Thromboangiitis obliterans (TAO), also known as Buerger’s Disease, is an occlusive vasculitis linked with high morbidity and amputation risk. To date, TAO is deemed incurable due to the lack of a definitive treatment. The immune system and inflammation are proposed to play a central role in TAO pathogenesis. Due to their immunomodulatory effects, mesenchymal stromal cells (MSCs) are the subject of intense research for the treatment of a wide range of immune-mediated diseases. Thus far, local intramuscular injections of autologous or allogeneic MSCs have shown promising results in TAO. However, sequential intravenous allogeneic MSC administration has not yet been explored, which we hypothesized could exert a systemic anti-inflammatory effect in the vasculature and modulate the immune response. Here, we report the first case of a TAO patient at amputation risk treated with four sequential intravenous infusions of bone marrow-derived allogeneic MSCs from a healthy donor. Following administration, there was significant regression of foot skin ulcers and improvements in rest pain, Walking Impairment Questionnaire scores, and quality of life. Sixteen months after the infusion, the patient had not required any further amputations. This report highlights the potential of sequential allogeneic MSC infusions as an effective treatment for TAO, warranting further studies to compare this approach with the more conventionally used intramuscular MSC administration and other cell-based therapies. Keywords: Allogeneic mesenchymal stromal cells, Thromboangiitis obliterans, Cell transplantation Introduction To date, the pathogenesis of TAO has not been fully Thromboangiitis obliterans (TAO), also known as Buerger’s elucidated. Smoking is considered the main precipitating disease, is an inflammatory occlusive disorder that affects factor of the disease which could trigger an immune re- small and medium sized peripheral blood vessels of the ex- sponse and inflammatory damage targeting vascular endo- tremities. It is characterized by hypercellular inflammatory thelial cells and leading to thrombosis . Indeed, several thrombotic occlusions of arteries and veins, which ultim- reports have provided insights into the immunopathogen- ately leads to vascular insufficiency, critical limb ischemia, esis of TAO, suggesting that the immune system plays a and amputation . This high-morbidity disease mainly af- critical role in the etiology of the disease [1, 3–5]. fects young male smokers, severely limiting their quality of Mesenchymal stromal cells (MSCs) are the subject of in- life. Although smoking cessation is the most effective thera- tense research over a wide range of conditions due to their peutic intervention, there is currently no definitive cure for angiogenic and immunomodulatory effects . Previous TAO . studies using MSCs for TAO have focused on their local effect after intramuscular administration [7, 8]. However, we hypothesized that their intravenous use could directly * Correspondence: firstname.lastname@example.org Department of Hematology, Cell Therapy Unit, IBSAL-Hospital Universitario act upon the mechanisms that underlie TAO pathogenesis de Salamanca, Paseo de San Vicente 58-182, 37007 Salamanca, Spain by exerting systemic anti-inflammatory effects in the vas- Faculty of Medicine, Universidad de Salamanca, Salamanca, Spain culature and modulating the response of the immune Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Martin-Rufino et al. Stem Cell Research & Therapy (2018) 9:150 Page 2 of 6 system. Sequential doses of intravenous MSCs have been and the pedal pulse was absent on examination. Remark- previously shown to be safe and potentially effective in the ably, all the dorsum of the right foot showed trophic treatment of cardiovascular conditions and immune com- changes with multiple punctate ulcers (Fig. 1a, b). The plications, such as graft-versus-host disease (GVHD), ankle-brachial index (ABI) was 0.66. The patient’streat- through systemic immunomodulatory mechanisms [9, ment included clopidogrel, pentoxifylline, amlodipine, and 10]. In addition, the use of allogeneic MSCs could over- buprenorphine transdermal patches. come the problems of autologous MSCs in inflammatory The Walking Impairment Questionnaire (WIQ) was diseases in which they are dysfunctional . used to quantitatively assess the impact of MSC treat- To the best of our knowledge, this is the first report of a ment on the patient’s walking capability. Each WIQ TAO patient treated with sequential intravenous infusions metric is scored from 0 (total incapacity) to 100 (full of allogeneic MSCs. The patient, who had critical limb is- capacity) . The patient’s WIQ distance score was 54, chemia and was at amputation risk, had exhausted all the speed score 31, and the climbing score 67. available therapeutic options and received intravenous TheEuropean Qualityof Life—5dimensions (EQ-5D) allogeneic MSCs under a compassionate use program. questionnaire was used to assess changes in the patient’s health-related quality of life. The patient’s EQ-5D descrip- Methods tive system score was 0.72 out of 1, which analyzes mobil- Patient and pretreatment assessment ity, self-care, usual activities, pain/discomfort, and anxiety/ A 41-year-old man, diagnosed with TAO and suffering depression. The EQ-5D visual analogue scale (VAS) score from critical chronic ischemia and ulcerous lesions on his was 70, with 0 and 100 being the ‘worst’ and the ‘best’ im- right lower leg, was referred to the Angiology and Vascu- aginable health states, respectively . lar Surgery Department to assess his eligibility for treat- Magnetic resonance angiography (MRA) studies re- ment with MSCs under a compassionate use program. He vealed a right patent femoral artery until the origin of had developed ulcers and critical ischemia on the left the popliteal artery, which displayed multiple critical lower leg, despite smoking cessation, 8 years before. A left stenoses and a complete occlusion. Abundant collateral lumbar sympatectomy and the implantation of an epidural circulation and tortuous corkscrew-like vessels, charac- spinal cord neurostimulator had been performed, but a teristic features of the disease, were also present. The left transtibial amputation was necessary 4 years before right posterior tibial artery was the only clearly identifi- after an unsuccessful femoropopliteal bypass. able distal branch (Fig. 2a, b). During our initial consultation, the patient complained In the patient’s situation, no therapeutic alternatives of severe rest pain and paresthetic symptoms in his right were available to stop the progression of trophic changes lower limb. The extremity displayed pallor and coolness or irrepressible pain, so ongoing clinical worsening could Fig. 1 The patient’s right foot before MSC treatment. Prior to intravenous allogeneic MSC sequential infusions, trophic changes and multiple punctate ulcers were visible in the patient’s right foot (a). Close-up view of the right foot dorsum (b) Martin-Rufino et al. Stem Cell Research & Therapy (2018) 9:150 Page 3 of 6 Fig. 2 MRA of the patient’s right lower limb. Subtracted maximum intensity projection (MIP) contrast-enhanced MRA of the thighs (a) and the right calf (b). Arrows indicate multiple critical stenoses in the right limb in a and characteristic corkscrew-like collateral vessels in b have soon required a new amputation. Therefore, due to cycle (−80 °C to 37 °C). Platelets were used irrespective the rationale indicated above, sequential intravenous in- of their ABO type. Samples underwent centrifugation at fusions of allogeneic MSCs were planned. 900 g for 30 min; the supernatants were used as a sup- plement and heparin (2 IU/mL of medium) was added MSC production to prevent gel formation. All the procedures that follow were in accordance with After expansion, a total dose of 3.40 × 10 MSCs was fro- the ethical standards of the Helsinki Declaration and were zeninfourcryopreservationbags using a rate-controlled approved by the Ethics Committee of the Hospital Univer- freezing device (CM-2010 Biological Freezer, Carburos sitario de Salamanca. Informed consent was obtained from Medica, Madrid, Spain). Cells were cryopreserved in 10% the MSC-treated TAO patient and the MSC donor. MSCs dimethyl sulfoxide (DMSO) and 90% human AB plasma so- were expanded following Good Manufacturing Practice lution and stored in liquid nitrogen until thawing. guidelines in the Spanish Medicines Agency-accredited Cell Production Unit of the Hospital Universitario de Sala- manca, as previously described . MSC characterization Briefly, following standard operating procedures, 85 mL MSCs were characterized by flow cytometric analysis of of bone marrow were harvested from a healthy MSC-associated cell surface markers and multilineage 42-year-old female donor. A total of 3.04 × 10 mono- differentiation assays, as previously described . Add- nuclear cells were isolated by Ficoll-Paque itionally, MSCs were karyotyped following our previ- density-gradient centrifugation (GE Healthcare BioSci- ously reported optimized procedure . ences, AB, Uppsala, Sweden). Following resuspension, MSC-associated cell surface marker analysis was mononuclear cells were plated in noncoated polystyrene performed by incubating 200,000 MSCs with a com- flasks (Corning Costar, Celbio, Milan, Italy) in modified bination of antibodies: CD90-fluorescein isothiocyan- Eagle’smedium-α, supplemented with 1% penicillin/ ate (FITC), CD73-phycoerythrin (PE), CD45-peridinin streptomycin (Pen/Strep; Gibco, Paisley, UK) and 5% chlorophyll protein (PerCP), CD34-FITC, platelet lysate. MSCs were expanded at 37 °C, 5% CO , CD19-PerCP, CD166-PE, HLA-DR PerCP.Cy5.5, and 90% relative humidity. The medium was changed CD14-PE (all manufactured by Becton-Dickinson BD, twice a week. San Diego, CA), CD44-FICT (Immunostep, Salamanca, Human platelet lysate was obtained by pooling 4 or 5 Spain), and CD105-allophycocyanin (R&D Systems, Minne- platelet units (300–400 mL/unit) after a freeze/thaw apolis, MN). Cells were analyzed using a FACSCalibur Martin-Rufino et al. Stem Cell Research & Therapy (2018) 9:150 Page 4 of 6 7 6 cytometer (BD Biosciences, San Jose, CA) and the Infinicyt 8.5 × 10 cells (1.06 × 10 cells/kg). To prevent Software (Cytognos, Salamanca, Spain). DMSO-derived toxicity, intravenous premedication with MSC phenotype was confirmed by successfully inducing 100 mg hydrocortisone sodium phosphate, 10 mg dex- differentiation into adipogenic, chondrogenic, and osteo- chlorpheniramine, and 1 g paracetamol was adminis- genic mesenchymal lineages. All multilineage differentiation tered, and vital signs were monitored after infusion. The assays were conducted in Miltenyi Biotec differentiation scheme employed followed the PEI-06-076 Investiga- media (Bergisch Gladbach, Germany). To verify osteogenic tional Medicinal Product Dossier (IMPD), approved by differentiation, alkaline phosphatase activity was measured the Spanish Medicines Agency (AEMPS) to produce after NBT/BCIP staining (nitroblue tetrazolium chloride/ allogeneic MSCs for immune-based diseases, as previ- 5-bromo-4-chloro-3-indolyl-phosphate) (Roche, Basel, ously published . Switzerland). Adipogenic differentiation was verified by Oil Red O staining (Certistain Merck KGaA, Darmstadt, Results Germany). Finally, chondrogenic differentiation was verified No adverse effects or signs of allograft rejection were de- by immunostaining using a collagen type II-specific mouse tected following any of the four intravenous allogeneic anti-human monoclonal antibody (Chemicon International, MSC infusions. Hofheim, Germany). Release testing before administration Three months after the treatment, a regression of comprised morphology (fibroblast-like adherent cells), trophic changes in the patient’s right foot could be ob- viability (> 70% live cells measured by Trypan Blue served (Fig. 3). Six months after the infusions, the pa- + + + assay), purity by FACS (> 70% CD90 ,CD44 ,CD73 , tient had almost complete ulcer remission and + − − − − CD166 ,CD34 ,CD45 ,CD19 ,CD14 ,and restitution of the skin integrity (Fig. 3). HLA-DR cells), normal karyotype, osteogenic and In the 5-month follow-up examination, the patient re- adipogenic differentiation assays, and mycoplasma and ported a reduction in rest pain and the disappearance of sterility testing. Cell viability was 100% before freezing paresthesia. The pedal pulse was again palpable and an and > 90% after thawing in all infusions. ABI of 0.47 was measured. However, ABI correlation with functional performance is controversial . Infusion The patient’s WIQ distance score increased from 54 to Four intravenous MSC infusions were performed in 64 out of 100, and the speed and climbing scores total, on days 1, 4, 11, and 18. Each dose contained remained unaltered. Furthermore, the EQ-5D scores Fig. 3 The patient’s right foot after four sequential intravenous infusions of allogeneic MSCs. A significant regression of trophic changes was observed 3 months after the last infusion. Near complete remission of skin lesions and ulcers was seen 6 months after the treatment Martin-Rufino et al. Stem Cell Research & Therapy (2018) 9:150 Page 5 of 6 improved from 0.72 to 0.83 out of 1 in the descriptive central role in the pathogenesis of TAO has been attrib- system and to 90 out of 100 in the VAS, revealing a uted to the immune system, based on the existence of marked quality of life improvement (Fig. 4). An MRA immunocompetent cells in acute lesions and the identifi- performed 5 months after the first infusion showed no cation of elevated pro- and anti-inflammatory cytokines significant changes in the vasculature. and autoantibodies in patient sera . Recently, it has Ten months after finishing the treatment, a new spinal been proposed that smoking, the main risk factor of the cord neurostimulator was implanted to improve pain disease, could induce IL-33-mediated immune responses management. Sixteen months after the MSC infusions, that would result in vascular endothelial damage with the patient had not required any further major nor subsequent thrombosis and ischemia . Therefore, se- minor amputations. quential intravenous infusions of allogeneic MSCs could Since the donor and the recipient only shared two out potentially target the mechanisms underlying TAO of six HLA antigens (one HLA-A and one HLA-DR immunopathogenesis. antigen by conventional serological studies), the patient’s To the best of our knowledge, this is the first descrip- serum was screened for the presence of anti-HLA anti- tion of a TAO patient treated with sequential intraven- bodies (IgG isotype) by Luminex® single antigen deter- ous infusions of allogeneic MSCs. A recent report has mination. No anti-HLA antibodies were detected. demonstrated that, when injected intravenously, MSCs become trapped in the lungs and phagocytized by cells Discussion of the innate immune system. These cells rapidly distrib- The patient was referred to our hospital in the same ute the MSC effect to distant body organs after clinical situation that resulted in the amputation of his polarization towards an immunoregulatory phenotype left leg several years before, with no therapeutic options . The immunomodulatory properties of MSCs after left in the event of clinical worsening. After the joint as- intravenous infusion have been highlighted in numerous sessment of the patient between the Vascular Surgery disease models and clinical studies, and have shown Department and the Cell Therapy Unit, which had col- enormous potential in the treatment of laborated in the past for the treatment of critical limb is- immune-mediated diseases, such as GVHD [6, 17, 18]. chemia patients, the administration of MSCs under With hundreds of completed or ongoing clinical trials compassionate use was decided. In this report, the qual- , allogeneic MSCs are considered safe and may be ity of life and clinical improvement suggest that sequen- superior to autologous MSCs that are dysfunctional in tial intravenous infusions of allogeneic MSCs might be inflammatory diseases . Furthermore, this report em- an effective treatment for TAO. In the future, clinical tri- phasizes the advantages of the intravenous route as a als with larger patient numbers are needed to demon- convenient method for the administration of multiple strate safety and efficacy, which would also be an MSC doses in the outpatient setting. opportunity to perform additional studies to assess the immune-system changes induced by MSCs in this clin- Conclusion ical entity. This report shows that sequential intravenous adminis- The therapeutic approach in this patient sought a sys- tration of allogeneic MSCs might be an effective treat- temic immunomodulatory effect on the vasculature and ment for TAO. Our results warrant further studies to the immune system, rather than a local one, through the compare this approach with the more conventionally sequential intravenous infusions of allogeneic MSCs. A used intramuscular MSC administration and with other cell-based therapies in order to determine the most ef- fective approach for TAO in larger clinical trials. Abbreviations ABI: Ankle-brachial index; EQ-5D: European Quality of Life—5 dimensions; GVHD: Graft-versus-host disease; MRA: Magnetic resonance angiography; MSC: Mesenchymal stromal cell; TAO: Thromboangiitis obliterans; VAS: Visual analogue scale; WIQ: Walking Impairment Questionnaire Acknowledgments We are grateful to Teresa Garcia, Eva Lorenzo, and Manuel Herrero for their technical assistance in cell production. Fig. 4 Walking Impairment Questionnaire (WIQ) and European Quality of Life–5 dimensions (EQ-5D) scores before and 5 months Funding after intravenous allogeneic mesenchymal stromal cell (MSC) No specific funding was received from any bodies in the public, commercial infusions. DS descriptive system, VAS visual analogue scale or not-for-profit sectors to carry out the work described in this article. Martin-Rufino et al. Stem Cell Research & Therapy (2018) 9:150 Page 6 of 6 Availability of data and materials mesenchymal stem cells in patients with severe Buerger’s disease. Cell Med. The datasets used and/or analyzed during the current study are available 2017;9(3):87–102. from the corresponding author on reasonable request. 9. Luger D, Lipinski MJ, Westman PC, Glover DK, Dimastromatteo J, Frias JC, et al. Intravenously-delivered mesenchymal stem cells: systemic anti-inflammatory effects improve left ventricular dysfunction in acute myocardial infarction and Authors’ contributions ischemic cardiomyopathy. Circ Res. 2017;120(10):1598–613. JDMR contributed to data analysis and interpretation, data collection, 10. Sánchez-Guijo F, Caballero-Velázquez T, López-Villar O, Redondo A, Parody literature review, and drafted the manuscript. FSL, RFS, and FSG contributed R, Martínez C, et al. Sequential third-party mesenchymal stromal cell therapy to the conception and design of the study. AMR and EMV contributed to for refractory acute graft-versus-host disease. Biol Blood Marrow Transplant. MSC production and characterization. RR participated in data collection and 2014;20(10):1580–5. interpretation. FSL and FSG oversaw the clinical procedure, and FSG also 11. Wang J, Liao L, Wang S, Tan J. Cell therapy with autologous mesenchymal participated in the analysis and interpretation of results. All authors critically stem cells—how the disease process impacts clinical considerations. reviewed and approved the final manuscript. Cytotherapy. 2013;15(8):893–904. 12. Nicolaï SPA, Kruidenier LM, Rouwet EV, Graffius K, Prins MH, Teijink JAW. The Ethics approval and consent to participate walking impairment questionnaire: an effective tool to assess the effect of All the procedures reported were in accordance with the ethical standards of treatment in patients with intermittent claudication. J Vasc Surg. 2009;50(1):89–94. the 1975 Helsinki Declaration and its later amendments and were approved 13. Rabin R, De Charro F. EQ-5D: a measure of health status from the EuroQol by the Ethics Committee of the Hospital Universitario de Salamanca (Spain). Group. Ann Med. 2001;33:337–43. Informed consent was obtained from the MSC-treated TAO patient and the 14. Muntión S, Sánchez-Guijo FM, Carrancio S, Villarón E, López O, Diez- MSC donor. Appropriate approval was also obtained from the Spanish Medi- Campelo M, et al. Optimisation of mesenchymal stromal cells karyotyping cines Agency (AEMPS). analysis: implications for clinical use. Transfus Med. 2012;22(2):122–7. 15. Coutinho T, Rooke TW, Kullo IJ. Arterial dysfunction and functional Consent for publication performance in patients with peripheral artery disease: a review. Vasc Med. The patient’s written informed consent for publication was obtained. 2011;16:203–11. 16. de Witte SFH, Luk F, Sierra Parraga JM, Gargesha M, Merino A, Korevaar SS, Competing interests et al. Immunomodulation by therapeutic mesenchymal stromal cells (MSC) The authors declare that they have no competing interests. is triggered through phagocytosis of MSC by monocytic cells. Stem Cells. 2018;36:602–15. 17. Amorin B, Alegretti AP, Valim V, Pezzi A, Laureano AM, da Silva MAL, et al. Publisher’sNote Mesenchymal stem cell therapy and acute graft-versus-host disease: a Springer Nature remains neutral with regard to jurisdictional claims in review. Hum Cell. 2014;27(4):137–50. published maps and institutional affiliations. 18. Zhao Q, Ren H, Han Z. Mesenchymal stem cells: immunomodulatory capability and clinical potential in immune diseases. J Cell Immunother. Author details 2016;2(1):3–20. Department of Hematology, Cell Therapy Unit, IBSAL-Hospital Universitario 19. Galderisi U, Squillaro T, Peluso G. Clinical trials with mesenchymal stem cells: de Salamanca, Paseo de San Vicente 58-182, 37007 Salamanca, Spain. an update. Cell Transplant. 2016;25:829–48. Department of Angiology and Vascular Surgery, IBSAL-Hospital Universitario de Salamanca, Salamanca, Spain. Faculty of Medicine, Universidad de Salamanca, Salamanca, Spain. Department of Radiology, Hospital de Leon, Leon, Spain. Department of Angiology and Vascular Surgery, Hospital de Leon, Leon, Spain. 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Published: May 30, 2018
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