SEARCH

SEARCH BY CITATION

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
sc-12-0650_sm_SupplFigure1.tif2264KFigure S1. Schematic illustration of the key genetic players used for imaging the emergence of Oct-4 GFP+ cells. A) Compound mice harboring Rosa26-rtTA and Oct-4 GFP alleles were used as donors for all experiments. rtTA is expressed but inactive in the absence of dox. Oct-4 locus is inactive. B) GMPs were introduced with the reprogramming factors by a poly-cistronic lentivirus. Reprogramming factors are not expressed in the absence of dox. Oct-4 locus is inactive. C) Adding dox activates rtTA and induces factor expression. Oct-4 locus undergoes epigenetic changes, preparing for potential activation. D) In rare cells, Oct-4 locus is activated and GFP protein is made. Microscopy detects the GFP+ cells at various reprogramming stages.
sc-12-0650_sm_SupplFigure2.tif1778KFigure S2. Schematic illustration of the scanning and stitching strategy. Camera photographs consecutive frames in a scanning motion with the dashed arrows showing its scanning path. It records signals from DIC and GFP (also RFP channels when necessary) at regular time intervals of 5-30 minutes. Image acquisition initiates about one hour after adding dox and continues for 3- 5 days, when GFP+ cells emerge and develop into ESC-like colonies. During post experiment image analysis, the neighboring frames were stitched together to synthesize a large field of view. This large field is necessary for capturing low frequency events and tracking the complete trajectory of a reprogramming lineage given the active cell migration. An area containing a reprogramming event could be zoomed in and examined in close detail. Shown is a hypothetical 10×10 synthetic field.
sc-12-0650_sm_SupplFigure3.pdf858KFigure S3. The interface of the custom software used for image processing, analysis and annotation. This software is coded in Matlab with graphical interface. A) is for image processing and B) for image annotation.
sc-12-0650_sm_SupplFigure4.tif1226KFigure S4. Schematic illustration of the formation of mixed colony containing both GFP+ and GFP− cells. This involves using GMPs from two genetically distinct mice, one that contains the Oct-4 GFP reporter and the other that does not. Both types of GMPs contain the same rtTA transgene at the Rosa26 locus to allow dox-inducible factor expression. Since all colonies from the Oct-4 GFP reporter containing mice are entirely GFP+, and all colonies from the non reporter containing GMPs are GFP−, three types of colonies are expected: all GFP+, all GFP− and a mix of GFP+ and GFP− if clonal mixing occurs during reprogramming.
sc-12-0650_sm_SupplFigure5.tif2754KFigure S5. A) Background fluorescence with immuno-fluorescence staining and detected by confocal microscopy. Staining was performed by omitting the primary E-cadherin antibody and processed in parallel. Similar background level of fluorescence is seen with GMPs and non reprogrammed cells as shown in Fig.5F top row and Fig.6F (Cre+), demonstrating a lack of Ecadherin expression in those cells. B) A conditional allele allows for dox inducible inactivation of the E-cadherin gene. PCR detection of the recombined/inactivated E-cadherin allele (Δ) in Cre+ cells when Dox was added.
sc-12-0650_sm_SupplMovie1.wmv31123KMovie S1. Time-lapse image series of a representative 10×10 scan acquired at 30 min intervals. Signals from DIC and GFP were recorded, stitched and overlayed. There were no GFP+ cells during the early part of the image series. GFP+ cells/colonies start to be visible around 60 hours. Image quality is reduced to show the overview of the entire synthetic field. Time stamps are in hours:minutes format. Bar = 100 μm.
sc-12-0650_sm_SupplMovie2.wmv11545KMovie S2. Time-lapse image series acquired at 15 minutes intervals, which were processed and annotated to allow tracing reprogramming to a founder cell. High resolution images were acquired by time-lapse imaging, processed and annotated. Images in the beginning show cells of hematopoietic morphology, which lack detectible Oct-4 GFP expression. Arrows follow the movement of cell(s) that developed into GFP+ colonies at the end of image acquisition. The founder cell is followed by the yellow arrow, which split and established two similar sized colonies at the end of image acquisition. This is a typical example of sister colony formation, with some minor satellite colonies seen as well (not marked). Overlayed images of DIC and GFP channels are shown. Time stamps are in hours:minutes format. Bar = 100 μm.
sc-12-0650_sm_SupplMovie3.wmv26299KMovie S3. Time-lapse image series at 5 minutes intervals, capturing a mixed colony formation by cell migration. GMPs from Rosa26-rtTA mice with or without the Oct-4 GFP reporter were induced to reprogram on the same culture dish. Time-lapse observation captured the formation of one mixed colony. The arrows track the movement of different cells as they continued the reprogramming process. They entered the field of view from distinct locations at different times. The migration brought them into close proximity of each other. The small cell clusters originated from these cells continued to grow and compact without distinguishing the cells of different origins. The resulted colony contained both GFP+ and GFP− cells, originating from cells indicated by the pink, yellow and blue arrows. Also present in view are colonies consisting of only GFP+ cells and of only GFP− cells (not marked). Time stamps are in hours:minutes format. Bar = 100 μm.
sc-12-0650_sm_SupplMovie4.wmv22782KMovie S4. Time-lapse image series at 15 minutes intervals, capturing the dispersing of a colony. Left: The founder cell divided on the 2nd frame of acquisition (00:30) and its two immediate daughters are followed by the yellow and blue arrows. All cells in view marked by arrows are descendents of the same founder. Addition of a new colored arrow indicates the birth of a visually distinct new daughter cell. Note the complex splitting, migrating and mixing behavior. Some arrows disappeared in the movie, which indicate either apoptotic appearance of the cell or loss of Oct4-GFP, or becoming out of view. Note the appearance of several dumbbell intermediates. Around 62 hours, two of the larger colonies disappeared suddenly from view. Images from another field (Right) show one of these colonies landed a remote site and continued its development afterwards. The identity of the break-away small cell cluster was informed by its shape and the timing when it disappeared from view in one field and appeared in another field. Overlayed images of DIC and GFP channels are shown. Time stamps are in hours:minutes format. Bar = 100 μm.
sc-12-0650_sm_SupplMovie5.wmv5963KMovie S5. Time-lapse image series at 30 minutes intervals, capturing the dispersing of a colony. Large yellow arrow follows the main reprogramming colony. Various colored smaller arrows point to the break-away cells, which continued to grow and form smaller “satellite” colonies. Overlayed images of DIC and GFP channels are shown. Time stamps are in hours:minutes format. Bar = 100 μm.
sc-12-0650_sm_SupplMovie6.wmv20091KMovie 6-8. Pluripotent cells of non-hematopoietic origin display similar mixing and dispersing behaviors. Pluripotent cells were prepared by reprogramming MEFs from the Oct-4 GFP mice. The pluripotent state is similarly indicated by the expression of Oct-4 GFP. Single cells were plated on feeder cells following trypsinization. Movie S6: Clonal mixing is seen with MEF-derived iPSCs. Two Oct4-GFP+ cells migrated toward each other and merged to form one colony. Movie S7: Colony dispersal is seen with MEF-derived iPSCs. Note the dispersing behavior of the colony growing on the bottom half of the imaging field. A non-dispersing colony is also in view. Movie S8: Clonal mixing is seen with ESCs. ESCs from wild type C57Bl6 mice were transduced with a retrovirus that expresses either GFP or dsRed. Fluorescently marked cultures were trypsinized to single cells and co-plated at 1:1 ratio on feeder cells. Cell migration yielded colonies that contain both GFP+ and dsRed+ cells. The migratory behaviors from these pluripotent cells are highly similar to those seen with GMP-derived pluripotent cells. Time stamps are in days:hours:minutes format. Bars = 50 μm.
sc-12-0650_sm_SupplMovie7.wmv38966KMovie 6-8. Pluripotent cells of non-hematopoietic origin display similar mixing and dispersing behaviors. Pluripotent cells were prepared by reprogramming MEFs from the Oct-4 GFP mice. The pluripotent state is similarly indicated by the expression of Oct-4 GFP. Single cells were plated on feeder cells following trypsinization. Movie S6: Clonal mixing is seen with MEF-derived iPSCs. Two Oct4-GFP+ cells migrated toward each other and merged to form one colony. Movie S7: Colony dispersal is seen with MEF-derived iPSCs. Note the dispersing behavior of the colony growing on the bottom half of the imaging field. A non-dispersing colony is also in view. Movie S8: Clonal mixing is seen with ESCs. ESCs from wild type C57Bl6 mice were transduced with a retrovirus that expresses either GFP or dsRed. Fluorescently marked cultures were trypsinized to single cells and co-plated at 1:1 ratio on feeder cells. Cell migration yielded colonies that contain both GFP+ and dsRed+ cells. The migratory behaviors from these pluripotent cells are highly similar to those seen with GMP-derived pluripotent cells. Time stamps are in days:hours:minutes format. Bars = 50 μm.
sc-12-0650_sm_SupplMovie8.wmv17113KMovie 6-8. Pluripotent cells of non-hematopoietic origin display similar mixing and dispersing behaviors. Pluripotent cells were prepared by reprogramming MEFs from the Oct-4 GFP mice. The pluripotent state is similarly indicated by the expression of Oct-4 GFP. Single cells were plated on feeder cells following trypsinization. Movie S6: Clonal mixing is seen with MEF-derived iPSCs. Two Oct4-GFP+ cells migrated toward each other and merged to form one colony. Movie S7: Colony dispersal is seen with MEF-derived iPSCs. Note the dispersing behavior of the colony growing on the bottom half of the imaging field. A non-dispersing colony is also in view. Movie S8: Clonal mixing is seen with ESCs. ESCs from wild type C57Bl6 mice were transduced with a retrovirus that expresses either GFP or dsRed. Fluorescently marked cultures were trypsinized to single cells and co-plated at 1:1 ratio on feeder cells. Cell migration yielded colonies that contain both GFP+ and dsRed+ cells. The migratory behaviors from these pluripotent cells are highly similar to those seen with GMP-derived pluripotent cells. Time stamps are in days:hours:minutes format. Bars = 50 μm.
sc-12-0650_sm_SupplMovie9.wmv2035KMovie S9. Time-lapse image series at 15 minutes intervals, revealing reprogramming in the presence of an shRNA targeting E-cadherin. GMPs were transduced with OSKM together with a lentivirus expressing E-cadherin shRNA plus tdTomato. Red fluorescence indicates shRNA expressing cells. At the beginning, the large blue arrow points to a single cell that gave rise to all the later cells marked by arrows. The two yellow flashes indicate two times of cell division in this traced lineage. Out of the 4 daughter cells from these 2 divisions, only one of the lineages is marked out for easier view. All the cells from the later divisions are marked by blue arrows. With each cell division, the arrow becomes one size smaller. Note the absence of the 2- cell dumbbell intermediate as well as compaction among the cells. Weak GFP+ (seen as a yellowish hue), loosely scattered cells were present at the end of image acquisition. Overlayed images of DIC, GFP and tdTomato channels are shown. Time stamps are in hours:minutes format. Bar = 100 μm.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.