Ratiometric distribution of OCT4 during stem cell division controls the balance between self-renewal and differentiation

A fundamental challenge in cell biology is to understand how an individual cell commits to a particular fate1–3. A classic example is the fate decision between self-renewal and differentiation, which plays an essential role in the biology of human embryonic stem cells (hESCs)4. Despite significant advances in our understanding of development of the human embryo5,6, it is still unclear how, and when, an individual stem cell makes the decision to differentiate. Here, we used time-lapse fluorescence microscopy to capture differentiation of hESCs to trophoblast—the first cell fate decision during mammalian development. By tracing the histories of both self-renewing and differentiating cells, we found that each population displayed distinct levels of the pluripotency factor OCT4 long before they were exposed to a differentiation stimulus. The levels of OCT4 were lineage dependent; however, each mother cell distributed unequal levels of OCT4 to its daughter cells randomly during cell division. The resulting ratio of OCT4 between daughter cells— established immediately after division—determined downstream fates: cells receiving a greater ratio of maternal OCT4 showed sustained increases in OCT4 and a reduced capacity to differentiate. We propose a simple formula, pdaughter = (pmother)r, that successfully predicts the probability that a daughter cell will differentiate based on its mother cell’s differentiation probability and its inherited ratio of OCT4. Our study reveals that the balance between self-renewal and differentiation is altered by the ratiometric distribution of OCT4 during cell division. These findings imply that a stem cell’s fate is already largely determined by the time the cell is born.

bone morphogenetic protein 4 (BMP4) 9 . After 24 h of BMP4 treatment, quantitative immunofluorescence (IF) reveals two emerging populations of cells: a pluripotent population with low CDX2 expression that retains the ability to differentiate into other cell types (Extended Data Fig. 1); and a differentiating population of cells with reduced OCT4 expression, increased CDX2 expression, and enlarged morphology (Figs. 1b-c). BMP4-treated hESC colonies adopted a radially symmetric pattern of differentiation that resembles the human gastrula 10 (Extended Data Fig. 2). This spatial configuration-with pluripotent cells located at the interior of the colony and differentiated cells near the periphery-was recently shown to arise from a gradient of receptor polarization and diffusible factors 6 . However, the self-organization of a seemingly uniform starting population still raises the fundamental question: how does a single stem cell choose between self-renewal and differentiation?
To address this question, we developed a fluorescent reporter system to monitor expression of human OCT4, a canonical marker of the pluripotent state 11 , in live hESCs. We used CRISPR-mediated genome editing to fuse a monomeric red fluorescent protein (mCherry) to the endogenous OCT4 protein in WA09 (H9) hESCs and isolated a clonal population of single-allele knock-in reporter cells ( Fig. 1d and Extended Data). The OCT4-mCherry fusion protein showed accurate co-localization with the endogenous OCT4 protein; similar degradation kinetics; and the same chromatin binding pattern near the promoters of OCT4 target genes (Extended Data Fig. 3). Moreover, cells bearing the OCT4-mCherry reporter were competent to differentiate into multiple differentiated cell types (Extended Data Fig. 4), and time-lapse imaging did not alter their proliferation characteristics (Extended Data Fig. 5). For each cell, we calculated a single OCT4 expression level by averaging OCT4-mCherry intensity over its cell cycle duration (Fig. 1e-f). In addition, we examined the time-series profile of OCT4 dynamics for individual cells and found that the majority of hESCs (68%) displayed sporadic bursts of OCT4 expression that lasted ~1.5 h, with some cells showing as many as 7 bursts (Fig. 1g). Finally, we calculated individual cell cycle durations, which ranged from 10-24 h with a mean duration of 14.6 h (Fig. 1h), consistent with the reported population doubling time of ~16 h 12 . Thus, our reporter system enabled the reliable analysis of single-cell OCT4 dynamics in hESCs and revealed considerable heterogeneity in untreated stem cells.
With this system in place, we set out to capture the fate decisions of hESCs in real time.
First, we performed time-lapse fluorescence imaging of H9 OCT4-mCherry hESCs for 42 h under basal conditions (Fig. 2a). We then treated these cells with 100 ng/mL BMP4 to induce differentiation while continuing to monitor their responses. Within 12 h of treatment, each cell began to follow one of two distinct fate paths: sustained accumulation of OCT4; or a precipitous decrease in OCT4. After 24 h, cells were fixed and stained for expression of CDX2 to determine their final differentiation status (Fig. 2b). We imposed a strict cutoff to classify each cell as either pluripotent or differentiated based on its OCT4 and CDX2 expression levels. By fitting the data in Fig. 2b to a 2-component Gaussian distribution (Extended Data Fig. 6), we selected only those cells that belonged exclusively to either the pluripotent distribution ( < 0.01) or the and "second cousin" cells ( Fig. 3b). Both sister and cousin cells, but not second cousins, were more similar than randomly paired cells, indicating that similarity in OCT4 levels can persist for at least two cell cycle generations 16 . Suspecting that each cell division event introduced variability in OCT4 levels, we detected a strong correlation between the number of cell divisions and the difference in OCT4 levels between all pairs of cells (Extended Data Fig. 7). Thus, OCT4 levels are heritable from mother to daughter cell, but each division event introduces incremental variability in OCT4 expression levels.
Close examination of cell division events at high temporal resolution revealed the precise time during which variability in OCT4 levels arises during cell division. As cells entered mitosis, OCT4 became strongly associated with the condensed chromosomes ( Fig. 3c, left panel). This compacted state persisted throughout anaphase until the two daughter chromatids could be visibly distinguished. We used this first time point-before cytokinesis was complete-to quantify the levels of OCT4 in both newly born daughter cells (Fig. 3c, center panel).
Comparison of OCT4-mCherry intensity between daughter cells revealed that the distribution of OCT4 was not perfectly symmetric but instead adopted a bell-shaped distribution that was centered around a mean ratio of 1 ( = 1/1) (Fig. 3d). Approximately 38% of divisions produced daughter cells with = 5/6 or a more extreme ratio; 12% of divisions resulted in = 3/4 or a more extreme ratio; and 3% of division events resulted in = 1/2 or a more extreme ratio.
These differences in OCT4 ratios were not due to measurement error because the distribution of between sisters remained consistent for several hours after cytokinesis both before and after BMP4 treatment (see below). In addition, we determined the half-life of OCT4 to be ~8 h (Extended Data Fig. 3), making it unlikely that asymmetric ratios were due to stochastic differences in protein degradation during the first 5 minutes of daughter cell lifetime. Moreover, OCT4 ratios were not correlated with nuclear area or radial position within the colony (Extended Data Fig. 8). Thus, significant differences in OCT4 protein levels between sister-cell pairs were established at the moment of cell division.
We next tested whether the ratio of OCT4 inherited by a particular daughter cell influenced its downstream behavior. By comparing OCT4-mCherry intensities between sister chromatids at the moment of cell division, we found that the inherited ratio of OCT4 established within the first 5 minutes of daughter cell separation was predictive of the final OCT4 level in each cell ( Fig. 3e). Daughter cells receiving the larger proportion of OCT4 ( > 1) showed increased levels of OCT4 relative to the mother cell, whereas daughters receiving the smaller proportion of OCT4 ( < 1) showed permanent decreases in OCT4. This trend was also observed after differentiation (Fig. 3e, right panel) and became stronger as more time elapsed after cell division (Extended Data Fig. 9). Furthermore, the ratio of OCT4 immediately after division predicted the difference in final OCT4 expression between sister cell pairs (Extended Data Fig. 10). Thus, the ratio of OCT4 established immediately after cell division-before the nuclear envelope is formed-determined the amount of OCT4 that was maintained throughout the lifetime of a cell.
To summarize thus far, differences in OCT4 expression levels arise through asymmetric distribution of OCT4 to daughter cells ( Fig. 3c-e). Precise levels of OCT4 are transmitted from mother to daughter cells as reflected by both the similarity among cells that share a common lineage ( Fig. 3a-b) as well as the observation that most progenitor cells (89%) give rise to a group of cells with the same fate (Fig. 2a). Moreover, OCT4 levels are strongly predictive of cell fate decisions (Fig. 2c). Taken together, these results suggest that a cell's probability of differentiation has both a heritable component-transmitted through the mother cell-and a random component that depends on the inherited ratio of OCT4 at the moment of cell division.
This behavior can be expressed in a simple formula (Fig. 4a) in which the probability that a daughter cell will differentiate, ℎ , is equal to the probability that its mother cell will give rise to differentiated cells, ℎ , raised to the power of , the inherited ratio of OCT4: Here, ℎ can represent either the probability that a daughter cell has differentiated (as shown in Fig. 2b) or the probability that the daughter cell will give rise to differentiated daughter cells (i.e., pro-differentiated, green circles in Fig. 2c). As such, Equation 1 is a recursive formula that can be used to assign a probability to every cell in a lineage tree. By fitting Equation 1 to our measured values of and , we assigned a differentiation probability to each cell in a proliferating population including those cells that were observed before the differentiation stimulus was given (Fig. 4b).
As expected, pro-pluripotent cells had ratio while the pro-differentiated population showed the least sensitivity ( Fig. 4d).
Mechanistically, this increase in sensitivity to OCT4 at intermediate levels is consistent with the finding that the OCT4 and CDX2 transcription factors are reciprocally inhibitory 8 . Such a "double-negative feedback loop" would lead to a bistable system 17 in which a small change in OCT4 levels drives cells strongly toward either pluripotency or differentiation (Fig. 4e).
In conclusion, we developed an endogenous fluorescent reporter for the canonical pluripotency factor OCT4 to capture differentiation of human embryonic stem cells in real time.
We found that the decision to differentiate to trophoblast is largely determined before the differentiation stimulus is presented to cells and can be predicted by a cell's preexisting OCT4 levels, bursting frequency, and cell cycle duration. These results in human cells harmonize with studies of mouse ESCs in which differences in OCT4 expression 14,18-20 and degradation kinetics 21,22 are associated with different developmental fate decisions. However, we identify a precise window of time during which these differences arise by showing that the strongest predictor of trophoblast fate-OCT4 levels-is established during cell division through ratiometric distribution of OCT4 to daughter cells. Thus, a cell's probability to differentiate is a mixture of both heritable and random components. These observations challenge a prevailing view of cell fate decisions by suggesting that the fate of a cell is already largely determined by the time it emerges from its mother cell. As such, the concept of pluripotency-currently defined as the capacity of a given cell to undergo differentiation-may be more properly comprehended as a heritable trait that applies to an entire lineage of proliferating stem cells.       Fig. 6). d, A uorescent mCherry coding sequence was introduced into endogenous OCT4 locus of H9 hESCs using CRISPR-mediated homologous recombination. e, Filmstrip of OCT4 dynamics in an undi erentiated hESC throughout its cell cycle duration. Yellow outlines indicate the region used to quantify mean nuclear uorescence intensity. f, Distribution of OCT4 levels in individual hESCs. A single OCT4 level was quanti ed for each cell by averaging the mCherry intensity over the lifetime of the cell. g, Single-cell traces of OCT4 signaling. The length of each cell's trace indicates its cell cycle duration. h, Distribution of cell cycle durations for 120 hESCs.  Fig. 6). hESCs that could be assigned to either distribution with >99% con dence (red and green dots, p di < 0.01 or p di > 0.99) were considered for pro-fate analysis. Cells that did not reach this threshold (gray dots) were ot used to determine pro-fate. Because cells in the nal frame were assigned to only pluripotent or di erentiated categories, pro-mixed cells yellow traces in panel a) do not persist to the nal frame.   was used recursively to assign a di erentiation probability to each cell based on the nal measured di erentiation probabilities p di (see Fig. 2b) and the inherited ratio of OCT4 for each cell. Cells are grouped according to their experimentally determined pro-fate. c, Theoretical predictions of Equation 1 for multiple values of maternal di erentiation probability p mother . The line is steepest at p mother = 1/e ≈ 0.368. d, Sensitivity of pro-fate groups to inherited OCT4 ratio. The change in nal OCT4 levels is plotted against the ratio of OCT4 inherited in pro-pluripotent, pro-mixed, or pro-di erentiated populations. e, Model for stem cell fate decisions altered by asymmetric distribution of OCT4 during cell division. ** p < 0.005, *** p < 0.0005. R, Pearson correlation; P, P-value; s, slope of best t line representing sensitivity to r. Change in OCT4 level   whereas cousin cells are separated by 3 division events. As the number of division events increases, the difference in OCT4 levels off because each division event is equally likely to increase or decrease the difference in OCT4 levels between two given cells.   OCT4 levels between sister-cell pairs were calculated as described in Fig. 1 of the main text.