Assessment of induced allelopathy in crop-weed co-culture with rye-pigweed model.

This study evaluates induced allelopathy in a rye-pigweed model driven by rye's (Secale cereale L.) allelopathic potential as a cover crop and pigweed's (Amaranthus retroflexus L.) notoriety as a weed. The response of rye towards pigweed's presence in terms of benzoxazinoids (BXs) provides valuable insight into induced allelopathy for crop improvement. In the 2 week plant stage, pigweed experiences a significant reduction in growth in rye's presence, implying allelopathic effects. Rye exhibits increased seedling length and BXs upsurge in response to pigweed presence. These trends persist in the 4 week plant stage, emphasizing robust allelopathic effects and the importance of different co-culture arrangements. Germination experiments show rye's ability to germinate in the presence of pigweed, while pigweed exhibits reduced germination with rye. High-performance liquid chromatography with diode-array detection (HPLC-DAD) analysis identifies allelopathic compounds (BXs), 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) in rye. Rye significantly increases BX production in response to pigweed, age-dependently. Furthermore, pigweed plants are screened for possible BX uptake from the rhizosphere. Results suggest that allelopathy in rye-pigweed co-cultures is influenced by seed timing, and age-dependent dynamics of plants' allelopathic compounds, providing a foundation for further investigations into chemical and ecological processes in crop-weed interactions.

An In Vitro Model of Blood-Brain Barrier for Studies on HIV Neuroinflammation and CNS Antibody Penetration.

The blood-brain barrier (BBB) is one of several barriers between the brain and the peripheral blood system to maintain homeostasis. Understanding the interactions between infectious agents such as human immunodeficiency virus type 1 (HIV-1), which are capable of traversing the BBB and causing neuroinflammation requires modeling an authentic BBB in vitro. Such an in vitro BBB model also helps develop means of targeting viruses that reside in the brain via natural immune effectors such as antibodies. The BBB consists of human brain microvascular endothelial cells (HBMECs), astrocytes, and pericytes. Here we report in vitro methods to establish a dual-cell BBB model consisting of primary HBMECs and primary astrocytes to measure the integrity of the BBB and antibody penetration of the BBB, as well as a method to establish a single cell BBB model to study the impact of HIV-1 infected medium on the integrity of such a BBB.

Monocytes prevent apoptosis of iPSCs and promote differentiation of kidney organoids.

BACKGROUND: Induced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols having been developed, further improvements are needed to overcome existing limitations and enable a wider application of this model. One of the approaches to improve the differentiation of renal organoids in vitro is to include in the system cell types important for kidney organogenesis in vivo, such as macrophages. Another approach could be to improve cell survival. Mesodermal lineage differentiation is the common initial step of the reported protocols. The glycogen synthase kinase-3 (GSK-3) activity inhibitor, CHIR99021 (CHIR), is applied to induce mesodermal differentiation. It has been reported that CHIR simultaneously induces iPSCs apoptosis that can compromise cell differentiation. We thought to interfere with CHIR-induced apoptosis of iPSCs using rapamycin. METHODS: Differentiation of kidney organoids from human iPSCs was performed. Cell survival and autophagy were analyzed using Cell counting kit 8 (CCK8) kit and Autophagy detection kit. Cells were treated with rapamycin or co-cultured with human monocytes isolated from peripheral blood or iPSCs-macrophages using a transwell co-culture system. Monocyte-derived extracellular vesicles (EVs) were isolated using polyethylene glycol precipitation. Expression of apoptotic markers cleaved Caspase 3, Poly [ADP-ribose] polymerase 1 (PARP-1) and markers of differentiation T-Box Transcription Factor 6 (TBX6), odd-skipped related 1 (OSR1), Nephrin, E-Cadherin, Paired box gene 2 (Pax2) and GATA Binding Protein 3 (Gata3) was assessed by RT-PCR and western blotting. Organoids were imaged by 3D-confocal microscopy. RESULTS: We observed that CHIR induced apoptosis of iPSCs during the initial stage of renal organoid differentiation. Underlying mechanisms implied the accumulation of reactive oxygen species and decreased autophagy. Activation of autophagy by rapamacin and by an indirect co-culture of differentiating iPSCs with iPSCs-macrophages and human peripheral blood monocytes prevented apoptosis induced by CHIR. Furthermore, monocytes (but not rapamycin) strongly promoted expression of renal differentiation markers and organoids development via released extracellular vesicles. CONCLUSION: Our data suggest that co-culturing of iPSCs with human monocytes strongly improves differentiation of kidney organoids. An underlying mechanism of monocytic action implies, but not limited to, an increased autophagy in CHIR-treated iPSCs. Our findings enhance the utility of kidney organoid models.

A pancreatic cancer organoid-in-matrix platform shows distinct sensitivities to T cell killing.

Poor treatment responses of pancreatic ductal adenocarcinoma (PDAC) are in large part due to tumor heterogeneity and an immunosuppressive desmoplastic tumor stroma that impacts interactions with cells in the tumor microenvironment (TME). Thus, there is a pressing need for models to probe the contributions of cellular and noncellular crosstalk. Organoids are promising model systems with the potential to generate a plethora of data including phenotypic, transcriptomic and genomic characterization but still require improvements in culture conditions mimicking the TME. Here, we describe an INTERaction with Organoid-in-MatriX ("InterOMaX") model system, that presents a 3D co-culture-based platform for investigating matrix-dependent cellular crosstalk. We describe its potential to uncover new molecular mechanisms of T cell responses to murine KPC (LSL-KrasG12D/+27/Trp53tm1Tyj/J/p48Cre/+) PDAC cells as well as PDAC patient-derived organoids (PDOs). For this, a customizable matrix and homogenously sized organoid-in-matrix positioning of cancer cells were designed based on a standardized agarose microwell chip array system and established for co-culture with T cells and inclusion of stromal cells. We describe the detection and orthogonal analysis of murine and human PDAC cell populations with distinct sensitivity to T cell killing that is corroborated in vivo. By enabling both identification and validation of gene candidates for T cell resistance, this platform sets the stage for better mechanistic understanding of cancer cell-intrinsic resistance phenotypes in PDAC.

Improvement of cell growth in green algae Chlamydomonas reinhardtii through co-cultivation with yeast Saccharomyces cerevisiae.

PURPOSE: CO2 fixation methods using green algae have attracted considerable attention because they can be applied for the fixation of dilute CO2 in the atmosphere. However, green algae generally exhibit low CO2 fixation efficiency under atmospheric conditions. Therefore, it is a challenge to improve the CO2 fixation efficiency of green algae under atmospheric conditions. Co-cultivation of certain microalgae with heterotrophic microorganisms can increase the growth potential of microalgae under atmospheric conditions. The objective of this study was to determine the culture conditions under which the growth potential of green algae Chlamydomonas reinhardtii is enhanced by co-culturing with the yeast Saccharomyces cerevisiae, and to identify the cause of the enhanced growth potential. RESULTS: When C. reinhardtii and S. cerevisiae were co-cultured with an initial green algae to yeast inoculum ratio of 1:3, the cell concentration of C. reinhardtii reached 133 × 105 cells/mL on day 18 of culture, which was 1.5 times higher than that of the monoculture. Transcriptome analysis revealed that the expression levels of 363 green algae and 815 yeast genes were altered through co-cultivation. These included genes responsible for ammonium transport and CO2 enrichment mechanism in green algae and the genes responsible for glycolysis and stress responses in yeast. CONCLUSION: We successfully increased C. reinhardtii growth potential by co-culturing it with S. cerevisiae. The main reasons for this are likely to be an increase in inorganic nitrogen available to green algae via yeast metabolism and an increase in energy available for green algae growth instead of CO2 enrichment.

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research.

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating drug efficacy and toxicity, and tissue bioengineering. However, applying these models for the described reasons may be limited because of the three-dimensional (3D) nature of these models. For example, using 3D enteroid culture systems to study digestive diseases is challenging due to the inaccessibility of the intestinal lumen and its secreted substances. Indeed, stimulation of 3D organoids with pathogens requires either luminal microinjection, mechanical disruption of the 3D structure, or generation of apical-out enteroids. Moreover, these organoids cannot be co-cultured with immune and stromal cells, limiting in-depth mechanistic analysis into pathophysiological dynamics. To circumvent this, we optimized a bovine primary cell two-dimensional (2D) enteroid-derived monolayer culture system, allowing co-culture with other relevant cell types. Ileal crypts isolated from healthy adult cattle were cultured to generate 3D organoids that were cryopreserved for future use. A 2D monolayer was created using revived 3D enteroids that were passaged and disrupted to yield single cells, which were seeded on basement membrane extract-coated transwell cell culture inserts, thereby exposing their apical surface. The intestinal monolayer polarity, cellular differentiation, and barrier function were characterized using immunofluorescence microscopy and measuring transepithelial electrical resistance. Stimulation of the apical surface of the monolayer revealed the expected functionality of the monolayer, as demonstrated by cytokine secretion from both apical and basal compartments. The described 2D enteroid-derived monolayer model holds great promise in investigating host-pathogen interactions and intestinal physiology, drug development, and regenerative medicine.

Potency assay to predict the anti-inflammatory capacity of a cell therapy product for macrophage-driven diseases: overcoming the challenges of assay development and validation.

BACKGROUND: Given the high level of product complexity and limited regulatory guidance, designing and implementing appropriate potency assays is often the most challenging part of establishing a quality control testing matrix for a cell-based medicinal product. Among the most elusive tasks are the selection of suitable read-out parameters, the development of assay designs that most closely model the pathophysiological conditions, and the validation of the methods. Here we describe these challenges and how they were addressed in developing an assay that measures the anti-inflammatory potency of mesenchymal stromal cells (MSCs) in an M1 macrophage-dominated inflammatory environment. METHODS: An in vitro inflammation model was established by coculturing skin-derived ABCB5+ MSCs with THP-1 monocyte-derived M1-polarized macrophages. Readout was the amount of interleukin 1 receptor antagonist (IL-1RA) secreted by the MSCs in the coculture, measured by an enzyme-linked immunosorbent assay. RESULTS: IL-1RA was quantified with guideline-concordant selectivity, accuracy and precision over a relevant concentration range. Consistent induction of the macrophage markers CD36 and CD80 indicated successful macrophage differentiation and M1 polarization of THP-1 cells, which was functionally confirmed by release of proinflammatory tumor necrosis factor α. Testing a wide range of MSC/macrophage ratios revealed the optimal ratio for near-maximal stimulation of MSCs to secrete IL-1RA, providing absolute maximum levels per individual MSC that can be used for future comparison with clinical efficacy. Batch release testing of 71 consecutively manufactured MSC batches showed a low overall failure rate and a high comparability between donors. CONCLUSIONS: We describe the systematic development and validation of a therapeutically relevant, straightforward, robust and reproducible potency assay to measure the immunomodulatory capacity of MSCs in M1 macrophage-driven inflammation. The insights into the challenges and how they were addressed may also be helpful to developers of potency assays related to other cellular functions and clinical indications.

3D Printing of a Vascularized Mini-Liver Based on the Size-Dependent Functional Enhancements of Cell Spheroids for Rescue of Liver Failure.

The emerging stem cell-derived hepatocyte-like cells (HLCs) are the alternative cell sources of hepatocytes for treatment of highly lethal acute liver failure (ALF). However, the hostile local environment and the immature cell differentiation may compromise their therapeutic efficacy. To this end, human adipose-derived mesenchymal stromal/stem cells (hASCs) are engineered into different-sized multicellular spheroids and co-cultured with 3D coaxially and hexagonally patterned human umbilical vein endothelial cells (HUVECs) in a liver lobule-like manner to enhance their hepatic differentiation efficiency. It is found that small-sized hASC spheroids, with a diameter of ≈50 µm, show superior pro-angiogenic effects and hepatic differentiation compared to the other counterparts. The size-dependent functional enhancements are mediated by the Wnt signaling pathway. Meanwhile, co-culture of hASCs with HUVECs, at a HUVECs/hASCs seeding density ratio of 2:1, distinctly promotes hepatic differentiation and vascularization both in vitro and in vivo, especially when endothelial cells are patterned into hollow hexagons. After subcutaneous implantation, the mini-liver, consisting of HLC spheroids and 3D-printed interconnected vasculatures, can effectively improve liver regeneration in two ALF animal models through amelioration of local oxidative stress and inflammation, reduction of liver necrosis, as well as increase of cell proliferation, thereby showing great promise for clinical translation.

Co-Culturing Rat Dorsal Root Ganglion Neurons With Rat Schwann Cells Protects Them Against the Cytotoxic Effects of Silver and Gold Nanoparticles.

Previous studies using monotypic nerve cell cultures have shown that nanoparticles induced neurotoxic effects on nerve cells. Interactions between neurons and Schwann cells may protect against the neurotoxicity of nanoparticles. In this study, we developed a co-culture model consisting of immortalized rat dorsal root ganglion (DRG) neurons and rat Schwann cells and employed it to investigate our hypothesis that co-culturing DRG neurons with Schwann cells imparts protection on them against neurotoxicity induced by silver or gold nanoparticles. Our results indicated that neurons survived better in co-cultures when they were exposed to these nanoparticles at the higher concentrations compared to when they were exposed to these nanoparticles at the same concentrations in monotypic cultures. Synapsin I expression was increased in DRG neurons when they were co-cultured with Schwann cells and treated with or without nanoparticles. Glial fibrillary acidic protein (GFAP) expression was increased in Schwann cells when they were co-cultured with DRG neurons and treated with nanoparticles. Furthermore, we found co-culturing with Schwann cells stimulated neurofilament polymerization in DRG neurons and produced the morphological differentiation. Silver nanoparticles induced morphological disorganization in monotypic cultures. However, there were more cells displaying normal morphology in co-cultures than in monotypic cultures. All of these results suggested that co-culturing DRG neurons with Schwann cells imparted some protection on them against neurotoxicity induced by silver or gold nanoparticles, and altering the expression of neurofilament-L, synapsin I, and GFAP could account for the phenomenon of protection in co-cultures.

Impact of Co-Culture on the Metabolism of Marine Microorganisms.

Natural products from plants have been listed for hundreds of years as a source of biologically active molecules. In recent years, the marine environment has demonstrated its ability to provide new structural entities. More than 70% of our planet's surface is covered by oceans, and with the technical advances in diving and remotely operated vehicles, it is becoming easier to collect samples. Although the risk of rediscovery is significant, the discovery of silent gene clusters and innovative analytical techniques has renewed interest in natural product research. Different strategies have been proposed to activate these silent genes, including co-culture, or mixed fermentation, a cultivation-based approach. This review highlights the potential of co-culture of marine microorganisms to induce the production of new metabolites as well as to increase the yields of respective target metabolites with pharmacological potential, and moreover to indirectly improve the biological activity of a crude extract.

Differential Expression Profiling of microRNAs in Human Placenta-Derived Mesenchymal Stem Cells Cocultured with Grooved Porous Hydroxyapatite Scaffolds.

Scaffold materials used for bone defect repair are often limited by osteogenic efficacy. Moreover, microRNAs (miRNAs) are involved in regulating the expression of osteogenic-related genes. In previous studies, we verified the enhancement of osteogenesis using a grooved porous hydroxyapatite scaffold (HAG). In the present study, we analyzed the contribution of HAG to the osteogenic differentiation of human placenta-derived mesenchymal stem cells (hPMSCs) from the perspective of miRNA differential expression. Furthermore, results showed that miRNAs were differentially expressed in the osteogenic differentiation of hPMSCs cocultured with HAG. In detail, 16 miRNAs were significantly upregulated and 29 miRNAs were downregulated with HAG. In addition, bioinformatics analyses showed that the differentially expressed miRNAs were enriched in a variety of biological processes, including signal transduction, cell metabolism, cell junctions, cell development and differentiation, and that they were associated with osteogenic differentiation through axon guidance, mitogen-activated protein kinase, and the transforming growth factor beta signaling pathway. Furthermore, multiple potential target genes of these miRNAs were closely related to osteogenic differentiation. Importantly, overexpression of miR-146a-5p (an upregulated miRNA) promoted the osteogenic differentiation of hPMSCs, and miR-145-5p overexpression (a downregulated miRNA) inhibited the osteogenic differentiation of hPMSCs.

Synthetic biology-powered microbial co-culture strategy and application of bacterial cellulose-based composite materials.

Bacterial cellulose (BC) is a carbohydrate polymer that is widely produced by diverse bacteria with unique structural features and properties. BC has a high water-retention capacity, appealing mechanical properties, and higher purity compared to plant cellulose. Due to these attractive properties, BC has been exploited in many applications, including biomedical treatment, textiles, architecture, and environmental engineering. Recently, taking advantage of synthetic biology concepts and techniques, the integration of genetically engineered microorganisms and BC produces composite materials with intriguing characteristics, for example, responsiveness to stimuli and capability to regenerate. These synthetic biology-inspired BC-based composite materials greatly extend the scopes of applications of BC. In this review, we discuss the construction and application of BC-based composite materials with an emphasis on those produced by genetically engineered microorganisms and by microbial co-culture approaches. We highlight the advantages of the synthetic biology-inspired fabrication strategy of BC-based composites and consider the challenges that need to be addressed by future work.

Effects of Cell Density and Microenvironment on Stem Cell Mitochondria Transfer among Human Adipose-Derived Stem Cells and HEK293 Tumorigenic Cells.

Stem cells (SC) are largely known for their potential to restore damaged tissue through various known mechanisms. Among these mechanisms is their ability to transfer healthy mitochondria to injured cells to rescue them. This mitochondrial transfer plays a critical role in the healing process. To determine the optimal parameters for inducing mitochondrial transfer between cells, we assessed mitochondrial transfer as a function of seeding density and in two-dimensional (2D) and semi three-dimensional (2.5D) culture models. Since mitochondrial transfer can occur through direct contact or secretion, the 2.5D culture model utilizes collagen to provide cells with a more physiologically relevant extracellular matrix and offers a more realistic representation of cell attachment and movement. Results demonstrate the dependence of mitochondrial transfer on cell density and the distance between donor and recipient cell. Furthermore, the differences found between the transfer of mitochondria in 2D and 2.5D microenvironments suggest an optimal mode of mitochondria transport. Using these parameters, we explored the effects on mitochondrial transfer between SCs and tumorigenic cells. HEK293 (HEK) is an immortalized cell line derived from human embryonic kidney cells which grow rapidly and form tumors in culture. Consequently, HEKs have been deemed tumorigenic and are widely used in cancer research. We observed mitochondrial transfer from SCs to HEK cells at significantly higher transfer rates when compared to a SC-SC co-culture system. Interestingly, our results also revealed an increase in the migratory ability of HEK cells when cultured with SCs. As more researchers find co-localization of stem cells and tumors in the human body, these results could be used to better understand their biological relationship and lead to enhanced therapeutic applications.

Pro- and Anti-Inflammatory Cytokines in the Context of NK Cell-Trophoblast Interactions.

During pregnancy, uterine NK cells interact with trophoblast cells. In addition to contact interactions, uterine NK cells are influenced by cytokines, which are secreted by the cells of the decidua microenvironment. Cytokines can affect the phenotypic characteristics of NK cells and change their functional activity. An imbalance of pro- and anti-inflammatory signals can lead to the development of reproductive pathology. The aim of this study was to assess the effects of cytokines on NK cells in the presence of trophoblast cells in an in vitro model. We used TNFα, IFNγ, TGFß and IL-10; the NK-92 cell line; and peripheral blood NK cells (pNKs) from healthy, non-pregnant women. For trophoblast cells, the JEG-3 cell line was used. In the monoculture of NK-92 cells, TNFα caused a decrease in CD56 expression. In the coculture of NK cells with JEG-3 cells, TNFα increased the expression of NKG2C and NKG2A by NK-92 cells. Under the influence of TGFß, the expression of CD56 increased and the expression of NKp30 decreased in the monoculture. After the preliminary cultivation of NK-92 cells in the presence of TGFß, their cytotoxicity increased. In the case of adding TGFß to the PBMC culture, as well as coculturing PBMCs and JEG-3 cells, the expression of CD56 and NKp44 by pNK cells was reduced. The differences in the effects of TGFß in the model using NK-92 cells and pNK cells may be associated with the possible influence of monocytes or other lymphoid cells from the mononuclear fraction.

[3D Tumor organoid models produced by cellular capsules technology CCT]. / Production d'organoïdes tumoraux 3D par la technologie des capsules cellulaires TCC.

Monolayer cultures of cell lines and derived-patient cells have long been the in vitro model of choice in oncology. In particular, these models have made it possible to decipher the mechanisms that determine tumor proliferation and invasion. However these 2D models are insufficient because they do not take into account the spatial organization of cells and their interactions with each other or with the extracellular matrix. In the context of cancer, there is a need to develop new 3D (tumoroid) models in order to gain a better understanding of the development of these pathologies but also to assess the penetration of drugs through a tissue and the associated cellular response. We present here the cell capsule technology (CCT), which allows the production of different tumoroid models: simple or more complex 3D culture models including co-culture of tumor cells with components of the microenvironment (fibroblasts, matrix, etc.). The development of these new 3D culture systems now makes it possible to propose refined physiopathological models that will allow the implementation of improved targeted therapeutic strategies.

Effect of passage number of genetically modified TGF-ß3 expressing primary chondrocytes on the chondrogenesis of ATDC5 cells in a 3D coculture system.

Due to the lack of blood vessels, nerves and lymphatics, articular cartilage is difficult to repair once damaged. Tissue engineering is considered to be a potential strategy for cartilage regeneration. Successful tissue engineering strategies depend on the effective combination of biomaterials, seed cells and biological factors. In our previous study, a genetically modified coculture system with chondrocytes and ATDC5 cells in an alginate hydrogel has exhibited a superior ability to enhance chondrogenesis. In this study, we further evaluated the influence of chondrocytes at various passages on chondrogenesis in the coculture system. The results demonstrated that transfection efficiency was hardly influenced by the passage number of chondrocytes. The coculture system with passage 5 (P5) chondrocytes had a better effect on chondrogenesis of ATDC5 cells, while chondrocytes in this coculture system presented higher levels of dedifferentiation than other groups with P1 or P3 chondrocytes. Therefore, P5 chondrocytes were shown to be more suitable for the coculture system, as they accumulated in sufficient cell numbers with more passages and had a higher level of dedifferentiation, which was prone to form a favorable niche for chondrogenesis of ATDC5 cells. This study may provide fresh insights for future cartilage tissue engineering strategies with a combination of a coculture system and advanced biomaterials.

Co-culture of mesenchymal stem cell spheres with hematopoietic stem cells under hypoxia: a cost-effective method to maintain self-renewal and homing marker expression.

BACKGROUND: Hematopoietic stem cell (HSC) transplantation is considered a possible treatment option capable of curing various diseases. The aim of this study was the co-culturing of mesenchymal stem cell (MSC) spheres with HSCs under hypoxic condition to enhance the proliferation, self-renewal, stemness, and homing capacities of HSCs. METHODS AND RESULTS: HSCs were expanded after being subjected to different conditions including cytokines without feeder (Cyto), co-culturing with adherent MSCs (MSC), co-culturing with adherent MSCs + hypoxia (MSC + Hyp), co-culturing with MSCs spheres (Sph-MSC), co-culturing with MSCs spheres + hypoxia (Sph-MSC + Hyp), co-culturing with MSC spheres + cytokines (Sph-MSC + Cyto). After 10 days, total nucleated cell (TNC) and CD34+/CD38- cell counts, colony-forming unit assay (CFU), long-term culture initiating cell (LTC-IC), the expression of endothelial protein C receptor (EPCR), nucleostemin (NS), nuclear factor I/X (Nfix) CXCR4, and VLA-4 were evaluated. The TNC, CD34+/CD38- cell count, CFU, and LTC-IC were higher in the Sph-MSC + Hyp and Sph-MSC + Cyto groups as compared with those of the MSC + Hyp group (P < 0.001). The expanded HSCs co-cultured with MSC spheres in combination with hypoxia expressed more EPCR, CXCR4, VLA-4, NS, and Nfix mRNA. The protein expression was also more up-regulated in the Sph-MSC + Cyto and Sph-MSC + Hyp groups. CONCLUSION: Co-culturing HSCs with MSC spheres under hypoxic condition not only leads to higher cellular yield but also increases the expression of self-renewal and homing genes. Therefore, we suggest this approach as a simple and non-expensive strategy that might improve the transplantation efficiency of HSCs.

Vascularization of iNSC spheroid in a 3D spheroid-on-a-chip platform enhances neural maturation.

In vitro platforms for studying the human brain have been developed, and brain organoids derived from stem cells have been studied. However, current organoid models lack three-dimensional (3D) vascular networks, limiting organoid proliferation, differentiation, and apoptosis. In this study, we created a 3D model of vascularized spheroid cells using an injection-molded microfluidic chip. We cocultured spheroids derived from induced neural stem cells (iNSCs) with perfusable blood vessels. Gene expression analysis and immunostaining revealed that the vascular network greatly enhanced spheroid differentiation and reduced apoptosis. This platform can be used to further study the functional and structural interactions between blood vessels and neural spheroids, and ultimately to simulate brain development and disease.

Skeletal muscle-adipose cocultured tissue fabricated using cell-laden microfibers and a hydrogel sheet.

The emerging interest in skeletal muscle tissue originates from its unique properties that control body movements. In particular, recent research advances in engineered skeletal muscle tissue have broadened the possibilities of applications in nonclinical models. However, due to the lack of adipose tissue, current engineered skeletal muscle tissue has the limitation of satisfying in vivo-like position and proportion of intermuscular fat. Adipose tissue within the skeletal muscle affects their functional properties. Here, a fabrication method for cocultured tissue composed of skeletal muscle and adipose tissues is proposed to reproduce the functional and morphological characteristics of muscle. By implementing prematured adipose microfibers in a myoblast-laden hydrogel sheet, both the accumulation of large lipid droplets and control of the position of adipose tissue within the skeletal muscle tissue becomes feasible. The findings of this study provide helpful information regarding engineered skeletal muscle, which has strong potential in drug screening models.

Stem cells from human exfoliated deciduous teeth (SHED) have mitochondrial transfer ability in stromal-derived inducing activity (SDIA) co-culture system.

Stem cells from human exfoliated deciduous teeth (SHED) have stromal-derived inducing activity (SDIA): which means these stromal cells induce neural differentiation where they are used as a substratum for embryonic stem cell (ESCs) culture. Recent studies show that mitochondria or mitochondrial products, as paracrine factors, can be released and transferred from one cell to another. With this information, we were curious to know whether in the SDIA co-culture system, SHED release or donate their mitochondria to ESCs. For this purpose, before co-culture, SHED s' mitochondria and ESCs s' cell membranes were separately labeled with specific fluorescent probes. After co-culture, SHED s' mitochondria were tracked by fluorescent microscope and flow cytometry analysis. Co-culture also performed in the presence of inhibitors that block probable transfer pathways suchlike tunneling nanotubes, gap junctions or vesicles. Results showed that mitochondrial transfer takes place from SHED to ESCs. This transfer partly occurs by tunneling nanotubes and not through gap junctions or vesicles; also was not dependent on intracellular calcium level. This kind of horizontal gene transfer may open a new prospect for further research on probable role of mitochondria on fate choice and neural induction processes.