2.1.1 Stem cells and small cells
Generally speaking, stem cells are small. The ratio of cytoplasm and nuclear is one of remarkable indexes of stem cells. However, the isolation method of only small cells is yet established.
2.1.2 Stem cells and sphere formation
Sphere formation is recognized as one of the results of stem cell property since stem cell is recognized that they have strong proliferative potential and self-renewal potency. Recently, many reports demonstrated that various adult tissues contained sphere forming cells. Cells derived from many adult tissues, including retina, brain, cornea, olfactory neuroepithelium, pancreas, skin, muscle and bone marrow have been propagated as non-adherent clusters or spheres, as have embryonic stem cells.
Cells contained within the spheres described in those reports, exhibit neural lineage markers and appear to possess varying degrees of stem cell potency. We believe that adult stem cells described in various reports represent the same adult stem cells at different stages of development, expressing different degrees of potency. Sphere forming cells are much more immature than previously expected. We hypothesized that adult stem cells procured from any tissue, endoderm, mesoderm or ectoderm, could demonstrate a wide multipotency and cross germ layers when maintained in appropriate environments. Therefore, we first investigated how to isolate small cells. And then we examined if they had sphere forming potency in the serum-free condition.
2.2.1 Isolation of small cells
Bone marrow, lung, muscle and spinal cord tissues were procured from 3- to 4-week-old C57BL/6J mice as described below. Bone marrow was acquired by flushing femur and tibia with culture media using an insulin syringe. Cells were plated at 1x10^ cells/cm" in F12/DMEM (1:1, v/v) supplemented with 2% B27, 20ng/ml bFGF and lOng/ml EGF
To isolate small cells from murin bone marrow, the following three types of methods were examined.
A. Cell sorter
Forward scatter was calibrated by size-defined beads. Less than 8 micro meters in diameter cells were isolated.
B. Optimistic<→Osmotic> pressure
Bone marrow cells were exposed to low optimistic<→osmotic> pressure liquid to destroy mature cells.
C. Trituration using thin-glass pipette
Standard glass pipettes were burned and stretched out to make thin tips. Mature cells were passed through thin glass pipettes many times and destroyed by mechanical stress. Obtained small cells were cultured in serum free medium, and appeared spheres were counted as a number of stem cells.
2.2.2 Characterization of small cells
Immunohistochemistry. Protein expression was assessed using immunohistochemical techniques as described below. Each slide was incubated with anti-c-kit rat monoclonal antibody, anti-Sca-1 rat monoclonal antibody or anti-E-cadherin rat monoclonal antibody. After washing with PBS, the cells were incubated with goat anti-rat IgG Texas Red-conjugated antibodies and goat anti-rat IgG Fluorescein-conjugated antibodies. SSEA-1 and Alkaline phosphatase (AP) staining was performed using the ES cell detection Kit
Single sphere RT-PCR. Single spheres were individually collected under the microscope. Total RNA was extracted from each single sphere, and then subjected to oligo-dT-primed reverse transcription (RT). RT-PCR was performed using TaqDNA polymerase on an iCycler for 35 cycles.
2.3.1 Sphere forming from isolated cells
Trituration and optimistic<→osmotic> pressure produced spheres. Whereas, cell sorter did not produced any spheres. Trituration produced most number of spheres. Therefore, we utilized trituration as the isolation method of small cells.
2.3.2 Effect of trituration
After the trituration, the population of small cells was increased comparing to native bone marrow cells (Fig. 3A and B). However not all of cells over 8 micro meters in diameter disappeared. Triturated cells formed spheres during culture.Interestingly, spheres were consisted of only small cells (Fig. 3C). Thus, it was demonstrated that trituration enabled to culture and grow only small cells.