- Email: [email protected]
Will not be presented
S32
Poster Abstracts
complies with the regulations. Finding qualified staff for regulatory affairs positions can be a challenge. For those seeking a position in regulatory affairs, the environment appears to be disorganized and poorly defined. Obvious career paths and other requirements are unclear. What are employers looking for in their regulatory staff? Where are the jobs? What are the requirements? Method: We determined the job postings to be reviewed and analyzed for position title, position level, industry type, required education, years of experience, certification requirements, location, and key words. We were looking for consistency and important distinctions to help us understand the requirements for regulatory professionals. Results: On November 20, 2014, 129 jobs were posted on the website of the Regulatory Affairs Professional Society. Regulatory related positions are difficult to define and have many inconsistencies. Over one third of the posted positions were not actually regulatory positions. The majority of postings required a 4-year degree (80%), with few requiring terminal degrees (6%). The minimum desired years of experience was one year, with the range being 1 to 15 years. Twenty-three percent of the positions were located in California while most of the remaining positions were located in the Northeast. Keyword frequency showed that CMC, global (which included international), FDA and the various FDA submission types were among the most frequently cited words. Conclusion: New ways to organize the positions are needed as regulatory has many areas across the drug and device development process. Most job postings do not list starting pay levels which contribute to the confusion. This will improve the quality of candidates and encourage more people to intentionally enter the field if it is better defined. 95 WILL NOT BE PRESENTED
98 USING ABCG2 TO IDENTIFY CLONOGENIC KERATINOCYTES IN HUMAN INTERFOLLICULAR EPIDERMIS FOR WOUND HEALING APPLICATION A Chua, D Ma, ST Lee Plastic, Reconstructive & Aesthetic Surgery, Singapore General Hospital, Singapore, Singapore ABCG2, a member of the ATP binding cassette (ABC) transporters, has been recognized as a universal marker for stem/progenitor cells in many tissues and organs. This study investigated the expression of ABCG2 in the human interfollicular epidermis, and its potential as a surface marker for identifying clonogenic keratinocytes. Immunofluorescent and western blot studies showed that the ABCG2 was exclusively localized in the basal layer of human epidermis. Flow cytometry analysis showed approximately 2.1-3.3% of keratinocytes in interfollicular epidermis express ABCG2, and this cell population expresses p63, b1-integrin, a6-integrin, cytokeratin14, but not CD34, CD71, C-kit and involucrin. The ABCG2-positive keratinocytes showed significant higher colony forming efficiency (CFE) than ABCG2-negative keratinocytes, and extensive proliferation capacity in long-term in-vitro culture. With clonal analysis, most of the freshly isolated ABCG2-positive keratinocytes formed holoclones and fully stratified epidermis in organotypic model. We also used nude mice as an animal wound healing model to study the cultivated human epithelial grafts. We found that cultured human epithelial grafts derived from ABCG2-positive keratinocytes can be grafted onto full-thickness skin wound, and formed new epidermis. However for ABCG2-negative group, the grafts failed to survive. In long term observation after transplantation, the regenerated epidermis derived from ABCG2-positive keratinocytes showed thick epidermal cell layers, including stratum corneum and rete ridge in the basal layer. In summary, ABCG2-positive human keratinocytes can potentially be used to enrich for epidermal stem cells in wound healing application such as extensive burns.
96 WILL NOT BE PRESENTED 97 DEVELOPMENT OF SUCCESSFUL BIO-PRESERVATION STRATEGIES FOR CELL THERAPY MEDICINAL PRODUCTS F D’Agostino1,2, E Martin1, J Loughlin2 1 Biopharmaceutical Bioprocessing Technology Center, Newcastle University, Newcastle upon Tyne, United Kingdom, 2Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom The International Society for Cellular Therapy estimated that 1058 clinical trials are ongoing for novel cell-based therapies. As these aim to be a cure and not just a treatment for diseases, the cell therapy industry holds the promise of revolutionising current healthcare. This is increasingly important given the aging of the world population and has triggered huge investments by many Governments around the world, particularly in the UK, Germany, US, Japan and Korea. The inherent nature of the cells makes their manufacturing, clinical development and transport very challenging. Unlike traditional biopharmaceuticals, cells undergo stimuli during transport which can affect the potency of the medicinal product. Current approaches are mainly based on cryopreservation, where cells are cooled to e80 C and toxic cryoprotectants, such as DMSO, are added to the product. However, this method cannot always be implemented and many alternative approaches are under investigation. Bio-preservation strategy design is an important part of a cell therapy process development and is also linked to product approval from regulatory bodies and to cost of the therapy. In order to successfully validate the use of Tower Cold Chain’s containers for the transport of cell therapy products, we started by studying the combined effect of temperature and oxygen partial pressure in a controlled environment over a simulated shelf life window of 5 and 7 days. Two cell therapy models have been investigated: scaffold free bone marrow derived cartilage discs and undifferentiated mesenchymal stem cells. Preliminary results show that temperature and oxygen partial pressure can modulate both metabolic activity and gene expression profile of both cell therapy models. We also developed a methodology that can be used for investigating multi factorial effects on the potency of the cell therapy medicinal product which is based on statistical design of experiment.
99 MSC IMMUNOMODULATION AND ARYL HYDROCARBONS RECEPTOR ACTIVATION BY TRYPTOPHAN CATABOLITES CN Lewis1,2, R Chinnadurai2, J Galipeau2,3 1 Medical Scientist Training Program, Emory University School of Medicine, Atlanta, Georgia, United States, 2Hematology and Medical Oncology, Winship Cancer Institute, Atlanta, Georgia, United States, 3Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, United States The catabolism of Trp by indoleamine 2,3-dioxygenase (IDO) is a key correlate of the immunomodulation by human mesenchymal stromal cells (MSCs). AHR is a cytosolic protein expressed by a variety of cells, that upon activation translocates to the nucleus to initiate transcription at aryl hydrocarbon response elements (AHREs). Since MSCs are known to express AHR at a basal state, we hypothesized that the catalytic activity of IDO and signal transduction via AHR are linked, as intracellular signals which deploy MSC suppressive properties. To test this, we treated MSCs with aromatic hydrocarbons known to activate AHR: TCDD or FICZ (10nM), IFNg (50ng/mL). We showed human MSCs express AHR basally, and that expression does not change during 48h of treatment with IFNg; whereas under the same conditions IFNg induced expression of IDO by 100,000-fold. Upon treatment of resting MSCs (IDO negative) with TCDD or FICZ, we observed 50- to 100-fold induction of Cyp1a1/Cyp1b1 mRNA, reflective of AHRE activation. We next examined the response of MSCs after 48h treatment with 1MT, Trp, kynurenine (Kyn) or kynurenic acid (KynAc), the latter two molecules being the first and second IDO-catalyzed Trp metabolites. We observed 1MT and KynAc both induced Cyp1a1/Cyp1b1 expression by 20to 60-fold. These data support the theory that IDO-catabolism of Trp generates endogenous ligands (i.e. KynAc) which can directly activate AHR signaling. We performed T cell proliferation assays in the presence of MSCs, and observed Trp catabolites augment the immunosuppressive function of MSCs (independently of PDL1 or IDO expression). Our findings suggest that endogenous AHR signals arising from IDO catalysis may play a role in MSC immunomodulation. This observation supports a novel paradigm for intracellular signaling arising from catabolic conversion of Trp to AHR ligands and induction of immunomodulatory properties. 100 WILL NOT BE PRESENTED
Poster Abstracts
complies with the regulations. Finding qualified staff for regulatory affairs positions can be a challenge. For those seeking a position in regulatory affairs, the environment appears to be disorganized and poorly defined. Obvious career paths and other requirements are unclear. What are employers looking for in their regulatory staff? Where are the jobs? What are the requirements? Method: We determined the job postings to be reviewed and analyzed for position title, position level, industry type, required education, years of experience, certification requirements, location, and key words. We were looking for consistency and important distinctions to help us understand the requirements for regulatory professionals. Results: On November 20, 2014, 129 jobs were posted on the website of the Regulatory Affairs Professional Society. Regulatory related positions are difficult to define and have many inconsistencies. Over one third of the posted positions were not actually regulatory positions. The majority of postings required a 4-year degree (80%), with few requiring terminal degrees (6%). The minimum desired years of experience was one year, with the range being 1 to 15 years. Twenty-three percent of the positions were located in California while most of the remaining positions were located in the Northeast. Keyword frequency showed that CMC, global (which included international), FDA and the various FDA submission types were among the most frequently cited words. Conclusion: New ways to organize the positions are needed as regulatory has many areas across the drug and device development process. Most job postings do not list starting pay levels which contribute to the confusion. This will improve the quality of candidates and encourage more people to intentionally enter the field if it is better defined. 95 WILL NOT BE PRESENTED
98 USING ABCG2 TO IDENTIFY CLONOGENIC KERATINOCYTES IN HUMAN INTERFOLLICULAR EPIDERMIS FOR WOUND HEALING APPLICATION A Chua, D Ma, ST Lee Plastic, Reconstructive & Aesthetic Surgery, Singapore General Hospital, Singapore, Singapore ABCG2, a member of the ATP binding cassette (ABC) transporters, has been recognized as a universal marker for stem/progenitor cells in many tissues and organs. This study investigated the expression of ABCG2 in the human interfollicular epidermis, and its potential as a surface marker for identifying clonogenic keratinocytes. Immunofluorescent and western blot studies showed that the ABCG2 was exclusively localized in the basal layer of human epidermis. Flow cytometry analysis showed approximately 2.1-3.3% of keratinocytes in interfollicular epidermis express ABCG2, and this cell population expresses p63, b1-integrin, a6-integrin, cytokeratin14, but not CD34, CD71, C-kit and involucrin. The ABCG2-positive keratinocytes showed significant higher colony forming efficiency (CFE) than ABCG2-negative keratinocytes, and extensive proliferation capacity in long-term in-vitro culture. With clonal analysis, most of the freshly isolated ABCG2-positive keratinocytes formed holoclones and fully stratified epidermis in organotypic model. We also used nude mice as an animal wound healing model to study the cultivated human epithelial grafts. We found that cultured human epithelial grafts derived from ABCG2-positive keratinocytes can be grafted onto full-thickness skin wound, and formed new epidermis. However for ABCG2-negative group, the grafts failed to survive. In long term observation after transplantation, the regenerated epidermis derived from ABCG2-positive keratinocytes showed thick epidermal cell layers, including stratum corneum and rete ridge in the basal layer. In summary, ABCG2-positive human keratinocytes can potentially be used to enrich for epidermal stem cells in wound healing application such as extensive burns.
96 WILL NOT BE PRESENTED 97 DEVELOPMENT OF SUCCESSFUL BIO-PRESERVATION STRATEGIES FOR CELL THERAPY MEDICINAL PRODUCTS F D’Agostino1,2, E Martin1, J Loughlin2 1 Biopharmaceutical Bioprocessing Technology Center, Newcastle University, Newcastle upon Tyne, United Kingdom, 2Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom The International Society for Cellular Therapy estimated that 1058 clinical trials are ongoing for novel cell-based therapies. As these aim to be a cure and not just a treatment for diseases, the cell therapy industry holds the promise of revolutionising current healthcare. This is increasingly important given the aging of the world population and has triggered huge investments by many Governments around the world, particularly in the UK, Germany, US, Japan and Korea. The inherent nature of the cells makes their manufacturing, clinical development and transport very challenging. Unlike traditional biopharmaceuticals, cells undergo stimuli during transport which can affect the potency of the medicinal product. Current approaches are mainly based on cryopreservation, where cells are cooled to e80 C and toxic cryoprotectants, such as DMSO, are added to the product. However, this method cannot always be implemented and many alternative approaches are under investigation. Bio-preservation strategy design is an important part of a cell therapy process development and is also linked to product approval from regulatory bodies and to cost of the therapy. In order to successfully validate the use of Tower Cold Chain’s containers for the transport of cell therapy products, we started by studying the combined effect of temperature and oxygen partial pressure in a controlled environment over a simulated shelf life window of 5 and 7 days. Two cell therapy models have been investigated: scaffold free bone marrow derived cartilage discs and undifferentiated mesenchymal stem cells. Preliminary results show that temperature and oxygen partial pressure can modulate both metabolic activity and gene expression profile of both cell therapy models. We also developed a methodology that can be used for investigating multi factorial effects on the potency of the cell therapy medicinal product which is based on statistical design of experiment.
99 MSC IMMUNOMODULATION AND ARYL HYDROCARBONS RECEPTOR ACTIVATION BY TRYPTOPHAN CATABOLITES CN Lewis1,2, R Chinnadurai2, J Galipeau2,3 1 Medical Scientist Training Program, Emory University School of Medicine, Atlanta, Georgia, United States, 2Hematology and Medical Oncology, Winship Cancer Institute, Atlanta, Georgia, United States, 3Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, United States The catabolism of Trp by indoleamine 2,3-dioxygenase (IDO) is a key correlate of the immunomodulation by human mesenchymal stromal cells (MSCs). AHR is a cytosolic protein expressed by a variety of cells, that upon activation translocates to the nucleus to initiate transcription at aryl hydrocarbon response elements (AHREs). Since MSCs are known to express AHR at a basal state, we hypothesized that the catalytic activity of IDO and signal transduction via AHR are linked, as intracellular signals which deploy MSC suppressive properties. To test this, we treated MSCs with aromatic hydrocarbons known to activate AHR: TCDD or FICZ (10nM), IFNg (50ng/mL). We showed human MSCs express AHR basally, and that expression does not change during 48h of treatment with IFNg; whereas under the same conditions IFNg induced expression of IDO by 100,000-fold. Upon treatment of resting MSCs (IDO negative) with TCDD or FICZ, we observed 50- to 100-fold induction of Cyp1a1/Cyp1b1 mRNA, reflective of AHRE activation. We next examined the response of MSCs after 48h treatment with 1MT, Trp, kynurenine (Kyn) or kynurenic acid (KynAc), the latter two molecules being the first and second IDO-catalyzed Trp metabolites. We observed 1MT and KynAc both induced Cyp1a1/Cyp1b1 expression by 20to 60-fold. These data support the theory that IDO-catabolism of Trp generates endogenous ligands (i.e. KynAc) which can directly activate AHR signaling. We performed T cell proliferation assays in the presence of MSCs, and observed Trp catabolites augment the immunosuppressive function of MSCs (independently of PDL1 or IDO expression). Our findings suggest that endogenous AHR signals arising from IDO catalysis may play a role in MSC immunomodulation. This observation supports a novel paradigm for intracellular signaling arising from catabolic conversion of Trp to AHR ligands and induction of immunomodulatory properties. 100 WILL NOT BE PRESENTED