immunode tection and histological analysisto assay the affects of heterochronic local and systemic environments on stem cell regenerative potential, hesc, hmsc, and myof iber - derived precursor cell cultures were fixed with 70 % etoh / pbs at 4 °c, and were analyzed by indirect immunoflu orescence. combinations of antibod ies were used to co - stain cultures and histosections, in order to determine the percentages of cells that proliferated or differentiated and to disting uish hescs from mouse cells. antibod ies to the myogenic transcription factors, myf5 / pax7, the intermediate filament protein, desmin, and the marker of newly formed myotubes, emyhc, were used to reveal commitment to myogenic differentiation. cell commitment to this differentiation program was assessed by the efficiency of myotube formation, estimated by the number of nuclei per myotube. ki67, a cell cycle related nuclear protein consistently absent in quiescent cells, was used as a marker for proliferation. whereas ki67 appears in all active phases of the cell cycle, brdu staining allowed exclusive detection of cells in s-phase, thereby enabling ac curate quantification of dna synthesis. in select cultures, 10 µm brdu was added for 2 h prior to fixation. brdu - specific immunostaining required nuclear permeabilization with treatment of 4n hcl. hescs were disting uished from mouse cells by using a species - specific antibod y to the cell - surface marker m - cadherin for murine and the nuclear marker numa for human cells. antibod ies to oct4 were used as a marker of hesc self - renewal / pluripotency. following permeabilization in pbs, + 1 % fbs, + 0. 25 % trit on x - 100, cells were incubated with primary antibod ies ( concentration determined as per manufacturer ' s recommendations ) for 1 h at room temperature in pbs, + 1 % fbs, washed several times, and then incubated with fluorophore-conjugated, species - specific secondary antibod ies ( diluted 1: 500 in pbs + 1 % fbs ) for 1 h at room temperature. for histological analysis, dissected muscle was treated in a 25 % sucrose / pbs solution, frozen in oct compound ( tissue tek ) and cryosectioned at 10 µm. immunostaining was performed in the manner described above, or h & e staining of cryosections was performed. nuclei were visualized by hoechst staining for all immunostains. samples were analyzed at room temperature by using a zeiss axioscope 40 fluorescent microscope, and imaged with an axiocan mrc camera and axiovision software. all images depict identical microscope fields at ×20 magnification, unless otherwise noted. reagents antibod ies to oct4 ( ab18976 ), brdu ( bu1 / 75 ( icr1 ), and ki67 ( ab15580 ) were purchased from abcam ( cambridge, ma, usa ). antibod y to m - cadherin ( clone 12g4 ) was acquired from upstate biotechnology ( lake placid, ny, usa ), and numa antibod y ( catalog number na09l ) from emd biosciences ( san diego, ca, usa ). antibod y to developmental emyhc ( clone rnmy2 / 9d2 ) was acquired from vector laboratories ( burlingame, ca, usa ). myf5 ( gtx77876 ) and pax7 ( gtx77888 ) antibod ies were obtained from genetex ( san antonio, tx, usa ). desmin antibod ies ( clone de - u - 10 and catalog number d8281 ), brdu labeling reagent and fk506 ( catalog number f4679 ) were obtained from sigma. fluorophore-conjugated secondary antibod ies ( alexa fluor ) were obtained from molecular probes ( eugene, or, usa ). statistical analysesa minimum of three replicates were undertaken for each experimental condition. quantified data are presented as means ± se. significance testing was performed using one - way analysis of variance ( anova ) to compare data from different experimental groups. p values of < 0. 05 were considered as statistically significant. backgroundhuman embryonic stem cells provide access to the earliest stages of human development and may serve as a source of specialized cells for regenerative medicine. thus, it becomes crucial to develop protocols for the directed differentiation of embryonic stem cells into tissue - restricted precursors. methods and findingshere, we present culture conditions for the derivation of unlimited numbers of pure mesenchymal precursors from human embryonic stem cells and demonstrate multiline age differentiation into fat, cartilage, bone, and skeletal muscle cells. conclusionour findings will help to elucidate the mechanism of mesoderm specification during embryonic stem cell differentiation and provide a platform to efficiently generate specialized human mesenchymal cell types for future clinical applications. lorenz studer and colleagues describe the use of embryonic stem cells to derive mesenchymal precursors and then fat, cartilage, bone, and skeletal muscle cells. embryonic stem ( es ) cells are pluripotent cells derived from the inner cell mass of the blastocyst that can be maintained in culture for an extended period of time without losing differentiation potential. the successful isolation of human es cells ( hescs ) has raised the hope that these cells may provide a universal tissue source to treat many human diseases. however, directed differentiation of hescs into specific tissue types poses a formidable challenge. protocols are currently available for only a few cell types, mostly of neural identity [ 1 – 3 ], and differentiation into many of the cell types derived from the paraxial mesoderm has not been reported, with the exception of a recent study indicating osteoblastic differentiation [ 4 ]. mesenchymal stem cells ( mscs ) have been isolated from the adult bone marrow [ 5 ], adipose tissue [ 6 ], and dermis and other connective tissues [ 7 ]. harvesting mscs from any of these sources requires invasive procedures and the availability of a suitable donor. the number of mscs that can be obtained from a single donor is limited, and the capacity of these cells for long - term proliferation is rather poor. in contrast, hescs could provide an unlimited number of specialized cells. in this study, we present techniques for the generation and purification of mesenchymal precursors from hescs and their directed differentiation in vitro into various mesenchymal derivatives, including skeletal myoblasts. our isolation method for mesenchymal precursors is the first example, to our knowledge, of efficiently deriving structures of the paraxial mesoderm from es cells, and further highlights the potential of hescs for basic biology and regenerative medicine. cell culture and facsundifferentiated hescs, h1 ( wa - 01, xy, passages 40 – 65 ) and h9 ( wa - 09, xx, passages 35 – 45 ), were cultured on mitotically inactivated mouse embryonic fibroblasts ( specialty media, phillipsburg, new jersey, united states ) and maintained under growth conditions and passaging techniques described previously [ 3 ]. op9 cells were maintained in alpha mem medium containing 20 % fetal bovine serum ( fbs ) and 2 mm l-glutamine. mesenchymal differentiation was induced by plating 10 × 103 to 25 × 103 cells / cm2 on a monolayer of op9 cells in the presence of 20 % heat - inactivated fbs in alpha mem medium. flow - activated cell sorting ( facs ) ( cd73 - pe; pharmingen, san diego, california, united states ) was performed on a moflo ( cytomation, fort collins, colorado, united states ). all human es cell – derived mesenchymal precursor cell ( hesmpc ) lines in this study are of polyclonal origin. primary human bone marrow – derived mscs and primary human foreskin fibroblasts ( both from poietics, cambrex, east rutherford, new jersey, united states ) were grown in alpha mem medium containing 10 % fbs and 2 mm l-glutamine. adipocytic differentiationhesmpcs are grown to confluence followed by exposure to 1 mm dexamethasone, 10 μg / ml insulin, and 0. 5 mm isobutylxanthine ( all from sigma, st. louis, missouri, united states ) in alpha mem medium containing 10 % fbs for 2 – 4 wk. data were confirmed in hesmpc - h1. 1, - h1. 2, - h1. 3, and - h9. 1 ( hesmpc - h1. 4 was not tested ).