Characterization of iPSC-derived midbrain organoids from Parkinson’s disease patient Yunsu Bang, Juhyun Choi, Ki Soon Kim, Jong Gu Lee, Woo yong Oh, Yoonsook Lee Clinical Research Division, NIFDS, MFDS, Osong Health Technology Administration Complex, 187, Osongsaengmyeong 2-ro, Osong, Heungdeok, Cheongju, 28159, Korea Abstract Parkinson's disease(PD) is the one of common neurodegenerative diseases, that results from the loss of neuromelanin(NM)-containing dopaminergic(DA) neurons. To overcome the limitations of animal degenerative models for human phenotypes, human induced Pluripotent Stem
Cell(iPSCs) are being widely used nowadays. In this study, we investigated the characterization of midbrain organoids(MOs) whether these can be the proper model for research and drug screening on neurodegenerative diseases. At first, we differentiated MOs from human iPSCs by 3D culture system. And then we checked the identified markers for representing DA neurons on culture day 0, 24, 44, 64, 84 by real time PCR, WB, and IHC. The differentiated MOs showed that DA progenitors(FoxA2, LMX1A and LMX1B), neuronal microtubule(Tuj1, MAP2) and DA neuron(TH, GIRK2) markers including accumulation of
alpha-synuclein. First of all, the NM granules were observed in organoids, not shown in 2D-cultured iPSCs. NM granules of patient's MOs were detected lower than those of normals', that shows the major characteristic of PD. Our study showed that the MOs had several features of the human midbrain. Even though more assessments remain to be elucidated, we propose that iPSCs-derived organoids are useful tool for researches. Material and Methods Analysis of differentiation To confirm the expression of differentiation markers, such as Oct4, NANOG, LMX1A, TH and Tuj1, Real-Time PCR, Western blotting
and Immunohistochemistry were performed. The PCR test was performed following the ssoadvanced universal probes supermix(Bio-Rad) protocol and used following the primers, GAPDH, OCT4, NANOG, LMX1A, TH and Tuj1(Applied biosystems). The expression of proteins was assessed with antibodies such as OCT4(Cell Signaling Technology), LMX1B(Abcam), TH(Abcam), α-synuclein(Abcam), Dopamine(Abcam) and actin(Sigma) by western blot analysis and immunohistochemistry. L-DOPA treatment L-DOPA is metabolized to dopamine in dopaminergic neurons. We treated the cells with excess L-DOPA and observed the remaining
dopamine in the cells accumulated as neuromelanin. L-DOPA is diluted with organoid culture medium to a final concentration of 50 μM for more than 1 month. Results Differentiation of human midbrain organoids Midbrain organoid differentiation was induced using modifications to the Junghyun Jo’s protocol as below(Figure 1). Induced Pluripotent Stem Cells(iPSCs) were obtained from normal and Parkinson’s disease patient. A. B. Figure 3. Final markers for DA of normal and patient’s organoids by immunohistochemistry . Immunohistological staining of midbrain DA markers, TH, Tuj1, neurotransmitter
dopamine and α-synculein in the human midbrain organoids frozen sections. TH and Tuj1 positive cells were more observed in organoid derived from normal than PD patient, whereas dopamine and α-synculein positive cells were more detected in PD patient derived organoid. Figure 1. Protocol of human midbrain organoids(hMOs) differentiation. A) Brief overall schematic of the differentiation process; B) Schematic diagram showing the reagents used at each stage A. B. during the differentiation process. This protocol is a modified version of Junghyun Jo’s protocol(Cell Stem Cell, 2016) Characterization
of human midbrain organoids The differentiation markers were confirmed by real-time PCR, western blotting and immunohistochemistry. For further confirm of the presence of dopaminergic neurons, accumulation of neuromelanin was observed by reaction to excess L-DOPA. As results in the pathology in humans, the accumulation of alpha-synuclein in normal organoid was more stained black than that of PD patient’s organoid. A. B. Figure 4. L-DOPA response and neuromelanin staining of normal and patient organoids. A) After treatment of 50 μM L-DOPA more than 1 month, the accumulation of neuromelanin in
normal organoid was higher than that of patient’s organoid. B) The neuromelanin staining(black, arrow) of normal organoid was higher than that of patient’s organoid. Conclusion ○ The expression of mRNA and protein markers of each differentiation step was identified. The accumulation of α-synuclein, representative PD pathology, was shown Figure 2. Analysis of mRNA and protein expression at each differentiation steps by real time PCR and higher in PD patient-derived organoids than that in normal organoid. western blotting. ○ Neuromelanin was observed in the 3D organoids, not shown in 2D
differentiated DAs. A) Real time PCR results for undifferentiation(OCT4, NANOG), neural(TUBB3) and DA markers(LMX1A, TH) during differentiation of dopaminergic neurons. Expression levels of each gene were normalized to ○ The neuromelanin accumulation of PD patient’s organoid was less detected than that of GAPDH level. B) Western blotting results for undiffentiation(OCT4), DA markers(LMX1B, TH, α-synuclein) normal’s organoid, despite the same concentration of L-DOPA were treated in both during differentiation of dopaminergic neurons. organoids. Acknowledgement This research was supported by a
grant 19181MFDS424 from Ministry of Food and Drug Safety in 2019. [R. Organoid-1] Characterization of iPSC-derived midbrain organoids from Parkinson's disease patient Yunsu Bang¹˙#˙*, Juhyun Choi¹˙#, Ki Soon Kim¹, Jong Gu Lee¹, Woo Yong Oh¹, Yoonsook Lee¹˙* ¹Clinical Research Division, NIFDS, MFDS, Cheongju 28159, Korea Parkinson's disease(PD) is the one of common neurodegenerative diseases, that results from the loss of neuromelanin(NM)-containing dopaminergic(DA) neurons. To overcome the limitations of animal degenerative models for human phenotypes, human induced Pluripotent Stem
Cell(iPSCs) are being widely used nowdays. In this study, we investigated the characterization of midbrain organoids(MOs) whether these can be the proper model for research and drug screening on neurodegenerative diseases. At first, we differentiated MOs from human iPSCs by 3D culture system. And then we checked the identified markers for representing DA neurons on culture day 0, 24, 44, 64, 84 by real time PCR, WB, and IHC. The differentiated MOs showed that DA progenitors(FoxA2, LMX1A and LMX1B), neuronal microtubule(Tuj1, MAP2) and DA neuron(TH, GIRK2) markers including accumulation of
alpha-synuclein. First of all, the NM granules were observed in organoids, not shown in 2D-cultured iPSCs. NM granules of patient's MOs were detected lower than those of normals', that shows the major characteristic of PD. Our study showed that the MOs had several features of the human midbrain. Even though more assessments remain to be elucidated, we propose that iPSCs-derived organoids are useful tool for researches. Comparative Proteomic Analysis and Evaluation of Anticancer Drug Responses Using Patient-Derived Tumor Organoids Yeon-Jin Chu, Hyuna Kim, Hyeong Ho Cho, Ki Soon Kim, Donghyun
Park, Woo Yong Oh, Yoonsook Lee* Clinical Research Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, 187 Osongsaengmyeong2-ro, Osong, Heungdeok, Cheongju, 28159, Korea ABSTRACT Tumor organoids using three-dimensional(3D) culture system are well known to reflect the heterogeneity and biological properties of patients compared with cancer cell lines using two- dimensional(2D) culture system. However, there are few studies of cancer mechanism and drug effects using tumor organoids. In this study, we
investigated the effects and the alteration of proteins by anticancer drug using patient-derived tumor organoids. At first, we cultured patient-derived tumor organoids isolated from colorectal cancer specimens using matrigel. Each tumor organoids were shown different growth properties and proteins expression level. Then, we evaluated the cytotoxicity of 5-Fluorouracil(5-FU) in tumor organoids. We found that 5-FU induced cell death was caused by apoptosis through the expression of apoptotic proteins. Based on these data, we tried to find proteins associated with 5-FU resistance using 2-
dimensional gel electrophoresis(2DE) and matrix-assisted laser desorption/degradation time of flight(MALDI-TOF). Most of proteins were involved in metabolism, synthesis/degradation of protein, cytoskeleton, redox reaction, etc. We measured mRNA and protein levels of them, and selected candidates for biomarker of 5-FU resistance. Our data shows that tumor organoids can be useful tool of tumor studies for evaluation and investigation of anti-cancer drug. INTRODUCTION EMBO J. 38: e101654 (2019) Tumor organoid cultures have enabled several observations: Science. 364:952-955. (2019) J Hematol
Oncol. 11:116. (2018) iii) Interpatient variation is captured and maintained. iii) Organoids can typically be derived from patient material with high efficiency and can be xenotransplanted. iii) Tumor organoids can faithfully report the drug response of the corresponding patient. iv) Drug sensitivities of patient-derived tumor organoids can be recapitulated in patient-derived xenograft(PDX) settings. Tumor organoids are well known to reflect the heterogeneity and biological properties of patients compared with cancer cell lines using 2D culture system. Therapeutic regimen of colorectal
cancer FOLFIRI Leucovorin calcium, 5-FU, Irinotecan ⇒ In this study, we selected 5-FU as main anticancer drug because most of therapeutic regimens have based on 5-FU. FOLFIRI-BEVACIZUMAB Leucovorin calcium, 5-FU, Irinotecan, bevacizumab Here, we tried to evaluate anticancer drug effects and investigate the molecules FOLFIRI-CETUXIMAB Leucovorin calcium, 5-FU, Irinotecan, Cetuximab FOLFOX Leucovorin calcium, 5-FU, Oxaliplatin associated with 5-FU resistance using comparative proteomic analysis FU-LV Leucovorin calcium, 5-FU in patient-derived tumor organoids. XELIRI Capecitabine(Xeloda),
Irinotecan XELOX Capecitabine(Xeloda), Oxaliplatin RESULTS I. Construction and characterization of patient-derived III. Investigation of proteins associated with 5-FU tumor organoids using colorectal cancer tissues sensitivity using 2-DE analysis and MALDI-TOF No. Gender Age TNM Stage MSS/MSI Table 1. Clinical information of colorectal A ① ② ③ #7 M 63 T3/N2b/M1 4 MSS patients who provided tumor tissue. #12 F 66 T3/N1a/M0 2 MSS TNM: T; invasion depth of tumor T1(submucosa), T2(muscular layer), T3(pericolic adipose tissue), T4(penetrate #15 F 71 T3/N2b/M1 3 MSS the serosa), N; nodal metastasis
of tumor N0(no metastasis #16 M 70 T1/N0/M0 3 MSS to lymph node), N1a(meta to one lymph node), N1b(2-3), N2a(4-6), N2b(>=7), M; distant metastasis of tumor M0(no #250 M 57 T4/N1a/M0 3 MSS metastasis to distant organ), M1(metastasis to other disant organ). #254 M 39 T3/N2a/M0 3 MSS MSI(microsatellite instability): MSI-H(MSI-High), HSI-L(MSI- #255 M 53 T3/N0/M0 2 MSI-H Low), MSS(microsatellite stable) Construction of patient-derived tumor organoids using colorectal B cancer specimens ① ② ③ ① ② ③ ① ② ③ ③ 1001 17 5719 A B ② 7007 119 6014 #7 #12 #15 #16 ① 1012 6709 5007 40X 40X 40X 40X #250 #254
#255 Figure 4. Different expression of proteins associated with 5-FU sensitivity. Results of 2-DE image(A) and differentially expressed protein spots(B). B A 5-FU (-) 5-FU (+) 40X 40X 40X S R S R Figure 1. Morphology(A) and growth rate(B) of patient-derived tumor organoids. #12 #254 #15 #250 #255 #7 #12 #254 #15 #250 #255 #7 1001 II. Evaluation and analysis of 5-FU drug response in β-actin #12 #254 #15 #250 #255 #7 #12 #254 #15 #250 #255 #7 patient-derived tumor organoids #12 #254 #15 #250 #255 #12 #254 #15 #250 #255 6709 A B 5-FU (-) 5-FU (+) 5-FU (-) 5-FU (+) #12 #254 #15 #250 #255 #12 #254
#15 #250 #255 #7 5719 #7 #12 #12 #12 #254 #15 #250 #255 #12 #2
