Study On Self - Renewal And Differentiation Of Embryonic Stem Cells

The thesis contains two parts of research projects.Part1:The function of miR23a～27a～24 in embryonic stem cell differentiationEmbryonic stem cells (ESCs) retain unlimited self-renewal potential in tissue culture and their propagation is characterized by a short cell cycle. Under appropriate conditions in vitro, ESCs can be induced to differentiate into all somatic cell types. These lineage commitments involve the silencing of self-renewal program and the activation of lineage-specific programs. Accumulating evidence reveals that microRNAs (miRNAs) play important roles in control of pluripotent stem cell state. ESCs lacking of key enzymes in the miRNA biogenesis pathway, such as Dicer or Dgcr8, show deficiency in self-renewal and differentiation. A number of miRNAs have been reported to participate in regulating ESC self-renewal and differentiation. However, miRNAs which can suppress self-renewal and may contribute to lineage commitment of ESCs have not been fully addressed.In previous research, we have confirmed that miR-27a and miR-24 directly target the critical pluripotency transcription factors and signal transducers to suppress ESC self-renewal program. In addition, the efficiency of iPSC generation is improved evidently when miR-27a or miR-24 is suppressed in mouse embryonic fibroblasts (MEFs). In consideration of important roles of miR-27a and miR-24 in ESC differentiation and somatic cell reprogramming, we are curious about whether miR-27a and miR-24 are indispensable for ESC differentiation. Five miRNAs (miR-23a,23b,27a, 27b,24) constitute mouse miR-23-27-24 clusters, which locate on two chromosomes as miR-23a cluster and miR-23b cluster separately. Their analogous functions in ESCs and similar sequence at the seed region suggest that the members may be functional compensative or redundant. So we generated miR-23a cluster and/or miR-23b cluster bi-allelic knockout V6.5 ESC clones using the newly developed powerful CRISPR/Cas9 genome editing system. The ESC clone carrying all four alleles deletions and one ESC clone having miR-23a cluster homologous knockout were selected for further study and named as DKO and KO ESC clones separately. PCR assay and further sequencing showed that both of the two clusters were bi-allelic knocked out in DKO ESC clone, while miR-23a cluster alone was ablated in KO ESC clone. All mature miR-23, miR-27 and miR-24 were undetectable in DKO ESC clone, while in KO ESC clone, mature miR-23b and miR-27b were normally expressed and miR-24 was reduced. Both KO and DKO ESCs were morphologically normal and expressed ESC-specific markers. Analyses of the potential off targets regions showed that none of the 19 predicted sites were truly modified by CRISPR/Cas9 in both DKO and KO ESC clones.We use EB formation to assess the differentiation capacity of the knockout ESCs. The up-regulation of Brachyury and Hand1 was delayed and was significantly lower than wild-type levels in EBs derived from DKO cells. By contrast, the expression of Nestin and Foxa2 was increased obviously. Meanwhile, the decline of Oct4 during EB formation was also delayed in the DKO cells. These results indicate that miR-23clusters are required for silence of ESC self-renewal program and formation of early mesoderm. To further demonstrate their indispensable roles in an exact lineage differentiation, we conducted cardiac differentiation of ESCs. None of DKO ESC derived EBs with spontaneous contraction was observed during the whole differentiation period. The morphology of DKO ESC-derived EBs was also significantly different from that of wildtype ESCs and showed many bulgy bubbles. These data correlated with the mRNA and protein expression level of the cardiac marker genes. Real-time PCR revealed markedly lower expression of Nkx2.5, Tbx5, a-MHC, b-MHC and cTnT in differentiating DKO and KO cultures compared with wild-type ESC derivations. Immunofluorescence analysis of differentiation cultures revealed that DKO ESCs could not differentiate and form typical myofi lament, with few expression of the three cardiac markers actinin, ANP and troponin I.Teratomas normally consist of a heterogeneous mix of differentiated cell types and usually are used to test the pluripotency of stem cells in vivo. In accordance with the defects in EB and cardiac differentiation, the teratomas produced with DKO ESCs grew slowly and were significantly lighter than that derived from wild-type ESCs. Some large undifferentiated regions indicative with Oct4 expression were detected in DKO and KO ESC-derived teratomas, which were occasional in the wild-type derivations. Moreover, teratomas derived from DKO ESCs largely appeared ectoderm structures especially neuronal rosette, while mesoderm cells such as adipocyte, cartilage and muscle cells were distinctly less than that of wild-type ESCs. Our results indicate that miR-23a and miR-23b cluster are indispensable for ESC differentiation especially the mesoderm differentiation in vivo. The detailed mechanism of their regulatory role in ESC mesoderm differentiation and cardiomyocyte differentiation will be studied further. In addition, no significant defects in differentiation of single miR-23a cluster knockout ESCs suggested functional redundancy of miRNA families or miRNA clusters. To may be effective to decipher function of miRNAs in biological processes. Our study reveals a feasible and efficient approach to remove all the redundant miRNAs in ESCs and demonstrates its usefulness in elucidating miRNA function.Part2:Establishment and application of a haploid embryonic stem cell mutant library Loss-of-function genetic screens using mammalian cell lines are valuable tools to identify genes required for many cellular processes. Genome-wide libraries of homozygous mutant cells are the substrates for conducting these screens, but the diploid nature of mammalian genomes hampers the generation of these mutants. Recently, a near-haploid human leukemia cell line and its derivatives have been used to isolate loss-of-function mutants and to perform several genetic screens successfully. This cell line has great potential for functional genomics, but the phenotypes that can be screened are limited to those accessible in transformed lymphoid cells.In 2011. two groups reported the isolation and establishment of haploid mouse ES cell lines, which resemble the diploid ES cells in their proliferation and pluripotent capacity. Haploid ES cells were also applied to generate the large pools of random mutations by combining with retroviral or transposon-based mutagenesis and to conduct a few small-or large-scale genetic screens. However, all these screens were performed in mixed pools of a vast number of mutant cells, indicating the null mutants of interest must be selected positively, either using a lethal drug or indirectly using an artificial cellular reporter construct. Thus, the application of haploid ES cells in genetic screening is still limited. An arrayed clonal library of haploid ES cells, each with a different gene mutation, could certainly facilitate recessive genetic screens.PB transposons have high chromosomal integration efficiencies in ES cells and show less bias for genomic hot spots than retroviral vectors. We electroporated haESC OG3 with PBDGTV and hyperPBase to generate four independent mutant libraries, HPL1-HPL4, containing approximately 60,000 puromycin-resistant colonies. The transposon copy number was determined by Southern blot analysis on dozens of individual clones randomly picked from HPL1 library. The majority of these cell clones contain a single-copy insertion of the transposon. To investigate the percentage of homozygous mutations in the mutant library, we identified the transposon-host junction in them and performed PCR analysis with site-specific primers separately. Of the 35 individual cell lines analyzed, thirty-one clones (>85%) carried homozygous insertions. To explore the genomic coverage of these libraries, we used Splinkerette-PCR combined with massively parallel sequencing to assess the overall mutants present in the mutation libraries. We could unambiguously identify 63,684 insertions and approximately 55% of insertions occurred in intragenic regions and promoter regions, encompassing 18,841 different genes in total. Taken together, these data indicate that HPLs is an eligible resource for the generation of arrayed homozygous mutant ES cell clones.The primary small-scale screens (92 clones/96-well plate,5 plates in total) were conducted in 96-well plates under two different differentiation-permissive conditions. Three days later, the majority of these cell clones had differentiated as WT haploid cells do, but individual mutant clones still showed ES cell colony morphology and expressed GFP at a relatively high level at one or two conditions. We extracted genomic DNA from these undifferentiated clones and identified the PB transposon insertion sites in them by Sp-PCR followed by sequencing. Nineteen insertions could be mapped to the reference mouse genome (GRCm38.p3), and 11 of them are located in encoding genes.Among these candidate "exit-from-pluripotency" factors, we noticed that two genes, Smgl and Sema5a were previously reported to be implicated in ESC differentiation and in embryonic developmental processes. SMG1, a PI3K-related kinase, was also found in a large-scale small interfering RNA (siRNA) screen for differentiation regulators. Smgl-deficient mice died in embryonic day 8.5 (E8.5) and knockdown of Smgl in ES cells also blocked their differentiation. The initial evidence for the role of Sema5 A (semF) in embryogenesis was demonstrated the expression of Sema5A in specific regions of mouse embryos, demarcating distinct compartments of the developing somites or the undifferentiated neuroepithelium. Sema5a-null mice died between E11.5 and E12.5, and the complexity of the hierarchically organized branches of the cranial cardinal veins was decreased. These results indicate an essential role of Sema5A during embryonic development. Obviously, an ongoing expansion of the arrayed mutant libraries could increase the readout of screening. Overall, this study provides a practical and versatile platform for a wide variety of phenotype-driven genetic screening studies...