Osteogenic differentiation of human mesenchymal stromal cells and fibroblasts differs depending on tissue origin and replicative senescence.

The need for an autologous cell source for bone tissue engineering and medical applications has led researchers to explore multipotent mesenchymal stromal cells (MSC), which show stem cell plasticity, in various human tissues. However, MSC with different tissue origins vary in their biological properties and their capability for osteogenic differentiation. Furthermore, MSC-based therapies require large-scale ex vivo expansion, accompanied by cell type-specific replicative senescence, which affects osteogenic differentiation. To elucidate cell type-specific differences in the osteogenic differentiation potential and replicative senescence, we analysed the impact of BMP and TGF-ß signaling in adipose-derived stromal cells (ASC), fibroblasts (FB), and dental pulp stromal cells (DSC). We used inhibitors of BMP and TGF-ß signaling, such as SB431542, dorsomorphin and/or a supplemental addition of BMP-2. The expression of high-affinity binding receptors for BMP-2 and calcium deposition with alizarin red S were evaluated to assess osteogenic differentiation potential. Our study demonstrated that TGF-ß signaling inhibits osteogenic differentiation of ASC, DSC and FB in the early cell culture passages. Moreover, DSC had the best osteogenic differentiation potential and an activation of BMP signaling with BMP-2 could further enhance this capacity. This phenomenon is likely due to an increased expression of activin receptor-like kinase-3 and -6. However, in DSC with replicative senescence (in cell culture passage 10), osteogenic differentiation sharply decreased, and the simultaneous use of BMP-2 and SB431542 did not result in further improvement of this process. In comparison, ASC retain a similar osteogenic differentiation potential regardless of whether they were in the early (cell culture passage 3) or later (cell culture passage 10) stages. Our study elucidated that ASC, DSC, and FB vary functionally in their osteogenic differentiation, depending on their tissue origin and replicative senescence. Therefore, our study provides important insights for cell-based therapies to optimize prospective bone tissue engineering strategies.

Porcine endometrial 3D co-culture: Morphological changes in 3D endometrium tissues according to hormonal changes.

Cells cultured as monolayers proliferate well, but do not sustain their differentiation characteristics. Previous studies have investigated the interactions between cells and growth factors or cytokines by establishing either in vivo or in vitro three-dimensional (3D) cultures. Using porcine uterine epithelial cells and endometrial cells, the current study was designed to develop a 3D uterine culture system and investigate the response to hormone treatment. Formation of the 3D uterine model was similar to that of uterus from the group supplemented with calcium and magnesium, and the addition of these ions altered the spectrum of basement membrane degrading enzyme expression and activity. In particular, the epithelial cell junctions in the 3D model most closely resembled those of an actual uterus when the medium was supplemented with calcium and magnesium; the intercellular basement membrane structure was also tall under these conditions. The study confirmed that Casp-3 expression was lowest in the P4 (progesterone) treatment group, and this hormone was the most potent stimulus for formation of the endometrial cell layer. Therefore, the addition of calcium and magnesium plays an important role in the formation of a 3D uterine model, and the addition of P4 hormone mimics uterine thickening by stimulating growth of the epithelial cell layer.

Dynamic microfactories co-encapsulating osteoblastic and adipose-derived stromal cells for the biofabrication of bone units.

Cells with differentiation potential into mesodermal types are the focus of emerging bone tissue engineering (TE) strategies as an alternative autologous source. When the source of cells is extremely limited or not readily accessible, such as in severe injuries, a tissue biopsy may not yield the required number of viable cells. In line, adipose-derived stromal cells (ASCs) quickly became attractive for bone TE, since they can be easily and repeatably harvested using minimally invasive techniques with low morbidity. Inspired by the multiphenotypic cellular environment of bone, we propose the co-encapsulation of ASCs and osteoblasts (OBs) in self-regulated liquefied and multilayered microcapsules. We explore the unique architecture of such hybrid units to provide a dynamic environment using a simple culture in spinner flasks. Results show that microtissues were successfully obtained inside the proposed microcapsules with an appropriate diffusion of essential molecules for cell survival and signaling. Remarkably, microcapsules cultured in the absence of supplemental osteogenic differentiation factors presented osteopontin immunofluorescence, evidencing that the combined effect of the dynamic environment, and the paracrine signaling between ASCs and OBs may prompt the development of bone-like microtissues. Furthermore, microcapsules cultured under dynamic environment presented an enhanced mineralized matrix and a more organized extracellular matrix ultrastructure compared to static cultures used as control. Altogether, data in this study unveil an effective engineered bioencapsulation strategy for the in vitro production of bone-like microtissues in a more realistic and cost-effective manner. Accordingly, we intend to use the proposed system as hybrid devices implantable by minimally invasive procedures for bone TE applications.

Three-dimensional culture of endometrial cells from domestic cats: A new in vitro platform for assessing plastic toxicity.

Plastic polymers can be combined with additives that modify physical properties and stability of the material. However, the biocompatibility of those additives is not well known. The objective of the study was to characterize the impact of zinc stearate-a common additive-through the development of a novel three-dimensional (3-D) in vitro platform with endometrial cells from domestic cats. Epithelial and stromal cells from adult uteri were isolated and cultured in medium supplemented with 3% Matrigel for two weeks in plastic tissue culture dishes that had been identified as polystyrene with and without zinc stearate by Raman, FTIR, and X-ray fluorescence spectroscopies. Three-dimensional cell structures that were obtained were measured and categorized by shape. Cell viability, proliferation, differentiation, organization, and apoptosis then were assessed by immuno-staining. Results indicated that zinc stearate did not affect 3-D endometrial cell structure morphology, viability, or cellular composition. This first study of a new in vitro platform will be useful for studies testing the influence of other additives, drugs, or exogenous hormones.

Upregulation of mitotic bookmarking factors during enhanced proliferation of human stromal cells in human platelet lysate.

BACKGROUND: Innovative human stromal cell therapeutics require xeno-free culture conditions. Various formulations of human platelet lysate (HPL) are efficient alternatives for fetal bovine serum (FBS). However, a consistent lack of standardized manufacturing protocols and quality criteria hampers comparability of HPL-products. Aim of this study was to compare the biochemical composition of three differential HPL-preparations with FBS and to investigate their impact on stromal cell biology. METHODS: Stromal cells were isolated from bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC) and cultured in medium supplemented with pooled HPL (pHPL), fibrinogen-depleted serum-converted pHPL (pHPLS), mechanically fibrinogen-depleted pHPL (mcpHPL) and FBS. Biochemical parameters were analyzed in comparison to standard values in whole blood. Distinct growth factors and cytokines were measured by bead-based multiplex technology. Flow cytometry of stromal cell immunophenotype, in vitro differentiation, and mRNA expression analysis of transcription factors SOX2, KLF4, cMYC, OCT4 and NANOG were performed. RESULTS: Biochemical parameters were comparable in all pHPL preparations, but to some extent different to FBS. Total protein, glucose, cholesterol and Na+ were elevated in pHPL preparations, K+ and Fe3+ levels were higher in FBS. Compared to FBS, pHPL-based media significantly enhanced stromal cell propagation. Characteristic immunophenotype and in vitro differentiation potential were maintained in all four culture conditions. The analysis of growth factors and cytokines revealed distinct levels depending on the pre-existence in pHPL, consumption or secretion by the stromal cells. Interestingly, mRNA expression of the transcription and mitotic bookmarking factors cMYC and KLF4 was significantly enhanced in a source dependent manner in stromal cells cultured in pHPL- compared to FBS-supplemented media. SOX2 mRNA expression of all stromal cell types was increased in all pHPL culture conditions. CONCLUSION: All pHPL-supplemented media equally supported proliferation of WAT- and UC-derived stromal cells significantly better than FBS. Mitotic bookmarking factors, known to enable a quick re-entry to the cell cycle, were significantly enhanced in pHPL-expanded cells. Our results support a better characterization and standardization of humanized culture media for stromal cell-based medicinal products.

Protective role of GPR120 in the maintenance of pregnancy by promoting decidualization via regulation of glucose metabolism.

BACKGROUND: Intake of ω-3 PUFAs have been demonstrated to have positive effects on pregnancy outcome, whose receptor, GPR120, regulates several cellular functions including differentiation, metabolism and immune reaction. However, whether GPR120 is involved in decidualization and pregnancy remains unknown. METHODS: Decidua tissue from women with normal pregnancy and spontaneous abortion were collected to determine the expression profile of GPR120. Abortion mouse models and artificially induced deciduoma in mice were established to evaluate the effect of GPR120 on pregnancy outcome and in vivo decidualization. HESCs and primary DSCs were used to explore the roles of GPR120 in decidualization and mechanisms involved. FINDINGS: We found that GPR120 functioned to promote decidualization by upregulating glucose uptake and pentose-phosphate pathway (PPP) of human endometrial stromal cells. Firstly, the expression of GPR120 in decidua of spontaneous abortion was downregulated compared to normal decidua. Lack of GPR120 predisposed mice to LPS or RU486 induced abortion. Decidualization was augmented by GPR120 via improving GLUT1-mediated glucose uptake and G6PD- mediated PPP. FOXO1 was upregulated by GPR120 via activation of ERK1/2 and AMPK signaling and increased the expression of GLUT1. Furthermore, the expression of chemokines and cytokines in decidual stromal cells was enhanced by GPR120. Lastly, GPR120 agonist ameliorated LPS-induced abortion in the mice. INTERPRETATION: GPR120 plays significant roles in decidualization and the maintenance of pregnancy, which might be a potential target for diagnosis and treatment of spontaneous abortion. FUND: Ministry of Science and Technology of China, National Natural Science Foundation of China, the Program of Science and Technology Commission of Shanghai Municipality.

Comparative Efficacy of Autologous Stromal Vascular Fraction and Autologous Adipose-Derived Mesenchymal Stem Cells Combined With Hyaluronic Acid for the Treatment of Sheep Osteoarthritis.

The current study explored whether intra-articular (IA) injection of autologous adipose mesenchymal stem cells (ASCs) combined with hyaluronic acid (HA) achieved better therapeutic efficacy than autologous stromal vascular fraction (SVF) combined with HA to prevent osteoarthritis (OA) progression and determined how long autologous ASCs combined with HA must remain in the joint to observe efficacy. OA models were established by performing anterior cruciate ligament transection (ACLT) and medial meniscectomy (MM). Autologous SVF (1×107 mononuclear cells), autologous low-dose ASCs (1×107), and autologous high-dose ASCs (5×107) combined with HA, and HA alone, or saline alone were injected into the OA model animals at 12 and 15 weeks after surgery, respectively. Compared with SVF+HA treatment, low-dose ASC+HA treatment yielded better magnetic resonance imaging (MRI) scores and macroscopic results, while the cartilage thickness of the tibial plateau did not differ between low, high ASC+HA and SVF+HA treatments detected by micro-computed tomography (µCT). Immunohistochemistry revealed that high-dose ASC+HA treatment rescued hypertrophic chondrocytes expressing collagen X in the deep area of articular cartilage. Western blotting analysis indicated the high- and low-dose ASC+HA groups expressed more collagen X than did the SVF+HA group. Enzyme-linked immunosorbent assay showed treatment with both ASC+HA and SVF+HA resulted in differing anti-inflammatory and trophic effects. Moreover, superparamagnetic iron oxide particle (SPIO)-labeled autologous ASC signals were detected by MRI at 2 and 18 weeks post-injection and were found in the lateral meniscus at 2 weeks and in the marrow cavity of the femoral condyle at 18 weeks post-injection. Thus, IA injection of autologous ASC+HA may demonstrate better efficacy than autologous SVF+HA in blocking OA progression and promoting cartilage regeneration, and autologous ASCs (5×107 cells) combined with HA potentially survive for at least 18 weeks after IA injection.

Photobiomodulation of freshly isolated human adipose tissue-derived stromal vascular fraction cells by pulsed light-emitting diodes for direct clinical application.

A highly interesting source for adult stem cells is adipose tissue, from which the stromal vascular fraction (SVF)-a heterogeneous cell population including the adipose-derived stromal/stem cells-can be obtained. To enhance the regenerative potential of freshly isolated SVF cells, low-level light therapy (LLLT) was used. The effects of pulsed blue (475 nm), green (516 nm), and red (635 nm) light from light-emitting diodes applied on freshly isolated SVF were analysed regarding cell phenotype, cell number, viability, adenosine triphosphate content, cytotoxicity, and proliferation but also osteogenic, adipogenic, and proangiogenic differentiation potential. The colony-forming unit fibroblast assay revealed a significantly increased colony size after LLLT with red light compared with untreated cells, whereas the frequency of colony-forming cells was not affected. LLLT with green and red light resulted in a stronger capacity to form vascular tubes by SVF when cultured within 3D fibrin matrices compared with untreated cells, which was corroborated by increased number and length of the single tubes and a significantly higher concentration of vascular endothelial growth factor. Our study showed beneficial effects after LLLT on the vascularization potential and proliferation capacity of SVF cells. Therefore, LLLT using pulsed light-emitting diode light might represent a new approach for activation of freshly isolated SVF cells for direct clinical application.

Qualitative and quantitative prediction of human in vivo metabolic pathways in a human hepatocyte-murine stromal cell co-culture model.

1. This study assessed the value of a static in vitro human hepatocyte-murine stromal cell co-culture model to qualitatively and quantitatively predict human in vivo metabolic clearance pathways using 14C-labeled test compounds and compared these results to an in vitro suspended human hepatocyte model and the in vivo human 14C ADME studies. 2. Test compounds represented a diverse set of clearance pathways (Phase I and Phase II). Compounds were incubated for 4 h in suspended human hepatocytes and for 24 and 168 h in the human co-culture model. Multivariate analysis revealed that long-term (168 h) incubation of test compounds in the co-culture had reasonable quantitative prediction of the in vivo human clearance pathways as compared to the 4 h suspended hepatocytes or the 24 h co-culture incubation. 3. In vivo and in vitro disconnects were observed in cases where extra-hepatic metabolism or urinary excretion was observed in vivo. Differences in the relative percentages of Phase I and Phase II metabolites observed were likely due to microbial ß-glucuronidase hydrolysis of conjugates and microflora mediated metabolism in the gut not present in the in vitro systems.

Andrographolide Promotes Neural Differentiation of Rat Adipose Tissue-Derived Stromal Cells through Wnt/ß-Catenin Signaling Pathway.

Adipose tissue-derived stromal cells (ADSCs) are a high-yield source of pluripotent stem cells for use in cell-based therapies. We explored the effect of andrographolide (ANDRO, one of the ingredients of the medicinal herb extract) on the neural differentiation of rat ADSCs and associated molecular mechanisms. We observed that rat ADSCs were small and spindle-shaped and expressed multiple stem cell markers including nestin. They were multipotent as evidenced by adipogenic, osteogenic, chondrogenic, and neural differentiation under appropriate conditions. The proportion of cells exhibiting neural-like morphology was higher, and neurites developed faster in the ANDRO group than in the control group in the same neural differentiation medium. Expression levels of the neural lineage markers MAP2, tau, GFAP, and ß-tubulin III were higher in the ANDRO group. ANDRO induced a concentration-dependent increase in Wnt/ß-catenin signaling as evidenced by the enhanced expression of nuclear ß-catenin and the inhibited form of GSK-3ß (pSer9). Thus, this study shows for the first time how by enhancing the neural differentiation of ADSCs we expect that ANDRO pretreatment may increase the efficacy of adult stem cell transplantation in nervous system diseases, but more exploration is needed.

Maternal Retinoids Increase PDGFRα+ Progenitor Population and Beige Adipogenesis in Progeny by Stimulating Vascular Development.

Maternal vitamin A intake varies but its impact on offspring metabolic health is unknown. Here we found that maternal vitamin A or retinoic acid (RA) administration expanded PDGFRα+ adipose progenitor population in progeny, accompanied by increased blood vessel density and enhanced brown-like (beige) phenotype in adipose tissue, protecting offspring from obesity. Blockage of retinoic acid signaling by either BMS493 or negative RA receptor (RARαDN) over-expression abolished the increase in blood vessel density, adipose progenitor population, and beige adipogenesis stimulated by RA. Furthermore, RA-induced beige adipogenesis was blocked following vascular endothelial growth factor receptor (VEGFR) 2 knock out in PDGFRα+ cells, suggesting its mediatory role. Our data reveal an intrinsic link between maternal retinoid level and offspring health via promoting beige adipogenesis. Thus, enhancing maternal retinoids is an amiable therapeutic strategy to prevent obesity in offspring, especially for those born to obese mothers which account for one third of all pregnancies.

Zebrafish Caudal Haematopoietic Embryonic Stromal Tissue (CHEST) Cells Support Haematopoiesis.

Haematopoiesis is an essential process in early vertebrate development that occurs in different distinct spatial locations in the embryo that shift over time. These different sites have distinct functions: in some anatomical locations specific hematopoietic stem and progenitor cells (HSPCs) are generated de novo. In others, HSPCs expand. HSPCs differentiate and renew in other locations, ensuring homeostatic maintenance. These niches primarily control haematopoiesis through a combination of cell-to-cell signalling and cytokine secretion that elicit unique biological effects in progenitors. To understand the molecular signals generated by these niches, we report the generation of caudal hematopoietic embryonic stromal tissue (CHEST) cells from 72-hours post fertilization (hpf) caudal hematopoietic tissue (CHT), the site of embryonic HSPC expansion in fish. CHEST cells are a primary cell line with perivascular endothelial properties that expand hematopoietic cells in vitro. Morphological and transcript analysis of these cultures indicates lymphoid, myeloid, and erythroid differentiation, indicating that CHEST cells are a useful tool for identifying molecular signals critical for HSPC proliferation and differentiation in the zebrafish. These findings permit comparison with other temporally and spatially distinct haematopoietic-supportive zebrafish niches, as well as with mammalian haematopoietic-supportive cells to further the understanding of the evolution of the vertebrate hematopoietic system.

Effects of Lycium barbarum polysaccharides on the damage to human endometrial stromal cells induced by hydrogen peroxide.

Previous studies have shown that Lycium barbarum polysaccharides (LBPs) serve an important role in antioxidant activity to protect the cells and tissues. However, the specific mechanism of LBPs in the prevention of endometrial damage remains to be elucidated. Using morphological observation, cell proliferation assay, the detection of superoxide dismutase (SOD) activity and the content of malondialdehyde (MDA) in cell culture supernatant fluid, the detection by western blot analysis and reverse transcription-quantitative polymerase chain reaction of the mRNA and protein expression levels of caspase­3 and Bcl­2 in endometrial stromal cells (ESCs), it was demonstrated that, in vitro, hydrogen peroxide (H2O2)-induced death of ESCs, increased the content of MDA and decreased the activity of SOD, and decreased the expression of Bcl-2 and increased the expression of caspase­3. LBPs can inhibit H2O2­induced cell death of ESCs, decrease the content of MDA in ESCs and increase the activity of SOD, as well as increasing the expression of Bcl­2 and decreasing the expression levels of caspase­3. These findings suggested that LBPs can inhibit H2O2­induced apoptosis of EECs and that LBPs are able to offer a significant protection against oxidative stress to ESCs.

Fingerprinting of neurotoxic compounds using a mouse embryonic stem cell dual luminescence reporter assay.

Identification of neurotoxic drugs and environmental chemicals is an important challenge. However, only few tools to address this topic are available. The aim of this study was to develop a neurotoxicity/developmental neurotoxicity (DNT) test system, using the pluripotent mouse embryonic stem cell line CGR8 (ESCs). The test system uses ESCs at two differentiation stages: undifferentiated ESCs and ESC-derived neurons. Under each condition, concentration-response curves were obtained for three parameters: activity of the tubulin alpha 1 promoter (typically activated in early neurons), activity of the elongation factor 1 alpha promoter (active in all cells), and total DNA content (proportional to the number of surviving cells). We tested 37 compounds from the ESNATS test battery, which includes polypeptide hormones, environmental pollutants (including methylmercury), and clinically used drugs (including valproic acid and tyrosine kinase inhibitors). Different classes of compounds showed distinct concentration-response profiles. Plotting of the lowest observed adverse effect concentrations (LOAEL) of the neuronal promoter activity against the general promoter activity or against cytotoxicity, allowed the differentiation between neurotoxic/DNT substances and non-neurotoxic controls. Reporter activity responses in neurons were more susceptible to neurotoxic compounds than the reporter activities in ESCs from which they were derived. To relate the effective/toxic concentrations found in our study to relevant in vivo concentrations, we used a reverse pharmacokinetic modeling approach for three exemplary compounds (teriflunomide, geldanamycin, abiraterone). The dual luminescence reporter assay described in this study allows high-throughput, and should be particularly useful for the prioritization of the neurotoxic potential of a large number of compounds.

Angiogenic Synergistic Effect of Adipose-Derived Stromal Cell Spheroids with Low-Level Light Therapy in a Model of Acute Skin Flap Ischemia.

Human adipose-derived mesenchymal stem cells (hASCs) are an attractive cell source for tissue engineering. However, one obstacle to this approach is that the transplanted hASC population can decline rapidly in the recipient tissue. The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted spheroid hASCs in skin flaps of mice. hASCs were cultured in monolayers or spheroids. LLLT, hASCs, spheroids and spheroids transplanted with LLLT were applied to the skin flaps. Healing of the skin flaps was assessed by gross evaluation and by hematoxylin and eosin staining and elastin van Gieson staining. Compared with the spheroid group, skin flap healing was enhanced in the spheroid + LLLT group, including the neovascularization and regeneration of skin appendages. The survival of hASCs was enhanced by decreased apoptosis of hASCs in the skin flaps of the spheroid + LLLT group. The secretion of growth factors was stimulated in the spheroid + LLLT group compared with the ASC and spheroid groups. These data suggest that LLLT was an effective biostimulator of spheroid hASCs in the skin flaps, enhancing the survival of hASCs and stimulating the secretion of growth factors.

Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats.

Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug.

Property of Human Bone Marrow Stromal Cells Derived From Bone Fragments Removed in Sagittal Split Ramus Osteotomy.

Bone tissue engineering is in the process of making the shift from bench to bed. Organ as a cell source is important for tissue engineering. The appropriate cells should be harvested without invasiveness and ethical problems. The authors focused on mandibular cortex bone fragments removed in sagittal split ramus osteotomy as a cell source for bone tissue engineering. These bone fragments were discarded after surgery until now. Bone marrow stromal cells (BMSCs) were harvested from inside of bone fragments, which is an endosteal region. Endosteal region is known to be a hematopoietic stem cell niche and harbors osteoblasts, preosteoblasts, and mesenchymal stem cells (MSCs). Bone marrow stromal cells could be cultured easily, and grew rapidly in vitro under ordinary serum-supplemented culture condition. The expression pattern of surface markers of BMSCs was the same as that of MSCs. Bone marrow stromal cells could differentiated into multiple mesenchymal lineages (osteoblasts, adipocytes, chondrocytes, and smooth muscle cells). These results indicated the existence of MSCs in BMSCs. The osteoblastic characters of BMSCs were examined more closely. Bone marrow stromal cells showed a high alkaline phosphatase activity, and expressed osteoblastic markers (PTHr, bone sialoprotein, Type I collagen, Rnut-related transcription factor 2, and osteocalcin). In transplantation experiments, BMSCs generated ectopic bone tissues on the border of hydroxyapatite scaffold without osteogenic differentiation-inducing agents such as dexamethasone (Dex) or bone morphogenetic protein. The results of this study suggest that mandibular cortex bone fragments removed in sagittal split ramus osteotomy are a good cell source for bone tissue engineering.

Multilayered, Hyaluronic Acid-Based Hydrogel Formulations Suitable for Automated 3D High Throughput Drug Screening of Cancer-Stromal Cell Cocultures.

Validation of a high-throughput compatible 3D hyaluronic acid hydrogel coculture of cancer cells with stromal cells. The multilayered hyaluronic acid hydrogels improve drug screening predictability as evaluated with a panel of clinically relevant chemotherapeutics in both prostate and endometrial cancer cell lines compared to 2D culture.

Advances in umbilical cord blood stem cell expansion and clinical translation.

Umbilical cord blood (CB) is a rich source of hematopoietic stem cells (HSCs) with important applications in allogeneic stem cell transplantation. However, the low numbers of hematopoietic stem and progenitor cells (HSPCs) in banked units remain a major limitation. Protocols developed for HSPC expansion ex vivo or to improve HSPC homing to the marrow represent solutions to overcome this shortcoming. In recent decades, wide arrays of functionally divergent approaches were developed for the amplification of HSPCs. These include optimization of cytokine cocktails, coculture systems, small molecules, and delivery systems for HSPC-expansion genes. Herein, we review past and current strategies, focusing on studies that characterize the contribution of expanded CB HSPC to short- and long-term engraftment in transplantation models or in clinical trials. Also discussed are homing effectors used to promote engraftment. In summary, these studies underscore that early-acting cytokines alone can expand HSPC with short-term engraftment activity, but that robust expansion of HSPCs with long-term engraftment necessitates the synergistic action of multiple HSC-expansion agonists. In support of this, early clinical trials based on cytokine-driven HSPC-expansion protocols delivered disappointing results, whereas recent trials based on the synergistic action of cytokines and HSPC-expansion agonists reported significant improvements in engraftment and therapeutic outcomes. Conversely, molecules that enhance homing of HSPC may represent a complementary approach to improve and perhaps accelerate engraftment. Optimization of the next generation of HSPC-expansion and priming strategies should support a paradigm shift in CB transplantation in which smaller, better matched units may preferentially be used.

Osteoclastogenesis/osteoblastogenesis using human bone marrow-derived cocultures on nanotopographical polymer surfaces.

BACKGROUND: Optimised nanotopography with controlled disorder (NSQ50) has been shown to stimulate osteogenesis and new bone formation in vitro. Following osteointegration the implant interface must undergo constant remodeling without inducing immune response. AIM: We aimed to assess the effect of nanotopography on bone remodelling using osteoclast and osteoblast cocultures. MATERIALS & METHODS: We developed a novel osteoblast/osteoclast coculture using solely human bone marrow derived mesenchymal and hematopeotic progenitor cells without extraneous supplementation. The coculture was been applied to NSQ50 or flat control polycarbonate substrates and assessed using immunohistochemical and immunofluorescent microscopy, scanning electron microscopy and quantitative reverse-transcription PCR methods. RESULTS: These confirm the presence of mature osteoclasts, osteoblasts and bone formation in coculture. Osteoblast differentiation increased on NSQ50, with no significant difference in osteoclast differentiation. CONCLUSION: Controlled disorder nanotopography appears to be selectively bioactive. We recommend this coculture method to be a better in vitro approximation of the osseous environment encountered by implants.