
Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease. Because the therapy and/or therapeutics for PD are still challenging, generating better models to recapitulate PD is imperative. Until now, the neuroblastoma cell line SH-SY5Y and immortalized Lund human mesencephalic (LUHMES) cells have been extensively used as
Besides the hiPSC-based disease model, the technology of direct reprogramming or transdifferentiation also has been adopted in disease modeling. Conversion of fibroblast to specific neurons shows impressive efficiency and phenotype (6–8). In 2010, induced neuron (iN) have been successfully converted from mouse fibroblasts by ectopic expression of three transcription factors—i.e., Brn2, Ascl1, and Myt1 (6). The iNs show electrophysiological currents
In this study, we optimized the protocol to generate hiNPC and described a sequential characterization procedure. Using our method, we successfully generated hiNPCs from a
All human fibroblast cell lines used in this study were obtained from the Coriell Institute (USA). All information about the cell lines is summarized in Table 1. The cells were cultured in human fibroblast medium (MEM medium supplemented with 10% FBS, 1× sodium pyruvate, and 1× MEM-NEAA; Thermo Fisher Scientific, USA). For reprogramming purposes, the human fibroblasts were allowed exemption from IRB review by Public Institutional Review Board Designated by Ministry of Health and Welfare (P01-201802-31-001).
The reprogramming of fibroblasts to hiNPC was performed as previously described (21), with some modifications. Briefly, 30,000 human fibroblasts cells/well were plated onto Geltrex coated 24-well plates. The next day, human fibroblasts were transduced with Sendai virus (SeV) mixtures (CytoTune™-iPS 2.0 Sendai reprogramming kit, Thermo Fisher Scientific), according to the manufacturer’s instruction. After 24 h, cells were washed with Dulbecco’s Phosphate-Buffered Saline (DPBS, Welgene, Korea) and replaced with human neural reprogramming medium which consisted of 1:1 mixture of advanced DMEM/F-12 and Neurobasal medium, 0.05% AlbuMAX, 1× N2, 1× B27, 2 mM Glutamax, 0.11 mM 2-mercaptoethanol (Thermo Fisher Scientific), 3.0
For PI staining, the hiNPCs were harvested using Accutase, and fixed with ice-cold 70% ethanol (Millipore) at 4°C overnight. The starting fibroblast cells were used as a control. Next, the cells were washed twice with DPBS, followed by incubation with solutions of 25
The G2019S mutation in
hiNPCs were plated onto Geltrex-coated coverslips and supplemented with a neuronal differentiation medium, which was comprised of DMEM/F-12 (Thermo Fisher Scientific) medium supplemented with B27 without Vitamin A, 50
Immunocytochemistry was performed as described previously (21). Briefly, the cultured cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, USA) for 10 min, followed by washing with DPBS. Next, the cells were blocked and permeabilized with 3% bovine serum albumin (BSA, Thermo Fisher Scientific) and 0.3% Triton X-100 (Sigma-Aldrich) in DPBS for 1 h at room temperature. All samples were then incubated with primary antibody solution overnight at 4°C. The next day, after washes with 0.1% BSA in DPBS, samples were incubated with Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature. Images were captured using a Fluoview FV1000 confocal microscope (Olympus, Japan). The antibodies used in this experiment are listed in Supplementary Table S2.
Karyotyping of hiNPCs was conducted by Gendix (Seoul, Korea). A STR array was performed as previously described (33). Briefly, genomic DNA was extracted from hiNPCs and their parental fibroblast cells using a DNeasy Blood and Tissue kit (Qiagen, Germany), according to the manufacturer’s instructions. The STR array was analyzed by Humanpass (Seoul, Korea).
Detection of mycoplasma in cells was performed as previously described (34). Briefly, the cell pellets were collected by centrifugation, lysed at 55°C for 3 h, followed by 1 h incubation at 95°C with proteinase K (Sigma-Aldrich). PCR was performed using the extracted DNA as PCR templates. The primer sequences used in this experiment are listed in Supplementary Table S1.
RT-PCR analysis was performed as previously described (17). Total RNA was extracted using the RNeasy mini kit with a QIAshredder and DNase I (Qiagen) to avoid genomic DNA contamination. The RNA was reverse-transcribed using an iScript cDNA synthesis kit (Bio-Rad, USA), according to the manufacturer’s instructions. Next, the PCR reaction was performed using a 1:50 dilution of the cDNA template with an iQ SYBR Green supermix (Bio-Rad). Glyceraldehyde 3-phosphate dehydrogenase (
Cells were treated with ice-cold sample lysis buffer consisting of 1% Triton X-100 (Sigma-Aldrich), 5 mM ethylenediaminetetraacetic acid (EDTA, Thermo Fischer Scientific), 1 mM phenylmethanesulfonylfluoride (PMSF, Thermo Fischer Scientific), and Xpert Protease Inhibitor Cocktail Solution (GenDEPOT, USA) in DPBS. Protein extracts were quantified with Protein Assay Dye Reagent Concentrate (Bio-Rad). An equal amount of total protein was separated by SDS-PAGE. All samples were then transferred to PVDF membrane (Bio-Rad) using a Wet/Tank Blotting System (Bio-Rad). The membranes were incubated first with blocking solution (Difco™ Skim milk, BD, USA), then primary antibodies were added, followed by the addition of horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, USA). For detection of the oxidized signals from HRP, we added substrates (ECLTM Select Western Blotting Detection Reagent, GE Healthcare, USA). The HRP images of protein bands were acquired by a LAS-3000 Imager (Fujifilm, Japan). Primary antibodies used in this experiment are listed in Supplementary Table S2.
hiNPCs were seeded in 96-well plates, and 5
We generated hiNPCs from fibroblasts of familial L2GS PD (FPD-hiNPC), sporadic PD (SPD-hiNPC) and two healthy donors (WT-hiNPC) by the PDR approach (Fig. 1a). After reprogramming, we manually picked some colonies and expanded them (Fig. 1b). To obtain intact hiNPC, we performed a step-wise analysis, as shown in Supplementary Fig. S1. Because we sometimes observed tetra ploidy in reprogrammed cells and previously aneuploid chromosomes often arise in reprogrammed cells (35), we first analyzed the ploidy of hiNPCs by simple PI staining-based flow cytometry to select diploid cells. We found that all established lines are diploid as the unreprogrammed starting fibroblasts (Fig. 2a). Second, we analyzed key markers of NPCs by immunocytochemistry, as shown in Fig. 2b. We selected the hiNPCs that expressed neural cadherin (N-CAD), PAX6, PLZF, and ZO1, as previously described (17, 21). Because almost all PAX6-expressing cells simultaneously expressed Ki-67, a cell cycle marker, we were able to confirm active proliferation of the cells as they were observed in culture. Because we wanted to confirm that the hiNPCs did not have the G2019S mutation in the
Next, we checked the differentiation potential of hiNPC candidates. We differentiated the cells spontaneously using neuronal differentiation medium. We observed that neurite outgrowths started within 3 days, and long and arborized neurites were observed after 21 days of differentiation (Fig. 1b). Because we sought to use the hiNPCs as a PD model, differentiation to dopaminergic neurons (DN) are a critical characteristic. We observed that tyrosine hydroxylase (TH) was co-stained with a pan-neuronal marker (TUJ1) in the differentiated cells from all hiNPCs. We also observed an astrocyte marker, GFAP, and a mature neuronal marker, MAP2, after 21 days of differentiation (Fig. 2d). Consistent with the immunocytochemistry results, the mRNA expression of mature neuronal markers such as
After characterization and confirmation of biological functions of the reprogrammed cells, we tested several basic requirements before cryopreservation. Because chromosomal anomaly often occurs in stem cells (37), karyotyping and G-banding analysis are useful to promptly check the genomic integrity. We found that all hiNPCs maintained a normal karyotype after culturing over eight passages (Fig. 3a). Because we usually reprogram multiple independent cells in a single batch experiment, contamination of other cells could be a potential risk. Thus, short tandem repeat (STR) analysis is important and always required after reprogramming and before banking to confirm clonality of the reprogrammed cells. We confirmed that all hiNPCs showed the same STR profile as that of the parental fibroblasts respectively (Fig. 3b). Finally, we confirmed the absence of mycoplasma by PCR amplification of the specific rRNA region of mycoplasma (Fig. 3c) (34). As above, we checked the characteristics of hiNPCs using NPC markers, differentiation potential and neuronal functionality after spontaneous differentiation using neuronal markers. To determine if the hiNPCs were adequate for long-term storage, the integrity of the reprogrammed cells was also checked. In our experience, we could efficiently analyze the hiNPC candidates with minimal effort by the proposed characterization flowchart (Supplementary Fig. S1).
To establish the cellular model of PD, various stress reagents have been widely used to mimic the vulnerability of DNs (38–40). One of the proteasome inhibitors, MG132, impairs the intracellular protein clearance system such as the ubiquitin proteasome system, resulting in cell death (41, 42). Because PD patient-derived cells showed more severe cell death by proteasomal stress than healthy controls, proteasome inhibitors has been widely used for PD modeling (39, 40, 42). To confirm whether our hiNPCs represent a PD phenotype such as apoptosis, we treated them with MG132 or DMSO (Fig. 4a). As expected, we found more apoptotic cells in FPD-hiNPCs and surprisingly in SPD-hiNPCs than in WT-hiNPCs by MG132 treatment whereas we did not observe the difference of cell death in all DMSO controls (Fig. 4b). To quantify cell death, we performed WST based cell viability assay and immunoblot for cleaved CASPASE3 (cCASP3). Consistent with the morphology of cells, when the DMSO control was set at 100% in each cell line respectively, FPD- and SPD-hiNPCs exhibited significantly decreased cell viability (27.5±0.4% and 30.4±2.1% respectively) than WT1-, WT2-hiNPCs (39.1±1.0% and 38.8±1.1% respectively) by MG132 treatment (Fig. 4c). We also confirmed increased expression of cCASP3 in FPD- and SPD-hiNPCs relative to WT1- and WT2-hiNPCs by MG132 treatment (Fig. 4d, 4e). These results demonstrate that PD patients-derived hiNPCs are more sensitive to the proteasome stress than healthy controls, consequently resulting in more cell death in hiNPCs derived from PD patients than hiNPCs derived from healthy donors. Thus, our hiNPCs are useful to model both familial and sporadic PD and could be used to develop various other PD modeling paradigms.
Here, we optimized a direct reprogramming protocol for hiNPCs and proposed a step-wise characterization process. We also showed that hiNPCs are adequate and acceptable resources for an
This work was supported by the KRIBB Research Initiative and Stem Cell Research Program through the National Research Foundation of Korea funded by the Ministry of Science and ICT (2013M3A9B4076483, 2015M3A9C7030128, 2018M3A9H3023077, and 2016K1A3A1A61006001) and a grant from Ministry of Food and Drug Safety in 2018 (18172MFDS182).
The authors have no conflicting financial interest.
Supplementary data including two tables and one figure can be found with this article online at
http://pdf.medrang.co.kr/paper/pdf/IJSC/IJSC-12-s19075.pdf.
Direct reprogramming to generate hiNPCs. (a) Schematic diagram to show direct reprogramming of fibroblasts to hiNPCs. (b) Representative bright field images of fibroblasts, a reprogrammed hiNPC colony, clonally expanded hiNPCs, and spontaneously differentiated cells from hiNPCs. Scale bars represent 100
Characterization of hiNPC lines. (a) Flow cytometry to detect ploidy of PI stained hiNPCs. Human fibroblasts from healthy donors were used as a 2n control. WT1, WT2, FPD, and SPD represent AG02261-hiNPC, GM01680-hiNPC, ND38262-hiNPC, and AG20446-hiNPC, respectively. (b) Immunocytochemistry for key NPC markers in hiNPCs. Ho. represents Hoechst33342 for staining nuclei. Scale bars represent 50
Quality check of hiNPC lines before cryopreservation. (a) Karyotypes of established hiNPC lines at passage 9, 13, 8, and 13 of WT1-, WT2-, FPD-, and SPD-hiNPC, respectively. (b) STR analysis comparing starting fibroblasts and their corresponding hiNPCs. (c) Mycoplasma test by PCR. A 100 bp ladder was used.
hiNPCs as a PD model. (a) Schematic diagram for PD modeling. (b) Representative bright field images of hiNPCs after treatment with MG132. Scale bars represent 200
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