Intravenous multipotent adult progenitor cell treatment decreases inflammation leading to functional recovery following spinal cord injury.

Following spinal cord injury (SCI), immune-mediated secondary processes exacerbate the extent of permanent neurological deficits. We investigated the capacity of adult bone marrow-derived stem cells, which exhibit immunomodulatory properties, to alter inflammation and promote recovery following SCI. In vitro, we show that human multipotent adult progenitor cells (MAPCs) have the ability to modulate macrophage activation, and prior exposure to MAPC secreted factors can reduce macrophage-mediated axonal dieback of dystrophic axons. Using a contusion model of SCI, we found that intravenous delivery of MAPCs one day, but not immediately, after SCI significantly improves urinary and locomotor recovery, which was associated with marked spinal cord tissue sparing. Intravenous MAPCs altered the immune response in the spinal cord and periphery, however biodistribution studies revealed that no MAPCs were found in the cord and instead preferentially homed to the spleen. Our results demonstrate that MAPCs exert their primary effects in the periphery and provide strong support for the use of these cells in acute human contusive SCI.

Development of a surrogate angiogenic potency assay for clinical-grade stem cell production.

BACKGROUND AIMS: Clinical results from acute myocardial infarction (AMI) patients treated with MultiStem®, a large-scale expanded adherent multipotent progenitor cell population (MAPC), have demonstrated a strong safety and benefit profile for these cells. The mechanism of benefit with MAPC treatment is a result, in part, of its ability to induce neovascularization through trophic support. Production of clinical-grade stem cell products requires the development of lot-release criteria based on potency assays that directly reflect the fundamental mechanistic pathway underlying the therapeutic response to verify manufacturing process consistency and product potency. METHODS AND RESULTS: Using an in vitro endothelial tube formation assay, a potency assay has been developed that reflects MAPC pro-angiogenic activity. Serum-free conditioned media collected from MAPC culture induced endothelial tube formation. A proteomic survey of angiogenic factors produced by the cells in vitro revealed candidate factors linked to angiogenic potency. Three cytokines, chemokine (C-X-C motif) ligand 5 (CXCL5), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF), were required for this angiogenic activity. Depletion of any of these factors from the media prevented tube formation, while adding back increasing amounts of these cytokines into the depleted serum-free conditioned media established the lower limits of each of the cytokines required to induce angiogenesis. CONCLUSIONS: A necessary threshold of angiogenic factor expression was established using an in vitro angiogenesis assay. By correlating the levels of the cytokines required to induce tube formation in vitro with levels of the factors found in the spent media from manufacturing production runs, detection of these factors was identified as a surrogate potency assay with defined pass/fail criteria.

Multipotent adult progenitor cells prevent macrophage-mediated axonal dieback and promote regrowth after spinal cord injury.

Macrophage-mediated axonal dieback presents an additional challenge to regenerating axons after spinal cord injury. Adult adherent stem cells are known to have immunomodulatory capabilities, but their potential to ameliorate this detrimental inflammation-related process has not been investigated. Using an in vitro model of axonal dieback as well as an adult rat dorsal column crush model of spinal cord injury, we found that multipotent adult progenitor cells (MAPCs) can affect both macrophages and dystrophic neurons simultaneously. MAPCs significantly decrease MMP-9 (matrix metalloproteinase-9) release from macrophages, effectively preventing induction of axonal dieback. MAPCs also induce a shift in macrophages from an M1, or "classically activated" proinflammatory state, to an M2, or "alternatively activated" antiinflammatory state. In addition to these effects on macrophages, MAPCs promote sensory neurite outgrowth, induce sprouting, and further enable axons to overcome the negative effects of macrophages as well as inhibitory proteoglycans in their environment by increasing their intrinsic growth capacity. Our results demonstrate that MAPCs have therapeutic benefits after spinal cord injury and provide specific evidence that adult stem cells exert positive immunomodulatory and neurotrophic influences.

Intravenous multipotent adult progenitor cell therapy for traumatic brain injury: preserving the blood brain barrier via an interaction with splenocytes.

Recent investigation has shown an interaction between transplanted progenitor cells and resident splenocytes leading to the modulation of the immunologic response in neurological injury. We hypothesize that the intravenous injection of multipotent adult progenitor cells (MAPC) confers neurovascular protection after traumatic brain injury through an interaction with resident splenocytes, subsequently leading to preservation of the blood brain barrier. Four groups of rats underwent controlled cortical impact injury (3 groups) or sham injury (1 group). MAPC were injected via the tail vein at two doses (2*10(6) MAPC/kg or 10*10(6) MAPC/kg) 2 and 24h after injury. Blood brain barrier permeability was assessed by measuring Evans blue dye extravasation (n=6/group). Additionally, splenic mass was measured (n=12/group) followed by splenocyte characterization (n=9/group) including: cell cycle analysis (n=6/group), apoptosis index (n=6/group), cell proliferation (n=6/group), and inflammatory cytokine measurements (n=6/group). Vascular architecture was determined by immunohistochemistry (n=3/group). Traumatic brain injury results in a decrease in splenic mass and increased blood brain barrier permeability. Intravenous infusion of MAPC preserved splenic mass and returned blood brain barrier permeability towards control sham injured levels. Splenocyte characterization indicated an increase in the number and proliferative rate of CD4+ T cells as well as an increase in IL-4 and IL-10 production in stimulated splenocytes isolated from the MAPC treatment groups. Immunohistochemistry demonstrated stabilization of the vascular architecture in the peri-lesion area. Traumatic brain injury causes a reduction in splenic mass that correlates with an increase in circulating immune cells leading to increased blood brain barrier permeability. The intravenous injection of MAPC preserves splenic mass and the integrity of the blood brain barrier. Furthermore, the co-localization of transplanted MAPC and resident CD4+ splenocytes is associated with a global increase in IL-4 and IL-10 production and stabilization of the cerebral microvasculature tight junction proteins.

Crocidolite asbestos and SV40 are cocarcinogens in human mesothelial cells and in causing mesothelioma in hamsters.

Only a fraction of subjects exposed to asbestos develop malignant mesothelioma (MM), suggesting that additional factors may render some individuals more susceptible. We tested the hypothesis that asbestos and Simian virus (SV40) are cocarcinogens. Asbestos and SV40 in combination had a costimulatory effect in inducing ERK1/2 phosphorylation and activator protein-1 (AP-1) activity in both primary Syrian hamster mesothelial cells (SHM) and primary human mesothelial cells (HM). Ap-1 activity caused the expression and activation of matrix metalloprotease (MMP)-1 and MMP-9, which in turn led to cell invasion. Experiments using siRNA and chemical inhibitors confirmed the specificity of these results. The same effects were observed in HM and SHM. Experiments in hamsters showed strong cocarcinogenesis between asbestos and SV40: SV40 did not cause MM, asbestos caused MM in 20% of hamsters, and asbestos and SV40 together caused MM in 90% of hamsters. Significantly lower amounts of asbestos were sufficient to cause MM in animals infected with SV40. Our results indicate that mineral fibers and viruses can be cocarcinogens and suggest that lower amounts of asbestos may be sufficient to cause MM in individuals infected with SV40.

Some oral poliovirus vaccines were contaminated with infectious SV40 after 1961.

Some polio vaccines prepared from 1954 to 1961 were contaminated with infectious SV40. It has been assumed that all polio vaccines were SV40 free in the United States after 1961 and in other countries after 1962. Following a WHO requirement that was prompted by the detection of SV40 in some human tumors, we conducted a multilaboratory study to test for SV40 polio vaccines prepared after 1961. Vaccine samples from 13 countries and the WHO seed were initially tested by PCR. The possible presence of intact and/or infectious SV40 DNA in PCR-positive samples was tested by transfection and infection of permissive CV-1 cells. All results were verified by immunohistochemistry, cloning, and sequencing. All the vaccines were SV40 free, except for vaccines from a major eastern European manufacturer that contained infectious SV40. We determined that the procedure used by this manufacturer to inactivate SV40 in oral poliovirus vaccine seed stocks based on heat inactivation in the presence of MgCl2 did not completely inactivate SV40. These SV40-contaminated vaccines were produced from early 1960s to about 1978 and were used throughout the world. Our findings underscore the potential risks of using primary monkey cells for preparing poliovirus vaccines, because of the possible contamination with SV40 or other monkey viruses, and emphasize the importance of using well-characterized cell substrates that are free from adventitious agents. Moreover, our results indicate possible geographic differences in SV40 exposure and offer a possible explanation for the different percentage of SV40-positive tumors detected in some laboratories.

Transforming growth factor-beta1 in a sterilized tissue derived from the pig small intestine submucosa.

The extracellular matrices of connective tissues contain growth factors such as transforming growth factor (TGF)-beta1. The possibility arises, therefore, that animal connective tissues that have been excised and rendered acellular in the sterilization, lyophilization, and other preparative processes for human use may still retain active growth factors that could contribute to the clinical efficacy of the product. We therefore analyzed 4M guanidine HCl extracts of a sterilized, acellular matrix, Oasis Wound Matrix, for the presence of TGF-beta1 by a sandwich enzyme-linked immunosorbent assay using the soluble type II receptor for TGF-beta to capture the growth factor, and for biological activity by testing the capacity of the extracts to inhibit 3[H]thymidine incorporation into Mv1Lu cells (mink lung epithelial cells). The enzyme-linked immunosorbent assay determined that TGF-beta1 was present at a concentration of 710.9 +/- 157.7 pg/g dry weight of tissue in Oasis and 768.1 +/- 182.1 pg/g dry weight of tissue in SIS (porcine small intestinal submucosa), the disinfected precursor of Oasis. The growth inhibition assays demonstrated that the Oasis extracts inhibited the proliferation of Mv1Lu cells in culture, consistent with the TGF-beta1 in the material having biological activity. Most of the TGF-beta1 survives the sterilization and lyophilization processes in the preparation of the Oasis Wound Matrix, and is functional in its ability to bind to its receptor and, apparently, in its capacity to inhibit growth.

Different susceptibility of human mesothelial cells to polyomavirus infection and malignant transformation.

SV40 has been associated with mesothelioma development. The possible role of the closely related human polyomaviruses JC virus (JCV) and BK virus (BKV) in mesothelioma remained unclear. We found that JCV did not infect human mesothelial cells. BKV and SV40 infected mesothelial cells, expressed viral oncoproteins, and caused similar alterations of key cell regulatory genes. BKV replicated faster than SV40 and caused mesothelial cell lysis, not cellular transformation. SV40 did not lyse mesothelial cells and caused a high rate of transformation. These findings provide a rationale for the observation that SV40 is found in mesothelioma, rather than the ubiquitous human JCV and BKV.