A Highly Efficient Method to Differentiate Smooth Muscle Cells From Human Embryonic Stem Cells
To the Editor:
The molecular mechanisms and the control of smooth muscle cell (SMC) differentiation have been extensively investigated because of its therapeutic potential.1 To date, different cell types have been used to study SMC differentiation, including a variety of mouse embryonic stem cells,2 adult stem cells,3,4 and others.5 Because several fundamental differences exist between mouse and human embryonic development,6 lack of a good model system to study human SMC differentiation has hampered the progress of translating SMC knowledge to novel clinical therapies.
Human embryonic stem (hES) cells provide a valuable source of cells for studying human cell differentiation and developing therapeutic potentials in regenerative medicine. Since the initial report describing the derivation of hES cells,7 a variety of studies have established in vitro differentiation strategies to several lineages. Recently, it has been demonstrated that vascular progenitors derived from hES cells could be differentiated into endothelial cells and SMCs by endothelial growth medium with VEGF and PDGF, respectively.8 In the present study, we demonstrate a highly efficient and feasible cell culture–based methodology to differentiate hES cells to the SMC lineage by using a combination of cell culture medium and extracellular matrix environment.
Two well-studied, NIH-approved, human ES cell lines (H1 and H9) were induced to differentiate after the strategy shown in supplemental Figure I (available online at http://atvb.ahajournals. org). Throughout this process, undifferentiated hES cells underwent complex morphological changes (supplemental Figure II). Cells derived from the outgrowth of embryoid bodies (EBs) were subsequently plated in growth conditions (GC), consisting of smooth muscle growth medium (SMGM) and matrigel-coated plates. As a result, a morphologically homogeneous cell population was achieved that remained so with culture time and passages (supplemental Figure IIE). On the other hand, these homogeneous cells underwent a dramatic morphological change when switching GC to differentiation conditions (DC), consisting of DMEM +5% FBS and a gelatin-coated surface. Cells growing in DC became larger and displayed an elongated spindle-shaped morphology, the characteristics of SMC-like cells (supplemental Figure IIF).
To characterize these cells as SMC-like cells, SMC markers were examined by immunohistochemical staining. As shown in Figure A, hES-derived cells growing in DC displayed smooth muscle (SM)-myosin heavy chain (SMMHC) and SM-α-actin expression. Fluorescence-activated cell sorter (FACS) analysis showed that both SMMHC- and SM-α-actin–positive cells significantly increased after the growth environment was switched to DC for 5 days (55.26±8.02% and 96.81±2.07%, respectively), compared with cells growing in GC (4.54±0.92% and 9.99±1.68, respectively, n=3, P<0.01) (supplemental Figure III).
To further demonstrate the effect of culture conditions on SMC differentiation, the expression of SMC-specific genes was determined by quantitative real-time reverse transcription polymerase chain reaction (PCR) (qRT-PCR). Myocardin, SM-α-actin, calponin, smoothelin, SMMHC, SM22α, and telokin were significantly upregulated in hES cell–derived culture in DC for 5 days (supplemental Figure IV). Despite an increased expression of Flk1, a marker for cardiovascular progenitor cells,9 it was determined that endothelial cell markers (such as CD31 and Tie2) are not increased in this differentiation protocol. Consistent with the qRT-PCR data, Western blot analyses demonstrated that expression levels of SM-α-actin, h-caldesmon, and SMMHC were upregulated in a time-dependent manner in DC (Figure, B).
It is well-established that cultured SMCs are able to contract in response to external ligands.4 First, carbachol, a muscarinic agonist, was used to evaluate the contraction of derived SMC-like cells. Cells were treated for 1 minute with carbachol and observed microscopically up to 30 minutes. A large proportion of cells growing in DC changed shape in response to carbachol treatment (arrows in Figure, C and supplemental Movie I). Also, a similar response to KCl was observed in these SMC-like cells (supplemental Movie II and arrows in supplemental Figure V).
Taken together, the described results clearly indicate that this differentiation approach results in a highly efficient achievement of human SMCs as determined by the acquisition of SMC characteristics including expression of specific markers and functional reactivity in response to contractile agents. This simple and efficient cell model system will provide a useful tool to study human SMC differentiation and vascular development as well as potential therapeutic targets for treating vascular diseases.
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