Supplemental Research Design and Methods:

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Supplemental Research Design and Methods:
Transgenic Animals. Transgenic Pdx1cre, Ins2cre and Z/EG offspring were
genotyped by tail biopsy genomic DNA PCR using Platinum Taq DNA
Polymerase (Invitrogen, Carlsbad, CA) with the GAPDH, Z/EG, Ins2cre and
Pdx1cre primer sets (See Supplemental Table 1.), as per manufacturers
conditions using the following cycling protocol: 1) 94C – 2 min, 2) 94C – 30sec,
59C – 30sec, 72C – 30sec (35 cycles), 3) 72C – 10 min. PCR products were
visualized on a 2% agarose gel. For Cre recombinase-mediated lineage tracing,
Ins2cre or Pdx1cre and Z/EG animals were bred and resultant double transgenic
offspring were used in the described experiments.
Isolation and culture of mouse islet-enriched pancreatic fractions.
Adult mice (10-24 weeks) were anesthetized by an IP dose of Avertin
(250mg/kg), after which the abdomen was opened and the pancreas was
perfused via the duct with 2.5mL Liberase RI (Roche, Indianapolis, IN) in HBSS
(Mediatech, Herndon, VA) + 0.1mg/mL DNase I (Roche), 25mM HEPES
(Mediatech) and digested at 37C for 14min. Digested pancreata were washed
3X in HBSS + 10% fetal bovine serum (FBS) (HyClone, Logan, UT) and islets
were purified using a continuous density gradient as described (1). Harvested
Islet-enriched fractions were cultured overnight in culture medium (CM)
consisting of 60:40 low-glucose DMEM (Mediatech):MCDB201 (Sigma, St. Louis,
MO) + 2% FBS (HyClone, Logan, UT), 10ng/ml human EGF (Sigma), 10ng/ml
human PDGF-BB (R&D Systems, Minneapolis, MN), 1000U/mL Lif (Chemicon,
Temecula, CA), 10,000 I.U. Penicillin/mL, 10,000 µg/mL Streptomycin
(Mediatech), 10-4 M ascorbic acid 2-phosphate (Sigma), 25mM HEPES, 0.1mM
2-Mercaptoethanol (Invitrogen) and 1mg/ml bovine serum albumin (Sigma) in
60mm hydrophobic dishes (Sarstedt, Newton, NC) at 37C and 5% CO2. For
fibroblast-like cell expansion, overnight cultured islet fractions were seeded at 1020 islets/cm2 in tissue cultured treated dishes (BD Biosciences, San Jose, CA)
coated with 10µg/mL fibronectin (Sigma). Total islet counts were determined by
staining with Dithizone (Sigma). To test fibroblast-like cell growth under
published media conditions, islet were harvested and purified as described above
and were cultured using media as described (2; 3) in uncoated tissue culturetreated dishes. For both media formulations, complete media changes were
made every 2-3 days.
For differentiation of Sca1+ fibroblast-like progenitors, 5x104 cells/cm2 were
seeded in expansion media overnight, followed by serum and cytokine-free CM
supplemented with 20ng/ml human HGF (Sigma), 200nM dexamethasone
(Sigma), and 0.25X ITS (Sigma). Alternatively, individual or combinations of
growth factors were added to serum and cytokine-free CM including 100ng/ml
Activin, 50ng/ml BMP4, 50ng/ml Betacellulin, 50ng/ml GDF11 (all from R&D
Systems), and 10nM Exendin-4 (Sigma). Differentiations revealing robust lipid
structures were evaluated by light microscopy and Oil red O staining (Chemicon)
as per the manufacturer’s protocol.
Standard PCR and Quantitative Real-Time-PCR. For real-time RT-qPCR, 510ng cDNA was amplified using SYBR Green PCR Master mix with an ABI
Prism 7700 (Applied Biosystems, Foster City, CA) under the following conditions:
1) 50C – 2min 2) 95C – 10min 3) 95C – 15sec , 59C – 60sec (40 cycles), 4)
95C – 15sec, 59C – 20sec, 95C – 15sec (Dissociation Curve Analysis). In
addition, PCR products were verified by gel electrophoresis. Primers used for
standard and real-time qPCR are listed in Supplemental Table 1 (below). For
detection of genomic excision within the Z/EG reporter construct, standard PCR
was performed on purified genomic DNA using Platinum Taq DNA Polymerase
(Invitrogen) with the GAPDH, CAAGS-LacZ and CAAGS-GFP primer sets under
the following conditions: 1) 95°C – 2 min, 2) 95°C – 30sec, 59°C – 30sec, 72°C –
60sec (40 cycles), 3) 72°C – 10 min. PCR products were visualized on a 2%
agarose (Invitrogen) gel. For real-time qPCR quantitation of excision, 100ng
genomic DNA was amplified using Platinum SYBR Green PCR Master mix
(Invitrogen) with the GAPDH and CAAGS-GFP primer sets under the following
conditions: 1) 50C – 2min 2) 95C – 10min 3) 95C – 15sec , 59C – 30sec,
72C – 30sec (40 cycles), 4) 95C – 15sec, 59C – 20sec, 95C – 15sec
(Dissociation Curve Analysis).
References
1. Guo Z, Wu T, Sozen H, Pan Y, Heuss N, Kalscheuer H, Sutherland DE, Blazar BR,
Hering BJ: A substantial level of donor hematopoietic chimerism is required to protect
donor-specific islet grafts in diabetic NOD mice. Transplantation 75:909-915, 2003
2. Gershengorn MC, Hardikar AA, Wei C, Geras-Raaka E, Marcus-Samuels B, Raaka
BM: Epithelial-to-mesenchymal transition generates proliferative human islet precursor
cells. Science 306:2261-2264, 2004
3. Ouziel-Yahalom L, Zalzman M, Anker-Kitai L, Knoller S, Bar Y, Glandt M, Herold K,
Efrat S: Expansion and redifferentiation of adult human pancreatic islet cells. Biochem
Biophys Res Commun 341:291-298, 2006
Supplemental Table 1. PCR primers
Target
Sequence
Actb
F: TGTTACCAACTGGGACGACA
R: GGGGTGTTGAAGGTCTCAAA
F: GCCCTACAGACCATGAAACAAG
R: GTGAAACAGACTTCCTGGTCCT
F: CTTTATGGTGTGGGCCAAAG
R: CTTCTCTGCCAAGGTCAACG
F: CCGCAATCTCAGAACCTCAT
R: AGGCTGCTAGCCACACTGTT
F: CCCGGGACTTAACTGTAACG
R: TCATGTTGCTCACGGAAGAG
F: AGGCTGAGAACTCTCGCTTG
R: ATTAGGCAAGGGGGAAGAGA
F: CTCCCTGGACTTGGATGGTA
R: ACGCTGGTTCTTCAAGGTGT
F: ATCGAGATCACCACCTACCG
R: TGAAGCCAGGGCTAGTGAGT
F: CGAGGCACTCAAGGAAGAAC
R: GCTGAGGTCCTGAGATTTGG
F: ACCCTCCCGAGATTACAACC
R: CAAGGCGTGTTCTGTCTCAA
F: ACTGTGAAGGGACGGTCAAC
R: GGAGCAGCAGGATCAGAATC
F: TCAGGGAGCCCATTACTGTC
R: GTTGTCCACCATCGCTTCTT
F: CCACTACCAGCAGTCGATGA
R: GCCTGTCTGTCCTCTTCCAG
F: AGCACAGGTCAGATGCAGTG
R: GTCTCGTCCTCCATGCAGTT
F: CGGTACAGCAGCAATGGTAA
R: TGGATATTCCCTGACCCAGT
F: GGTTGCTTTGGCTATCGTCT
R: ATTACCTGCCGAAACCTCCT
F: AACTTAACCTAGGCGTCGCA
R: CATTCGCTTGGCATCAGAAGC
F: CAAAGCCACGGATCAATCTT
R: CCCGGGAATAGTGAAACTGA
F: TCTACGACAGCAGCGACAAC
R: GCTTTGGAGAAGAGCACTCG
F: ACTTGGCAGGACCAGAGAGA
Afp
Sox17
Hnf1β
Hnf3β
Nes
CK7
CK8
CK18
CK19
Cdh1
Car2
Cldn3
Cldn4
Ocldn
Muc1
Pdx1
NeuroD
Nkx2.2
Nkx6.1
Amplicon Size
(bp)
165
149
121
116
152
167
229
151
130
160
123
98
126
164
165
119
145
168
220
Pax6
Ins1
Ins2
Vim
Acta2
Cdh2
Csf1
Zeb
Snail1
Snail2
CAAGS-LACZ
CAAGS-GFP
GAPDH
Z/EG
Ins2cre
Pdx1cre
R: AGAGTTCGGGTCCAGAGGTT
F: AACAACCTGCCTATGCAACC
R: ACTTGGACGGGAACTGACAC
F: TGTTGGTGCACTTCCTACCC
R: CACTTGTGGGTCCTCCACTT
F: GACCCACAAGTGGCACAAC
R: TCTACAATGCCACGCTTCTG
F: ATGCTTCTCTGGCACGTCTT
R: AGCCACGCTTTCATACTGCT
F: CTGACAGAGGCACCACTGAA
R: CATCTCCAGAGTCCAGCACA
F: ATCAAAGCCTGGGACGTATG
R: TGATGATGTCCCCAGTCTCA
F: GCTGGATGATCCTGTTTGCT
R: TCATGGAAAGTTCGGACACA
F: CCACTGTGGAGGACCAGAAT
R: CTCGTGAGGCCTCTTACCTG
F: TGGAAAGGCCTTCTCTAGGC
R: CTTCACATCCGAGTGGGTTT
F: TGCCCAGTCTAGGAAATCGT
R: GAGAAAGGCTTTTCCCCAGT
F: GTTCGGCTTCTGGCGTGT
R: GTTGCACCACAGATGAAACG
F: GTTCGGCTTCTGGCGTGT
R: GTAGGTCAGGGTGGTCACGA
F: CATGGCCTTCCGTGTTCCTA
R: CTGGTCCTCAGTGTAGCCCAA
F: ACTATCCCGACCGCCTTACT
R: CTGTAGCGGCTGATGTTGAA
F: CTCTGGCCATCTGCTGATCC
R: CGCCGCATAACCAGTGAAAC
F: ACTACATCTTGAGTTGCAGGC
R: CCTGTTTTGCACGTTCACCG
224
206
182
101
206
160
158
198
164
148
186
750
500
151
175
550
700
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