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Laterally confined growth of cells induces nuclear reprogramming in the absence of exogenous biochemical factors

Significance In this study, we demonstrate a platform for reprogramming somatic cells with high efficiency in the absence of exogenous reprogramming factors. Sustained laterally confined growth of cells on micropatterned substrates results in sequential changes to the nucleus and chromatin with each cell division, leading to the progressive erasure of lineage specific characteristics and incorporation of pluripotency. After 10 days of confined growth, the cells exhibit stemness and have multilineage differentiation potential. Our observation highlights a previously unknown role of mechanical constraints in nuclear reprogramming. Our method provides a unique approach to greatly improve stem cell technologies for developing patient specific disease models and regenerative medicine.

40 μl of 20% Triton X-100 was added and incubated at 37˚C for 60 min with constant agitation. 30 μl of HindIII (50 U/μl; Thermo Fisher Scientific) was added and incubated at 37˚C for overnight with constant agitation. Nuclei were washed with PBS and blocked with 5% BSA for 1 hr at RT before immunostaining. Nuclei were then washed with 5% BSA, scraped and collected in a tube. Coupling with beads: Dynabeads coupled with Anti-Rabbit (M-280; Thermo Fisher Scientific) were re-suspended in 1 ml of washing Buffer (Ca 2+ and Mg 2+ free PBS supplemented with 0.1% bovine serum albumin (BSA) and 2 mM EDTA, pH 7.4). Dynabeads were washed using the DynaMagTM-2 Magnet. A solution of 5% BSA and ChIp grade Anti-Histone 3 (acetyl K9) Rabbit pAb (Abcam) was added to the beads and incubated with gentle tilting and rotation at RT for one hour. Unbound Anti-Histone 3 (acetyl K9) Rabbit pAb was removed using the DynaMagTM-2 Magnet. Dynabeads were then washed with 5% BSA to ensure there was not any unbound Anti-Histone 3 (acetyl K9) Rabbit pAb remaining. These Dynabeads were then resuspended in the nuclei in 5% BSA and incubated for over 12 hours at 4°C. The product obtained after the incubation was a tertiary complex comprised of Dynabeads coated with Anti-Rabbit, bound to Anti-Histone 3 (acetyl K9) Rabbit pAb, which was further bound to chromatin associated with Histone 3 (acetyl K9). The beads were washed with PBS to ensure that any chromatin not associated with Histone 3 (acetyl K9) was removed. Reverse crosslinking: Reverse crosslinking was performed using 5ul of Proteinase K (PK; Thermo Fisher Scientific) in 200ul of PK buffer (30mM Tris (pH8.0), 10mM EDTA, 1% SDS) incubated at 65˚C for at least 90 min. Using the DynaMagTM-2 Magnet the supernatant was separated from the Dynabeads. 50 μL of PK buffer was added to the bead fraction to elute any remaining DNA. The supernatant collected was purified using Qiagen PCR clean-up to concentrate the DNA. This DNA was further amplified using the REPLI-g Single Cell Kit (Qiagen). The amplified DNA was analyzed using designed primers for EMT EpiTect ChIP qPCR array (Qiagen). The Mouse Epithelial to Mesenchymal Transition (EMT) Epitect ChIP qPCR Array was used to measure histone acetylation changes at certain promoters. This array profiles 84 genes representing epithelial genes, mesenchymal genes, proliferation genes, transcription factors etc involved in EMT.
Chromosome Fluorescence in situ hybridization (FISH): Cells were washed with 1× PBS to remove cell culture medium followed by incubation on ice for 5-8 minutes, with 0.25 % Triton in CSK buffer (100 mM NaCl, 300 mM Sucrose, 3 mM MgCl2, 10 mM PIPES with pH 6.8). Cells were then fixed with 4 % PFA (Paraformaldehyde) for 10 minutes, briefly rinsed with 0.1 M Tris-HCl followed by 1× PBS wash. This was followed by permeabilization with 0.5 % Triton for 10-15 minutes. Overnight incubation in 20% glycerol at 4 °C, and then 5 -6 freeze-thaw cycles in liquid nitrogen followed. After this, cells were washed with 1× PBS a few times, before and after treatment with 0.01 % HCl for 5-10 minutes, followed by digestion with 0.002 % porcine pepsin (Sigma Aldrich, USA) in 0.01N HCl at 37 °C for 4 minutes. Cells were then fixed with 1 % PFA for 4 minutes, briefly rinsed in 1× PBS before being treated with RNAse (from Promega, USA, 200 μg/ml made in 2× SSC-0.3M sodium chloride and 30mM trisodium citrate) at 37°C for 15-20 minutes. The cells were then washed with 2× SSC and equilibrated in 50 % Formamide / 2× SSC [(pH 7.4) overnight at 4 °C. Hybridization was set up the following day. Chromosome paints (Chrombios, Germany) tagged with different fluorophores were thawed to room temperature, and mixed with hybridization buffer provided by the supplier. Cells were denatured in 50 % Formamide / 2× SSC at 85 °C for 2-3 minutes and then incubated with the fluorescently labeled mouse chromosome FISH probe mix; the slides were then sealed with a Sigmacote (Sigma-Aldrich) coated hydrophobic coverslip and rubber cement to incubate overnight in a moist chamber at 37 °C with shaking. Chromosomes were pair-wisely painted and 3 representative pairs were selected for analysis: Chr11-Chr15, Chr5-Chr9 and Chr3-Chr6. At the end of the incubation period, slides were washed thrice each in 50 % Formamide / 2× SSC at 45 °C and 0.1× SSC at 60 °C. Finally, the nuclei were stained with Hoechst 33342 (Sigma-Aldrich) for 10 minutes and then mounted with Prolong Gold antifade mounting medium (Thermo Fisher Scientific), sealed with a coverslip, and imaged.
Endoderm Differentiation: The ten-day old spheroids (developed from NIH3T3 cells) were collected from the RE micropattern by mild trypsinization (30 seconds -1 minute). The collected spheroids were plated onto 100 mm gelatinized tissue culture dishes in 1 10 dilutions and incubated at 37°C with 5% CO2 with 15% FBS, 0.1 mM non-essential amino acids, 1 mM MTG, 2 mM of L-glutamine, 10 −7 M of all-trans-retinoic acid (Sigma) and 25 ng/ml of human basic fibroblast growth factor (bFGF) (Stem Cell Technologies), and high glucose DMEM for 3 days. The cells generated in previous step were then maintained at 37°C with 5% CO2 with 15% FBS containing high glucose DMEM supplemented with 0.1 mM nonessential amino acids, 1 mM MTG, 2 mM of L-glutamine in the presence of 25 ng/ml of bFGF for up to 14 days (2). At this time, the cells were trypsinized, collected, seeded onto petri dishes, and incubated for 6 days in suspension at 37°C with 5% CO2 with DMEM/F12 (1:1) basal media (GIBCO) supplemented with 1x N2 supplement-A, bFGF, 1x B27 supplement (GIBCO) to induce formation of cell clusters. The cell clusters from previous step were cultured for 6 days at 37°C with 5% CO2 in the presence of 2×10 −3 M dibutyryl cAMP in DMEM/F12 (1:1) basal media (GIBCO) supplemented supplemented with 1x N2 supplement and 10 mM nicotinamide.

Dopaminergic neuronal differentiation:
As illustrated in SI Appendix, Fig. S19, the isolated ten day old spheroids were cultured on Pluronic-F127 treated bacterial culture plate in prepared ESC media for 4 days at 37 °C and 5% CO2. On day 12, media was changed by allowing the spheroids suspension to stand for approximately 5 minutes in a 15 mL tube. After settling down of spheroids, old media was replaced with fresh media and this was followed by gentle pipetting and the transfer of spheroids back into the original plates. On day 14, spheroids were collected as mentioned above and transferred on 6 well tissue culture plate and cultured for 2 hours in ESC media. On day 15, the ESC media was replaced with ITS/Fibronectin media and cultured in it for another 6-8 days at 37 °C and 5% CO2 with media replacement every alternative day. ITS/Fibronectin media was prepared with DMEM/F-12, glucose, L-glutamine, NaHCO3, ITS supplement and fibronectin according to manufacturer's protocol. After 6-8 days, cells were typsinized and seeded on Poly-L-Lysine/Fibronectin-coated coverslips and cultured in N-2 MAX/FGF basic/FGF-8b/ Shh-N/Ascorbic Acid Media. In this condition, cells were cultured for 4-6 days with media replacement every other day. Cells from this stage were cultured in N-2 MAX/Ascorbic Acid Media (without growth factors) for 10-15 days with every alternative day media replacement. After 10-15 days, cells were immuno-stained for dopaminergic neurons by staining with Tyrosine Hydroxylase and Neuron-specific β-III Tubulin antibodies. For control differentiation assay, these spheroids were cultured under similar conditions but in absence of dopaminergic neuronal differentiation factors Mammosphere culture condition: For MCF-7 mammosphere culture, cells were suspended at 1× 10 5 cells/mL and seeded into ultra-low attachment surface (0.2% Pluronic F127 treated surface). In serum free DMEM/F12 (1:1) supplemented with 10 ng/mL basic fibroblast growth factor (b-FGF, R&D Biosystem), 20 ng/mL epidermal growth factor (EGF, R&D Biosystem), 5 µg/mL ITS (insulin + transferrin + selenium, GIBCO), with B27 (GIBCO) (3). Every two days 2mL of fresh media was replaced. Cells grown in these conditions becomes non-adherent spherical clusters of cells (named ''mammospheres'') were collected on day 10 s by gentle centrifugation.

Image Analysis:
Nuclear area fluctuation and dynamics: ImageJ was used to calculate the projected nuclear area fluctuation (PNAF) of H2B-EGFP nucleus and generate time-lapse merged nuclear periphery data. PNAFs were combined from multiple cells and time points for each condition to obtain a normal distribution. SD (σ) of such distribution indicates the amplitude (in percentage) of area fluctuations and was performed in ORIGIN. The nuclear circularity for fibroblasts seeded onto micropattern were calculated according to the formula, circularity = 4πA/P 2 , where A is nucleus projection area and P is nucleus perimeter. The corresponding projected area and perimeter were calculated in ImageJ. In order to estimate nuclear orientation for fibroblasts seeded onto micropattern, the angle between major axis and cell basal surface plane was calculated using custom written code in MATLAB. Confocal images of the nucleus stained with DAPI were used for this purpose.
The mean square displacement (MSD) is calculated for the centroid of each nucleus by finding the average displacement between two time points with a time interval τ = nΔt, where n takes integer values (1 to N), as follows: Here r is the position vector of the particle at each time point and N is the total number of measured points.
Chromosome Intermingling: A semi-automated algorithm written in MATLAB (Mathworks, USA) was used to analyze 3D chromosome FISH images, where both nuclei and chromosomes were manually selected according to the signal distribution on the Z-maximum projected images. Maximum projected masks for nuclei and chromosomes were then generated. This was used to multiply the entire Z-stack of the nucleus and chromosome to remove the background noise, which would undermine the segmentation accuracy. For 3D segmentation, the mean and standard deviation of intensity was computed throughout the entire Z-stack. The criteria for setting a pixel as 1 or 0 is based on the mean ± (standard deviation × a), where a is a value to adjust the criteria. Pixels above mean ± (standard deviation × a), were set as 1, and those below were set as 0. The segmentation procedure was monitored by merging the outline of the segmented object with the original object. This resulted in best 3D masking for both nucleus and chromosomes. To compute the intermingling degree, the intermingling volume between two chromosomes was first estimated. To estimate the intermingling volume, the segmented images of the two chromosomes were multiplied, and only the overlapping region resulted in pixels with value of 1. The number of pixels with value 1 represents intermingling volume.