Isolation and characterization of human spermatogonial stem cells

Abstract

Background: To isolate and characterization of human spermatogonial stem cells from stem spermatogonium.

Methods: The disassociation of spermatogonial stem cells (SSCs) were performed using enzymatic digestion of type I collagenase and trypsin. The SSCs were isolated by using Percoll density gradient centrifugation, followed by differential surface-attachment method. Octamer-4(OCT4)-positive SSC cells were further identified using immunofluorescence staining and flow cytometry technques. The purity of the human SSCs was also determined, and a co-culture system for SSCs and Sertoli cells was established.

Results: The cell viability was 91.07% for the suspension of human spermatogonial stem cells dissociated using a two-step enzymatic digestion process. The cells isolated from Percoll density gradient coupled with differential surface-attachement purification were OCT4 positive, indicating the cells were human spermatogonial stem cells. The purity of isolated human spermatogonial stem cells was 86.7% as assessed by flow cytometry. The isolated SSCs were shown to form stable human spermatogonial stem cell colonies on the feeder layer of the Sertoli cells.

Conclusions: The two-step enzyme digestion (by type I collagenase and trypsin) process is an economical, simple and reproducible technique for isolating human spermatogonial stem cells. With little contamination and less cell damage, this method facilitates isolated human spermatogonial stem cells to form a stable cell colony on the supporting cell layer.

Figure 1

Isolated SSCs tend to congregate and form small cell clusters.

Figure 2

Immunofluorecent staining of SSCs, detecte Oct-4 positive cells. A: After Isolating, cells observed under inverted phase contrast microscope(×100)B: Show green fluorescent cells are Oct-4 positive cells observed under Immune fluorescence microscope(×100).C: Show green fluorescent cells observed under Immune fluorescence microscope(×800).D: The negative control groups of rabbit serum replace antibody, cells observed under Immune fluorescence microscope(×100).

Figure 3

Immunofluorecent staining of SSCs, detected SSEA-4 positive cells. Cells were observed under immune fluorescence microscope(×400).

Figure 4

SSCs observed under inverted phase contrast microscope at different times. a, b, c: SSCs at 48 h of culture on the feeder layer of Sertoli cells (×100, ×200, ×400, respectively); d: SSCs at 30 days of culture on the feeder layer of Sertoli cells (×100).

Figure 5

Statistical analysis of MTT values (72 h vs. 24 h, 48 h and 96 h, P < 0.01) was performed. The A is absorption value at a wavelength of 570 nm. Human SSCs in vitro has a value-added short period, but then gradually reduced the number of cells. The results shown are from three trials.

Figure 6

a, b, c: SSC populations at 24 h, 72 h, and 144 h of culturing, respectively, using an inverted phase contrast microscope (×400). The largest number of cells in 72 h, 144 h at least.

Figure 7

Oct-4 positive cells detected using flow cytometry. Two clusters of cells were segregated, of which the positive cells accounted for 86.7%.