Characteristics and Immunomodulating Functions of Adipose-Derived and Bone Marrow-Derived Mesenchymal Stem Cells Across Defined Human Leukocyte Antigen Barriers

BackgroundVascularized composite allotransplantation opens new possibilities in reconstructive transplantation such as hand or face transplants. Lifelong immunosuppression and its side-effects are the main drawbacks of this procedure. Mesenchymal stem cells (MSCs) have clinically useful immunomodulatory effects and may be able to reduce the burden of chronic immunosuppression. Herein, we assess and compare characteristics and immunomodulatory capacities of bone marrow- and adipose tissue-derived MSCs isolated from the same human individual across defined human leukocyte antigen (HLA) barriers.Materials and methodsSamples of omental (o.) adipose tissue, subcutaneous (s.c.) adipose tissue, and bone marrow aspirate from 10 human organ donors were retrieved and MSCs isolated. Cells were characterized by flow cytometry and differentiated in three lineages: adipogenic, osteogenic, and chondrogenic. In mixed lymphocyte reactions, the ability of adipose-derived mesenchymal stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (BMSCs) to suppress the immune response was assessed and compared within individual donors. HLA mismatched or mitogen stimulations were analyzed in co-culture with different MSC concentrations. Supernatants were analyzed for cytokine contents.ResultsAll cell types, s.c.ASC, o.ASC, and BMSC demonstrated individual differentiation potential and cell surface markers. Immunomodulating effects were dependent on dose and cell passage. Proliferation of responder cells was most effectively suppressed by s.c.ASCs and combination with BMSC resulted in highly efficient immunomodulation. Immunomodulation was not cell contact-dependent and cells demonstrated a specific cytokine secretion.ConclusionWhen human ASCs and BMSCs are isolated from the same individual, both show effective immunomodulation across defined HLA barriers in vitro. We demonstrate a synergistic effect when cells from the same biologic system were combined. This cell contact-independent function underlines the potential of clinical systemic application of MSCs

Injectable Hydrogel Guides Neurons Growth with Specific Directionality

Visual disabilities affect more than 250 million people, with 43 million suffering from irreversible blindness. The eyes are an extension of the central nervous system which cannot regenerate. Neural tissue engineering is a potential method to cure the disease. Injectability is a desirable property for tissue engineering scaffolds which can eliminate some surgical procedures and reduce possible complications and health risks. We report the development of the anisotropic structured hydrogel scaffold created by a co-injection of cellulose nanofiber (CNF) solution and co-polypeptide solution. The positively charged poly (L-lysine)-r-poly(L-glutamic acid) with 20 mol% of glutamic acid (PLLGA) is crosslinked with negatively charged CNF while promoting cellular activity from the acid nerve stimulate. We found that CNF easily aligns under shear forces from injection and is able to form hydrogel with an ordered structure. Hydrogel is mechanically strong and able to support, guide, and stimulate neurite growth. The anisotropy of our hydrogel was quantitatively determined in situ by 2D optical microscopy and 3D X-ray tomography. The effects of PLLGA:CNF blend ratios on cell viability, neurite growth, and neuronal signaling are systematically investigated in this study. We determined the optimal blend composition for stimulating directional neurite growth yielded a 16% increase in length compared with control, reaching anisotropy of 30.30% at 10°/57.58% at 30°. Using measurements of calcium signaling in vitro, we found a 2.45-fold increase vs. control. Based on our results, we conclude this novel material and unique injection method has a high potential for application in neural tissue engineering

Tacrolimus before CTLA4Ig and rapamycin promotes vascularized composite allograft survival in MGH miniature swine

BACKGROUND We evaluated the outcome of vertical rectus abdominus myocutaneous flap (VRAM) allotransplantation in a mini-pig model, using a combined co-stimulation blockade (Co-SB) and mechanistic target of rapamycin inhibition (mTORi)-based regimen, with or without preceding calcineurin inhibition (CNI). MATERIALS AND METHODS VRAM allotransplants were performed between SLA-mismatched MGH miniature swine. Group A (n = 2) was treated continuously with the mTOR inhibitor rapamycin from day -1 in combination with the Co-SB agent cytotoxic T lymphocyte antigen 4-Ig (CTLA4-Ig) from post-operative day (POD) 0. In group B (n = 3), animals received tacrolimus daily from POD 0 to POD 13, followed by rapamycin daily from POD 7 and CTLA4-Ig weekly from POD 7-28. Graft rejection was determined by Banff criteria and host cellular and humoral immunity monitored. RESULTS In group A, allografts developed grade-I acute rejection by POD 2 and POD 7, and reached grade-IV by POD 17 and POD 20, respectively. By contrast, in group B, two allografts demonstrated grade-I rejection on POD 30 and grade-IV on POD 74, while the third exhibited grade-I rejection starting on POD 50, though this animal had to be euthanized on POD 58 due to Pneumocystis jirovecii infection. Time-to-event incidence of grade-I rejection was significantly lower in group A compared to group B. During the first 3 weeks post-transplant, no significant differences in anti-donor immunity were observed between the groups. CONCLUSION A short course of CNI, followed by combined Co-SB and mTORi significantly delays acute rejection of VRAM allografts in SLA-mismatched miniature swine

An Elastomeric Polymer Matrix, PEUU-Tac, Delivers Bioactive Tacrolimus Transdurally to the CNS in Rat

Central nervous system (CNS) neurons fail to regrow injured axons, often resulting in permanently lost neurologic function. Tacrolimus is an FDA-approved immunosuppressive drug with known neuroprotective and neuroregenerative properties in the CNS. However, tacrolimus is typically administered systemically and blood levels required to effectively treat CNS injuries can lead to lethal, off-target organ toxicity. Thus, delivering tacrolimus locally to CNS tissues may provide therapeutic control over tacrolimus levels in CNS tissues while minimizing off-target toxicity. Herein we show an electrospun poly(ester urethane) urea and tacrolimus elastomeric matrix (PEUU-Tac) can deliver tacrolimus trans-durally to CNS tissues. In an acute CNS ischemia model in rat, the optic nerve (ON) was clamped for 10s and then PEUU-Tac was used as an ON wrap and sutured around the injury site. Tacrolimus was detected in PEUU-Tac wrapped ONs at 24 h and 14 days, without significant increases in tacrolimus blood levels. Similar to systemically administered tacrolimus, PEUU-Tac locally decreased glial fibrillary acidic protein (GFAP) at the injury site and increased growth associated protein-43 (GAP-43) expression in ischemic ONs from the globe to the chiasm, consistent with decreased astrogliosis and increased retinal ganglion cell (RGC) axon growth signaling pathways. These initial results suggest PEUU-Tac is a biocompatible elastic matrix that delivers bioactive tacrolimus trans-durally to CNS tissues without significantly increasing tacrolimus blood levels and off-target toxicity

Heterotopic Transplantation of Allogeneic Vertical Rectus Abdominis Myocutaneous Flaps in Miniature Swine

BACKGROUND Vascularized composite tissue allotransplantation (VCA) opens new possibilities for reconstruction of complex tissue defects, including upper extremity and facial transplantation. The main challenges in VCA transplantation are the side effects of long-term immunosuppression and chronic graft rejection. Translational preclinical animal models are crucial for VCA research to improve clinical outcomes and to study underlying immunologic mechanisms. Herein, we describe a novel, large animal, non-bone-bearing VCA model in inbred, swine leukocyte antigen-typed miniature swine. METHODS Transplantation of vertical rectus abdominis myocutaneous (VRAM) flaps was performed between fully swine leukocyte antigen-mismatched miniature swine. The flaps were transferred to the posterolateral aspect of the neck of recipients and anastomosed to the common carotid artery and internal jugular vein. Different immunosuppressive drug regimens were used. Clinical graft evaluation was performed daily, and punch biopsies were taken for histology. RESULTS Ten VRAM transplants were performed. The mean ischemia time was 89.4 min (SD ± 47), mean pedicle length 7.5 cm (SD ± 2), mean venous diameter 2.5 mm (SD ± 0.4), and mean arterial diameter 2.2 mm (SD ± 0.3). Follow-up demonstrated good correlation between clinical appearance and progression of graft rejection confirmed by histologic assessment. Complications were intraoperative cardiac arrest in one recipient and one flap loss due to venous compromise. CONCLUSIONS VRAM transplantation in miniature swine is an appropriate preclinical VCA model, with the advantage of good clinical and histologic correlation during the course of rejection, as well as easy access to the graft. The availability of inbred, haplotyped animals allows studies across different major histocompatibility complex barriers in a non-bone-bearing VCA