Unlike previously reported methods which typically rely on the addition of soluble factors to affect pluripotent stem cell differentiation, the present study reports an alternative approach using a biomaterial coating on a cell culture plate made of fibrillar collagen Type I to promote the derivation of MSC-like cells. Both hESCs and hiPSCs were subjected to MSC derivation on a fibrillar type I collagen coating prepared by self-assembly from collagen solutions on non-treated tissue culture plastic which thereby reproduces physiological Type I collagen, i.e. collagen with individual fibrils 200–300 nm in diameter and several µm in length 
. In the present study, homogenous spindle-shaped cells were obtained after 10 days in the absence of complex growth factors, cytokines or complicated manipulation of the cultures when grown on biomimetic fibrillar collagen. The present method successfully avoided the necessity of time consuming serial passaging and associated cell sorting or enrichment, and simply, in one step, homogenous MSC-like cells were obtained. The spindle-shaped cells and their subcultures showed characteristic MSC surface markers including being positive for CD73, CD90, CD105, CD146 and CD166, and negative for hematopoietic markers CD34 and CD45. The cells also were capable of tri-lineage differentiation, indicating their multi-potency, as shown through in vitro
studies. Thus the derived cells are referred to as MSC-like cells.
It is noteworthy that the MSC-like cells derived on the Type I collagen coating displayed a unique growth pattern over time, in that at 10 days the cells were found growing in isolated, separate colonies mainly located near the edge of the culture wells rather than growing as a uniform monolayer of cells. In contrast, on tissue culture treated polystyrene (i.e., the conventional culture surface), neither hESCs nor hiPSCs differentiated into spindle-shaped cells, although they did have more robust sustained attachment and growth than Type I collagen over a large area of the plate. It is not yet known why the cells on collagen preferentially colonized the edges better than other regions of the collagen-coated well although the collagen coating may be slightly thicker at the plate edges. Increasing the seeding density from 15,000 to 50,000/cm2 did not change the growth pattern in our study, i.e., by day 10 the cells repeatedly formed separate colonies on collagen instead of a continuous monolayer, implying other mechanisms than cell density were contributing to the unique growth pattern.
We did not yet identify the mechanisms underlying the enhanced derivation process on Type I collagen in the present study. However, other relevant studies provide clues that EMT might have played a fundamental role in the derivation process. EMT is known for its roles in embryogenesis, organ development, wound healing and tissue regeneration 
. There is evidence that hESCs can develop an epithelial phenotype after monolayer culture or from migration out of embryoid bodies which then undergo EMT to acquire mesenchymal phenotype 
. In the present study, the single cells plated for 10 days on tissue culture plates did have epithelial cell-like characteristics such as close cell packing and tight junction-like structures ( inset). Cells on collagen coatings did not show such an overt epithelial-like phenotype possibly due to their rapid transition to a mesenchymal phenotype stimulated by the fibrillar collagen coating. A rapid transition to MSC-like cells could also have arisen from a possible selection process whereby slightly differentiated cells with less-embryonic, more mature cell surface receptors were preferentially able to attach to the Type I collagen. Type I collagen is known to be able to stimulate and promote EMT through integrin-mediated signaling pathways 
and retain osteogenic differentiation potential of human adult MSCs during ex vivo expansion 
. This postulated mechanism could be addressed with the application of integrin-blocking antibodies and will be examined in future studies.
Dissociation of hESCs and hiPSCs into single cells was a necessary step in the present method as evidenced by the failure of the experiments in which traditional multi-cellular colonies were plated. Directly plating colonies of hESCs and hiPSCs, did not give rise to the spindle-shaped like cells on either the tissue culture polystyrene or the collagen coating. Instead, the cell colonies tended to develop into a mixture of various cell types judging from the heterogeneous cell morphologies. The non-dissociated colonies preserved the tight cell-cell interactions resembling the conditions for EB formation, which might have similarly favored spontaneous differentiation. We postulate that a separation of the cells from each other forced a direct interaction of the cell surface receptors with the substrate materials, rather than to each other, thereby enhancing the effect of the substrate in the differentiation process. The success of the biomaterials substrata to direct the differentiation process demonstrated here further strengthens the growing body of literature indicating that biomaterials, much like the extracellular matrix, can direct stem cell fate 
Dexamethasone was present in the derivation medium, and might also have positively contributed to the mesenchymal differentiation. Dexamethasone is commonly used in osteogeneic, chondrogeneic and adipogenic cultures 
and is also used for promoting in vitro
proliferation of MSCs by boosting the effects of other growth factors 
. The addition of dexamethasone might have expedited the differentiation process through enhancement of the activity of the growth factors produced by the cells themselves or present in the fetal bovine serum.
The tri-lineage differentiation potential of the derived MSC-like cells was demonstrated through in vitro
studies. Chondrogenic differentiation was easily achieved as evidenced by the strong gene expression of cartilage matrix components including aggrecan and collagen type II and transcription factor SOX9. Histological examination displayed a prevalent distribution of acidic glycosaminoglycans in the pellet. Significant production of aggrecan and collagen type I was evidenced by immunohistochemical staining. The adipogenic culture conditions induced apparent lipid accumulation in the cytoplasm and adipocyte gene expression after 21 days, although PPARγ expression was unexpectedly low given the amount of lipid droplets in the cells. Osteogenic differentiation was demonstrated by mRNA level gene expression of RUNX2, COL1A1 and ALP. ALP was significantly up-regulated in response to exposure to osteogenic medium and resulted in mineral deposition. However, late stage osteoblast markers were not present in the cultures as evidenced by the lack of osteocalcin and bone sialoprotein gene expression (the Ct
of real-time PCR reaction was more than 35). The inability of human marrow derived MSCs to mature into late-stage matrix producing osteoblasts in vitro
has also been observed by other researchers 
. The similar expression of RUNX2 and COL1A1 mRNA before and after osteogenic differentiation observed in these MSC-like cells derived from human pluripotent stem cells is consistent with a previous observation during osteogenic differentiation of human bone marrow derived MSCs 
. Osteogenic differentiation of the MSCs can be associated with increases in DNA-binding activity of Runx2 without changes in mRNA level expression 
. It should be noted that the differentiation potential of human adult MSCs is donor dependent 
, source tissue dependent 
and that repeated passaging leads to a reduced osteogenic differentiation capacity 
. Recently it was shown that only 2 of 10 donors had a strong chondrogenic potential 
. Since there is no standard adult MSC cell source with a standard gene expression profile at a given time point, adult MSCs were not included as controls in these in vitro
studies. Based on these promising in vitro
assay results, in vivo
differentiation studies should be pursued to conclusively demonstrate the multi-potentiality and lack of teratoma formation of these MSC-like cells.
In conclusion, a one-step MSC derivation method from pluripotent stem cells is reported in which singly dissociated H9 hESCs and YK26 iPSCs are cultured on biomimetic fibrillar Type I collagen coatings to obtain MSC-like cells. Both H9-hESCs and YK26-iPS cells responded similarly when subjected to the present MSC derivation process on fibrillar Type I collagen. The derived MSC-like cells from both cell lines displayed high expression levels of surface markers typical of MSCs, and the cells demonstrated in vitro tri-lineage differentiation potential including osteogenesis, chondrogenesis and adipogenesis. In vivo studies are needed to fully confirm the ability of these progenitors to function adequately in a therapeutic setting, nonetheless multipotent stem cells with properties resembling that of MSCs were successfully obtained. The biomimetic fibrillar type I collagen coating played a necessary role in the mesenchymal derivation as verified by the absence of the transition to MSC-like cells on controls of tissue culture treated polystyrene, although the detailed mechanism is not yet known. Most importantly, the new method presented is not only a convenient and efficient way to induce mesenchymal-like stem cells from hES and iPS cells, but also demonstrates the successful strategy of using an appropriate biomaterial matrix to influence and direct stem cell differentiation.