Culture of undifferentiated human ES cells
H9 hES cells (WiCell, Madison, WI) were cultured on a feeder layer of irradiated murine embryonic fibroblasts (MEFs) using hES cell culture medium consisting of 80% Dulbecco’s modified Eagle’s medium/F-12 (Invitrogen, Carlsbad, CA), 20% knock-out serum replacement (Invitrogen), 1 mM L-glutamine, 1% nonessential amino acids, 0.1 mM β-mercaptoethanol, and 8 ng/ml basic fibroblast growth factor (Invitrogen). Cells were passaged every 4–6 days using Collagenase IV (Invitrogen). Prior to transplantation, cells were moved to a Matrigel (BD Biosciences, San Jose, CA) coated plate and cultured for two passages under feeder free conditions using mTeSR-1 (Stemcell Technologies, Vancouver, Canada) to eliminate presence of MEFs.
Lentiviral transduction of human ES cells with a double-fusion reporter gene
ES cells of the H9 cell line were transduced with a self inactivating lentiviral vector carrying a human ubiquitin promoter driving firefly luciferase and enhanced green fluorescence protein (Fluc-eGFP) at a multiplicity of infection (MOI) of 10. After two rounds of cell sorting for eGFP by a Vantage SE cell sorter (Becton Dickinson Immunocytometry Systems, San Jose, CA), a population of human ES cells stably expressing the DF construct was isolated and expanded on feeder layers of MEFs as described above.
Preparation of varying numbers of human ES cells for cardiac and skeletal muscle transplantation
Human ES cells were harvested from feeder free Matrigel coated plates by treating cells with cell dissociation buffer (Invitrogen) for 10 minutes. Cells were counted using a hemocytometer and suspended in PBS at a concentration of 1 × 106 cells/30 µl PBS. Serial dilution was subsequently used to obtain 1 × 105, 1 × 100, 1×103 and 1 × 102 cells per 30 µl PBS. ES cells were kept on ice for <45 min for optimal viability prior to injection.
Cardiac and skeletal muscle transplantation of DF positive human ES cells
All animal study protocols were approved by the Stanford Animal Research Committee. Surgical procedures were performed on 8–10 week old female immunocompromised SCID beige mice (Charles River Laboratories, Inc.,) by a single experienced micro-surgeon. Briefly, animals were knocked down using 2% isoflurane. Mice were intubated, ventilated and anesthesia was maintained with inhaled isoflurane (1% to 2%). An aseptic left thoracotomy was performed and the pericardium was opened. Mice were subsequently injected intramyocardially with 1 × 106, 1 × 105, 1 × 104, 1 × 103 or 1 × 102 human ES cells suspended in 30 µl of PBS (n = 7 per group). For skeletal muscle injections, mice were knocked down and maintained on anesthesia using 1–2% isoflurane. Human ES cells were suspended in 30 µl of Matrigel and injected directly into the gastrocnemius muscles of recipient mice using a 28.5-gauge insulin syringe. Mice were divided into the following groups (n = 7 each): 1 × 106, 1 × 105, 1 × 104, 1 × 103 and 1 × 102 cells.
Optical bioluminescence imaging (BLI) of DF positive human ES cell transplanted animals
BLI was performed on all animals using a Xenogen IVIS system. All animals were imaged on days 0, 2, 7, 14, 21, 28, 49 and 56 following cell transplantation. The reporter probe D-Luciferin (375 mg/kg) was administered via intra-peritoneal injection 10 minutes prior to image acquisition, after which animals were imaged for 20 min using 1 second to 5 minute acquisition intervals repetitively for the study period. Region of interest (ROI) were drawn over the signals using the Igor image analysis software (Wavemetrics, Lake Oswego, OR). BLI signal was standardized for acquisition time and quantified in units of maximum photons per second per square centimeter per steridian (photons/sec/cm2/sr).
Postmortem immunohistochemical staining
Animals were sacrificed according to protocols approved by the Stanford Animal Research Committee after the duration of the study. Teratomas and major organs (heart, liver, kidney, spleen) were explanted and processed for H&E staining. For animals that received hindlimb injection, the gastrocnemius muscle was dissected, sectioned and stained with H&E. Slides were interpreted by an expert pathologist (A.J.C.).
Data are given as mean ± SEM. For statistical analysis, the two-tailed Student’s t-test was used. Differences were considered significant at p < 0.05.