The objective of this work is to investigate the effect of devitalized human mesenchymal stem cells (hMSCs) and endothelial colony‐forming cells (ECFCs) seeded on mineralized nanofiber microsheets on protein release, osteogenesis, vasculogenesis, and macrophage polarization. Calcium phosphate nanocrystals are grown on the surface of aligned, functionalized nanofiber microsheets. The microsheets are seeded with hMSCs, ECFCs, or a mixture of hMSCs + ECFCs, cultured for cell attachment, differentiated to the osteogenic or vasculogenic lineage, and devitalized by lyophilization. The release kinetic of total protein, bone morphogenetic protein‐2 (BMP2), and vascular endothelial growth factor (VEGF) from the devitalized microsheets is measured. Next, hMSCs and/or ECFCs are seeded on the devitalized cell microsheets and cultured in the absence of osteo‐/vasculoinductive factors to determine the effect of devitalized cell microsheets on hMSC/ECFC differentiation. Human macrophages are seeded on the microsheets to determine the effect of devitalized cells on macrophage polarization. Based on the results, devitalized undifferentiated hMSC and vasculogenic‐differentiated ECFC microsheets have highest sustained release of BMP2 and VEGF, respectively. The devitalized hMSC microsheets do not affect M2 macrophage polarization while vascular‐differentiated, devitalized ECFC microsheets do not affect M1 polarization. Both groups stimulate higher M2 macrophage polarization compared to M1.
The pathology of neurological disorders is associated with the loss of neuronal and glial cells that results in functional impairments. Human neural stem cells (hNSCs), due to their self‐renewing and multipotent characteristics, possess enormous tissue‐specific regenerative potential. However, the efficacy of clinical applications is restricted due to the lack of standardized in vitro cell production methods with the capability of generating hNSC populations with well‐defined cellular compositions. At any point, a population of hNSCs may include undifferentiated stem cells, intermediate and terminally differentiated progenies, and dead cells. Due to the plasticity of hNSCs, environmental cues play crucial roles in determining the cellular composition of hNSC cultures over time. Here, we investigated the independent and synergistic effect of three important environmental factors (i.e., culture dimensionality, oxygen concentration, and growth factors) on the survival, renewal potential, and differentiation of hNSCs. Our experimental design included two dimensional (2D) versus three dimensional (3D) cultures and normoxic (21% O2) versus hypoxic (3% O2) conditions in the presence and absence of epidermal growth factor (EGF) and fibroblast growth factor‐2 (FGF‐2). Additionally, we discuss the feasibility of mathematical models that predict hNSC growth and differentiation under these culture conditions by adopting a negative feedback regulatory term. Our results indicate that the synergistic effect of culture dimensionality and hypoxic oxygen concentration in the presence of growth factors enhances the proliferation of viable, undifferentiated hNSCs. Moreover, the same synergistic effect in the absence of growth factors promotes the differentiation of hNSCs. Biotechnol. Bioeng. 2017;114: 1096–1106. © 2016 Wiley Periodicals, Inc.
- NSF-PAR ID:
- 10037814
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology and Bioengineering
- Volume:
- 114
- Issue:
- 5
- ISSN:
- 0006-3592
- Page Range / eLocation ID:
- p. 1096-1106
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
-
more » « less
Abstract Human intestinal enteroids derived from adult stem cells offer a relevant ex vivo system to study biological processes of the human gut. They recreate cellular and functional features of the intestinal epithelium of the small intestine (enteroids) or colon (colonoids) albeit limited by the lack of associated cell types that help maintain tissue homeostasis and respond to external challenges. In the gut, innate immune cells interact with the epithelium, support barrier function, and deploy effector functions. We have established a co‐culture system of enteroid/colonoid monolayers and underlying macrophages and polymorphonuclear neutrophils to recapitulate the cellular framework of the human intestinal epithelial niche. Enteroids are generated from biopsies or resected tissue from any segment of the human gut and maintained in long‐term cultures as three‐dimensional structures through supplementation of stem cell growth factors. Immune cells are isolated from fresh human whole blood or frozen peripheral blood mononuclear cells (PBMC). Monocytes from PBMC are differentiated into macrophages by cytokine stimulation prior to co‐culture. The methods are divided into the two main components of the model: (1) generating enteroid/colonoid monolayers and isolating immune cells and (2) assembly of enteroid/colonoid‐immune cell co‐cultures with separate apical and basolateral compartments. Co‐cultures containing macrophages can be maintained for 48 hr while those involving neutrophils, due to their shorter life span, remain viable for 4 hr. Enteroid‐immune co‐cultures enable multiple outcome measures, including transepithelial resistance, production of cytokines/chemokines, phenotypic analysis of immune cells, tissue immunofluorescence imaging, protein or mRNA expression, antigen or microbe uptake, and other cellular functions. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : Seeding enteroid fragments onto Transwells for monolayer formationAlternate Protocol : Seeding enteroid fragments for monolayer formation using triturationBasic Protocol 2 : Isolation of monocytes and derivation of immune cells from human peripheral bloodBasic Protocol 3 : Isolation of neutrophils from human peripheral bloodBasic Protocol 4 : Assembly of enteroid/macrophage or enteroid/neutrophil co‐culture -
more » « less
Abstract Background Mesenchymal stem cells (MSCs) secrete a diversity of factors with broad therapeutic potential, yet current culture methods limit potency outcomes. In this study, we used topographical cues on polystyrene films to investigate their impact on the secretory profile and potency of bone marrow-derived MSCs (hBM-MSCs). hBM-MSCs from four donors were cultured on topographic substrates depicting defined roughness, curvature, grooves and various levels of wettability.
Methods The topographical PS-based array was developed using razor printing, polishing and plasma treatment methods. hBM-MSCs from four donors were purchased from RoosterBio and used in co-culture with peripheral blood mononuclear cells (PBMCs) from Cell Applications Inc. in an immunopotency assay to measure immunosuppressive capacity. Cells were cultured on low serum (2%) for 24–48 h prior to analysis. Image-based analysis was used for cell quantification and morphology assessment. Metabolic activity of BM-hMSCs was measured as the mitochondrial oxygen consumption rate using an extracellular flux analyzer. Conditioned media samples of BM-hMSCs were used to quantify secreted factors, and the data were analyzed using R statistics. Enriched bioprocesses were identify using the Gene Ontology tool
enrichGO from theclusterprofiler. One-way and two-way ANOVAs were carried out to identify significant changes between the conditions. Results were deemed statistically significant for combinedP < 0.05 for at least three independent experiments.Results Cell viability was not significantly affected in the topographical substrates, and cell elongation was enhanced at least twofold in microgrooves and surfaces with a low contact angle. Increased cell elongation correlated with a metabolic shift from oxidative phosphorylation to a glycolytic state which is indicative of a high-energy state. Differential protein expression and gene ontology analyses identified bioprocesses enriched across donors associated with immune modulation and tissue regeneration. The growth of peripheral blood mononuclear cells (PBMCs) was suppressed in hBM-MSCs co-cultures, confirming enhanced immunosuppressive potency. YAP/TAZ levels were found to be reduced on these topographies confirming a mechanosensing effect on cells and suggesting a potential role in the immunomodulatory function of hMSCs.
Conclusions This work demonstrates the potential of topographical cues as a culture strategy to improve the secretory capacity and enrich for an immunomodulatory phenotype in hBM-MSCs.
-
more » « less
Abstract Aim Numerous studies have shown that H2S serves as an acute oxygen sensor in a variety of cells. We hypothesize that H2S also serves in extended oxygen sensing.
Methods Here, we compare the effects of extended exposure (24‐48 hours) to varying O2tensions on H2S and polysulphide metabolism in human embryonic kidney (HEK 293), human adenocarcinomic alveolar basal epithelial (A549), human colon cancer (HTC116), bovine pulmonary artery smooth muscle, human umbilical‐derived mesenchymal stromal (stem) cells and porcine tracheal epithelium (PTE) using sulphur‐specific fluorophores and fluorometry or confocal microscopy.
Results All cells continuously produced H2S in 21% O2and H2S production was increased at lower O2tensions. Decreasing O2from 21% to 10%, 5% and 1% O2progressively increased H2S production in HEK293 cells and this was partially inhibited by a combination of inhibitors of H2S biosynthesis, aminooxyacetate, propargyl glycine and compound 3. Mitochondria appeared to be the source of much of this increase in HEK 293 cells. H2S production in all other cells and PTE increased when O2was lowered from 21% to 5% except for HTC116 cells where 1% O2was necessary to increase H2S, presumably reflecting the hypoxic environment in vivo. Polysulphides (H2Sn, where n = 2‐7), the key signalling metabolite of H2S also appeared to increase in many cells although this was often masked by high endogenous polysulphide concentrations.
Conclusion These results show that cellular H2S is increased during extended hypoxia and they suggest this is a continuously active O2‐sensing mechanism in a variety of cells.
-
more » « less
Abstract Controlled differentiation of mesenchymal stem cells (MSCs) into the chondrogenic lineage is crucial for in vitro generation of neocartilage, yet achieving it remains challenging. Traditional protocols for MSC differentiation using exogenous inductive molecules, such as transforming growth factor-β, fall short in meeting the needs of clinical applications because they yield differentiated cells that exhibit hypertrophic characteristics and subsequently facilitate endochondral bone formation. The objective of the current study was to deliver endogenous inductive factors from juvenile articular chondrocytes to bone marrow-derived MSCs to drive MSC chondrogenic differentiation through cocultivation of the two cell types in the absence of direct physical contact and exogenous stimulators. An initial chondrocyte/MSC ratio of 63:1 was identified as the appropriate proportion of the two cell populations to ensure that coculture-driven MSC-differentiated (CDMD) cells replicated the cellular morphology, behavior, and phenotype of articular chondrocytes. In a three-dimensional agarose system, CDMD cells were further shown to develop into robust neocartilage structurally and mechanically stronger than chondrocyte-laden constructs and with reduced hypertrophic potential. Although MSCs tended to lose the ability to express CD44, an important regulator in cartilage biology, during the coculture induction, CDMD cells regained this function in the three-dimensional tissue cultivation. The present work establishes a chondrocyte/MSC coculture model that serves as a template to better understand chondrocyte-driven MSC differentiation and provides insights for improved strategies to develop clinically relevant cartilage tissue replacements.
