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, USA). SSEA-1 and SSEA-4 were supplied by the Developmental Studies Hybridoma Bank, University of Iowa, USA; TRA-1-60 and Oct-4 were supplied by Chemicon.

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 (Fermentas, USA), and 1??PCR reaction buffer. The sequences of the primer were as following: Oct4 (forward, cgtgaagctggagaaggagaagctg; reverse, caagggccgcagcttacacatgttc), Rex1 (forward, ctgaagaaacgggcaaagac; reverse, gaacattcaaggg-agcttgc), and Nanog (forward, caaaggcaaacaacccactt; reverse, ctggatgttctgggtctggt) (Invitrogen). ??-actin (200 bp) was used as positive control. PCR started with an initial denaturation at 95?C for 10 minutes, followed by 38 amplification cycles (94?C for 45 seconds, 56?C for 45 seconds, and 72?C for 45 seconds), followed by 7 minutes at 72?C. The PCR products were electrophoresed on 1.5% agarose gels stained with ethidium bromide.

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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