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1 New strategies for diagnostic, therapeutic and clinical care in The new biotechnologies Project Classification IRG: Project Classification SS: Bioengineering Sciences and Technologies Gene and Drug Delivery Systems - GDD Project Keyword 1: Project Keyword 2: Project Keyword 3: Delivery of nucleic acids, peptide/protein complexes, vaccines, genes, small molecules, antibiotics and other drugs and biomaterials. Generation of Red Blood Cells (RBCs) from Cord Blood (CB) Induced Pluripotent Stem Cells (ipscs) Blood Group Systems Project Request: Animals: X Humans: X Clinical trial: The project has already been presented: Project code reference: Dichiaro che il progetto ha per oggetto prodotti o idee già coperte da brevetto industriale da parte della ditta co-finanziatrice o comunque sulla quale gravino diritti di un soggetto giuridico diverso dal DI. Overall Summary The transfusion of red blood cells(rbcs) represents the most important supportive therapy now consolidated and universally adopted in many clinical settings for the management of acute and chronic anemias. Difficulties and limitations still occur, such as long-term blood product shortage, blood donor aging, infectious agents, and the inescapable blood group polymorphism barrier. Functional RBCs production has been achieved from different sources of hematopoietic stem cells, but these cells are unable to alleviate the ongoing need for donors because of their limited in vitro expansion capabilities. Human induced pluripotent stem cells (hipscs) can be propagated in vitro indefinitely, providing a potentially unlimited and donor-less source for therapeutic purposes. Blood group type specific-ipscs generated from selected cord blood(cb) units could offer an inexhaustible source of RBCs that may be used for patients with alloimmunization or a rare phenotype of different ethnic groups. Background / State of Art Medical progress, specifically in the fields of hematology/oncology, transplantations and elective surgery, has led to an ever-increasing demand for erythrocytes for transfusion. In fact, in clinical practice, transfusion of RBCs is widely used to treat conditions associated with inheritance anemias, blood loss,marrow failure and transplant engraftment.more recently, systems for in vitro production of RBCs from hematopoietic progenitors have been developed. In this new and promising scientific scenario, CB stem cells have several distinct advantages over other haematopoietic stem cell sources: they can be cryopreserved and stored for years without any loss of viability and self renewal capability. Recent reports of successful production of functional RBCs from CB-hiPSCs provide evidence for the feasibility of such approach. ipscs represent a potentially unlimited source of RBCs and open the door to the revolutionary development of a new generation of allogeneic transfusion products. 1 / 22

2 Project Code: Hyphotesis and Specific AIMS Hyphotesis and Significance: The main steps of this project are: 1. Collection of CB units with RBC rare and uncommon blood group phenotypes. 2. Generation of transgene free CB-iPSCs from CD34+ cord blood stem cells by ectopic expression of the four Yamanaka factors (Oct4,Sox2,Klf4.c-Myc). 3. Differentiation of human CB-iPSC into early erythroid progenitors. 4. Production of RBCs through terminal differentiation and enucleation of CB-iPSCs derived erythroid cells. 5. Characterization and functional validation of CB-iPSCs derived RBCs with serological and molecular investigation for blood group typing, and with biochemical, chromatographic and electrophoretic analysis for evaluating the erythrocyte protein membrane and metabolism. Specific Aim: Generate ipscs derived from cells of umbilical cord blood that are selected for rare antigens or uncommon phenotypes of blood group systems. Umbilical cord blood is a tissue rich in stem cells and readily available. In comparison with stem cells isolated from aged individuals, cord blood stem cells are expected to be superior because nuclear and mitochondrial mutations tend to accumulate in adult stem cells and differentiated somatic lineages over an organism s lifetime. Analyze gene expression of ipscs for evaluating the efficiency of generation and the genetic stability of the cells. Generate hematopoietic intermediate with subsequent enucleation and differentiation in RBCs. Assess their functional parameters and antigenic typing. Experimental Design Aim 1: CB units will be selected for some rare blood group antigens (<1/5000) or uncommon combination phenotypes (<1/1000) by serological and extended molecular typing. CD34+ stem cells will be isolated from CB units by immunomagnetic selection and reprogrammed into hipscs using the four Yamanaka factors (Oct4,Sox2,Klf4.c-Myc). For generation of transgene-free CB-iPSCs, we will use the Sendai virus system (SeV), as an highly efficient and safe method. In contrast with many available protocols, the SeV vectors are nonintegrating and remain in the cytoplasm (zero footprints). In addition, the host cell can be cleared of the vectors and reprogramming genes by temperature sensitivity. CD34+ stem cells will be transduced with the four SeVs and after two days of incubation will be seeded into imefs culture dishes in medium supplemented with hfgf until colonies appearance. When colonies are ready for transfer, they will be manually picked and transferred separately onto new imefs plates for expansion and characterization. Experimental Design Aim 2: CB-iPSCs will be completely characterized for pluripotency using gene expression analysis, immunofluorescence and differentiation ability in vitro and in vivo. Each CB-iPSC line will be subjected to alkaline phosphatase staining, a marker commonly used as a quick test for the pluripotent state. The expression analysis of pluripotency associated genes such as Oct4, Nanog, Sox2, SSEA4, Rex1, Tra-1-81 and TRA1-60 will be performed both by immunostaining and quantitative RT-PCR. The expression of the Sendai virus genome and transgenes in the vector-free CB-iPSCs will be determined by RT-PCR in order to confirm the complete absence of viral vectors. 2 / 22

3 Project Code: The ability to differentiate into all three lineages (endoderm, mesoderm and ectoderm), a key characteristic of ipscs, will be tested by immunohistochemistry both in vitro (EBs formation assay) and in vivo (teratoma formation). Karyotype analysis of CB-iPSCs will be performed to check the correct number and morphology of chromosomes, since both reprogramming and extended culture in vitro can expose cells to high selection pressures, resulting in genomic instability. Experimental Design Aim 3: After complete characterization for pluripotency, CB-iPSCs will be differentiated into enucleated erythrocytes and we will use a five step approach to achieve hematopoietic differentiation of CB-iPSCs. In the first step, CB-iPSCs will be co-cultured on OP9 stromal cells to induce their differentiation into hematopoietic stem cells. In the second step, CD34+ cells will be sorted from the co-culture population prior to seeding them in media supplemented with cytokines and BMP-4, which promotes hematopoietic differentiation. In the third step, the erythroid progenitors will be stimulated using the IGF-1, necessary for normal erythroid differentiation. In the fourth step, terminal erythroid maturation will be induced by co-culture with MS-5 stromal cells, in serum-free medium containing Epo and hemin. The addiction of hemin supposedly improves the iron transport, whereas stromal cells have been shown to promote terminal differentiation and enucleation of erythroid cells. Full enucleation and maturation of induced erythroid cells will be also assessed in NOD-SCID mice in vivo, after the final coculture with MS-5 stroma, to assess the ability of this cells to complete differentiation in terminally and functionally generated erythrocytes. The erythroid cells will be then analyzed by flow cytometry for the CD34, CD45, CD71 (tranferrin), and CD235a (glycophorin A) antigens. Cell morphology analysis will be done using Wright Giemsa staining at different time points during the liquid culture. Hemoglobin concentration will be measured by spectrophotometry at each stage of erythroid maturation. To confirm the erythroid lineage, quantitative real time RT-PCR on total RNA and high-performance liquid chromatography (HPLC) analysis on cell lysates will be performed at each stage of differentiation. The following analyses on generated red cells will be carried out: the evaluation of metabolic (percentage of hemolysis and blood gas analysis) and physical parameters (automated blood count, erythrocyte osmotic fragility, hemoglobin fractions and enzyme activity analysis), the determination of surface blood group alloantigens (extended typing with immunohematological investigation) and membrane protein expression (SDS-PAGE assay). Finally, a transplant mouse model will be developed. The generated human RBCs will be injected in mice and then flow cytometry will be performed 2h and 24h in blood samples after transfusion to check the ratio of human/murine RBCs. Preliminary Data: We have generated CB-iPSCs lines both from CD133+ and CD34+ stem cells of different cord blood units by retroviralmediated overexpression of the pluripotency-associated trascription factors (OCT4,SOX2,KLF4,and c-myc). CB-iPSCs lines were characterized for expression of pluripotency-associated genes, surface markers and differentiation ability in vitro. All the CB-iPSCs lines tested showed strong alkaline phosphatase and expression of pluripotency markers such as OCT4, SOX2, TRA-1-81, TRA-1-60, SSEA3, SSEA4, and NANOG, revealed by immunofluorescence. Quantitative RT-PCR showed that all CB-iPSCs lines tested expressed a set of pluripotency genes, including OCT4, SOX2, NANOG, CRIPTO and REX1. Expression levels of the transgenes were determined by qpcr and showed a varied degree of silencing between different clones and individual transgenes. The differentiation potential of CB-iPSCs lines was evaluated in vitro showing that CBiPSCs were able to form embryoid bodies (EBs) with high efficiency. CB-iPSCs derived EBs were able to differentiate into derivatives of the three embryonic germ layers, including Tuj1-positive ectoderm, a-fetoprotein (AFP) and FoxA2-positive 3 / 22

4 Project Code: endoderm and a-sarcomeric actin (ASA) and GATA4-positive mesoderm. Finally,cytogenetic analysis showed that CBiPSCs lines maintained a normal 46XY or 46XX karyotype for more than 10 passages. Picture to support preliminary data: C:\Users\ANTONELLA\Desktop\VF ipscs Project\VF FIGURE_1.pdf Metodologies and statistical analyses: For generation of CB-iPSCs we will use different cell culture techniques such as separation and culture of haematopoietic stem cells, somatic and stromal cells culture, virus mediated gene transduction, ipscs derivation and culture and embryoid bodies formation assay. Molecular characterization of CB-iPSCs will be performed by extraction and quantification of DNA, RNA and proteins, RTqPCR, nucleid acids and proteins electrophoresis, immunofluorescence and Karyotype analysis. Characterization of erythroid cells will be done by flow cytometry, light microscopy,wright Giemsa staining, spectrophotometry, quantitative real time RT-PCR and high-performance liquid chromatography analysis ( HPLC). For extended blood group typing will be use some serological (tube test, microplate and column agglutination with anti-sera of different companies) and molecular techniques with SSP-PCR and DNA microarray assay (Beadchip system). For physical parameters will be used an osmometer for erythrocyte osmotic fragility, an instrument (differential Ph technology) for G6PD and PK enzyme activity measurement, HPLC instrument for hemoglobin fractions analysis and SDS- PAGE assay for membrane protein expression. In vitro generated red cells from CB-iPSCs and their intermediates will be compared to native red cells obtained from the same CB in terms of metabolic and physical parameters, expression of membrane protein and of sample-specific profiles of red cell blood group antigens (using a scoring system ranging from 0 to 4). Parametric and non-parametric tests will be used for comparisons as appropriate and a p < 0.05 will be considered as significant. Expected outcomes, possible problems and solutions: We expect to generate ipscs from cells of umbilical cord blood units selected for rare antigens or uncommon blood group phenotype. We plan to reprogram CD34+ fraction using the Sendai virus system (SeV), an highly efficient and safe method. The CB-iPSCs will be characterized for pluripotency and then differentiated into enucleated functional RBCs. Finally, the produced RBCs will be tested both in vitro and in vivo. Possible difficulties could occur during the differentiation process and could affect the phenotype and the functionality of produced red cells. Therefore, rigorous studies must be conducted to fully characterize these in vitro generated RBCs including their membrane surface potential, liability, and half-life in vivo, in addition to profiling their hemoglobin packing, gas exchange properties, and immunogenicities 4 / 22

5 Significance and Innovation CB-iPSCs derived RBCs could represent a new allogeneic transfusion products.the reprogramming of CB-iPSCs using the SeV system has a significant advantage over presently available methods for its safety, efficiency and convenience. In fact, the SeV allows transgenes expression without modification of the host genome, so the resulting ipscs are genetically intact and carry the same genome DNA as the original cells. The use of young CB stem cells for the PSCs generation represents an evident advantage over the use of adult somatic cell types, since they are readily available and are already banked along with immunological information. In contrast to other ex vivo stem-cell-derived cellular therapeutics, tumorigenesis is not a concern, as RBCs can be irradiated without marked adverse effects on in vivo function. In vitro large-scale cultured RBC production could provide potential and effective applications for alloimmunized patients or with a rare blood group of various populations. Description of the complementary and sinergy research team The coordination of this project will concentrate in promoting the natural flow of information between the research groups in order to focus on the main goals of the project. The main flow of information will run from the research group (Dr. Matteocci and Dr. Fazzina) that will select cord blood units with rare or uncommon rare blood group pheno-genotyping (Dr. Matteocci- Prof. Grammatico) to generate the CB-iPSCs and further differentiate them into erythroid progenitors and RBCs (Dr. Fazzina). Once the erythroid progenitors and RBCs are generated, the cells will be passed to Prof. Grammatico for molecular investigation in collaboration with Dr. Fazzina which will also perform further characterization analysis of CB-iPSCs derived erythroid cells. Next, the produced RBCs will be analyzed by evaluation of metabolic and physical parameters, determination of blood group alloantigens and protein expression (Dr. Matteocci), in order to correlate the findings with the clinical evidences. Finally, Prof. Pierelli will set up an animal model to assess RBCs functionality in vivo in collaboration with Dr. Fazzina. According to the description of the project the research teams will perform the different goals following the established time line. The coordinating team will be in charge to ensure that the different objectives will be achieved. Dr Matteocci (PI) will be also responsible for statistical analysis, final report and publications. 5 / 22

6 Bibliography Generation and characterization of erythroid cells from human embryonic stem cells and induced pluripotent stem cells: an overview. Chang KH. et al. Stem Cells Int. 2011;2011: Generation of red blood cells from human induced pluripotent stem cells. J. Dias et al. Stem Cells and Dev vol. 20, no. 9, pp , Banking of pluripotent adult stem cells as an unlimited source for red blood cell production: potential applications for alloimmunized patients and rare blood challenges. Peyrard T. et al. Transfus Med Rev Jul;25(3): Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine. Lapillonne H. et al. Haematologica Oct;95(10): Production of erythroid cells from human embryonic stem cells (hesc) and human induced pluripotent stem cells (hips). Anstee DJ. Transfus Clin Biol Sep;17(3):104-9 Efficient generation of transgene-free human induced pluripotent stem cells (ipscs) by temperature-sensitive Sendai virus vectors. Ban H. et al. Proc Natl Acad Sci U S A Aug 23;108(34): Human cord blood reprogrammed into embryonic-like stem cells A. Giorgetti, R. Fazzina, M. Li, J. C. I. Belmonte ISBT Science Series May Volume 6, Issue 1. Generation of induced pluripotent stem cells from human cord blood cells with only two factors:oct4 and Sox2. Giorgetti A.et al. JC.Nat Protoc Apr;5(4): Timeline / Deliverables / Payable Milestones The timeline will consist of the achievement of the following milestones at 18 months and 36 months and all the included points will be considered as specific deliverables. For a more detailed plan during time of the project see also the attached GANTT chart. Finally, the payable milestones are the following: 1. Generation of human ipsc from CD34+ of selected umbilical cord blood units for some RBC blood group phenotypes. 2. Characterization of CB-iPSCs for pluripotency in vitro and in vivo 3. Differentiation of CB-iPSCs into early erythroid progenitors 4. Generation of enucleated RBCs from CB-iPSCS derived erythroid progenitors 5. Characterization of RBCs in vitro by morphology, flow cytometric and molecular analysis 6. Evaluation of metabolic and physical parameters, determination of blood group alloantigens and membrane protein expression of RBCs 7. Hemoglobin characterization and functional assay in vitro 8. Functional assays in vivo Milestones 18 month 6 / 22

7 1. Generation of human ipsc from CD34+ of selected umbilical cord blood units for some RBC blood group phenotypes. 2. Characterization of CB-iPSCs for pluripotency in vitro and in vivo 3. Differentiation of CB-iPSCs into early erythroid progenitors Milestones 36 month 1. Generation of enucleated RBCs from CB-iPSCS derived erythroid progenitors 2. Characterization of RBCs in vitro by morphology, flow cytometric and molecular analysis 3. Evaluation of metabolic and physical parameters, determination of blood group alloantigens and membrane protein expression of RBCs 4. Hemoglobin characterization and functional assay in vitro 5. Functional assays in vivo Gantt chart C:\Users\ANTONELLA\Desktop\VF ipscs Project\VF GANNT_Progetto_iPSCS.xls 7 / 22

8 Equipment and resources available Sterile cell culture hood Inverted microscope and fluorescence microscope Cell culture incubator and water bath Benchtop Centrifuges -20 C and -80 C Freezers Liquid Nitrogen tank CR Thermal cycler MACS Cell Separation kit Flow cytometer Manual pipettes HPLC System (Variant II-Biorad) Cord blood units Osmometer (Osmored-Eurospital) Instrument for G6PD and PK enzyme activity measurement (CL-10 Plus-Biocontrol) Automatic analyzer Galileo/Neo (Immucor) with microplate and solid phase methods for typing of AB0, Rh, Kell, Duffy, Kidd, MNSs, Lewis, P1, Lutheran, Kp blood group systems; Anti-sera mono and polyclonal, and card for extended blood typing: Biotest, Biorad, Diagast, Diamed, Johnson & Johnson, Formedic, Grifols, Menarini, Pieco, Beta Diagnostici; Sample Incubator 37 C; Sample centrifuge; Card incubator and centrifuge; Agglutinoscope; Microscope; Manual DNA extraction kit-qiagen; Automatic DNA extractor -PROMEGA MAXWELL 16 Amplification instrument - PCR Gene Amp PCR System 9700 gold Applied Biosystem Amplification instrument -PCR Veriti 96 well Thermal Cycler Applied Biosystem Electrophoresis tanks-biorad Electrophoretic gel reader-gel Doc Biorad BAG kits: BAGene ABO-Type/Variant, RH-Type, Partial D Type, Weak D Type Inno-Train kits: Blood Group Ready Gene-ABO/subtype, CDE, KKD, MNS, Rare Screen DNA microarray complete system: HEA BEADCHIP (Bioarray Solution-Immucor) Blood count analyzer Consumables. 8 / 22

9 Translational relevance and impact for the National Health System (SSN) Our aim is to demonstrate that CB-iPSCs can be maintained indefinitely in vitro and can provide a potentially inexhaustible source blood donor-free to produce high volumes of mature and functional RBCs with potential applications in chronic transfusion therapy with complex alloimmunization or with rare blood group. Our main objective is to have at one's disposal an unlimited source of stem cells of specific blood group phenotypes to generate RBCs in vitro, in quantities equal to those provided by a standard RBC concentrate (2 10/12cells). We consider that a very limited number of RBC clones would be able to not only cover the need for the management of most alloimmunized patients and those with a rare blood group but also to efficiently allow the prevention of RBC sensitization in multiply transfused patients with hemoglobinopathies or hematological diseases of different ethnicities. The generation of CB-iPSCS will open new perspectives in the field of regenerative medicine. Proposed total budget ( Euro ) Costs Budget Year 1 Budget Year 2 Budget Year 3 TOTAL BUDGET Co-Funding List of costs proposed for funding to the moh 1 Staff salary 0,00 0,00 0,00 0,00 0,00 2 Researchers contracts , , , ,00 0, ,00 3a Equipment (leasing) 0, ,00 0, ,00 0, ,00 3b Supplies , , , ,00 0, ,00 3c Model costs , , , ,00 0, ,00 4 Subcontracts 0,00 0,00 0,00 0,00 0,00 0,00 5 Patient costs 0,00 0,00 0,00 0,00 0,00 0,00 6 IT services and data bases 1.000, , , ,00 0, ,00 7 Travels 2.000, , , ,00 0, ,00 8 Pubblication costs 1.000, , , ,00 0, ,00 9 Overheads , , , ,00 0, ,00 10 Coordination costs 1.000, , , ,00 0, ,00 Total , , , ,00 0, ,00 Report the Co-Funding Contributor: 9 / 22

10 Budget Justification 1 Staff salary None 2 Researchers contracts Three consecutive annual contracts for an expert researcher in ipscs generation and establishment/ evaluation of animal models. 3a Equipment (leasing) 3b Supplies Spectrophotometer, microscopes and electrophoresis equipment (SDS PAGE). Materials and reagents for: CB collection, analysis, CB-iPSCs generation, re-analysis; cell cultures; molecular biology; serology (blood group typing) physical and biochemical determinations. 3c Model costs Purchase and establishment of the NOD-SCID animal model to assess the rate of enucleation of induced red cells. 4 Subcontracts None 5 Patient costs None 6 IT services and data bases Purchase of a dedicated PC and softwares to generate plots and basic statistics. 7 Travels Meeting and congress participation of investigators. 8 Pubblication costs Costs for manuscript preparation, additional statistics and other general costs associated with publications. 9 Overheads Economical resources to be assigned to hospitals and institutions of investigators for general functioning of operating units. 10 Coordination costs Secretarial and administrative activities, fax use, photocopies, teaching materials, advice for English language, other. 10 / 22

11 Proposed total budget UO1 Institution: (Euro) Costs Budget Year 1 Budget Year 2 Budget Year 3 TOTAL BUDGET Co-Funding List of costs proposed for funding to the moh 1 Staff salary 0,00 0,00 0,00 0,00 0,00 2 Researchers contracts 0,00 0,00 0,00 0,00 0,00 0,00 3a Equipment (leasing) 0, ,00 0, ,00 0, ,00 3b Supplies , , , ,00 0, ,00 3c Model costs 0,00 0,00 0,00 0,00 0,00 0,00 4 Subcontracts 0,00 0,00 0,00 0,00 0,00 0,00 5 Patient costs 0,00 0,00 0,00 0,00 0,00 0,00 6 IT services and data bases 1.000,00 0,00 0, ,00 0, ,00 7 Travels 1.000, , , ,00 0, ,00 8 Pubblication costs 500,00 500,00 500, ,00 0, ,00 9 Overheads 7.500, , , ,00 0, ,00 10 Coordination costs 1.000, , , ,00 0, ,00 Total , , , ,00 0, ,00 Report the Co-Funding Contributor: Proposed total budget UO2 Institution: S. Camillo Hospital, Rome (Euro) Costs Budget Year 1 Budget Year 2 Budget Year 3 TOTAL BUDGET Co-Funding List of costs proposed for funding to the moh 1 Staff salary 0,00 0,00 0,00 0,00 0,00 2 Researchers contracts 0,00 0,00 0,00 0,00 0,00 0,00 3a Equipment (leasing) 0,00 0,00 0,00 0,00 0,00 0,00 3b Supplies , , , ,00 0, ,00 3c Model costs 0,00 0,00 0,00 0,00 0,00 0,00 4 Subcontracts 0,00 0,00 0,00 0,00 0,00 0,00 5 Patient costs 0,00 0,00 0,00 0,00 0,00 0,00 6 IT services and data bases 0, ,00 0, ,00 0, ,00 7 Travels 0,00 0,00 0,00 0,00 0,00 0,00 11 / 22

12 Costs Budget Year 1 Budget Year 2 Budget Year 3 TOTAL BUDGET Co-Funding List of costs proposed for funding to the moh 8 Pubblication costs 0,00 0,00 0,00 0,00 0,00 0,00 9 Overheads 0,00 0,00 0,00 0,00 0,00 0,00 10 Coordination costs 0,00 0,00 0,00 0,00 Total , , , ,00 0, ,00 Report the Co-Funding Contributor: Proposed total budget UO3 Institution: Sapienza University, Rome (Euro) Costs Budget Year 1 Budget Year 2 Budget Year 3 TOTAL BUDGET Co-Funding List of costs proposed for funding to the moh 1 Staff salary 0,00 0,00 0,00 0,00 0,00 2 Researchers contracts , , , ,00 0, ,00 3a Equipment (leasing) 0,00 0,00 0,00 0,00 0,00 0,00 3b Supplies 0,00 0,00 0,00 0,00 0,00 0,00 3c Model costs , , , ,00 0, ,00 4 Subcontracts 0,00 0,00 0,00 0,00 0,00 0,00 5 Patient costs 0,00 0,00 0,00 0,00 0,00 0,00 6 IT services and data bases 0,00 0, , ,00 0, ,00 7 Travels 1.000, , , ,00 0, ,00 8 Pubblication costs 500,00 500,00 500, ,00 0, ,00 9 Overheads 2.500, , , ,00 0, ,00 10 Coordination costs 0,00 0,00 0,00 0,00 Total , , , ,00 0, ,00 Report the Co-Funding Contributor: 12 / 22

13 Name PRINCIPAL INVESTIGATOR PROFILE Institution Department/Unit Position Title S. Camillo Hospital, Rome Transfusion Medicine Service Red Cell and Platelet Immunohematology Laboratory Head of Red Cell and Platelet Immunohematology Laboratory of Immunohematology and Transfusion Medicine Unit Education/Training - Institution and Location Degree Year(s) Field of study Sapienza University, Rome, Italy 20th October Specialization in Hematology Sapienza University, Rome, Italy 5th November Degree in Medicine and Surgery 1994 Hematology, Immunohematology and Transfusion Medicine 1990 Hematology Personal Statement This project aims to generate hipscs derived from CB cells selected for some blood group antigens, with subsequent production of red blood cells (RBCs). Matteocci Antonella, MD, PI: Manages extended blood group typing and metabolic and physical parameters analysis. Is responsible for statistical analysis, final report and publications. Fazzina Raffaella, BD: Manages generation and differentiation of CB-iPSCs into RBCs. Grammatico Paola, BD: Collaborates for bio-molecular analysis. Pierelli Luca, MD: Assesses in NOD-SCID mice the full enucleation and maturation of red cells. 13 / 22

14 Project Code: Positions and Honors Institution Division / Research group Location Position From year To year S. Camillo Hospital Immunohematology Transfusion Medicine Unit Rome, Italy MD, Head of Red Cell and Platelet Immunohematology Laboratory S. Camillo Hospital CAPE: Center of Blood Component Collection and Production Rome, Italy MD, Red Cell Immunohematology Laboratory S. Camillo Hospital Hematology Unit Rome Italy MD, Five Grants from ADMO/DIMOS Italian Red Cross Transfusion Service of S. Camillo Hospital Rome Italy MD, Professional Collaboration Italian Red Cross CNTS: National Center of Blood Transfusion Rome, Italy MD, Red Cell Immunohematology Laboratory Awards and Honors Official H index: 2.0 Awards and Honors: Two scientific recognitions from SIMTI for two multicentric studies: 1) A. Matteocci, M. Albano, C. Damico et al: Immunizzazioni ed eventi avversi alla trasfusione di sangue in pazienti talassemici: esperienza del Gruppo di Studio Cooperativo Laziale della Talassemia. XXXVII National Congress of SIMTI, Paestum 4-7 October 2006, Italy. 2) A. Matteocci, A. Moscetti, S. Ferrari et al: Studio multicentrico sulle varianti dell antigene D nella popolazione italiana: dati preliminari del Progetto Arcobaleno. XXXVIII National Congress of SIMTI, Rimini September 2008, Italy. Other CV Informations: Working Activity Manager of the Red Cell and Platelet Laboratory at S. Camillo Hospital in Rome. Coordinator of the -Progetto Arcobaleno- (Italian RhD variants). PI in funded project (Ministry of Health) - RH Project: Advanced Serological and Molecular Investigation for the RhD Variants Definition - code RF Scientific Activity Teacher of university and training courses. Co-relator of many graduation theses. Participation to many Conventions and Courses. Reviewer of scientific manuscripts for SIMTI 14 / 22

15 Publications of numerous abstracts and articles. Selected peer-reviewed publications Best publications with bibliographic data Publications N of Citations Impact Factor Scocchera R, Iudicone P, Matteocci A, Terlizzi F, Piccari M, Dionisi MG, Fioravanti D, Guglielmetti M, Mannella E.: Determinazione dei livelli di citochine in concentrati eritrocitari sottoposti a filtrazione e lavaggio. La Trasfusione del Sangue, vol 47, pag , A. Matteocci, R. Scocchera, P. Cianciulli et al: Reazioni trasfusionali febbrili non emolitiche e citochine in pazienti beta-talassemici. La Trasfusione del Sangue; 47, , Palange M, Dionisi MG, Matteocci A, Berchicci G, Maldone O, Mazzoli C, Scappucci W.: La Fase Solida nell'identificazione dei Fenotipi Dmosaic. La Trasfusione del Sangue, vol 46, num 2, pag , Datturi T, Grilli MA, Carlucci F, Matteocci A, De Felicis D, Di Nuccio A, Podagrosi D.: L'esame emocromocitometrico nei donatori di sangue. La Trasfusione del Sangue: vol 42, n 2, pag , 1998 A. De Laurenzi, A. Matteocci. A. Lanti et al.: Amphotericin B Prophylaxis against invasive fungal infections in neutropenic patients: a single Center Experience from 1980 to Infection; 24, , Best publications on the same topic of the project proposal Publications N of Citations Impact Factor Giorgetti A, Marchetto MC, Li M, Yu D, Fazzina R et al: Cord blood-derived neuronal cells by ectopic expression of Sox2 and c-myc. Proc Natl Acad Sci U S A Jul 31;109(31): doi: /pnas Epub 2012 Jul 18. Giorgetti A, Fazzina R, Li M, Belmonte JCI: Human cord blood reprogrammed into embryonic-like stem cells. ISBT Science Series (affiliated to Vox sanguinis) ; 6: , 2011 A. Matteocci, MB Rondinelli, P. Mastromonaco et al: Paziente con anticorpo raro anti-pp1pk: diagnostica immunoematologica e assistenza trasfusionale integrata. Blood Transfusion; 9, S4, 121, A. Matteocci, T. Mancuso, A. Moscetti et al: Tipizzazione eritrocitaria estesa: integrazione dei sistemi Bioarray Solutions e Galileo. Blood Transfusion; 9, S4, 110, Moscetti A., Matteocci A., Valentini T, Collaretti A., Spaccino C., Trabace S, Grammatico P., Pierelli L. Sierologia e biologia molecolare: integrazione di due tecnologie per la tipizzazione dei sistemi gruppoematici. Blood Transfusion; 8, S2, 115, / 22

16 Most recent publications Publications N of Citations Impact Factor R. Mancini, L. Marinelli, N. Mirante, A. Gallo, A. Matteocci et al: Evaluation of haemoglobin, haematocrit, haemolysis, residual protein content and leucocytes in 345 red blood cell concentrates used for the treatment of patients with ß-thalassaemia. Blood Transfusion: DOI / , Matteocci A, Moscetti A, Spaccino C: Phylogenesis of African RhD clusters: identification of related variants and anti-d immunization in Italy. Vox Sanguinis; 103, S1,193, A. Matteocci, C. Spaccino, A. Moscetti et al: RHD variants in Italian population. Transfusion; 51, 3S, 148, Matteocci, A. Moscetti, C. Spaccino et al: Widespread study on the RHD variants in Italy. Vox Sanguinis; 101, S1, 19, A. Matteocci, A. Collaretti, K. Castagna et al: Detection of platelet autoantibodies: a retrospective data analysis using the PakAuto assay in the diagnosis of AITP. Vox Sanguinis; 101 Suppl 1, 277, / 22

17 Biographical Sketch Contributors 1 Name: Prof.ssa Paola Grammatico, BD Institution and Position Title: S. Camillo Hospital, Rome Director of Medical Genetics Unit Personal Statement: This project aims to generate hipscs derived from CB cells selected for some blood group antigens, with subsequent production of red blood cells (RBCs). Matteocci Antonella, MD, PI: Manages extended blood group typing and metabolic and physical parameters analysis. Is responsible for statistical analysis, final report and publications. Fazzina Raffaella, BD: Manages generation and differentiation of CB-iPSCs into RBCs. Grammatico Paola, BD: Collaborates for bio-molecular analysis. Pierelli Luca, MD: Assesses in NOD-SCID mice the full enucleation and maturation of red cells. Institution Division / Research group Location Position From year To year Sapienza University Medical Genetics Unit Rome, Italy Associate Professor S. Camillo Hospital Ethical Committee Rome, Italy President S. Camillo Hospital Medical Genetics Unit Rome, Italy BD, Director Awards and Honors Official H index: 22.0 Awards and Honors: None 17 / 22

18 Biographical Sketch Contributors 2 Name: Prof. Luca Pierelli, MD Institution and Position Title: Sapienza University, Rome Associate Professor -Department of Experimental Medicine, School of Medicine and Odontoiatrics Personal Statement: This project aims to generate hipscs derived from CB cells selected for some blood group antigens, with subsequent production of red blood cells (RBCs). Matteocci Antonella, MD, PI: Manages extended blood group typing and metabolic and physical parameters analysis. Is responsible for statistical analysis, final report and publications. Fazzina Raffaella, BD: Manages generation and differentiation of CB-iPSCs into RBCs. Grammatico Paola, BD: Collaborates for bio-molecular analysis. Pierelli Luca, MD: Assesses in NOD-SCID mice the full enucleation and maturation of red cells. Institution Division / Research group Location Position From year To year ASL Viterbo Immunohematology and Transfusion Medicine Unit and then Transfusion Department Nord (2006) Viterbo, Italy MD, Director Sapienza University Sapienza University School of Medicine (in convention with S. Camillo Hospital) Department of Experimental Medicine, School of Medicine and Odontoiatrics Immunohematology Transfusion Medicine Unit and Transfusion Department Roma and then Ovest (2010) Rome, Italy Associate Professor Rome, Italy MD, Director Awards and Honors Official H index: 28.0 Awards and Honors: None 18 / 22

19 19 / 22

20 Biographical Sketch Contributors 3 Name: D.ssa Raffaella Fazzina, BD Institution and Position Title: Sapienza University, Rome BD, Department of Experimental Medicine, School of Medicine and Odontoiatrics Personal Statement: This project aims to generate hipscs derived from CB cells selected for some blood group antigens, with subsequent production of red blood cells (RBCs). Matteocci Antonella, MD, PI: Manages extended blood group typing and metabolic and physical parameters analysis. Is responsible for statistical analysis, final report and publications. Fazzina Raffaella, BD: Manages generation and differentiation of CB-iPSCs into RBCs. Grammatico Paola, BD: Collaborates for bio-molecular analysis. Pierelli Luca, MD: Assesses in NOD-SCID mice the full enucleation and maturation of red cells. Institution Division / Research group Location Position From year To year Sapienza University and S. Camillo Hospital Department of Experimental Medicine, School of Medicine and Odontoiatrics and Immunohematology and Transfusion Medicine Unit Rome, Italy Collaboration to plan and start a program of CB-IPSCs generation for large-scale production of intermediate cells of hematopoiesis and terminally differentiated into myeloid and erythroid cells. Since June Center of Regenerative Medicine (CMR) Laboratory of Stem Cells and ipscs Barcelona, Spain Research Assistant with projects: 1) Generation of ipscs from umbilical cord blood tem cell ) Transdifferentation of CD133+ cord blood stem cells into neurons Until February 2013 University of Illinois Department of Pathology and Cancer Center (Laboratory of G. Nucifora) Chicago, USA Research fellowship with projects: 1) In vivo and in vitro characterization of M5 infant myeloid acute leukemia 2) Clinical and molecular characterization of adult myelodisplastic syndrome Bologna University Laboratory of Pediatric Oncohematology Bologna, Italy Research fellowship with project: Developing new strategies of nucleic acids therapy in vitro and in vivo for infant leukemia scarring MLL fusion oncogenes / 22

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