(A) FACS (MoFlo, Cytomation) for the isolation of CD73+ precursors (right) and isotype control (left).
(B) Flow cytometry analysis of the CD73+ hESMPC population for various markers characteristic of MSCs, including CD44, CD73, CD105, CD166, VCAM, ICAM-1, CD29, and STRO-1.
(C) Immunocytochemistry of hESMPCs for MSC markers (VCAM, STRO-1, CD73, and CD105). The cells also express vimentin and alpha smooth muscle actin. Scale bar = 50 μm.
(D) Venn diagram presenting the overlap among transcripts selectively expressed in hESMPC-H1.2, hESMPC-H9.1, and primary adult human MSCs.

(A) Adipocytic differentiation in the presence of dexamethasone, insulin, and isobutylxanthine. Adipocytic characterization by Oil Red O staining and RT-PCR analysis for PPARγ.
(B) Chondrocytic differentiation in the presence of TGF-β3 and ascorbic acid. Chondrocytic characterization by Alcian Blue staining and RT-PCR for aggrecan and collagen II.
(C) Osteogenic differentiation in the presence of β-glycerolphosphate, dexamethasone, and ascorbic acid. Osteocytic characterization by von Kossa staining and RT-PCR for bone-specific alkaline phosphatase (ALP) and bone sialoprotein (BSP).
(D) Phase-contrast image of hESMPCs and RT-PCR for the ES cell markers Nanog and Oct-4 in hESMPC-H1.1 and -H9.1 compared with undifferentiated H1 hESCs.
Scale bar = 50 μm for all panels.

(A) Immunocytochemistry for MyoD (red) and fast-switch myosin (green). RT-PCR for MyoD in human skeletal muscle as a positive control (hSM), and in hESMPC-H9.1 cells differentiated for 10 d in the presence of C2C12-conditioned medium (hESMPC).
(B) Myotube formation induced at high cell densities in the presence of C2C12 cells. Myotube characterization by immunocytochemistry for MF20 against sarcomeric myosin (green) and human nuclear antigen (hNA, red). Left panel: Control undifferentiated hESCs (H9) do not fuse with C2C12. Right panel: Under identical culture conditions, hESMPCs (line 9.1) efficiently fuse with C2C12 cells, forming myotubes containing human nuclei. RT-PCR for human specific muscle transcripts myosin heavy chain IIa (MYHC-2) and MyoD in C2C12 cells, in human skeletal muscle as positive control (huSM), and in hESMPC-H9.1 cells cocultured with C2C12 cells.