- Email: [email protected]
Optimized Method to Determine Infarct Size and Stem Cell Engraftment in Rodent Hearts Subjected to Ischemia-Reperfusion Injury
S52
Poster Abstracts
nofluorescence staining. These colonies were then subjected to Embryoid Bodies (EB) formation and trans-differentiation into mesoderm lineage cells. Presence of three germ layers in EB were assessed by immunofluorescence staining. Trans-differentiation into mesodermal lineage cells was demonstrated with Alizarin Red S for osteocytes and Oil O red for adiocytes. Results: Embryonic Stem Cells (ESCs) like colonies appeared at day 15 and were morphologically different from the parental cancer cells. Specific pluripotency markers were detected in ESCs like colonies. Three germ layers specific markers were detected in EB. OSCC-IPSCs were shown to transdifferentiate into adipocytes and osteocytes confirmed by respective tissue staining protocol. Conclusion: OSCC cells were successfully reprogrammed into iPSCs using Yamanaka’s approach, as evidenced by ESC-like morphology of the colonies, presence of pluripotency markers, EB formation with three germ layers, and capacity to trans-differentiate. H103-iPSCs can be passaged in-vitro above 20 passages without change of properties, indicating their self-renewal capacity. 144 AMNIOTIC EXOSOMES—THE WAY FORWARD? R. Lim1,2, S. Lau1, J. Tan1,2, D. Zhu1,2, E. Wallace1,2 1 The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Victoria, Australia, 2Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia Background: Mechanistically, exosomes operate as complex vectors that contain parental cell material. They can contain proteins and genetic material, which are then transferred to their target cells. While there is now growing literature on MSC-derived exosomes, there has been an absolute paucity of information on exosomes from human amnion epithelial cells (hAECs). Aims: To isolate and characterise the cargo of exosomes obtained from primary hAECs. Methods: Exosomes from hAECs were isolated using serial ultracentrifugation and purity was assessed using electron microscopy and ImageJ analysis. Proteomic analysis was performed using an in-solution trypsin digest followed by liquid chromatography and mass spectrometry. Identification of miRNAs was performed using TaqMan array followed by qPCR on Biomark HD chips (Fluidigm). In vitro assessment was performed using T cell suppression and macrophage phagocytosis assays. In vivo assessment was performed by administering either 10 ug or 50 ug of amniotic exosomes intranasally to C57Bl6 mice aged 6–8 months, 7 days following bleomycin challenge. Results: We observed that amniotic exosomes inhibit CD3/CD28 induced T cell proliferation and increased macrophage phagocytosis in a dose-dependent manner. When administered to bleomycin challenged animals, we observed that both doses of amniotic exosomes reduced fibrosis to a similar extent (p < 0.05) and this effect coincided with the reduction of activated myofibroblasts in lung tissues (p < 0.05). We also noted that exosome administration increased endogenous lung progenitor cells at the terminal bronchioles (i.e. bronchioalveolar stem cells). Conclusion: Amniotic exosomes contain cargo consistent with their biological properties and their potential as a method of cell-free warrants further exploration.
in co-cultures with stromal cells might be affected by the choice of the scaffold material. The organoid culture model presented in this work consist of adiposederived stem cells (ASC) and endothelial cells. Endothelial cells in the coculture self-assemble into sprouts that interconnect to the vascular network. Mesenchymal stem cells create the microenvironment just tens of micrometers thick with “hill and valley” topography that facilitate the ingrowth of capillaries into the organoid. After 7 days in culture, ASCs that are in close contact with capillaries differentiate into the smooth muscle cells and stabilize the vessel wall. This quasi-three dimensional system enables studying extracellular matrix deposition, cell-cell interactions and also vascular lumen formation. Supported by the Grant Agency of the Czech Rep. (grants No. P108/12/G108 and P108/12/1168), by the Technology Agency of the Czech Rep. (grant No.TA04011345) and the Agency for Healthcare Research, Ministry of Health of the Czech Republic (grants 15-33018A and 15-29153A). 146 INTRACORONARY BONE MARROW-DERIVED MESENCHYMAL STROMAL STEM CELLS INJECTION FOR ISCHEMIC CARDIOMYOPATHY PATIENT: A CASE REPORT FROM MAKATI MEDICAL CENTER, PHILIPPINES F. Chung, M. de Jesus, K. Semon, J. Santiago, C. Trinidad, M. See, G. Lopez, F. Lopez Cellular Therapeutics Center, Makati Medical Center, Makati, Manila, Philippines A 63-year-old male was diagnosed with coronary artery disease in 2001. After Coronary Artery Bypass Grafting in 2011 and coronary angioplasty in 2012, his 2D-Echocardiogram (2D-ECHO) and cardiac MRI findings showed persistent wall motion abnormalities, global hypokinesia and LV EF of 42 in August 2014. After approval from the Ethics Committee to pursue autologous stem cell therapy for ischemic cardiomyopathy, the patient underwent eligibility assessment. Bone marrow aspiration was carried out in October 2014 after signing the informed consent. The bone marrow aspirates were then evaluated by the Cellular Therapeutics Center in accordance to acceptance criteria set by the Center. Bone marrow-derived mesenchymal stromal cells (BMSCs) were cultured in class 100 clean room facility. After 51 days, BMSCs (passage 3) were evaluated according to its trilineage capacity, flow cytometric assays and sterility following the guidelines set by the International Society for Cellular Therapy. BMSCs (1 × 108) were injected via intracoronary and the patient was monitored in the hospital for 24 h. After BMSCs injection, patient was monitored via 2D-ECHO, MPI and cardiac MRI; three months, six months and 12 months, respectively. The results revealed increase in LV EF from 42 to 48 based on 2D-ECHO; based on MPI study, decrease in scar size from 18 to 6 cm2; decrease in extent of scar from 11 to 3%; based on cardiac MRI dilated LV improved in comparison with September 2014 vs December 2015; decrease in apical inferior infarct presently less than 25% compared to 100% of the wall. No safety issues were noted during the 12-month study period. Our report suggests a potential clinical utility of autologous BMSCs for ischemic cardiomyopathy.
145 CAPILLARY FORMATION IN ORGANOID CELL CULTURE MODEL J. Zarubova1, M. Molitor2, L. Bacakova1 1 Department of Biomaterials and Tissue Engineering, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic, 2Department of Plastic Surgery, Bulovka Hospital in Prague, Prague, Czech Republic
147 OPTIMIZED METHOD TO DETERMINE INFARCT SIZE AND STEM CELL ENGRAFTMENT IN RODENT HEARTS SUBJECTED TO ISCHEMIA-REPERFUSION INJURY M. Ciuffreda1,2, G. Malpasso1,2, M. Mura1,2, F. Pisano1,2, M. Gnecchi1,2 1 Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, 2Medicina Molecolare, Università degli Studi di Pavia, Pavia, Italy
One of the major limitations in construction of larger tissue replacements is cell death resulting from the inability of nutrients to diffuse across longer distances inside a scaffold. In fact, within a body most cells are found no more than 100–200 μm from the nearest capillary that enables sufficient diffusion of oxygen, nutrients and waste products. To better understand the process of capillary formation, reliable in vitro model is needed. In vitro assays that are nowadays used usually concentrate on one particular step in the angiogenic process such as cell proliferation or migration and therefore lack the desired complexity. Data obtained from 3D systems consisting of extracellular matrix scaffolds seeded with endothelial cells
Introduction and aims: Cell therapy is a promising approach to treat myocardial infarction (MI). Infarct size (IS) measurement is the best method to quantify myocardial damage. A reliable and easy method to estimate IS and a reproducible way to track the fate of the injected cells are of pivotal importance to determine the efficacy of cell therapy. Accordingly, we aimed to optimize a method allowing quantification of both IS and cell engraftment on the same heart. Methods: Rats were subjected to ischemia/reperfusion injury. After reperfusion, rat mesenchymal stem cells expressing the green fluorescent protein (GFP) or labelled with Vibrant CM-DiI (V-CM-Dil) were injected at the infarct border
22nd Annual ISCT Meeting
zone. Rats were sacrificed after two or 30 days, for IS and/or cell engraftment evaluation. The area at risk (AR) was assessed either by Evans blue or by fluorescent microspheres (FMic). IS was expressed as percentage of AR not uptaking TTC. Stem cell engraftment was evaluated at 30 days by immunohistochemistry. Results: Calculation of infarcted area resulted easier, faster and more reliable when using the FMic compared with Evans blue. In many samples, the dye permeated into the tissue colouring the heart in blue and resulting in a difficult identification of non-ischemic area. At the contrary, delimitation of the AR was easier and reproducible when using FMic. Histology showed high quality Hematoxylin&Eosin and α-sarcomeric actin only in hearts injected with FMic. At two days, both tracking methods were reliable. At day 30, cells labelled with the V-CM-Dil were promptly identified, while the GFP signal was dim even when the fluorescence was amplified using anti-GFP antibodies. Conclusions: Our study provides evidences suggesting that the use of FMic represents a better alternative compared with Evans blue for AR/IS quantification. We also demonstrated that lipophilic dyes seem to work better than GFP for cell tracking.
148 ADVANCED THERAPY MEDICINAL PRODUCTS FOR UNMET CLINICAL NEEDS: BONE MARROW-DERIVED MESENCHYMAL STEM CELL TO TREAT AIRWAY DEFECTS T. Montemurro1, S. Budelli1, M. Viganò1, C. Lavazza1, E. Montelatici1, L. Lazzari1, F. Petrella2, L. Spaggiari2,3, R. Giordano1 1 Cell Factory “F. Calori”, Unit of Cellular Therapy and Cryobiology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy, 2 Department of Thoracic Surgery, European Institute of Oncology, Milan, Italy, 3School of Medicine, University of Milan, Milan, Italy Bronchopleural fistula (BF) after pneumonectomy is an infrequent but severe condition in which standard reparative approaches are usually frustrating. Under hospital exemption and Ethics Committee approval, our hospital-based GMPapproved facility provided autologous bone marrow (BM) mesenchymal stem cells (MSC) manufactured as advanced therapy medicinal products to treat a BF occurred after extrapleural pneumonectomy in a patient affected by malignant mesothelioma. BM was obtained by puncture of the posterior iliac crest in asepsis and was directly seeded in alphaMEM supplemented with 10% qualified fetal bovine serum in a closed system (Cellstack, Macopharma). At passage 1 cultured cells were harvested, counted and cryopreserved in plastic bags at the target cell dose. The final product was tested for the following quality controls (specifications in brackets): viability with PI- staining (>80%), purity with flow cytometry (CD90+/105+/CD73+/45− > 80%), sterility following Ph. Eu. 2.6.27 (no growth), endotoxins following Ph. Eu. 2.6.14 (<0.25 EU/ml), mycoplasma following Ph. Eu. 2.6.7 (no growth) and kariotyope (46,XY). Before injection, 10x106 BMMSC were thawed at 37°C, washed and resuspended in normal saline solution (5 mL final volume). The final product, in a sterile closed syringe, was sent to the clinical department and injected in the pars membranacea of the right main bronchus stump, as close as possible to the orifice, by an endoscopic needle as already described (Petrella et al, NEJM, 2015). Sixty days later, bronchoscopy disclosed a complete healing of the resection line, with the previous orifice not longer visible. The immunocytochemical stain showed a condition consistent with repair. This application of well-established procedure is an innovative, safe, and non-invasive method for closing small caliber fistula after pneumonectomy, and provides the evidence that BMMSC may be effective in restoring otherwise untreatable airway defects.
149 BONE MARROW-DERIVED MESENCHYMAL STEM CELL THERAPY FOR MYOCARDIAL INFARCTION: A SYSTEMATIC REVIEW AND META-ANALYSIS H. Jeong, H. Yim, Y. Cho, S. Jeong, I. Oh The Catholic University of Korea, Seoul, Republic of Korea Introduction: Recent studies suggest that the bone marrow-derived mononuclear cells may improve left ventricular function in patients with myocardial infarction (MI). However, there is still argumentative for the safety and efficacy of bone marrow-derived mesenchymal stem cell (BM-MSC) in the treatment of patients with myocardial infarction. Thus, we conducted systematic review and meta-analysis to assess the efficacy and safety also, we evaluated the mag-
S53
nitude of the treatment effect of BM-MSCs as time progressed in the treatment of patients with myocardial infarction. Methods: We searched Medline, EMBASE, and Cochrane databases from inception to November 2015 for studies of BM-MSC transplantation in patients with MI. The articles included in the search were restricted to the English language, studies compared with placebo as a control group, and efficacy evaluation using cardiac function. Article selection and data extraction were conducted by two authors independently with standard methods. Outcome data were analyzed by using Review Manager 5.3, and presented as weighted mean differences and 95% confidence intervals. Data were pooled by use of the DerSimonianLaird random-effects model because of the high degree of heterogeneity. Results: We included 9 studies in the final meta-analysis to summarize the efficacy of BM-MSC for MI patient. Compared with the control group, BMMSC transplantation improved left ventricular ejection fraction (LVEF) by 3.03 (95%CI: 2.34–3.68) at 6 months. 6-min walking distance increased 310 cm (95%CI: −14.2–76.3 cm) and wall motion index score slightly decreased at 6 month, however it is not statistically significant. Mean changes LVEF at 3, 6, 12, 24 months 10.5%, 6.9%, 6.8%, and 4.3%, respectively. Mean relative changes from baseline of cardiac function during 24 months. LVEF increased peak at 3 month and showed decreased trend as time goes on. Conclusion: The findings of this meta-analysis indicate that BM-MSCs may be beneficial in improving heart function in the treatment of MI. However, this improvement was not maintained in long term follow-up analyses. This research was supported by a grant (10172MFDS993) from Ministry of Food & Drug Safety in 2014. 150 IMPROVING THE CARDIOPROTECTIVE AND REGENERATIVE PROPERTIES OF BONE MARROW DERIVED MESENCHYMAL STEM CELLS THROUGH THE OVEREXPRESSION OF IGF1 AND BMP2 M. Mura1,2, M. Ciuffreda1, F. Pisano1, G. Malpasso1, M. Gnecchi1,2 1 Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, 2Molecular Medicine, University of Pavia, Pavia, Italy Background: The effects of bone marrow derived mesenchymal stem cells (BMMSC) on ischemic hearts are limited by poor engraftment and low regenerative capacity. Moreover, the cardioprotective paracrine properties are impaired by donor age. Here, we attempted to overcome these hurdles by overexpressing IGF1 and BMP2 (IB), two factors involved in cardiac differentiation and cytoprotection. Methods: Rat BM-MSC were transduced with a novel lentiviral vector encoding either GFP (GFP-MSC) or both IGF1 and BMP2 (IB-MSC). Autocrine cytoprotection was evaluated after MSC exposure to hypoxia. The cytoprotective effects of medium conditioned by GFP-MSC (GFP-CM) or by IB-MSC (IBCM) were compared with control medium (CTRL-M) on cardiac cells (H9c2) exposed to hypoxia/reoxygenation (H/R). Paracrine stem cell activation was evaluated on rat cardiac stem cells (CSC). Viability, cytotoxicity and apoptosis were measured by dedicated assays. The expression of cardiac genes was tested by qPCR, immunoblot and immunostaining. In vivo, rats were subjected to ischemia/ reperfusion injury (I/R) and treated with saline, GFP- or IB-MSC. Cardiac function and histological analysis were performed 2 and 30 days after I/R. Results: Compared with GFP-MSC, IB-MSC showed higher expression of cardiac genes and transcription factors. IB-MSC showed increased viability (+26.8%, p < 0.01), reduced apoptosis (−62.9%, p < 0.001) and cytotoxicity (−22.9%, p < 0.05) vs GFP-MSC after H/R. IB-CM increased H9c2 viability (+21.4% vs GFP-CM, p < 0.01) and reduced apoptosis (−60.4% vs GFPMSC, p < 0.001) after H/R. Furthermore, CSC fed with IB-CM showed increased proliferation and up-regulation of cardiac genes. In vivo, IB-MSC engrafted better (+81% vs GFP-MSC, p < 0.01) and significantly reduced myocardial infarct size of 31% vs GFP-MSC and 40% vs saline (p < 0.01). Finally, IB-MSC improved ejection fraction at 30d (p < 0.05 vs GFP-MSC and saline). Conclusions: IB overexpression commits BM-MSC toward the cardiac lineage and concomitantly increases their resistance to hypoxia and their paracrine effects. Most importantly, IB-MSC lead to myocardial repair after I/R. 151 UMBILICAL CORD DERIVED SUB-EPITHELIAL CELLS IMPROVE HEART FUNCTION POST MYOCARDIAL INFARCTION C. Bartlett, D Atkinson, R. Walker, F. Silva, A. Patel University of Utah, Salt Lake City, Utah, USA
Poster Abstracts
nofluorescence staining. These colonies were then subjected to Embryoid Bodies (EB) formation and trans-differentiation into mesoderm lineage cells. Presence of three germ layers in EB were assessed by immunofluorescence staining. Trans-differentiation into mesodermal lineage cells was demonstrated with Alizarin Red S for osteocytes and Oil O red for adiocytes. Results: Embryonic Stem Cells (ESCs) like colonies appeared at day 15 and were morphologically different from the parental cancer cells. Specific pluripotency markers were detected in ESCs like colonies. Three germ layers specific markers were detected in EB. OSCC-IPSCs were shown to transdifferentiate into adipocytes and osteocytes confirmed by respective tissue staining protocol. Conclusion: OSCC cells were successfully reprogrammed into iPSCs using Yamanaka’s approach, as evidenced by ESC-like morphology of the colonies, presence of pluripotency markers, EB formation with three germ layers, and capacity to trans-differentiate. H103-iPSCs can be passaged in-vitro above 20 passages without change of properties, indicating their self-renewal capacity. 144 AMNIOTIC EXOSOMES—THE WAY FORWARD? R. Lim1,2, S. Lau1, J. Tan1,2, D. Zhu1,2, E. Wallace1,2 1 The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Victoria, Australia, 2Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia Background: Mechanistically, exosomes operate as complex vectors that contain parental cell material. They can contain proteins and genetic material, which are then transferred to their target cells. While there is now growing literature on MSC-derived exosomes, there has been an absolute paucity of information on exosomes from human amnion epithelial cells (hAECs). Aims: To isolate and characterise the cargo of exosomes obtained from primary hAECs. Methods: Exosomes from hAECs were isolated using serial ultracentrifugation and purity was assessed using electron microscopy and ImageJ analysis. Proteomic analysis was performed using an in-solution trypsin digest followed by liquid chromatography and mass spectrometry. Identification of miRNAs was performed using TaqMan array followed by qPCR on Biomark HD chips (Fluidigm). In vitro assessment was performed using T cell suppression and macrophage phagocytosis assays. In vivo assessment was performed by administering either 10 ug or 50 ug of amniotic exosomes intranasally to C57Bl6 mice aged 6–8 months, 7 days following bleomycin challenge. Results: We observed that amniotic exosomes inhibit CD3/CD28 induced T cell proliferation and increased macrophage phagocytosis in a dose-dependent manner. When administered to bleomycin challenged animals, we observed that both doses of amniotic exosomes reduced fibrosis to a similar extent (p < 0.05) and this effect coincided with the reduction of activated myofibroblasts in lung tissues (p < 0.05). We also noted that exosome administration increased endogenous lung progenitor cells at the terminal bronchioles (i.e. bronchioalveolar stem cells). Conclusion: Amniotic exosomes contain cargo consistent with their biological properties and their potential as a method of cell-free warrants further exploration.
in co-cultures with stromal cells might be affected by the choice of the scaffold material. The organoid culture model presented in this work consist of adiposederived stem cells (ASC) and endothelial cells. Endothelial cells in the coculture self-assemble into sprouts that interconnect to the vascular network. Mesenchymal stem cells create the microenvironment just tens of micrometers thick with “hill and valley” topography that facilitate the ingrowth of capillaries into the organoid. After 7 days in culture, ASCs that are in close contact with capillaries differentiate into the smooth muscle cells and stabilize the vessel wall. This quasi-three dimensional system enables studying extracellular matrix deposition, cell-cell interactions and also vascular lumen formation. Supported by the Grant Agency of the Czech Rep. (grants No. P108/12/G108 and P108/12/1168), by the Technology Agency of the Czech Rep. (grant No.TA04011345) and the Agency for Healthcare Research, Ministry of Health of the Czech Republic (grants 15-33018A and 15-29153A). 146 INTRACORONARY BONE MARROW-DERIVED MESENCHYMAL STROMAL STEM CELLS INJECTION FOR ISCHEMIC CARDIOMYOPATHY PATIENT: A CASE REPORT FROM MAKATI MEDICAL CENTER, PHILIPPINES F. Chung, M. de Jesus, K. Semon, J. Santiago, C. Trinidad, M. See, G. Lopez, F. Lopez Cellular Therapeutics Center, Makati Medical Center, Makati, Manila, Philippines A 63-year-old male was diagnosed with coronary artery disease in 2001. After Coronary Artery Bypass Grafting in 2011 and coronary angioplasty in 2012, his 2D-Echocardiogram (2D-ECHO) and cardiac MRI findings showed persistent wall motion abnormalities, global hypokinesia and LV EF of 42 in August 2014. After approval from the Ethics Committee to pursue autologous stem cell therapy for ischemic cardiomyopathy, the patient underwent eligibility assessment. Bone marrow aspiration was carried out in October 2014 after signing the informed consent. The bone marrow aspirates were then evaluated by the Cellular Therapeutics Center in accordance to acceptance criteria set by the Center. Bone marrow-derived mesenchymal stromal cells (BMSCs) were cultured in class 100 clean room facility. After 51 days, BMSCs (passage 3) were evaluated according to its trilineage capacity, flow cytometric assays and sterility following the guidelines set by the International Society for Cellular Therapy. BMSCs (1 × 108) were injected via intracoronary and the patient was monitored in the hospital for 24 h. After BMSCs injection, patient was monitored via 2D-ECHO, MPI and cardiac MRI; three months, six months and 12 months, respectively. The results revealed increase in LV EF from 42 to 48 based on 2D-ECHO; based on MPI study, decrease in scar size from 18 to 6 cm2; decrease in extent of scar from 11 to 3%; based on cardiac MRI dilated LV improved in comparison with September 2014 vs December 2015; decrease in apical inferior infarct presently less than 25% compared to 100% of the wall. No safety issues were noted during the 12-month study period. Our report suggests a potential clinical utility of autologous BMSCs for ischemic cardiomyopathy.
145 CAPILLARY FORMATION IN ORGANOID CELL CULTURE MODEL J. Zarubova1, M. Molitor2, L. Bacakova1 1 Department of Biomaterials and Tissue Engineering, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic, 2Department of Plastic Surgery, Bulovka Hospital in Prague, Prague, Czech Republic
147 OPTIMIZED METHOD TO DETERMINE INFARCT SIZE AND STEM CELL ENGRAFTMENT IN RODENT HEARTS SUBJECTED TO ISCHEMIA-REPERFUSION INJURY M. Ciuffreda1,2, G. Malpasso1,2, M. Mura1,2, F. Pisano1,2, M. Gnecchi1,2 1 Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, 2Medicina Molecolare, Università degli Studi di Pavia, Pavia, Italy
One of the major limitations in construction of larger tissue replacements is cell death resulting from the inability of nutrients to diffuse across longer distances inside a scaffold. In fact, within a body most cells are found no more than 100–200 μm from the nearest capillary that enables sufficient diffusion of oxygen, nutrients and waste products. To better understand the process of capillary formation, reliable in vitro model is needed. In vitro assays that are nowadays used usually concentrate on one particular step in the angiogenic process such as cell proliferation or migration and therefore lack the desired complexity. Data obtained from 3D systems consisting of extracellular matrix scaffolds seeded with endothelial cells
Introduction and aims: Cell therapy is a promising approach to treat myocardial infarction (MI). Infarct size (IS) measurement is the best method to quantify myocardial damage. A reliable and easy method to estimate IS and a reproducible way to track the fate of the injected cells are of pivotal importance to determine the efficacy of cell therapy. Accordingly, we aimed to optimize a method allowing quantification of both IS and cell engraftment on the same heart. Methods: Rats were subjected to ischemia/reperfusion injury. After reperfusion, rat mesenchymal stem cells expressing the green fluorescent protein (GFP) or labelled with Vibrant CM-DiI (V-CM-Dil) were injected at the infarct border
22nd Annual ISCT Meeting
zone. Rats were sacrificed after two or 30 days, for IS and/or cell engraftment evaluation. The area at risk (AR) was assessed either by Evans blue or by fluorescent microspheres (FMic). IS was expressed as percentage of AR not uptaking TTC. Stem cell engraftment was evaluated at 30 days by immunohistochemistry. Results: Calculation of infarcted area resulted easier, faster and more reliable when using the FMic compared with Evans blue. In many samples, the dye permeated into the tissue colouring the heart in blue and resulting in a difficult identification of non-ischemic area. At the contrary, delimitation of the AR was easier and reproducible when using FMic. Histology showed high quality Hematoxylin&Eosin and α-sarcomeric actin only in hearts injected with FMic. At two days, both tracking methods were reliable. At day 30, cells labelled with the V-CM-Dil were promptly identified, while the GFP signal was dim even when the fluorescence was amplified using anti-GFP antibodies. Conclusions: Our study provides evidences suggesting that the use of FMic represents a better alternative compared with Evans blue for AR/IS quantification. We also demonstrated that lipophilic dyes seem to work better than GFP for cell tracking.
148 ADVANCED THERAPY MEDICINAL PRODUCTS FOR UNMET CLINICAL NEEDS: BONE MARROW-DERIVED MESENCHYMAL STEM CELL TO TREAT AIRWAY DEFECTS T. Montemurro1, S. Budelli1, M. Viganò1, C. Lavazza1, E. Montelatici1, L. Lazzari1, F. Petrella2, L. Spaggiari2,3, R. Giordano1 1 Cell Factory “F. Calori”, Unit of Cellular Therapy and Cryobiology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy, 2 Department of Thoracic Surgery, European Institute of Oncology, Milan, Italy, 3School of Medicine, University of Milan, Milan, Italy Bronchopleural fistula (BF) after pneumonectomy is an infrequent but severe condition in which standard reparative approaches are usually frustrating. Under hospital exemption and Ethics Committee approval, our hospital-based GMPapproved facility provided autologous bone marrow (BM) mesenchymal stem cells (MSC) manufactured as advanced therapy medicinal products to treat a BF occurred after extrapleural pneumonectomy in a patient affected by malignant mesothelioma. BM was obtained by puncture of the posterior iliac crest in asepsis and was directly seeded in alphaMEM supplemented with 10% qualified fetal bovine serum in a closed system (Cellstack, Macopharma). At passage 1 cultured cells were harvested, counted and cryopreserved in plastic bags at the target cell dose. The final product was tested for the following quality controls (specifications in brackets): viability with PI- staining (>80%), purity with flow cytometry (CD90+/105+/CD73+/45− > 80%), sterility following Ph. Eu. 2.6.27 (no growth), endotoxins following Ph. Eu. 2.6.14 (<0.25 EU/ml), mycoplasma following Ph. Eu. 2.6.7 (no growth) and kariotyope (46,XY). Before injection, 10x106 BMMSC were thawed at 37°C, washed and resuspended in normal saline solution (5 mL final volume). The final product, in a sterile closed syringe, was sent to the clinical department and injected in the pars membranacea of the right main bronchus stump, as close as possible to the orifice, by an endoscopic needle as already described (Petrella et al, NEJM, 2015). Sixty days later, bronchoscopy disclosed a complete healing of the resection line, with the previous orifice not longer visible. The immunocytochemical stain showed a condition consistent with repair. This application of well-established procedure is an innovative, safe, and non-invasive method for closing small caliber fistula after pneumonectomy, and provides the evidence that BMMSC may be effective in restoring otherwise untreatable airway defects.
149 BONE MARROW-DERIVED MESENCHYMAL STEM CELL THERAPY FOR MYOCARDIAL INFARCTION: A SYSTEMATIC REVIEW AND META-ANALYSIS H. Jeong, H. Yim, Y. Cho, S. Jeong, I. Oh The Catholic University of Korea, Seoul, Republic of Korea Introduction: Recent studies suggest that the bone marrow-derived mononuclear cells may improve left ventricular function in patients with myocardial infarction (MI). However, there is still argumentative for the safety and efficacy of bone marrow-derived mesenchymal stem cell (BM-MSC) in the treatment of patients with myocardial infarction. Thus, we conducted systematic review and meta-analysis to assess the efficacy and safety also, we evaluated the mag-
S53
nitude of the treatment effect of BM-MSCs as time progressed in the treatment of patients with myocardial infarction. Methods: We searched Medline, EMBASE, and Cochrane databases from inception to November 2015 for studies of BM-MSC transplantation in patients with MI. The articles included in the search were restricted to the English language, studies compared with placebo as a control group, and efficacy evaluation using cardiac function. Article selection and data extraction were conducted by two authors independently with standard methods. Outcome data were analyzed by using Review Manager 5.3, and presented as weighted mean differences and 95% confidence intervals. Data were pooled by use of the DerSimonianLaird random-effects model because of the high degree of heterogeneity. Results: We included 9 studies in the final meta-analysis to summarize the efficacy of BM-MSC for MI patient. Compared with the control group, BMMSC transplantation improved left ventricular ejection fraction (LVEF) by 3.03 (95%CI: 2.34–3.68) at 6 months. 6-min walking distance increased 310 cm (95%CI: −14.2–76.3 cm) and wall motion index score slightly decreased at 6 month, however it is not statistically significant. Mean changes LVEF at 3, 6, 12, 24 months 10.5%, 6.9%, 6.8%, and 4.3%, respectively. Mean relative changes from baseline of cardiac function during 24 months. LVEF increased peak at 3 month and showed decreased trend as time goes on. Conclusion: The findings of this meta-analysis indicate that BM-MSCs may be beneficial in improving heart function in the treatment of MI. However, this improvement was not maintained in long term follow-up analyses. This research was supported by a grant (10172MFDS993) from Ministry of Food & Drug Safety in 2014. 150 IMPROVING THE CARDIOPROTECTIVE AND REGENERATIVE PROPERTIES OF BONE MARROW DERIVED MESENCHYMAL STEM CELLS THROUGH THE OVEREXPRESSION OF IGF1 AND BMP2 M. Mura1,2, M. Ciuffreda1, F. Pisano1, G. Malpasso1, M. Gnecchi1,2 1 Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, 2Molecular Medicine, University of Pavia, Pavia, Italy Background: The effects of bone marrow derived mesenchymal stem cells (BMMSC) on ischemic hearts are limited by poor engraftment and low regenerative capacity. Moreover, the cardioprotective paracrine properties are impaired by donor age. Here, we attempted to overcome these hurdles by overexpressing IGF1 and BMP2 (IB), two factors involved in cardiac differentiation and cytoprotection. Methods: Rat BM-MSC were transduced with a novel lentiviral vector encoding either GFP (GFP-MSC) or both IGF1 and BMP2 (IB-MSC). Autocrine cytoprotection was evaluated after MSC exposure to hypoxia. The cytoprotective effects of medium conditioned by GFP-MSC (GFP-CM) or by IB-MSC (IBCM) were compared with control medium (CTRL-M) on cardiac cells (H9c2) exposed to hypoxia/reoxygenation (H/R). Paracrine stem cell activation was evaluated on rat cardiac stem cells (CSC). Viability, cytotoxicity and apoptosis were measured by dedicated assays. The expression of cardiac genes was tested by qPCR, immunoblot and immunostaining. In vivo, rats were subjected to ischemia/ reperfusion injury (I/R) and treated with saline, GFP- or IB-MSC. Cardiac function and histological analysis were performed 2 and 30 days after I/R. Results: Compared with GFP-MSC, IB-MSC showed higher expression of cardiac genes and transcription factors. IB-MSC showed increased viability (+26.8%, p < 0.01), reduced apoptosis (−62.9%, p < 0.001) and cytotoxicity (−22.9%, p < 0.05) vs GFP-MSC after H/R. IB-CM increased H9c2 viability (+21.4% vs GFP-CM, p < 0.01) and reduced apoptosis (−60.4% vs GFPMSC, p < 0.001) after H/R. Furthermore, CSC fed with IB-CM showed increased proliferation and up-regulation of cardiac genes. In vivo, IB-MSC engrafted better (+81% vs GFP-MSC, p < 0.01) and significantly reduced myocardial infarct size of 31% vs GFP-MSC and 40% vs saline (p < 0.01). Finally, IB-MSC improved ejection fraction at 30d (p < 0.05 vs GFP-MSC and saline). Conclusions: IB overexpression commits BM-MSC toward the cardiac lineage and concomitantly increases their resistance to hypoxia and their paracrine effects. Most importantly, IB-MSC lead to myocardial repair after I/R. 151 UMBILICAL CORD DERIVED SUB-EPITHELIAL CELLS IMPROVE HEART FUNCTION POST MYOCARDIAL INFARCTION C. Bartlett, D Atkinson, R. Walker, F. Silva, A. Patel University of Utah, Salt Lake City, Utah, USA