Mesenchymal Stem Cells Induce a Reversible Suppression of Alloimmune Reactions.

Objective Bone marrow contains pluripotent mesenchymal stem cells (MSC) that form bone, cartilage, adipose tissue and muscle. These cells are not immunogenic and escape recognition by alloreactive T cells and natural killer cells. MSC can be readily isolated from bone marrow and expanded ex vivo without modification in phenotype or loss of function. They are capable of differentiating along multiple mesenchymal lineages, play a crucial role in the bone marrow microenvironment and have profound immunosuppressive properties. In the present investigation we analysed the antiproliferative capacity of MSC using primary and secondary immune responses in vitro.

Methods MSC were isolated from heparinized bone marrow of healthy donors (n=5). Bone marrow aspirates were layered onto a Percoll cushion (density 1,073g/ml) and the MSC-enriched mononuclear fraction was collected, washed and resuspended in Dulbecco modified Eagle medium with low glucose and 10% fetal bovine serum. The cells were plated at 2x10⁵/cm² and maintained at 37°C in a humidified atmosphere and subcultured prior to confluency.

MSC stained positive for specific markers as SH2, SH3 and SH4 but were negative for CD14, CD34 and CD45. Immunsuppressive effects were assessed by adding third party MSC to primary mixed lymphocyte reactions (MLR) in decreasing concentrations (10%, 1%, 0,1%) on days 0–5. Cell proliferation was measured on day 6 by means of an 18-hour pulse with 3H-thymidine (3H-TdR). Secondary MLRs were performed by restimulating MSC co-cultured MLRs with the primary PBMC on day 10. In addition secondary MLRs were also tested in presence of MSC and cultured for 1, 2, 3 and 6 days in 96-well plates and proliferation was detected by 3H-TdR uptake.

Results Primary MLRs show significant inhibition of proliferation with 10% MSC added on day 0 (85% inhibition), 1 (69% inhibition) or 2 (70% inhibition) (p<0,01). Similar effects were observed by adding 1% MSC on day 0 (42% inhibition) and 1 (10% inhibition). Co-cultivation with MSC over 10 days lead to delayed proliferation in secondary MLRs with peak on day 3. Secondary MLRs were also inhibited by the addition of 1% (47% inhibition) and 10% (56% inhibition) MSC independent from co-cultivation with MSC in the primary culture (11% and 44% inhibition respectively). Restimulation with third party PBMC on day 10 lead to a primary response with a proliferation peak after 6 days.

Conclusions Our findings emphasize the immunosuppressive effects of MSC. We observed that MSC-induced inhibition of primary MLRs was reversible in secondary MLRs. This clearly demonstrates that MSC do not induce tolerance and could be used in a clinical setting due to their immunomodulatory properties.