Methods

Cell Culture and FACS
Undifferentiated 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 Differentiation
hESMPCs 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).

Chondrocytic Differentiation
Differentiation of hESMPCs was induced in pellet culture [5] by exposure to 10 ng/ml TGF-β3 (R & D Systems, Minneapolis, Minnesota, United States) and 200 μM ascorbic acid (Sigma) in alpha MEM medium containing 10% FBS for 3–4 wk. Data were confirmed in hESMPC-H1.1, -H1.3, and -H9.1 (hESMPC-H1.2 and -H1.4 were not tested).

Osteogenic Differentiation
hESMPCs were plated at low density (1 × 103 to 2.5 × 103 cells/cm2) on tissue-culture-treated dishes in the presence of 10 mM β-glycerol phosphate (Sigma), 0.1 μM dexamethasone, and 200 μM ascorbic acid in alpha MEM medium containing 10% FBS for 3–4 wk. Data were confirmed in hESMPC-H1.1, -H1.3, and -H9.1 (hESMPC-H1.2 and -H1.4 were not tested).

Myogenic Differentiation
Confluent hESMPCs were maintained for 2–3 wk in alpha MEM medium with 20% heat-inactivated FBS. More rapid induction was observed in the presence of medium conditioned for 24 h by differentiated C2C12 cells. Coculture of hESMPCs and C2C12 cells was carried out in alpha MEM with 3% horse serum and 1% FBS [8]. Data were confirmed in hESMPC-H1.3, -H1.4, and -H9.1 (hESMPC-H1.1 and -H1.2 were not tested).

Cytochemistry
Immunocytochemistry for all surface markers was performed on live cells. Monoclonal antibodies VCAM, STRO-1, ICAM-1(CD54), CD105, CD29, and MF20 were from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, Iowa, United States); CD73, CD44, and ALCAM(CD166) were from BD Biosciences Pharmingen (San Diego, California, United States). All other immunocytochemical analyses were performed after fixation in 4% paraformaldehyde and 0.15% picric acid, followed by permeabilization in 0.3% Triton X100. Polyclonal antibodies used were MyoD (Santa Cruz Biotechnology, Santa Cruz, California, United States) and nestin (gift from R. McKay); monoclonal antibodies were vimentin, alpha smooth muscle actin, fast-switch myosin, pan-cytokeratin (all from Sigma), and human nuclear antigen (Chemicon, Temecula, California, United States).
Alkaline phosphatase reaction was performed using a commercially available kit (Kit-86; Sigma) and the mineral was stained with silver nitrate according to the von Kossa method. Fat granules were visualized by Oil Red O staining solution (Sigma). Alcian Blue (Sigma) was used to detect extracellular matrix proteoglycans in chondrogenic cultures.

Gene-Expression Analyses

RT-PCR analysis
Total RNA was extracted by using the RNeasy kit and DNase I treatment (Qiagen, Valencia, California, United States). Total RNA (2 μg each) was reverse transcribed (SuperScript; Invitrogen, Carlsbad, California, United States). PCR conditions were optimized and linear amplification range was determined for each primer by varying annealing temperature and cycle number. PCR products were identified by size, and identity was confirmed by DNA sequencing. Primer sequences, cycle numbers, and annealing temperatures are provided in Table S1.

Affymetrix analysis
Total RNA (5 μg) from primary MSCs, from hESMPC-H9.1, hESMPC-H1.2, and three samples of undifferentiated hESCs (H1; passages 42–46), were processed by the Memorial Sloan-Kettering Cancer Center Genomics Core Facility and hybridized on Affymetrix (Santa Clara, California, United States) U133A human oligonucleotide arrays. Data were analyzed using MAS5.0 (Affymetrix) software. Transcripts selectively expressed in each of the mesenchymal cell populations (MSC, hESMPC-H9.1, and hESMPC-H1.2) were defined as those called “increased” by the MAS5.0 algorithm in each of three comparisons with independent samples of undifferentiated hESCs. A Venn diagram was generated to visualize overlap in gene expression. Further statistical analyses were performed as described below.