hES cells constitute a valuable model system in developmental studies and in the search for new regenerative therapies. Various stem cell types utilize BMP signals in a multitude of ways in order to define their fates 
. BMPs are known to be involved in several types of differentiation processes and the use of BMPs in differentiation of pluripotent cells is a powerful tool in biological and medical research 
. The development of new molecules with increased activity is, therefore, a priority in medical and pharmaceutical research. The use of these BMPs with increased activity would permit the development of more efficient protocols and increase the productivity of the existent ones.
In this line of investigation, increased activity of BMP heterodimers has been previously reported; co-expression of BMP-2 with BMP-6 or BMP-7 yields heterodimeric BMPs with a specific activity about 20-fold higher than BMP homodimers in in vitro
alkaline phosphatase induction assay, and 5- to 10-fold more potent than BMP-2 in inducing cartilage and bone in an in vivo
. Other heterodimers, as BMP-4/7 or BMP-7/GDF-7, also showed enhanced and/or novel properties in the context of embryo development 
. Therefore, BMP heterodimers could be used as a natural choice for substituting BMP homodimers in in vitro
or in vivo
assays. The production of BMP heterodimers in an Escherichia coli
cell expression system and subsequent chemical refolding in vitro
allowed us to work with high concentrations of purified protein while avoiding the low yield problems associated with a mammalian cell expression system 
The BMP-2/6 heterodimer consists of one BMP-2 and one BMP-6 subunit. Our experiments confirm the increased activity of BMP-2/6 heterodimer relative to the homodimeric counterparts BMP-2 or BMP-6, which support the hypothesis that such heterodimeric forms are likely to have natural biological functions. Previously BMP-2/7 heterodimer failed to exert an increased effect on hES cells growing on MEFs when compared to BMP-2 
, indicating that culture conditions and/or conditioned media could play an important role in determining the fate of BMP-differentiated cells.
BMP-2 and BMP-6 are both inducers of osteogenesis in human mesenchymal stem cells 
and endogenous BMP-2 and BMP-6 cooperatively play pivotal roles in bone formation under both physiological and pathological conditions 
. Nevertheless, type I and type II receptor utilization differs significantly between BMP-2/BMP-4 and BMP-6/BMP-7. BMP-4 is known to bind to BMPR1A and/or BMPR1B, whereas BMP-6 and BMP-7 preferentially bind to ACVR1 
. Also a greater reliance of BMPR2 is observed for BMP-2 (or BMP-4) relative to BMP-6 (or BMP-7), whereas ACVR2A is more critical to BMP-6 for signaling 
. Unlike the homodimers, we recently reported that BMP-2/6 heterodimer exhibits a relatively high affinity for each receptor type extracellular domain (ECD) as measured by surface plasmon resonance 
. Correlated to this, in several in vitro
experiments we have observed stronger activity of BMP-2/6 than that of BMP-2 or BMP-6 
. Thus, the more potent inducer activity of BMP-2/6 on hES cells is also likely derived from higher affinity to both type I and type II receptors. In the present study we have observed an increased expression of BMPR2
that could be responsible for the stronger effect of BMP-2 when compared to BMP-6.
For differentiation analysis we used a combination of markers of the three embryonic layers: ectoderm, mesoderm and endoderm. None of the BMPs we examined exclusively direct differentiation to only one cell type, but rather alter the relative proportions of a specific cell type. By quantification of the expression of differentiation markers, we identified BMP-2/6 as a better inducer of trophoblast and endoderm differentiation of hES cells than BMP-2 or BMP-6. BMP-2/6 is more effective than either BMP-2 or BMP-6 in inducing the expression of trophectodermal (CDX2), mesendodermal (T), and endodermal (SOX17, GATA6, AFP, CXCR4) markers, including the expression of BMPR2 receptor. These results support an increased bioactivity of BMP-2/6 in the described stem cell culture system, and it is being confirmed using other hES cell lines.
The increase in CDX2
expression indicates trophectodermal differentiation and suggests that in feeder-free conditions BMP-2 induces trophoblast, as observed previously with BMP-4 
. Markers of endoderm (both extraembryonic and definitive) were also strongly induced. The definitive endoderm forms during gastrulation and replaces the extraembryonic visceral endoderm. Definitive endoderm is the precursor of several organs (pancreas, liver) and in vitro
differentiation to those cell types is of great use in medical and biological studies. To generate definitive endoderm, 100 ng/ml of Activin A has been traditionally used in unconditioned medium 
, revealing a central role of TGFβ family pathways on this type of differentiation. Feeder layer-free conditions are also better systems for differentiation of hES cells into definitive endoderm 
. Our results demonstrate that the heterodimer BMP-2/6 is better inductor of endodermal markers than the BMP-2 homodimer, including definitive endoderm. It suggests that BMP-2/6 could replace BMP-2 as the inductor in protocols of BMP-2-guided differentiation in vitro
BMP receptors are present on hES cells and BMP signaling can induce expression of BMP ligands, forming a positive feedback loop in cells from various species, including human 
. Specifically, BMP-2 can induce its own expression in human embryonic carcinoma cells 
. Treatment of human pluripotent cells with BMP-2 leads to the accumulation of transcripts for this factor consistent with a positive feedback model 
, but an increased level of BMP2
gene transcription in hES cells had not been reported. In our experiments we observed an increased expression of both BMP2
and the type II receptor BMPR2
in cells treated with BMP-2 and BMP-2/6. Up-regulation of the expression of both ligand and receptor is a strong evidence of a positive feedback of BMP-2 signaling and differential induction of BMPR2
could be also a factor in the increased biological activity of BMP-2/6 in hES cells. These results should be confirmed in the future by analyzing levels of both proteins in hES cells after BMP treatment.
BMPs play an important role during all stages of embryonic development, and although only two major signaling pathways have been characterized (p38 and Smad pathways), the BMP signaling is complex and includes cross-talk with other major signaling pathways and negative feedback mechanisms 
. It has been reported that the initiation of Smad-dependent and Smad-independent signaling by BMP-2 depends on BMP-receptor complexes 
. BMP-2/6 possesses a higher affinity to both receptors type I and II than its homodimeric counterparts 
, and this new biological property could be responsible for the increased activation of Smad-dependent and Smad-independent signaling observed. We detected increased levels of SMAD1/5 phosphorylation induced by BMP-2/6, which confirms the recent reports using diverse experimental models, as luciferase reporter assay, osteogenic differentiation-inducing activity and chondrogenic activity 
. Furthermore, increases in SMAD1 phosphorylation have been previously reported in the early events of BMP-induced hES cell differentiation 
. Further study of BMP-2/6-activated signaling pathways would be necessary to fully understand its increased potency in hES cell differentiation.
Fibroblasts secrete multiple growth factors, including FGFs, TGFβs, Activins, Wnts and antagonists of BMP signaling 
. Of those, FGF2 has the greatest effect in promoting hES cell self-renewal, interrupting BMP signaling either by preventing the nuclear translocation of phosphorylated SMAD1 
or by repressing SMAD1 activity in the nucleus 
. Suppressed BMP signaling remains a consistent hallmark of current methods of hES cell culture. When we cultured H9 cells in a modified TeSR1 medium without FGF2, cells differentiated quickly after splitting and this differentiation was not blocked by noggin (data not shown), but blocked by 100 ng/ml Activin A, suggesting a compensatory effect due to the activation of the SMAD2/3 pathway 
. Depletion of FGF2 has a more drastic effect than TGFβ1, as FGF2 is also involved in cell proliferation 
and trace levels of TGFβ1 are present in Growth Factor-Reduced Matrigel coating. Our results confirm that both FGF2 and TGFβ1 in mTeSR1 are necessary for long term maintenance of H9 cells in mTeSR1. Further analysis of this involvement should be performed to improve and manipulate BMP-directed differentiation of hES cells in vitro
FGF2 depletion also affected BMP-2 and BMP-2/6-induced differentiation by diminishing levels of expression of all the differentiation markers analyzed, both trophectodermal and endodermal. Presence of FGF2 was necessary for driving BMP-2 or BMP-2/6-induced differentiation to endoderm in mTeSR1. When FGF2 was absent in mTeSR1, however, morphological differentiation appeared but the pattern of expression of differentiation markers after incubation with BMP-2 or BMP-2/6 completely changed. We hypothesize that FGF2 inhibition of neural differentiation could be a requirement for efficient BMP-induced differentiation to endoderm. Further investigation in this sense is required to clarify the involvement of FGF2 in BMP-induced differentiation in mTeSR1.
We analyzed the biological activity of BMP-2/6 for inducing differentiation of hES cells by measuring the expression of differentiation markers using qPCR and flow cytometry. Both BMP-2 and BMP-6 induce differentiation of hES cells, but the heterodimer BMP-2/6 is a more efficient inductor of expression of differentiation markers and percentage of CXCR4-positive definitive endoderm cells. It suggests that BMP-2/6 is a better candidate than BMP-2 as inductor in protocols of BMP-2-guided differentiation in vitro, as well as possible applications of BMP-2/6 to treat bone injury substituting BMP-2 as active molecule.