Riccardo Schweizer

Paracrine effects of mesenchymal stem cells enhance vascular regeneration in ischemic murine skin

New theories on the regeneration of ischemic vasculature have emerged indicating a pivotal role of adult stem cells. The aim of this study was to investigate homing and hemodynamic effects of circulating bone marrow-derived mesenchymal stem cells (MSCs) in a critically ischemic murine skin flap model. Bone marrow-derived mesenchymal stem cells (Lin(-)CD105(+)) were harvested from GFP(+)-donor mice and transferred to wildtype C57BL/6 mice. Animals receiving GFP(+)-fibroblasts served as a control group. Laser scanning confocal microscopy and intravital fluorescence microscopy were used for morphological analysis, monitoring and quantitative assessment of the stem cell homing and microhemodynamics over two weeks. Immunohistochemical staining was performed for GFP, eNOS, iNOS, VEGF. Tissue viability was analyzed by TUNEL-assay. We were able to visualize perivascular homing of MSCs in vivo. After 4 days, MSCs aligned along the vascular wall without undergoing endothelial or smooth muscle cell differentiation during the observation period. The gradual increase in arterial vascular resistance observed in the control group was abolished after MSC administration (P<0.01). At capillary level, a strong angiogenic response was found from day 7 onwards. Functional capillary density was raised in the MSC group to 197% compared to 132% in the control group (P<0.01). Paracrine expression of VEGF and iNOS, but not eNOS could be shown in the MSC group but not in the controls. In conclusion, we demonstrated that circulating bone marrow-derived MSCs home to perivascular sites in critically ischemic tissue, exhibits paracrine function and augment microhemodynamics. These effects were mediated through arteriogenesis and angiogenesis, which contributed to vascular regeneration.

Adipose- and Bone Marrow-Derived Mesenchymal Stem Cells Prolong Graft Survival in Vascularized Composite Allotransplantation

Background Strategies aiming at minimization or elimination of systemic immunosuppression are key immediate goals for clinical expansion of vascularized composite allotransplantation (VCA). We compared the in vitro and in vivo immunomodulatory efficacy of adipose-derived mesenchymal stem cells (AD-MSCs) and bone marrow (BM)–derived MSCs in a rat VCA model. Methods Both cell types were tested in vitro for suppressor function using mixed lymphocyte reactivity assays. AD-MSCs or BM-MSCs were administered intravenously (1 × 106 or 5 × 106 cells/animal) to Lewis rat recipients of mismatched Brown Norway hindlimb transplants. Short course tacrolimus (FK-506) monotherapy was withdrawn at postoperative day 21. In vivo regulatory T-cell induction, peripheral blood chimerism, and microchimerism in lymphatic organs were analyzed. Results AD-MSCs and BM-MSCs exhibited strong dose-dependent suppressor function in vitro, which was significantly more pronounced for AD cells. In vivo, all animals revealed peripheral multi-lineage chimerism at four weeks (P < 0.01) independent of cell type and dosage. Regulatory T-cell levels were increased with both cell types, the most in AD-MSC groups. These immunomodulatory effects were only transient. MSC treatment resulted in long-term (>120 day) allograft survival in 47% of the animals, which correlated with durable microchimerism in BM and spleen. Conclusions AD-MSCs and BM-MSCs exert immunomodulatory effects that prolong survival of immunogenic skin-bearing VCA grafts with short course (21 day) tacrolimus induction therapy. The in vivo findings in terms of allograft survival did not reflect superior immunomodulatory characteristics of AD-MSCs found in vitro.

The Role of Adipose-Derived Stem Cells in Breast Cancer Progression and Metastasis

Conventional breast cancer extirpation involves resection of parts of or the whole gland, resulting in asymmetry and disfiguration. Given the unsatisfactory aesthetic outcomes, patients often desire postmastectomy reconstructive procedures. Autologous fat grafting has been proposed for reconstructive purposes for decades to restore form and anatomy after mastectomy. Fat has the inherent advantage of being autologous tissue and the most natural-appearing filler, but given its inconsistent engraftment and retention rates, it lacks reliability. Implementation of autologous fat grafts with cellular adjuncts, such as multipotent adipose-derived stem cells (ADSCs), has shown promising results. However, it is pertinent and critical to question whether these cells could promote any residual tumor cells to proliferate, differentiate, or metastasize or even induce de novo carcinogenesis. Thus far, preclinical and clinical study findings are discordant. A trend towards potential promotion of both breast cancer growth and invasion by ADSCs found in basic science studies was indeed not confirmed in clinical trials. Whether experimental findings eventually correlate with or will be predictive of clinical outcomes remains unclear. Herein, we aimed to concisely review current experimental findings on the interaction of mesenchymal stem cells and breast cancer, mainly focusing on ADSCs as a promising tool for regenerative medicine, and discuss the implications in clinical translation.

Human adipose-derived mesenchymal stromal cells may promote breast cancer progression and metastatic spread

Background: Stem cell–enriched fat grafting has been proposed as a potential therapy for reconstructive, restorative, or enhancement-related procedures of the breast. Its role in postoncologic breast reconstruction is still emerging, with concerns about safety. The authors investigated the dose-dependent interaction between human adipose-derived mesenchymal stromal cells (AD-MSCs) and human breast cancer cell (BCC) lines [MDA-MB-231 (MDA) and MCF-7 (MCF)] focusing on tumor microenvironment, tumor growth, and metastatic spread. Methods: Adipose-derived mesenchymal stromal cell influence on viability and factor expression [regulated on activation, normal T cell expressed and secreted (RANTES), tumor necrosis factor-&agr;, and eotaxin) of breast cancer cells was studied in vitro using direct and indirect co-culture systems. Groups were formed according to adipose-derived mesenchymal stromal cell–to–cancer cell number ratio [MDA/MCF only, AD-MSClow/(MDA/MCF), and AD-MSChigh/(MDA/MCF)]. A humanized orthotopic murine cancer model was used to evaluate breast cancer progression and metastasis (n = 10/group). Cells were injected into the mammary pad in different ratios and animals were monitored over 42 days. Microdialysis was performed to analyze RANTES levels in the tumor microenvironment (days 21 and 42). Primary and metastatic tumors were weighed and analyzed for oncogene, growth factor, and metastatic marker expression. Results: MDA cell viability increased from 45.5 percent to 95.5 percent in presence of adipose-derived mesenchymal stromal cells in vitro. In vivo, animals with AD-MSChigh showed increased mean tumor weight (MDA, p < 0.01; MCF versus controls, p < 0.05) and metastatic occurrence (40 percent in MDA; 30 percent in MCF versus 0 percent in controls). Cytokine analysis revealed switching of MCF tumor phenotype to a more malignant type in the presence of adipose-derived mesenchymal stromal cells. Conclusion: Human adipose-derived mesenchymal stromal cells may promote progression and metastatic spread in breast cancer through a switch to a more malignant phenotype with worse prognosis.

Botulinum toxin A and B raise blood flow and increase survival of critically ischemic skin flaps

BACKGROUND Botulinum toxin (BTX) A and B are commonly used for aesthetic indications and in neuromuscular disorders. New concepts seek to prove efficacy of BTX for critical tissue perfusion. Our aim was to evaluate BTX A and B in a mouse model of critical flap ischemia for preoperative and intraoperative application. METHODS BTX A and B were applied on the vascular pedicle of an axial pattern flap in mice preoperatively or intraoperatively. Blood flow, tissue oxygenation, tissue metabolism, flap necrosis rate, apoptosis assay, and RhoA and eNOS expression were endpoints. RESULTS Blood-flow measurements 1 d after the flap operation revealed a significant reduction to 53% in the control group, while flow was maintained or increased in all BTX groups (103%-129%). Over 5 d all BTX groups showed significant increase in blood flow to 166-187% (P < 0.01). Microdialysis revealed an increase of glucose and reduced lactate/pyruvate ratio and glycerol levels in the flap tissue of all BTX groups. This resulted in significantly improved tissue survival in all BTX groups compared with the control group (62% ± 10%; all P < 0.01): BTX A preconditioning (84% ± 5%), BTX A application intraoperatively (88% ± 4%), BTX B preconditioning (91% ± 4%), and intraoperative BTX B treatment (92% ± 5%). This was confirmed by TUNEL assay. Immunofluorescence demonstrated RhoA and eNOS expression in BTX groups. All BTX applications were similarly effective, despite pharmacologic dissimilarities and different timing. CONCLUSIONS In conclusion, we were able to show on a vascular, tissue, cell, and molecular level that BTX injection to the feeding arteries supports flap survival through ameliorated blood flow and oxygen delivery.

Are cultured mesenchymal stromal cells an option for immunomodulation in transplantation

Mesenchymal stromal cells (MSCs) are under investigation for clinical application. Despite approval by the United States Food and Drug Administration for MSC use in pediatric steroid-refractory acute GvHD after allogeneic hematopoietic stem cell transplantation (Parekkadan and Milwid, 2010) uncertainty about the fate of MSCs after infusion remains thus far. Clinical trials have provided evidence for high response rates, efficacy, and safety leading to mortality reduction after MSC treatment of GvHD (Bernardo et al., 2010). Recently de Girolamo et al. (2012) have reviewed clinical observations and critical aspects extensively. Systemic immunomodulation following MSC treatment has been demonstrated (Dander et al., 2012; Zanotti et al., 2013), regardless of the many unresolved questions including possible entrapment in the lungs and liver, homing to sites of inflammation or trauma, and the relevance of chimerism. Recently, Eggenhofer et al. (2012) presented evidence in a mouse model that cultured bone-marrow derived MSCs are entrapped in the lungs after intravenous infusion. These results confirm that, in addition to a need for greater understanding of their functional and immunologic characteristics, there is also a need to investigate the migratory properties of cultured MSCs in circulation prior to clinical implementation. We congratulate Eggenhofer et al. for their experimental insights. Herein, we propose to summarize some salient aspects of existing literature evidence and our own experience in response to some comments and conclusions made by the authors. The present study supports the findings from Fischer et al. (2009), who have described a first-pass effect in the lung capillaries for MSCs. These authors used MSCs up to passage 4 and could demonstrate that cells from a second bolus injection passed the lungs more efficiently. This study elegantly could show a dependency of MSCs and their ability to pass the lung filter on size and surface antigens. In rodent and swine transplant models, intravenous delivery of MSCs has been shown to achieve long-term peripheral blood chimerism. Some of these studies (Kuo et al., 2009, 2011; Pan et al., 2010) prove that MSCs survived for months or long-term in the periphery without complete entrapment in the pulmonary capillary bed. However, they also confirmed on histopathology that homing of MSCs to lungs does occur (Kuo et al., 2009). In our own experiments, we investigated sites of vascular regeneration in a critically perfused skin-flap model in immunocompetent mice (C57BL/6) after transplantation of fluorescent allogeneic MSCs. Freshly isolated Lin−CD105+ bone-marrow derived MSCs (2 × 105/animal in 100 μm 0.9% NaCl via tail vein injection), were infused via tail vein injection. MSCs exhibited perivascular homing remote to the lungs and liver as well as paracrine expression of growth factors mediating vascular regeneration in specific sites. We were able to visualize MSCs in vivo by intravital fluorescence microscopy and laser scanning confocal microscopy and post mortem histologically in the peripheral tissue (Schlosser et al., 2012). Over time, cell numbers increased but they did not change their morphology (Figure ​(Figure1).1). Yet, we could not differentiate whether this was due to local proliferation or further recruitment of MSCs in these experiments. Figure 1 (A) Fluorescent MSC (yellow) after perivascular homing to critically ischemic skin. 3D reconstruction from Laser-Scanning Confocal Microscopy. (B) Fluorescent MSCs (green) 14 days after tailvein injection and homing to critically ischemic skin. (Freshly ... Cultured MSCs may not be phenotypically distinguishable from fibroblasts and may even share similar surface antigens or differentiation potential (Hematti, 2012). With regards to cultured fibroblasts, we found that these cells induced lethal pulmonary embolism if infused too quickly (<1 min; own unpublished data) but slow IV injection was consistent with survival. The findings by Eggenhofer et al. (2012) of predominant lung entrapment explain our observational findings of mortality. However, in other studies, Schlosser et al. (2012) reported that entrapped fibroblasts exhibit regenerative effects over critically perfused skin. These findings may indirectly support conclusions of Eggenhofer et al. that MSCs could mediate distant effects via endocrine mechanisms. In the past, it has been shown that the duration and degree of cell expansion and culture has a clear impact on MSC morphology, differentiation, viability, and migratory properties (Wagner et al., 2010). Freshly isolated MSCs show superior homing ability compared to expanded cells (Rombouts and Ploemacher, 2003), which might be due to their size (own unpublished data; Fischer et al., 2009) as well as unique homing factors. Importantly, MSCs not only undergo phenotypic changes in culture and during passage (size, morphology, and cell surface marker expression) (Wagner et al., 2010; Hematti, 2012), but also lose capacity for functional proliferation and differentiation potential (Vacanti et al., 2005; Wagner et al., 2010). In addition, their ability for cytokine production is altered (Banfi et al., 2002; Vacanti et al., 2005). To avoid the first pass effect and consequent pulmonary capillary entrapment following MSC transplantation, Zonta et al. (2010) suggest an arterial route of access. They delivered MSCs to the renal artery during kidney transplantation in rodents and reported favorable recovery of kidney function as opposed to the intravenous route. Arterial application might thus enable direct delivery to the capillary bed of the graft with reduced cell loss through entrapment and consequent unwarranted systemic effects. Pulmonary and hepatic entrapment of MSCs has been intensely debated and studied for years. The study by Eggenhofer et al. is the first to lucidly demonstrate that cultured MSCs undergo significant entrapment in the lung after intravenous application. It still remains speculative: (1) If the degree of this phenomenon varies with the size of MSCs infused (based on passage cycle or culture denominators); (2) if there are long-term effects on lung function due to the entrapped cells and; (3) if the immunological efficacy of MSCs could be improved through direct arterial delivery to the graft or specific end organs. There is some evidence that the loss of cells through a first pass effect is indeed lower with freshly isolated MSCs indicating a link to smaller cell size or possibly related to enhanced viability and homing capacity. Taken together, studies comparing effects of fresh isolated MSCs delivered intra-arterially to the graft or in proximity to the end organ to those secondary to passaged MSCs delivered via a peripheral intravenous route may be important to define if indeed this is a technical or procedural consideration essential for incorporation into pre-clinical protocols to optimize overall outcomes.

Premise and promise of mesenchymal stem cell-based therapies in clinical vascularized composite allotransplantation.

Purpose of reviewOver the past decade, clinical vascularized composite allotransplantation (VCA) has enabled functional and quality of life restoration in a wide range of indications secondary to devastating tissue loss. However, the spectre of toxicity and long-term complications of chronic immunosuppression has curtailed the momentum of VCA. This study summarizes the literature evidence behind successful mesenchymal stem cell (MSC)-based cell therapies highlighting their multipronged immunomodulatory, restorative and regenerative characteristics with special emphasis towards VCA applications. Recent findingsExperimental and clinical studies in solid organs and VCA have confirmed that MSCs facilitate immunosuppression-free allograft survival or tolerance, stimulate peripheral nerve regeneration, attenuate ischaemia-reperfusion injury, and improve tissue healing after surgery. It has been hypothesized that MSC-induced long-term operational tolerance in experimental VCA is mediated by induction of mixed donor-specific chimerism and regulatory T-cell mechanisms. All these characteristics of MSCs could thus help expand the scope and clinical feasibility of VCA. SummaryCellular therapies, especially those focusing on MSCs, are emerging in solid organ transplantation including VCA. Although some clinical trials have begun to assess the effects of MSCs in solid organ transplantation, much scientific domain remains uncharted, especially for VCA.

The Influence of Timing and Frequency of Adipose-Derived Mesenchymal Stem Cell Therapy on Immunomodulation Outcomes After Vascularized Composite Allotransplantation.

Background Cellular therapies for immunomodulation in vascularized composite allotransplantation (VCA) have gained importance due to their potential for minimization of immunosuppression. Adipose-derived (AD) mesenchymal stem cells (MSCs) especially have shown encouraging potential. We investigated the influence of timing and frequency of AD-MSC treatment on immunologic and graft survival as well as graft vasculopathy outcomes after VCA. Methods Lewis rats received full-mismatched Brown Norway rat hindlimb transplants. Recipient animals were assigned to groups receiving donor-derived AD-MSCs (106 cells/animal) either on postoperative day (POD) 1, POD 4, or repeatedly on POD 4, 8, and 15, and compared to untreated controls. Results Although AD-MSC administration on POD 1 or POD 4, 8, and 15 resulted in 50% long-term graft acceptance, recipients treated on POD 4, and controls rejected before POD 50. All treated animals revealed peripheral blood chimerism (4 weeks), most pronounced after repetitive cell administration (12.92% vs 5.03% [POD 1] vs 6.31% [POD 4]; P < 0.05; all P < 0.01 vs control 1.45%). Chimerism was associated with the generation of regulatory T cells (CD4+CD25highFoxP3+). In vitro mixed lymphocyte reactions revealed modulation of the recipient immune response after AD-MSC treatment. Graft arteries at end point revealed significant differences of arterial intimal thickness between rejecting and AD-MSC–treated animals (P < 0.01). Conclusions Taken together, our results point to the potential for repetitive AD-MSC administration in improving outcomes after VCA. Future studies are warranted into optimization of the dosing and frequency of AD-MSC therapy, either alone or used in, combination with other cell therapies (such as hematopoietic stem cells or bone marrow–derived MSC or dendritic cells) for optimization of appropriate conditioning or maintenance regimens.

Effects of Immunosuppressive Drugs on Viability and Susceptibility of Adipose- and Bone Marrow-Derived Mesenchymal Stem Cells

The immunomodulatory potential of cell therapies using adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs) has been studied in vascularized composite allotransplantation (VCA). Most cell therapy-based experimental and clinical protocols integrate some degree of recipient conditioning/induction with antibodies or other immunosuppressive agents. We investigated the susceptibility of ASCs and BM-MSCs to anti-lymphocyte serum (ALS) and tacrolimus. Rat ASCs and BM-MSCs were exposed to varying concentrations of tacrolimus and ALS in vitro. Serum from ALS-treated animals was added to cell cultures. Viability, susceptibility, and cytotoxicity parameters were evaluated. ALS inhibited ASC and BM-MSC viability and susceptibility in vitro in a dose-dependent manner. ASCs were more susceptible to both ALS and tacrolimus than BM-MSCs. Trypsinized and adherent ASCs were significantly smaller than BM-MSCs. This is the first report on the viability and susceptibility characteristics of BM-MSCs or ASCs to collateral effects of ALS and tacrolimus. These in vitro insights may impact choice of cell type as well as concomitant conditioning agents and the logistical coordination of the timing, dosing, and frequency of drug or cell therapy in solid organ transplantation or VCA protocols.

Morphology and Hemodynamics during Vascular Regeneration in Critically Ischemic Murine Skin Studied by Intravital Microscopy Techniques

Background: With the understanding of angiogenesis and arteriogenesis, new theories about the orchestration of these processes have emerged. The aim of this study was to develop an in vivo model that enables visualization of vascular regenerating mechanisms by intravital microscopy techniques in collateral arteriolar flap vascularity. Methods: A dorsal skin flap (15 × 30 mm) was created in mice and fixed into a skinfold chamber to allow for assessment of morphology and microhemodynamics by intravital fluorescence microscopy (IVFM). Laser scanning confocal microscopy (LSCM) was utilized for three-dimensional reconstruction of the microvascular architecture. Results: Flap tpO2 was 5.3 ± 0.9 versus 30.5 ± 1.2 mm Hg in controls (p < 0.01). The collateral arterioles in the flap tissue were dilated (29.4 ± 5.3 µm; p < 0.01 vs. controls) and lengthened in a tortuous manner (tortuosity index 1.00 on day 1 vs. 1.35± 0.05 on day 12; p < 0.01). Functional capillary density was increased from 121.00 ± 25 to 170 ± 30 cm/cm2 (day 12; p < 0.01) as a result of angiogenesis. Morphological evidence of angiogenesis on capillary level and vascular remodeling on arteriolar level could be demonstrated by IVFM and LSCM. Conclusions: Present intravital microscopy techniques offer unique opportunities to study structural changes and hemodynamic effects of vascular regeneration in this extended axial pattern flap model.