Mesenchymal Stem Cell Spheroids Research Articles

In Situ Formation of Proangiogenic Mesenchymal Stem Cell Spheroids in Hyaluronic Acid/Alginate Core/Shell Microcapsules

Mesenchymal-stem-cell (MSC)-based therapies have been recognized as promising strategies for the treatment of various injuries or diseases due to their unique characteristics, such as self-renewal, differentiation potential, and secretion of various bioactive molecules. However, MSC transplantation often results in low efficacy, including a cell viability loss and a low therapeutic activity. Alternatively, MSC spheroids have been studied to improve the viability and therapeutic activity of MSCs. Also, microencapsulation of cells can protect and retain the cells from harsh environments after transplantation. Here, MSC spheroids were formed in hyaluronic acid/alginate (HA@Alg) core-shell microcapsules and employed for neovascularization. A well-defined core-shell structure of HA@Alg microcapsules was produced by optimizing various electrospraying conditions. MSC spheroids could be spontaneously formed in the HA core of the microcapsules after 1 day of incubation. An enhanced secretion of various growth factors was found from MSC spheroids in HA@Alg. In vivo plug assay revealed the significant promotion of angiogenesis by MSC spheroids in HA@Alg compared to that by the controls (i.e., MSCs and MSC spheroids), which is likely due to the better retention of MSC spheroid forms in the microcapsules. Thus, the HA@Alg microcapsules embedding MSC spheroids will be greatly beneficial for various stem cell-based therapies.

Scalable and cost-effective generation of osteogenic micro-tissues through the incorporation of inorganic microparticles within mesenchymal stem cell spheroids

Mesenchymal stem cells (MSCs) are considered primary candidates for treating complex bone defects in cell-based therapy and tissue engineering. Compared with monolayer cultures, spheroid cultures of MSCs (mesenspheres) are favorable due to their increased potential for differentiation, extracellular matrix (ECM) synthesis, paracrine activity, and in vivo engraftment. Here, we present a strategy for the incorporation of microparticles for the fabrication of osteogenic micro-tissues from mesenspheres in a cost-effective and scalable manner. A facile method was developed to synthesize mineral microparticles with cell-sized spherical shape, biphasic calcium phosphate composition (hydroxyapatite and β-tricalcium phosphate), and a microporous structure. Calcium phosphate microparticles (CMPs) were incorporated within the mesenspheres through mixing with the single cells during cell aggregation. Interestingly, the osteogenic genes were upregulated significantly (collagen type 1 (Col 1) 30-fold, osteopontin (OPN) 10-fold, and osteocalcin (OCN) 3-fold) after 14 days of culture with the incorporated CMPs, while no significant upregulation was observed with the incorporation of gelatin microparticles. The porous structure of the CMPs was exploited for loading and sustained release of an angiogenic small molecule. Dimethyloxaloylglycine (DMOG) was loaded efficiently onto the CMPs (loading efficiency: 65.32 ± 6%) and showed a sustained release profile over 12 days. Upon incorporation of the DMOG-loaded CMPs (DCMPs) within the mesenspheres, a similar osteogenic differentiation and an upregulation in angiogenic genes (VEGF 5-fold and kinase insert domain (KDR) 2-fold) were observed after 14 days of culture. These trends were also observed in immunostaining analysis. To evaluate scalable production of the osteogenic micro-tissues, the incorporation of microparticles was performed during cell aggregation in a spinner flask. The DCMPs were efficiently incorporated and directed the mesenspheres toward osteogenesis and angiogenesis. Finally, the DCMP mesenspheres were loaded within a three-dimensional printed cell trapper and transplanted into a critical-sized defect in a rat model. Computed tomography and histological analysis showed significant bone formation with blood vessel reconstruction after 8 weeks in this group. Taken together, we provide a scalable and cost-effective approach for fabrication of osteogenic micro-tissues, as building blocks of macro-tissues, that can address the large amounts of cells required for cell-based therapies.

Microfluidic Approach to Generate a Tadpole-Egg-Shaped Alginate Fiber and Its Application in Tissue Engineering.

Herein, we introduce a facile microfluidic technique to produce a hybrid alginate fiber with a tadpole-egg shape. A triple-flow polydimethylsiloxane microfluidic device was constructed to allow the formation of oil droplets inside the alginate stream and was instantaneously gelated with the coaxially adjacent CaCl2. The fiber entrapping the uniform oil droplets was dehydrated, leading to the formation of a distinct tadpole-egg-shaped structure. A series of diverse fiber architectures was fabricated in a controlled manner based on the flow rates of the relevant flows. The tadpole-egg-shaped alginate fibers were employed as building blocks to create a three-dimensional microwell template for cell cultures. First, the tadpole-egg-shaped alginate fibers containing the oil droplets were half-dipped into a melted agarose solution. After the solidification of the agarose gel, the alginate fibers were degraded by an ethylenediaminetetraacetic acid (EDTA) solution to generate the hemispherical microwells. Mesenchymal stem cells (MSCs) were cultured in the microwells to generate spheroids, which were induced into chondrocytes using transforming growth factor-β3. The formed MSC spheroids exhibited a relatively high ratio of cell viability with more than 95% live cells after 14 days of culture. The success of the chondrogenic differentiation was proven based on staining (Safranin O) and the glycosaminoglycan levels. The latter was significantly higher in spheroids that were induced to form chondrocytes compared to those that were not induced after 21 days of differentiation. Second, we investigated the potential of the tadpole-egg-shaped alginate fibers as microcarriers for applications in drug delivery and implantable technologies. It was revealed that the degradation of the Ca-alginate wall of the hybrid fibers to release the oil droplets required an EDTA solution with a concentration of 500 mM for a 15 min period. This result can be used to further develop the tadpole-egg-shaped alginate fibers as uniform microcarriers with multiple compartments.

The Role of the BMP Pathway in Sustaining CML Stem Cells in the Bone Marrow Niche

Despite improved patient outcome using tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) patients require life-long treatment due to leukemic stem cell (LSC) persistence. LSCs reside together with mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) in the bone marrow (BM) niche, which they modify to their advantage whilst impairing normal hematopoiesis. To date it has proved difficult to understand how LSCs both dominate and alter the niche, and to effectively target LSCs with current therapies. Recent studies have shown intrinsic and extrinsic deregulation of the bone morphogenetic protein (BMP) pathway in CML. These cells show altered stem cell fate, persistence, and response to BMP receptor (BMPR) antagonists, which affect cell behavior including cell cycle, apoptosis, and expansion (Laperrousaz et al. 2013; Grockowiak et al. 2017; Zylbersztejn et al. 2018; Toofan et al. 2018). We compared TKIs alone and in combination with a BMPR inhibitor to gain improved insights into BCR-ABL1 dependent and independent regulatory mechanisms within the niche environment using this therapeutic approach. K562 CML cell line and CML CD34+ primary cells were used in this study in combination with HS5 stromal cell co-culture. CML cells were treated with single or combination treatments of imatinib, the dual SRC-ABL1 TKI saracatinib, and the BMPR inhibitor dorsomorphin for 4h or 72h with and without BMP4 stimulation/co-culture. HSC and LSC interactions were also investigated using our artificial 3D BM niche model which comprises of magnetically levitated MSC spheroids embedded in medical-grade collagen type I, mimicking the BM biological and mechanical microenvironment, along with a high throughput microfluidic MSC spheroid formation system for drug testing. Kinase screens were performed on CML CD34+ cells using a chip-based microarray assay (PamGene) following 4h drug treatments. Phosphorylation data were then utilized for upstream kinase and pathway analysis using the metacore platform. This analysis facilitated identification of proteins showing a change in expression of ≥0.5-fold across all (n=3) patient samples, which was validated by qPCR and immunoblotting. Fluidigm multiplex qPCR was utilized to assess changes in expression of early response, self-renewal and differentiation genes. Flow cytometry was performed to investigate apoptosis, cell cycle progression and proliferation, alongside colony assays of primary CD34+CP-CML samples (n=3) following treatment. We demonstrated a synergistic mode of action upon inhibition of the BMP pathway in combination with TKI treatment, resulting in increased apoptosis (p< 0.001), altered cell cycle (G2-M, p< 0.01), fewer cell divisions, and a reduction in CD34+cells. Primary patient samples displayed differential gene expression in relation to response for genes involved in cell cycle (CDKN1A, CDKN2B, RB1; p< 0.05), self-renewal (PBX1; p< 0.01) and cell survival (GATA1, CKIT, p< 0.05). Kinase prediction analysis identified kinases involved in cell growth, development, differentiation, apoptosis, and cell-cell adhesion in treated sample lysates, with consequent pathway analysis highlighting transcription factors, ETS1, TP53 and C-MYC as main regulators across all common pathways. QPCR identified significant changes in expression of ETS1 (p< 0.001) and C-MYC (p< 0.01) following treatment. Within the top 10 deregulated pathways based on PamGene TK profiling we also identified GAB1 and GAB2 as common effectors, known to play an important role in growth and differentiation of myeloid cells, and which were demonstrated by immunoblotting to be considerably downregulated following 72h single and dual treatments in K562. Co-culture of CD34+ cells on stroma was chemo-protective, however dual treatment was still able to elicit strong anti-proliferative effects. We are currently investigating these promising findings in more detail using our 3D niche model and microfluidic spheroid platform. Taken together, these results provide vital insights into the mechanisms by which CML cells respond to current treatments, which is critical for improving therapeutic approaches and avoiding patient resistance or relapse. A combinatorial approach targeting the BMP pathway with BMP antagonists or small molecule inhibitors together with second-generation TKIs could open up new therapeutic possibilities. Disclosures No relevant conflicts of interest to declare.

3D Printed Cartilage-Like Tissue Constructs with Spatially Controlled Mechanical Properties.

Developing biomimetic cartilaginous tissues that support locomotion while maintaining chondrogenic behavior is a major challenge in the tissue engineering field. Specifically, while locomotive forces demand tissues with strong mechanical properties, chondrogenesis requires a soft microenvironment. To address this challenge, 3D cartilage-like tissue is bioprinted using two biomaterials with different mechanical properties: a hard biomaterial to reflect the macromechanical properties of native cartilage, and a soft biomaterial to create a chondrogenic microenvironment. To this end, a hard biomaterial (MPa order compressive modulus) composed of an interpenetrating polymer network (IPN) of polyethylene glycol (PEG) and alginate hydrogel is developed as an extracellular matrix (ECM) with self-healing properties, but low diffusive capacity. Within this bath supplemented with thrombin, fibrinogen containing human mesenchymal stem cell (hMSC) spheroids is bioprinted forming fibrin, as the soft biomaterial (kPa order compressive modulus) to simulate cartilage's pericellular matrix and allow a fast diffusion of nutrients. The bioprinted hMSC spheroids improve viability and chondrogenic-like behavior without adversely affecting the macromechanical properties of the tissue. Therefore, the ability to print locally soft and cell stimulating microenvironments inside of a mechanically robust hydrogel is demonstrated, thereby uncoupling the micro- and macromechanical properties of the 3D printed tissues such as cartilage.

Intraportally delivered stem cell spheroids localize in the liver and protect hepatocytes against GalN/LPS-induced fulminant hepatic toxicity

BackgroundSystemic inflammatory response syndrome (SIRS) is common in severe fulminant hepatic failure (FHF) and has a high mortality rate (20–50%) due to irreversible cerebral edema or sepsis. Stem cell-based treatment has emerged as a promising alternative therapeutic strategy to prolong the survival of patients suffering from FHF via the inhibition of SIRS due to their immunomodulatory effects.Methods3D spheroids of adipose-derived mesenchymal stem cells (3D-ADSC) were prepared by the hanging drop method. The efficacy of the 3D-ADSC to rescue FHF was evaluated in a d-galactosamine/lipopolysaccharide (GalN/LPS)-induced mouse model of FHF via intraportal transplantation of the spheroids.ResultsIntraportally delivered 3D-ADSC better engrafted and localized into the damaged livers compared to 2D-cultured adipose-derived mesenchymal stem cells (2D-ADSC). Transplantation of 3D-ADSC rescued 50% of mice from FHF-induced lethality, whereas only 20% of mice survived when 2D-ADSC were transplanted. The improved transplantation outcomes correlated with the enhanced immunomodulatory effect of 3D-ADSC in the liver microenvironment.ConclusionThe study shows that the transplantation of optimized 3D-ADSC can efficiently ameliorate GalN/LPS-induced FHF due to improved viability, resistance to exogenous ROS, and enhanced immunomodulatory effects of 3D-ADSC.

Serum-Free Culture System for Spontaneous Human Mesenchymal Stem Cell Spheroid Formation.

Human mesenchymal stem cells (hMSCs) are widely used in clinical research because of their multipotential, immunomodulatory, and reparative properties. Previous studies determined that hMSC spheroids from a three-dimensional (3D) culture possess higher therapeutic efficacy than conventional hMSCs from a monolayer (2D) culture. To date, various 3D culture methods have been developed to form hMSC spheroids but most of them used culture medium containing fetal bovine serum (FBS), which is not suitable for further clinical use. Here, we demonstrate that dissociated single MSCs seeded in induced pluripotent stem medium (MiPS) adhere loosely to the dish and spontaneously migrate to form spheroids during day 3 to day 6. Through component deletion screening and complementation experiments, the knockout serum replacement (KSR) was identified as necessary and sufficient for hMSC spheroid formation. Transcriptome analysis showed that the overall expression profiles were highly similar between 2D culture with FBS and KSR-derived spheroids. Interestingly, genes related to inflammatory response, immune response, and angiogenesis were upregulated in spheroids at day 6 and qPCR results further validated the increased expression level of related genes, including STC1, CCL7, HGF, IL24, and TGFB3. When spheroids were replated in normal FBS medium, cells formed a typical spindle-shaped morphology and FACS results showed that the recovered cells retained MSC-specific surface markers, such as CD73, CD90, and CD105. In summary, we developed a practical and convenient method to generate hMSC spheroids for clinical research and therapy.

Harnessing Stem Cells and Neurotrophic Factors with Novel Technologies in the Treatment of Parkinson's Disease.

Parkinson's disease (PD) is characterized by loss of dopaminergic neurons, oxidative stress and neuroinflammation. The classical therapeutic approach with L-DOPA is not able to control motor symptoms in the long term, thus new disease-modifying or neuroprotective treatments are urgently required in order to match such yet unmet clinical needs. Success in cell-based therapy has been accomplished at a clinical level with human fetal mesencephalic tissue, but ethical issues and a shortage of organs clearly underline the need for novel sources of dopaminergic neurons. Mesenchymal stem cells (MSCs) can be obtained from different adult and fetal tissues that are normally discarded as waste, including adipose tissue, placenta, umbilical cord, and dental tissues. Their neuroregenerative, anti-inflammatory and immunomodulatory properties are mainly mediated by the secretion of an array of bioactive molecules and are heightened when MSCs form tri-dimensional structures called spheroids. Not only can MSCs spontaneously produce neurotrophic factors (NFs) but they can be engineered to synthetize and secrete them in vivo. The aim of this review is to provide a picture of results gained with MSC secretome and spheroids in PD, as well as the possibility of harnessing MSC-based therapy with the use of nano- and micro-structured materials for NF delivery.

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Efficacy of 3D Culture Priming is Maintained in Human Mesenchymal Stem Cells after Extensive Expansion of the Cells.

The use of non-optimal preparations of mesenchymal stem cells (MSCs), such as extensively expanded cells, might be necessary to obtain the large numbers of cells needed for many clinical applications. We previously demonstrated that minimally expanded (early passage) MSCs can be pre-activated as spheroids to produce potentially therapeutic factors in 3D cultures. Here, we used extensively expanded (late passage) MSCs and studied their 3D-culture activation potential. MSCs were culture-expanded as 2D monolayers, and cells from various passages were activated by 3D culture in hanging drops with either fetal bovine serum (FBS)-containing media or a more clinically-applicable animal product-free (xeno-free) media. Gene expression analyses demonstrated that MSC spheroids prepared from passage 3, 5, and 7 cells were similar to each other but different from 2D MSCs. Furthermore, the expression of notable anti-inflammatory/immune-modulatory factors cyclooxygenase-2 (PTGS2), TNF alpha induced protein 6 (TNFAIP6), and stanniocalcin 1 (STC-1) were up-regulated in all spheroid preparations. This was confirmed by the detection of secreted prostaglandin E2 (PGE-2), tumor necrosis factor-stimulated gene 6 (TSG-6, and STC-1. This study demonstrated that extensively expanded MSCs can be activated in 3D culture through spheroid formation in both FBS-containing and xeno-free media. This work highlights the possibility of activating otherwise less useable MSC preparations through 3D culture generating large numbers of potentially therapeutic MSCs.

Characterization of Placental Mesenchymal Stem Cells Spheroids after Generation, Hypothermic and Subnormothermic Storage

Background. Placental mesenchymal stem cells application is a promising area of cell therapy for number diseases. Their using in the form of spheroids results in enhancement of the therapeutic potential, improve the cells survival after transplantation. Medical purposes require the characterization and development of spheroids storage technology. Objective. The aim of the paper is to characterize placental mesenchymal stem cells spheroids after generation and low-temperature storage. Methods . Spheroids were produced by a hanging drop method from placental mesenchymal stem cells, chara­cterized after production and after subnormothermic (20 °C) and hypothermic (4 °C) storage for up to 96 h. The structure, metabolic activity of spheroids according to the data of the MTT test and the resazurin restoration test were studied. Results . The hanging drop method makes it possible to produce from 1×10 6 placental mesenchymal stem cells about 825.3 ± 56.2 spheroids in the size of 50–150 microns and 62.3 ± 5.1 spheroids of 150–300 microns in size, having a spherical shape, not stained with trypan blue, stained with neutral red and fluorescein diacetate. The spheroids adhere to the plastic and destroying through short-time centrifugation. During subnormothermic storage spheroids, structural characteristics and metabolic activity change after 48 h, during hypothermic – after 24 h of storage. Conclusions. The hanging drop method makes it possible to produce about 900 spheroids from 1 million placental MSCs with stable metabolic activity. For medical purposes, subornorthomeric (20 °C) storage not more than 24 h is recommended for placental MSC spheroid. Longer storage leads to a sharp decreasing of their metabolic activity and destroying to the separated cells.

Synthesis and secretome release by human bone marrow mesenchymal stem cell spheroids within three-dimensional collagen hydrogels: Integrating experiments and modelling.

Myocardial infarction results in loss of cardiac cell types, inflammation, extracellular matrix (ECM) degradation, and fibrotic scar. Transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) is being explored as they could differentiate into cardiomyocyte-like cells, integrate into host tissue, and enhance resident cell activity. The ability of these cells to restore lost ECM, remodel the inflammatory scar tissue, and repair the injured myocardium remains unexplored. We here elucidated the synthesis and deposition of ECM (e.g., elastin, sulfated glycosaminoglycans, hyaluronan, collagen type III, laminin, fibrillin, lysyl oxidase, and nitric oxide synthases), matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), and other secretome (cytokines, chemokines, and growth factors) in adult human BM-MSC spheroid cultures within three-dimensional collagen gels. The roles of species-specific type I collagen and 5-azacytadine were assessed over a 28-day period. Results revealed that human collagen (but not rat-derived) suppressed MSC proliferation and survival, and MSCs synthesized and released a variety of ECM proteins and secretome over the 28 days. Matrix deposition is at least an order of magnitude lower than their release levels at every time point, most possibly due to elevated MMP levels and interleukins with a concomitant decrease in TIMPs. Matrix synthesis over the 28-day period was fitted to a competitive inhibition form of Michaelis-Menten kinetics, and the production and decay rates of ECM, MMPs, and TIMPs, along with the kinetic model parameters quantified. Such an integrated experimental and modelling approach would help elucidate the critical roles of various parameters (e.g., cell encapsulation and delivery vehicles) in stem cell-based transplantation therapies.

Continuous Mechanical Indexing of Single-Cell Spheroids Using a Robot-Integrated Microfluidic Chip

In single-cell/spheroid analyses, flow cytometry plays a key role in high-throughput measurements. Among the considerable numbers of indexes available for a target evaluation, mechanical characteristics such as a Young's modulus have been focused upon as new indexes related to physiological condition. However, a continuous mechanical indexing system has yet to be achieved due to the difficulty in flow control, which brings about a fluctuation of the force sensor probe. In this letter, we propose an automated mechanical indexing system of spheroids. By utilizing a combination of two syringe pumps, we succeeded in positioning the target spheroids without an undesirable pinching. We conducted experiments on the continuous mechanical indexing of single-cell spheroids using 26 mesenchymal stem cell spheroids. The evaluation results of positioning showed that the error in the position control was 14.1 $\mu$ m with a standard deviation of 87.7 $\mu$ m, which corresponded to a measurement error of the Young's modulus of under 2%. In addition, we succeeded in achieving a measurement throughput of 3.14 spheroids per minute, which is approximately 2 times faster than a manual operation. Finally, we demonstrated the on-chip sorting of single spheroids based on the mechanical index to show the adaptability of the proposed method toward the flow cytometry.

Dynamic Mechanics-Modulated Hydrogels to Regulate the Differentiation of Stem-Cell Spheroids in Soft Microniches and Modeling of the Nonlinear Behavior.

Although mechanisms of how physical forces convert into biochemical signals are increasingly understood, it is still unknown how soft cues guide cell behavior. Herein, it is shown that the commitment and differentiation of encapsulating human mesenchymal stem cell (hMSC) spheroids in thermosensitive 3D hydrogels are simply altered by interpenetrating poly(N-isopropylacrylamide-co-2-hydroxyethyl methacrylate) (NIPAM-HEMA) nanogel to a gelatin methacryloyl (GelMA) network. This cell-laden hydrogel provides dynamic mechanics with covalent crosslinking coordinated reversible physical networks, which can regulate hMSCs in situ by reversibly stiffening soft niches via multicyclic temperature changes from 25 to 37 °C. The spreading of hMSC spheroids in the hydrogel is strongly dependent on myosin-dependent traction stress with dynamic mechanical stimuli through focal adhesion kinase (FAK) signaling. Notably, the dynamic microenvironment gradually influences the expression and distribution from the basal to apical side of nuclear lamin A/C and increases the Yes-associated protein (YAP) nuclear localization with cycles, which ultimately favors hMSCs undergoing osteogenesis (but not adipogenesis) in the soft microniche. Moreover, it is demonstrated that the viscoelastic behavior of the soft microniche can be guided by temperature through a nonlinear model. These findings highlight the central roles of the dynamic relationship between the biomechanical signals and mechanosensitive transcriptional regulators in cellular mechanosensing.

GENERATION OF THREE-DIMENSIONAL SPHEROIDS FROM MOUSE MESENCHYMAL STEM CELL

In comparison with two-dimensional (2D) culture, three-dimensional (3D) cultures of mesenchymal stem cells (MSCs) in spheroids (3D spheroids) offer a more physiologically realistic condition. Accordingly, 3D culture has been used to improve the immunomodulatory properties of MSCs and their survival after transplantation. Several methods have been developed to generate 3D spheroids of MSCs to date; however, these methods are time-consuming or expensive, thus limiting their wide application. In this study, we compared two inexpensive and simple culture methods for the generation of 3D spheroids from the MSCs of mouse compact bone: pellet culture and rotating vessels using low-binding 96-well plates. The effectiveness of the two methods was determined by the number of obtained spheroids, their diameter, and the expression of the MSC marker CD44 using morphometric and immunofluorescent methods. A higher number of homogeneous 3D spheroids was obtained from the MSCs of compact bone using a rotating vessel compared to that obtained with pellet culture. The immunofluorescence assay further showed that the developed 3D spheroids highly expressed CD44 on the cell surface. Thus, we suggest that the method of rotating vessels is effective for generating 3D spheroids from the MSCs of compact bone. This method can be used to promote research on the biology of stem cells and develop new therapeutic approaches in cell therapy and regenerative medicine.

PF466 POTENTIAL COMBINATIONAL THERAPY OF LEUKEMIC STEM CELLS USING BONE MORPHOGENETIC PROTEIN AND TYROSINE KINASE INHIBITORS

Background:Chronic myeloid leukemia (CML) disease outcomes have improved since the introduction of tyrosine kinase inhibitors (TKIs), however a proportion of patients will have TKI resistance and TKIs are not curative in the majority of patients. Low‐levels of Bcr‐Abl transcripts persist and a major clinical aim is to understand mechanism underlying disease persistence and the role of leukemic stem cells (LSCs) in this. To date it has proved difficult to both understand how LSCs dominate and alter the niche, and to target LSCs with current therapies. Previous studies have highlighted the importance of the bone morphogenetic protein (BMP) pathway in CML LSCs with altered stem cell fate, persistence and the response to BMP antagonists affecting cell behavior with altered cell cycle, apoptosis and expansion (Laperrousaz, B. et al, Blood, 2013; Grockowiak, E. et al, Blood, 2017).Aims:To investigate the effect of BMP pathway inhibition in combination with TKIs in primary CML samples to improve insights and identify new potential therapeutic approaches.Methods:Primary CML CD34+cells were treated with Imatinib (IM) or Saracatinib (SC), a Src/Abl dual inhibitor, alone or in combination with BMP inhibitor Dorsomorphin (DOR). Additionally, cell cultures were supplemented with soluble BMP4 or co‐cultured with the human stromal cell line HS‐5. Apoptosis, cell cycle and proliferation were investigated by flow cytometry. RNA was harvested, cDNA generated and pre‐amplified prior to the performance of multiplex Real‐Time PCR (Fluidigm) on 89 lineage‐specific, cell cycle, proliferation, and survival genes. Furthermore, we investigated tyrosine kinase activity in cell lysates of primary CML patient samples post single or dual treatment of SC and DOR using a chip‐based microarray assay from PamGene containing 196 peptides. Kinase phosphorylation activities measured on peptides were further evaluated by relating them to kinases that are able to phosphorylate a particular site on the peptide sequence.Results:Combinatorial treatments targeting the BMP pathway together with TKIs display a synergistic mode of action. Increases in late apoptosis (p < 0.0002), altered cell cycle (G2‐M, p = 0.0058), fewer cell divisions, and reduced numbers of CD34+cells were observed in dual treatments. BMP4 supplementation revealed similar differences whereas stroma co‐cultures displayed a protective effect. Furthermore, multiplex quantitative PCR has revealed significant deregulation of genes involved in cell cycle,apoptosis and differentiation, such as CDKN1a, CDKN2b, BCL6, GATA1 and SMAD5 showing differential expression in relation to response. Upstream kinase analysis using the PamGene platform, revealed kinases involved in cell growth, development, differentiation, apoptosis and cell‐cell adhesion. Amongst the top 20 potential differentially expressed kinases after SC treatment, were the expected members of the Src family kinases. DOR treatment revealed an up regulation of the largest known subfamily of receptor tyrosine kinases, the Ephrin receptors and Met, a single pass tyrosine kinase receptor that had already been identified as a potential therapy target in CML for IM resistant cells.Summary/Conclusion:Our results highlight the potential of a combined approach of targeting the BMP pathway with BMP antagonists or small inhibitors together with TKIs, which could open up new therapeutic possibilities. Future experiments will assess the potential of combinatorial treatment in a 3D bone marrow model, comprising magnetically levitated Mesenchymal Stem Cell spheroids embedded in medical‐grade collagen type I.

Engineered Mesenchymal Stem Cell/Nanomedicine Spheroid as an Active Drug Delivery Platform for Combinational Glioblastoma Therapy.

Mesenchymal stem cell (MSC) has been increasingly applied to cancer therapy because of its tumor-tropic capability. However, short retention at target tissue and limited payload option hinder the progress of MSC-based cancer therapy. Herein, we proposed a hybrid spheroid/nanomedicine system, comprising MSC spheroid entrapping drug-loaded nanocomposite, to address these limitations. Spheroid formulation enhanced MSC's tumor tropism and facilitated loading of different types of therapeutic payloads. This system acted as an active drug delivery platform seeking and specifically targeting glioblastoma cells. It enabled effective delivery of combinational protein and chemotherapeutic drugs by engineered MSC and nanocomposite, respectively. In an in vivo migration model, the hybrid spheroid showed higher nanocomposite retention in the tumor tissue compared with the single MSC approach, leading to enhanced tumor inhibition in a heterotopic glioblastoma murine model. Taken together, this system integrates the merits of cell- and nanoparticle- mediated drug delivery with the tumor-homing characteristics of MSC to advance targeted combinational cancer therapy.

Comparison of Immunosuppressive and Angiogenic Properties of Human Amnion-Derived Mesenchymal Stem Cells between 2D and 3D Culture Systems.

The secretion of potential therapeutic factors by mesenchymal stem cells (MSCs) has aroused much interest given the benefits that it can bring in the field of regenerative medicine. Indeed, the in vitro multipotency of these cells and the secretive capacity of both angiogenic and immunomodulatory factors suggest a role in tissue repair and regeneration. However, during culture, MSCs rapidly lose the expression of key transcription factors associated with multipotency and self-renewal, as well as the ability to produce functional paracrine factors. In our study, we show that a three-dimensional (3D) culture method is effective to induce MSC spheroid formation, to maintain the multipotency and to improve the paracrine activity of a specific population of human amnion-derived MSCs (hAMSCs). The regenerative potential of both 3D culture-derived conditioned medium (3D CM) and their exosomes (EXO) was assessed against 2D culture products. In particular, tubulogenesis assays revealed increased capillary maturation in the presence of 3D CM compared with both 2D CM and 2D EXO. Furthermore, 3D CM had a greater effect on inhibition of PBMC proliferation than both 2D CM and 2D EXO. To support this data, hAMSC spheroids kept in our 3D culture system remained viable and multipotent and secreted considerable amounts of both angiogenic and immunosuppressive factors, which were detected at lower levels in 2D cultures. This work reveals the placenta as an important source of MSCs that can be used for eventual clinical applications as cell-free therapies.

Cellular Spheroids of Mesenchymal Stem Cells and Their Perspectives in Future Healthcare

Intrinsic cellular properties of several types of cells are dramatically altered as the culture condition shifts from two-dimensional (2D) to three-dimensional (3D) environment. Currently, several lines of evidence have demonstrated the therapeutic potential of mesenchymal stem cells (MSCs) in regenerative medicine. MSCs not only replenish the lost cells, they also promote the regeneration of impaired tissues by modulating the immune responses. Following the development of 3D cell culture, the enhanced therapeutic efficacy of spheroid-forming MSCs have been identified in several animal disease models by promoting differentiation or trophic factor secretion, as compared to planar-cultured MSCs. Due to the complicated and multifunctional applications in the medical field, MSCs are recently named as medicinal signaling cells. In this review, we summarize the predominant differences of cell–environment interactions for the MSC spheroids formed by chitosan-based substrates and other scaffold-free approaches. Furthermore, several important physical and chemical factors affecting cell behaviors in the cell spheroids are discussed. Currently, the understanding of MSCs spheroid interactions is continuously expanding. Overall, this article aims to review the broad advantages and perspectives of MSC spheroids in regenerative medicine and in future healthcare.

3D-cultured human placenta-derived mesenchymal stem cell spheroids enhance ovary function by inducing folliculogenesis

Placenta-derived mesenchymal stem cells (PD-MSCs) have numerous advantages over other adult MSCs that make them an attractive cell source for regenerative medicine. Here, we demonstrate the therapeutic effect of PD-MSCs in ovariectomized (Ovx) rats and compare their efficacy when generated via a conventional monolayer culture system (2D, naïve) and a spheroid culture system (3D, spheroid). PD-MSC transplantation significantly increased the estradiol level in Ovx rats compared with the non-transplantation (NTx) group. In particular, the estradiol level in the Spheroid group was significantly higher than that in the Naïve group at 2 weeks. Spheroid PD-MSCs exhibited a significantly higher efficiency of engraftment onto ovarian tissues at 2 weeks. The mRNA and protein expression levels of Nanos3, Nobox, and Lhx8 were also significantly increased in the Spheroid group compared with those in the NTx group at 1 and 2 weeks. These results suggest that PD-MSC transplantation can restore ovarian function in Ovx rats by increasing estrogen production and enhancing folliculogenesis-related gene expression levels and further indicate that spheroid-cultured PD-MSCs have enhanced therapeutic potential via increased engraftment efficiency. These findings improve our understanding of stem-cell-based therapies for reproductive systems and may suggest new avenues for developing efficient therapies using 3D cultivation systems.