Cell culture
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Human THP-1 monocytic cells were subcultured in RPMI 1640 as described previously in detail 6 cells/ml in RPMI 1640 supplemented with 10% FBS and 5 µg/ml gentamicin.
- After 24 h, 1 or 10 µg/ml globular adiponectin was added and the cells were incubated for 6 to 24 h. Globular adiponectin is a recombinant protein derived from human globular domain adiponectin cDNA expressed in Escherichia coli.
- This protein was endotoxin free (<2 EU/µg) according to the manufacturer.
- In addition, treatment of cells with globular adiponectin (10 µg/ml) in the presence of polymyxin B (50 µg/ml) did not affect globular adiponectin-related TNFα expression (data not shown).
- The ox-LDL incubation experiments were performed like previously described 6 cells/ml in glucose-free RPMI 1640 supplemented with 10% FBS, 5 µg/ml gentamicin, and 5.5 mM D-glucose in a 5% CO2 incubator at 37°C.
- After 24 h, 9.5 mM D-glucose…
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Human THP-1 monocytic cells were subcultured in RPMI 1640 as described previously in detail 6 cells/ml in RPMI 1640 supplemented with 10% FBS and 5 µg/ml gentamicin.
- After 24 h, 1 or 10 µg/ml globular adiponectin was added and the cells were incubated for 6 to 24 h. Globular adiponectin is a recombinant protein derived from human globular domain adiponectin cDNA expressed in Escherichia coli.
- This protein was endotoxin free (<2 EU/µg) according to the manufacturer.
- In addition, treatment of cells with globular adiponectin (10 µg/ml) in the presence of polymyxin B (50 µg/ml) did not affect globular adiponectin-related TNFα expression (data not shown).
- The ox-LDL incubation experiments were performed like previously described 6 cells/ml in glucose-free RPMI 1640 supplemented with 10% FBS, 5 µg/ml gentamicin, and 5.5 mM D-glucose in a 5% CO2 incubator at 37°C.
- After 24 h, 9.5 mM D-glucose or 9.5 mM D-mannitol (osmotic control) was added and incubated for 24 h under normal growth conditions.
- For IL-6 experiments, THP-1 cells were stimulated with 100 ng/ml recombinant IL-6 for 24 h.
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Cell culture
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Human THP-1 monocytic cells were subcultured in RPMI 1640 as described previously in detail 6 cells/ml in RPMI 1640 supplemented with 10% FBS and 5 µg/ml gentamicin.
- After 24 h, 1 or 10 µg/ml globular adiponectin was added and the cells were incubated for 6 to 24 h. Globular adiponectin is a recombinant protein derived from human globular domain adiponectin cDNA expressed in Escherichia coli.
- This protein was endotoxin free (<2 EU/µg) according to the manufacturer.
- For glucose incubation experiments, cells were cultured at a density of 1×106 cells/ml in glucose-free RPMI 1640 supplemented with 10% FBS, 5 µg/ml gentamicin, and 5.5 mM D-glucose in a 5% CO2 incubator at 37°C.
- After 24 h, 9.5 mM D-glucose or 9.5 mM D-mannitol (osmotic control) was added and incubated for 24 h under normal growth conditions.
- Cell viability, as determined by trypan blue exclusion, was…
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Human THP-1 monocytic cells were subcultured in RPMI 1640 as described previously in detail 6 cells/ml in RPMI 1640 supplemented with 10% FBS and 5 µg/ml gentamicin.
- After 24 h, 1 or 10 µg/ml globular adiponectin was added and the cells were incubated for 6 to 24 h. Globular adiponectin is a recombinant protein derived from human globular domain adiponectin cDNA expressed in Escherichia coli.
- This protein was endotoxin free (<2 EU/µg) according to the manufacturer.
- For glucose incubation experiments, cells were cultured at a density of 1×106 cells/ml in glucose-free RPMI 1640 supplemented with 10% FBS, 5 µg/ml gentamicin, and 5.5 mM D-glucose in a 5% CO2 incubator at 37°C.
- After 24 h, 9.5 mM D-glucose or 9.5 mM D-mannitol (osmotic control) was added and incubated for 24 h under normal growth conditions.
- Cell viability, as determined by trypan blue exclusion, was >80%.
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Growth Factors
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ecombinant human Noggin/Fc chimera and neutralizing anti-human /4 antibody from& , and from.
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Crypt isolation and organoid culture
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Organ culture of freshly isolated human small intestinal biopsies was performed in medium'>RPMI medium .
- For organoid culture, crypts were isolated from the small intestine of mice and cultured for a minimum of 7 days as previously described.
- In brief, crypts were isolated by incubating pieces of small intestine in isolation buffer (phosphate buffered saline without calcium and magnesium (PBSO), 2 mM EDTA).
- Crypts were then transferred into matrigel in 48 well plates and 350 μl culture medium (Advanced ME/F12 , containing HEPES (10 mM, PAA), GlutaMax (2 mM), Penicillin (100 U/ml), Streptomycin (100 μg/ml), murine EGF (50 ng/ml), recombinant human-spondin (1 μg/ml& ), N2 Supplement 1x , B27 Supplement 1x , 1 mM N-acetylcystein and recombinant murine Noggin (100 ng/ml)).
- Organoid growth was monitored by light microscopy.
- In some experiments, human biopsies or organoids were treated with recombinant mouse TNF-α (25 ng/ml), recombinant human TNF-α (50 ng/ml),…
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Organ culture of freshly isolated human small intestinal biopsies was performed in medium'>RPMI medium .
- For organoid culture, crypts were isolated from the small intestine of mice and cultured for a minimum of 7 days as previously described.
- In brief, crypts were isolated by incubating pieces of small intestine in isolation buffer (phosphate buffered saline without calcium and magnesium (PBSO), 2 mM EDTA).
- Crypts were then transferred into matrigel in 48 well plates and 350 μl culture medium (Advanced ME/F12 , containing HEPES (10 mM, PAA), GlutaMax (2 mM), Penicillin (100 U/ml), Streptomycin (100 μg/ml), murine EGF (50 ng/ml), recombinant human-spondin (1 μg/ml& ), N2 Supplement 1x , B27 Supplement 1x , 1 mM N-acetylcystein and recombinant murine Noggin (100 ng/ml)).
- Organoid growth was monitored by light microscopy.
- In some experiments, human biopsies or organoids were treated with recombinant mouse TNF-α (25 ng/ml), recombinant human TNF-α (50 ng/ml), necrostatin-1 (30 μM) or caspase-8 inhibitor (50 μM ).
- Cell Viability of organoids was analyzed indirectly by quantification of relative ATP level with the CellTiter-Glo assay fromaccording to the manufacturer's instructions.
- Luminescence was measured on the microplate reader infinite M200 .
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mTORC1 signaling in Paneth cells mediates the effects of calorie restriction on ISC function a. Schematic of the Tet-ON human Rheb2 transgene (Rheb-tg).
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Organoid potential of intestinal crypts (n=3) (i) and AL Lgr5hi ISCs with Paneth cells (n=5) (j) from R, CR, or CR+R treated mice.
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Cell culture
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DCs were generated from mouse spleens as described elsewhere Swap-70 and Swap-70 mice were homogenized through a cell strainer.
- After red blood cell lysis, cells were washed once and seeded at a density of 1×106 cells/ml in MEM supplemented with 10% fetal calf serum (FCS) , 1% Penicillin/Streptomycin (10000 U/ml/10 mg/ml ), 50 µM 2-mercaptoethanol, 10% GM-CSF supernatant obtained from J558 cells, and 1 ng/ml recombinant human TGF-β (& or) into a 24 well plate (1 ml/well) at 37°C in a humidified atmosphere and 5% CO2.
- Every 4 to 5 days half of the medium was changed.
- DCs were used at days 12 to 16 of culture at which point 80 to 90% of the cells were CD11c positive as analyzed by FACS (data not shown).
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Crypt isolation, Enteroid Culture and Analysis
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The pelleted epithelium was re-suspended directly into hESC Matrigel supplemented with 100 ngmL−1 recombinant mouse Noggin , 500 ngmL−1 recombinant human-Spondin , 50ngmL−1 recombinant mouse EGF , 100 ngmL−1 recombinant human Wnt3a (& systems), 10 µM Y-27632 , 10 µM SB202190 , and 500 nM LY2157299 .
- Between 50 and 200 crypt/villi units were plated in 50 uL of matrigel on a 48 well plate.
- After allowing the matrix to polymerize for 30 min at 37°C, each well was overlaid with 500 µL of Advanced DMEM/F12 containing the supplements 1× N-2 supplement , 1× B-27 supplement minus vitamin A , 1× Glutamax , 100 µgmL−1 penicillin/streptomycin, 1 mM Hepes buffer , and 1 mM N-Acetylcysteine.
- Growth factors were added to the media 48 hr after plating and every 72 hr following that.
- The entire volume of media was changed 72 hr following plating and every 72 hr after that.
- Every 1–2 weeks organoids…
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The pelleted epithelium was re-suspended directly into hESC Matrigel supplemented with 100 ngmL−1 recombinant mouse Noggin , 500 ngmL−1 recombinant human-Spondin , 50ngmL−1 recombinant mouse EGF , 100 ngmL−1 recombinant human Wnt3a (& systems), 10 µM Y-27632 , 10 µM SB202190 , and 500 nM LY2157299 .
- Between 50 and 200 crypt/villi units were plated in 50 uL of matrigel on a 48 well plate.
- After allowing the matrix to polymerize for 30 min at 37°C, each well was overlaid with 500 µL of Advanced DMEM/F12 containing the supplements 1× N-2 supplement , 1× B-27 supplement minus vitamin A , 1× Glutamax , 100 µgmL−1 penicillin/streptomycin, 1 mM Hepes buffer , and 1 mM N-Acetylcysteine.
- Growth factors were added to the media 48 hr after plating and every 72 hr following that.
- The entire volume of media was changed 72 hr following plating and every 72 hr after that.
- Every 1–2 weeks organoids were passaged at a 1∶5 ratio by mechanical dissociation, pelleting, and re-plating the pellet, into new Matrigel containing the growth supplements described above.
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Induction of cholangiocytic cyst formation by human iPS cell-derived CD13highCD133+ cells
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Colonies derived from CD13highCD133+ cells were passaged and trypsinized using 0.05% trypsin-EDTA, washed in DMEM containing 10% FBS, and then counted.
- The cells were then combined with an extracellular matrix gel consisting of a mixture of 40% collagen type-I and 40% Matrigel , and cultured in 24-well culture plates (1500 cells/50 µl extracellular matrix gel/well).
- After the 10 min incubation, culture medium was added, followed by incubation for 10–12 days with medium changes every 3 days.
- The culture medium was a 1∶1 mixture of medium'>H-CFU-C medium and MEM/F-12 supplemented with 2% B27 supplement, 0.25 µM A-83-01, 10 µM Y-27632, 20 ng/ml EGF, 40 ng/ml HGF, 40 ng/ml recombinant human Wnt-3a , and 100 ng/ml recombinant human-spondin 1 .
- Cysts in gels were stained according to previously described methods
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Colony forming analysis
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celltype'>Leukemic cell lines, MNCs from the patients with, or healthy controls were infected with or without the indicated viruses (50 MOI) and seeded in triplicate in a mixture containing 1.35% methylcellulose in medium'>Iscove's medium'>modified medium'>Dulbecco's medium (IMDM;) supplemented with 20% fetal bovine serum and10−4 M 2-Mercaptoethanol .
- For colony assay of bone marrow cells, 3 U/mL recombinant human (rh) erythropoietin, 50 ng/mL rh stem cell factor, 30 ng/mL rh granulocyte macrophage–colony-stimulating factor, and 10 ng/mL rh interleukin-3 were added to the methylcellulose medium.
- The colonies were evaluated under a microscope on day 12 of culture.
- In contrast, leukemic cell lines were seeded in cytokine-free mixture as described previously [
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Induced pluripotent stem cell-directed differentiation
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For hepatocyte differentiation, post-excised iPSCs were grown on Matrigel as stated above until reaching a 60 to 70% confluence upon which endoderm induction was initiated by replacing the post-excised iPSCs for 24 hours with RPMI 1640 medium , supplemented with 0.5 mg/ml albumin fraction V , and 100 ng/ml Activin A .
- On the following 2 days, 0.1 and 1% insulin–transferrin–selenium were added to the medium, respectively.
- Post-excised iPSCs were then cultured in hepatocyte culture medium containing 30 ng/ml fibroblast growth factor-4 and 20 ng/ml BMP2 for 4 days.
- The now-differentiated cells were then incubated in hepatocyte culture medium'>medium containing 20 ng/ml hematopoietic growth factor and 20 ng/ml keratinocyte growth factor for 6 days, in hepatocyte culture medium'>medium containing 10 ng/ml oncostatin-M plus 0.1 μM dexamethasone…
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For hepatocyte differentiation, post-excised iPSCs were grown on Matrigel as stated above until reaching a 60 to 70% confluence upon which endoderm induction was initiated by replacing the post-excised iPSCs for 24 hours with RPMI 1640 medium , supplemented with 0.5 mg/ml albumin fraction V , and 100 ng/ml Activin A .
- On the following 2 days, 0.1 and 1% insulin–transferrin–selenium were added to the medium, respectively.
- Post-excised iPSCs were then cultured in hepatocyte culture medium containing 30 ng/ml fibroblast growth factor-4 and 20 ng/ml BMP2 for 4 days.
- The now-differentiated cells were then incubated in hepatocyte culture medium'>medium containing 20 ng/ml hematopoietic growth factor and 20 ng/ml keratinocyte growth factor for 6 days, in hepatocyte culture medium'>medium containing 10 ng/ml oncostatin-M plus 0.1 μM dexamethasone for 5 days, and in MEM containing , , nonessential amino acid, and β-mercaptoethanol (all from) for 3 more days.
- Media were changed daily during differentiation [
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