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. 2018 Feb;154:60-73.
doi: 10.1016/j.biomaterials.2017.10.038. Epub 2017 Oct 26.

Towards a Defined ECM and Small Molecule Based Monolayer Culture System for the Expansion of Mouse and Human Intestinal Stem Cells

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Free PMC article

Towards a Defined ECM and Small Molecule Based Monolayer Culture System for the Expansion of Mouse and Human Intestinal Stem Cells

Zhixiang Tong et al. Biomaterials. .
Free PMC article

Abstract

Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5+ population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2β1, integrin β4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP+ cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5+ ISCs. Considering the key roles Lgr5+ ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy).

Keywords: BLT sandwich culture; Integrin α2β1; Intestinal stem cells; Lgr5.

Conflict of interest statement

Disclosure Statement

No competing financial interests exist. J.M.K. R.S.L. and X.Y. hold equity in Frequency Therapeutics, a company that has an option to license IP generated by J.M.K., R.S.L. and X.Y. and that may benefit financially if the IP is licensed and further validated.

Figures

Figure 1
Figure 1
Schematic of the 2D BLT Sandwich culture system. Intestinal crypts isolated from Lgr5-GFP donor mice or human intestinal biopsies are directly seeded onto Col IV-coated transwells. Alternatively, the isolated crypts can be pre-expanded in Matrigel to form Lgr5+ ISCs-enriched organoids following established methods [1]. The organoids are then mechanically dissociated into ISC-containing IEC clusters and seeded onto Col IV-coated transwells. Next, a thin layer of type I collagen solution is gently overlaid onto the settled cells and gelled, resulting in a sandwich configuration comprising Col IV coating/IECs/Col I gel overlay. Chemically defined ISC maintenance media containing essential signaling modulators (i.e. CV + BMP inhibitor LDN) is then added into the transwell insert. An epithelial monolayer containing high levels of Lgr5+ ISCs is rapidly generated via the combination of potent signaling factors and unique presentation of defined ECM cues.
Figure 2
Figure 2
Identification of Col IV as a Matrigel alternative coating for the maximal attachment of IECs. A: Define BM ECMs with different coating concentrations that are most supportive for the initial attachment of single IECs on transwells. a–e: 2, 10, 50, 100 and 150 μg/mL. Two hours after seeding, the transwells were gently washed with 500 μL cold PBS for 3 times to remove unattached cells. The number of attached IECs per well on each coating was measured by Cyquant DNA quantification assay and normalized to the coating with 30X dilution of Matrigel in F12 media, i.e. Matri-coating (red bar). Transwells coated with 0.1% BSA was included as negative control. Data represents mean ± SD, n = 2–3 wells, representative from three independent trials. B: Towards understanding the mechanism of Col IV mediated single IEC attachment: image quantification for the number of attached IECs after 2 hour incubation on Col IV (100 μg/mL)-coated transwells. Transwells in all groups were pre-coated with Col IV prior to cell seeding, except the BSA coating group (seeded with untreated IECs) highlighted in grey bar. Single IECs derived from Matrigel-expanded SI organoids were pretreated with integrin α2, β1 antibodies, their combination and isotype controls, as well as integrin α2β1 binding peptide (DGEA) and the scrambled peptide sequence (RGES). To further confirm that the Col IV-mediated cell adhesion is integrin- or divalent cation-dependent, IECs were also seeded in the presence of 2 mM EDTA for the 2 hour incubation. Mean ± SEM, n = 10–12 fields of view per group, representative from three independent trials with single IECs derived from different passages in Matrigel.
Figure 3
Figure 3
Optimization of the BLT Sandwich culture system using mouse SI organoids under different culture settings for 4 days. A: Sandwich configuration composed of Col IV thin coating (100 μg/mL) and Col I gel overlay synergistically enhances the expansion of Lgr5+ cells. Representative confocal images (A) show the morphology and presence of Lgr5-GFP in IEC monolayer on transwells under different culture settings with or without Col IV coating and Col I overlay. B1–2: Col IV coating concentration dictates the number of Lgr5-GFP+ cells (B1, normalized to no Col IV coating control) and the percentage of Lgr5/Ki67 co-positive population (B2). n = 3–4 wells, data represents mean ± SD from one representative trial out of three independent trials using SI organoids derived from three donor mice.
Figure 4
Figure 4
Characterization of the BLT Sandwich culture system. A: Representative phase contrast and fluorescence images showing the proliferation and presence of Lgr5-GFP of IEC monolayer at day 4. Contour of epithelial island/monolayer is delineated by dashed lines in phase contrast images for better visualization. Zoomed-in view depicts the undulating pattern of IEC monolayer/sheet that is closely associated with Lgr5-GFP+ cells. B1: Confocal images comparing the expression of Lgr5 and Ki67 between 2D Sandwich and 3D Matrigel culture systems at day 4 (a); B2–3: Flow cytometry results comparing the level of Ki67+ cells (B2) and Lgr5/Ki67 co-positive cells (B3) between the two culture systems. Mean ± SEM, averaged from n = 3–4 independent trials with different SI organoid donor mice.
Figure 5
Figure 5
Validation of the multi-lineage differentiation potential of BLT Sandwich culture-expanded ISCs. A1–4: qPCR analysis of relative mRNA expression of Lgr5 and mature IEC markers (Alpi, Lyz1, Muc2 and ChgA) from monolayer IECs (with ~35% baseline Lgr5-GFP level) treated with indicated differentiation cocktails for 3 days with ENR being present in all conditions. mRNA levels of target genes were normalized to ENR-alone conditions. Mean ± SEM, n = 3 independent trials each with 2–3 biological repeats. **, ***: significantly different versus ENR-alone control. B: Colorimetric staining (Alpi) and immunocytochemistry staining of mature IEC lineage markers after 3-day differentiation. Representative images from three independent trials with n = 2 wells (8–10 images) each condition. Scale bars for all Alpi images: 500 μm.
Figure 6
Figure 6
(A) BLT Sandwich culture is capable of expanding freshly isolated mouse SI, colon crypts and (B) patient-derived SI crypts. Equal number of freshly isolated crypts were evenly seeded into 2D Sandwich and 3D Matrigel systems and cultured for 4 days (mouse) or 6 days (human). A1: Confocal images showing the morphology and Lgr5-GFP expression of SI and colonic IECs in 2D and 3D systems. Percentage of Lgr5-GFP+ cells was noted inside each picture in green. A2: Flow cytometry comparing Ki67 expression of IECs expanded from both systems. A3: Comparison of the fold expansion of mouse SI and colonic Lgr5-GFP+ cells between 2D and 3D systems. Mean ± SEM, n = 4 donor mice each with 3–5 wells/group. B1: Photographs showing the proliferation and morphological change of human SI IECs cultured under both systems for 6 days. Insets show the growth of a representative single crypt into a monolayer patch with diameter ~ 1 mm at day 6 (scale bar: 100 μm). B2: Flow cytometry comparing Ki67 expression between both systems at day 6 (representative from n = 3 patients). B3: qPCR analysis comparing the mRNA expression of key IEC lineage markers between both systems at day 6. Mean ± SEM, n = 3 trials using freshly isolated SI crypts from three patients. ** p<0.01: significantly different between 2D and 3D cultures.
Figure 7
Figure 7
Positive effect of LDN-193189 (L) on the expansion of Lgr5-GFP+ cells cultured from freshly isolated mouse SI crypts for 4 days in 2D BLT Sandwich culture under different culture media conditions. A: Number of Lgr5-GFP+ cells normalized to the control without LDN in response to different concentrations of LDN in ISC maintenance media (i.e. ENR+CV). B: Percentage of resulting Lgr5-GFP+ cells under different media conditions; C: Normalized number of Lgr5-GFP+ cells (to ENR-alone control) under different conditions with or without CV, LDN (200 nM) and Noggin (N). Data represents mean ± SEM, n = 3–4 independent trials with different donor mice.

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