Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Tulane University

Tulane University School of Medicine

The use of bone marrow-derived human multipotent stromal cells (hMSC) in cell-based therapies has dramatically increased in recent years, as researchers have exploited the ability of these cells to migrate to sites of tissue injury, inflammation, and tumors. Our group established that hMSC respond to "danger" signals--by-products of damaged, infected or inflamed tissues--via activation of Toll-like receptors (TLRs). However, little is known regarding downstream signaling mediated by TLRs in hMSC.

Toll-like receptor 3 and suppressor of cytokine signaling proteins regulate CXCR4 and CXCR7 expression in bone marrow-derived human multipotent stromal cells.

Currently, patients with end-stage lung disease are limited to lung transplantation as their only treatment option. Unfortunately, the lungs available for transplantation are few. Moreover, transplant recipients require life-long immune suppression to tolerate the transplanted lung. A promising alternative therapeutic strategy is decellularization of whole lungs, which permits the isolation of an intact scaffold comprised of innate extracellular matrix (ECM) that can theoretically be recellularized with autologous stem or progenitor cells to yield a functional lung. Nonhuman primates (NHP) provide a highly relevant preclinical model with which to assess the feasibility of recellularized lung scaffolds for human lung transplantation. Our laboratory has successfully accomplished lung decellularization and initial stem cell inoculation of the resulting ECM scaffold in an NHP model. Decellularization of normal adult rhesus macaque lungs as well as the biology of the resulting acellular matrix have been extensively characterized. Acellular NHP matrices retained the anatomical and ultrastructural properties of native lungs with minimal effect on the content, organization, and appearance of ECM components, including collagen types I and IV, laminin, fibronectin, and sulfated glycosaminoglycans (GAG), due to decellularization. Proteomics analysis showed enrichment of ECM proteins in total tissue extracts due to the removal of cells and cellular proteins by decellularization. Cellular DNA was effectively removed after decellularization (∼92% reduction), and the remaining nuclear material was found to be highly disorganized, very-low-molecular-weight fragments. Both bone marrow- and adipose-derived mesenchymal stem cells (MSC) attach to the decellularized lung matrix and can be maintained within this environment in vitro, suggesting that these cells may be promising candidates and useful tools for lung regeneration. Analysis of decellularized lung slice cultures to which MSC were seeded showed that the cells attached to the decellularized matrix, elongated, and proliferated in culture. Future investigations will focus on optimizing the recellularization of NHP lung scaffolds toward the goal of regenerating pulmonary tissue. Bringing this technology to eventual human clinical application will provide patients with an alternative therapeutic strategy as well as significantly reduce the demand for transplantable organs and patient wait-list time.

A nonhuman primate model of lung regeneration: detergent-mediated decellularization and initial in vitro recellularization with mesenchymal stem cells.

Cell-based therapies (CBTs) are quickly taking hold as a revolutionary new approach to treat many human diseases. Among the cells used in these treatments, multipotent mesenchymal stromal cells, also often and imprecisely termed mesenchymal stem cells (MSC), are widely used because they are considered clinically safe, unique in their immune-modulating capabilities, easily obtained from adult tissues, and quickly expanded as well as stored. However, despite these established advantages, there are limiting factors to employing MSCs in these therapeutic strategies. Foremost is the lack of a general consensus on a definition of these cells, marring efforts to prepare homogeneous lots and more importantly complicating their in vitro and in vivo investigation. Furthermore, although one of the most profound clinical effects of MSC intravenous administration is the modulation of host immune responses, no adequate ex vivo assays exist to consistently predict the therapeutic effect of each MSC lot in the treated patient. Until these issues are addressed, this very promising and safe new therapeutic approach cannot be used to its full advantage. However, these confounding issues do present exciting opportunities. The first is an opportunity to discover unknown aspects of host immune responses because the unique effect driven by MSC infusion on a patient's immunity has not yet been identified. In addition, there is an opportunity to develop methods, tests, and tools to better define MSCs and MSC-based therapy and provide consistency in preparation and effect. To this end, my laboratory recently developed a new approach to induce uniform pro-inflammatory MSC1 and anti-inflammatory MSC2 phenotypes from bone marrow-derived MSC preparations. I anticipate that MSC1 and MSC2 provide convenient tools with which to address some of these limitations and will help advance safe and effective CBTs for human disease.

New Cell-Based Therapy Paradigm: Induction of Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells into Pro-Inflammatory MSC1 and Anti-inflammatory MSC2 Phenotypes.

Currently, there are many promising clinical trials using mesenchymal stem cells (MSCs) in cell-based therapies of numerous diseases. Increasingly, however, there is a concern over the use of MSCs because they home to tumors and can support tumor growth and metastasis. For instance, we established that MSCs in the ovarian tumor microenvironment promoted tumor growth and favored angiogenesis. In parallel studies, we also developed a new approach to induce the conventional mixed pool of MSCs into two uniform but distinct phenotypes we termed MSC1 and MSC2.

Mesenchymal stem cell 1 (MSC1)-based therapy attenuates tumor growth whereas MSC2-treatment promotes tumor growth and metastasis.

Mesenchymal stem cells (MSCs) are very attractive candidates in cell-based strategies that target inflammatory diseases. Preclinical animal studies and many clinical trials have demonstrated that human MSCs can be safely administered and that they modify the inflammatory process in the targeted injured tissue. Our laboratory developed a novel method that optimizes the anti-inflammatory effects of MSCs. We termed the cells prepared by this method MSC2. In this study, we determined the effects of MSC2-based therapies on an inflammation-linked painful diabetic peripheral neuropathy (pDPN) mouse model. Streptozotocin-induced diabetic mice were treated with conventionally prepared MSCs, MSC2, or vehicle at three specific time points. Prior to each treatment, responses to radiant heat (Hargreaves) and mechanical stimuli (von Frey) were measured. Blood serum from each animal was collected at the end of the study to compare levels of inflammatory markers between the treatment groups. We observed that MSC2-treated mice had significant improvement in behavioral assays compared with the vehicle and MSC groups, and moreover these responses did not differ from the observations seen in the healthy wild-type control group. Mice treated with conventional MSCs showed significant improvement in the radiant heat assay, but not in the von Frey test. Additionally, mice treated with MSC2 had decreased serum levels in many proinflammatory cytokines compared with the values measured in the MSC- or vehicle-treated groups. These findings indicate that MSC2-based therapy is a new anti-inflammatory treatment to consider in the management of pDPN.

Anti-inflammatory mesenchymal stem cells (MSC2) attenuate symptoms of painful diabetic peripheral neuropathy.

INTRODUCTION: Adipose-derived stem cells (ASCs) have emerged as important regulators of inflammatory/immune responses in vitro and in vivo and represent attractive candidates for cell-based therapies for diseases that involve excessive inflammation. Acute lung injury (ALI) is an inflammatory condition for which treatment is mainly supportive due to lack of effective therapies. In this study, the therapeutic effects of ASC-based therapy were assessed in vivo by comparison of the anti-inflammatory properties of both human and murine ASCs in a mouse model of lipopolysaccharide (LPS)-induced ALI. METHODS: Human ASCs (hASCs) or mouse ASCs (mASCs) were delivered to C57Bl/6 mice (7.5 × 105 total cells/mouse) by oropharyngeal aspiration (OA) four hours after the animals were challenged with lipopolysaccharide (15 mg/kg). Mice were sacrificed 24 and 72 hours after LPS exposure, and lung histology examined for evaluation of inflammation and injury. Bronchoalveolar lavage fluid (BALF) was analyzed to determine total and differential cell counts, total protein and albumin concentrations, and myeloperoxidase (MPO) activity. Cytokine expression in the injured lungs was measured at the steady-state mRNA levels and protein levels for assessment of the degree of lung inflammation. RESULTS: Both human and mouse ASC treatments provided protective anti-inflammatory responses. There were decreased levels of leukocyte (for example neutrophil) migration into the alveoli, total protein and albumin concentrations in BALF, and MPO activity after the induction of ALI following both therapies. Additionally, cell therapy with both cell types effectively suppressed the expression of proinflammatory cytokines and increased the anti-inflammatory cytokine interleukin 10 (IL-10). Overall, the syngeneic mASC therapy had a more potent therapeutic effect than the xenogeneic hASC therapy in this model. CONCLUSIONS: Treatment with hASCs or mASCs significantly attenuated LPS-induced acute lung injury in mice. These results suggest a potential benefit for using an ASC-based therapy to treat clinical ALI and may possibly prevent the development of acute respiratory distress syndrome (ARDS).

Aline M. Betancourt

Center for Stem Cell Research and Regenerative Medicine

Tulane University

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

Aline M. Betancourt

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Tulane University

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

Center for Stem Cell Research and Regenerative Medicine