Background

MicroRNAs are non-coding small RNAs that reportedly regulate mRNA targets or protein translation of various tissues in physiological and pathological contexts. This study was undertaken to characterise the contributions of microRNA-29a (miR-29a) to the progression of estrogen deficiency-mediated excessive osteoclast resorption and bone loss.

Mesenchymal stem cells (MSCs) are believed to be immune-privileged due to lack of antigen-presenting-cell related markers, however, evidence suggests that MSCs are immunogenic and are attacked by the immune system. Our research investigates the hypothesis that there are differences between MSC clones from the same individual in terms of their morphology, proliferation, differentiation and immune profile. Our goal is to discover immune-privileged stem cells, which can act as a universal allogenic mesenchymal stem cell donor to facilitate bone ingrowth for osteosarcoma patients status post tumor excision and prosthesis implantation.

Serial dilutions of bone-marrow derived (BMMSCs) and adipose derived mesenchymal stem cells (ADMSCs) from same animal were carried out in order to isolate single-cell clones. From a single animal we obtained 3 clones from BMMSCs and 3 from ADMSCs. This procedure was repeated for another other 2 animals. The proliferation rate and cell doubling time of each clonal culture was measured. The proliferation rate of mixed clonal cultures was also measured. The tri-differentiation potential of the clonal cultures was compared and a comparison was also made with the original isolates from bone marrow and fat. The immune-privileged properties were measured by flow cytometry and immuno-staining for the major histocompatibility complex (MHC) antigens. To measure the immune response a mixed leucocyte reaction was used but where leucocytes from a different individual were mixed with the clonal MSC cells.

All isolates were able to differentiate into osteoblasts, chondrocytes and adipocytes. All clonal cultures revealed significantly different proliferation rates and doubling times when compared with each other and with mixed cultures. All clonal cultures showed different surface marker presentations, which included differences in the expression of MHC antigens. One clone isolated from ADMSCs showed lack of MHCI and MHCII. Our mixed leucocyte reaction and MHC staining showed variety of immune-modulation and this was related to the expression of the MHC antigens.

All clones tri-differentiated and therefore show a degree of ‘stemness’. MSCs are generally are believed not to express MHC II and to be immune-privileged. However, this study shows that the expression of these antigens in clones isolated from bone marrow and from fat is variable. A heterogeneous result indicates individual differences between MSCs, even from same origin. The immune response elicited by MSCs is complicated. MSCs have been shown to release interleukin 10, which could inhibit the immune response but on the other hand interferon-gamma could enhance MHCII presentation in some MSCs. Our results confirmed our hypothesis because clonal cultures isolated from different sources of MSCs in the same animal showed significant differences in proliferation rate, morphology and surface marker presentation. Mesenchymal stem cells are not immunogenic or immune-privileged. Individual differences highlighted through single-cell clonal cultures may be the key to finding a universal immune-privileged MSCs for allogeneic transplantation.

Mesenchymal stem cells (MSCs) are usually believed to be immune-privileged. However, immunogenic MSCs were also reported. We hypothesize that there are differences between MSC clones from the same individual in terms of their morphology, proliferation, differentiation and immunogenicity. Our goal is to discover immune-privileged stem cells for universal allogenic MSCs transplantation.

Serial dilutions of bone-marrow derived (BMMSCs) and adipose derived mesenchymal stem cells (ADMSCs) from same animal were carried out to isolate single-cell clones. From a single animal we obtained 3 clones from BMMSCs and 3 from ADMSCs. The proliferation rate of each clonal culture and mixed clonal culture were measured. The tri-differentiation potential of the clonal cultures was compared, as well as with the original isolates from bone marrow and fat. The immune-privileged properties were measured by flow cytometry and immuno-staining for the major histocompatibility complex (MHC) antigens. Mixed leucocyte reaction (MLR) were also performed to investigate immunogenicity.

Tri-differentiation was confirmed in all isolates. All clonal cultures revealed significant different morphology and proliferation rates, compared with each other and mixed cultures. All clonal cultures showed different surface markers, inclusive of MHC antigens. One clone from ADMSCs showed lack of MHC antigens. Our MLR and MHC staining disclosed variety of immune properties.

All clones tri-differentiated which indicated a degree of ‘stemness’. MSCs are generally believed not to express MHC II, resulting in immune-privileged. Our results confirmed our hypothesis because clonal cultures isolated from different origins of same animal show differences in morphology, proliferation rate, and surface marker presentation. Individual immune differences highlighted through single-cell clonal cultures may be crucial to find universal immune-privileged MSCs as universal allogeneic donor.