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n3:pmcid: PMC0
bibo:doi: 10.1038%2Fncb3104
n6:contains: _:vb38007497 _:vb38007506 _:vb38007531 _:vb38007540

Subject Item: _:vb38007497
rdf:type: n6:Section
dc:title: introduction
n6:contains: _:vb38007498 _:vb38007499 _:vb38007500 _:vb38007501 _:vb38007502 _:vb38007503 _:vb38007504 _:vb38007505

Subject Item: _:vb38007498
rdf:type: n3:Context
rdf:value: in vivo lineage tracing remains an important technique for the analysis of stem cell behavior, developments in primary stem cell culture have expanded the stem cell biologist's toolkit to include powerful, complementary in vitro assays >>1<<.
n3:mentions: n2:23744940

Subject Item: _:vb38007499
rdf:type: n3:Context
rdf:value: Lgr5high or Sox9low ISCs are capable of forming “enteroid” structures in vitro, demonstrating multipotency and self-renewal >>2<<-4. In vivo, ISCs are closely associated with Paneth cells (PCs), which function as niche cells and express soluble and insoluble factors associated with stemness, such as Wnt and Notch ligands 5, 6.
n3:mentions: n2:19329995 n2:17934449

Subject Item: _:vb38007500
rdf:type: n3:Context
rdf:value: In vivo, ISCs are closely associated with Paneth cells (PCs), which function as niche cells and express soluble and insoluble factors associated with stemness, such as Wnt and Notch ligands 5, >>6<<. PCs have been shown to increase the efficiency of enteroid formation by ISCs in vitro 6. However, these studies relied on the co-culture of hundreds of ISCs with hundreds of PCs, and may not reflect physiological normal conditions in
n3:mentions: n2:21113151

Subject Item: _:vb38007501
rdf:type: n3:Context
rdf:value: PCs have been shown to increase the efficiency of enteroid formation by ISCs in vitro >>6<<. However, these studies relied on the co-culture of hundreds of ISCs with hundreds of PCs, and may not reflect physiological normal conditions in single intestinal crypts, where much smaller numbers of ISCs (∼15) and PCs (∼8) interact 6-8.
n3:mentions: n2:21113151

Subject Item: _:vb38007502
rdf:type: n3:Context
rdf:value: However, these studies relied on the co-culture of hundreds of ISCs with hundreds of PCs, and may not reflect physiological normal conditions in single intestinal crypts, where much smaller numbers of ISCs (∼15) and PCs (∼8) interact >>6<<-8.
n3:mentions: n2:21113151 n2:20887898 n2:19228882

Subject Item: _:vb38007503
rdf:type: n3:Context
rdf:value: Additionally, the field currently lacks a robust assay to study ISCs at the clonal level, a tool that has driven the understanding of stem cell niches in the hematopoietic system and mammary glands >>9<<, 10. Array-based technologies are emerging as a powerful method to study the functional characteristics of single and/or small numbers of stem cells in the hematopoietic system, and hold similar promise for epithelial tissues such as the
n3:mentions: n2:16397499

Subject Item: _:vb38007504
rdf:type: n3:Context
rdf:value: Additionally, the field currently lacks a robust assay to study ISCs at the clonal level, a tool that has driven the understanding of stem cell niches in the hematopoietic system and mammary glands 9, >>10<<. Array-based technologies are emerging as a powerful method to study the functional characteristics of single and/or small numbers of stem cells in the hematopoietic system, and hold similar promise for epithelial tissues such as the
n3:mentions: n2:13776896

Subject Item: _:vb38007505
rdf:type: n3:Context
rdf:value: technologies are emerging as a powerful method to study the functional characteristics of single and/or small numbers of stem cells in the hematopoietic system, and hold similar promise for epithelial tissues such as the intestine >>11<<. In the present study, we describe a platform to study large numbers of single ISCs simultaneously, either at the clonal level or in the presence of niche cells.
n3:mentions: n2:21602799

Subject Item: _:vb38007506
rdf:type: n6:Section
dc:title: results
n6:contains: _:vb38007528 _:vb38007529 _:vb38007530 _:vb38007524 _:vb38007525 _:vb38007526 _:vb38007527 _:vb38007520 _:vb38007521 _:vb38007522 _:vb38007523 _:vb38007516 _:vb38007517 _:vb38007518 _:vb38007519 _:vb38007512 _:vb38007513 _:vb38007514 _:vb38007515 _:vb38007508 _:vb38007509 _:vb38007510 _:vb38007511 _:vb38007507

Subject Item: _:vb38007507
rdf:type: n3:Context
rdf:value: We hypothesized that previously described polydimethylsiloxane (PDMS)/polystyrene “microraft arrays” (MRAs) could be utilized to isolate and culture single ISCs in three-dimensional ECM (Fig. 1A-C) >>12<<. Since ISCs require several days to develop into enteroids, MRAs had to be amenable to media changes 3, 4.
n3:mentions: n2:20838672

Subject Item: _:vb38007508
rdf:type: n3:Context
rdf:value: Since ISCs require several days to develop into enteroids, MRAs had to be amenable to media changes 3, >>4<<. To meet these requirements, polycarbonate cassettes, with dividers to create multiple media reservoirs, were bonded to MRAs (Fig. 1A,B, Supplementary Fig. 1H). Cassettes were fabricated with two or four culture chambers (∼2,500 or 5,000
n3:mentions: n2:19329995

Subject Item: _:vb38007509
rdf:type: n3:Context
rdf:value: To facilitate tracking of isolated cells in MRAs, Sox9EGFP mice were crossed to CAGDsRed mice, which express the DsRed fluorescent transgene ubiquitously across all cell and tissue types (Fig. 1D) 3, >>13<<. Sox9EGFPlow:DsRed+ ISCs were plated in a single culture chamber of a two-chamber MRA, randomly seeded into microwells by centrifugation, and overlaid with Matrigel and ISC-supporting growth factors (Fig.
n3:mentions: n2:15593332

Subject Item: _:vb38007510
rdf:type: n3:Context
rdf:value: tissue types (Fig. 1D) 3, 13. Sox9EGFPlow:DsRed+ ISCs were plated in a single culture chamber of a two-chamber MRA, randomly seeded into microwells by centrifugation, and overlaid with Matrigel and ISC-supporting growth factors (Fig. 1E) >>6<<, 14. This resulted in a random distribution of ISCs across MRAs, with microwells containing one, multiple, or no ISCs (Fig 1F-H).
n3:mentions: n2:21113151

Subject Item: _:vb38007511
rdf:type: n3:Context
rdf:value: types (Fig. 1D) 3, 13. Sox9EGFPlow:DsRed+ ISCs were plated in a single culture chamber of a two-chamber MRA, randomly seeded into microwells by centrifugation, and overlaid with Matrigel and ISC-supporting growth factors (Fig. 1E) 6, >>14<<. This resulted in a random distribution of ISCs across MRAs, with microwells containing one, multiple, or no ISCs (Fig 1F-H).
n3:mentions: n2:23644405

Subject Item: _:vb38007512
rdf:type: n3:Context
rdf:value: For biocompatibility experiments, we utilized high efficiency ISC culture methods that drive high Wnt and Notch signaling >>14<<. Tile scanning of the MRA in the dsRed wavelength immediately after plating and at 48hrs revealed that isolated ISCs had begun to produce primitive enteroids, indicative of biocompatibility (Fig. 1F-K).
n3:mentions: n2:23644405

Subject Item: _:vb38007513
rdf:type: n3:Context
rdf:value: Conventional ISC cultures are capable of supporting enteroid growth for many weeks >>4<<. ISCs were maintained up to 8 weeks in MRAs, with retention of enteroids in their original microwells (Fig.
n3:mentions: n2:19329995

Subject Item: _:vb38007514
rdf:type: n3:Context
rdf:value: To achieve this, we developed an image analysis computational pipeline (Fig. 2; Supplementary Methods) >>15<<.
n3:mentions: n2:17076895

Subject Item: _:vb38007515
rdf:type: n3:Context
rdf:value: Previous studies have isolated PCs by FACS of CD24High:SSCHigh populations >>6<<. However, CD24 is also expressed on ISCs, TAs, and enteroendocrine cells 3, 14, 16-18.
n3:mentions: n2:21113151

Subject Item: _:vb38007516
rdf:type: n3:Context
rdf:value: However, CD24 is also expressed on ISCs, TAs, and enteroendocrine cells 3, >>14<<, 16-18. Since the experimental approach of MRA cultures examines events on a “per well” basis, it was critical to refine isolation procedures for PCs to meet purity requirements of clonal and microscale co-cultures and avoid artifactual
n3:mentions: n2:23644405

Subject Item: _:vb38007517
rdf:type: n3:Context
rdf:value: However, CD24 is also expressed on ISCs, TAs, and enteroendocrine cells 3, 14, >>16<<-18. Since the experimental approach of MRA cultures examines events on a “per well” basis, it was critical to refine isolation procedures for PCs to meet purity requirements of clonal and microscale co-cultures and avoid artifactual
n3:mentions: n2:22464327

Subject Item: _:vb38007518
rdf:type: n3:Context
rdf:value: Previous characterization of the Sox9EGFP mouse model demonstrated that Sox9 is expressed at different levels in ISCs, progenitors, and enteroendocrine cells, but that the Sox9EGFP transgene is preferentially silenced in PCs >>7<<. We exploited this property to isolate a highly pure population of PCs by FACS exclusion of Sox9EGFP populations. PCs were FACS-isolated using CD24High:SSCHigh parameters, and the additional exclusion of all Sox9EGFP-positive cells
n3:mentions: n2:19228882

Subject Item: _:vb38007519
rdf:type: n3:Context
rdf:value: and large cell size (Fig. 3A). Importantly, all isolated cells expressed the PC-related gene Defcr-rs (Fig. 3B,C). Lgr5 was detected in some cells, consistent with recent reports on Lgr5 expression in a subset of PCs in vivo (Fig. 3C) >>19<<. Similarly, while all cells were negative for the enteroendocrine transcript Tac1, expression of Chga was observed in a single PC, consistent with reports of PC progenitors expressing this enteroendocrine marker (Fig.
n3:mentions: n2:23446353

Subject Item: _:vb38007520
rdf:type: n3:Context
rdf:value: Similarly, while all cells were negative for the enteroendocrine transcript Tac1, expression of Chga was observed in a single PC, consistent with reports of PC progenitors expressing this enteroendocrine marker (Fig. 3C) >>19<<.
n3:mentions: n2:23446353

Subject Item: _:vb38007521
rdf:type: n3:Context
rdf:value: Emerging evidence demonstrates that multiple intestinal progenitor cell populations are capable of dedifferentiating and functioning as ISCs in vitro and in vivo >>19<<-21. To address the possibility that enteroids might form from early progenitors in the PC population, 2,810 individual PCs were examined in subsequent in vitro experiments.
n3:mentions: n2:23446353 n2:23273993

Subject Item: _:vb38007522
rdf:type: n3:Context
rdf:value: have speculated that PC-secreted WNTs are responsible for enhancing ISC growth in vitro, ISC-PC co-culture experiments were carried out in the absence of exogenous WNT, to avoid “masking” the potential impact of PCs on enteroid formation >>4<<-6. The GSK3β-inhibitor CHIR99021, a WNT agonist, was also excluded from co-culture experiments.
n3:mentions: n2:21113151 n2:19329995

Subject Item: _:vb38007523
rdf:type: n3:Context
rdf:value: Previous studies have suggested that cell-to-cell contact between ISCs and PCs may influence enteroid formation, but this has not been formally tested by comparison between touching and non-touching ISCs and PCs >>6<<. Using the same data generated in our ISC-PC co-culture experiments, we reanalyzed initial MRA contents to classify microwells by cell-to-cell contacts at t=0hr and correlated this status with enteroid formation outcome (Fig.
n3:mentions: n2:21113151

Subject Item: _:vb38007524
rdf:type: n3:Context
rdf:value: Previous studies demonstrate significant enteroid movement and merger in vitro >>6<<. To further examine cell movement in the MRA platform, we performed time-lapse imaging of microwells.
n3:mentions: n2:21113151

Subject Item: _:vb38007525
rdf:type: n3:Context
rdf:value: single ISC and enteroid retrieval for downstream analysis, MRAs were modified so that standard polystyrene rafts at the bottom of each microwell were replaced with magnetized rafts, as recently described (Supplementary Fig. 5A) >>12<<, 22. A raft release device was fitted to a 10X objective to liberate the rafts from the PDMS wells, and a magnetic wand was used to retrieve rafts for transfer to RNA lysis buffer (Supplementary Fig.
n3:mentions: n2:20838672

Subject Item: _:vb38007526
rdf:type: n3:Context
rdf:value: crypt-base columnar ISC markers Olfm4 and Smoc2 were strongly detected in single ISCs, a number of cells were negative for putative “+4” ISC markers Bmi1, Hopx, and Tert, contrary to studies conducted on populations of Lgr5high cells >>23<<-26. To test if this finding was reflective of a transcriptional response to ISC culture conditions, we compared gene expression profiles of single Lgr5high cells sorted directly into lysis buffer with those exposed to Matrigel culture
n3:mentions: n2:22190486 n2:22075725 n2:22692129 n2:21173232

Subject Item: _:vb38007527
rdf:type: n3:Context
rdf:value: Subsequently, Dll1 expression initiates in a majority of enteroids at 48-hours, consistent with the emergence of secretory progenitor populations 21, >>28<<. Early expression of Sis appears to be coincident with upregulation Hes1, a known driver of enterocyte fate, while Muc2 and Chga are upregulated at later time points, coincident with increased Atoh1 expression 29, 30. By 10 days in
n3:mentions: n2:21915337

Subject Item: _:vb38007528
rdf:type: n3:Context
rdf:value: Early expression of Sis appears to be coincident with upregulation Hes1, a known driver of enterocyte fate, while Muc2 and Chga are upregulated at later time points, coincident with increased Atoh1 expression >>29<<, 30. By 10 days in culture, enteroids are enriched for the expression of transcripts associated with absorptive enterocytes (Sis), goblet cells (Muc2), PCs (Lyz, Defr-rs1), and endocrine cells (Chga, Chgb), consistent with a fully
n3:mentions: n2:10615124

Subject Item: _:vb38007529
rdf:type: n3:Context
rdf:value: Early expression of Sis appears to be coincident with upregulation Hes1, a known driver of enterocyte fate, while Muc2 and Chga are upregulated at later time points, coincident with increased Atoh1 expression 29, >>30<<. By 10 days in culture, enteroids are enriched for the expression of transcripts associated with absorptive enterocytes (Sis), goblet cells (Muc2), PCs (Lyz, Defr-rs1), and endocrine cells (Chga, Chgb), consistent with a fully developed
n3:mentions: n2:11739954

Subject Item: _:vb38007530
rdf:type: n3:Context
rdf:value: are enriched for the expression of transcripts associated with absorptive enterocytes (Sis), goblet cells (Muc2), PCs (Lyz, Defr-rs1), and endocrine cells (Chga, Chgb), consistent with a fully developed organoid phenotype (Fig. 6B,C) >>4<<.
n3:mentions: n2:19329995

Subject Item: _:vb38007531
rdf:type: n6:Section
dc:title: discussion
n6:contains: _:vb38007532 _:vb38007533 _:vb38007534 _:vb38007535 _:vb38007536 _:vb38007537 _:vb38007538 _:vb38007539

Subject Item: _:vb38007532
rdf:type: n3:Context
rdf:value: As an alternative approach, in vitro techniques that rely on the co-culture of isolated stem and niche cell populations have recently been used to assess the impact of individual niche components on stem cell behavior >>6<<, 31. However, these methods commonly rely on large numbers of cells, which may not reflect physiologically relevant niche behavior, and are not amenable to high-throughput studies. Here, we present an array-based platform that facilitates
n3:mentions: n2:21113151

Subject Item: _:vb38007533
rdf:type: n3:Context
rdf:value: As an alternative approach, in vitro techniques that rely on the co-culture of isolated stem and niche cell populations have recently been used to assess the impact of individual niche components on stem cell behavior 6, >>31<<. However, these methods commonly rely on large numbers of cells, which may not reflect physiologically relevant niche behavior, and are not amenable to high-throughput studies. Here, we present an array-based platform that facilitates the
n3:mentions: n2:23040482

Subject Item: _:vb38007534
rdf:type: n3:Context
rdf:value: PCs express soluble and insoluble ISC niche signaling components, including Wnt and Notch ligands 5, >>6<<. In the present study, we examine the impact of PC presence and contact with ISCs on enteroid formation.
n3:mentions: n2:21113151

Subject Item: _:vb38007535
rdf:type: n3:Context
rdf:value: These results support in vivo findings that stemness is most strongly correlated cells that exist in intimate contact with PCs, and provide insight to the functional role of PCs in maintaining stemness >>6<<. Importantly, the MRA platform was critical in testing dose and contact-dependency of PCs in a microscale format.
n3:mentions: n2:21113151

Subject Item: _:vb38007536
rdf:type: n3:Context
rdf:value: Together with methodology for low-input RNA-seq, MRAs potentiate screening of genetic mutants and drugs/small molecules at the genomic level >>32<<.
n3:mentions: n2:24141493

Subject Item: _:vb38007537
rdf:type: n3:Context
rdf:value: Array-based stem cell culture platforms are growing in use and present an efficient and cost-effective alternative to conventional cell culture >>33<<, 34. However, most platforms are not amenable to long-term cultures, such as required for the development of ISC-derived enteroids and other self-assembled, stem cell derived organoids 35-38.
n3:mentions: n2:21983923

Subject Item: _:vb38007538
rdf:type: n3:Context
rdf:value: Array-based stem cell culture platforms are growing in use and present an efficient and cost-effective alternative to conventional cell culture 33, >>34<<. However, most platforms are not amenable to long-term cultures, such as required for the development of ISC-derived enteroids and other self-assembled, stem cell derived organoids 35-38.
n3:mentions: n2:22307554

Subject Item: _:vb38007539
rdf:type: n3:Context
rdf:value: However, most platforms are not amenable to long-term cultures, such as required for the development of ISC-derived enteroids and other self-assembled, stem cell derived organoids >>35<<-38. MRAs facilitate the culture of thousands of primary stem cells over many days and weeks as well as high throughput reconstitution of the stem cell niche at physiologically relevant cell numbers. The power of the MRA platform is
n3:mentions: n2:23995685 n2:21889923 n2:22036745 n2:21475194

Subject Item: _:vb38007540
rdf:type: n6:Section
dc:title: experimental procedures
n6:contains: _:vb38007552 _:vb38007553 _:vb38007541 _:vb38007542 _:vb38007543 _:vb38007548 _:vb38007549 _:vb38007550 _:vb38007551 _:vb38007544 _:vb38007545 _:vb38007546 _:vb38007547

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rdf:value: Sox9EGFP transgenic mice were originally generated by the GENSAT Brain Atlas Project, and are maintained on an outbred CD-1 background >>39<<. Lgr5EGFP-CreERT2 mice were obtained from Jackson Labs (stock number: 008875, Jackson Laboratory, Bar Harbor, ME) 40.
n3:mentions: n2:14586460

Subject Item: _:vb38007542
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rdf:value: Lgr5EGFP-CreERT2 mice were obtained from Jackson Labs (stock number: 008875, Jackson Laboratory, Bar Harbor, ME) >>40<<. Male and female mice were used. For microwell array experiments requiring constitutive expression of dsRed in isolated ISCs, heterozygous Sox9EGFP mice were bred to homozygous CAGdsRed mice to produce Sox9EGFP:CAGdsRed offspring
n3:mentions: n2:17934449

Subject Item: _:vb38007543
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rdf:value: Lgr5EGFP-CreERT2 mice were genotyped by previously described PCR protocols >>40<<. All experiments were conducted on adult mice between 8 and 16 weeks of age.
n3:mentions: n2:17934449

Subject Item: _:vb38007544
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rdf:value: growth factors, with some modification, at the time of plating: 15μM JAGGED-1 peptide (AnaSpec, San Jose, CA), 750ng/mL EGF (R&D, Minneapolis, MN), 100ng/mL NOGGIN (Peprotech, Rocky Hill, NJ), and 500nM LY2157299 (Selleck Chemicals) >>41<<. Initial media in validation experiments contained 2.5μM CHIR99021 (Selleck Chemicals) and 2.5μM Thiazovivin (Selleck Chemicals).
n3:mentions: n2:23644405

Subject Item: _:vb38007545
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rdf:value: In ISC/PC co-culture experiments, growth factors, minus JAGGED-1 peptide and Y27632, were supplemented every two days, and media was changed every four days, as previously described >>42<<, 43.
n3:mentions: n2:21113151

Subject Item: _:vb38007546
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rdf:value: In ISC/PC co-culture experiments, growth factors, minus JAGGED-1 peptide and Y27632, were supplemented every two days, and media was changed every four days, as previously described 42, >>43<<.
n3:mentions: n2:19329995

Subject Item: _:vb38007547
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rdf:value: Scanned images were stitched into a single composite image using the open source image analysis suite FIJI >>44<< and then segmented into address-associated individual well images using an algorithm, “Segmenter.
n3:mentions: n2:22743772

Subject Item: _:vb38007548
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rdf:value: An SU-8 master mold with an array of microposts was fabricated using standard photolithography with 100 μm thick SU-8 as described previously >>45<<. The SU-8 master was coated by octyltrichlorosilane using vapor deposition to render the surface of the master non-sticky to PDMS 46.
n3:mentions: n2:17949059

Subject Item: _:vb38007549
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rdf:value: The SU-8 master was coated by octyltrichlorosilane using vapor deposition to render the surface of the master non-sticky to PDMS >>46<<. Clean glass slides (75 mm × 50 mm × 1 mm) were spin coated with a 15-μm thick layer of poly(acrylic acid) (PAA) by using 50 wt% solution and a spin speed of 2000 rpm, followed by a 1 hr bake on a 100°C hotplate to remove the water. The
n3:mentions: n2:20838672

Subject Item: _:vb38007550
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rdf:value: This phenomenon is called discontinuous dewetting >>47<<. The array was then placed in a 95°C oven overnight to evaporate the GBL solvent, and pockets of polystyrene solution shrunk into solid polystyrene microrafts with a concave geometry (supplemental material, Fig.
n3:mentions: n2:21644640

Subject Item: _:vb38007551
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rdf:value: Epithelial cells were isolated from whole murine intestines, as previously described, with some modifications >>48<<. Briefly, intestines were opened longitudinally, rinsed in DPBS (Life Technologies, Grand Island, NY), minced, and incubated in 3mM EDTA (Sigma, St Louis, MO) in DPBS for 45min at 4°C with gentle agitation.
n3:mentions: n2:22610555

Subject Item: _:vb38007552
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rdf:value: FACS was conducted on a MoFloXDP (Beckman Coulter, Pasadena, CA) and Sox9EGFP and Lgr5EGFP cells were isolated as previously described >>40<<, 48, 49. Paneth cells were isolated by high expression of CD24, high side-scatter (SSC), and exclusion of CD45, with or without exclusion of Sox9EGFP (supplemental material, Fig.
n3:mentions: n2:17934449

Subject Item: _:vb38007553
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rdf:value: FACS was conducted on a MoFloXDP (Beckman Coulter, Pasadena, CA) and Sox9EGFP and Lgr5EGFP cells were isolated as previously described 40, >>48<<, 49. Paneth cells were isolated by high expression of CD24, high side-scatter (SSC), and exclusion of CD45, with or without exclusion of Sox9EGFP (supplemental material, Fig.
n3:mentions: n2:22610555

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