Growth of Human Pluripotent Stem Cells Using Functional Human Extracellular Matrix : Human Embryonic Stem Cell Protocols

The use of animal products in the derivation and maintenance of human pluripotent stem cells (hPSCs) limits their possible applications in research and in clinics. Thus, one of the major goals in regenerative medicine is the establishment of animal-free conditions to support the culture and differentiation of human stem cells. Human fibroblasts produce an extracellular matrix (ECM) which can be extracted without the use of detergents, sterilized, and then used to coat tissue culture plates. We have shown that human embryonic stem cells (hESCs) grown on this matrix maintain their pluripotency in the presence of medium conditioned by fibroblast cells, and that these cells maintain expression of surface proteins (SSEA4, Tra1-60, Tra1-81), alkaline phosphatase activity, and specific intracellular markers (Nanog, Oct-4, Tert, FoxD3) in hESCs. This growth system reduces exposure of hPSCs to feeder layers and animal ingredients, thereby limiting the risk of pathogenic contamination and additionally, facilitating their manipulation. Herein we present an improved version of our previous protocol for extracting ECM from human foreskin fibroblast using a different buffer. Our new hypotonic shock method is detergent-free, reduces costs, and preserves the integrity of the extracted ECM. This improved protocol has been validated for undifferentiated-state hPSC maintenance (more than 40 passages), stem cell differentiation, and for cell migration assays.Peer reviewe

Identification Of Mitotically Competent SOX2+ Cells In White Matter Of Normal Human Adult Brain

SOX2 expression is linked to the undifferentiated state of stem cells in mammalian neurogenic niches. While its expression has been reported in the adult human subventricular zone (SVZ), to date it has not been detected in adult human white matter. Here we describe a population of SOX2+ cells from the white matter of the adult human temporal lobe, which proliferate and express glial markers in vitro

A Versatile Open-Source Printhead for Low-Cost 3D Microextrusion-Based Bioprinting

Three-dimensional (3D) bioprinting promises to be essential in tissue engineering for solving the rising demand for organs and tissues. Some bioprinters are commercially available, but their impact on the field of Tissue engineering (TE) is still limited due to their cost or difficulty to tune. Herein, we present a low-cost easy-to-build printhead for microextrusion-based bioprinting (MEBB) that can be installed in many desktop 3D printers to transform them into 3D bioprinters. We can extrude bioinks with precise control of print temperature between 2–60 °C. We validated the versatility of the printhead, by assembling it in three low-cost open-source desktop 3D printers. Multiple units of the printhead can also be easily put together in a single printer carriage for building a multi-material 3D bioprinter. Print resolution was evaluated by creating representative calibration models at different temperatures using natural hydrogels such as gelatin and alginate, and synthetic ones like poloxamer. Using one of the three modified low-cost 3D printers, we successfully printed cell-laden lattice constructs with cell viabilities higher than 90% after 24-h post printing. Controlling temperature and pressure according to the rheological properties of the bioinks was essential in achieving optimal printability and great cell viability. The cost per unit of our device, which can be used with syringes of different volume, is less expensive than any other commercially available product. These data demonstrate an affordable open-source printhead with the potential to become a reliable alternative to commercial bioprinters for any laboratory

A Versatile Open-Source Printhead for Low-Cost 3D Microextrusion-Based Bioprinting

Three-dimensional (3D) bioprinting promises to be essential in tissue engineering for solving the rising demand for organs and tissues. Some bioprinters are commercially available, but their impact on the field of Tissue engineering (TE) is still limited due to their cost or difficulty to tune. Herein, we present a low-cost easy-to-build printhead for microextrusion-based bioprinting (MEBB) that can be installed in many desktop 3D printers to transform them into 3D bioprinters. We can extrude bioinks with precise control of print temperature between 2–60 °C. We validated the versatility of the printhead, by assembling it in three low-cost open-source desktop 3D printers. Multiple units of the printhead can also be easily put together in a single printer carriage for building a multi-material 3D bioprinter. Print resolution was evaluated by creating representative calibration models at different temperatures using natural hydrogels such as gelatin and alginate, and synthetic ones like poloxamer. Using one of the three modified low-cost 3D printers, we successfully printed cell-laden lattice constructs with cell viabilities higher than 90% after 24-h post printing. Controlling temperature and pressure according to the rheological properties of the bioinks was essential in achieving optimal printability and great cell viability. The cost per unit of our device, which can be used with syringes of different volume, is less expensive than any other commercially available product. These data demonstrate an affordable open-source printhead with the potential to become a reliable alternative to commercial bioprinters for any laboratory

Different gDNA content in the subpopulations of Prostate Cancer Extracellular Vesicles: Apoptotic bodies, Microvesicles and Exosomes

Peer reviewe

Distinct prostate cancer-related mRNA cargo in extracellular vesicle subsets from prostate cell lines

Background: Multiple types of extracellular vesicles (EVs), including microvesicles (MVs) and exosomes (EXOs), are released by all cells constituting part of the cellular EV secretome. The bioactive cargo of EVs can be shuffled between cells and consists of lipids, metabolites, proteins, and nucleic acids, including multiple RNA species from non-coding RNAs to messenger RNAs (mRNAs). In this study, we hypothesized that the mRNA cargo of EVs could differ based on the EV cellular origin and subpopulation analyzed. Methods: We isolated MVs and EXOs from PC-3 and LNCaP prostate cancer cells by differential centrifugation and compared them to EVs derived from the benign PNT2 prostate cells. The relative mRNA levels of 84 prostate cancer-related genes were investigated and validated using quantitative reverse transcription PCR arrays. Results: Based on the mRNA abundance, MVs rather than EXOs were enriched in the analyzed transcripts, providing a snapshot of the tumor transcriptome. LNCaP MVs specifically contained significantly increased mRNA levels of NK3 Homeobox 1 (NKX3-1), transmembrane protease serine 2 (TMPRSS2), and tumor protein 53 (TP53) genes, whereas PC-3 MVs carried increased mRNA levels of several genes including, caveolin-2 (CAV2), glutathione S-transferase pi 1 (GSTP1), pescadillo ribosomal biogenesis factor 1 (PES1), calmodulin regulated spectrin associated protein 1 (CAMSAP1), zinc-finger protein 185 (ZNF185), and others compared to PNT2 MVs. Additionally, ETS variant 1 (ETV1) and fatty acid synthase (FASN) mRNAs identified in LNCaP-and PC-3-derived MVs highly correlated with prostate cancer progression. Conclusions: Our study provides new understandings of the variability of the mRNA cargo of MVs and EXOs from different cell lines despite same cancer origin, which is essential to better understand the the proportion of the cell transcriptome that can be detected within EVs and to evaluate their role in disease diagnosis.Peer reviewe

Potential Therapeutic Effects of the Neural Stem Cell-Targeting Antibody Nilo1 in Patient-Derived Glioblastoma Stem Cells

Glioblastoma (GBM) is the most devastating and least treatable brain tumor with median survivalPeer reviewe

Human stem cell decorated nanocellulose threads for biomedical applications

Abstract Upon surgery, local inflammatory reactions and postoperative infections cause complications, morbidity, and mortality. Delivery of human adipose mesenchymal stem cells (hASC) into the wounds is an efficient and safe means to reduce inflammation and promote wound healing. However, administration of stem cells by injection often results in low cell retention, and the cells deposit in other organs, reducing the efficiency of the therapy. Thus, it is essential to improve cell delivery to the target area using carriers to which the cells have a high affinity. Moreover, the application of hASC in surgery has typically relied on animal-origin components, which may induce immune reactions or even transmit infections due to pathogens. To solve these issues, we first show that native cellulose nanofibers (nanofibrillated cellulose, NFC) extracted from plants allow preparation of glutaraldehyde cross-linked threads (NFC-X) with high mechanical strength even under the wet cell culture or surgery conditions, characteristically challenging for cellulosic materials. Secondly, using a xenogeneic free protocol for isolation and maintenance of hASC, we demonstrate that cells adhere, migrate and proliferate on the NFC-X, even without surface modifiers. Cross-linked threads were not found to induce toxicity on the cells and, importantly, hASC attached on NFC-X maintained their undifferentiated state and preserved their bioactivity. After intradermal suturing with the hASC decorated NFC-X threads in an ex vivo experiment, cells remained attached to the multifilament sutures without displaying morphological changes or reducing their metabolic activity. Finally, as NFC-X optionally allows facile surface tailoring if needed, we anticipate that stem-cell-decorated NFC-X opens a versatile generic platform as a surgical bionanomaterial for fighting postoperative inflammation and chronic wound healing problems.Peer reviewe

Hippo pathway effectors control cardiac progenitor cell fate by acting as dynamic sensors of substrate mechanics and nanostructure

Stem cell responsiveness to extracellular matrix (ECM) composition and mechanical cues has been the subject of a number of investigations so far, yet the molecular mechanisms underlying stem cell mechano-biology still need full clarification. Here we demonstrate that the paralog proteins YAP and TAZ exert a crucial role in adult cardiac progenitor cell mechano-sensing and fate decision. Cardiac progenitors respond to dynamic modifications in substrate rigidity and nanopattern by promptly changing YAP/TAZ intracellular localization. We identify a novel activity of YAP and TAZ in the regulation of tubulogenesis in 3D environments and highlight a role for YAP/TAZ in cardiac progenitor proliferation and differentiation. Furthermore, we show that YAP/TAZ expression is triggered in the heart cells located at the infarct border zone. Our results suggest a fundamental role for the YAP/TAZ axis in the response of resident progenitor cells to the modifications in microenvironment nanostructure and mechanics, thereby contributing to the maintenance of myocardial homeostasis in the adult heart. These proteins are indicated as potential targets to control cardiac progenitor cell fate by materials design