244. Development and Qualification of Assays for Human Embryonic Stem Cell (hESC)-Derived Cardiomyocytes Intended for Clinical Use

Cell Therapies I HSV-TK phosphorylates the prodrug ganciclovir (GCV), an analog of guanosine nucleoside. The phosphorylated GCV is incorporated into host DNA and terminates the elongation of DNA strands, resulting in cell death. The therapeutic range of GCV is narrow and the administration of GCV often induces adverse reactions. Aiming at a more potent and thus less toxic TK, mutations in the HSV-TK had been screened in previous studies. However, no studies have been conducted on the direct comparison of the efficacy of mutant TKs for iPS cells. In the current study, we examined some TK mutants with substitution at the nucleoside binding site that had been reported before (Black, et al., 1996; Black, et al., 2001; Mercer, et al., 2002; Balzarini, et al., 2006). We made iPS cell clones that constitutively express individual mutant TK. Treatment of the TK-iPS clones with GCV revealed that cells with mutant SR11 (IF160 LL+A168Y), SR39 (LIF159IFL+AL168FM), Q7530 (IF160LL+AL168YF), and A168H were 10-fold more sensitive to GCV than wild type TK. Some other mutants that had been shown to have an enhanced killing activity in their original reports did not show an advantage over the wild type. These results suggested that TK mutants were more potent than the wild type and have to be screened and selected for a specific purpose.

244. Development and Qualification of Assays for Human Embryonic Stem Cell (hESC)-Derived Cardiomyocytes Intended for Clinical Use

Jennil Patel, Wei Dang, Aparna Krishnan, Derek Kong, David Hsu, Larry Couture Center for Biomedicine and Genetics, Beckman Research Institute of City of Hope, Duarte, CA Stem cell therapy holds the promise for numerous degenerative diseases and injuries. Human embryonic stem cells (hESCs) are the starting cell population for many of these potential therapies since they are capable of seemingly indefinite proliferation in the pluripotent state and have the ability to differentiate into all cell types found in the adult. It is vital that the cellular therapeutics derived from these highly proliferative cells are well characterized prior to clinical use and the assays used to characterize these cellular products are well developed. At the Center of Biomedicine and Genetics (CBG), we have manufactured under cGMP numerous hESC-differentiated products including neuron stem cells (NSC), neuronal progenitor cells (NPC), retinal pigment epithelium (RPE) cells, dopaminergic neurons and cardiomyocytes intended for pre-clinical and early phase clinical studies. We have developed a systematic approach to characterize hESC-derived cell products using well developed technologies such as real-time quantitative polymerase chain reaction (RT-qPCR) and flow cytometry. We have standardized the development and qualification of each assay which involves the selection and banking of positive and negative controls, selection and banking of critical reagents, determination of assay conditions, qualification of assay, generation of assay qualification report and standard operating procedure (SOP). In addition, we have established a general guideline to assist the selection of appropriate markers for different products. Here we describe the application of this systematic approach to develop and qualify identity and purity assays for hESC-derived cardiomyocytes produced through hESC differentiation procedure in suspension culture. Selection of markers for the characterization of cardiomyocyte products will be discussed, and development and qualification of the assays will be reported.

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245. MicroRNA Expression in Bone MarrowDerived Human MSCs

Ian H. Bellayr1, Jennifer G. Catalano2, Raj K. Puri1 Division of Cellular and Gene Therapies, US Food and Drug Administration, Silver Spring, MD, 2Office of Industries, US International Trade Commission, Washington, DC 1

Multipotent stromal cells (MSCs) are being studied in the field of regenerative medicine for their capacity for multi-differentiation. These cells can be isolated from multiple tissue types. The current literature indicates that MSCs have an immunoregulatory capacity, which can suppress the immune system. MicroRNAs (miRNAs) are short non-coding RNAs that are responsible for regulating gene expression. Through targeting binding to gene transcripts, miRNAs have been observed to impact MSC function such as proliferation, differentiation, migration and apoptosis. Studies have shown that various miRNAs are expressed in MSCs; however, the impact of cellular expansion and donor variability on the miRNA expression is not well understood. Six commercially available MSC lines were expanded from passage 3 to 7 and their miRNA expression was evaluated using microarray technology. Statistical analyses of our data revealed that 71 miRNAs out of 939 examined were expressed by this set of MSC lines at all passages and the expression of 13 miRNAs were significantly different between passage 3 and 7. The expression of six miRNAs with the largest fold changes was further evaluated using RT-qPCR for both the original MSC lines and a second set of seven MSC lines expanded from passage 4 to 8. By RT-qPCR only 2 miRNAs, miR-638 and miR-572 were upregulated at passage 7 compared to passage 3 for the original MSC lines by 1.71 and 1.54 fold, respectively; and upregulated at passage 8 compared to passage 4 for the second set of MSC lines, 1.34 and 1.59 fold, respectively. These 2 miRNAs distinguish aging MSCs expanded in culture. These novel results may be useful in establishing critical quality attributes for limiting clinical applications of MSCs beyond specific cellular expansion protocols.

246. Characterization of Peripheral Cardiac Progenitors

José R. Navarro-Betancourt1, Salomón Hernández1, Felipe Masso2, Luis A. Lara-Martínez1, Alejandra Contreras-Ramos3, Andrés R. Martinez-Calderón1, Victor M. Arenas1, José L. Aceves-Chimal4 1 Molecular Biology, Universidad Panamericana, Mexico City, Mexico, 2Cellular Biology, National Institute of Cardiology “Ignacio Chávez”, Mexico City, Mexico, 3Cellular Biology, Mexico Children’s Hospital “Federico Gómez”, Mexico City, Mexico, 4 Cardiothoracic Surgery, “20 de Noviembre” National Medical Centre, Mexico City, Mexico Introduction We propose that cells expressing cardiomyocytespecific structural proteins have a specific cardiac differentiation commitment and that an immunophenotype distinguished by the presence of cardiac stem cell (CSC) related surface markers, namely CD117, CD34 and Sca-1, identifies peripheral cardiac progenitors, therefore we explored the presence of these attributes in bone marrowderived cells, aiming to establish an association between cardiac differentiation potential and a particular cellular profile. Methods Whole bone marrow was extracted from Wistar rat femur, cells were isolated through enzymatic desegregation and plated for incubation. Twenty-four hours later non-adherent supernatant cells were recovered and re-seeded, seventy-two hours later adherent cells were harvested and underwent dilution cloning. Flow cytometry was used to characterize each clone searching particularly for the surface markers CD117, CD34 and Sca-1. The subpopulation expressing the highest levels of these markers was isolated and labeled bone marrow supernatant (BMSN). Subsequently, a complete immunophenotypic profile of BMSN was performed, their capacity to differentiate into Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy