Spatio-temporal analyses ofOCT4expression and fate transitions in human embryonic stem cells

Abstract

OCT4 is one of the transcription factors required to maintain an undifferentiated state in human embryonic stem cells (hESCs). Thus, it is crucial to understand how OCT4 transcription is regulated both at the single-cell and colony level. Here we analyse the changes of OCT4-mCherry intensity expression in hESCs in the presence and absence of the BMP4 morphogenetic protein.We show that OCT4 expression is dynamic, reaching a maximum response 10 h after BMP4 treatment. We obtain the stationary probability distributions that govern the hESCs transitions amongst the different cell states in the presence/absence of BMP4 and establish the times at which the hESCs, that lead to differentiated and pluripotent cells, cluster in the colony. Furthermore, by quantifying the similarities between the OCT4 expression amongst neighbouring hESCs, we show that hESCs express, on average, similar values in their local neighbourhood within the first two days of the experiment and before BMP4 treatment. These results are relevant for the development of mathematical and computational models of adherent hESC colonies. Introduction 1 Human pluripotent stem cells (hPSCs), encompassing human embryonic stem cells 2 (hESCs) and human induced pluripotent stem cells (hiPSCs) self-renew indefinitely while 3 maintaining the property to give rise, under differentiation conditions, to almost any cell 4 type in the human body [1,2]. The scientific viewpoint on self-renewal and differentiation 5 of hESCs is established in a regulatory network whose components are a core set of 6 pluripotency transcription factors (TFs) expressed to maintain self-renewal and suppress 7 differentiation [3,4]. Amongst the most important TFs that preserve the undifferentiated 8 1/21 state in hESCs are NANOG, OCT4 and SOX2 [3, 5]. During development, these TFs 9become expressed at different levels and initiate differentiation towards specific cell 10 lineages following signalling cues [6]. 11 Several experiments have been performed to quantify the behaviour and joint influence 12 of each TF in the pluripotent cell [7][8][9][10][11]. Their results indicate that the expression of the 13 TFs proteins are highly variable both at the single-cell (time) and colony-level (space) 14 and are subjected to intrinsic noise due to a finite copy of genes, interactions at the 15 molecular level or due to randomness present in the extracellular environment [12][13][14]. 16 Thus, heterogeneity and stochasticity are inherent properties of pluripotent stem cell 17 populations [13,15,16], that hinder their clonal expansion in culture [17][18][19], but in vivo 18 promote the regionalisation and specification of the early blastocyst stage [20]. 19OCT4, acting in conjunction with other core members of the pluripotent regulatory 20 network, (e.g. SOX2, NANOG), activates both protein-coding genes and non-coding 21 RNAs necessary to maintain pluripotency [9]. Furthermore, besides being an essential 22 TF for the pluripotent state, OCT4 is also key to induce the reprogramming of somatic 23 cells...