Cell Therapy Center Research Articles

Terminal differentiation induction as DNA damage response in hematopoietic stem cells by GADD45A.

Hematopoietic stem cells (HSCs) sustain lifelong blood cell regeneration by balancing their ability for self-renewal with their ability to differentiate into all blood cell types. To prevent organ exhaustion and malignant transformation, long-lived HSCs, in particular, must be protected from exogenous and endogenous stress, which cause severe DNA damage. When DNA is damaged, distinct DNA repair mechanisms and cell fate controls occur in adult HSCs compared with committed cells. Growth arrest and DNA damage-inducible 45 alpha (GADD45A) is known to coordinate a variety of cellular stress responses, indicating the molecule is an important stress mediator. So far, the function of GADD45A in hematopoietic stem and progenitor cells is controversial and appears highly dependent on the cell type and stress stimulus. Recent studies have analyzed its role in cell fate decision control of prospectively isolated HSCs and have revealed unexpected functions of GADD45A, as discussed here. The upregulation of GADD45A by DNA damage-causing conditions results in enhanced HSC differentiation, probably to efficiently eliminate aberrant HSCs from the system. These findings, in concert with a few studies on other stem cell systems, have led us to propose DNA damage-induced differentiation as a novel DNA damage response mechanism in stem cells that circumvents the fatal consequences of cumulative DNA damage in the stem cell compartment.

Safety and efficacy of human umbilical cord-derived mesenchymal stem cells in patients with Alzheimer′s disease: study protocol for an open-label self-control trial

Background: Animal model experiments have demonstrated the efficacy and safety of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in the treatment of Alzheimer's disease. However, the clinical efficacy of hUC-MSCs in the treatment of Alzheimer's disease in humans remains unclear. This study protocol for a phase I/II clinical trial was designed to investigate the clinical safety (primary outcome) and efficacy (secondary outcome) of hUC-MSCs in the clinical treatment of Alzheimer's disease. Methods/Design: This open-label, self-control, single-center prospective phase I/II clinical trial will be performed at the Department of Hematopoietic Stem Cell Transplantation, Cell and Gene Therapy Center, the 307 th Hospital of Chinese People's Liberation Army, China. Thirty patients with moderate to severe Alzheimer's disease will be included. The primary outcome is the number of patients who have adverse events at 10 weeks and 1 year after hUM-MSCs therapy. Secondary outcomes include cognitive function, overall clinical status, daily living activities, neuropsychiatric symptoms, blood biochemical Alzheimer's disease markers. Discussion: The findings from this trial will provide valuable evidence for the use of hUM-MSCs in the clinical treatment of Alzheimer's disease. Trial registration: ClinicalTrial.gov identifier: NCT01547689; registered on 5 March 2012. This study protocol was approved by the Ethics Committee of the 307 th Hospital of Chinese People's Liberation Army.

Cohn de Laval Award Lectureship: The Science Behind the Success Development of a Continuous Flow Blood Cell Separator

On behalf of Dr. Jeane Hester, I would like to thank everyone in the American Apheresis Society and World Apheresis Society involved with the nomination and presentation of this award. She is truly honored by the recognition of her peers and wishes that she could be here to thank each and every one in person. The reason I am standing here goes back 43 years to July of 1971 when a new ‘crop’ of oncology fellows came to MD Anderson Cancer Center and the Department of Developmental Therapeutics. The chairmen of the department were Emil Frei and Emil J Frereich, with Ken McCredie as the head of the then fledgling apheresis center and I was the tech working in Ken’s research lab. Back then it was somewhat rare to have a female physician in the mix, so word traveled quickly about the tall attractive ‘fellow’ who was winning everyone over with her personality, commitment to her profession and, above all, dedication to her patients. As a fellow, part of the responsibility was to choose a research project and work in the lab in their free time. Several months later this here-to-for phantom Dr. Hester appeared in my lab to discuss where she could work on her project regarding Leukoagglutins in Transfusion. So we began sharing the lab, I used it during the day and she used it at night and occasionally we shared! To paraphrase the last line from Casablanca as Bogart and Claude Rains walk into the night: “This was the beginning of a beautiful friendship”. From that time forward, we worked together on many projects until her retirement from MD Anderson in 1995. The most notable achievement was the development of the first blood cell separators to use disposable channels and all the associated outcomes – the calcium citrate binding incorporated into the software, calcium replacement therapy used during collection, bag parameters for the storage and cryopreservation of cells and optimization of the software for the various procedures. The initial development was accomplished through collaboration with the Biomedical Division of IBM and moving forward she collaborated with GAMBRO COBE and until her 2nd retirement this year with TerumoBCT. The very first 2997 manufactured was called the ‘Genie’ and was donated to Dr. Hester’s Apheresis Unit. It was marketed as the IBM 2997, followed by the next generation – the COBE Spectra. Fast forward 30 years and today, this same blood cell separator is the work horse of apheresis, performing stem cell collections, plasma depletions, plateletpheresis, WBC reductions, and RBC depletions. Like all of us, the Spectra will be retired to make room for a newer model, but the scientific principals established during the development of the continuous flow separators (2990 – 2997 – Spectra) serve as the foundation for the future in apheresis. On a personal note, this award has been a time-traveling experience for Jeane and me. We are back 30+ years where she is drafting the speech and I am preparing the slides of our data. Only back then, it was truly hand-produced 35 mm slides and at least now I have the modern technology of PowerPoint. However, several of the slides and photos presented are digitized from the original 35 mm slide. The speech I will be giving has been written by Dr. Hester as if she were here presenting it herself. Many of you who know her will be able to pick up on several of the ‘mantras’ that she often presented and challenged at meetings, such as. . .‘Showme the data’. She is fiercely protective and loving, steadfast when right and can be stubbornly tenacious and I am proud to call her mentor and friend. – April G. Durett, MSc, Manager, Flow Cytometry Facility, Center for Cell & Gene Therapy I will now channel Dr. Hester in Houston, TX, and present her talk. International Forum: from the WAA Congress in San Francisco, 2014. * Corresponding author. Canadian Apheresis Group. E-mail address: cag@cagcanada.ca (G. Rock).

Generation of Antigen-Specific Cytotoxic T Lymphocytes with Peripheral B Cells Activated By α-Galactosylceramide

Dendritic cells (DCs) are well known as the most potent professional antigen presenting cells (APCs). Nonetheless, the use of these cells in immunotherapy has been limited due to the time consuming and laborious steps required to generate DCs from monocytes in vitro. Therefore, alternative APCs has drawn much attention because of their relative convenience in manipulation.In this study, the efficacy of B cells as APCs, as compared to DCs, in induction of cytotoxic T lymphocytes (CTLs) against cytomegalovirus (CMV)-specific antigens was evaluated. B cells were isolated by depletion of peripheral blood mononuclear cell (PBMCs) from healthy individuals with MACS system, loaded with α-galactosylceramide (α-GalCer) for inducing B cell activation, and nucleofected with CMV-antigen coding plasmid DNA, pCK-pp65-IRES-IE1. As other APCs, monocyte-derived DCs were induced with various cytokines (GM-CSF, IL-4, IL-1b, TNF-a), for 6 days and nucleofected with the same plasmid DNA. Ag-nucleofected B cells or DCs were cocultured with T cells for 14 days in vitro. The cells were harvested and subsequently immunoassayed.Proliferation of cells was more expanded by about 25~32% in CMV-CTLs induced by DCs compared to of B cells, but there was no significant difference in immunogenicity between CMV-CTLs induced with B cells and DCs. Compared to CMV-CTLs induced by DCs, the CTLs induced by α-GalCer-loaded B cells induced similar cytotoxicity against CMV antigen (Ag) in vitro. The CMV-CTLs by α-GalCer-loaded B cells recognized CMV antigen pp65 (median 88 SFC/105) and IE-1 (median 86 SFC/105) in donor 1, and CMV antigen pp65 (median 31 SFC/105) and IE-1 (median 37 SFC/105) in donor 2. Similarly, the CMV-CTLs by DCs recognized CMV antigen pp65 (median 133 SFC/105) and IE-1 (median 32 SFC/105) in donor 1, and CMV antigen pp65 (median 37 SFC/105) and IE-1 (median 43 SFC/105) in donor 2.Immunogenicities of both CTLs were similar not only on IFN-γ ELISPOT (Enzyme-linked immunospot) assay but also on cytotoxicity assay. The CMV-CTLs by α-GalCer-loaded B cells have killing activity against CMV antigen pp65 (100%, at E:T ratio 10:1) and IE1 (85%, at E:T ratio 10:1) in donor 1, and CMV antigen pp65 (69%, at E:T ratio 10:1) and IE1 (27%, at E:T ratio 10:1) in donor 2. Also, the CMV-CTLs by DCs show killing activity against CMV antigen pp65 (100%, at E:T ratio 10:1) and IE1 (42%, at E:T ratio 10:1) in donor 1, and CMV antigen pp65 (88%, at E:T ratio 10:1) and IE1 (64%, at E:T ratio 10:1) in donor 2.These observations suggest that α-GalCer-loaded B cells could be used in general as APCs instead of DCs. Using the B cells as APCs have several benefits such as cost-effectiveness, less time-consuming, and less laborious compared to when DCs are used. Furthermore, nucleofection technique might be useful in delivering antigen-coding DNA, not only for virus antigens but also for tumor antigens, directly into the nucleus. Our results demonstrate that α-GalCer-loaded B cells could be potent APCs in generating antigen-specific CTLs for cellular vaccines and adoptive immunotherapy.Acknowledgment: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012R1A1A2008316) and we thank Ann M. Leen, Helen E. Heslop and Malcomn K. Brenner from Center for Cell and Gene Therapy, Baylor College of Medicine Center for their kind help. DisclosuresNo relevant conflicts of interest to declare.

Cell Therapy in Cardiovascular Disease

Recently, cell therapy has sparked a revolution in ischemic heart disease that will in the future help clinicians to cure patients. Earlier investigations in animal models and clinical trials have suggested that positive paracrine effects such as neoangiogenesis and anti-apoptotic can improve myocardial function. In this regard the Royan cell therapy center designed a few trials in collaboration with multi hospitals such as Baqiyatallah, Shahid Lavasani, Tehran Heart Center, Shahid rajaee, Masih daneshvari, Imam Reza, Razavi and Sasan from 2006. Their results were interesting. However, cardiac stem cell therapy still faces great challenges in optimizing the treatment of patients. Keyword: Cardiovascular disease, Cell therapy.

An Update from the United States National Heart, Lung, and Blood Institute‐funded Production Assistance for Cellular Therapies (PACT) Program: A Decade of Cell Therapy

Recognizing the challenges faced by researchers and clinicians working in the field of cellular therapy, the National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health, established the Production Assistance for Cellular Therapies (PACT) program in 2003 and expanded it in 2010. The PACT program provides both clinical product manufacturing support that furthers the mission of NHLBI in the areas of cardiac, lung, and blood diseases and broad support of translational development across all disease areas to serve the entire cell therapy community. The program also provides access to expertise in project management, regulatory affairs, and quality assurance and control. Education initiatives include webinars, cell processing facility-hosted workshops, national workshops, and active participation and leadership within the cell therapy community through collaboration with other cell therapy organizations and academia. So far, over 650 PACT-manufactured cell therapy products have been administered in 32 clinical trials for a range of illnesses and diseases such as acute myocardial infarction, sickle cell disease, and graft-versus-host disease.

Efficacy of fetal stem cells in Duchenne muscular dystrophy therapy

Efficacy of fetal stem cells in Duchenne muscular dystrophy therapy Nataliia Sych,1 Mariya Klunnik,1 Olena Ivankova,1 Irina Matyaschuk,1 Mariya Demchuk,1 Alla Novytska,1 Inna Arkhipenko,2 Iuliia Shalita,2 Dario Siniscalco31Clinical Department, 2Biotechnology Department, Cell Therapy Center EmCell, Kiev, Ukraine; 3Biomedical Centre for Autism Research and Therapy, Bari, ItalyAbstract: The absence of effective treatment methods for Duchenne muscular dystrophy (DMD) calls for new therapeutic approaches. One of the promising treatment methods for DMD is stem cell therapy. This study demonstrates the impact of fetal stem cells (FSCs) on functional capacity and life quality of DMD patients and the ability of FSCs to prevent DMD-related complications in order to inhibit the disease progression. FSC therapy substantially improves functional capacity, life quality, left ventricular ejection fraction, and forced vital capacity of the lungs of DMD patients; this was confirmed by comparison of neurological, laboratory (aspartate aminotransferase, alanine aminotransferase, creatine phosphokinase, and lactate dehydrogenase), and morphofunctional findings (left ventricular ejection fraction and forced vital capacity) in DMD patients before the treatment, and 6 and 12 months after the FSC treatment.Keywords: life quality, heart failure, respiratory failure, cell therapy

A New Class of Antigen T-Cells That Redirect Bystander T-Cells to CD19 Positive Malignancies

A New Class of Antigen T-Cells That Redirect Bystander TCells to CD19 Positive Malignancies Mireya Paulina Velasquez , Kota Iwahori , Sunitha Kakarla , Caroline Arber , Tania Rodriguez-Cruz , Claudia Gerken , Xiao-Tong Song , Stephen Gottschalk . Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX; Pediatrics, Baylor College of Medicine, Houston, TX; Pediatrics, Pathology and Immunology, Interdepartmental Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, TX; Pathology and Immunology, Baylor College of Medicine, Houston, TX

Celgene Partners with Bluebird Bio to Advance CAR T-Cell Cancer Therapies

Celgene has partnered with Bluebird bio to discover, develop and commercialise novel disease-altering gene therapies in oncology that utilise a patient’s own genetically modified T-cells, known as chimeric antigen receptor (CAR) T-cells, to target and destroy cancer cells selectively. Celgene has the option to license collaboration products on completion of each Phase I study; bluebird will be responsible for R&D up until this point. In a separate deal, Celgene has also formed a collaboration with the Center for Cell and Gene Therapy in the CAR T-cell field.

Cytotoxicity of Substituted Benzimidazolyl Curcumin Mimics Against Multi-Drug Resistance Cancer Cell

Cell Therapy and Tissue Engineering Center, Yonsei University Wonju College of Medicine, Wonju 220-701, Korea Department of Basic Science, Kwandong University College of Medicine, Gangneung 210-701, Korea Department of Basic Science, Yonsei University Wonju College of Medicine, Wonju 220-701, Korea E-mail: ahn0707@yonsei.ac.kr Marine Chemomics Laboratory, Natural Medicine Center, Korea Institute of Science and Technology, Gangneung 210-340, Korea Department of Pharmacology, Kwandong University College of Medicine, Gangneung 210-701, Korea E-mail: sjlee@kwandong.ac.kr Received December 26, 2012, Accepted January 21, 2013

MicroRNA-137 is downregulated in glioblastoma and inhibits the stemness of glioma stem cells by targeting RTVP-1

Glioblastomas (GBM), the most common and aggressive malignant astrocytic tumors, contain a small subpopulation of cancer stem cells (GSCs) that are implicated in therapeutic resistance and tumor recurrence. Here, we study the expression and function of miR-137, a putative suppressor miRNA, in GBM and GSCs. We found that the expression of miR-137 was significantly lower in GBM and GSCs compared to normal brains and neural stem cells (NSCs) and that the miR-137 promoter was hypermethylated in the GBM specimens. The expression of miR-137 was increased in differentiated NSCs and GSCs and overexpression of miR-137 promoted the neural differentiation of both cell types. Moreover, pre-miR-137 significantly decreased the self-renewal of GSCs and the stem cell markers Oct4, Nanog, Sox2 and Shh. We identified RTVP-1 as a novel target of miR-137 in GSCs; transfection of the cells with miR-137 decreased the expression of RTVP-1 and the luciferase activity of RTVP-1 3'-UTR reporter plasmid. Furthermore, overexpression of RTVP-1 plasmid lacking its 3'-UTR abrogated the inhibitory effect of miR-137 on the self-renewal of GSCs. Silencing of RTVP-1 decreased the self-renewal of GSCs and the expression of CXCR4 and overexpression of CXCR4 abrogated the inhibitory effect of RTVP-1 silencing on GSC self-renewal. These results demonstrate that miR-137 is downregulated in GBM probably due to promoter hypermethylation. miR-137 inhibits GSC self-renewal and promotes their differentiation by targeting RTVP-1 which downregulates CXCR4. Thus, miR-137 and RTVP-1 are attractive therapeutic targets for the eradication of GSCs and for the treatment of GBM.

REXIC Project: Retrospective Cross-Sectional Study of Documentation of Informed Consent for Research Biobanking in A Public Research and Teaching Hospital.

BackgroundThe Center for Transfusion Medicine, Cell Therapy and Cryobiology, Milan, Northern Italy, is the headquarter of the POLI-MI biobank. It co-ordinates the biobank activities of the Fondazione Ca’ Granda Ospedale Maggiore Policlinico of Milan. Such activities require specific safeguarding of donors’ rights and protection of sensitive and genetic data. The Fondazione Ca’ Granda Ospedale Maggiore Policlinico has set up a project on informed consent with the aim of developing awareness and understanding of this issue. Within this project, it has been decided to evaluate how consent for biobanking material is expressed.Design and methodsThe aim of the study was to evaluate the quality and completeness of consent to biobanking in the POLI-MI biobank. This was a retrospective study carried out in 2012 on samples of consent declarations collected by biobank units in 2011. Some units used a single, standard consent model available from a previous POLI-MI biobank workgroup. Other units used models which had been previouly formulated. Evaluation was made using a form that indicated the essential elements of consent.ResultsA total of 48 consent declarations were collected using the single, standard model and 84 were collected using other models. The consent declarations that used the single, standard model were found to be the most complete and were filled in better than other models.ConclusionsProgressive adoption of a simple, standard consent model is expected to improve the quality of consent acquisition. Regular audit of the compliance of consent practices with ethical and legal requirements is mandatory to improve the quality of research biobanking.

Compassionate use of cell products

Compassionate use of cell products

The predictive role of a�-double negative T cells in clinical outcome of patients with lymphoma: preliminary data of prospective study.

The predictive role of a�-double negative T cells in clinical outcome of patients with lymphoma: preliminary data of prospective study.

Expanded Natural Killer (NK) Cells for Immunotherapy: Fresh and Made to Order

AbstractAbstract 1912 IntroductionRemarkable increases in the dose and activity of NK cells can be achieved by co-culture with the HLA class I deficient cell line K562 that has been genetically modified to express membrane-bound IL15 and the co-stimulatory molecule 41BB-ligand (K562-mb15-41BBL; Fujisaki et al. Cancer Res. 2009;69:4010–4017). We are conducting a clinical trial utilizing these ex-vivo expanded NK cells (ENK) which are produced at the Center for Cell and Gene Therapy (CAGT) at Baylor and then shipped to the University of Arkansas for Medical Sciences (UAMS) for infusion to high-risk relapsed multiple myeloma (MM) patients using the NHLBI-PACT mechanism. Here we report on the characteristics of the ENK cell products sent fresh versus frozen. MethodsApheresis products were collected from MM patients or healthy donors (HD), cryopreserved, and then shipped to CAGT for GMP grade production, as described (Lapteva et al. Cytotherapy 2012; in press). Briefly, mononuclear cells from thawed and ficolled apheresis products were cultured in Stem Cell Growth Medium (CellGenix) supplemented with 10% fetal bovine serum and 10 U/mL IL2 with stimulator cells at a ratio of 1 NK cell to 10 irradiated K562-mb15-41BBL cells (developed at St. Jude Children’s Research Hospital, Memphis, TN). Cells were harvested on day 8–9; products from HD were CD3-depleted. Clinical-grade products were shipped to UAMS overnight either cryopreserved in a dry shipper (n=7) or fresh in 5% human serum albumin on cold packs at 1–11°C (n=4). Cell purity, expression of activating molecules, and viability by 7AAD exclusion was assessed by flow cytometry. Standard 4h chromium-release assays were used to assess potency against K562 cells at a 20:1 ENK: K562 ratio. Student’s t-Test was used to determine significance. ResultsFrom 0.9–1.5×107 starting NK cells, the total number of ENK cells produced was 5.4×109 (range 1.8–24×109). The fold NK-cell expansion was significantly lower for MM patients (n=5, median 22, 12–70 fold) than for HD (n=6, median 95, 31–160 fold; p<0.05). At harvest, median CD3+/CD56+ NK cell purity was 70% (52–88); CD3 depletion of HD products increased CD3+/CD56+ purity to 93% (86–95) resulting in a median CD3+/CD56- T cell content of 0.02% (0.04–1.02). Overall, median viability was 93% (67–98) and potency (defined as lysis of K562 cells at a 20:1 E:T ratio) was 74% (26–92). One product derived from a patient with 21% CD138+ MM cells in the apheresis collection had low expansion (12-fold), viability (66.7%) and potency (26%). For cryopreserved products, viability immediately after thawing was acceptable (median 94%, 75–99) but recovery of viable cells varied from 61% to 100% and thawed ENK failed to lyse K562 cells unless rested overnight. Further, recovery was extremely poor after overnight incubation (median 16%, 10–21). We therefore validated shipment of fresh ENK products. In contrast with frozen NK cells, the median recovery for fresh clinical products post-shipping was 101% (87–151). We confirmed that NK purity, viability, potency and expression of the key activating molecules NKG2D, NKp30, NKp44 and CD226 were retained up to 48h after transfer. ENK further increased by 34% after 72h in vitro incubation in the presence of IL2. Significant in vivo expansion of ENK was observed after infusion of fresh ENK cell products (n=3) but not after infusion of thawed products (n=3, see separate abstract). An additional advantage was that the fresh cells arrived ready to infuse and changes in release criteria relying on rapid and in process testing significantly reduced the time from apheresis collection to ENK infusion, an important consideration when treating high-risk MM patients who can experience rapid disease progression. ConclusionWe conclude that large numbers of clinical grade ENK cells can be generated from both MM patient and HD derived apheresis products by co-culture with IL2 and K562-mb15-41BBL although less vigorous expansion was observed with patient-derived cells. Upon thawing, cryopreserved ENK cells exhibited inferior recovery and potency, and survived poorly during further in vitro culture. In contrast, freshly formulated and shipped ENK cells have excellent recovery and retain cytolytic ability. Robust in vivo expansion was only seen after infusion of fresh ENK cells. Production assistance by CAGT allowed for the rapid implementation of a novel therapy utilizing fresh ENK cells for poor prognosis MM patients. Disclosures:No relevant conflicts of interest to declare.

Fresh Ex Vivo Expanded Natural Killer Cells Demonstrate Robust Proliferation in Vivo in High-Risk Relapsed Multiple Myeloma (MM) Patients

AbstractAbstract 579Stimulation of NK cells with the HLA-deficient cell line K562 genetically modified to express 41BB-ligand and interleukin (IL) 15 allows for the production of large numbers of highly activated NK cells for therapy (Fujisaki et al, Cancer Res. 2009;69:4010–4017). We tested the safety, persistence and anti-MM activity of these ex-vivo expanded NK cells (ENK) in patients with high-risk MM relapsing after AUTO-PBSCT. Apheresis products were obtained from MM patients (AUTO-ENK) or from killer immunoglobulin-like receptor-ligand (KIR-L) mismatched haplo-identical family donors (ALLO-ENK). The preparative regimen comprised bortezomib only (Bor, days −9, −6, and −2) or Bor and immunosuppression with cyclophosphamide (Cy, day −7) and fludarabine (Flu, days −5 to −2), see Table 1. ENK cells were produced at the Center for Cell and Gene Therapy (CAGT) in Houston, shipped to UAMS either cryopreserved or fresh (see separate abstract), infused on day 0 and followed by daily administrations of 3×106 units of IL2 (13 doses total) to support ENK cell persistence in vivo. Flow cytometry was used to determine immunophenotype and the absolute number of NK and T cells in blood. Chimerism was assessed by PCR for donor specific short tandem repeats. Standard 4h chromium release assays were used to assess potency of ENK against MM cells. Of 6 patients treated, ENK cell doses of 2–8×107/kg (with T cell doses in ALLO-ENK products limited to <5×105/kg) were safely infused. IL2 therapy-induced rigors and grade II fever were the most common adverse events. No in vivo expansion was observed in the 3 patients receiving cryopreserved ENK products. One patient had stable disease for 3 months followed by slow disease progression. The other two patients experienced early progression, with one succumbing to MM. The 3 subsequent patients received fresh ENK products and transient expansion in vivo was observed in all 3, peaking at day 7 (absolute number of NK cells/μl blood: 512, 547, 153; NK cell percentage of lymphocytes: 49%, 92%, 64%). For the fresh ALLO-ENK cell recipient we confirmed that the in vivo expanded cells (Figure 1A) were derived from the donor (> 92–95% donor chimerism on days 5, 7 and 9). Furthermore, NK cells isolated from the blood on days 5 and 7 avidly killed recipient MM cells (Figure 1B). Unfortunately, this patient died of septic shock 11 days post-infusion and clinical response could not be evaluated. While non-expanded NK cells expressed low levels of CD26 and CD70, these molecules were highly expressed on ENK cells and NK cells in the PB post-ENK cell infusion, suggesting that CD26 and CD70 can be used to track the infused cells. AUTO-ENK cells failed to kill recipient MM in vitro but significant killing of third party MM cells (43%, 63%) and K562 (80%, 78%) was observed (effector: target ratio of 10:1). In keeping with this, the two patients who received fresh AUTO-ENK had progressive disease. Patients receiving Cy/Flu had increased serum levels of the NK cell growth factor IL15 (median 34 vs. 4pg/ml, respectively). High frequencies of CD25+/Foxp3+ T regulatory cells (24%, 44%, 47% of the CD3+/4+ lymphocytes) were observed 14 days after ENK infusion in 3/4 patients studied and we are investigating whether these cells were involved in ENK cell suppression. In conclusion, this study has established the safety and feasibility of infusing up to 8×107/kg ENK cells. We only observed dramatic in vivo expansion of fresh ENK suggesting that infusing fresh rather than freeze-thawed ENK cells is critical. ALLO-ENK cells isolated from the PB post-infusion killed recipient MM cells. In contrast, this was not observed with AUTO-ENK cells suggesting that further optimization of the AUTO-ENK cell approach may be required to overcome KIR-L induced inhibition. In addition, studies are under way to determine whether suppression by T regulatory cells, soluble factors, or suboptimal IL15 surge may prove important in modulating ENK activity and in vivo persistence.Table 1ENK product type and preparative regimenBortezomib (Bor), Cyclophosphamide (Cy), Fludarabine (Flu).ENKPatientDonorPreparationFreeze-Thawed ENK1AUTOBor/Cy/Flu2ALLOBor/Cy/Flu3AUTOBor/Cy/FluFresh ENK4AUTOBor5ALLOBor/Cy/Flu6AUTOBor [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Establishing a stem cell culture laboratory for clinical trials.

Adult stem/progenitor cells are found in different human tissues. An in vitro cell culture is needed for their isolation or for their expansion when they are not available in a sufficient quantity to regenerate damaged organs and tissues. The level of complexity of these new technologies requires adequate facilities, qualified personnel with experience in cell culture techniques, assessment of quality and clear protocols for cell production. The rules for the implementation of cell therapy centers involve national and international standards of good manufacturing practices. However, such standards are not uniform, reflecting the diversity of technical and scientific development. Here standards from the United States, the European Union and Brazil are analyzed. Moreover, practical solutions encountered for the implementation of a cell therapy center appropriate for the preparation and supply of cultured cells for clinical studies are described. Development stages involved the planning and preparation of the project, the construction of the facility, standardization of laboratory procedures and development of systems to prevent cross contamination. Combining the theoretical knowledge of research centers involved in the study of cells with the practical experience of blood therapy services that manage structures for cell transplantation is presented as the best potential for synergy to meet the demands to implement cell therapy centers.

Two-step generation of induced pluripotent stem cells from mouse fibroblasts using Id3 and Oct4

Laboratory of Cell Function Regulation, College of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea Cell Therapy Center, College of Medicine, Yonsei University, Seoul, Republic of Korea Department of Pathology, College of Medicine, Korea University Guro Hospital, Seoul, Republic of Korea Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea

CD39 Expression in Mesenchymal Stromal Cells

National Institute of Science and Technology in Stem Cell and Cell Therapy Center for Cell Therapy and Regional Blood Center Faculty of Medicine, University of São Paulo, Ribeirão Preto, Brazil

A phase I/II study of adenovirus-mediated interleukin-12 gene therapy for hormone refractory prostate cancer: An interim report.

148 Background: A phase I/II study of adenovirus-mediated interleukin-12 (IL–12) gene therapy for castration resistant prostate cancer has been initiated from May 2008 in Okayama University Hospital. Interim results of 10 cases including a positive clinical response are reported. Methods: Major inclusion criteria was a recurrence of prostate cancer following hormonal therapy with/without metastasis. Replication–defective adenovirus vector expressing IL–12 (Adv.IL–12) was injected directly into the prostate (or metastatic lesion in patients who received prostatectomy) in escalating doses from 1.0×1010 to 5.0×1012 viral particle (vp). GMP grade vector was produced at Center for Cell and Gene Therapy, Baylor College of Medicine (Director: Malcolm K. Brenner). Each patient received total of 3 times viral injections every 4 weeks. In addition to the safety profile, cancer control efficacy and immunological changes after viral injections were evaluated. Results: Ten patients received the gene therapy in doses of 1.0×1010 to 5.0×1011 vp from May 2008 to October 2010. 9 cases completed, and 1 patient dropped out the study because of rapid cancer progression. Major adverse effects are following: urinary retention (G1), trasaminase elevation (G3), fever up (G3). These adverse effects were rapidly disappeared after proper treatments. One patient with highest dose (5.0×1011 vp) showed some clinical positive responses (PSA decrease, shrinkage of lymphnode metastasis), therefore received an additional viral injection. Temporal decreases just after each vector injections and gradual increases at the peak of 7-14 days after each vector injections in some immunocompetent cells (CD3+CD19–, CD3–CD19– CD16+CD56+, CD3+CD4–CD8–) were seen in relatively high dose group. Elevations of serum cytokines were observed in parallel with vector dose escalation. Conclusions: This interim report suggests the favorable safety profile and positive clinical responses in a clinical trial of Adv.IL–12 gene therapy for hormone refractory prostate cancer in higher doses of viral vectors. Further evaluations in the next higher vector doses are needed and in progress. No significant financial relationships to disclose.