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Apolipoprotein E expression pattern in human induced pluripotent stem cells during in vitro neural induction.

ABSTRACT: Apolipoprotein E (APOE) is a multifunctional protein that plays significant roles in important cellular mechanisms in peripheral tissues and is as well expressed in the central nervous system, notably by adult neural stem cells (NSCs) in the hippocampus. Evidence from animal studies suggest that APOE is critical for adult NSC maintenance. However, whether APOE has the potential to play a similar role in human NSCs has not been directly investigated. To address this question, we conducted a focused study characterising APOE gene and protein expression in an in vitro model of neural differentiation utilising human induced pluripotent stem cells. We found that APOE gene expression was dramatically decreased as the cells became more differentiated, indicating that APOE expression levels reflect the degree of cellular differentiation during neural induction. Furthermore, qualitative analysis results of immunocytochemistry showed that intracellular localisation of APOE protein becomes more pronounced as neural differentiation progresses. Taken together, our findings suggest a potential role for APOE in human NSC maintenance and justify further investigations being carried out to understand whether changes in APOE levels can directly impact the neurogenic capacity of human stem cells.

SUBMITTER: Lee H

PROVIDER: S-EPMC7355222 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Apolipoprotein E expression pattern in human induced pluripotent stem cells during <i>in vitro</i> neural induction.

Lee Hyunah H Nowosiad Paulina P Dutan Polit Lucia M LM Price Jack J Srivastava Deepak P DP Thuret Sandrine S

F1000Research 20200512

Apolipoprotein E (APOE) is a multifunctional protein that plays significant roles in important cellular mechanisms in peripheral tissues and is as well expressed in the central nervous system, notably by adult neural stem cells (NSCs) in the hippocampus. Evidence from animal studies suggest that APOE is critical for adult NSC maintenance. However, whether APOE has the potential to play a similar role in human NSCs has not been directly investigated. To address this question, we conducted a focus ...[more]

PMID: 32685135

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Project description:IntroductionNeural induction of human induced pluripotent stem cells represents a critical switch in cell state during which pluripotency is lost and commitment to a neural lineage is initiated. Although many of the key transcription factors involved in neural induction are known, we know little of the temporal and causal relationships that are required for this state transition.MethodsHere, we have carried out a longitudinal analysis of the transcriptome of human iPSCs undergoing neural induction. Using the temporal relationships between the changing profile of key transcription factors and subsequent changes in their target gene expression profiles, we have identified distinct functional modules operative throughout neural induction.ResultsIn addition to modules that govern loss of pluripotency and gain of neural ectoderm identity, we discover other modules governing cell cycle and metabolism. Strikingly, some of these functional modules are retained throughout neural induction, even though the gene membership of the module changes. Systems analysis identifies other modules associated with cell fate commitment, genome integrity, stress response and lineage specification. We then focussed on OTX2, one of the most precociously activated transcription factors during neural induction. Our temporal analysis of OTX2 target gene expression identified several OTX2 regulated gene modules representing protein remodelling, RNA splicing and RNA processing. Further CRISPRi inhibition of OTX2 prior to neural induction promotes an accelerated loss of pluripotency and a precocious and aberrant neural induction disrupting some of the previously identified modules.DiscussionWe infer that OTX2 has a diverse role during neural induction and regulates many of the biological processes that are required for loss of pluripotency and gain of neural identity. This dynamical analysis of transcriptional changes provides a unique perspective of the widespread remodelling of the cell machinery that occurs during neural induction of human iPSCs.

Project description:BACKGROUND:In mammals, specification of primordial germ cells (PGCs) is established in the early post-implantation embryo. The bone morphogenetic protein (BMP)-SMAD and WNT3-?-catenin signaling initiate the gene regulatory network for PGC specification. The activation of SOX17-BLIMP1 axis is critical for human PGC program. Moreover, EpCAM and INTEGRIN?6 were identified as surface markers of human PGC-like cells (PGCLCs) recently. However, the signaling mechanism for PGC specification in non-rodent mammals remains to be clarified. METHODS:We differentiated human induced pluripotent stem cells (hiPSCs) into PGCLCs in vitro in response to Activin A and BMP4. The percentage of EpCAM/INTEGRIN?6 double-positive cells (PGCLCs) was analyzed by flow cytometry. The expression of PGC genes was evaluated by qRT-PCR and immunofluorescence. The expression dynamic of multi-lineage genes during the differentiation process was evaluated by qRT-PCR. RESULTS:Under the stimulation for PGCLC induction, the embryoids derived from hiPSCs initiated significant upregulation of the early PGC genes (BLIMP1, TFAP2C, and NANOS3), but maintained low or no levels of DPPA3 and late PGC genes (DAZL and DDX4). The percentage of EpCAM/INTEGRIN?6 double-positive PGCLCs reached the highest at day 6 of induction. After pre-induction, the incipient mesoderm-like cells (iMeLCs) upregulated most of the mesoderm genes (EOMES, T, MSXI, RUNX2, and MIXL1). The differentiating embryoids showed high levels of key pluripotency genes, OCT4 and NANOG, but became negative for SOX2. In contrast to iMeLCs, the differentiating embryoids downregulated mesoderm genes RUNX2 and EOMES, and ectoderm gene PAX6, but increased the expression of endoderm gene SOX17. CONCLUSIONS:During PGCLC induction process in vitro, the differentiating embryoids not only activated the PGC-related genes, but also displayed complex regulation of pluripotency genes and multi-lineage genes. These results would be meaningful for future research investigating the regulation of human early germ line development.

Dataset Information

Apolipoprotein E expression pattern in human induced pluripotent stem cells during in vitro neural induction.

Publications

Apolipoprotein E expression pattern in human induced pluripotent stem cells during <i>in vitro</i> neural induction.

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