Enhanced early chondrogenesis in articular defects following arthroscopic mesenchymal stem cell implantation in an equine model

Mesenchymal stem cells (MSCs) provide an important source of pluripotent cells for musculoskeletal tissue repair. This study examined the impact of MSC implantation on cartilage healing characteristics in a large animal model. Twelve full-thickness 15-mm cartilage lesions in the femoropatellar articulations of six young mature horses were repaired by injection of a self-polymerizing autogenous fibrin vehicle containing mesenchymal stem cells, or autogenous fibrin alone in control joints. Arthroscopic second look and defect biopsy was obtained at 30 days, and all animals were euthanized 8 months after repair. Cartilage repair tissue and surrounding cartilage were assessed by histology, histochemistry, collagen type I and type II immunohistochemistry, collagen type II in situ hybridization, and matrix biochemical assays. Arthroscopic scores for MSC-implanted defects were significantly improved at the 30-day arthroscopic assessment. Biopsy showed MSC-implanted defects contained increased fibrous tissue with several defects containing predominantly type II collagen. Long-term assessment revealed repair tissue filled grafted and control lesions at 8 months, with no significant difference between stem cell-treated and control defects. Collagen type II and proteoglycan content in MSC-implanted and control defects were similar. Mesenchymal stem cell grafts improved the early healing response, but did not significantly enhance the long-term histologic appearance or biochemical composition of full-thickness cartilage lesions.

MRI indices of functional recovery following intracerebral implantation of a human neural stem cell line in a pre-clinical model of ischaemic stroke

Stem cell therapy for ischaemic stroke is an emerging field in light of an increasing number of patients surviving with permanent disability. Several allogenic and autologous cells types are now in clinical trials with preliminary evidence of safety. Some clinical studies have reported functional improvements in some patients. After initial safety evaluation in a Phase 1 study, the conditionally immortalised human neural stem cell line CTX0E03 is currently in a Phase 2 clinical trial (PISCES-II). Previous pre-clinical studies conducted by ReNeuron Ltd, showed evidence of functional recovery in the Bilateral Asymmetry test up to 6 weeks following transplantation into rodent brain, 4 weeks after middle cerebral artery occlusion. Resting-state fMRI is increasingly used to investigate brain function in health and disease, and may also act as a predictor of recovery due to known network changes in the post-stroke recovery period. Resting-state methods have also been applied to non-human primates and rodents which have been found to have analogous resting-state networks to humans. The sensorimotor resting-state network of rodents is impaired following experimental focal ischaemia of the middle cerebral artery territory. However, the effects of stem cell implantation on brain functional networks has not previously been investigated. Prior studies assessed sensorimotor function following sub-cortical implantation of CTX0E03 cells in the rodent post-stroke brain but with no MRI assessments of functional improvements. This thesis presents research on the effect of sub-cortical implantation of CTX0E03 cells on the resting- state sensorimotor network and sensorimotor deficits in the rat following experimental stroke, using protocols based on previous work with this cell line. The work in this thesis identified functional tests of appropriate sensitivity for long-term dysfunction suitable for this laboratory, and investigated non-invasive monitoring of physiological variables required to optimize BOLD signal stability within a high-field MRI scanner. Following experimental stroke, rats demonstrated expected sensorimotor dysfunction and changes in the resting-state sensorimotor network. CTX0E03 cells did not improve post-stroke functional outcome (compared to previous studies) and with no changes in resting-state sensorimotor network activity. However, in control animals, we observed changes in functional networks due to the stereotaxic procedure. This illustrates the sensitivity of resting-state fMRI to stereotaxic procedures. We hypothesise that the damage caused by cell or vehicle implantation may have prevented functional and network recovery which has not been previously identified due to the application of different functional tests. The findings in this thesis represent one of few pre-clinical studies in resting-state fMRI network changes post-stroke and the only to date applying this technique to evaluate functional outcomes following a clinically applicable human neural stem cell treatment for ischaemic stroke. It was found that injury caused by stereotaxic injection should be taken into account when assessing the effectiveness of treatment.

Implantation of osteogenic differentiated donor mesenchymal stem cells causes recruitment of host cells

The interaction between host and donor cells is believed to play an important role in osteogenesis. However, it is still unclear how donor osteogenic cells behave and interact with host cells in vivo. The purpose of this study was to track the interactions between transplanted osteogenic cells and host cells during osteogenesis. In vitro migration assay was carried out to investigate the ability of osteogenic differentiated humanmesenchymal stemcells (O-hMSCs) to recruit MSCs. At the in vivo level, O-hMSCs were implanted subcutaneously or into skull defects in severe combined immunodeficient (SCID) mice. New bone formation was observed bymicro-CT and histological procedures. In situ hybridization (ISH) against human Alu sequences was performed to distinguish donor osteogenic cells from host cells. In vitro migration assay revealed an increased migration potential of MSCs by co-culturing with O-hMSCs. In agreement with the results of in vitro studies, ISH against human Alu sequences showed that host mouse MSCs migrated in large numbers into the transplantation site in response to O-hMSCs. Interestingly, host cells recruited by O-hMSCs were the major cell populations in newly formed bone tissues, indicating that O-hMSCs can trigger and initiate osteogenesis when transplanted in orthotopic sites. The observations fromthis study demonstrated that in vitro induced O-hMSCs were able to attract hostMSCs in vivo andwere involved inosteogenesis togetherwith host cells,whichmay be of importance for bone tissue-engineering applications.

An adipoinductive role of inflammation in adipose tissue engineering : key factors in the early development of engineered soft tissues

Tissue engineering and cell implantation therapies are gaining popularity because of their potential to repair and regenerate tissues and organs. To investigate the role of inflammatory cytokines in new tissue development in engineered tissues, we have characterized the nature and timing of cell populations forming new adipose tissue in a mouse tissue engineering chamber (TEC) and characterized the gene and protein expression of cytokines in the newly developing tissues. EGFP-labeled bone marrow transplant mice and MacGreen mice were implanted with TEC for periods ranging from 0.5 days to 6 weeks. Tissues were collected at various time points and assessed for cytokine expression through ELISA and mRNA analysis or labeled for specific cell populations in the TEC. Macrophage-derived factors, such as monocyte chemotactic protein-1 (MCP-1), appear to induce adipogenesis by recruiting macrophages and bone marrow-derived precursor cells to the TEC at early time points, with a second wave of nonbone marrow-derived progenitors. Gene expression analysis suggests that TNFα, LCN-2, and Interleukin 1β are important in early stages of neo-adipogenesis. Increasing platelet-derived growth factor and vascular endothelial cell growth factor expression at early time points correlates with preadipocyte proliferation and induction of angiogenesis. This study provides new information about key elements that are involved in early development of new adipose tissue.

A Bruch’s membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a similar thickness as Bruch’s membrane (3 μm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell). Cultures established on either material developed a cobblestoned morphology with partial pigmentation within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na+/K+-ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned medium collected from above and below both membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrate that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model.

INTRAOCULAR TRANSMISSIBLE VENEREAL TUMOR IN A DOG

Tumor venéreo transmissível (TVT) é uma neoplasia de células redondas que acomete a mucosa genital externa de cães, machos e fêmeas. A transmissão decorre da implantação de células tumorais durante o coito, brigas ou interações entre animais portadores e susceptíveis. Existem relatos referindo-se a localizações atípicas do TVT, mas metástases raramente ocorrem. O presente relato descreve um caso incomum de TVT, com acometimento intra-ocular e metástases nos linfonodos ingüinais, num cão Terrier Brasileiro, com seis anos de idade. O animal apresentava massas anormais de tecido no olho direito, extremidade do pênis e aumento de volume de linfonodos da região ingüinal. A histopatologia do globo ocular e as citologias da massa peniana e dos nódulos subcutâneos evidenciaram aspectos citológicos semelhantes, caracterizados por células redondas com núcleo grande e nucléolo proeminente localizado centralmente. O citoplasma apresentou-se pálido e com presença de vacúolos pequenos e arredondados. O diagnóstico de TVT com acometimento intra-ocular e metástases em linfonodos foi baseado nos achados clínicos, citológicos e histopatológicos.

Use of power doppler for the assessment of vascularization in equine tendinitis treated with mesenchymal stem cells

Tendinous lesions are very common in athlete horses. The process of tendon healing is slow and the quality of the new tissue is often inferior to the original, leading in many cases to recurrence of the lesion. One of the main reasons for the limited healing capacity of tendons is its poor vascularization. At present, cell therapy is used in equine practice for the treatment of several disorders including tendinitis, desmitis and joint disease. However, there is little information regarding the mechanisms of action of these cells during tissue repair. It is known that Mesenchymal Stem Cells (MSCs) release several growth factors at the site of implantation, some of which promote angiogenesis. Comparison of blood flow using power Doppler ultrasonography was performed after the induction superficial digital flexor tendon tendinitis and implantation of adipose tissue-derived MSCs in order to analyze the effect of cell therapy on tendon neovascularization. For quantification of blood vessel histopathological examinations were conducted. Increased blood flow and number of vessels was observed in treated tendons up to 30 days after cell implantation, suggesting promotion of angiogenesis by the cell therapy.

Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

Background: The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells. Methods: A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses. Results: Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles. Conclusion: We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.

Deletion of CD39 inhibits development of metastatic liver cancer

New vessel formation and tumor infiltrating lymphocytes (TIL) influence host responses to malignant tissues. Extracellular adenosine-mediated pathways promote both vascular endothelial cell proliferation and inhibit cytotoxic T cells, thereby potentiating cancer growth. CD39 is the dominant ectonucleotidase of vascular and T regulatory cells and has the potential to generate high levels of adenosine locally. We have previously shown that deletion of Cd39 results in angiogenic failure and T regulatory cell dysfunction with loss of immune suppressive functions. Aim: Investigate impact of CD39 upon development of hepatic metastases. Methods and Results: We studied the development of metastatic liver deposits following portal vein infusion of 1.5x105 melanoma B16/F10 cells, with luciferase expression, in wild type and Cd39-null C57BL/6 mice (n=24). Tumor formation in liver was directly examined and animals imaged at days 7-17 after tumor cell implantation. As predicted, the formation of hepatic malignant foci was markedly suppressed in Cd39-null mice, at all time points examined. To test whether the major impact of Cd39-deletion was upon neovasculature formation or immune responsiveness, adoptive transfer experiments were conducted. Bone marrow transplants (BMT) from Cd39-null or wild type BL/6 mice were placed in lethally irradiated control and/or null mice, in a crossover manner (total n=24 for each group, respectively). Eight weeks postadoptive transfer, melanoma cells were infused via portal vein as before and tumor growth studied. The Cd39-null mice that received wild type BMT mirrored the wild type phenotype with progressive tumor growth observed (n=8 per time point; p=0.015). In contrast, metastases were significantly inhibited in both number and size and ultimately became necrotic in the wild type mice that had received Cd39-null BMT. Conclusions: Bone marrow derived cells mediate the major inhibitory effects of CD39 deletion on tumor growth. Pharmacological inhibition of CD39 may find utility as an adjunct therapy in the management of hepatic malignancy.

Taurolidine in the prevention and therapy of lung metastases

OBJECTIVE: During surgery for colon carcinoma, tumour cells may spread into the blood and may lead to the development of distant metastases. The most frequent sites of metastases are the liver and lungs. A new therapeutic approach is required to prevent tumour implantation of freely circulating tumour cells during and after surgery and to treat established metastases. The aim of this prospective study was to observe the influence of long-term intravenous taurolidine on the development of lung metastases after intravenous injection of colon adenocarcinoma cells. METHODS: Tumour cells (DHD/K12/TRb colon adenocarcinoma cell line, 1 x 10(6) cells) were injected into the right vena jugularis interna of BDIX rats. The animals (n=13) were randomised into three groups: group 1: tumour cell implantation without taurolidine application (control group); group 2: tumour cell implantation and simultaneous start of the taurolidine injection through osmotic pump, removal of the osmotic pump on day 7; group 3: tumour cell implantation on day 0 and start of the taurolidine injection through osmotic pump on day 14. RESULTS: In the taurolidine groups, the number and size of lung metastases were significantly lower compared to the control group (p=0.018; p=0.018 and p=0.036; p=0.018). Although the results of the intravenous long-term therapy with taurolidine in group 2 did not reach statistical significance in comparison with the results of group 3, a positive trend was revealed: The mean number of metastases in group 2 was 18.2 versus 28.2 in group 3. CONCLUSIONS: The application of taurolidine tends to prevent the development of lung metastases. Furthermore, taurolidine seems to reduce established lung metastases in this in vivo model. Taurolidine may offer additional therapeutic options in patients with colon adenocarcinoma.

Gene gun-mediated skin transfection with interleukin 12 gene results in regression of established primary and metastatic murine tumors.

Particle-mediated (gene gun) in vivo delivery of the murine interleukin 12 (IL-12) gene in an expression plasmid was evaluated for antitumor activity. Transfer of IL-12 cDNA into epidermal cells overlying an implanted intradermal tumor resulted in detectable levels (266.0 +/- 27.8 pg) of the transgenic protein at the skin tissue treatment site. Despite these low levels of transgenic IL-12, complete regression of established tumors (0.4-0.8 cm in diameter) was achieved in mice bearing Renca, MethA, SA-1, or L5178Y syngeneic tumors. Only one to four treatments with IL-12 cDNA-coated particles, starting on day 7 after tumor cell implantation, were required to achieve complete tumor regression. This antitumor effect was CD8+ T cell-dependent and led to the generation of tumor-specific immunological memory. By using a metastatic P815 tumor model, we further showed that a delivery of IL-12 cDNA into the skin overlying an advanced intradermal tumor, followed by tumor excision and three additional IL-12 gene transfections, could significantly inhibit systemic metastases, resulting in extended survival of test mice. These results suggest that gene gun-mediated in vivo delivery of IL-12 cDNA should be further developed for potential clinical testing as an approach for human cancer gene therapy.

Treatment of a human breast cancer xenograft with an adenovirus vector containing an interferon gene results in rapid regression due to viral oncolysis and gene therapy.

Treatment of a human breast cancer cell line (MDA-MB-435) in nude mice with a recombinant adenovirus containing the human interferon (IFN) consensus gene, IFN-con1 (ad5/IFN), resulted in tumor regression in 100% of the animals. Tumor regression occurred when virus was injected either within 24 hr of tumor cell implantation or with established tumors. However, regression of the tumor was also observed in controls in which either the wild-type virus or a recombinant virus containing the luciferase gene was used, although tumor growth was not completely suppressed. Tumor regression was accompanied by a decrease in p53 expression. Two other tumors, the human myelogenous leukemic cell line K562 and the hamster melanoma tumor RPMI 1846, also responded to treatment but only with ad5/IFN. In the case of K562 tumors, there was complete regression of the tumor, and tumors derived from RPMI 1846 showed partial regression. We propose that the complete regression of the breast cancer with the recombinant virus ad5/IFN was the result of two events: viral oncolysis in which tumor cells are being selectively lysed by the replication-competent virus and the enhanced effect of expression of the IFN-con1 gene. K562 and RPMI 1846 tumors regressed only as a result of IFN gene therapy. This was confirmed by in vitro analysis. Our results indicate that a combination of viral oncolysis with a virus of low pathogenicity, itself resistant to the effects of IFN and IFN gene therapy, might be a fruitful approach to the treatment of a variety of different tumors, in particular breast cancers.

Cell adhesion and growth on surfaces modified by plasma and ion implantation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Transcriptome Analysis Reveals New Insights into the Modulation of Endometrial Stromal Cell Receptive Phenotype by Embryo-Derived Signals Interleukin-1 and Human Chorionic Gonadotropin: Possible Involvement in Early Embryo Implantation

The presence of the conceptus in uterine cavity necessitates an elaborate network of interactions between the implanting embryo and a receptive endometrial tissue. We believe that embryo-derived signals play an important role in the remodeling and the extension of endometrial receptivity period. Our previous studies provided original evidence that human Chorionic Gonadotropin (hCG) modulates and potentiates endometrial epithelial as well as stromal cell responsiveness to interleukin 1 (IL1), one of the earliest embryonic signals, which may represent a novel pathway by which the embryo favors its own implantation and growth within the maternal endometrial host. The present study was designed to gain a broader understanding of hCG impact on the modulation of endometrial cell receptivity, and in particular, cell responsiveness to IL1 and the acquisition of growth-promoting phenotype capable of receiving, sustaining, and promoting early and crucial steps of embryonic development. Our results showed significant changes in the expression of genes involved in cell proliferation, immune modulation, tissue remodeling, apoptotic and angiogenic processes. This points to a relevant impact of these embryonic signals on the receptivity of the maternal endometrium, its adaptation to the implanting embryo and the creation of an environment that is favorable for the implantation and the growth of this latter within a new and likely hostile host tissue. Interestingly our data further identified a complex interaction between IL1 and hCG, which, despite a synergistic action on several significant endometrial target genes, may encompass a tight control of endogenous IL1 and extends to other IL1 family members.