Molecular cloning. All DNA constructs were cloned using the Golden Gate cloning system [39]. The genes of interest were cloned into Level 0 and Level 1 backbones obtained from a MoClo Toolkit (AddGene Kit #1000000044). BpiI (BbsI) and Eco31I (BsaI) restriction endonucleases (Thermo Scientific, Waltham, MA, USA) and T4 DNA ligase (Evrogen) were used for the MoClo cloning procedure.
MPP8 reader domain DNA (aa 55-117) was amplified from human placenta сDNA (Takara Bio, USA) with specific primers based on available sequence NM_017520.4 and cloned into Level 0. ATOH1 DNA fragment was amplified from Addgene plasmid pTet-O-ATOH1-T2A-PuroR (Addgene #162342), and put under the control of human Phosphoglycerate Kinase (hPGK) promoter in a lentiviral vector. Transfection efficiency of this plasmid was visualized using mTagBFP2 protein fused to T2A peptide GSGEGRGSLLTCGDVEENPGP. All plasmids were made using a custom backbone based on pRRLSIN.cPPT.EF1 vector, kindly provided by Dr. D. Trono, Lausanne.
RT-PCR. RNA was isolated from iPSCs at various stages of differentiation using the RNA Solo kit (Evrogen). Complementary DNA (cDNA) was synthesized from the isolated RNA utilizing Magnus Reverse transcriptase (Evrogen). The reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using the OneTube RT-PCR SYBR kit (Evrogen) with the following primer:
NES for CAACAGCGACGGAGGTCTC
NES rev GCCTCTACGCTCTCTTCTTTGA
TUBB3 for CCGAAGCCAGCAGTGTCTAA
TUBB3 rev AAGACAGAGACAGGAGCAGC
SOX1 for AATACTGGAGACGAACGCC
SOX1 rev AACCCAAGTCTGGTGTCAGC
MAP2 for CTCAGCACCGCTAACAGAGG
MAP2 rev CATTGGCGCTTCGGACAAG
MSI for GGGACTCAGTTGGCAGACTAC
MSI rev CTGGTCCATGAAAGTGACGAA
ENO2 for CCGGGAACTCAGACCTCATC
ENO2 rev CTCTGCACCTAGTCGCATGG
S100B for GAAGGGAGGGAGACAAGCAC
S100B rev TCGTGGCAGGCAGTAGTAAC
MAPT for TTTGGTGGTGGTTAGAGATATGC
MAPT rev CCGAGGTGCGTGAAGAAATG
Cell culture and cell line generation. HEK293T cells were cultured at 37 °C (5% CO2) in DMEM (PanEco, Moscow, Russia) supplemented with 10% fetal bovine serum (BioSera, Nuaille, France), 100 U/mL penicillin, and 100 mg/mL streptomycin (PanEco). For the live cell imaging experiments, the DMEM was replaced by imaging media: MEM (PanEco) supplemented with 10% fetal bovine serum (BioSera) and 20 mM HEPES (Corning, New York, NY, USA). Initially, a culture of induced pluripotent stem cells (iPSCs), iPS-KYOU, has been obtained at the Shinya Yamanaka laboratory (Kyoto University, Japan) by the reprogramming of adult female skin fibroblasts. The iPS-KYOU cell line was purchased from the ATCC cell bank (KYOU-DXR0109B, ATCC® ACS-1023™). IPSC were cultured in mTeSR (StemCell Technologies, USA) with daily medium changes for optimal growth and Matrigel (Corning, USA) as the surface coating matrix. The stable cell line IPSC MPP8-NeonGreen was registered in the database hPSCreg, the information is available at https://hpscreg.eu/cell-line/KUIFMSi004-A-1.
For transient transfection of HEK293T cells with various GEEP plasmids, PEI 25K (Polysciences, USA) was used according to the manufacturer's instructions. Stably transduced iPSC MPP8-Green cell line was obtained by lentiviral transduction. Vector particles were generated by PEI 25K (Polysciences, USA) transient transfection of HEK293T cells. Twenty-four hours before transfection, 1.5×106 HEK293T cells were seeded into a 60 mm culture dish. The total of 2 μg and 0.6 μg of the two packaging plasmids pR8.91 and pMD.G, respectively, and 6 μg of MPP8-mNeonGreen-NLS-MPP8 or ATOH1-t2a-TagBFP2 were used for transfection. The DNA-PEI mixture was incubated for 20 min at room temperature and then was added dropwise. After 4 hours, the medium was replaced with 2 ml of fresh DMEM. Twenty-four hours afterwards the medium containing lentiviral vector particles was filtered (0.45-µm filter) and concentrated by ultracentrifugation at 100,000 g (Beckman, USA) at 4°C for 3 hours. The pellet was resuspended in 500 µl mTeSR (StemCell Technologies, USA) and used for transduction of iPS cells. To create stable cell lines, lentiviral particles were added to 1x105 iPSCs or HEK293T. Then transduced cells were sorted with Bio-Rad S3e cell sorter (USA) using mNeonGreen as a selective marker. Lentiviruses containing ATOH1-t2a-TagBFP2 were used for experiments with differentiation of iPS cells into neurons. The ATOH1 lentiviruses were made using a similar protocol as MPP8-mNeonGreen-MPP8. Concentrated ATOH1 viruses were added to iPS cells on day 0, and after 24 hours, the virus-containing medium was replaced with fresh mTesR medium.
Live-cell imaging. For the live cell imaging experiments, iPS cells were seeded on glass-bottomed 35 × 10 mm dishes (SPL Life Sciences, Gyeonggi-do, Korea) and were incubated at 37°C (5% CO2) overnight. The next day, the cells were transduced with ATOH1-t2a-TagBFP2 lentiviruses. Imaging experiments were performed using a BZ-9000 inverted fluorescence microscope (Keyence, Osaka, Japan) with a 60 × PlanApo 1.40 NA oil objective (Nikon, Melville, NY, USA). TexasRed OP66838 BZ filter (Keyence, Osaka, Japan) was used to induce red fluorescence, a GFP-BP OP66836 BZ filter (Keyence) was used to induce the mNeonGreen fluorescence, a 49021-ET-EBFP2/Coumarin/Attenuated DAPI filter (Chroma Technology) was used to induce the TagBFP2 fluorescence.
Fixed-cell immunofluorescence. Cells were seeded and grown as described above, fixed in 4% formaldehyde in PBS for 15 min at room temperature, washed three times in PBS, permeabilized for 20 min at room temperature in PBS supplemented with 0.1% Triton X-100 (Helicon, USA), and incubated for 1 hour with 1% BSA (Sigma, USA) in PBS for blocking. Corresponding primary antibodies anti-H3K9me3: ab39161 (Active Motif), anti-Oct4: PAA424Hu01 (Cloud-Clone Corp.), anti-Sox2: AF7950 (Affinity), anti-SSEA4: RGK25601 (AntibodySystem), anti-TRA 1-60: Ab16288 (Abcam), anti-tubb3: AF700 (Affinity), and secondary antibodies (Alexa Fluor 488 anti-mouse and 568 anti-rabbit IgGs from ThermoFisher) were diluted in PBS containing 0.02% BSA. Primary antibody incubations were performed 1hour at room temperature and secondary antibody incubations were performed also for 1hour at room temperature. Cells were washed with PBS and imaged in imaging media using a BZ-9000 inverted fluorescence microscope (Keyence, Osaka, Japan).
Image and data analysis. LiveMIEL analysis of fluorescent microscopic images included nuclei segmentation, features extraction from segmented single nuclei images and data clustering. Features of nuclear morphology and texture features (98 features) were calculated for single nuclei images using custom algorithms and libraries scikit, mahotas. Thus, a set of 98-dimensional feature vectors describing single nuclei images was obtained. To improve clustering, feature vectors were averaged over N nuclei within the same class (N = 30, 40; 200-350 nuclei per class).
Segmentation. Segmentation of single objects (nuclei, cells) from fluorescent microscopic images was carried out by applying bandpass and watershed segmentation to the images followed by removal of false-positive objects. The pixel values of the image were normalized by intensity to [0; 1], performed noise removal (image smoothing) and background subtraction using Gaussian blur: image convolution with a blur kernel to remove noise (low σ values) and with a blur kernel to subtract background (high σ values). Image areas were considered segmented with a pixel value above a specified threshold. Watershed segmentation was used to separate closely spaced objects in the original image mask obtained via bandpass filtering. Segmented objects with a maximum intensity value below μ * k (μ is the average value of the intensity of an image pixel with a mask; the values of the k coefficient varied within [1.2; 1.9] or with too small an image area were considered false-positive and were removed. For further processing tasks each segmented object (nucleus, cell) with its coordinates in the original image were saved.
Image alignment. Alignment of nuclei images in different fluorescent channels was performed to obtain additional information about the start of cell differentiation processes in each cell. The nuclei for which the Euclidean distance between the centers of mass in non-segmented images did not exceed half the radius of the bigger nucleus were considered matched.
Data processing. Single nuclei image was represented using a feature vector. The following image features were calculated: Haralick features (13 features), Threshold Adjacency Statistic (TAS) (54 features), Zernike moments (25 features), coordinates of the center of mass of the nucleus image (2 features) and the custom statistic of chromatin distribution - the minimum, maximum, average and total areas of chromatin dots within nucleus, normalized to the area of the nucleus (4 features). Segmentation of chromatin dots within the nucleus was done using the same segmentation strategy as for nuclei segmentation from the original images. In total, 98 features were identified for each nucleus. Raw feature values were normalized by z-scoring to the zero mean and unit variance.
Data dimensionality reduction. To reduce the number of features extracted from images, the Principal Component Analysis (PCA) was used. The applicability of the PCA was tested by calculating explained variance ratio for the first 10 principal components. Two first principal components PC1 and PC2 were displayed as 2D scatter plots.
Clustering. Data clustering was conducted using K-means and EM algorithms (scikit-learn library). To estimate the optimal number of clusters silhouette coefficient analysis was used. For evaluation, the average value of the silhouette coefficient was calculated, and the optimal number of clusters was chosen, which corresponded to the average value of the silhouette coefficient tends to 1 and the presence in all clusters of objects with values of the silhouette coefficient above the average value. Hopkins statistic was calculated to assess the cluster tendency of a data set (1/2 corresponds to random objects distribution, 1 indicates if objects are aggregated).