Derivation and characterization of a ES-like cell line from indian catfish Heteropneustes fossilis blastulas

Abstract

A cell line designated as HFB-ES was established from blastula stage embryos of H. fossilis (Singhi). The embryonic cells were harvested and maintained in Leibovitz's medium supplemented with 15% fetal bovine serum. The cell line had been subcultured for more than 90 passages in a period of 24 months. HFB-ES cells were able to grow at temperatures between 25 and 35°C with an optimum temperature of 28°C. The growth rate of HFB-ES was proportional to FBS concentration, with optimum growth seen at 15% FBS concentration. The originality of the cell line was confirmed by sequencing of cytochrome oxidase c subunit I (COI), cytochrome b gene, and microsatellite DNA profile. Results of chromosome complements of HFB showed normal karyo-morphology with 56 (2n) diploid number of chromosomes after 40 passages which indicated that the developed cell line is chromosomally stable. The pluripotency of HFB was demonstrated by alkaline phosphatase activity and Oct-4 gene expression. Expression of GFP reporter gene was successful in HFB-ES. These results indicated that HFB-ES could be utilized for future gene expression studies.

Figure 1

Developmental stages of H. fossilis embryonic cells (4x).

Figure 2

Development of embryonic cell line from blastula stage embryo of H. fossilis. (a) Confluent monolayer (10x). (b) Cells after 50 passages, showing polygonal cells (20x).

Figure 3

Evaluation of growth parameters of HFB-ES. (a) Effect of temperature. (b) Effect of FBS concentration (%).

Figure 4

Embryonic cells cryopreserved (150 days). (a) Immediately after thawing. (b) Cryopreserved embryonic cells after 24 h of thawing. (10x).

Figure 5

Confirmation of originality of cells: relatedness drawn between HFB-ES cells and H. fossilis based on the (a) partial COI sequences. (b) Cytochrome b mtDNA genes and C. batrachus sequences obtained from NCBI database were used as outgroup.

Figure 6

Karyotype of H. fossilis embryonic cell. (a) Metaphase spread and (b) karyotype, m = metacentric, sm = submetacentric, st = subtelocentric, and t = telocentric. Bar—5 μm.

Figure 7

Frequency distribution of chromosomes in metaphase spreads of HFES cells.

Figure 8

Embryoid body formation in embryonic cells of H. fossilis after retinoic acid treatment. White arrowheads showing embryoid bodies (100x).

Figure 9

GFP expression in HFB-ES using phMGFP vector. Transfection was done with 2.0 μg vector/100 μL of cells. White arrowheads show the expression of GFP in transfected cells (100x).

Figure 10

Immunofluorescence shows the expression of Oct-4 protein in embryonic cells. Blue colour dot showed the DAPI stained nucleus and green colour shows the expression of Oct-4 (1.5 μg/mL concentration of Oct-4 antibody used) (100x).

Figure 11

Alkaline phosphatase (AP) staining showed the pluripotency in embryonic cells (HFB-ES). White arrowheads show the purple coloured AP positive embryonic cells after 24 h of incubation (20x).