In the mammalian genome, the clustered protocadherin (cPcdh) locus is a paradigm of stochastic gene expression with the potential to express a different cPcdh combination in every neuron. Here, we report a limited version established during the transition from the naive to the primed states of human cell pluripotency that reduces by orders of magnitude the combinatorial potential in the cPcdh locus. It is a chromatin-based mechanism that increases the frequency of stochastic selection of a subset of cPcdh promoters upon differentiation to fetal-like neurons in monolayers, organoids, or the rat spinal cord. Signs of similar preferential selections can be observed in the brain throughout fetal development, disappearing after birth, but not in a condition of delayed maturation such as Down Syndrome. We therefore propose that pluripotent cells impose a pattern of limited cPcdh diversity on neurons that increases the likelihood of cPcdh repetition until these cells acquire adult-like maturation.
Overall design: Lines/sublines: The hiPSC1-8 sublines were generated from single cells of the parent CVB hiPSC line. Six of these sublines were genetically modified (hiPSC2-7) and two were cases of failed genome editing (hiPSC1 and 8); please see Supplementary Table S1 in the associated publication for more details. Initially, these modifications should be irrelevant to the study of the cPcdh locus. The. hiPSC1 1.1-6 sublines were generated from the hiPSC1 subline and all underwent a process of genome editing, which again should be irrelevant to the study of the cPcdh locus (see Supplementary Table S1 in the associated publication for more details). The hiPSC 1.7-1.9 sublines were also generated from single cells of the parent CVB hiPSC line without undergoing any genome editing protocol. The hiPSC 1.13-15 sublines were generated from single cells of the parent CVI hiPSC line also without undergoing any genome editing protocol. The parent CVB and CVI lines were generated in the same reprogramming process and corresponded to two independent colonies. The hESC 1.7-1.9 sublines were generated from single cells of the parent HUES9 hESC line without undergoing any genome editing protocol. The HUES9 hESC 1.8 1.1-8 sublines were generated from single cells of the HUES9 1.8 subline after a process of primed-to-naive-to-reprimed conversion. All hiPSC/hESC lines/sublines were cultured in the primed state unless indicated (when indicated, some lines/sublines were cultured under naive-inducing conditions (5iLA protocol, and when returned to the primed state, the conditions were referred to as 're-primed'). For mouse cells, the 46C mESC 1.1-1.6 sublines were generated from single cells of the parent 46C mESC line. The R1 mESC 1.4/1.7 and 2.10/2.13 sublines were generated from single cells of the parent R1 mESC line. The R1 EpiSC 1.1-1.15 sublines were generated from single cells of the parent R1 mESC line after conversion to EpiSCs. The parental R1 mESC and EpiSC populations have also been included here. ChIP-seq data: All ChIP-seq experiments were performed in pluripotent stem cells (hiPSCs/hESCs/mESCs/EpiSCs). Most profiles were based on H3K4me3, but many others also on CTCF, Rad1, H3K4me2, H3K9me3, H3K9ac, H2A.Z, REST/NRSF, SIN3A, and JMJD2A. RNA-seq data: triplicate differentiation replicates of neurons generated from NPCs derived from hiPSC1-8 (n=24); cortical neurons generated from hiPSC1/3/5 (n=3); 10-month-old cortical organoids generated from the CVB line (n=1, pool of organoids); and primed, naive, and re-primed HUES9 1.8 subline (n=1 each). scRNA-seq data (SMART-seq+NEXTERA; full transcript): 79 single cells of neurons generated from NPCs of the hiPSC1 line (54 to 71), hiPSC2 (1 to 51), hiPSC6 (88 to 96), and hiPSC7 (72 to 87). The libraries were sequenced twice, in one case the libraries from all cells were pooled before cleaning and measuring DNA concentration, whereas in the other case the libraries were pooled after cleaning. and measuring DNA concentration (so equal amounts were mixed).
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