Lung on a chip: Co-culture of alveolar epithelial cells and bone marrow derived stromal stem cells in a microfluidic system

Background: Microfluidics system are novel tools to study cell-cell interactions in vitro. This project focuses on the development of a new microfluidic device to co-culture alveolar epithelial cells and mesenchymal stem cells to study cellular interactions involved in healing the injured alveolar epithelium. Methods: Microfluidic systems in polydimethylsiloxane were fabricated by soft lithography. The alveolar A549 epithelial cells were seeded and injury tests were made on the cells by perfusion with media containing H2O2 or bleomycin during 6 or 18hrs. Rat Bone marrow derived stromal cells (BMSC) were then introduced into the system and cell-cell interaction was studied over 24 hrs. Results: A successful co-culture of A549 alveolar epithelial cells and BMS was achieved in the microfluidic system. The seeded alveolar epithelial cells and BMSC adhered to the bottom surface of the microfluidic device and proliferated under constant perfusion. Epithelial injury to mimic mechanisms seen in idiopathic pulmonary fibrosis was induced in the microchannels by perfusing with H2O2 or bleomycin. Migration of BMSC towards the injured epithelium was observed as well as cell-cell interaction between the two cell types was also seen. Conclusion: We demonstrate a novel microfluidic device aimed at showing interactions between different cell types on the basis of a changing microenvironment. Also we were able to confirm interaction between injured alvolar epithelium and BMSC, and showed that BMSC try to heal the injured epitelium.

Comparative analysis of canine monocyte- and bone-marrow-derived dendritic cells

Dendritic cells (DC) represent a heterogeneous cell family of major importance for innate immune responses against pathogens and antigen presentation during infection, cancer, allergy and autoimmunity. The aim of the present study was to characterize canine DC generated in vitro with respect to their phenotype, responsiveness to toll-like receptor (TLR) ligands and T-cell stimulatory capacity. DC were derived from monocytes (MoDC) and from bone marrow hematopoietic cells cultured with either Flt3-ligand (FL-BMDC) or with GM-CSF (GM-BMDC). All three methods generated cells with typical DC morphology that expressed CD1c, CD11c and CD14, similar to macrophages. However, CD40 was only found on DC, CD206 on MPhi and BMDC, but not on monocytes and MoDC. CD1c was not found on monocytes but on all in vitro differentiated cells. FL-BMDC and GM-BMDC were partially positive for CD4 and CD8. CD45RA was expressed on a subset of FL-BMDC but not on MoDC and GM-BMDC. MoDC and FL-DC responded well to TLR ligands including poly-IC (TLR2), Pam3Cys (TLR3), LPS (TLR4) and imiquimod (TLR7) by up-regulating MHC II and CD86. The generated DC and MPhi showed a stimulatory capacity for lymphocytes, which increased upon maturation with LPS. Taken together, our results are the basis for further characterization of canine DC subsets with respect to their role in inflammation and immune responses.

The Effect Of Osteoprotection On Immune Reconstitution Post Bone Marrow Transplant

Specialized microenvironments have been known to strongly influence stem cell fate in hematopoiesis. The interplay between osteolineage cells, specifically the mature osteoblast, and the hematopoietic stem cell (HSC) niche have been of particular note. Recently, preliminary unpublished data obtained in the Scadden laboratory suggests the critical role of the osteoblast in regulating T cells. The goal of this project was to initially determine whether stimulating the osteoblast in the HSC niche leads to increased immune reconstitution after hematopoietic stem cell transplant (HSCT). These results indicated that while bone manipulation pre-transplant may have a positive effect on T and B lymphocyte cell recovery, bone manipulation post-transplant seems to have a suppressing effect. Additionally, stimulation of the osteoblast may have an inhibitory effect on the regeneration of GR1+ myeloid cells. Based on these results, we then sought to determine how osteoprotection pre-HSCT modifies the kinetics of graft-versus-host disease (GVHD) and impacts the regeneration of immune cells. The data from this phase of my experiment suggests a possible immediate benefit in stimulation of the osteoblast in response to GVHD prior to HSCT. The overall results from my thesis project demonstrate a promising relationship between pre-HSCT stimulation of the osteoblast and lymphocyte recovery post-HSCT. ¿

Equine peripheral blood-derived progenitors in comparison to bone marrow-derived mesenchymal stem cells

Fibroblast-like cells isolated from peripheral blood of human, canine, guinea pig, and rat have been demonstrated to possess the capacity to differentiate into several mesenchymal lineages. The aim of this work was to investigate the possibility of isolating pluripotent precursor cells from equine peripheral blood and compare them with equine bone marrow-derived mesenchymal stem cells. Human mesenchymal stem cells (MSCs) were used as a control for cell multipotency assessment. Venous blood (n = 33) and bone marrow (n = 5) were obtained from adult horses. Mononuclear cells were obtained by Ficoll gradient centrifugation and cultured in monolayer, and adherent fibroblast-like cells were tested for their differentiation potential. Chondrogenic differentiation was performed in serum-free medium in pellet cultures as a three-dimensional model, whereas osteogenic and adipogenic differentiation were induced in monolayer culture. Evidence for differentiation was made via biochemical, histological, and reverse transcription-polymerase chain reaction evaluations. Fibroblast-like cells were observed on day 10 in 12 out of 33 samples and were allowed to proliferate until confluence. Equine peripheral blood-derived cells had osteogenic and adipogenic differentiation capacities comparable to cells derived from bone marrow. Both cell types showed a limited capacity to produce lipid droplets compared to human MSCs. This result may be due to the assay conditions, which are established for human MSCs from bone marrow and may not be optimal for equine progenitor cells. Bone marrow-derived equine and human MSCs could be induced to develop cartilage, whereas equine peripheral blood progenitors did not show any capacity to produce cartilage at the histological level. In conclusion, equine peripheral blood-derived fibroblast-like cells can differentiate into distinct mesenchymal lineages but have less multipotency than bone marrow-derived MSCs under the conditions used in this study.

Retinal pigment epithelium damage enhances expression of chemoattractants and migration of bone marrow-derived stem cells

PURPOSE: To characterize chemoattractants expressed by the retinal pigment epithelium (RPE) after sodium iodate (NaIO3)-induced damage and to investigate whether ocular-committed stem cells preexist in the bone marrow (BM) and migrate in response to the chemoattractive signals expressed by the damaged RPE. METHODS: C57/BL6 mice were treated with a single intravenous injection of NaIO3 (50 mg/kg) to create RPE damage. At different time points real-time RT-PCR, ELISA, and immunohistochemistry were used to identify chemoattractants secreted in the subretinal space. Conditioned medium from NaIO3-treated mouse RPE was used in an in vitro assay to assess chemotaxis of stem cell antigen-1 positive (Sca-1+) BM mononuclear cells (MNCs). The expression of early ocular markers (MITF, Pax-6, Six-3, Otx) in migrated cells and in MNCs isolated from granulocyte colony-stimulating factor (G-CSF) and Flt3 ligand (FL)-mobilized and nonmobilized peripheral blood (PB) was analyzed by real-time RT-PCR. RESULTS: mRNA for stromal cell-derived factor-1 (SDF-1), C3, hepatocyte growth factor (HGF), and leukemia inhibitory factor (LIF) was significantly increased, and higher SDF-1 and C3 protein secretion from the RPE was found after NaIO3 treatment. A higher number of BMMNCs expressing early ocular markers migrated to conditioned medium from damaged retina. There was also increased expression of early ocular markers in PBMNCs after mobilization. CONCLUSIONS: Damaged RPE secretes cytokines that have been shown to serve as chemoattractants for BM-derived stem cells (BMSCs). Retina-committed stem cells appear to reside in the BM and can be mobilized into the PB by G-CSF and FL. These stem cells may have the potential to serve as an endogenous source for tissue regeneration after RPE damage.

Mandibular reconstruction using a combination graft of rhBMP-2 with bone marrow cells expanded in vitro

BACKGROUND: The aim of this study was to evaluate the efficacy of a combination graft, using recombinant human bone morphogenetic protein-2 (rhBMP-2) and culture-expanded cells derived from bone marrow, for bone regeneration in a nonhuman primate mandible. METHODS: Five Japanese monkeys were used. Three milliliters of bone marrow was obtained from the tibia and plated into culture flasks. Adherent cells were cultured until near confluence; then, the proliferated cells were transferred to a three-dimensional culture system using collagen beads as the cell carrier. The medium was supplemented with ascorbic acid, beta-glycerophosphate, and dexamethasone to promote osteoblastic differentiation. After further proliferation on beads, the cells were mixed with a collagen sponge that was impregnated with rhBMP-2 and grafted into surgically created segmental bone defects of the mandibles. Three animals received this treatment, and either culture-expanded cells alone or collagen beads without cells were implanted into the remaining two monkeys as controls. The animals were killed 24 weeks after surgery, and the results were assessed by radiographic and histologic evaluation. RESULTS: The combination graft of culture-expanded bone marrow cells with rhBMP-2 in a collagen sponge regenerated the mandibular bone completely. By contrast, the graft of culture-expanded cells alone resulted in only a small amount of bone formation, and the implantation of collagen beads alone led to no bone formation. CONCLUSION: The combination graft of rhBMP-2 and culture-expanded cells, which requires only a small amount of bone marrow, is a reliable method for the reconstruction of segmental bone defects of the mandible.

MRI characteristics and histology of bone marrow lesions in dogs with experimentally induced osteoarthritis

Signal changes within the bone marrow adjacent to osteoarthritic joints are commonly seen on magnetic resonance (MR) images in humans and in dogs. The histological nature of these lesions is poorly known. In this study, we describe the MR imaging of bone marrow lesions adjacent to the stifle joints of dogs with experimental osteoarthritis over 13 months. Histology of the proximal tibia at the end of the study was compared with the last MR imaging findings. In five adult dogs, the left cranial cruciate ligament was transected. Post-operatively, MR imaging was performed at 1, 2, 3, 4, 6, 8, and 13 months. Dogs were euthanised after 13 months and histological specimen of the proximal tibia were evaluated. Bone marrow edema like MR imaging signal changes were seen in every MR examination of all dogs in one or more locations of the proximal tibia and the distal femur. Lesions varied in size and location throughout the whole study with the exception of constantly seen lesions in the epiphyseal and metaphyseal region at the level of the tibial eminence. On histology, hematopoiesis and myxomatous transformation of the bone marrow and/or intertrabecular fibrosis without signs of bone marrow edema were consistent findings in the areas corresponding to the MR imaging signal changes. We conclude that within the bone marrow, zones of increased signal intensity on fat suppressed MR images do not necessarily represent edema but can be due to cellular infiltration. Contrary to humans, hematopoiesis is seen in bone marrow edema-like lesions in this canine model of osteoarthritis.

Endogenous bone marrow derived cells express retinal pigment epithelium cell markers and migrate to focal areas of RPE damage

PURPOSE: The aim of the present study was to investigate whether bone marrow-derived cells (BMCs) can be induced to express retinal pigment epithelial (RPE) cell markers in vitro and can home to the site of RPE damage after mobilization and express markers of RPE lineage in vivo. METHODS: Adult RPE cells were cocultured with green fluorescence protein (GFP)-labeled stem cell antigen-1 positive (Sca-1(+)) BMCs for 1, 2, and 3 weeks. Cell morphology and expression of RPE-specific markers and markers for other retinal cell types were studied. Using an animal model of sodium iodate (NaIO(3))-induced RPE degeneration, BMCs were mobilized into the peripheral circulation by granulocyte-colony stimulating factor, flt3 ligand, or both. Immunocytochemistry was used to identify and characterize BMCs in the subretinal space in C57BL/6 wild-type (wt) mice and GFP chimeric mice. RESULTS: In vitro, BMCs changed from round to flattened, polygonal cells and expressed cytokeratin, RPE65, and microphthalmia transcription factor (MITF) when cocultured in direct cell-cell contact with RPE. In vivo, BMCs were identified in the subretinal space as Sca-1(+) or c-kit(+) cells. They were also double labeled for GFP and RPE65 or MITF. These cells formed a monolayer on the Bruch membrane in focal areas of RPE damage. CONCLUSIONS: Thus, it appears that BMCs, when mobilized into the peripheral circulation, can home to focal areas of RPE damage and express cell markers of RPE lineage. The use of endogenous BMCs to replace damaged retinal tissue opens new possibilities for cell replacement therapy in ophthalmology.

Sensitivity of osteoblasts, fibroblasts, bone marrow cells, and dendritic cells to 5-aminolevulinic acid based photodynamic therapy

INTRODUCTION: Photodynamic therapy with 5-aminolevulinic acid (5-ALA-PDT) exerts cell type specific effects on target cells. Since chondrocytes were found to be more resistant than osteoblasts to 5-ALA-PDT, the pre-treatment of osteochondral grafts with 5-ALA-PDT may represent a means to devitalize the osseous portion while maintaining functional cartilage. The present study was designed to determine the effects of 5-ALA-PDT in vitro on cell populations residing in skeletal tissues. METHODS: Osteoblasts, fibroblasts, bone marrow cells, and dendritic cells were incubated with 0.5 mM 5-ALA for 4 h. Protoporphyrin IX (PpIX) accumulation and after exposure to light cellular functions were assessed for up to 6 days. RESULTS: Accumulation of PpIX reached a plateau at 0.5 mM in osteoblasts, fibroblasts, and dendritic cells, and at 2.0 mM in bone marrow cells. At 0.5 mM 5-ALA, similar responses to illumination were observed in all cells with a survival rate of less than 12% at a light dose of 20 J/cm(2). The function of osteoblasts (proliferation, levels of mRNA encoding collagen type I, alkaline phosphatase activity) and fibroblasts (proliferation, levels of mRNAs encoding collagens type I and III) was not affected, when the cells were treated with 5-ALA and light doses of < or =10 J/cm(2). Paralleling the reduction of viable cells after 5-ALA-PDT, the capacity of dendritic cells to stimulate T cells in a mixed leukocyte reaction decreased to 4+/-2% at 20 J/cm(2). CONCLUSION: The investigated cell types were sensitive to 5-ALA-PDT and the residual cell debris did not elicit an allogenic response. These findings, together with the resistance of chondrocytes to 5-ALA-PDT, encourage the further investigation of this protocol in the pretreatment of osteochondral allografts.