The development of resistance to chemotherapy is a major obstacle for lasting effective treatment of cancer. Here, we demonstrate that endogenous mesenchymal stem cells (MSCs) become activated during treatment with platinum analogs and secrete factors that protect tumor cells against a range of chemotherapeutics. Through a metabolomics approach, we identified two distinct platinum-induced polyunsaturated fatty acids (PIFAs), 12-oxo-5,8,10-heptadecatrienoic acid (KHT) and hexadeca-4,7,10,13-tetraenoic acid (16:4(n-3)), that in minute quantities induce resistance to a broad spectrum of chemotherapeutic agents. Interestingly, blocking central enzymes involved in the production of these PIFAs (cyclooxygenase-1 and thromboxane synthase) prevents MSC-induced resistance. Our findings show that MSCs are potent mediators of resistance to chemotherapy and reveal targets to enhance chemotherapy efficacy in patients.

Osteochondral defects are prone to induce osteoarthritic degenerative changes. Many tissue-engineering approaches that aim to generate osteochondral implants suffer from poor tissue formation and compromised integration. This illustrates the need for further improvement of heterogeneous tissue constructs. Engineering of these structures is expected to profit from strategies addressing the complexity of tissue organization and the simultaneous use of multiple cell types. Moreover, this enables the investigation of the effects of three-dimensional (3D) organization and architecture on tissue function. In the present study, we characterize the use of a 3D fiber deposition (3DF) technique for the fabrication of cell-laden, heterogeneous hydrogel constructs for potential use as osteochondral grafts. Changing fiber spacing or angle of fiber deposition yielded scaffolds of varying porosity and elastic modulus. We encapsulated and printed fluorescently labeled human chondrocytes and osteogenic progenitors in alginate hydrogel yielding scaffolds of 1×2 cm with different parts for both cell types. Cell viability remained high throughout the printing process, and cells remained in their compartment of the printed scaffold for the whole culture period. Moreover, distinctive tissue formation was observed, both in vitro after 3 weeks and in vivo (6 weeks subcutaneously in immunodeficient mice), at different locations within one construct. These results demonstrate the possibility of manufacturing viable centimeter-scaled structured tissues by the 3DF technique, which could potentially be used for the repair of osteochondral defects.

Earlier, we have shown that the increased cartilage production in pellet co-cultures of chondrocytes and bone marrow-derived mesenchymal stem cells (BM-MSCs) is due to a trophic role of the MSC in stimulating chondrocyte proliferation and matrix production rather than MSCs actively undergoing chondrogenic differentiation. These studies were performed in a culture medium that was not compatible with the chondrogenic differentiation of MSCs. In this study, we tested whether the trophic role of the MSCs is dependent on culturing co-culture pellets in a medium that is compatible with the chondrogenic differentiation of MSCs. In addition, we investigated whether the trophic role of the MSCs is dependent on their origins or is a more general characteristic of MSCs. Human BM-MSCs and bovine primary chondrocytes were co-cultured in a medium that was compatible with the chondrogenic differentiation of MSCs. Enhanced matrix production was confirmed by glycosaminoglycans (GAG) quantification. A species-specific quantitative polymerase chain reaction demonstrated that the cartilage matrix was mainly of bovine origin, indicative of a lack of the chondrogenic differentiation of MSCs. In addition, pellet co-cultures were overgrown by bovine cells over time. To test the influence of origin on MSCs' trophic effects, the MSCs isolated from adipose tissue and the synovial membrane were co-cultured with human primary chondrocytes, and their activity was compared with BM-MSCs, which served as control. GAG quantification again confirmed increased cartilage matrix production, irrespective of the source of the MSCs. EdU staining combined with cell tracking revealed an increased proliferation of chondrocytes in each condition. Irrespective of the MSC source, a short tandem repeat analysis of genomic DNA showed a decrease in MSCs in the co-culture over time. Our results clearly demonstrate that in co-culture pellets, the MSCs stimulate cartilage formation due to a trophic effect on the chondrocytes rather than differentiating into chondrocytes, irrespective of culture condition or origin. This implies that the trophic effect of MSCs in co-cultures is a general phenomenon with potential implications for use in cartilage repair strategies. Introduction Despite the success of autologous chondrocyte implantation in treating large-size cartilage defects, there are some disadvantages of this treatment that limit its broader clinical application. One major issue is the requirement of relatively large quantities of chondrocytes from the patient, 1 which are obtained by in vitro expansion. The partial replacement of the chondrocytes with mesenchymal stem cells (MSC) has been proposed to tackle this problem. Many studies have evaluated the feasibility of this idea by the coculturing of MSCs and chondrocytes.2 Indeed, cartilage matrix deposition was found to be improved in the cocultures of MSCs and chondrocytes compared with the cultures of pure chondrocytes or MSCs. 3,4 In our previous report, 5 we have shown that pellet co-cultures of bovine pri...

In patients undergoing maxillary sinus floor elevation (MSFE) for dental implant placement, bone substitutes are currently evaluated as alternatives for autologous bone. However, bone substitutes have only osteoconductive properties and lack osteoinductive potential. Therefore, this phase I study evaluated the potential additive effect on bone regeneration by the addition of freshly isolated, autologous but heterologous stromal vascular fraction (SVF), which is highly enriched with adipose stromal/ stem cells when compared with native adipose tissue. From 10 patients, SVF was procured using automatic processing, seeded on either b-tricalcium phosphate (n = 5) or biphasic calcium phosphate carriers (n = 5), and used for MSFE in a one-step surgical procedure. Primary objectives were feasibility and safety. The secondary objective was efficacy, evaluated by using biopsies of the augmented area taken 6 months postoperatively, concomitant with dental implant placement. Biopsies were assessed for bone, graft, and osteoid volumes. No adverse effects were reported during the procedure or follow-up ( ‡3 years). Bone and osteoid percentages were higher in study biopsies (SVF supplemented) than in control biopsies (ceramic only on contralateral side), in particular in b-tricalcium phosphatetreated patients. Paired analysis on the six bilaterally treated patients revealed markedly higher bone and osteoid volumes using microcomputed tomography or histomorphometric evaluations, demonstrating an additive effect of SVF supplementation, independent of the bone substitute. This study demonstrated for the first time the feasibility, safety, and potential efficacy of SVF seeded on bone substitutes for MSFE, providing the first step toward a novel treatment concept that might offer broad potential for SVF-based regenerative medicine applications. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:1362-1374

Adult stem cells, or mesenchymal stromal cells (MSCs), are of great potential for cell therapy and tissue-engineering applications. However, for therapeutic use, these cells need to be isolated from tissue or a biopsy and efficiently expanded, as they cannot be harvested in sufficient quantities from the body. In our opinion, efficient expansion of MSCs can be achieved in a microcarrier-based cultivation system. This study selected a suitable microcarrier for human bone marrow-derived stromal cells (HBMSCs), optimized cell-seeding strategies by varying serum concentrations, and optimized dynamic expansion of the HBMSCs in a microcarrier-based spinner flask cultivation system by applying various feeding regimes. Cytodex 1 microcarriers in combination with a lowserum concentration (0-5%) in the medium resulted in the highest seeding efficiency for the HBMSCs. Subsequently, significant expansion of the HBMSCs on these carriers has been observed. The highest number of HBMSCs population doublings (4.8 doublings) was obtained by a combination of 50% medium refreshment combined with addition of 30% medium containing microcarriers every 3 days. Exponential cell growth was observed for at least 9 days after seeding, provided that sufficient nutrients (such as glucose) were present, metabolite concentrations (such as ammonia) were kept below growth-inhibitory concentrations and adequate surface area was present for the cells. After dynamic expansion of the HBMSCs, the cells retained their differentiation potential and their cell surface markers, indicating that HBMSCs expansion on Cytodex 1 microcarriers did not alter the phenotypic properties of the cells.

Interactions within the hematopoietic niche in the BM microenvironment are essential for maintenance of the stem cell pool. In addition, this niche is thought to serve as a sanctuary site for malignant progenitors during chemotherapy. Therapy resistance induced by interactions with the BM microenvironment is a major drawback in the treatment of hematologic malignancies and bone-metastasizing solid tumors. To date, studying these interactions was hampered by the lack of adequate in vivo models that simulate the human situation. In the present study, we describe a unique human-mouse hybrid model that allows engraftment and outgrowth of normal and malignant hematopoietic progenitors by implementing a technology for generating a human bone environment. Using luciferase gene marking of patient-derived multiple myeloma cells and bioluminescent imaging, we were able to follow pMM cells outgrowth and to visualize the effect of treatment. Therapeutic interventions in this model IntroductionIn the BM, specialized microenvironments such as hematopoietic niches regulate hematopoiesis. Within these niches, hematopoietic stem cells (HSCs) are present in a complex network consisting of mesenchymal stromal cells (MSCs), osteoblasts, osteoclasts, endothelial cells, and adipocytes embedded in an extracellular matrix. The bidirectional interactions with the hematopoietic niche are essential for HSC maintenance and function. [1][2][3][4] The BM niche is also thought to serve as a sanctuary site for leukemic stem cells (LSCs), which, in addition to their immortalizing genetic events, highly depend on interaction with the microenvironment to survive and proliferate. 5,6 Although the majority of leukemias initially respond to therapeutic intervention, relapse rates are high. [7][8][9] There is increasing evidence that the tumor niche plays a crucial role in the survival and drug resistance of LSCs. Interactions with the niche provide signals protecting the LSCs from apoptosis and eventually leading to the selection and outgrowth of a resistant cell. 10-13 Therefore, it is apparent that the hematopoietic niche plays an important role in hematopoietic development and in chemotherapy resistance of BM-localized leukemic and solid tumors.Although our understanding of how the BM niche regulates HSC self-renewal and confers therapy resistance has advanced greatly over the past years, most of this knowledge is based on genetic loss-of-function or gain-of-function murine models. 1,2,10,11,14 However, these murine models do not simulate human physiology and much of the constituents of the human hematopoietic niche remain largely unclear. [14][15][16] This emphasizes the need for more suitable models that recapitulate the human BM microenvironment and, very importantly, facilitate the engraftment and outgrowth of normal HSCs and patient-derived tumor cells within these protected sites.In the present study, we describe a unique humanized model that implements a novel scaffold-based technology for generating a human bone environment in RAG 2 Ϫ/Ϫ ...

One of the applications of bone marrow stromal cells (BMSCs) that are produced by ex vivo expansion is for use in in vivo bone tissue engineering. Cultured stromal cells are a mixture of cells at different stages of commitment and expansion capability, leading to a heterogeneous cell population that each time can differ in the potential to form in vivo bone. A parameter that predicts for in vivo bone forming capacity is thus far lacking. We employed single colony-derived BMSC cultures to identify such predictive parameters. Using limiting dilution, we have produced sixteen single CFU-F derived BMSC cultures from human bone marrow and found that only five of these formed bone in vivo. The single colony-derived BMSC strains were tested for proliferation, osteogenic-, adipogenic- and chondrogenic differentiation capacity and the expression of a variety of associated markers. The only robust predictors of in vivo bone forming capacity were the induction of alkaline phosphatase, (ALP) mRNA levels and ALP activity during in vitro osteogenic differentiation. The predictive value of in vitro ALP induction was confirmed by analyzing "bulk-cultured" BMSCs from various bone marrow biopsies. Our findings show that in BMSCs, the additional increase in ALP levels over basal levels during in vitro osteogenic differentiation is predictive of in vivo performance.

Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.