Cultured mesenchymal stroma cells (MSCs) have a high potential for novel cell therapy approaches in clinical transplantation due to their intriguing properties, i.e. high proliferation and multi-lineage differentiation capacity, stroma support function and immune-modulation 
Currently, bone marrow cells are the major source for MSC therapies. However, bone marrow MSCs – as other MSC preparations derived from single donor adult stroma stem cell sources – are primary cells, which have certain properties that unfortunately limit a broader clinical application in larger cohorts of patients. First, the availability of primary bone marrow cells is dependent on (healthy volunteer) donors, who are willing to undergo a relatively harmless but nevertheless invasive bone marrow harvest procedure. Secondly, and importantly, the number of BM-MSCs that can be generated from a single donor is limited and usually does not allow treating more than one or two patients. Furthermore, growth characteristics and cell yields of BM-MSC preparations vary among individual preparations and MSC proliferation is donor age dependent 
. In addition, we and others have reported biological differences even within BM-MSCs culture-derived from a single donor 
, indicating that standardization of a primary adult stem cell-derived MSC products is difficult. Last, BM-MSCs can only be passaged for a limited number of times, after which they show a marked reduction in proliferation and differentiation potential. In addition, genetic instability and the risk of developing chromosomal aberrations increase with prolonged time in culture 
Therefore, an alternative and unlimited source of MSC with high potential for standardization for cell-based therapies is highly desirable. Recently developed hES cell-derived MSCs may represent such a potential alternative MSC source. These cells, named hES-MP002.5 cells, were derived by direct differentiation of hES cells in a feeder-free culture system using DMEM medium containing 10% FBS and 10 ng/ml basic fibroblast growth factor (bFGF). Under these conditions, growth of a stromal cell population with typical MSC properties was observed after two to three passages 
We report herein a detailed biological study of these cells, demonstrating that hES cell-derived MSCs have a similar morphology and a typical MSC surface marker expression pattern compared to standard human bone marrow-derived MSCs 
, which is in accordance with recently reported results 
. The only difference between the two cell types was the bimodal expression of CD90 in hES-MP002.5 cells, which has also been reported for human placenta-derived MSCs 
. Interestingly, CD90 expression has been demonstrated to decrease with osteoblastic differentiation 
and thus, CD90 expression differences may reflect differences in differentiation potential towards the osteoblastic lineage within hES-MP002.5 cells. We have not formally addressed possible heretogeneity of ES-MSC in the current work and, thus, these important considerations remain to be investigated.
hES-MP002.5 cells in our experiments showed a lower CFU-F potential compared with BM-MSCs, which might be due to differences in passage number. However, considering the various CFU-F numbers reported previously for BM-MSCs 
, hES-MP002.5 cells still showed reasonable CFU-F generation capability even in higher passages.
The in vitro
hES-MP002.5 cells differentiation potential towards chondrogenesis was comparable with that of BM-MSCs, whereas on the other hand hES-MP002.5 cells showed higher osteogenic potential and a different pattern of adipocytic cells. These results are in line with previous reports with this specific hES-MP002.5 cells cell line as well as with the results obtained with other hES-derived MSCs 
. It was furthermore shown that the superior osteogenic potential of hES-MP002.5 cells is due to signaling pathway differences 
, however, the reason for the differences in adipogenic differentiation has not yet been identified and needs to be investigated further.
Recent studies indicate that MSCs are key constituents of the bone marrow microenvironment, thus playing an important role for the regulation of hematopoietic stem cell (HSC) self-renewal and differentiation 
. Because of their known stroma supporting capacity, BM-MSCs have been suggested to be able to enhance HSC engraftment after transplantation 
. Accordingly, studies in humans as well as xenotransplantation experiments showed that co-transplantation of human MSCs and HSCs resulted in increased chimerism and accelerated hematopoietic recovery 
. Interestingly, bone marrow homing of in vitro
cultured MSCs have – with the exception of ex vivo
homing receptor-engineered MSCs 
– not yet been demonstrated conclusively. Thus, effects on hematopoiesis observed after i.v. administration are likely to be due to the paracrine factors released by cultured MSCs.
Accordingly, our results demonstrate that neither hES-MP002.5 cells nor BM-MSCs homed to the bone marrow of NSG mice after intravenous injection, at least not to such an extent that homing would have been detectable by flow cytometry analysis of stably-transfected GFP+ MSCs.
On the other hand, when MSCs were injected intra-femorally, both hES-MP002.5 cells and BM-MSCs engrafted, and localization of the transplanted cells was comparable with the distribution of primary BM-MSCs in-situ, i.e. cells were primarily found perivascularly and also endosteally 
The hematopoietic stroma capacity of the two cell populations was tested in-vitro
with the LTC-IC assay and in-vivo
by co-transplantation of human CD34 hematopoietic cells (i.v.) and stroma cells (i.f.) into immunodeficient NSG mice. The results of both assays clearly demonstrated that hES-MP002.5 cells and BM-MSCs have potent stroma supporting capacity. It is worth mentioning that the injection of stromal cells in one femur also enhanced hematopoiesis in the non-stroma-injected femur when compared to control mice without stroma cell support. This observation strengthens the assumption that transplanted stroma cells do not only act locally by direct interaction with hematopoietic cells but also act in a paracrine fashion to enhance hematopoiesis even in a distant, non-stroma-injected site. An alternative explanation would be that intrafemorally injected stromal cells migrated and homed to the opposite femur. However, this appears to be rather unlikely given the negative results of our migration experiments. The potent stroma-supportive activity of hES-MP002.5 cells is an important property of this novel type of mesenchymal ES-cell derived cells. However, whether or not all ES-cell derived mesenchymal cell lines share this property has not yet been reported and, certainly, this is a relevant topic for future investigations 
One of the most prominent current clinical applications of BM-MSCs is their use as immune-modulators in diseases such as Graft versus Host Disease (GvHD), inflammatory bowel disease, and others 
. MSCs exert their immune effects by direct cell-cell contact as well as by other mechanisms, such as production of transforming growth factor β-1 (TGF-β1), indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), nitric oxide (NO), and other substance as well as by recruiting other immune-suppressing networks 
Our results demonstrate that hES-cell derived stromal cells – at least in vitro – have only little or no effect on allo-antigen as well as mitogen-stimulated lymphocyte proliferation (). Even when pretreated with interferon-γ, which was previously shown to increase HLA-DR expression and to enhance immune-suppressive activity of fetal MSCs 
, hES-MP002.5 cells remained non-immunosuppressive. Accordingly, others have shown that HLA-DR expression of hES-MP002.5 cells does not increase upon interferon-γ treatment 
. Thus, in contrast to BM-MSCs, hES-MP002.5 cells are most likely not suitable for cell therapy approaches that aim to modulate immune function.
However, there are other clinical applications which do not depend on the immune-suppressive effects of MSCs and for some applications, this “side effect” might even be harmful. For example, there is no need for immune-modulation when administering MSCs to accelerate hematopoietic recovery after autologous transplantation. Furthermore, patients undergoing allogeneic transplantation for high-risk leukemia would certainly profit from the stroma-supporting effects of additionally transplanted MSCs leading to a faster hematopoietic recovery 
. However, additional MSC-induced immune suppression in the early post-transplant phase might dampen NK graft-versus tumor activity, thus increasing the risk for relapse 
. In addition, based on their ability to migrate to and invade tumor tissues, MSCs have also been proposed as cellular vector systems in anti-tumor therapy approaches, e.g. for the delivery of oncolytic viruses or other substances with anti-cancer activity 
. For theses applications the use of immuno-suppressive MSC preparations would post a safety concern as this may affect the host anti-tumor immune response possibly resulting in enhanced tumor growth and metastasis 
. Non-immunosuppressive MSC products such as the hES-MP002.5 cells described herein, would here certainly be the preferable choice of cells over MSC preparations with immune-suppressive potential. However, whether or not non-immunosuppressive hES-derived MSC can be transplanted across HLA barriers in a similar way as bone marrow-derived cells is not clear at the moment. Our experiments showed that both cell types lack expression of class II HLA molecules, however, transplantibility of hES-MP cells remains to be investigated in appropriately designed studies.
In summary, our study shows that the recently developed hES cell-derived MSC cell line hES-MP002.5 cells possess similar biological and functional properties compared to conventional BM-MSCs, except for the immune-modulatory effects. Based on the fact that hES-MP002.5 cells can be reliably and safely produced from established ES cell lines, hES-MP002.5 cells are an attractive unlimited source for stroma transplantation approaches in clinical situations when immune suppression is either not required or even potentially dangerous.