MSCs have recently been used experimentally in clinical trials of cardiovascular repair (15
), treatment of lung fibrosis (17
), spinal cord injury (18
) and bone and cartilage repair (4
) as a stem cell source. MSCs have been successfully used for therapeutic application when they are derived from bone marrow (20
Generally, an accepted range of HSC dose for engraftment after bone marrow transplantation is 3-4×106 CD34+ cells/kg of recipient body weight for humans. Considering that a mouse weighs about 30 g on average, we postulated that 9-12×104 CD34+ cells should be a reasonable dose range for a mouse, and therefore infused 1×105 CD34+ cells.
Usually, the fluorescence intensity of CD34+ cells when conjugated with FITC is weaker than that when conjugated with PE using flow cytometry. Therefore, it is a matter of course that the percentage of CD34+ fraction may is different to some extent according to the conjugated materials even though the testing samples are exactly the same. Authors usually evaluate the purity of overall CD34+ cell fraction stained with PE according to the manufacturer's protocol of CD34 sort kit (Miltenyi Biotec, Germany). is intended to show the percentage of CD34+/CD38- fraction which is known to be more primitive and have higher engraftment potential than CD34+/CD38+ fraction using PE-conjugated CD38 and FITC-conjugated CD34.
At present we do not know how long MSCs will maintain innate characteristics including the ability to differentiate and cytokine production; nor do we know what the best composition culture media should contain for maintaining MSCs. We used early MSCs not exceeding 3 passages based on the assumption that early passage cells would be more likely to have the innate characteristics of MSCs.
Cotransplantation of HSCs and MSCs enhanced engraftment as the dose of the MSCs were increased up until a 1:8 ratio. The percent donor chimerism in the 1:16 group, however, was lower than that of the 1:8 group even though it was still higher than the HSCs only group as shown in the experiment 3. These findings suggest that HSCs and MSCs have a threshold ratio of HSCs to MSCs for enhancing engraftment when cotransplanted into a NOD/SCID mouse. Our findings suggest that the optimal ratio of HSCs to MSCs in cotransplantation might be between 1:8 and 1:16 and we are conducting further experiments using these ratios. We confirmed MSCs engraftment enhancing ability in 3 experiments. Each experiment was performed with bone marrow-derived MSCs obtained from a different volunteer donor, and showed different enhancing abilities. For example, the mean fold-increase of the 1:4 ratio group was 3.99 compared with the HSCs only group in the experiment 1, while it was 7.21 in the experiment 2, and 1.21 in the experiment 3. The CD34+ cells were also derived from different donors; however, it is unlikely that the quality of MSCs is the sole determinant of engraftment efficiency. It is clear that the CD34+ cell dose has been shown to be strongly associated with engraftment kinetics regardless of individual differences (21
In addition, the proliferation rate and morphology of the 3 MSCs experiments differed when observed during cell expansion before cotransplantation (data not shown). Therefore, our findings suggest that the engraftment enhancing ability of MSCs might have considerable difference according to the MSC donor.
Our study demonstrated that MSCs could enhance engraftment in a dose-dependent manner up to a certain level in NOD/SCID mice. Future studies should help elucidate further mechanisms associated with engraftment such as secretion of specific MSC-cytokines that help enhance engraftment. For clinical application to human patients characteristics of MSCs including immunosuppression and HLA restriction require further explanation.