Supplemental Material Small Intestinal Stem Cell Identity Is Maintained with Functional Paneth Cells in Heterotopically Grafted Epithelium onto Colon Masayoshi Fukuda, Tomohiro Mizutani, Wakana Mochizuki, Taichi Matsumoto, Kengo Nozaki, Yuriko Sakamaki, Shizuko Ichinose, Yukinori Okada, Toshihiro Tanaka, Mamoru Watanabe, Tetsuya Nakamura Supplemental Materials and Methods Supplemental Figures and Figure Legends: Supplemental Figure S1 Colonic epithelial injury model used in the present study Supplemental Figure S2 Freshly isolated SI epithelia also maintain SI phenotype when grafted Supplemental Figure S3 Subepithelium of the graft contains multiple cellular components of recipient origin Supplemental Figure S4 Multi-differentiation and self-renewal capabilities of grafted cells at 4 months post-transplantation Supplemental Figure S5 Laser capture microdissection and following microarray analysis Supplemental Table S1 List of genes differentially expressed between the graft and normal colonic epithelium
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Supplemental Material
Small Intestinal Stem Cell Identity Is Maintained with Functional Paneth Cells
Supplemental Materials and Methods Supplemental Figures and Figure Legends: Supplemental Figure S1 Colonic epithelial injury model used in the present study Supplemental Figure S2 Freshly isolated SI epithelia also maintain SI phenotype when grafted Supplemental Figure S3 Subepithelium of the graft contains multiple cellular components of recipient origin Supplemental Figure S4 Multi-differentiation and self-renewal capabilities of grafted cells at 4 months post-transplantation Supplemental Figure S5 Laser capture microdissection and following microarray analysis Supplemental Table S1 List of genes differentially expressed between the graft and normal colonic epithelium
Supplemental Materials and Methods
Stereomicroscopy, Histology, and Immunohistochemistry
Whole distal colons of recipients and their fluorescence were imaged using a fluorescence stereomicroscope
system MVX10 (Olympus). For histology and immunohistochemistry, recipient colons were fixed overnight at 4˚C
in 4% paraformaldehyde, sequentially dehydrated in 10, 15 and 20% sucrose in PBS, and embedded in OCT
compound (Tissue Tek). Frozen sections of 6-mm thickness were cut, mounted on slide glasses, and examined
for their fluorescent signals in order to determine whether the section contains EGFP+ grafts. Sections containing
EGFP+ grafts were subjected to H&E staining or immunohistochemistry. The followings were used as antibodies
specific for each protein: Cdh (Cell Signaling); CA2 (Santa Cruz); CDX2 (Bio Genex); Ki67 (Dako Cytomation);
(Abcam); and CD31 (Abcam). In all immunofluorescent experiments, nuclei were counterstained with DAPI.
Sections obtained from some tissue blocks showed obvious but weak endogenous EGFP signals presumably
because of the variation in the rate and quality of fixation. In those cases, we performed immunofluorescence
staining of adjacent sections with anti GFP antibody (Invitrogen) and presented them to show the EGFP
fluorescence as a reference. Fluorescent images of sections were acquired using a DeltaVision system (Applied
Precision) where a fluorescent microscope IX-71 (Olympus) with objectives UplansApo 10x 0.4NA or UplansApo
20x 0.75NA (Olympus) is incorporated. If necessary, image processing was carried out using Adobe Photoshop
Elements 7.0 software. For quantification of cellular components, sections of proximal, middle, distal SI, colon,
and EGFP+ grafts were subjected to immunohistochemistry for ChgA, MUC2, or Lysozyme. Thirty crypt-villus
units or crypt units (colon), originating from 3 independent recipients or normal controls, were analyzed. The
numbers of ChgA+, MUC2+, or Lysozyme+ cells were counted and presented as mean cell counts per crypt-villus
or crypt unit. Statistical significance was determined by Student’s t test (p < 0.05).
Laser Capture Microdissection
Recipient mice at 4 weeks post-transplantation and C57BL normal control mice were sacrificed. The
graft-containing recipients’ tissues and control colon tissues were quickly washed in PBS, embedded in OCT
compound (Tissue Tek), rapidly frozen, and stored. For recipients’ colon experiments, we searched and located
the plane of specimens so that the section would contain EGFP+ grafts. From there onwards, serial cryosections
(10-mm thickness) of more than 30 slices were made. Fluorescent images of a part of these slices (1 every 7
sections) were acquired, and they were used as references for dissecting the EGFP+ epithelia from sections
located in between. Likewise, we also made ~ 30 sections from the distal colon of control mice. Sections were
stained with cresyl violet solution and Arcturus HistGene Staining Solution (Applied Biosystems). Grafted
epithelia in recipients or control colonic epithelia were microdissected using Micro Laser System MBIII
(PALM/ZEISS). Dissected epithelia obtained from serial sections (~ 30 sections) of each specimen were
combined and then total RNA extraction was performed by using RNeasy Plus Micro Kit (QUIAGEN). After
calculating RNA Integrity Number (RIN), we subjected one graft sample and one control colon sample, which
showed the highest RIN value, for the following gene expression analysis.
Microarray and Data Analysis
cDNA synthesis, amplification and labeling were performed with Ovation Pico WTA System V2 and Encore Biotin
Module (Nugen). Microarray hybridization was carried out onto GeneChip Mouse Gene 1.0 ST Array (Affymetrix).
The arrays were scanned with GeneChip Scanner 3000 7G (Affymetrix). Data files were imported to GeneSpring
GX 12.5 software (Agilent), and its Robust Multichip Average algorithm 16 (RMA16) was used for data
normalization. The bottom 20% of genes with the lowest expression levels was excluded from subsequent
analysis. Multiple probesets on Affymetrix microarrays are associated with transcript clusters. Transcript clusters
that showed > 3-fold differences in expression values between two samples are presented. For a gene or a group
of genes to which values are given by multiple transcript clusters, averages of the values for those clusters were
used for assessing fold enrichment. Detailed information on the probeset and transcript cluster grouping is
available on the NetAffyx site (http://www.affymetrix.com/estore/index.jsp) provided by the manufacturer.
Fukuda et al., Supplemental Figure S1
A
B
Supplemental Fig. S1 Colonic epithelial injury model used in the present study.(A) Devices used to generate distal colonic epithelial injury. Thin catheter equipped with two small rubber balloons used for topical EDTA treatment (left). After the EDTA treatment, balloons were deflated and the catheter was removed out of the colon. Epithelial abrasion was performed by using an electric brush (right), giving its rotational movement to scratch the colonic luminal surface. (B) Time course change of the tissue damage during recovery. Stereoscopic images (left) at Day 2, 14, 28 after injury are presented with their longitudinal sections (right). H&E-stained sections are shown with anal side on the left and oral side on the right. High-power views of areas in dotted rectangles are shown at the bottom. Scale bars show 1 mm.
Day 2
Day 14
Day 28
Day 2
Day 14
Day 28
Fukuda et al., Supplemental Figure S2
Supplemental Fig. S2Freshly isolated SI epithelia also maintain SI phenotype when grafted. SI epithelia were isolated from EGFP transgenic mice and transplanted immediately into recipients without culturing. Recipient colons at 2 weeks post-transplantation were analyzed. (A) immunostaining for Lysozyme (A) and endogenous EGFP fluorescence (A´) of the same section. (B) Section double stained with CDX2 (B) and CA2 (B´) is presented with the endogenous EGFP fluorescence of the same section (B´´). Note that the antigen retrieval procedure before immunostaining completely clears endogenous EGFP signals.Images are shown with DAPI staining. Dotted lines indicate borders between EGFP+ and EGFP- epithelia. Scale bars; 100 μm.
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Supplemental Fig. S3Subepithelium of the graft contains multiple cellular components of recipient origin. Immunohistochemical analysis of recipient colons at 4 weeks post-transplantation. Tissue sections were immunostained with anti-CD3 (A), anti-F4/80 (B), or anti-CD31 (C) antibody to detect T cells, monocyte/macrophage lineage of cells, and blood vessel endothelial cells, respectively. EGFP fluorescence of the same sections (A´, B´, and C´) is also shown. Arrowheads point to positively-stained cells residing within the subepithelia entirely surrounded by EGFP+ epithelia. Images are shown with DAPI staining. Dotted lines indicate borders between EGFP+ and EGFP- epithelia. Scale bars; 100 μm.
Fukuda et al., Supplemental Figure S3
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Fukuda et al., Supplemental Figure S4
Supplemental Fig. S4 Multi-differentiation and self-renewal capabilities of grafted cells at 4 months post-transplantation. (A) H&E staining of a section obtained from a recipient colon at 4 months (18 weeks) post-transplantation. (B,C) EGFP fluorescence (B) of the section adjacent to the one shown in (A) and its immunostaining for Lysozyme (C). High-power views of areas in dotted boxes in (A) and (C) are shown as (A´) and (C´), respectively. (D) Section double stained with CA2 (D) and sucrase isomaltase (SIase; D´) is presented with the endogenous EGFP fluorescence of the same section (D´´). Note that the antigen retrieval procedure before immunostaining completely clears endogenous EGFP signals. (E-H) Immunofluorescence for CDX2 (E), Ki67 (F), MUC2 (G), and ChgA (H). Arrowheads point to ChgA+ cells. Images of endogenous EGFP fluorescence of the same sections (E´, F´, G´) or an adjacent section (H´) are shown. Fluorescent images are shown with DAPI staining. Dotted lines indicate borders between EGFP+ and EGFP- epithelia. Scale bars; 100 μm.
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RNA Extraction
cDNA Amplification
Microarray Analysis
Post Dissection
Pre Dissection
Mesenchyme elimination
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Pre Dissection
Post Dissection
Graft Control Colon
Supplemental Fig. S5 Laser capture microdissection and following microarray analysis. The graft-containing tissues and control colon tissues were obtained from recipient mice (4 weeks post-transplantation) and normal syngeneic mice, respectively. To obtain donor-derived epithelia from recipients’ colon, serial sections were prepared per sample so that all sections contain EGFP+ epithelia. Every 6th section was used to detect EGFP+ signals as references for dissecting the EGFP+ epithelia from other sections located in between. Microdissected epithelia from multiple sections of each sample were combined and then total RNA was extracted. RNA was converted to cDNA, amplified, labeled, and used for GeneChip hybridization. Scale bars show 100 μm.
Fukuda et al., Supplemental Figure S5
No Affimetrix Transcripts Cluster ID Gene Symbol Gene Description Fold Change Graft/Colon1 10550131 Pla2g4c phospholipase A2, group IVC (cytosolic, calcium-independent) 129.04922 10419575 Ang4 angiogenin, ribonuclease A family, member 4 111.4434
Supplemental Table S1. List of genes differentially expressed between the graft and normal colonicepithelium.Listed in this table are the transcript clusters (See Experimental Procedures) that showed > 3-folddifferences in expression values between two samples. For a gene or a group of genes to which values aregiven by multiple transcript clusters, the average of values for those clusters was used for assessing foldenrichment and presented.
76 10546929 Cidec cell death-inducing DFFA-like effector c 5.398777 10570018 Tnfsf13b tumor necrosis factor (ligand) superfamily, member 13b 5.279478 10546685 Eif4e3 eukaryotic translation initiation factor 4E member 3 5.248479 10543319 Fam3c family with sequence similarity 3, member C 5.1963