Repurposing agricultural waste as low-cost cultured meat scaffolds

Growing meat in vitro using tissue engineering and bioproduction techniques (cellular agriculture) has become an increasingly promising solution to the global food security challenge. Our lab has established methods to cultivate bovine muscle tissue on decellularized plants, representing a viable low-cost, sustainable method to grow meat on edible scaffolds. Most work in this area has focused on the use of edible plant materials (i.e., spinach leaves, apple, broccoli) with inherent economic value. Harvest waste such as corn husk or jackfruit represent abundant sources of cellulose for scaffold production and may be a viable alternative. The present study aims to investigate production of cultured meat through tissue engineering and bioproduction on decellularized, edible samples of corn husk and jackfruit rind. Corn husks and jackfruit rinds were exposed to immersion decellularization. DNA quantification and histological analysis demonstrated sufficient decellularization (0.17 ± 0.06 and 0.07 ± 0.00 ug DNA/g tissue for corn husk and jackfruit rinds, respectively). Following decellularization, corn husk scaffold stiffnesses decreased from 56.67±16.71 MPa to 12.95±2.43 MPa in fiber-aligned direction, while jackfruit decreased from 7.54 ±2.42 MPa to 2.47±1.47 MPa. Seeded scaffolds with bovine satellite cells (BSCs) (11.45±2.24 ug/ul lysate/Gram) and avian (QM7s) (12.90±1.99 ug/ul lysate/Gram) demonstrated increased protein yields on jackfruit scaffolds. QM7 cultured on corn husk scaffolds yielded increased protein but PBSCs seeded on corn husks did not yield protein content higher than controls (QM7 on corn husk: 16.28±3.55, PBSCs on corn husks: 9.57±1.56 ug/ul lysate/Gram, control: 6.35±1.43 ug/ul lysate/Gram). Additionally, cell transfer from scaffold to scaffold (bead-to-bead transfer) was observed on corn husk scaffolds in a dynamic environment. These results suggest that decellularized harvest waste scaffolds may aid in realization of cultured meat products that will contribute to a more robust and environmentally sustainable food supply

Corrigendum to “Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction” [Dev. Biol. 276 (2004) 237–252]

AbstractTherapeutic cloning or nuclear transfer for stem cells (NTSC) seeks to overcome immune rejection through the development of embryonic stem cells (ES cells) derived from cloned blastocysts. The successful derivation of a human embryonic stem cell (hESC) line from blastocysts generated by somatic cell nuclear transfer (SCNT) provides proof-of-principle for “therapeutic cloning,” though immune matching of the differentiated NT-hES remains to be established. Here, in nonhuman primates (NHPs; rhesus and cynomologus macaques), the strategies used with human SCNT improve NHP-SCNT development significantly. Protocol improvements include the following: enucleation just prior to metaphase-II arrest; extrusion rather than extraction of the meiotic spindle-chromosome complex (SCC); nuclear transfer by electrofusion with simultaneous cytoplast activation; and sequential media. Embryo transfers (ET) of 135 SCNT-NHP into 25 staged surrogates did not result in convincing evidence of pregnancies after 30 days post-ET. These results demonstrate that (i) protocols optimized in humans generate preimplantation embryos in nonhuman primates; (ii) some, though perhaps not yet all, hurdles in deriving NT-nhpES cells from cloned macaque embryos (therapeutic cloning) have been overcome; (iii) reproductive cloning with SCNT-NHP embryos appears significantly less efficient than with fertilized embryos; (iv) therapeutic cloning with matured metaphase-II oocytes, aged oocytes, or “fertilization failures” might remain difficult since enucleation is optimally performed prior to metaphase-II arrest; and (v) challenges remain for producing reproductive successes since NT embryos appear inferior to fertilized ones due to spindle defects resulting from centrosome and motor deficiencies that produce aneuploid preimplantation embryos, among other anomalies including genomic imprinting, mitochondrial and cytoplasmic heterogeneities, cell cycle asynchronies, and improper nuclear reprogramming

Onset of transcription in bovine oocytes and preimplantation embryos

The transition from the maternal to embryonic control of early embryonic development (MET) in mammals is not fully understood. The objective of this study was to determine the amount of transcriptional activity in immature oocytes containing germinal vesicle (GV), mature metaphase II arrested oocytes (MII), 2-, 4- and 8-cell bovine embryos by labeling with 35S-UTP followed by isolation of total RNA and autoradiography. Expression of counts per minute (CPM) per cell showed that incorporation of 35S-UTP in GV oocytes was significantly higher than the background (P \u3c 0.01) and decreased sharply by the time the oocytes reached MII arrest. Incorporation significantly increased during the 2-cell stage and remained at the same level during the 4- and 8-cell stages. Uptake remained constant throughout different development stages (P \u3e 0.05) with the highest variability observed during the 2-cell stage. When CPM were expressed per oocyte or embryo incorporation remained high at the GV stage, decreased to the background levels at the time of MII and increased again at the 2-cell stage. It remained at the same level during the 4-cell stage but increased significantly for the second time during the 8-cell stage. Uptake remained at the same level until the 8-cell stage when a significant increase was observed. The negative controls showed a significantly lower amount of incorporation compared to the positive control (P \u3c 0.05). Similar results were observed by autoradiography. Our observations suggest that MET starts as early as the 2-cell stage in bovine embryos

FGF2-induced effects on transcriptome associated with regeneration competence in adult human fibroblasts

BACKGROUND: Adult human fibroblasts grown in low oxygen and with FGF2 supplementation have the capacity to tip the healing outcome of skeletal muscle injury – by favoring regeneration response in vivo over scar formation. Here, we compare the transcriptomes of control adult human dermal fibroblasts and induced regeneration-competent (iRC) fibroblasts to identify transcriptional changes that may be related to their regeneration competence. RESULTS: We identified a unique gene-expression profile that characterizes FGF2-induced iRC fibroblast phenotype. Significantly differentially expressed genes due to FGF2 treatment were identified and analyzed to determine overrepresented Gene Ontology terms. Genes belonging to extracellular matrix components, adhesion molecules, matrix remodelling, cytoskeleton, and cytokines were determined to be affected by FGF2 treatment. CONCLUSIONS: Transcriptome analysis comparing control adult human fibroblasts with FGF2-treated fibroblasts identified functional groups of genes that reflect transcriptional changes potentially contributing to their regeneration competence. This comparative transcriptome analysis should contribute new insights into genes that characterize cells with greater regenerative potential

Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species

The transfer of nuclei from one cell to another provides a powerful tool for studying the interactions between the cytoplasm of one cell and the nucleus of another. This study was designed to examine the ability of the bovine metaphase oocyte cytoplasm to support mitotic cell cycles under the direction of differentiated somatic cell nuclei of various mammalian species. Skin fibroblast cells from cows, sheep, pigs, monkeys, and rats were used as sources of donor nuclei. Nuclear transfer units produced by fusion of enucleated bovine oocytes and individual fibroblasts from all species examined underwent transition to interphase accompanied by nuclear swelling, further progression through the cell cycle, and completion of the first mitosis. Regardless of the species of donor fibroblasts used, some cleaving units progressed further and developed to advanced stages, as evidenced by continuation of cell proliferation and formation of a blastocoele cavity at the time appropriate for the donor fibroblast species. Although no pregnancies have been carried to term after transfer of embryos into surrogate animals, these observations suggest that mechanisms regulating early embryonic development may be conserved among mammalian species and that bovine oocyte cytoplasm can support the introduced differentiated nucleus regardless of chromosome number, species, or age of the donor fibroblast