Neurodegenerative diseases are caused by a loss of neurons within the central or peripheral nervous system, and is a chronic rare incurable disease for which there is no therapy for complete recovery. Therefore, research to treat neurodegenerative diseases using cell-based therapy has been actively ...
Neurodegenerative diseases are caused by a loss of neurons within the central or peripheral nervous system, and is a chronic rare incurable disease for which there is no therapy for complete recovery. Therefore, research to treat neurodegenerative diseases using cell-based therapy has been actively conducted recently. Especially, because of the regenerative potential of neural stem cell (NSC), cell-based therapy using NSCs is an emerging therapeutic approach targeting neurodegenerative diseases. Cell therapy products including NSCs belong to advanced biopharmaceuticals, and due to their unstable, complex characteristics, characterization and quality control(QC) of products are very important. However, there are currently few QC test methods to refer to for the development of NSCs as a therapeutics, which is an obstacle to the development of therapeutic agents. Therefore, in this study I try to provide characterization and QC test items that can be used for NSC based therapeutics.
I investigate literature research such as domestic and foreign guidelines and papers regarding the QC of cell therapy product, and some items were confirmed through experiments. Among several methods for obtaining NSCs, I used direct reprogramming technology. To generate NSC, human fibroblasts were directly reprogrammed by introducing pluripotent factors (Oct4, Sox2, Klf4, c-Myc) withSendai virus using optimized media and growth factors at each step. Using these NSCs, the applicability of characterization items was confirmed through experiments on the main QC items of cell therapeutics, such as identity, purity, and potency.
To identify the properties of NSCs, I assessed microscopic cell morphology, doubling time calculation, Short Tandem Repeat (STR) profiling. The expression of NSC-specific markers (Nestin, PAX6, SOX1, SOX2, and N-Cadherin) was also detected by immunostaining and qPCR.
Genetic stability is very important in cell therapy products. I have confirmed that the NSCs in this study have normal karyotype, and the removal of Sendai virus used during gene introduction was also confirmed. At the purity test, the detection of non-target cells and impurities during the process can be set. In this study, a test that detects induced pluripotent stem cell(iPSC) as non-target cells was set as the purity test. As a potency test, it is possible to set the differentiation ability of NSCs and quantify growth factors and neurotrophic factors secreted by NSCs. In this study, after differentiating NSCs into neurons, it was confirmed that NSCs differentiate into functional neurons by verifying the expression of neuronal-specific markers and electrophysiological activity of neurons.
Some of the various characterization tests such as identity, purity, and potency suggested in this study can be used as the QC test items of the drug substance and drug product. For the QC test item of the drug product, it can be used in the release test after verifying its validity through method validation
The characterization analysis and QC test items of NSCs presented in this study are expected to be used as basic data for NSCs to be developed as therapeutics for incurable neurological diseases.
Neurodegenerative diseases are caused by a loss of neurons within the central or peripheral nervous system, and is a chronic rare incurable disease for which there is no therapy for complete recovery. Therefore, research to treat neurodegenerative diseases using cell-based therapy has been actively conducted recently. Especially, because of the regenerative potential of neural stem cell (NSC), cell-based therapy using NSCs is an emerging therapeutic approach targeting neurodegenerative diseases. Cell therapy products including NSCs belong to advanced biopharmaceuticals, and due to their unstable, complex characteristics, characterization and quality control(QC) of products are very important. However, there are currently few QC test methods to refer to for the development of NSCs as a therapeutics, which is an obstacle to the development of therapeutic agents. Therefore, in this study I try to provide characterization and QC test items that can be used for NSC based therapeutics.
I investigate literature research such as domestic and foreign guidelines and papers regarding the QC of cell therapy product, and some items were confirmed through experiments. Among several methods for obtaining NSCs, I used direct reprogramming technology. To generate NSC, human fibroblasts were directly reprogrammed by introducing pluripotent factors (Oct4, Sox2, Klf4, c-Myc) withSendai virus using optimized media and growth factors at each step. Using these NSCs, the applicability of characterization items was confirmed through experiments on the main QC items of cell therapeutics, such as identity, purity, and potency.
To identify the properties of NSCs, I assessed microscopic cell morphology, doubling time calculation, Short Tandem Repeat (STR) profiling. The expression of NSC-specific markers (Nestin, PAX6, SOX1, SOX2, and N-Cadherin) was also detected by immunostaining and qPCR.
Genetic stability is very important in cell therapy products. I have confirmed that the NSCs in this study have normal karyotype, and the removal of Sendai virus used during gene introduction was also confirmed. At the purity test, the detection of non-target cells and impurities during the process can be set. In this study, a test that detects induced pluripotent stem cell(iPSC) as non-target cells was set as the purity test. As a potency test, it is possible to set the differentiation ability of NSCs and quantify growth factors and neurotrophic factors secreted by NSCs. In this study, after differentiating NSCs into neurons, it was confirmed that NSCs differentiate into functional neurons by verifying the expression of neuronal-specific markers and electrophysiological activity of neurons.
Some of the various characterization tests such as identity, purity, and potency suggested in this study can be used as the QC test items of the drug substance and drug product. For the QC test item of the drug product, it can be used in the release test after verifying its validity through method validation
The characterization analysis and QC test items of NSCs presented in this study are expected to be used as basic data for NSCs to be developed as therapeutics for incurable neurological diseases.
Keyword
#신경줄기세포치료제, 특성분석, 품질평가
※ AI-Helper는 부적절한 답변을 할 수 있습니다.