Mesenchymal Stem Cells

Probe libraries were designed and constructed as described in Raj et al.^{25 (link)}. Most libraries consisted of 48 probes of length 20bps, complementary to the CDS of each gene (Supplementary table). Lgr5 and Ki67 libraries consisted of 96 probes. Hybridizations were done overnight with three differentially labeled probes using Cy5, Alexa594 and TMR fluorophores. An additional FIT-C conjugated antibody for E-cadherin (BD Biosciences) was added to the hybridization mix and used for protein immunefluorescence. DAPI dye for nuclear staining was added during the washes. Images were taken with a Nikon Ti-E inverted fluorescence microscope equipped with a 100× oil-immersion objective and a Photometrics Pixis 1024 CCD camera using MetaMorph software (Molecular Devices, Downington, PA). The image-plane pixel dimension was 0.13 microns. Quantification was done on stacks of 6‒12 optical sections with Z-spacing of 0.3 microns, in which not more than a single cell was observed.
Dots were automatically detected using a custom Matlab program, implementing algorithms described in Raj et al^{25 (link)}. Briefly, the dot stack images were first filtered with a 3-dimensional Laplacian of Gaussian filter of size 15 pixels and standard deviation of 1.5 pixels. The number of connected components in binary thresholded images was then recorded for a uniform range of intensity thresholds and the threshold for which the number of components was least sensitive to threshold selection was used for dot detection (Fig. S1a–d). Automatic threshold selection was manually verified and corrected for errors (<5% of crypts). Background dots were detected according to size and by automatically identifying dots that appear in more than one channel (typically <1% of dots) and were removed. Such dots occasionally appeared in the surrounding mesenchymal cells but were rare in the epithelial cells. Bleed through of transcript signal between channels was minimal (Fig. S1e–g). Cell segmentation was performed manually on a maximal projection of the FIT-C channel. Cells at the lower 15 positions of the crypt were typically segmented. Transcript concentrations were obtained by dividing the number of transcripts per cell by the cell volume estimated as the product of the segmented area and the number of vertical stacks times a voxel size of 0.13um *0.13um *0.3um. Crypt apex and outline were manually marked and used to determine cell position along the crypt axis. For four genes – Olfm4,Dcamkl-1,Gob5 and Lysozyme, transcript abundance was too high in some of the cells to facilitate reliable dot counting. In these cells the cytoplasm was often uniformly fluorescent (Fig. 2cFig. S4g). Thus for these genes our dot counting algorithm underestimated the number of transcripts per cell.

For MEF isolation, chimeric embryos were isolated at E13.5, and the head and internal organs were removed. The remaining tissues were physically dissociated and incubated in trypsin at 37° C for 20 min after which cells were resuspended in MEF medium. 24 hours later puromycin (2μg/ml) was added and the cells were expanded for two passages before freezing or plating in doxycline containing media (2μg/ml) for reprogramming experiments. Somatic organs were isolated from 4–6 week-old transgenic mice. Epidermal keratinocytes and intestinal epithelium were isolated and cultured as previously described 6 (link). For mesenchymal stem cells (MSCs) and pro-B cells whole marrow was isolated from the femur and tibia after removal of the condyles at the growth plate by flushing with a syringe and 30-guague needle containing DMEM+5% FBS (Hyclone, Thermo Fisher Scientific). CD19+ pro-B cells were isolated by MACS cell separation (Miltenyibiotec Cat# 130-052-201) following manufactures instructions. Purified B cell subsets were resuspended in IMDM with 15% FCS as well as IL-4, IL-7, SCF (10 ng/ml each, Peprotech), doxycyline (2 μg/ml) and plated on OP9 bone marrow stromal cells (ATCC). Three days later the medium was changed to ESC medium plus Dox. Macrophage cells (CD11b+) from freshly isolated spleen were isolated by MACS cell separation and plated by a similar protocol described for CD19+ pro-B cells. Mesenchymal stem cells were selected through differential plating on tissue culture plates (10cm) for 72h in α-MEM supplemented with 15% FBS. Once plates reached a full monolayer cells were split into 6-well dishes and cultured in the presence of Dox. For isolation of liver cells mice were first perfused with 50 ml HBSS buffer (w/o Ca2+ and Mg2+) then 50 ml HBSS (w/o Ca2+ and Mg2+) containing collagenase (type IV) (Sigma Cat# C5138) (100U/ml). Liver was dissected away from surrounding tissues and dissociated in 10ml DAG media (phenol-red free EMEM Gibco-11054-020 and Bovine serum albumin (BSA) 1g/0.5L) and filtered two times through a sterile 100μM cell strainer. Liver cell preparations were centrifuged at 30 g for 3 minutes at 4 °C and the cells were washed two times with DAG media and then plated on γ-irradiated MEFs in ES media + Dox.