Final publication is available at http://www.sciencedirect.com/science/article/pii/S0006291X15310664 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19598号 / 医博第4105号 / 新制||医||1014(附属図書館) / 32634 / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 開 祐司, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Thymic stromal lymphopoietin (TSLP), an epithelial-derived cytokine, has a critical role in the development of allergic inflammatory responses and have been implicated in type 2 allergic disease, including asthma, allergic rhinitis, and atopic dermatitis. Genetic polymorphisms in the TSLP gene are among the most commonly cited variants associated with asthma and allergic disease, however, the functional effects of these polymorphism are not fully understood. The objective of this study was to investigate the role of a TSLP polymorphism in the Th2 inflammatory responses of the nasal epithelium, as well as in responding to nasal allergen provocation and intranasal corticosteroid treatment. We cultured nasal epithelial cells from allergic asthmatic subjects and examined cytokine and chemokine secretions and gene expression profiles in response to polyinosinic:polycytidylic acid treatment. To explore the functional consequences of the rs1837253 polymorphism we analyzed the two TSLP gene isoforms, as they have shown dichotomous effects, however, no associations were found between rs1837253 genotype and the expression of TSLP and gene isoforms. We did not find any associations of TSLP or cytokine production between genotypes, or in relation to response to nasal allergen challenge or corticosteroid treatment. Exploration of local and systemic effects of the rs1837253 SNP did not show any differences in response to INCS treatment in vitro or ex vivo. We did demonstrate that nasal epithelial cell-derived factors are capable of stimulating eosinophil/basophil colony forming units in the absence and presence of exogenous IL-3. Overall, the results indicate a role of the nasal epithelium in driving eosinophil/basophil differentiation and highlight the complexity of gene-environment interactions and the mechanisms of asthma and allergic inflammation. / Thesis / Master of Science (MSc)

Infections of fracture fixation implants and synovial structures are a primary cause of complications, increased treatment costs, and mortality in people and horses. Treatment failure is often due to biofilms that are communities of bacteria that are adhered to a surface or to each other and are surrounded in a self-secreted extracellular matrix. The biofilm matrix protects the indwelling bacteria from being killed by antibiotics and the immune system. Biofilms also stimulate chronic inflammation and tissue destruction, including peri-implant osteolysis and subsequent implant failure and chondromalacia with subsequent osteoarthritis. In horses, the resulting lameness, reduced athletic potential, and poor quality of life may necessitate euthanasia. Equine bone marrow-derived mesenchymal stromal cells (MSC) reduce inflammation and promote healing in musculoskeletal injuries and have recently been discovered to have antimicrobial properties. Equine MSC kill planktonic (free-floating) bacteria and prevent biofilm establishment in laboratory models. MSC from mice and people also promote the transition from acute inflammation to tissue regeneration (resolution of inflammation) by secretion of specialized pro-resolving lipid mediators (SPM). Whether equine MSC can disrupt established biofilms of orthopedic pathogens and modulate the inflammatory response to orthopedic biofilms is unknown. Using a novel biofilm-MSC co-culture model, our objectives were two-fold. We investigated whether MSC alone or with amikacin sulfate, an antibiotic used to treat equine orthopedic infections, could reduce biomass, pellicle size, and live bacteria of biofilms of orthopedic infectious agents S. aureus and E. coli. Next, we investigated whether MSC could modulate immune response to S. aureus biofilms by reducing secretion of pro-inflammatory cytokines by peripheral blood mononuclear cells (PBMC) and by secreting SPM. MSC demonstrated partial ability to reduce biofilms but performed differently on S. aureus versus E. coli biofilms. Co-culture of biofilms with MSC significantly reduced pellicle area of biofilms of both bacteria, reduced biomass of S. aureus biofilms, and killed live S. aureus bacteria. MSC combined with amikacin also significantly reduced S. aureus biomass to a greater extent compared to amikacin alone. The resolution in detecting differences between groups for E. coli was diminished because of high variation between biofilms treated with MSC between different donors and between control biofilms between experiments. Using the same experimental system, culture of S. aureus biofilms with MSC in the transwell inserts and PBMC in the bottom wells significantly reduced biofilm size compared to untreated biofilms. Co-culture of MSC and PBMC with S. aureus biofilms also significantly increased detection of multiple SPM on lipid chromatography-mass spectrometry compared to MSC or PBMC cultures alone. Using a commercial equine multiplex bead ELISA, multiple inflammatory cytokines and chemokines were increased when S. aureus biofilms were cultured with MSC and PBMC; however, these were not different from untreated biofilms. Our results indicate that the utility of MSC in combating orthopedic biofilm infections lies in their ability to disrupt the biofilm matrix and promote inflammation resolution. These findings support continued investigation into and optimization of the anti-biofilm mechanisms of MSC. / Doctor of Philosophy / Biofilms are coating layers made by bacteria to protect them from being killed by antibiotics or the immune system. Biofilms result in untreatable infection, chronic inflammation and tissue destruction in people and horses with bone and joint infections. The resulting complications, including pain, reduced mobility, and poor quality of life, may result in horses being euthanized. Equine bone marrow-derived mesenchymal stromal cells (MSC) kill free floating bacteria in laboratory models and reduce inflammation in orthopedic injuries. Whether MSC can disrupt formed biofilms and reduce inflammation resulting from biofilm infections is unknown. Using a laboratory model, our objectives were to determine: 1) whether MSC alone or with an antibiotic used to treat orthopedic infections in horses can disrupt biofilms and kill indwelling live bacteria of orthopedic infectious agents S. aureus and E. coli, and 2) whether MSC can modify the immune response to S. aureus biofilms. MSC demonstrated some biofilm reducing ability but performed differently on S. aureus versus E. coli biofilms. Specifically, MSC reduced the size of biofilms of both bacteria, reduced the coating layer of S. aureus biofilms alone and to a greater extent when combined with the antibiotic, and killed live S. aureus bacteria. Using the same system, culture of MSC with S. aureus biofilms and peripheral blood mononuclear cells (PBMC), a type of white blood cell, reduced biofilm size compared to controls. The addition of MSC and PBMC to S. aureus biofilms also increased detection of fatty acid-derived signals that promote resolution of inflammation, compared to controls. Multiple inflammatory cytokines and chemokines were increased with culture of MSC and PBMC with S. aureus biofilms but were not different from untreated biofilms. These results indicate that MSC may be useful to combat biofilm infections by breaking down the coating layer of biofilms and by promoting resolution of inflammation. Taken together, our results support continued investigation into the potential of MSC as a treatment for orthopedic biofilm infections. The potential of MSC to simultaneously break down biofilms and mitigate inflammation in orthopedic infections would improve cure rates and overall outcomes for horses and people afflicted with orthopedic biofilm infections.

Detrimental immune and inflammatory responses contribute to the pathogenesis of various conditions, including Crohn’s disease, Lupus, and sepsis.1,2,3 Unfortunately, novel treatments for detrimental immune and inflammatory responses have been met with little success. Mesenchymal stromal cells (MSCs) represent a promising cellular therapy to treat immune and inflammatory disorders due to their ability to suppress the immune system. However, despite their promise, clinical trials that have employed MSC cellular therapies have produced varying and sometimes conflicting results. These discrepancies have been partially attributed to the cellular heterogeneity within MSC populations. To address these discrepancies, I performed transcriptomic and proteomic analysis of MSCs with varying immunomodulatory capacity to identify robust immunomodulatory biomarkers and gain better mechanistic insights into MSC immunomodulatory function. In this study, MSCs with differing immunomodulatory function were identified and the effect of in vitro passaging and proinflammatory induction on immunomodulatory ability was characterized. To characterize MSC immunomodulatory control mechanisms, RNA sequencing and proteomic analyses were performed on MSCs with different immunomodulatory capabilities. These analyses enabled the identification of potential immunomodulatory biomarkers and regulatory mechanisms. Finally, to test the therapeutic efficacy of immunomodulatory MSC subpopulations, I developed a humanize mouse model for sepsis. Overall, this work contributes to our understanding of MSC immunomodulation and to the development of a robust MSC cellular therapeutics.

In the United States, nearly half a million bone graft operations are performed annually to repair defects arising from birth defects, trauma, and disease, making bone the second most transplanted tissue. Autogenous bone is the current gold standard for bone grafts; however it is in limited supply and results in a second injury at the donor site. A promising alternative is a tissue engineered bone graft composed of a biomaterial scaffold, pharmaceutics, and osteoprogenitor cells. One source of osteoprogenitor cells is bone marrow stroma, which can be obtained from the patient - minimizing the risk of an immune response - directed in vitro to proliferate, and differentiate into a bone-like tissue. To date, tissue engineered bone grafts have not been clinically effective; thus, strategies must be developed to improve efficacy. I hypothesize that to facilitate tissue healing in a manner similar to autogenous bone tissue engineering bone must possess a mineralized collagen matrix to support tissue integration, and angiogenic factors to stimulate vascular infiltration, and osteogenic factors to direct normal bone remodeling. I propose that these factors can be synthesized by osteoprogenitor cells in vitro when cultured under the appropriate conditions. Previous work has demonstrated that perfusion culture of osteoprogenitor cells within 3D scaffolds stimulates phenotypic markers of osteoblastic differentiation, but those studies did not determine whether the effects were a consequence of shear stress or increased nutrient availability. Consequently, this work has involved studies in a planar geometry, where nutrient effects are negligible. Three studies that characterize the effect of fluid flow on osteoblastic differentiation of osteoprogenitor cells are presented here. The objective of the first study was to determine the effect of shear stress magnitude on cell density and osteocalcin deposition. In this study, radial flow chambers were used to generate a spatially dependent range of shear stresses (0.36 to 2.7 dynes/cm2) across single substrates, and immunofluorescent techniques were used to assay cell phenotype as a function of shear stress. The objective of the second study was to determine the effect of the duration of fluid flow on cell density and phenotypic markers of differentiation. Here, parallel plate flow chambers were used to generate a single shear stress at the cell surface, and entire cell layers were assayed for expression of osteoblastic genes. The objective of the third study was to compare continuous and intermittent fluid flow strategies. In this study, a microprocessor-controlled actuator was added to the flow loop to periodically halt flow, and markers of mechanosensation and osteoblastic differentiation were measured. These studies demonstrated that shear stresses of 0.36 to 2.7 dynes/cm2 stimulate late phenotypic markers of osteoblastic differentiation but not cell proliferation. In addition, this osteogenic effect is sensitive to duration of fluid flow but insensitive to the magnitude of shear stress. Further, intermittent fluid flow enhances cell retention, biochemical markers of mechanotransduction, and synthesis of the angiogenic factor vascular endothelial growth factor (VEGF). Thus, these studies suggest that intermittent fluid flow may be an attractive component of a biodynamic bioreactor for in vitro manufacture of clinically effective tissue engineered bone grafts. Future studies will further investigate intermittent fluid flow strategies and three-dimensional studies with scaffolds suitable for bone tissue engineering. / Ph. D.

Bone is the second most transplanted tissue after blood and the need for bone graft materials continues to rise at an average annual growth rate of over 18%. An engineered bone substitute consisting of a bone-like extracellular matrix deposited on the internal pores of a resorbable biomaterial scaffold is postulated to stimulate normal bone remodeling when implanted in vivo. Part one of this engineering strategy, the deposition of bone-like extracellular matrix, can be achieved by the directed differentiation of progenitor cells such as bone marrow stromal cells (BMSCs). Part two of the engineering strategy, the biomaterial scaffold, can be fabricated with the appropriate mechanical properties using a synthetic polymer system with tunable properties like polyurethanes. Finally, BMSCs seeded within the biomaterial scaffold can be cultured in a perfusion flow bioreactor to stimulate osteoblastic differentiation and the deposition of bioactive factors. Using the three-part engineering strategy described, I hypothesize that the extracellular matrix produced by BMSCs can be modulated by two stimuli: the stiffness of the scaffold and perfusion flow. First, I propose that culturing BMSCs on polyurethane scaffolds with increasing stiffness will increase markers of osteoblastic differentiation. Secondly, I suggest that mechanically stimulating BMSCs with novel perfusion strategies will also increase markers of osteoblastic differentiation. In aim 1, a family of segmented degradable poly(esterurethane urea)s (PEUURs) were synthesized. The modulus of the PEUUR materials was systematically increased from 0.18 to 0.80 MPa by systematically increasing the molecular weight of the poly(ε-caprolactone) (PCL) soft segment from 1425 to 2700 Da. BMSCs were cultured on both rigid polymer films and on porous foam scaffolds to dissociate the effect of variation in polymer chemistry from the effect of scaffold modulus on cell phenotype. These studies demonstrated changes in osteoblastic differentiation as measured by prostaglandin E2 production, alkaline phosphatase activity (ALP) activity, and osteopontin gene expression. However, the increased levels of these phenotypic markers on the PCL 2700 material could not be attributed to scaffold chemistry or modulus. Instead, the differences may be related to polymer crystallinity or surface topography. In aim 2, novel dynamic perfusion strategies were used to investigate the influence of frequency on osteoblastic differentiation. BMSCs were seeded on porous foam scaffolds and exposed to both steady perfusion and pulsatile perfusion at 0.017, 0.050, and 0.083 Hz frequencies. The data presented here demonstrated that while some markers of osteoblastic phenotype such as ALP activity are enhanced by 0.05 Hz pulsatile flow over continuous flow, they are insensitive to frequency at low frequencies. Therefore, future studies will continue to investigate the effect of a larger range of frequencies. Additionally, fluid flow has also been shown to stimulate the deposition of bioactive factors such as BMP-2 and VEGF-A, and these growth factors are known to significantly enhance healing in bone defect models. Therefore, we plan to investigate the effect of dynamic flow strategies on the deposition of these bioactive factors. We propose that an engineered bone graft material containing a bone-like extracellular matrix and producing these growth factors will show more rapid formation of bone when implanted in vivo. / Ph. D.

Keratins are a family of proteins found within human hair, skin and nails, as well as a broad variety of animal tissue. Prior research suggests hydrogel constructs of keratin and keratin derivatives exhibit several mechanical and biological properties that support their use for tissue engineering and regenerative medicine applications. Microparticle formulations of these hydrogels are an intriguing delivery vehicle for drugs and cellular payloads for tissue engineering purposes due to the ability to exploit size, surface area, loading potential and importantly, non-invasive delivery (i.e. injection) of cells and biologics. Here we examine the water-in-oil emulsion synthesis procedure to produce keratin microparticles using an oxidized keratin derivative, keratose (KOS). Analyses of particle size, microstructure, and other characterization techniques were performed. Drug loading characteristics, release kinetics, and feasibility of use in two different microparticles was subsequently investigated, first using a model-drug and later testing an antibiotic payload on bacterial cultures to validate antibacterial applications. A suspension culture technique was developed to load bone marrow-derived mesenchymyal stromal cells (BM-MSCs), testing the capacity to maintain viability and express key protein-based factors in cell growth and development. Finally, we tested the in vitro effects of cell-loaded microparticles on the L6 skeletal muscle cell line to determine potentially beneficial outcomes for skeletal muscle tissue regeneration. Largely spherical particles with a porous internal structure were obtained, displaying hydrogel properties and forming viscoelastic gels with small differences between synthesis components (solvents, crosslinkers), generating tailorable properties. The uniquely fibrous microstructure of KOS particles may lend them to applications in rapid drug release or other payload delivery wherein a high level of biocompatibility is desired. Data showed an ability to inhibit bacterial growth in the emulsion-generated system, and thereby demonstrated the potential for a keratin-based microparticle construct to be used in wound healing applications. Dense cell populations were loaded onto particles. Particles maintained cell viability, even after freeze-thaw cycling, and provided a material substrate that supported cell attachment through the formation of focal adhesions. Finally, in vitro studies show that both KOS and BM-MSCs support varying aspects of skeletal muscle development, with combinatorial treatments of cell-loaded particles conferring the greatest growth responses. / Doctor of Philosophy / Keratins and keratin hydrogels may exhibit several properties that support their use for tissue engineering and regenerative medicine applications. Microparticle formulations of these hydrogels are an intriguing delivery vehicle for payloads for tissue engineering purposes. Here we examine the water-in-oil emulsion synthesis procedure to produce keratin microparticles that were analyzed based on drug loading characteristics. A suspension culture technique was developed to load bone marrow-derived mesenchymyal stromal cells (BM-MSCs). Finally, we tested these products to determine potentially beneficial outcomes for skeletal muscle tissue regeneration. Particles with a porous structure were obtained. The microstructure of these particles may lend them to applications in drug release or other payload delivery. Data showed an ability to load and unload specific drug payloads. Dense cell populations were loaded onto particles. Finally, studies show that both keratin and BM-MSCs support skeletal muscle development, with combinatorial treatments of cell-loaded particles conferring the greatest growth responses.

Falls among the elderly are a major source of injury, often leading to serious fractures, hospitalization, and death. Osteoporosis (OP) is a global problem intimately related with these fractures, characterized by reduced bone mass, increased bone fragility. There exists a high failure rate in the translation of treatments to osteoporotic populations. Mesenchymal stromal cell (MSC) transplantation as a therapeutic strategy for OP has not yet been examined in clinical trials. This may be attributed to the mixed findings of pre-clinical studies aimed at determining the efficacy of MSC therapy towards bone regeneration in OP. The most common animal model of OP is ovariectomy (OVX) that simulates post-menopausal estrogen loss. A plethora of bone regeneration studies have used OVX models with 12-16 weeks post-OVX periods and have generally reported positive results from a variety of treatment modalities, including MSC therapy. However, the use of the minimum post-OVX period may not be appropriate to reflect the global changes in regenerative potential of OP patients. In our research group's previous study, MSC were isolated from a minimum 60 week post-OVX rat model, representing a severe case of OP. The MSC isolated from these animals are a unique cell population that we expect may better represent the outcomes of autologous cell therapies for the older patient population in the clinic. In the present study, adipose and bone marrow derived MSC from OVX and age-matched animals were evaluated for their osteogenic and adipogenic differentiation potentials in culture through passage 10. Results from this study suggest that bone marrow derived-MSC maintain their phenotype and functionality more effectively than adipose derived-MSC in OP. Further investigations used regenerative medicine approaches for cell expansion on keratin protein coated microcarriers in static culture. Hair-derived keratin biomaterials have demonstrated their utility as carriers of biologics and drugs for tissue engineering. An optimal microcarrier was selected that demonstrated superior retention of the protein coating through electrostatic interactions and high cell viability. Finally, the integration of cell-microcarriers into a perfusion bioreactor system was explored. Preliminary results demonstrated the feasibility of MSC growth and differentiation on microcarrier based packed beds. Moreover, AD-MSC from OP rats were unresponsive to both inductive media and shear stress related osteogenic cues. These results highlight the complexity and challenges associated with the MSC regenerative strategy. / Doctor of Philosophy / Osteoporosis is a skeletal disease that results in reduced bone mass, increased bone fragility and fracture risk. Osteoporotic patients who experience falls suffer serious fractures, hospitalization, and poor bone healing. Several different therapies have been developed for the treatment of osteoporosis, though many are unable to translate from the bench to the clinical population. A popular treatment being investigated is the application of mesenchymal stromal cells (MSC) for fracture repair and the reversal of osteoporotic bone losses. However, cells isolated from aged and osteoporotic patients have been shown to have deficient bone forming properties. Nevertheless, animal models of osteoporosis applying this treatment report amelioration of bone loss. This work seeks to examine a more clinically relevant rat model of osteoporosis. Typical osteoporosis models use an ovariectomy procedure to simulate post-menopausal bone loss on relatively young animals and conduct short-term studies. These studies may not accurately reflect the global regenerative changes in osteoporosis patients or the impaired MSC properties. Adipose and bone marrow derived MSC from a long term ovariectomy model were investigated for their regenerative potentials. MSC growth and bone forming potential was evaluated on keratin protein coated microcarriers in both static and perfusion cultures. Results from this study suggest that bone marrow derived MSC maintain their phenotype and functionality more effectively than adipose derived MSC in osteoporosis. Further preliminary results demonstrated the feasibility of MSC growth and differentiation on microcarrier based packed beds. These results highlight the complexity and challenges associated with the MSC regenerative strategy.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecological malignancy due to the insufficient accurate screening programs for the early detection of EOC. To improve the accuracy of the early detection, there is a need to deeply understand the mechanism of EOC progression and the interaction between cancer cells with their unique microenvironment. Therefore, this work investigated the metabolic shift in the mouse model for progressive ovarian cancer, and evaluated the effects of hypoxic environment, spheroid formation as well as stromal vascular fractions (SVF) on the metabolic shift, proliferation rate, drug resistance and protein markers in functional categories. The results demonstrated an increasingly glycolytic nature of MOSE cells as they progress from a tumorigenic (MOSE-L) to a highly aggressive phenotype (MOSE-FFL), and also showed changes in metabolism during ovarian cancer spheroid formation with SVF under different oxygen levels. More specifically, the hypoxic environment enhanced glycolytic shift by upregulating the glucose uptake and lactate secretion, and the spheroid formation affected the cellular metabolism by increasing the lactate secretion to acidify local environments, modulating the expression of cell adhesion molecules to enhance cell motility and spheroids disaggregation, and up-regulating invasiveness markers and stemness makers to promote ovarian cancer aggressive potential. Hypoxia and spheroid formation decreased ovarian cancer cells growth but increased the chemoresistance, which leads to the promotion of aggressiveness and metastasis potential of ovarian cancer. SVF co-cultured spheroids further increased the glycolytic shift of the heterogeneous of ovarian cancer spheroids, induced the aggressive phenotype by elevating the corresponding protein markers. Decreasing the glycolytic shift and suppression of the proteins/pathways may be used to inhibit aggressiveness or metastatic potential of ovarian cancer heterogeneous of ovarian cancer spheroids, induced the aggressive phenotype by elevating the corresponding protein markers. Decreasing the glycolytic shift and suppression of the proteins/pathways may be used to inhibit aggressiveness or metastatic potential of ovarian cancer. / Master of Science / Epithelial ovarian cancer (EOC) is the leading cause of death from gynecological malignancy due to the usually late detection when the cancer has already spread throughout the peritoneal cavity. Physical, cellular and chemical factors can contribute to EOC progression and metastasis. Critical physical factors are the low oxygen content in the peritoneal cavity (hypoxia) that promotes tumor cells survival, and the formation of tumor spheres, which have been demonstrated to have a more aggressive phenotype. Moreover, obesity has been proposed to support ovarian cancer development and progression. Therefore, this work investigated the impact of oxygen deprivation, sphere formation, and white adipose tissue-derived stromal cells on ovarian cancer cells progression. The results showed that all these factors contribute to the aggressive potential of ovarian cancer cells by increasing the drug resistance, and modulation of cellular metabolism. The understanding of the interactions between ovarian cancer and other cells within their unique microenvironment may provide critical targets for chemotherapeutic interventions that are aimed to control the aggressiveness of ovarian metastases in their hypoxic tumor microenvironment, and enhance the life of women afflicted with ovarian cancer.

Células reticulares fibroblásticas (FRCs, gp38+ e CD31-) e células duplo negativas (DNCs, gp38- e CD31-) são células estromais encontradas em órgãos linfoides secundários, como linfonodos. Enquanto as FRCs têm sido amplamente estudadas, pouco se sabe ainda sobre DNCs. Apesar da função estrutural das FRCs nos linfonodos já estar bem estabelecida, estudos recentes indicam que as FRCs também desempenham um papel fundamental em processos imunológicos, por exemplo, migração celular, ativação e qualidade da resposta imune, além da participação na tolerância periférica. Outra célula estromal em constante estudo são as células-tronco mesenquimais (CTMs), principalmente encontradas na medula óssea. Estas células compartilham similaridades, como por exemplo; são células estromais encontradas em órgãos linfoides, apresentam morfologia e características semelhantes quando cultivadas in vitro e estão envolvidas na resposta imune por mecanismos semelhantes. As CTMs são provenientes de um órgão linfoide primário, cuja função principal não está relacionada à resposta imunológica, entretanto, de acordo com inúmeros trabalhos, estas células possuem capacidade de interferir na ativação de várias células do sistema imunológico. Portanto, nossa hipótese é de que as FRCs e DNCs, que se encontram em um órgão linfoide secundário, cuja função principal remete a resposta imunológica, apresentem também um papel regulador, descrito na literatura como tolerância periférica e contração de uma resposta imunológica já estabelecida. Em nosso estudo mostramos que FRCs e DNCs foram isoladas a partir de linfonodos humanos e devidamente caraterizadas. Evidenciamos que FRCs e DNCs atendem todos os critérios mínimos propostos pela sociedade internacional de terapia celular para serem consideradas células-tronco estromais. Além disso, mostramos que FRCs e DNCs influênciam a proliferação e a expressão de moléculas de homing em linfócitos alogênicos in vitro. Portanto, contribuimos de forma inédita para o entendimento funcional das FRCs e DNCs, visto que estudos em humanos envolvendo estas células são escassos / Fibroblastic reticular cells (FRCs, gp38+ e CD31-) and double-negative cells (DNCs, gp38- e CD31-) are stromal cells found in secondary lymphoid organs, such as lymph nodes. While the FRCs has been widely studied, little is known about DNCs. Despite the structural function of FRCs on lymph nodes is well established, recent studies indicate that FRCs also play a key role in immunological processes, for example, cell migration, immune response activation and quality, beyond their involvement in peripheral tolerance. Another stromal cell type in constant study are mesenchymal stem cells (MSCs), mainly found in bone marrow. These cells share similarities with FRCs and DNCs, for example; they are estromal cells found in lymphoid organs, they present similar morphology and characteristics when cultured in vitro and they are involved in the immune response by similar mechanisms. MSCs are derived from a primary lymphoid organ which the major function is not related to immune response, but according to numerous studies these cells have the capacity of the interfere on activation of various immune cells. Consequently, our hypothesis is that FRCs and DNCs, usually found in secondary lymphoid organ, display immune regulatory roles, which were described in the literature as peripheral tolerance and immune response contraction. In our study we showed that FRCs and DNCs were isolated from human lymph nodes and adequately characterized. We evidenced that FRCs and DNCs meet all minimum criteria proposed by the International Society of Cell Therapy to be considerate a stromal stem cell. Therefore, we contributed in an unpublished manner to the functional understanding of FRCs and DNCs, since human studies involving these cells are scarce