration process.</p>Dissertatio

Characterization of microRNAs and their targets in wild barley (Hordeum vulgare subsp. spontaneum) using deep sequencing

MicroRNAs (miRNA) are a class of small, endogenous RNAs that play a negative regulatory role in various developmental and metabolic processes of plants. Wild barley (Hordeum vulgare subsp. spontaneum) as the progenitor of cultivated barley (Hordeum vulgare subsp. vulgare), has served as a valuable germplasm resource for barley genetic improvement. To survey miRNAs in wild barley, we sequenced the small RNA library prepared from wild barley using the Illumina deep sequencing technology. A total of 70 known miRNAs and 18 putative novel miRNAs were identified. Sequence analysis revealed that all of the miRNAs identified in wild barley contained the highly conserved hairpin sequences found in barley cultivars. MiRNA target predictions showed that 12 out of 52 miRNA families were predicted to target transcription factors, including 8 highly conserved miRNA families in plants and 4 wheat-barley conserved miRNA families. In addition to transcription factors, other predicted target genes were involved in diverse physiological and metabolic processes and stress defense. Our study for the first time reported the large-scale investigation of small RNAs in wild barley, which will provide essential information for understanding the regulatory role of miRNAs in wild barley and also shed light on future practical utilization of miRNAs for barley improvement.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Direct comparison of β-glucan content in wild and cultivated barley

β-Glucan is an important chemical found in cereals, tremendously beneficial to human health. In this study, β-glucan contents in wild and cultivated barley from representative regions worldwide were investigated. Results exhibited that coefficient of variation of β-glucan content of wild and cultivated barley was 24.18% and 13.99%, respectively. The β-glucan contents of studied wild barley accessions were ranged from 3.26% to 7.67% while cultivated barley varieties were ranged from 2.68% to 4.74%. A significant difference of β-glucan content (p ≤ 0.001) was found between wild and cultivated barely. Wild barley showed a higher β-glucan content and variation than cultivated barley. This study gives an idea about elite germplasms for genetic improvement and shed light to trace barley domestication in relation to grain metabolite view

Transcriptional Dynamics of Grain Development in Barley (<i>Hordeum vulgare</i> L.)

Grain development, as a vital process in the crop&#8217;s life cycle, is crucial for determining crop quality and yield. However, the molecular basis and regulatory network of barley grain development is not well understood at present. Here, we investigated the transcriptional dynamics of barley grain development through RNA sequencing at four developmental phases, including early prestorage phase (3 days post anthesis (DPA)), late prestorage or transition phase (8 DPA), early storage phase (13 DPA), and levels off stages (18 DPA). Transcriptome profiling found that pronounced shifts occurred in the abundance of transcripts involved in both primary and secondary metabolism during grain development. The transcripts&#8217; activity was decreased during maturation while the largest divergence was observed between the transitions from prestorage phase to storage phase, which coincided with the physiological changes. Furthermore, the transcription factors, hormone signal transduction-related as well as sugar-metabolism-related genes, were found to play a crucial role in barley grain development. Finally, 4771 RNA editing events were identified in these four development stages, and most of the RNA editing genes were preferentially expressed at the prestore stage rather than in the store stage, which was significantly enriched in &#8220;essential&#8222; genes and plant hormone signal transduction pathway. These results suggested that RNA editing might act as a &#8216;regulator&#8217; to control grain development. This study systematically dissected the gene expression atlas of barley grain development through transcriptome analysis, which not only provided the potential targets for further functional studies, but also provided insights into the dynamics of gene regulation underlying grain development in barley and beyond