Adherent Stem Cells Research Articles

Catalysis-mediated dynamic ligand presentation regulates mechanosensing–metabolism coupling of stem cells

The dynamic presentation of ligands on biomaterial surfaces can greatly affect cellular behaviors. However, there have been no prior studies that utilize catalysis-mediated dynamic bonds for dynamic ligand presentation and explore its impact on cellular events. In this work we employ aniline as a catalyst to enhance the dynamics of the hydrazone bond in the tether structure of ligands attached to biomaterial substrate to study the impact of such controlled dynamic ligand presentation on the mechanosensing–metabolism coupling of adherent stem cells. Gold nanoparticles, decorated with polyethylene glycol (PEG)-hydrazide, are linked to benzaldehyde-modified glass substrates through hydrazone bonds. The catalytic aniline, combined with the cell adhesive arginyl-glycyl-aspartic acid (RGD) peptide, is conjugated on the nanoparticle surface to facilitate the disassociation and reassociation of hydrazone bonds in the RGD tether structure. This catalysis-mediated dynamic presentation of RGD effectively regulates the Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent mechanosensing and stimulates the actin cytoskeleton rearrangement in stem cells. The enhanced release of a glycolytic enzyme (Aldolase A) due to the cytoskeleton rearrangement leads to increased glycolysis, adenosine triphosphate (ATP) generation, oxidative phosphorylation, thereby contributing to the osteogenic differentiation of human mesenchymal stem cells (hMSCs). These findings highlight the importance of chemically manipulating the dynamic ligand presentation in regulating mechanosensing–metabolism coupling and the differentiation of stem cells.

Spatial heterogeneity analysis of seeding of human induced pluripotent stem cells for neuroectodermal differentiation

IntroductionPreparing a uniform cell population in high–density seeding of adherent human induced pluripotent stem cells (hiPSC) requires stable culture conditions and consistent culture operation. In this study, we evaluated cell distribution patterns by changing cell seeding operations and their impact on differentiation toward the neuroectodermal lineage. MethodsThe hiPSC line 201B7 was seeded at 1.23 × 105 cells/cm2 following a conventional operation, prolongated time of cell seeding suspension or vessel tilting during cell seeding operation. Fluorescent imaging of cell nuclei was performed 24 h following cell seeding and used for spatial heterogeneity analysis. Flow cytometric analysis was also performed seven days after cell differentiation induction toward neuroectodermal lineage. ResultsIndices for spatial heterogeneity following high–density cell seeding were proposed to assess cell distribution patterns. Global heterogeneity (HG) was shown to be mostly affected by vessel tilting during cell seeding operation, while local heterogeneity (HL) was affected by prolongated time of cell seeding suspension. Changes in both spatial heterogeneities in the hiPSC population resulted in a lower yield of target neuroectodermal cells compared with the control operation. ConclusionHigh–density hiPSC seeding is critical for achieving a higher yield of target cells of neuroectodermal lineage. Understanding the spatial heterogeneity in early stages detects errors in cell culture motion and predicts cell fate in later stages of cell culture.

Evaluation of nanoscale versus hybrid micro/nano surface topographies for endosseous implants

We examined the effect of a nanoscale titanium surface topography (D) versus two hybrid micro/nanoscale topographies (B and OS) on adherent mesenchymal stem cells (MSCs) and bone marrow derived macrophages (BMMs) function in cell culture and in vivo. In the in vitro study, compared to OS and B surfaces, D surface induced earlier and greater cell spreading, and earlier and profound mRNA expression of RUNX2, Osterix and BMP2 in MSCs. D surface induced earlier and higher expression of RUNX2 and BMP2 and lower expression of inflammatory genes in implant adherent cells in vivo. Measurement of osteogenesis at implant surfaces showed greater bone-to-implant contact at D versus OS surfaces after 21 days. We explored the cell population on the D and OS implant surfaces 24 h after placement using single-cell RNA sequencing and identified distinct cell clusters including macrophages, neutrophils and B cells. D surface induced lower expression and earlier reduction of inflammatory genes expression in BMMs in vitro. BMMs on D, B and OS surfaces demonstrated a marked increase of BMP2 expression after 1 and 3 days, and this increase was significantly higher on D surface at day 3. Our data implicates a dynamic process that may be influenced by nanotopography at multiple stages of osseointegration including initial immunomodulation, recruitment of MSCs and later osteoblastic differentiation leading to bone matrix production and mineralization. The results suggest that a nanoscale topography (D) favorably modulates adherent macrophage polarization toward anti-inflammatory and regenerative phenotypes and promotes the osteoinductive phenotype of adherent mesenchymal stem cells. Statement of significanceOur manuscript contains original data developed to define effects of a novel nanotopography on the process of osseointegration at the cell and tissue level. Few studies have compared the effects of a nanoscale surface versus the more typical hybrid micro/nano-scale surfaces used today. We have utilized single-cell RNA sequencing for the first time to identify earliest cell populations on implant surfaces in vivo. We provide data indicating that the nanoscale surface acts upon both osteoprogenitor and immune cell (macrophages) to alter the process of bone formation in a surface-specific manner. This work represents new observations regarding osseointegration and immunomodulation.

SAT272 Insulin Signaling In Hyperactivation Of The HPA Axis In Metabolic Diseases

Abstract Disclosure: D. Cozma: None. P. Siatra: None. I. Oikonomakos: None. D. Kalra: None. U.A. Friedrich: None. A. Dahl: None. S.R. Bornstein: None. S. Zeissig: None. C.L. Andoniadou: None. C. Steenblock: None. Stress can lead to an increase in body fat and obesity as main clinical conditions preceding the metabolic syndrome [1]. In metabolic stress, hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis has been observed, resulting in increased steroidogenesis and alteration of cortisol secretion [1]. This increased sensitivity of the HPA axis may be related to the development of comorbidities or severe illness in patients with metabolic diseases and vice versa. Disorders of the HPA axis, e.g., Cushing’s disease, resulting from overproduction of ACTH, share similar clinical symptoms with metabolic diseases [1]. The molecular signaling mechanisms that cause such changes in the HPA axis under metabolic stress remain elusive. Previous studies indicate a role of insulin in the regulation of adrenal steroidogenesis by upregulating the transcriptional activity of NR5A1 [2]. Here, we study the molecular mechanisms to insulin-induced hyperactivation of the HPA axis and characterize the molecular changes that lead to altered cortisol secretion. We generated non-adherent spheroids from primary adrenocortical cells in vitro and treated these with insulin. This resulted in an increase of spheroid diameter over time [Slope: 5.163 ± 0.994; P=0.0139; n=11], compared to control [Slope: -4.058 ± 0.987; P=0.0261; n=10], indicating greater cell expansion following insulin treatment. Similarly, repeating this experiment with adherent primary anterior pituitary stem cell colonies showed an increase in colony size when treated with insulin compared to control [Diameter: 1221.0 ± 290.9 µm vs. 914.5 ± 256.2 µm; P=0.0079; n=3]. This indicates that the stem cell populations of the adrenal and pituitary gland are responsive to insulin stimulation, likely leading to increased proliferation. This is complementary to our previous studies where we revealed increased secretion of aldosterone in insulin-stimulated adrenocortical cultures [3]. To identify signaling pathways related to insulin stimulation, we performed bulk RNA sequencing of in vitro primary cultures of adrenocortical and pituitary cells with and without insulin treatment. Analysis of these data sets reveals specific signaling pathways that are candidates in regulation of metabolic stress in vivo. Using mouse models of obesity and diabetes along with genetic lineage-tracing studies, we aim to decipher the role of these pathways on HPA axis stem/progenitor cells during metabolic stress. The outcomes will pave the way for a better understanding of HPA axis activity during metabolic stress adaption, and the role of the HPA axis in patients suffering from metabolic diseases.

Measuring Glutathione Regeneration Capacity in Stem Cells.

Glutathione (GSH) is a chief cellular antioxidant, affecting stem cell functions. The cellular GSH level is dynamically altered by the redox buffering system and transcription factors, including NRF2. Additionally, GSH is differentially regulated in each organelle. We previously reported a protocol for monitoring the real-time GSH levels in live stem cells using the reversible GSH sensor FreSHtracer. However, GSH-based stem cell analysis needs be comprehensive and organelle-specific. Hence, in this study, we demonstrate a detailed protocol to measure the GSH regeneration capacity (GRC) in living stem cells by measuring the intensities of the FreSHtracer and the mitochondrial GSH sensor MitoFreSHtracer using a high-content screening confocal microscope. This protocol typically analyses the GRC in approximately 4 h following the seeding of the cells onto plates. This protocol is simple and quantitative. With some minor modifications, it can be employed flexibly to measure the GRC for the whole-cell area or just the mitochondria in all adherent mammalian stem cells.

Metallurgical manipulation of surface Volta potential in bimetals and cell response of human mesenchymal stem cells

Bioelectricity plays an overriding role in directing cell migration, proliferation, differentiation etc. Tailoring the electro-extracellular environment through metallurgical manipulation could modulate the surrounding cell behaviors. In this study, different electric potential patterns, in terms of Volta potential distribution and gradient, were created on the metallic surface as an electric microenvironment, and their effects on adherent human mesenchymal stem cells were investigated. Periodically and randomly distributed Volta potential pattern, respectively, were generated on the surface through spark plasma sintering of two alternatively stacked dissimilar metals films and of a mixture of metallic powders. Actin cytoskeleton staining demonstrated that the Volta potential pattern strongly affected cell attachment and deformation. The cytoskeletons of cells were observed to elongate along the Volta potential gradient and across the border of adjacent regions with higher and lower potentials. Moreover, the steepest potential gradient resulting from the drastic compositional changes on the periodic borders gave rise to the strongest osteogenic tendency among all the samples. This study suggests that tailoring the Volta potential distribution and gradient of metallic biomaterials via metallurgical manipulation is a promising approach to activate surrounding cells, providing an extra degree of freedom for designing desirable bone-repairing metallic implants.

Novel Scaffold Based on Chitosan Hydrogels/Phthalated Cashew Gum for Supporting Human Dental Pulp Stem Cells.

Hydrogels are structures that have value for application in the area of tissue engineering because they mimic the extracellular matrix. Naturally obtained polysaccharides, such as chitosan (CH) and cashew gum, are materials with the ability to form polymeric networks due to their physicochemical properties. This research aimed to develop a scaffold based on chitosan and phthalated cashew tree gum and test it as a support for the growth of human mesenchymal stem cells. In this study, phthalation in cashew gum (PCG) was performed by using a solvent-free route. PCG-CH scaffold was developed by polyelectrolyte complexation, and its ability to support adherent stem cell growth was evaluated. The scaffold showed a high swelling rate. The pore sizes of the scaffold were analyzed by scanning electron microscopy. Human dental pulp stem cells (hDPSCs) were isolated, expanded, and characterized for their potential to differentiate into mesenchymal lineages and for their immunophenotypic profile. Isolated mesenchymal stem cells presented fibroblastoid morphology, plastic adhesion capacity, and differentiation in osteogenic, adipogenic, and chondrogenic lineages. Mesenchymal stem cells were cultured in scaffolds to assess cell adhesion and growth. The cells seeded on the scaffold showed typical morphology, attachment, and adequate distribution inside the matrix pores. Thus, cells seeded in the scaffold may improve the osteoinductive and osteoconductive properties of these biomaterials.

Co-Surfactant-Free Bioactive Protein Nanosheets for the Stabilization of Bioemulsions Enabling Adherent Cell Expansion.

Bioemulsions are attractive platforms for the scalable expansion of adherent cells and stem cells. In these systems, cell adhesion is enabled by the assembly of protein nanosheets that display high interfacial shear moduli and elasticity. However, to date, most successful systems reported to support cell adhesion at liquid substrates have been based on coassemblies of protein and reactive cosurfactants, which limit the translation of bioemulsions. In this report, we describe the design of protein nanosheets based on two globular proteins, bovine serum albumin (BSA) and β-lactoglobulin (BLG), biofunctionalized with RGDSP peptides to enable cell adhesion. The interfacial mechanics of BSA and BLG assemblies at fluorinated liquid-water interfaces is studied by interfacial shear rheology, with and without cosurfactant acyl chloride. Conformational changes associated with globular protein assembly are studied by circular dichroism and protein densities at fluorinated interfaces are evaluated via surface plasmon resonance. Biofunctionalization mediated by sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) is studied by fluorescence microscopy. On the basis of the relatively high elasticities observed in the case of BLG nanosheets, even in the absence of cosurfactant, the adhesion and proliferation of mesenchymal stem cells and human embryonic kidney (HEK) cells on bioemulsions stabilized by RGD-functionalized protein nanosheets is studied. To account for the high cell spreading and proliferation observed at these interfaces, despite initial moderate interfacial elasticities, the deposition of fibronectin fibers at the surface of corresponding microdroplets is characterized by immunostaining and confocal microscopy. These results demonstrate the feasibility of achieving high cell proliferation on bioemulsions with protein nanosheets assembled without cosurfactants and establish strategies for rational design of scaffolding proteins enabling the stabilization of interfaces with strong shear mechanics and elasticity, as well as bioactive and cell adhesive properties. Such protein nanosheets and bioemulsions are proposed to enable the development of new generations of bioreactors for the scale up of cell manufacturing.

Very Small Embryonic Like Stem Cells: A Review of Basic Science, Applications,and Potential Use in Orthopedics.

Many cell types have been studied and applied in cell therapy and RM. In orthopedics, Mesenchymal Stem Cells (MSC) are used to regenerate and repair bone, cartilage, and tendons. Identified in 1955, [1,2]. MSCs were first isolated from rat BM by Frieden stein in 1970 and characterized as non-hematopoietic, multipotent, plastic adherent, and fibroblastic-like stem cells [3].

Needle to needle robot-assisted manufacture of cell therapy products.

Advanced therapeutic medicinal products (ATMPs) have emerged as novel therapies for untreatable diseases, generating the need for large volumes of high‐quality, clinically‐compliant GMP cells to replace costly, high‐risk and limited scale manual expansion processes. We present the design of a fully automated, robot‐assisted platform incorporating the use of multiliter stirred tank bioreactors for scalable production of adherent human stem cells. The design addresses a needle‐to‐needle closed process incorporating automated bone marrow collection, cell isolation, expansion, and collection into cryovials for patient delivery. AUTOSTEM, a modular, adaptable, fully closed system ensures no direct operator interaction with biological material; all commands are performed through a graphic interface. Seeding of source material, process monitoring, feeding, sampling, harvesting and cryopreservation are automated within the closed platform, comprising two clean room levels enabling both open and closed processes. A bioprocess based on human MSCs expanded on microcarriers was used for proof of concept. Utilizing equivalent culture parameters, the AUTOSTEM robot‐assisted platform successfully performed cell expansion at the liter scale, generating results comparable to manual production, while maintaining cell quality postprocessing.

Can ECIS Biosensor Technology Be Used to Measure the Cellular Responses of Glioblastoma Stem Cells?

Glioblastoma is considered the most aggressive and lethal form of brain cancer. Glioblastoma tumours are complex, comprising a spectrum of oncogenically transformed cells displaying distinct phenotypes. These can be generated in culture and are called differentiated-glioblastoma cells and glioblastoma stem cells. These cells are phenotypically and functionally distinct, where the stem-like glioblastoma cells give rise to and perpetuate the tumour. Electric cell-substrate impedance sensing (ECIS) is a real-time, label-free, impedance-based method for the analysis of cellular behaviour, based on cellular adhesion. Therefore, we asked the question of whether ECIS was suitable for, and capable of measuring the adhesion of glioblastoma cells. The goal was to identify whether ECIS was capable of measuring glioblastoma cell adhesion, with a particular focus on the glioblastoma stem cells. We reveal that ECIS reliably measures adhesion of the differentiated glioblastoma cells on various array types. We also demonstrate the ability of ECIS to measure the migratory behaviour of differentiated glioblastoma cells onto ECIS electrodes post-ablation. Although the glioblastoma stem cells are adherent, ECIS is substantially less capable at reliably measuring their adhesion, compared with the differentiated counterparts. This means that ECIS has applicability for some glioblastoma cultures but much less utility for weakly adherent stem cell counterparts.

Droplet-based vitrification of adherent human induced pluripotent stem cells on alginate microcarrier influenced by adhesion time and matrix elasticity

The gold standard in cryopreservation is still conventional slow freezing of single cells or small aggregates in suspension, although major cell loss and limitation to non-specialised cell types in stem cell technology are known drawbacks. The requirement for rapidly available therapeutic and diagnostic cell types is increasing constantly. In the case of human induced pluripotent stem cells (hiPSCs) or their derivates, more sophisticated cryopreservation protocols are needed to address this demand. These should allow a preservation in their physiological, adherent state, an efficient re-cultivation and upscaling upon thawing towards high-throughput applications in cell therapies or disease modelling in drug discovery. Here, we present a novel vitrification-based method for adherent hiPSCs, designed for automated handling by microfluidic approaches and with ready-to-use potential e.g. in suspension-based bioreactors after thawing. Modifiable alginate microcarriers serve as a growth surface for adherent hiPSCs that were cultured in a suspension-based bioreactor and subsequently cryopreserved via droplet-based vitrification in comparison to conventional slow freezing. Soft (0.35%) versus stiff (0.65%) alginate microcarriers in concert with adhesion time variation have been examined. Findings revealed specific optimal conditions leading to an adhesion time and growth surface (matrix) elasticity dependent hypothesis on cryo-induced damaging regimes for adherent cell types. Deviations from the found optimum parameters give rise to membrane ruptures assessed via SEM and major cell loss after adherent vitrification. Applying the optimal conditions, droplet-based vitrification was superior to conventional slow freezing. A decreased microcarrier stiffness was found to outperform stiffer material regarding cell recovery, whereas the stemness characteristics of rewarmed hiPSCs were preserved.

Long term expansion profile of mesenchymal stromal cells at protein nanosheet-stabilised bioemulsions for next generation cell culture microcarriers.

Tremendous progress in the identification, isolation and expansion of stem cells has allowed their application in regenerative medicine and tissue engineering, and their use as advanced in vitro models. As a result, stem cell manufacturing increasingly requires scale up, parallelisation and automation. However, solid substrates currently used for the culture of adherent cells are poorly adapted for such applications, owing to their difficult processing from cell products, relatively high costs and their typical reliance on difficult to recycle plastics and microplastics. In this work, we show that bioemulsions formed of microdroplets stabilised by protein nanosheets displaying strong interfacial mechanics are well-suited for the scale up of adherent stem cells such as mesenchymal stromal cells (MSCs). We demonstrate that, over multiple passages (up to passage 10), MSCs retain comparable phenotypes when cultured on such bioemulsions, solid microcarriers (Synthemax II) and classic 2D tissue culture polystyrene. Phenotyping (cell proliferation, morphometry, flow cytometry and differentiation assays) of MSCs cultured for multiple passages on these systems indicate that, although stemness is lost at late passages when cultured on these different substrates, stem cell phenotypes remained comparable between different culture conditions, at any given passage. Hence our study validates the use of bioemulsions for the long term expansion of adherent stem cells and paves the way to the design of novel 3D bioreactors based on microdroplet microcarriers.

Photothermal release and recovery of mesenchymal stem cells from substrates functionalized with gold nanorods

Mesenchymal stem cell therapies show great promise in regenerative medicine. However, to generate clinically relevant numbers of these stem cells, significant in vitro expansion of the cells is required before transplantation into the affected wound or defect. The current gold standard protocol for recovering in vitro cultured cells involves treatment with enzymes such as trypsin which can affect the cell phenotype and ability to interact with the environment. Alternative enzyme free methods of adherent cell recovery have been investigated, but none match the convenience and performance of enzymatic detachment. In this work we have developed a synthetically simple, low cost cell culture substrate functionalized with gold nanorods that can support cell proliferation and detachment. When these nanorods are irradiated with biocompatible low intensity near infrared radiation (785 nm, 560 mWcm−2) they generate localized surface plasmon resonance induced nanoscale heating effects which trigger detachment of adherent mesenchymal stem cells. Through simulations and thermometry experiments we show that this localized heating is concentrated at the cell-nanorod interface, and that the stem cells detached using this technique show either similar or improved multipotency, viability and ability to differentiate into clinically desirable osteo and adipocytes, compared to enzymatically harvested cells. This proof-of-principle work shows that photothermally mediated cell detachment is a promising method for recovering mesenchymal stem cells from in vitro culture substrates, and paves the way for further studies to scale up this process and facilitate its clinical translation. Statement of significanceNew non-enzymatic methods of harvesting adherent cells without damaging or killing them are highly desirable in fields such as regenerative medicine. Here, we present a synthetically simple, non-toxic, infra-red induced method of harvesting mesenchymal stem cells from gold nanorod functionalized substrates. The detached cells retain their ability to differentiate into therapeutically valuable osteo and adipocytes. This work represents a significant improvement on similar cell harvesting studies due to: its simplicity; the use of clinically valuable stem cells as oppose to immortalized cell lines; and the extensive cellular characterization performed. Understanding, not just if cells live or die but how they proliferate and differentiate after photothermal detachment will be essential for the translation of this and similar techniques into commercial devices.

Large fuzzy biodegradable polyester microspheres with dopamine deposition enhance cell adhesion and bone regeneration in vivo

The biodegradable polymer microparticles with different surface morphology and chemical compositions may influence significantly the behaviors of cells, and thereby further the performance of tissue regeneration in vivo. In this study, multi-stage hierarchical textures of poly(D,L-lactic-co-glycolide) (PLGA)/PLGA-b-PEG (poly(ethylene glycol)) microspheres with a diameter as large as 50–100 μm are fabricated based on interfacial instability of an emulsion. The obtained fuzzy structures on the microspheres are sensitive to annealing, which are changed gradually to a smooth one after treatment at 37 °C for 6 d or 80 °C for 1 h. The surface microstructures that are chemically dominated by PEG can be stabilized against annealing by dopamine deposition. By the combination use of annealing and dopamine deposition, a series of microspheres with robust surface topologies are facilely prepared. The fuzzy microstructures and dopamine deposition show a synergetic role to enhance cell-material interaction, leading to a larger number of adherent bone marrow-derived mesenchymal stem cells (BMSCs), A549 and MC 3T3 cells. The fuzzy microspheres with dopamine deposition can significantly promote bone regeneration 12 w post surgery in vivo, as revealed by micro-CT, histological, western blotting and RT-PCR analyses.

Abstract P433: Multipotent Adult Progenitor Cells as a Highly Promising Therapy for Treatment of Intracerebral Hemorrhage

Background: Multipotent adult progenitor cells (MAPC) are an adherent adult stem cell being evaluated as a treatment for ischemic stroke in humans under the name MultiStem®. However, the efficacy of MAPC cells for the treatment of intracerebral hemorrhage (ICH), the most devastating form of stroke for which there is no effective treatment, is not clear Method: The therapeutic efficacy of MAPC administration was evaluated in both autologous blood injection (ABI) and collagenase (COL) rat ICH models. We treated rats intravenously with 1.2x10 6 cells (sub-optimal dose based on MAPC efficacy in ischemic stroke) and 1.2x10 7 cells (optimal dose) at either 2 or 24h after ICH, and used 2 different doses of collagenase to better understand the dose responses. Outcome measurements included 4 sensorimotor tests (up to 28d), ventricular hypertrophy, spleen size, and body weight (N=128 rats tested across 4 separate experiments). Results: MAPC offered a robust benefit in both ICH models in a dose-dependent fashion. (1) ABI model: at the sub-optimal dose MAPCs had no significant effect on behavioral performance, but effectively reduced ventricular hypertrophy. At an optimal dose, MAPCs at 2h or 24h after ICH, robustly reduced deficits in all 4 behavioral tests, and reduced ventricular hypertrophy by 59% and 35% in 2h and 24h post-treatment groups, respectively. No difference in body weight and spleen size was observed. (2) COL model: MAPC administered 2h after high collagenase dose, reduced hematoma volume (hemispheric hemoglobin level), as measured at 48h after collagenase injection. In addition, MAPC administration significantly reduced neurological deficit in the COL model. Conclusions: MAPC provide a uniquely robust therapeutic effect on clinically relevant neurological and morphological outcomes in two different ICH models. MAPC also reduced bleeding in the COL model, suggesting the potential for MAPC as a safe acute therapeutic treatment after ICH. In addition to having beneficial effects on recovery processes, MAPC could be further evaluated as a candidate to limit the hematoma enlargement during the initial postictal period. We are currently investigating the mechanism of MAPC-induced post-ICH recovery as well as hemostasis using tissue microarray analysis.

Design and development of a new ambr250\xae bioreactor vessel for improved cell and gene therapy applications

The emergence of cell and gene therapies has generated significant interest in their clinical and commercial potential. However, these therapies are prohibitively expensive to manufacture and can require extensive time for development due to our limited process knowledge and understanding. The automated ambr250® stirred-tank bioreactor platform provides an effective platform for high-throughput process development. However, the original dual pitched-blade 20 mm impeller and baffles proved sub-optimal for cell therapy candidates that require suspension of microcarriers (e.g. for the culture of adherent human mesenchymal stem cells) or other particles such as activating Dynabeads® (e.g. for the culture of human T-cells). We demonstrate the development of a new ambr250® stirred-tank bioreactor vessel which has been designed specifically to improve the suspension of microcarriers/beads and thereby improve the culture of such cellular systems. The new design is unbaffled and has a single, larger elephant ear impeller. We undertook a range of engineering and physical characterizations to determine which vessel and impeller configuration would be most suitable for suspension based on the minimum agitation speed (NJS) and associated specific power input (P/V)JS. A vessel (diameter, T, = 60 mm) without baffles and incorporating a single elephant ear impeller (diameter 30 mm and 45° pitch-blade angle) was selected as it had the lowest (P/V)JS and therefore potentially, based on Kolmogorov concepts, was the most flexible system. These experimentally-based conclusions were further validated firstly with computational fluid dynamic (CFD) simulations and secondly experimental studies involving the culture of both T-cells with Dynabeads® and hMSCs on microcarriers. The new ambr250® stirred-tank bioreactor successfully supported the culture of both cell types, with the T-cell culture demonstrating significant improvements compared to the original ambr250® and the hMSC-microcarrier culture gave significantly higher yields compared with spinner flask cultures. The new ambr250® bioreactor vessel design is an effective process development tool for cell and gene therapy candidates and potentially for autologous manufacture too.

Transcriptome Analysis Reveals High Similarities between Adult Human Cardiac Stem Cells and Neural Crest-Derived Stem Cells.

Simple SummaryThe regeneration of nearly all organs of the human body mainly depends on the functionality of adult stem cell populations that reside in their respective niches and can be activated upon injuries or other damages. These stem cell populations greatly differ in their expression profile of molecular markers, which greatly influences their potential use in regenerative medicine. Neural crest-derived stem cells are a prominent subpopulation of adult stem cells and are known for their high regenerative potential. Within this study, we compared two adult human stem cell populations, namely neural crest-derived inferior turbinate stem cells from the nasal cavity and human cardiac stem cells from the heart, using global gene expression profiling. Here, we found differences that correspond to the tissue sources of origin but also similarities in the expression of markers that are associated with the neural crest. Further classifying nasal stem cells and cardiac stem cells in a broader context, we identified clear similarities between both populations and other adherent stem cell populations compared to non-adherent progenitor cells of the blood system. The analyses provided here might help to understand the differences and similarities between different adult human stem cell populations.For the identification of a stem cell population, the comparison of transcriptome data enables the simultaneous analysis of tens of thousands of molecular markers and thus enables the precise distinction of even closely related populations. Here, we utilized global gene expression profiling to compare two adult human stem cell populations, namely neural crest-derived inferior turbinate stem cells (ITSCs) of the nasal cavity and human cardiac stem cells (hCSCs) from the heart auricle. We detected high similarities between the transcriptomes of both stem cell populations, particularly including a range of neural crest-associated genes. However, global gene expression likewise reflected differences between the stem cell populations with regard to their niches of origin. In a broader analysis, we further identified clear similarities between ITSCs, hCSCs and other adherent stem cell populations compared to non-adherent hematopoietic progenitor cells. In summary, our observations reveal high similarities between adult human cardiac stem cells and neural crest-derived stem cells from the nasal cavity, which include a shared relation to the neural crest. The analyses provided here may help to understand underlying molecular regulators determining differences between adult human stem cell populations.

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes.

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates the isolated patient's blood mononuclear cells (e.g., lymphocytes and monocytes) through an apheresis machine, co-cultures the patient's blood mononuclear cells with adherent cord blood-derived stem cells (CB-SC) in the SCE device, and then returns these "educated" immune cells to the patient's blood. Exosomes are nano-sized extracellular vesicles between 30‒150 nm existing in all biofluid and cell culture media. To further explore molecular mechanisms underlying SCE therapy and determine the actions of exosomes released from CB-SC, we investigate which cells phagocytize these exosomes during the treatment with CB-SC. By co-culturing Dio-labeled CB-SC-derived exosomes with human peripheral blood mononuclear cells (PBMC), we found that CB-SC-derived exosomes were predominantly taken up by human CD14-positive monocytes, leading to the differentiation of monocytes into type 2 macrophages (M2), with spindle-like morphology and expression of M2-associated surface molecular markers. Here, we present a protocol for the isolation and characterization of CB-SC-derived exosomes and the protocol for the co-culture of CB-SC-derived exosomes with human monocytes and the monitoring of M2 differentiation.

Cell quality evaluation with gene expression analysis of spheroids (3D) and adherent (2D) adipose stem cells.

Adipose stem cells (ASCs) represent a reliable source of stem cells with a widely demonstrated potential in regenerative medicine and tissue engineering applications. New recent insights suggest that three-dimensional (3D) models may closely mimic the native tissue properties; spheroids from adipose derived stem cells (SASCs) exhibit enhanced regenerative abilities compared with those of 2D models. Stem cell therapy success is determined by "cell-quality"; for this reason, the involvement of stress signals and cellular aging need to be further investigated. Here, we performed a comparative analysis of genes connected with stemness, aging, telomeric length and oxidative stress, in 3D and 2D primary cultures. The expression levels of stemness-related markers and anti-aging Sirtuin1 were significantly up-regulated (P<0.001) in SASCs-3D while gene expression of aging-related p16INK4a was increased in ASCs-2D (P<0.001). The 3D and 2D cultures also had a different gene expression profile for genes related to telomere maintenance (Shelterin complex, RNA Binding proteins and DNA repair genes) (P<0.01 and P<0.001) and oxidative stress (aldehyde dehydrogenase class1 and 3) (P<0.05, P<0.01 and P<0.001) and presented a striking large variation in their cellular redox state. Based on our findings, we propose a "cell quality" model of SASCs, highlighting a precise molecular expression of several genes involved with stemness (SOX2, POU5F1 and NANOG), anti-aging (SIRT1), oxidative stress (ALDH3) and telomeres maintenance.