Jonas T. Schnider

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 Short course tacrolimus (FK-506) monotherapy was withdrawn at postoperative day 21.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.

Perspectives on the Use of Mesenchymal Stem Cells in Vascularized Composite Allotransplantation

Reconstructive transplantation has emerged as clinical reality over the past decade. Long-term graft acceptance has been feasible in extremity and facial vascularized composite allotransplantation (VCA) under standard immunosuppression. Minimizing overall burden of lifelong immunosuppression is key to wider application of these non-life saving grafts. Allograft tolerance is the holy grail of many cell-based immunomodulatory strategies. Recent protocols using mesenchymal stem cells from bone marrow and adipose tissue offer promise and potential in VCA. This article provides an overview of the experimental basis, the scientific background and clinical applications of stem cell-based therapies in the field of reconstructive allotransplantation.

Review of the Early Diagnoses and Assessment of Rejection in Vascularized Composite Allotransplantation

The emerging field of vascular composite allotransplantation (VCA) has become a clinical reality. Building upon cutting edge understandings of transplant surgery and immunology, complex grafts such as hands and faces can now be transplanted with success. Many of the challenges that have historically been limiting factors in transplantation, such as rejection and the morbidity of immunosuppression, remain challenges in VCA. Because of the accessibility of most VCA grafts, and the highly immunogenic nature of the skin in particular, VCA has become the focal point for cross-disciplinary approaches to developing novel approaches for some of the most challenging immunological problems in transplantation, particularly the early diagnoses and assessment of rejection. This paper provides a historically oriented introduction to the field of organ transplantation and the evolution of VCA.

Hemoglobin vesicles improve wound healing and tissue survival in critically ischemic skin in mice

Local hypoxia, as due to trauma, surgery, or arterial occlusive disease, may severely jeopardize the survival of the affected tissue and its wound-healing capacity. Initially developed to replace blood transfusions, artificial oxygen carriers have emerged as oxygen therapeutics in such conditions. The aim of this study was to target primary wound healing and survival in critically ischemic skin by the systemic application of left-shifted liposomal hemoglobin vesicles (HbVs). This was tested in bilateral, cranially based dorsal skin flaps in mice treated with a HbV solution with an oxygen affinity that was increased to a P(50) (partial oxygen tension at which the hemoglobin becomes 50% saturated with oxygen) of 9 mmHg. Twenty percent of the total blood volume of the HbV solution was injected immediately and 24 h after surgery. On the first postoperative day, oxygen saturation in the critically ischemic middle flap portions was increased from 23% (untreated control) to 39% in the HbV-treated animals (P < 0.05). Six days postoperatively, flap tissue survival was increased from 33% (control) to 57% (P < 0.01) and primary healing of the ischemic wound margins from 6.6 to 12.7 mm (P < 0.05) after HbV injection. In addition, higher capillary counts and endothelial nitric oxide synthase expression (both P < 0.01) were found in the immunostained flap tissue. We conclude that left-shifted HbVs may ameliorate the survival and primary wound healing in critically ischemic skin, possibly mediated by endothelial nitric oxide synthase-induced neovascularization.

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.

Site-Specific Immunosuppression in Vascularized Composite Allotransplantation: Prospects and Potential

Skin is the most immunogenic component of a vascularized composite allograft (VCA) and is the primary trigger and target of rejection. The skin is directly accessible for visual monitoring of acute rejection (AR) and for directed biopsy, timely therapeutic intervention, and management of AR. Logically, antirejection drugs, biologics, or other agents delivered locally to the VCA may reduce the need for systemic immunosuppression with its adverse effects. Topical FK 506 (tacrolimus) and steroids have been used in clinical VCA as an adjunct to systemic therapy with unclear beneficial effects. However, there are no commercially available topical formulations for other widely used systemic immunosuppressive drugs such as mycophenolic acid, sirolimus, and everolimus. Investigating the site-specific therapeutic effects and efficacy of systemically active agents may enable optimizing the dosing, frequency, and duration of overall immunosuppression in VCA with minimization or elimination of long-term drug-related toxicity.

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.

Single Implantable FK506 Disk Prevents Rejection in Vascularized Composite Allotransplantation

Background: In vascularized composite allotransplantation, medication nonadherence leads to increased acute rejections. Improving medication adherence would improve overall allograft survival. Regionally delivered immunosuppression, targeted to sites of allorecognition, may reduce or eliminate the need for daily systemic immunosuppression. Methods: The authors developed biodegradable FK disks containing FK506-loaded double-walled microspheres and tested their efficacy at preventing rejection in a Brown-Norway–to-Lewis rat hindlimb transplantation model. In some experimental group animals, one FK disk was implanted subcutaneously either in native nontransplanted leg or in a transplanted allograft. Regular blood FK506 levels were measured. The endpoint was 180-day allograft survival or grade 3 rejection. At the endpoint, tissue FK506 levels were measured and mixed lymphocytic reaction was performed. Results: A single FK disk maintained systemic blood FK506 levels between 5 and 15 ng/ml for 146 ± 11.1 days. After that, the levels declined to less than 5 ng/ml through the endpoint. There was significantly increased FK506 concentration in groin lymph nodes draining the implanted FK disk. Compared with other groups, animals with an FK disk in the transplanted allograft had 100 percent allograft survival to more than 180 days despite subtherapeutic levels below 5 ng/ml. In these animals, significant T-cell hyporesponsiveness was seen in groin lymph nodes draining the FK disk compared with robust splenic T-cell proliferation. Conclusions: Sustained regional immunosuppression (with a single FK506 disk) maintained the allograft by means of a high regional concentration of FK506. Notably, this was achieved at subtherapeutic blood concentrations of FK506, without any further systemic FK506 administration.

Abstract P9: Non-Invasive Objective Evaluation of Peripheral Neuroregeneration Using Magnetic Resonance Diffusion Tensor Imaging

ConClusion: This study confirms feasibility of application of hES conduit for restoration of peripheral nerve defects. hES supported with hMSC demonstrated comparable functional outcomes to the autograft technique. The role of local hMSC application in nerve regeneration is currently under investigation. P9 non-invasive objective evaluation of Peripheral neuroregeneration Using Magnetic resonance diffusion tensor imaging