Isolation and Culture of Multipotent Stem Cells from Human Bone Morrow

Bone marrow contains three types of stem cells:

1. Hematopoietic stem cells give rise to the three classes of blood cells that are found in the circulation: white blood cells (leukocytes), red blood cells (erythrocytes), and platelets (thrombocytes).

2. Mesenchymal stem cells are found arrayed around the central sinus in the bone marrow. They have the capability to differentiate into osteoblasts, chondrocytes, myocytes, and many other types of cells. They also function as "gatekeeper" cells of the bone marrow.

3. Endothelial stem cells

The bone marrow stroma contains mesenchymal stem cells (also called marrow stromal cells).  These cells are multipotent stem cells that can differentiate into a variety of cell types.  Cell types that MSCs have been shown to differentiate into in vitro or in vivo include osteoblasts, chondrocytes, myocytes, adipocytes, and, as described lately, beta-pancreatic islets cells.  They can also trans-differentiate into neuronal cells.

Materials and Methods

1. Fresh unprocessed human bone marrow (BM) from young male donors was isolated.

2. BM was centrifuged at 350 g for 10 minutes to obtain cell pellets, which then were resuspended in 25 ml of Dulbecco’s PBS (DPBS) containing 0.5 M EDTA.

3. After centrifugation at 350 g for 7 minutes, the cells were resuspended in 5 ml DPBS containing 0.5 M EDTA and 20 ml of NH4Cl for induction of hemolysis.

4. After centrifugation and washing with DPBS containing 0.5 M EDTA, cells were filtered through a 40-μm nylon filter and plated in wells of 6-well plates that had been coated with fibronectin (100 μg/ml).

5. The cells were grown at a density of 5 × 106 per square centimeter in complete DMEM with low (1 g) glucose containing 17% of FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin and 2 mM glutamate at 37°C and 5% CO^₂.

6. Once the attached cells began to form colonies (4–6 days), the medium was replaced and the adherent cells were grown to 60% confluence.

7. The cells were reseeded in complete medium into 25-cm2 tissue culture flasks at a density of 1 × 10⁴ cells per square centimeter.

8. After the cells were 60% confluent, they were serially reseeded into 75-cm2 and 175-cm2 flasks at the same density.

9. After at least 2 passages of cultures in 175-cm² flasks, cells were labeled with CellTracker CM-DiI (DiI; Invitrogen Corp.), plated into the wells of a 96-well plate at a density of 0.5 cell per well by limiting-dilution method, and cultured with conditioned media, which were collected prior to limiting dilution, stored in –80°C, and filtered through a 0.22-μm sterile filter.

10. After the exclusion of wells containing more than 1 cell under fluorescent microscopy, the clones derived from a single cell were further cultured.

11. When these cells were grown to 40–50% confluence, cells from 1 well were reseeded into 1 well of a 6-well plate and thereafter serially reseeded in 25-cm², 75-cm², and 175-cm² flasks when cells reached 30% confluence.

12. When the cells reached a density of 4 × 10³ to 8 × 10³ cells per square centimeter in 175-cm2 flasks, they were replated at 1:40 to 1:10 dilution; this process was repeated through 140 population doublings (PDs).

13. All reseeding was performed in triplicate, and the fastest-growing clone was selected through 50–60 PDs.