Primary Human Mesenchymal Stromal Cells Research Articles

Characterization and biological evaluation of 3D printed composite ink consisting of collagen, hyaluronic acid and calcium phosphate for bone regeneration

In large bone defects the self-healing capacity is insufficient, and the current standard treatment, autologous bone grafting, has severe disadvantages such as limited availability and donor site morbidity. Alternatively, clinically available bone graft substitutes lack spatial control over scaffold architecture to anatomically match complicated bone defects. Therefore, the aim in this study was to develop a 3D printable composite biomaterial-ink to promote healing of large bone defects. The composite biomaterial-ink consisted of an organic biopolymer matrix with tyramine modified hyaluronic acid (THA) and collagen type I (Col) mixed with osteoinductive calcium phosphate particles (CaP). The biopolymer was combined with 0, 10, 20 and 30 % of either 45–63 µm or 45–106 µm CaP. µCT imaging showed a homogeneous distribution of CaP in the THA-Col hydrogel and all composites were 3D printable. In vitro cell activity assays revealed no indirect cytotoxicity using L929 cells and high cell cytocompatibility using human mesenchymal stromal cells (hMSCs). Additionally, all composites supported in vitro osteogenic differentiation of hMSCs. This study highlights the development of a 3D printable composite biomaterial-ink using CaP and THA-Col hydrogel that holds significant potential to be used as patient-specific bone graft substitute for the regeneration of large bone defects. Statement of significanceThis paper introduces a 3D printable composite biomaterial-ink made of osteoinductive calcium phosphate particles combined with matrix biopolymers collagen and hyaluronic acid, which was chemically modified to introduce shear thinning and shape fixation properties for 3D printing. The chemical modification only involves a small percentage of functional groups, preserving hyaluronan's biological properties. We demonstrated printability, the homogeneous distribution of the mineral phase, cytocompatibility and that the composites support osteogenesis of primary human mesenchymal stromal cells from multiple donors. The printability of the composite biomaterial-ink allows the creation of patient-specific implants with controlled geometry on porosity. This study contributes towards engineering personalized implants for replacing autologous bone grafting in all clinical situations where the bone self-healing capacity is insufficient.

Glutamine Addiction of Multiple Myeloma Cells Reprograms Mesenchymal Stromal Cell Enzymes and Transporters Towards a Pro-Tumor Phenotype

Metabolic alterations in cancer are not only functional to ensure malignant cell growth but also shape a pro-tumor behavior of normal cell populations in the tumor microenvironment. Multiple myeloma (MM) is the only human cancer that is both glutamine-addicted and glutamine-auxotroph, a feature that renders MM growth completely dependent upon extracellular glutamine availability. Indeed, plasma cells from most MM patients do not express Glutamine Synthetase (GS), the only enzyme able to synthetize glutamine from glutamate and ammonium, while express high levels of Glutaminase (GLS), which catalyze the first step of glutaminolysis by deamidating glutamine into glutamate and ammonium. As a consequence, the bone marrow (BM) plasma of MM patients has low-glutamine/high glutamate levels compared to patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS), as firstly demonstrated by our group. This particular metabolic microenvironment induces GS expression in mesenchymal stromal cells (MSCs) impairing osteoblast (OB) differentiation of MSCs, thus favoring MM bone lesions typical of active MM. The impact of these metabolic alterations on other BM cell populations is much less defined and it has been investigated in this study. For instance, although MM BM displays an oversized adipocyte population, which sustain MM growth by providing free fatty acids, the effects of MM metabolism on MSCs and their adipocyte differentiation remain to be characterized. First we show that 13C 5-Gln isotopomer distribution revealed that almost 50% of total glutamate in human myeloma cell lines (HMLCs) directly derives from glutamine deamidation by GLS. Furthermore, in standard culture conditions, HMLCs secreted glutamate likely through the SLC7A11 transporter, whose activity was higher in HMLCs than in MSCs. On the other hand, HMCLs were not able to take up glutamate from the extracellular space, while human primary MSCs isolated from healthy donors displayed high activity of the sodium-dependent glutamate transporters of the EAAT family. Moreover, glutamate uptake was higher in undifferentiated MSCs than in MSC incubated for 14 days in osteogenic medium (10 -8 M dexamethasone and 50 µg/ml ascorbic acid). Consistently, a public transcriptional profile of MSCs and OBs obtained from bone biopsies of healthy donors (n=7) or MM patients (n=16) revealed that the expression of the inward Glu transporter EAAT3 is higher in MSCs compared to OBs. In glutamine-free conditions, MSCs produced and secreted glutamine, a process boosted by extracellular glutamate in a dose-dependent manner. Glutamine secretion was hindered by either l-methionine sulfoximine (MSO), an irreversible inhibitor of GS, or D-aspartate (D-Asp), a high-affinity inhibitor of EAAT3 that hinders glutamate uptake. In a co-culture system, HMCLs induced the expression of SNAT5 glutamine efflux transporter in MSCs. Consistently, upon glutamine withdrawal, primary undifferentiated MSCs from healthy donors sustained HMCLs growth, while this nutritional support was markedly impaired by either inhibition or silencing of GS and EAAT3, with a substantial decrease in MM cell viability. Lastly, primary human MSCs were incubated under adipogenic conditions (0.5 mM 3-Isobutyl-1-methylxanthine, 5 µM indomethacin, 50 µM dexamethasone and 10 mg/ml human insulin) in the presence or in the absence of glutamine to mimic, respectively, normal or MM BM microenvironment. Glutamine deprivation increased lipogenesis, assessed with Oil Red O staining, as well as the expression of the adipocyte markers PPARG, LEP, and ADIPOQ. These data point to a MM-driven metabolic pro-tumor BM niche in which MSCs feed glutamine-addicted MM cells, and MSC differentiation is skewed from osteogenesis to adipogenesis. Several steps of these deranged pathways are amenable to pharmacological inhibition, pointing to possible novel therapeutic approaches to counteract MM growth and its effects on the BM niche.

Effects of single and repeated shock wave application on the osteogenic differentiation potential of human primary mesenchymal stromal cells and the osteoblastic cell line MG63 in vitro.

Introduction: Extracorporeal shock wave therapy is a non-invasive and effective option for treating various musculoskeletal disorders. Recent literature indicates that the parameters for extracorporeal shock wave therapy, such as the optimal intensity, treatment frequency, and localization, are yet to be determined. Studies reporting on the effects of shock wave application on primary mesenchymal stromal cells (MSCs) as well as osteoblastic cell lines in vitro are barely available and not standardized. Methods: In this study, we designed a special setup to precisely expose primary MSCs and the osteoblastic cell line MG63 to shock waves and subsequently analyzed the resulting cellular responses using standardized protocols to investigate their viability, proliferation behavior, cytokine secretion, and osteogenic differentiation potential in vitro. The shock wave transducer was coupled to a specifically designed water bath containing a 5mL tube holder. Primary human MSCs and MG63 cells were trypsinated and centrifuged in a 5mL tube and exposed to single and repeated shock wave application using different intensities and numbers of pulses. Results: Single treatment of MSCs using intensities 5, 10, 15, and 20 and pulse numbers 100, 250, 500, 750, and 1,000 at a constant pulse repetition frequency of 1Hz resulted in a decreased viability and proliferation of both cell types with an increase in the intensity and number of pulses compared to controls. No significant difference in the osteogenic differentiation was observed at different time intervals in both cell types when a single shock wave application was performed. However, repeated shock wave sessions over three consecutive days of primary MSCs using low intensity levels 0.1 and 1 showed significant osteogenic differentiation 4-fold higher than that of the extracted Alizarin Red S at day 14, whereas MG63 cells showed no significant osteogenic differentiation compared to their corresponding controls. More specifically, repeated shock wave application triggered a significant downregulation of COL1A1, upregulation of RUNX2, and sustained increase of OCN in primary MSCs but not in the cell line MG63 when induced toward the osteogenic differentiation. Discussion: The effects of shock wave application on MSCs make it an effective therapy in regenerative medicine. We established a protocol to analyze a standardized shock wave application on MSCs and were able to determine conditions that enhance the osteogenic differentiation of MSCs in vitro.

Size-tunable lipid vectors for controlled local delivery of siRNA from gene activated matrix

Tissue engineering aims to restore or replace different types of biological tissues through the association of cells, biologic factors and biomaterials. Currently, stem cells arise as a major cell source for many therapeutic indications, and their association with 3D scaffolds allow increasing regenerative medicine efficiency. In this context, the use of RNA interference to enhance or control stem cell differentiation into the desired phenotype appears as a promising strategy. However, achieving high transfection efficiency of cells in a 3D structure requires the use of a vector allowing for the spatiotemporally controlled release of the genetic material from these scaffolds. In this study, we report a new siRNA nanovector, called solvent exchange lipoplexe formulation (SELF), which has a tunable size, is stable over time in cell culture conditions and possess a high efficiency to transfect primary human mesenchymal stromal cells (hMSC). We associated SELFs with porous 3D collagen microspheres and demonstrated that the loading capacity and release kinetics were different depending on the size of the associated SELF. Interestingly, these different release profiles resulted in differences in the transfection kinetics of hMSCs. This original and unique type of gene activated matrix, with adaptable release kinetics, could be of interest for long-term and/or sequential transfection profiles of stem cells in 3D culture. Statement of SignificanceThis work combines the use of human mesenchymal stromal cell (hMSC) and gene therapy for tissue engineering. Here, a gene-activated matrix was elaborated with collagen microspheres supporting hMSCs and acting as a reservoir for transfection vectors. This injectable GAM allows for the local and sustained delivery of nucleic acids, hence long-lasting transfection of the supported cells. With the original synthesis protocol presented herein, the size of the nanocarriers can be easily adapted, resulting in different siRNA release profiles from the microspheres. Most interestingly, different siRNA release profiles gave rise to different cell transfection profiles as assessed by the downregulation of a target gene. This highlights the versatility of the system and its suitability for various pathophysiological needs in regenerative medicine.

DEAE-Dextran Enhances the Lentiviral Transduction of Primary Human Mesenchymal Stromal Cells from All Major Tissue Sources Without Affecting Their Proliferation and Phenotype

Genetic engineering of mesenchymal stromal cells (MSCs) is a tool widely used to explore MSC properties in vitro and in vivo. Lentiviral infection with the use of polycations as an adjuvant is a method that is commonly used to generate stably transduced cells. However, it is known that some polycations can negatively affect primary MSCs and to date, no study has explored the effect of different polycations on the transduction efficiency and properties of all main types of MSCs, namely those derived from umbilical cord, bone marrow and adipose tissue. Here we explore a range of polycations, using transduction protocols with and without spinoculation, to produce stably transduced MSCs from these three tissue sources. We identified that an overnight incubation with diethylaminoethyl-dextran (DEAE-Dextran) is the protocol associated with the best transduction efficiency without compromising the viability of the cells, and which worked consistently with lentiviral particles encoding for different transgenes. Transduced and sorted MSC populations revealed no significant changes in proliferation, morphology and expression of MSC markers compared to naïve MSCs. Following this study, we conclude that DEAE-Dextran is a polycation that can be successfully used to enhance the transduction of MSCs from all major tissue sources.

Inner strut morphology is the key parameter in producing highly porous and mechanically stable poly(ε-caprolactone) scaffolds via selective laser sintering

Selective laser sintering (SLS) is an established method to produce dimensionally accurate scaffolds for tissue engineering (TE) applications, especially in bone. In this context, the FDA-approved, biodegradable polymer poly(ε-caprolactone) (PCL) has been suggested as a suitable scaffold material. However, PCL scaffold mechanical stability – an attribute of particular importance in the field of bone TE – was not considered as a primary target for SLS process parameters optimization so far. Here, we investigated the influence of SLS process parameters on the sintered scaffolds with the aim of producing highly porous (>70% porosity) PCL scaffolds with sub-mm geometrical features for bone TE. Specifically, we studied the influence of laser power, beam compensation and laser beam diameter on the dimensional accuracy and mechanical stiffness of the produced PCL scaffolds. We found that the ratio between the diameter of the molten cross-section within scaffold struts and the outer strut diameter (including partially sintered particles) depended on the SLS process parameters. By maximizing this ratio, the mechanical stability could be optimized. The comparison with in silico predictions of scaffold mechanical stiffness revealed that the diameter of the molten cross-section within struts and not the strut diameter controlled the mechanical behaviour of the scaffold. These observations should be considered when evaluating the quality of the sintering process based on dimensional accuracy, especially for features <1 mm. Based on these findings, we suggested an approach to evaluate the sintering outcome and to define SLS process parameters that enable the production of highly porous scaffolds that are both dimensionally accurate and mechanically stable. Moreover, the cytocompatibility of PCL scaffolds was evaluated by elution tests with primary human mesenchymal stromal cells. No evidence of cytotoxicity was found in any of the investigated scaffolds, confirming the suitability of SLS as production technique of PCL scaffolds for bone TE over a wide range of SLS process parameters.

In Vivo Validation of Spray-Dried Mesoporous Bioactive Glass Microspheres Acting as Prolonged Local Release Systems for BMP-2 to Support Bone Regeneration

Bone morphogenetic protein-2 (BMP-2) is a known key mediator of physiological bone regeneration and is clinically approved for selected musculoskeletal interventions. Yet, broad usage of this growth factor is impeded due to side effects that are majorly evoked by high dosages and burst release kinetics. In this study, mesoporous bioactive glass microspheres (MBGs), produced by an aerosol-assisted spray-drying scalable process, were loaded with BMP-2 resulting in prolonged, low-dose BMP-2 release without affecting the material characteristics. In vitro, MBGs were found to be cytocompatible and to induce a pro-osteogenic response in primary human mesenchymal stromal cells (MSCs). In a pre-clinical rodent model, BMP-2 loaded MBGs significantly enhanced bone formation and influenced the microarchitecture of newly formed bone. The MBG carriers alone performed equal to the untreated (empty) control in most parameters tested, while additionally exerting mild pro-angiogenic effects. Using MBGs as a biocompatible, pro-regenerative carrier for local and sustained low dose BMP-2 release could limit side effects, thus enabling a safer usage of BMP-2 as a potent pro-osteogenic growth factor.

Multifunctional biomimetic hydrogel systems to boost the immunomodulatory potential of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) hold great therapeutic potential, in part because of their immunomodulatory properties. However, these properties can be transient and depend on multiple factors. Here, we developed a multifunctional hydrogel system to synergistically enhance the immunomodulatory properties of MSCs, using a combination of sustained inflammatory licensing and three-dimensional (3D) encapsulation in hydrogels with tunable mechanical properties. The immunomodulatory extracellular matrix hydrogels (iECM) consist of an interpenetrating network of click functionalized-alginate and fibrillar collagen, in which interferon γ (IFN-γ) loaded heparin-coated beads are incorporated. The 3D microenvironment significantly enhanced the expression of a wide panel of pivotal immunomodulatory genes in bone marrow-derived primary human MSCs (hMSCs), compared to two-dimensional (2D) tissue culture. Moreover, the inclusion of IFN-γ loaded heparin-coated beads prolonged the expression of key regulatory genes upregulated upon licensing, including indoleamine 2,3-dioxygenase 1 (IDO1) and galectin-9 (GAL9). At a protein level, iECM hydrogels enhanced the secretion of the licensing responsive factor Gal-9 by hMSCs. Its presence in hydrogel conditioned media confirmed the correct release and diffusion of the factors secreted by hMSCs from the system. Furthermore, co-culture of iECM-encapsulated hMSCs and activated human T cells resulted in suppressed proliferation, demonstrating direct regulation on immune cells. These data highlight the potential of iECM hydrogels to enhance the immunomodulatory properties of hMSCs in cell therapies.

Dosage and composition of bioactive glasses differentially regulate angiogenic and osteogenic response of human MSCs.

Vascularization of the fracture site and cell-mediated deposition of the mineralized matrix are crucial determinants for successful bone regeneration after injury. Ceramic biomaterials such as bioactive glasses (BAGs) that release bioactive ions have shown promising results in bone defect regeneration. However, it remains unclear how the dosage and composition of bioactive ions influence the angiogenic and osteogenic behavior of primary human mesenchymal stromal cells (MSCs). Here, we show that exposure to ionic dissolution products from 1393 and 45S5 BAGs can evoke distinct angiogenic and osteogenic responses from primary MSCs in a dose- and composition-dependent manner. Significantly higher concentrations of the pro-angiogenic factors VEGF, HGF, PIGF, angiopoietin, and angiogenin were detected in conditioned media (CM) from MSCs exposed to 45S5, but not 1393, BAGs. Application of this CM to human umbilical vein endothelial cells (HUVECs) resulted in robust 2D tube formation in vitro. Osteogenic differentiation of MSCs was assessed by gene expression analysis and mineralization assays. Low concentrations (0.1% w/v) of 1393 BAGs significantly enhanced the gene expression of RUNX2 and ALP and induced an earlier onset of matrix mineralization compared to all other groups. We further tested whether simultaneous exposure to both BAGs would improve both angiogenic secretion and osteogenic differentiation of MSCs, and did not find evidence to support this hypothesis. Our results provide evidence of BAG composition-dependent enhancement of primary human MSCs' regenerative function, besides also underlining the importance of an in vitro evaluation of the dose-response relationship to translate BAG based approaches into safe and effective clinical therapies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2827-2837, 2018., 2018.

GH prevents adipogenic differentiation of mesenchymal stromal stem cells derived from human trabecular bone via canonical Wnt signaling.

The imbalance between osteogenesis and adipogenesis, which naturally accompanies bone marrow senescence, may contribute to the development of bone-associated diseases, like osteoporosis. In the present study, using primary human mesenchymal stromal cells (hMSCs) isolated from trabecular bone, we assessed the possible effect of GH on hMSC differentiation potential into adipocytes. GH (5 ng/ml) significantly inhibited the lipid accumulation in hMSCs cultured for 14 days in lipogenic medium. GH decreased the expression of the adipogenic genes, CCAAT/enhancer-binding protein alpha (C/EBPα) and adiponectin (ADN) as well as the expression of two lipogenesis-related enzymes, lipoprotein lipase (LPL) and acethylCoA carboxylase (ACACA). In parallel, GH induced an increase in the gene expression and protein levels of osterix (OSX) and osteoprotegerin (OPG). These effects were ascribed to enhanced Wnt signaling as GH significantly reduced Wnt inhibitors, Dickkopf 1 (DKK1) and the secreted frizzled protein 2 (SFRP2), and increased the expression of an activator of Wnt, Wnt3. Accordingly, the expression of β-catenin and its nuclear levels were raised. Wnt involvement in GH anti-adipogenic effect was further confirmed by the silencing of β-catenin. In silenced hMSC, both the inhibitory effect of GH on the expression of the adipogenic genes, ADN and C/EBPα and the lipogenesis enzymes LPL and ACACA, were prevented together with the stimulatory effect of GH on the osteogenic genes OSX and OPG. The present study supports the hypothesis that when GH secretion declines as in aging, the fat in the bone-marrow cavities increases and the osteogenic capacity of the MSC pool is reduced due to a decrease in Wnt signaling.

Enhanced Biological Activity of BMP-2 Bound to Surface-Grafted Heparan Sulfate.

Over the last decade, there has been a growing interest in the development of new materials to improve bone morphogenetic protein-2 (BMP-2) delivery for tissue regeneration. This study reports the development and application of model surfaces that present BMP-2 via heparan sulfate (HS), a ubiquitous component of the extracellular matrix (ECM). On these surfaces, HS is grafted by its reducing end, to mimic the natural arrangement of HS proteoglycans in the ECM. The binding of each component on these biomimetic surfaces is highly controlled, in terms of stoichiometry of molecules and BMP-2/grafted-HS affinity, as determined by surface-sensitive techniques. For comparison, this study also uses surfaces presenting immobilized BMP-2 alone. Functional validations of the surfaces are performed using a murine myoblast cell line (C2C12) and primary human mesenchymal stromal cells. In both cell types, HS-bound BMP-2 and surface-immobilized BMP-2 significantly prolong SMAD 1/5 phosphorylation, compared to BMP-2 added to the culture media. Moreover, HS-bound BMP-2 enhances p-SMAD 1/5 levels in C2C12 cells and reduces noggin antagonistic activity. Thus, grafted HS positively affects BMP-2 cellular activity. This innovative surface design, which mimics natural interactions of growth factors with ECM components, constitutes a promising candidate for future regenerative medicine applications.

Efficient Transformation of Primary Human Mesenchymal Stromal Cells by Adenovirus Early Region 1 Oncogenes

It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type which is susceptible to HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells.

Superior Red Blood Cell Generation from Human Pluripotent Stem Cells Through a Novel Microcarrier-Based Embryoid Body Platform.

In vitro generation of red blood cells (RBCs) from human embryonic stem cells and human induced pluripotent stem cells appears to be a promising alternate approach to circumvent shortages in donor-derived blood supplies for clinical applications. Conventional methods for hematopoietic differentiation of human pluripotent stem cells (hPSC) rely on embryoid body (EB) formation and/or coculture with xenogeneic cell lines. However, most current methods for hPSC expansion and EB formation are not amenable for scale-up to levels required for large-scale RBC generation. Moreover, differentiation methods that rely on xenogenic cell lines would face obstacles for future clinical translation. In this study, we report the development of a serum-free and chemically defined microcarrier-based suspension culture platform for scalable hPSC expansion and EB formation. Improved survival and better quality EBs generated with the microcarrier-based method resulted in significantly improved mesoderm induction and, when combined with hematopoietic differentiation, resulted in at least a 6-fold improvement in hematopoietic precursor expansion, potentially culminating in a 80-fold improvement in the yield of RBC generation compared to a conventional EB-based differentiation method. In addition, we report efficient terminal maturation and generation of mature enucleated RBCs using a coculture system that comprised primary human mesenchymal stromal cells. The microcarrier-based platform could prove to be an appealing strategy for future scale-up of hPSC culture, EB generation, and large-scale generation of RBCs under defined and xeno-free conditions.

Effects of thromboprophylaxis on mesenchymal stromal cells during osteogenic differentiation: an in-vitro study comparing enoxaparin with rivaroxaban

BackgroundLow-molecular-weight heparins (e.g. Enoxaparin) are widely used to prevent venous thromboembolism after orthopaedic surgery, but there are reports about serious side effects including reduction in bone density and strength. In recent years new oral antithrombotic drugs (e.g. direct Factor Xa-inhibitor, Rivaroxaban) have been used to prevent venous thromboembolism. However, there is lack of information on the effects of these new drugs on human mesenchymal stromal cells during osteogenic differentiation and, therefore, effects during postoperative bone healing.MethodsWe evaluated the effects of Rivaroxaban and Enoxaparin on the proliferation, mRNA and surface receptor expression as well as differentiation capacity of primary human mesenchymal stromal cells during their osteogenic differentiation.ResultsEnoxaparin, but not Rivaroxaban treatment significantly increased human mesenchymal stromal cell (hMSC) proliferation during the first week of osteogenic differentiation while suppressing osteogenic marker genes, surface receptor expression and calcification.ConclusionsThis is the first paper to demonstrate that Rivaroxaban had no significant influence on hMSC differentiation towards the osteogenic lineage, indicating a less affected bone healing process compared with Enoxaparin in vitro. Based on these findings Rivaroxaban seems to be superior to Enoxaparin in early stages of bone healing in vitro.Electronic supplementary materialThe online version of this article (doi:10.1186/s12891-016-0966-2) contains supplementary material, which is available to authorized users.

Additive manufacturing of fibrous sub-micron poly(e-caprolactone) scaffolds for tissue engineering

Event Abstract Back to Event Additive manufacturing of fibrous sub-micron poly(ε-caprolactone) scaffolds for tissue engineering Gernot Hochleitner1, Tomasz Jüngst1, Toby D. Brown2, Kathrin Hahn1, Claus Moseke1, Franz Jakob3, Paul D. Dalton1 and Jürgen Groll1 1 University of Würzburg, Department for Functional Materials in Medicine and Dentistry, Germany 2 Queensland University of Technology, Institute of Health and Biomedical Innovation, Australia 3 University of Würzburg, Orthopedic Center for Musculoskeletal Research, Germany Introduction: The field of additive manufacturing (AM) has attracted increasing interest over the last decade, in part for tissue engineering (TE) approaches. In our study we use an emerging type of AM technology, termed Melt Electrospinning Writing (MEW), a process that combines the characteristic benefits of 3D printing and electrospinning. When using an electrohydrodynamic drawing process, thermoplastic melts can be deposited to well defined scaffold structures, even with sub-micron sized fiber diameters (817 ± 165 nm) as we recently observed[1],[2]. Materials and Methods: A custom-built device was used to manufacture scaffolds made of poly(ε-caprolactone) (PCL), a biodegradable polymer with a history of clinical use. In order to investigate the influence of the instrumental parameters, the feeding pressure (0.5–4.0 bar), heating temperature (80–120°C), acceleration voltage (2.0–10.0 kV), spinneret diameter (21 G; 23 G; 25 G; 27 G; 30 G; 33 G), collector distance (1–10 mm) and collector speed (1000–9000 mmmin-1) was systematically varied and optimized. To confirm cell adhesion to the scaffolds, we printed scaffolds directly onto hydrophilized (NCO-sP(EO-stat-PO)coated) glass slides and seeded primary human mesenchymal stromal cells (hMSC) to them afterwards[1]. Results and Discussion: Through the adjustment of instrumental parameters, PCL fibers with diameters ranging from 50 µm to the sub-micron level (817 ± 165 nm) can be deposited to highly uniform scaffolds (figure 1 A, B, C). Thus, MEW can be used to tailor scaffold architecture and to define its specific surface, which is of high interest for degradation or cell-interaction processes. Figure 1: A, B & C: MEW PCL scaffolds with a box structure consisting of 2x50 layers of sub-micron fliaments. D: HMSCs adhered to a scaffold after 4 days in vitro[1]. We observed excellent scaffold adhesion to the microscope slide that could withstand media changes and up to 2 weeks in vitro. The MSCs adhered well on the scaffolds, forming circular structures, even when the box spacing is only 90µm (figure 1 D). Further, the hydrophilic coatings of the glass slides not only prevented cell adhesion to the underlying surface, so that specific scaffold cell interactions could be observed[1]. Conclusion: While common AM methods, such as fused deposition modeling, allow a fabrication of fibers in a range of 100 µm microns and more, MEW can print sub-micron fibers with medical grade polymers. In contrast to solution electrospinning, drawing fibers from melts enables manufacturing without the use of often toxic solvents highly interesting for TE or medical approaches[1]. German Academic Exchange Service (DAAD, project No. 54417792); European research council (ERC consolidator grant Design2Heal, contract No. 617989); Professor Dietmar W Hutmacher from Institute of Health and Biomedical Innovation (Queensland University of Technology) for his support of TDB for MEW device construction; Björn Schulte from the Dept of Macromolecular Chemistry (RWTH Aachen) for SEC analysis

Cobblestone-area forming cells derived from patients with mantle cell lymphoma are enriched for CD133+ tumor-initiating cells.

Mantle cell lymphoma (MCL) is associated with a significant risk of therapeutic failure and disease relapse, but the biological origin of relapse is poorly understood. Here, we prospectively identify subpopulations of primary MCL cells with different biologic and immunophenotypic features. Using a simple culture system, we demonstrate that a subset of primary MCL cells co-cultured with either primary human mesenchymal stromal cells (hMSC) or murine MS-5 cells form in cobblestone-areas consisting of cells with a primitive immunophenotype (CD19−CD133+) containing the chromosomal translocation t (11;14)(q13;q32) characteristic of MCL. Limiting dilution serial transplantation experiments utilizing immunodeficient mice revealed that primary MCL engraftment was only observed when either unsorted or CD19−CD133+ cells were utilized. No engraftment was seen using the CD19+CD133− subpopulation. Our results establish that primary CD19−CD133+ MCL cells are a functionally distinct subpopulation of primary MCL cells enriched for MCL-initiating activity in immunodeficient mice. This rare subpopulation of MCL-initiating cells may play an important role in the pathogenesis of MCL.

Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells

The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.

Hypoxia Alters the Main Characteristics of the Hematopoietic Stem and Progenitor Cell Microenvironment in Vitro

Abstract 4803 Background:Hematopoietic stem and progenitor cells (HSPC) are located in a specialized microenvironment, called the stem cell niche, where their stem cell phenotype and differentiation are tightly regulated via interactions with the supporting mesenchymal stromal cells (MSC). These niches have been shown to be localized in regions with a lower oxygen tension which may also impact on the functional properties of MSC. For a better understanding to what extent hypoxia contributes to the establishment of an undifferentiated niche microenvironment that prevents inopportune differentiation of HSPC, we investigated MSC/HSPC co-cultures as well as MSC single cultures under low oxygen conditions. Design and Methods:Distribution, functional and phenotypical characteristics of CD34+ HSPC in hypoxic co-cultures (0.5% O2) were analyzed by flow cytometry. The effect of co-culture medium on the HSPC migration potential was tested in a transwell assay. The secretion of vascular endothelial growth factor A (VEGF-A), stromal-derived factor 1 (SDF-1), IL-6 and IL-8 by MSC was determined using ELISA whereas the expression of cell surface molecules was detected by flow cytometry. Moreover, the MSC proliferation as well as adipogenic and osteogenic differentiation was compared between hypoxic and normoxic culture conditions. Results:In the hypoxic co-culture, the adhesion of HSPC to the MSC layer was inhibited, whereas HSPC transmigration beneath the MSC layer was favoured. Increased VEGF-A secretion by MSC under hypoxic conditions, which enhanced the permeability of the MSC monolayer, was responsible for this effect. Furthermore, VEGF expression in hypoxic MSC was induced via hypoxia-inducible factor (HIF) signalling. Whereas IL-6 and IL-8 secretion were increased, SDF-1 expression by MSC was down-regulated under hypoxic conditions in a HIF-independent manner. The MSC immunophenotype which is characterized by expression of CD73, CD90, CD105, and CD166 was not significantly changed by hypoxia. Interestingly, a significant decrease of CD146 mRNA and protein expression levels was observed. The MSC proliferation was not significantly affected by lower oxygen tension. Culture of MSC in adipogenic induction medium for 14 days under hypoxia resulted in a reduced appearance of adipocyte-like cells containing lipid droplets and almost 50 % lower mRNA levels of fatty acid binding protein 2. The ALP activity as readout for osteogenic differentiation was decreased between 10% and 60% in hypoxic MSC. Conclusions:Low oxygen tension reduces the in vitro differentiation capacity and alters the cytokine secretion profile of primary human MSC. These functional changes may favour the homing and maintenance of quiescent HSC simulating the physiologically hypoxic niche conditions in vitro. Disclosures:No relevant conflicts of interest to declare.

Biphasic Electrical Targeting Plays a Significant Role in Schwann Cell Activation

Electrical stimulation (ES) is a promising technique for axonal regeneration of peripheral nerve injuries. However, long-term, continuous ES in the form of biphasic electric current (BEC) to stimulate axonal regeneration has rarely been attempted and the effects of BEC on Schwann cells are unknown. We hypothesized that long-term, continuous ES would trigger the activation of Schwann cells, and we therefore investigated the effect of BEC on the functional differentiation of primary human mesenchymal stromal cells (hMSCs) into Schwann cells, as well as the activity of primary Schwann cells. Differentiation of hMSCs into Schwann cells was determined by coculture with rat pheochromocytoma cells (PC12 cell line). We also investigated the in vivo effects of long-term ES (4 weeks) on axonal outgrowth of a severed sciatic nerve with a 7-mm gap after retraction of the nerve ends in rats by implanting an electronic device to serve as a neural conduit. PC12 cells cocultured with hMSCs electrically stimulated during culture in Schwann cell differentiation medium (Group I) had longer neurites and a greater percentage of PC12 cells were neurite-sprouting than when cocultured with hMSCs cultured in growth medium (control group) or unstimulated hMSCs in the same culture conditions as used for Group I (Group II). Group I cells showed significant upregulation of Schwann cell-related neurotrophic factors such as nerve growth factor and glial-derived neurotrophic factor compared to Group II cells at both the mRNA and protein levels. Primary Schwann cells responded to continuous BEC with increased proliferation and the induction of nerve growth factor and glial-derived neurotrophic factor, similar to Group I cells, and in addition, induction of brain-derived neurotrophic factor was observed. Immunohistochemical investigation of sciatic nerve regenerates revealed that BEC increased axonal outgrowth significantly. These results demonstrate that BEC enhanced the functional activity of Schwann cells via the induction of neurotrophic factor release and guide-increased axonal outgrowth in vivo. The effectiveness of long-term ES highlights the feasibility of a BEC-based therapeutic device to accelerate nerve regeneration of severed peripheral nerve injuries with a gap.

Mesenchymal stromal cells revert multiple myeloma cells to less differentiated phenotype by the combined activities of adhesive interactions and interleukin-6

Multiple myeloma is characterized by the malignant growth of immunoglobulin producing plasma cells, predominantly in the bone marrow. The effects of primary human mesenchymal stromal cells on the differentiation phenotype of multiple myeloma cells were studied by co-culture experiments. The incubation of multiple myeloma cells with bone marrow-derived mesenchymal stromal cells resulted in significant reduction of the expression of the predominant plasma cell differentiation markers CD38 and CD138, and cell surface immunoglobulin light chain. While the down-regulation of CD138 by stromal cells was completely dependent on their adhesive interactions with the multiple myeloma cells, interleukin-6 induced specific down-regulation of CD38. Mesenchymal stromal cells or their conditioned media inhibited the growth of multiple myeloma cell line, thereby reducing the overall amounts of secreted light chains. Analysis of primary multiple myeloma bone marrow samples reveled that the expression of CD38 on multiple myeloma cells was not affected by adhesive interactions. The ex vivo propagation of primary multiple myeloma cells resulted in significant increase in their differentiation markers. Overall, the data indicate that the bone marrow-derived mesenchymal stromal cells revert multiple myeloma cells to less differentiated phenotype by the combined activities of adhesive interactions and interleukin-6.