Establishment and characterization of human embryonic stem cell line (2)
Alkaline phosphatase activity assay
The activity
of alkaline phosphatase was assessed by histochemical staining. Human ES cells were fixed in 4% paraformaldehyde at room temperature
for 15 minutes, washed twice with PBS, stained with BCIP/NBT (Maxim Biotech Inc.) for 20-30 minutes and detected for colour reaction
by light microscopy. Human ES cells stained for alkaline phosphatase activity, but colonies spontaneously differentiated and feeder
layers did not.
Immunocytochemistry analysis
To detect the expression of SSEA-1, SSEA-4, TRA-1-60, and Oct-4, hES cells were processed
for immunocytochemistry using standard protocol from vendor's instructions. Briefly, undifferentiated hES cells grown on a layer of
mouse embryonic fibroblasts were fixed within 4% paraformaldehyde, and incubated with primary monoclonal antibodies at appropriate
dilution at 4?C. Then the cells were treated with fluorescent conjugated secondary antibody diluted at 1 : 500 (v/v) (Cy3 Jackson
Immuno Research,
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
Total RNA was extracted from
hES cells cultured on MEFs, or from control MEFs with Trizol reagent (Invitrogen) according to the vendor's protocol. DNase I was
used to eliminate genomic DNA contamination and ensure that the PCR product represented the specific mRNA species. cDNA were produced
by reverse transcription reaction with 500 ng of total RNA using oligo(dT) as primers (Invitrogen) and Moloney murine leukaemia virus
reverse transcriptase (Invitrogen). cDNA samples were amplified by PCR with DNA primers selective for the human genes. The PCR reaction
was carried out in a volume of 10 ??l containing 2 ??l of cDNA template, 2.5 mmol/L MgCl2, 200 ??mol/L of each deoxynucleotide triphosphate,
200 nmol/L of each primer, 0.05 U Taq DNA polymerase (
Karyotype analysis
For chromosome analysis, hES cells in the exponential growth stage
were treated for 2 hours with colcemid (0.1 ??g/ml). After colcemid treatment, digested single cells were karyotyped using G-band
method.
In vitro differentiation
To induce the formation of EBs, undifferentiated hES cell colonies in 6-well plates were digested
with trypsin/EDTA, then transferred to nonadhesive plastic petri dishes to allow aggregation and prevent adherence to the plate. The
EBs were cultured in EB medium consisting of 85% knockout DMEM supplemented with 15% knockout serum replacement, 2 mmol/L L-glutamine,
0.1 mmol/L ??-mercaptoethanol and 1% nonessential amino acids. The medium was changed every day.
For the detection of differentiated
derivatives by immunocytochemistry, EBs (day 7) were plated on 0.2% gelatin coated 6-well plates, grown in DMEM containing 10% FBS
to induce further differentiation for 14 days and fixed in 4% paraformaldehyde followed by fluorescent immunostaining. The following
antibodies were used: microtubule associated protein 2 and ??-smooth muscle actin (Sigma) and albumin (Dako).
Teratoma formation in
nude mice
During routine passage, the colonies were digested with trypsin EDTA solution for 1 to 3 minutes, and dissociated into clumps
of 50 to 100 cells. These clumps were collected and injected subcutaneously into the hind leg of four to five-week old nude mice (two
or more mice per cell line). Six weeks after transplantation, the teratoma became apparent and was removed, fixed for 4-8 hours in
4% paraformaldehyde, embedded in paraffin and cut in 10 ??m sections. After staining with haematoxylin and eosin, the sections were
observed under light microscope
References:HUANG Guo, LI Wei-qiang, CHEN Rui et al. Chinese MedicalJournal, 2007, Vol.120 No.7 :589-594
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