Cell lines and culture conditions
EKP cells derived from C57BL/6 mice were purchased from Millipore (Billerica, MA). NSCs, derived from Balb/C mice, were purchased from American Type Culture Collection (Manassas, VA). MSCs were obtained from freshly isolated bone marrow following established protocols (MSCs, Technical Manual, Stem Cell Technologies, Vancouver, BC, Canada). Wild-type, Casp3−/, −Casp7−/−, and Casp3−/−Casp7−/− double knockout MEFs were obtained from Dr. Richard Flavell of Yale University (New Haven, CT). EPK cells were maintained in Epidermal Keratinocyte medium from Millipore (catalog#CNT-02). NSC cells were grown in neural stem cell medium (Millipore, catalog#SCM003). Wild-type and caspase-knockout MEF cells were cultured in DMEM medium with 5% FBS.
Nude mice were obtained from National Cancer Institute (Bethesda, MD). Wild-type C57BL/6, −Casp3−/−, and −Casp7−/− mice, and enhanced green fluorescent protein (EGFP)-expressing transgenic mouse (all in C57/BL6 background) were obtained from the Jackson Laboratory (Bar Harbor, Maine, US).The transgenic mice were deposited by Dr. Richard Flavell. All animal procedures were approved by the University of Colorado Denver Institutional Animal Use and Care Committee. Mice were kept in the vivarium located on the Anschutz Medical Campus of UC Denver, Aurora, CO.
Genetic manipulation of the cell strains
For many experiments, we used the pLEX lentiviral vector system (52
) from Open Biosystems (Huntsville, AL) for transduction of various genes into MEF cells and various stem or progenitor cells. The genes transduced with pLEX include (i) firefly luciferase gene, which was transferred from the plasmid pGL4.31-luc2 from Promega (Madison, Wisoconsin); (ii) a truncated version of the mouse iPLA2
, which was amplified through RT-PCR from murine mRNA with the following primers:
We used the Pfx polymerase (Invitrogen, Carlsbad, CA) for the PCR amplification. The amplified fragment encodes aa453–679, which is equivalent to aa514–733 of human iPLA2(35
), of murine iPLA2
(accession# NM-016915). This fragment is a constitutively active, caspase-cleavage product (34
). The fragment was cloned into the Spe I and Not sites of a modified pLEX plasmid. The modified pLEX plasmid has an influenza hemagglutinin (HA) tag inserted between Not I and Mlu I (two of the unique restriction sites in pLEX) so that genes inserted into the NotI site can be fused with the HA tag.
We obtained lentiviral vectors encoding shRNA-encoding minigenes targeted against the murine iPLA2 gene from Open Biosystems (Huntsville, AL). These minigenes were carried in the pLKO.1 lentiviral vectors system. The most effective one had the following targeting sequence: 5’GCGTATGAAGGACGAGGTGTTTCTCGAGAAACACCTCGTCCTTCATACGTTTTTTG-3’ (catalog#Rmmu534-NM-016915 from Open Biosystems), in bold red are the sense and antisense targeting sequences and in underlined blue is loop sequence). We followed the manufacturer’s instructions to produce live recombinant lentiviral vectors in 293T cells.
Bioluminescence imaging of cultured cells
Proliferation of Fluc-labeled stem or progenitor cells on lethally irradiated feeder cells was tracked though bioluminescence imaging. Imaging of the cells was performed with an IVIS200 instrument from Caliper Life Sciences (Hopkinton, MA). Luciferin (obtained from Caliper Life Sciences) was added to the mediaat a concentration of 150 µg/ml. After 5–10 minutes of incubation, cells cultured in Petri dishes or multiwell plates were imaged in the instrument. Quantification of signal strength was performed with manufacturer supplied software.
Bioluminescence imaging of cells implanted in mice
Growth of Fluc-labeled stem or progenitor cells in vivo was followed through noninvasive bioluminescence imaging with the IVIS200 instrument. Mice were injected with 150 mg/kg of D-luciferin in 200 µl of PBS and imaged 10 minutes later. The time between injection and imaging was kept constant among different batches of mice.
Irradiation of cells and mice
X-ray irradiation of cells and mice were carried out in a RS-2000 Biological Irradiator [Rad Source Corporation (Atlanta, GA)] located in the vivarium of University of Colorado Denver Anschutz Medical Campus.. The dose rate for the machine is about 1 Gy/minute.
Measurement of arachidonic acid release
To measure arachidonic release, we plated cells in 6-well plates at a density of 2.0 × 105 cells/well. About 1.0 µCi of [3H]-archidonic acid (GE Healthcare Life Sciences) was added to the cells cultured in about 1 ml of serum-free DMEM with 0.5 mg/ml lipid-free bovine serum albumin (Sigma Chemical Co., St Louis, USA). After 16 hrs, the cells were washed with fresh media 3 times and incubated with 3 ml of DMEM supplemented with 5% serum. After 5 hrs, when archidonic acid in the supernatant reaches a steady state level, the cells were exposed to 8 Gy of x-rays. Supernatants were then removed at 4, 24, and 48 hrs from the cells and counted with a scintillation counter for quantification of [3H]-arachidonic acid.
ELISA measurement of PGE2
To evaluate the PGE2 secretion from cells, we plated about 4 × 104 cells/well in 24-well plates. Cells were cultured in 1 ml of DMEM supplemented with 2% fetal bovine serum. Cells were then irradiated with x-rays (10 Gy). Supernatant from the cells was removed before and 48 hrs after cellular irradiation. PGE2 in the supernatants was measured with an ELISA kit from R&D Systems (Minneapolis, MN, USA).
Skin excision wound healing assay
To characterize the rate of wound healing in different strains of mice, punch biopsies were created in the dorsal skin of mice. The biopsy procedure was carried out using VisiPunch devices from Huot Instruments (Menomonee Falls, WI, USA). Usually, two 4-mm excision wounds were created in the dorsal skin of each mouse. Monitoring of the rate of healing was performed by measuring the diameter of the wounds with a caliper every other day. For histological analysis, skin biopsies containing the full-thickness wounds were taken from mice sacrificed at different time points after generating the excision wounds. About 60 minutes before the sacrifice, mice were injected with BrdU at 50 mg/Kg. The tissue samples were paraffin-embedded at the Histology Core in the Dept of Dermatology at University of Colorado Denver.
For BrdU immunofluorescence staining, an Alexa Fluor 594 conjugated anti-BrdU, mouse monoclonal antibody from Invitrogen (Carlsbad, CA) was used at a dilution of 1:20. To quantify the number of cells positive for BrdU staining, four randomly chosen 100X fields were counted.
To analyze skin epithelial cells and keratinocytes through immunofluorescence staining, antibodies against cytokeratin 14 and cytokeratin 6, respectively, were used. These were rabbit polyclonal antibodies produced in-house and described previously (53
Partial hepatectomy-liver regeneration assay
To quantify liver regeneration, we adopted a published protocol (33
). Briefly, mice (8–10 weeks old) were anesthetized with isofluorane. After restraining the mouse, a 3-cm incision was created in the midline of abdominal skin and muscle to expose the xiphoid process. The median and left lateral lobes were tied around the top of the lobes using 4-0 silk surgical thread. The lobes were then removed surgically. This removes about 2/3 of the total liver weight. After suturing the abdomen closed, the mice were placed on a warm pad to recover. At different times after hepatectomy, mice were sacrificed for evaluation. About 60 minutes before the sacrifice, BrdU (50mg/kg) were injected i.p. into the mice to label proliferating cells. Regeneration rate of the liver was quantified with two methods.The weight of the two lobes of liver left intact in mice were determined and normalized to the weight of the same two lobes right after the initial surgery to derive a relative liver weight. In theory, the range should be from 1.0 (right after surgery) to 3.0 (100% recovery). Alternatively, immunofluorescence staining of BrdU-positive cells was performed in sections of paraffin-embedded liver tissues. Liver regeneration was then quantified through enumeration BrdU-positive cells in 4 randomly chosen 200x fields.
Modified DIVAA assay for tissue regeneration
To quantify the ability of different cells to induce tissue regeneration, we adapted the DIVAA™ from Trevigen (Gaithersburg, MD). The original purpose of this kit was to measure the ability of various growth factors to induced angiogenesis in vivo. The basic procedure involves mixing growth factors at different concentrations with basement membrane extract (BME) and loading the mixture (in a total volume of 20 µl) into silicone cylinders called the “angioreactor”. In our experiments, instead of mixing growth factors with the BME, we mixed various MEFs with BME. For each angioreactor, we used about 2 × 105 cells in a final volume of 20 µl. We surgically implanted up to four the angioreactors subcutaneously into each nude mouse. Because the angioreactor has only one sealed end, the host tissue and vasculature can grow into it. Two weeks after implantation, host mice were sacrificed and the angioreactors were removed. The contents of the angioreactors were then transferred to microtubes and their endothelial cell content was quantified by FITC-lectin staining following manufacturer (Trevigen)’s instructions.