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

Abbreviations

AD (Alzheimer’s disease); APOE (Apolipoprotein E); iPSCs (induced pluripotent stem cells); NSCs (neural stem cells)

Introduction

Apolipoprotein E (APOE) is a pleiotropic protein that plays an important role in lipid metabolism (Mahley & Rall, 2000) and is highly expressed in the brain mainly by glial cells (Boyles et al., 1985; Elshourbagy et al., 1985). Although the primary function of APOE is lipid transport, its expression is also found in other cell types outside the context of lipid metabolism in the brain (Liao et al., 2017). For example, a recent single-cell RNA sequencing study on human post-mortem Alzheimer’s disease (AD) brains showed that activated microglia (relevant to the disease state) express high levels of APOE unlike naïve microglia (relevant to healthy/homeostatic state) in the prefrontal cortex, indicating that APOE expression is associated with immune function (Mathys et al., 2019). Furthermore, neuronal APOE can also be expressed at high levels under stress conditions such as brain injury although APOE expression is normally low in healthy neurons (Mahley & Huang, 2012; Xu et al., 2006). Interestingly, APOE is highly expressed in nestin/glial fibrillary acidic protein (GFAP) double-positive neural stem cells (NSCs) in the adult hippocampus of mice, and one of the phenotypes characterised in APOE-null mice is the premature depletion of NSC pool in the hippocampus, suggesting that NSC maintenance requires APOE expression (Yang et al., 2011).

Although the existing literature suggest that APOE plays an important role in stem cell maintenance, one should note that the majority of these findings were generated from rodent models. Since NSCs obtained from different species have been shown to behave in fundamentally different ways (Mertens et al., 2013; Otani et al., 2016; Ray & Gage, 2006), characterisation of APOE expression in ‘human’ NSCs should be done prior to investigating its exact function. However, such evidence has not been reported to this date. To reduce this knowledge gap, we conducted a short study examining the expression pattern of APOE gene and protein in human induced pluripotent stem cells (iPSCs) undergoing neural induction in vitro. We found that gene expression is the highest in cells at the earliest stage of neural induction, whereas protein expression becomes more localised intracellularly, indicating that APOE expression pattern changes according to the differentiation state of cells.

Methods

A list of materials used in this study is presented in Table 1.

Results

To characterise the expression of APOE in human stem cells undergoing neural induction, an iPSC line derived from a neurotypical male with APOE3 homozygous genotype (CTR_M3_36S cell line) (Figure 1; (Lee, 2020b)) (Deans et al., 2017) were differentiated into neural lineages. Genotyping of CTR_M3_36S was performed using the method developed by Henderson and colleagues (Henderson et al., 2002), and CTR_M3_36S was confirmed to be homozygous for APOE3 by comparing the data with that of an APOE3 homozygous cell line (CTR_M1_04) that reported by Henderson and colleagues (Henderson et al., 2002, Figure 1). Neural induction into dorsal forebrain progenitors was performed using modified dual SMAD inhibition protocols (Cocks et al., 2014; Deans et al., 2017; Kathuria et al., 2018; Yu et al., 2014), where combinations of small molecule inhibitors were used to inhibit bone morphogenetic protein (BMP), transforming growth factor (TGF)-β, Wnt/β-catenin, and sonic hedgehog signalling pathways from D0 to D7 of neural induction (Figure 2A).

Figure 1. APOE genotyping of the cell line used in this study.

CTR_M3_36S human iPSC line derived from a neurotypical male is homozygous for APOE3 (denoted as M3 in this figure). CTR_M1_04 human iPSC line that was known to be homozygous for APOE3 was used as control (denoted as M1 in this figure). HhaI-digested PCR amplicons were run on a 3% agarose gel, and the band loci were compared with the data previously reported by Henderson and colleagues who developed this genotyping method (Henderson et al., 2002). The band loci for both CTR_M3_36S and CTR_M1_04 lines match with the homozygous APOE3 data reported by Henderson and colleagues (see Figure 1 of Henderson et al., 2002).

Figure 2. APOE gene expression changes according to the differentiation state of cells during in vitro directed differentiation.

A) Schematic diagram of directed differentiation. CTR_M3_36S iPSCs were maintained in stem cell maintenance medium after replating (D-2/-1). On D0 neural induction began by changing the stem cell maintenance medium to neural induction medium. N2:B27 was used from D8 onwards. Medium was changed every 24 hrs throughout the entire differentiation period. Neural passaging 1, 2, and 3 were carried out on D7, D12, and D15/16, respectively. Total RNA extraction was made on cells that were not used for neural passaging on D7, D12, D15/16, and D18/19. Neural induction medium composition for each differentiation lineage and N2:B27 medium composition are also shown. B) APOE gene expression is reduced along neural induction regardless of lineage. Real-time qPCR was performed on CTR_M3_36S iPSCs undergoing directed differentiation at D7, D12, D15/16, and D18/19. APOE expression was normalised to that of GAPDH. D7 samples were used as reference samples for each lineage. One-way ANOVA with Bonferroni correction. n = 3. Mean (bars) with S.E.M. (error bars) shown. **** ANOVA p-value < 0.0001. ns: non-significant after Bonferroni correction. DSi: dual SMAD inhibition. DS-Wi: DSi plus Wnt/β-catenin inhibition. DS-WHi: DS-Wi plus sonic hedgehog inhibition.

Gene expression analysis revealed that APOE expression was the highest at D7, and the drastic down-regulation of APOE from D7 > to D18/19 was observed regardless of the combination of small molecule inhibitors used from D0 to D7 (p < 0.0001) (Figure 2B, underlying data (Lee, 2020a)). Immunocytochemistry showed that D12 and D18/19 cells expressed SRY-box transcription factor 2 (SOX2), a NSC marker, and T-Box Brain Protein 2 (TBR2), a neural progenitor cell (NPC) marker, respectively, for all combinations of small molecule inhibitors used from D0 to D7. Qualitative analysis of immunocytochemistry results revealed that ApoE became more localised to the intracellular region at D18/19 compared to D12 (Figure 3; (Lee, 2020c; Lee, 2020d)).

Figure 3. APOE localisation pattern becomes more intracellular with in vitro directed differentiation.

APOE protein is more localised intracellularly in differentiated cells. (Left) Representative images of cells at D12 and D18/19 expressing SOX2 (NSC marker) and TBR2 (NPC marker), respectively. Insets show images of SOX2/TBR2 in green and APOE in red. Scale bars indicate 50 µm unless stated otherwise. (Right) Quantification of SOX2-/TBR2-positive cells and intracellular APOE-positive cells amongst SOX2-TBR2-positive cells. DSi: dual SMAD inhibition. DS-Wi: DSi plus Wnt/β-catenin inhibition. DS-WHi: DS-Wi plus sonic hedgehog inhibition.

Discussion

Unlike the existing animal models of APOE deficiency and humanised APOE expression where genetic modifications were introduced globally (whole body) rather than specifically to NSCs, the in vitro model used in this study allowed us to examine APOE expression pattern exclusively in stem cells that were pushed towards the neural lineage. Our findings demonstrate that in cells at the earliest stage of neurodevelopment, 1) human APOE gene expression is high, and 2) APOE protein is not clearly localised at the intracellular region. Various combinations of small molecule inhibitors did not alter these patterns of expression.

Although further investigations will be needed to understand the exact role of APOE in neurodevelopment, the existing literature seems to suggest that APOE can be both downstream and upstream of stem cell maintenance. For example, several chromatin precipitation studies have shown that POU class 5 homeobox 1 (POU5F1), SOX2, Kruppel like factor 4 (KLF4), MYC proto-oncogene (MYC) and Nanog Homeobox (NANOG) all bind to the promoter region of APOE, suggesting that APOE expression could be directly regulated by such stem cell maintenance factors (ENCODE Project Consortium, 2004; ENCODE Project Consortium, 2011; Kim et al., 2008; Lachmann et al., 2010; Liu et al., 2008; Marson et al., 2008). However, other evidence suggests that APOE itself could be a direct regulator of cell fate determination. Meyer and colleagues (Meyer et al., 2019) showed that changing the APOE genotype from ε4 (AD risk factor) to ε3 (neutral genotype) in human NPCs can suppress premature neuronal differentiation and maturation via increasing the transcription repressor activity of RE1 silencing transcription factor (REST). Interestingly, APOE mRNA levels were lower in ε4 NPCs compared to ε3 NPCs, suggesting higher APOE gene expression is indeed likely to be associated with the undifferentiated state of NPCs. As a follow-up to our findings and the existing literature, we propose that further investigations should be carried out to elucidate the role of APOE in stem cell maintenance. For example, one could examine whether prolonged expression and/or overexpression of APOE gene in human NPCs can suppress further differentiation in these cells.

In this study, qualitative analysis was performed on APOE immunocytochemistry results. As the cells became more differentiated from NSCs to NPCs, APOE localisation pattern became more clearly intracellular. To validate this observation, co-localisation analysis with cytoskeletal proteins (such as Tubulin beta-3 chain and Microtubule-associated protein 2) or with plasma membrane proteins (such as N-Cadherin) will be needed in future investigations. Furthermore, APOE has been shown to exist in both secreted and intracellular forms in the existing literature (Huang & Mahley, 2014). Therefore, it will also be important to examine which form of APOE is produced at each differentiation stage. It is possible that more APOE is secreted in undifferentiated cells compared to differentiated cells, which may not be fully captured using immunocytochemistry techniques performed on fixed cells. Interestingly, Gan and colleagues previously reported that APOE is indeed secreted by NSCs as well as NPCs, and secreted APOE was found to play a vital role in regulating NSC survival and neurosphere formation (Gan et al., 2011). Further investigations on changes of secreted and intracellular protein levels throughout neural differentiation will be able to clarify whether cells indeed produce different forms and levels of APOE depending on its differentiation state. This will in turn provide more definitive clues to whether APOE plays a stage-dependent role in neurodevelopment.

In this study, qualitative analysis was performed on APOE immunocytochemistry results. As the cells became more differentiated from NSCs to NPCs, APOE localisation pattern became more intracellular. To validate this observation, however, additional experiments with a more direct quantitative approach should be conducted. For example, APOE protein levels in various subcellular compartments could be measured and compared by performing Western Blot. Since APOE has been shown to exist in both secreted and intracellular forms (Huang & Mahley, 2014), it will be interesting to see which form of APOE is produced at each differentiation stage. It is possible that more APOE is secreted in undifferentiated cells compared to differentiated cells, which may not be fully captured using immunocytochemistry techniques performed on fixed cells. Interestingly, Gan and colleagues previously reported that APOE is indeed secreted by NSCs as well as NPCs, and secreted APOE was found to play a vital role in regulating NSC survival and neurosphere formation (Gan et al., 2011). Therefore, further investigations on secreted and intracellular APOE using quantitative approaches will be able to clarify whether cells indeed produce different forms and levels of APOE depending on its differentiation state. This will in turn provide more definitive clues to whether APOE plays a stage-dependent role in neurodevelopment.

One limitation of this study is that the time-dependent changes of differentiation markers such as SOX2 and TBR2 were not examined alongside APOE. It is worth noting, however, that TBR2 was shown to be capable of suppressing SOX2 expression during differentiation of NSCs to NPCs (Hodge et al., 2012). Given this information, it is unlikely that TBR2-positive cells observed in this study at D18/19 will simultaneously express high levels of SOX2. However, time-dependent changes of various markers of differentiation would add further validity to our observations and unequivocally clarify whether APOE expression is indeed correlated with the differentiation state of the cells. Another limitation of this study is that the exact locus of APOE expression could not be examined in detail using a standard epifluorescence microscope in this study. High-resolution microscopy techniques (such as confocal microscopy) would have been more ideal to identify the accurate loci of APOE expression and overcome the challenges of imaging densely packed cells at the earliest stages of neural induction (D0–D7). Further investigations with improved imaging capacity will therefore allow us to characterise APOE during the earlier stages of neural induction and hint at potential mechanisms underlying its role in neurodevelopment.

Since NSCs derived from iPSCs in vitro may not fully resemble the developmental and postnatal NSCs found in vivo, APOE expression should be further investigated in animal models of brain development as well. The most direct evidence of in vivo APOE expression in NSCs to this date comes from a study by Yang and colleagues, where Nestin-positive NSCs in the mouse developing dentate gyrus was isolated using fluorescence-activated cell sorting, and APOE expression was examined from as early as postnatal day 7 (P7) (Yang et al., 2011). NSCs at P7 had low expression of APOE which increased with the age of mice, and the deletion of APOE had detrimental effects on the maintenance of stem cells in the dentate gyrus. Although these findings clearly demonstrate the importance of APOE in brain development, the study had limitations in that prenatal NSCs were not examined, and functional studies of APOE were based on global rather than conditional knockouts. Furthermore, Yang and colleagues’ data cannot be directly compared with our dataset due to species difference and the lack of detailed characterisation of NSCs in this study. To address this knowledge gap, more data from both in vitro and in vivo samples derived from various species should be generated and compared against each other. We hope that our focused study has laid a strong foundation to such collaborative investigations that may be conducted in the future.

In conclusion, we report that human APOE gene expression levels are highly correlated with the undifferentiated state of cells during directed differentiation in vitro, and ApoE protein is localised more in the intracellular region in cells at later stages of differentiation. Combining our observations and previous evidence reported in the literature, we speculate that APOE has an important role in stem cell maintenance and propose that further investigations should be carried out to validate our findings including methods that were not employed in this study. Moreover, it would be interesting to examine the exact underlying mechanisms such as 1) whether APOE is an upstream or downstream factor of stem cell maintenance, and 2) whether APOE4 genotype and APOE loss-of-function would produce similar phenotypes.

Data availability