Sarah Steinbach - Academia.edu (original) (raw)
Papers by Sarah Steinbach
Successful completion of cytokinesis relies on addition of new membrane, and requires the recycli... more Successful completion of cytokinesis relies on addition of new membrane, and requires the recycling endosome regulator Rab11, which localizes to the midzone. Despite the critical role of Rab11 in this process, little is known about the formation and composition of Rab11-containing organelles. Here, we identify the phosphatidylinositol (PI) 4-kinase III � Four wheel drive (Fwd) as a key regulator of Rab11 during cytokinesis in Drosophila melanogaster spermatocytes. We show Fwd is required for synthesis of PI 4-phosphate (PI4P) on Golgi
Background: Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to b... more Background: Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not
Atherosclerosis Supplements
Circulation, 2016
Rationale: Sca-1 + progenitor cells in mouse aorta are known to generate vascular cells. However,... more Rationale: Sca-1 + progenitor cells in mouse aorta are known to generate vascular cells. However, their embryological origins and temporospatial abundance during development were not known. Objective: To describe stem cells of the mouse aorta and identify lineages of Sca-1 + progenitors. Methods & Results: Lineage-traced reporter mice at 8+ weeks of age reveal aortic stem cells, defined as self-renewing colony-forming cells with multi-lineage potential, to be most abundant in proximal aorta and predominantly of mesodermal origin ( T-Cre ), with little, if any, from neural crest ( Wnt1-Cre ). In distal aorta, the few stem cells that exist are strictly of mesodermal origin. Clonal cell colonies can be cultured in suspension from aorta-derived stem cells of somitic mesoderm origin ( Myf5-Cre ), and contain cells expressing stem cell marker Sox2. Using tamoxifen-inducible Myf5-Cre ER , we show that Sca-1 + aortic cells from somite are specified at E8.5, even before Myf5 + cells contribu...
Rationale: Few methods enable molecular and cellular studies of human vascular aging or type-2 di... more Rationale: Few methods enable molecular and cellular studies of human vascular aging or type-2 diabetes (T2D). Objective: To characterize functional responses of human vascular smooth muscle cells (VSMC) differentiated from progenitors found in skin. Methods and Results: Small biopsies (~1 cm 2 ) taken from the edges of leg or chest incisions of subjects (N=123; males 72%; mean age=63±12 years) undergoing cardiothoracic surgery were used to isolate skin cells and quantify the number of skin-derived precursors (SKPs) that can be cultured. Multivariate analysis revealed that the numbers of cells isolated from skin biopsies (normalized to skin weight) were reduced as a function of age, dyslipidemia and coronary artery disease (CAD). Although SKPs could be subsequently cultured from these biopsies and differentiated into VSMCs at high efficiency (>80% yield), the numbers of SKPs isolated from subjects with T2D were ~50% lower than those without T2D (cells/g: 0.22±0.04, N=46 vs. 0.52±...
Stem cells and development, Jan 2, 2018
Sca-1<sup>+</sup> progenitor cells in the adult mouse aorta are known to generate vas... more Sca-1<sup>+</sup> progenitor cells in the adult mouse aorta are known to generate vascular smooth muscle cells (VSMC), but their embryological origins and temporal abundance are not known. Using tamoxifen-inducible <i>Myf5-Cre</i><sup>ER</sup> mice, we demonstrate that Sca-1<sup>+</sup> adult aortic cells arise from the somitic mesoderm beginning at E8.5 and continue throughout somitogenesis. <i>Myf5</i> lineage-derived Sca-1<sup>+</sup> cells greatly expand in situ starting at 4 weeks of age and become a major source of aortic Sca-1<sup>+</sup> cells by 6 weeks of age. <i>Myf5</i>-derived adult aortic cells are capable of forming multicellular sphere-like structures <i>in vitro</i>, and express the pluripotency marker Sox2. Exposure to TGF-β3 induces these spheres to differentiate into calponin-expressing VSMC. Pulse-chase experiments using tamoxifen-inducible <i>Sox2-C...
Canadian Journal of Cardiology
Clinical Science
Objective: Few methods enable molecular and cellular studies of vascular aging or Type 2 diabetes... more Objective: Few methods enable molecular and cellular studies of vascular aging or Type 2 diabetes (T2D). Here, we report a new approach to studying human vascular smooth muscle cell (VSMC) pathophysiology by examining VSMCs differentiated from progenitors found in skin. Approach and results: Skin-derived precursors (SKPs) were cultured from biopsies (N=164, ∼1 cm2) taken from the edges of surgical incisions of older adults (N=158; males 72%; mean age 62.7 ± 13 years) undergoing cardiothoracic surgery, and differentiated into VSMCs at high efficiency (>80% yield). The number of SKPs isolated from subjects with T2D was ∼50% lower than those without T2D (cells/g: 0.18 ± 0.03, N=58 versus 0.40 ± 0.05, N=100, P<0.05). Importantly, SKP-derived VSMCs from subjects with T2D had higher Fluo-5F-determined baseline cytosolic Ca2+ concentrations (AU: 1,968 ± 160, N=7 versus 1,386 ± 170, N=13, P<0.05), and a trend toward greater Ca2+ cycling responses to norepinephrine (NE) (AUC: 177,20...
ACS Applied Materials & Interfaces, 2017
In the embryonic heart, electrical impulses propagate in a unidirectional manner from the sinus v... more In the embryonic heart, electrical impulses propagate in a unidirectional manner from the sinus venosus and appear to be involved in cardiogenesis. In this work, aligned and random polyaniline/polyetersulfone (PANI/PES) nanofibrous scaffolds doped by Camphor-10-sulfonic acid (β) (CPSA) were fabricated via electrospinning and used to conduct electrical impulses in a unidirectional and multidirectional fashion, respectively. A bioreactor was subsequently engineered to apply electrical impulses to cells cultured on PANI/PES scaffolds. We established cardiovascular disease-specific induced pluripotent stem cells (CVD-iPSCs) from the fibroblasts of patients undergoing cardiothoracic surgeries. The CVD-iPSCs were seeded onto the scaffolds, cultured in cardiomyocyte-inducing factors, and exposed to electrical impulses for 1 h/day, over a 15-day time period in the bioreactor. The application of the unidirectional electrical stimulation to the cells significantly increased the number of cardiac Troponin T (cTnT+) cells in comparison to multidirectional electrical stimulation using random fibrous scaffolds. This was confirmed by real-time polymerase chain reaction for cardiac-related transcription factors (NKX2.5, GATA4, and NPPA) and a cardiac-specific structural gene (TNNT2). Here we report for the first time that applying electrical pulses in a unidirectional manner mimicking the unidirectional wave of electrical stimulation in the heart, could increase the derivation of cardiomyocytes from CVD-iPSCs.
Circulation, Nov 23, 2010
Circulation, Nov 26, 2013
Methods, 2015
Transplantation of vascular smooth muscle cells (VSMCs) is a promising cellular therapy to promot... more Transplantation of vascular smooth muscle cells (VSMCs) is a promising cellular therapy to promote angiogenesis and wound healing. However, VSMCs are derived from diverse embryonic sources which may influence their role in the development of vascular disease and in its therapeutic modulation. Despite progress in understanding the mechanisms of VSMC differentiation, there remains a shortage of robust methods for generating lineage-specific VSMCs from pluripotent and adult stem/progenitor cells in serum-free conditions. Here we describe a method for differentiating pluripotent stem cells, such as embryonic and induced pluripotent stem cells, as well as skin-derived precursors, into lateral plate-derived VSMCs including &amp;amp;amp;#39;coronary-like&amp;amp;amp;#39; VSMCs and neural crest-derived VSMC, respectively. We believe this approach will have broad applications in modeling origin-specific disease vulnerability and in developing personalized cell-based vascular grafts for regenerative medicine.
Circulation, Nov 26, 2013
Canadian Journal of Cardiology, 2015
The Journal of Cell Biology, 2009
Fwd shuttles Rab11 to the cleavage furrow by both kinase-dependent and -independent mechanisms.
Circulation Research, 2011
Cell cycle progression in vascular smooth muscle cells (VSMCs) is a therapeutic target for resten... more Cell cycle progression in vascular smooth muscle cells (VSMCs) is a therapeutic target for restenosis. Objective: Having discovered that calmodulin (CaM)-dependent cyclin E/CDK2 activity underlies Ca 2؉-sensitive G 1-to-S phase transitions in VSMCs, we sought to explore the physiological importance of the CaM-cyclin E interaction. Methods and Results: A peptide based on the CaM binding sequence (CBS) of cyclin E was designed to interfere with CaM-cyclin E binding. Compared with control peptides, CBS blocked activating Thr160 phosphorylation of CDK2, decreased basal cyclin E/CDK2 activity, and eliminated Ca 2؉-sensitive cyclin E/CDK2 activity in nuclear extracts from mouse VSMCs. Nucleofection with CBS, or treatment with CBS conjugated to the HIV-1 TAT protein transduction domain to improve bioavailability, inhibited G 1-to-S cell cycle progression in a dose-dependent manner. These effects were not observed with control peptides. TAT-CBS inhibited 3 Hthymidine incorporation in primary human aortic SMCs (HA-SMCs) in vitro, manifested greater transduction into HA-SMCs compared with endothelial cells in vitro, and limited decreased SM22␣ expression, neointima formation, and medial thickening without affecting collagen deposition or reendothelialization in a mouse model of carotid artery injury in vivo. The antiproliferative effects of CBS remained evident in mouse embryonic fibroblasts derived from wild-type mice but not cyclin E1/E2 double knockout mice. Conclusions: A synthetic peptide designed to disrupt CaM-cyclin E binding inhibits Ca 2؉ /CaM-dependent CDK2 activity, cell cycle progression, and proliferation in VSMCs and limits arterial remodeling following injury. Importantly, this effect appears to be cyclin E-dependent and may form the basis of a potentially novel therapeutic approach for restenosis. (Circ Res. 2011;108:1053-1062.) Key Words: vascular smooth muscle cell Ⅲ cell cycle Ⅲ restenosis Ⅲ calmodulin Ⅲ cyclin E/CDK2 V ascular smooth muscle cells (VSMCs) normally proliferate at very low rates in the media of adult arteries, remaining in the growth arrested (G 0) phase of the cell cycle. A shift in the balance between growth stimulatory and inhibitory factors can lead to cell cycle reentry and transformation from contractile and quiescent to proliferative and synthetic phenotypes. Thus activated, VSMCs can remodel the artery by altering the extracellular matrix, replicating in the media, and migrating to the intima to undergo further cycles of proliferation. Indeed, unregulated proliferation of VSMCs is a principal mechanism underlying the pathogenesis of common vascular diseases, such as atherosclerosis and restenosis. 1,2 Decades of work have implicated ionic calcium (Ca 2ϩ) as a regulator of eukaryotic cell cycle progression. 3 In VSMCs, we previously made 3 related discoveries regarding Ca 2ϩ-mediated cell cycle regulation: (1) a coordinated increase in the free intracellular Ca 2ϩ concentration is required for G 1-to-S phase cell cycle transition 4,5 ; (2) this occurs through cell cycleassociated expression and activation of specific Ca 2ϩ pumps and channels 5-7 ; and (3) is at least partly mediated by Ca 2ϩ /calmodulin (CaM)-dependent cyclin E/CDK2 activity. 8 Our findings suggested that Ca 2ϩ sensitivity of the G 1-to-S phase cell cycle transition requires the direct binding of the major Ca 2ϩ signal transducer CaM to cyclin E, through a specific and conserved CaM-binding motif in cyclin E. The functional importance of this motif was accentuated by the observation that a cyclin E mutant lacking this motif was unable to produce Ca 2ϩ /CaM-stimulated activity of CDK2. 8
Cardiovascular Research, 2013
Time for primary review: 32 days Aims Despite the diverse developmental origins of vascular smoot... more Time for primary review: 32 days Aims Despite the diverse developmental origins of vascular smooth muscle cells (VSMCs), recent attempts to generate VSMCs from human embryonic stem cells (hESCs) differentiated along various lineages did not yield distinct cell phenotypes. The aim of this study was to derive and characterize functional coronary-like VSMCs from hESCs using serum-free cardiac-directed differentiation. Methods and results Embryoid bodies (EBs) from three pluripotent stem cell lines subjected to cardiac-directed differentiation in defined media were characterized over 30 days for VSMC-specific gene expression by qRT-PCR, immunofluorescence microscopy and fluorescence-activated cell sorting (FACS). EBs composed of cardiomyocytes, endothelial cells (ECs), fibroblasts, and VSMCs underwent FACS on d28 to reveal that the VSMCs form a distinct subpopulation, which migrate with ECs in an in vitro angiogenesis assay. To enrich for VSMCs, d28 EBs were dissociated and cultured as monolayers. Over several passages, mRNA and protein levels of cardiomyocyte, endothelial, and fibroblast markers were abolished, whereas those of mature VSMCs were unchanged. Vascular endothelial growth factor and basic fibroblast growth factor were critical for the separation of the cardiac and VSMC lineages in EBs, and for the enrichment of functional VSMCs in monolayer cultures. Calcium cycling and cell shortening responses to vasoconstrictors in hESC-derived VSMCs in vitro were indistinguishable from primary human coronary artery SMCs, and distinct from bladder and aorta SMCs. VSMCs identically derived from green fluorescent protein-expressing hESCs integrated in and contributed to new vessel formation in vivo. Conclusion The ability to generate hESC-derived functional human coronary-like VSMCs in serum-free conditions has implications for disease modelling, drug screening, and regenerative therapies.
Canadian Journal of Cardiology, 2013
Canadian Journal of Cardiology, 2012
Successful completion of cytokinesis relies on addition of new membrane, and requires the recycli... more Successful completion of cytokinesis relies on addition of new membrane, and requires the recycling endosome regulator Rab11, which localizes to the midzone. Despite the critical role of Rab11 in this process, little is known about the formation and composition of Rab11-containing organelles. Here, we identify the phosphatidylinositol (PI) 4-kinase III � Four wheel drive (Fwd) as a key regulator of Rab11 during cytokinesis in Drosophila melanogaster spermatocytes. We show Fwd is required for synthesis of PI 4-phosphate (PI4P) on Golgi
Background: Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to b... more Background: Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not
Atherosclerosis Supplements
Circulation, 2016
Rationale: Sca-1 + progenitor cells in mouse aorta are known to generate vascular cells. However,... more Rationale: Sca-1 + progenitor cells in mouse aorta are known to generate vascular cells. However, their embryological origins and temporospatial abundance during development were not known. Objective: To describe stem cells of the mouse aorta and identify lineages of Sca-1 + progenitors. Methods & Results: Lineage-traced reporter mice at 8+ weeks of age reveal aortic stem cells, defined as self-renewing colony-forming cells with multi-lineage potential, to be most abundant in proximal aorta and predominantly of mesodermal origin ( T-Cre ), with little, if any, from neural crest ( Wnt1-Cre ). In distal aorta, the few stem cells that exist are strictly of mesodermal origin. Clonal cell colonies can be cultured in suspension from aorta-derived stem cells of somitic mesoderm origin ( Myf5-Cre ), and contain cells expressing stem cell marker Sox2. Using tamoxifen-inducible Myf5-Cre ER , we show that Sca-1 + aortic cells from somite are specified at E8.5, even before Myf5 + cells contribu...
Rationale: Few methods enable molecular and cellular studies of human vascular aging or type-2 di... more Rationale: Few methods enable molecular and cellular studies of human vascular aging or type-2 diabetes (T2D). Objective: To characterize functional responses of human vascular smooth muscle cells (VSMC) differentiated from progenitors found in skin. Methods and Results: Small biopsies (~1 cm 2 ) taken from the edges of leg or chest incisions of subjects (N=123; males 72%; mean age=63±12 years) undergoing cardiothoracic surgery were used to isolate skin cells and quantify the number of skin-derived precursors (SKPs) that can be cultured. Multivariate analysis revealed that the numbers of cells isolated from skin biopsies (normalized to skin weight) were reduced as a function of age, dyslipidemia and coronary artery disease (CAD). Although SKPs could be subsequently cultured from these biopsies and differentiated into VSMCs at high efficiency (>80% yield), the numbers of SKPs isolated from subjects with T2D were ~50% lower than those without T2D (cells/g: 0.22±0.04, N=46 vs. 0.52±...
Stem cells and development, Jan 2, 2018
Sca-1<sup>+</sup> progenitor cells in the adult mouse aorta are known to generate vas... more Sca-1<sup>+</sup> progenitor cells in the adult mouse aorta are known to generate vascular smooth muscle cells (VSMC), but their embryological origins and temporal abundance are not known. Using tamoxifen-inducible <i>Myf5-Cre</i><sup>ER</sup> mice, we demonstrate that Sca-1<sup>+</sup> adult aortic cells arise from the somitic mesoderm beginning at E8.5 and continue throughout somitogenesis. <i>Myf5</i> lineage-derived Sca-1<sup>+</sup> cells greatly expand in situ starting at 4 weeks of age and become a major source of aortic Sca-1<sup>+</sup> cells by 6 weeks of age. <i>Myf5</i>-derived adult aortic cells are capable of forming multicellular sphere-like structures <i>in vitro</i>, and express the pluripotency marker Sox2. Exposure to TGF-β3 induces these spheres to differentiate into calponin-expressing VSMC. Pulse-chase experiments using tamoxifen-inducible <i>Sox2-C...
Canadian Journal of Cardiology
Clinical Science
Objective: Few methods enable molecular and cellular studies of vascular aging or Type 2 diabetes... more Objective: Few methods enable molecular and cellular studies of vascular aging or Type 2 diabetes (T2D). Here, we report a new approach to studying human vascular smooth muscle cell (VSMC) pathophysiology by examining VSMCs differentiated from progenitors found in skin. Approach and results: Skin-derived precursors (SKPs) were cultured from biopsies (N=164, ∼1 cm2) taken from the edges of surgical incisions of older adults (N=158; males 72%; mean age 62.7 ± 13 years) undergoing cardiothoracic surgery, and differentiated into VSMCs at high efficiency (>80% yield). The number of SKPs isolated from subjects with T2D was ∼50% lower than those without T2D (cells/g: 0.18 ± 0.03, N=58 versus 0.40 ± 0.05, N=100, P<0.05). Importantly, SKP-derived VSMCs from subjects with T2D had higher Fluo-5F-determined baseline cytosolic Ca2+ concentrations (AU: 1,968 ± 160, N=7 versus 1,386 ± 170, N=13, P<0.05), and a trend toward greater Ca2+ cycling responses to norepinephrine (NE) (AUC: 177,20...
ACS Applied Materials & Interfaces, 2017
In the embryonic heart, electrical impulses propagate in a unidirectional manner from the sinus v... more In the embryonic heart, electrical impulses propagate in a unidirectional manner from the sinus venosus and appear to be involved in cardiogenesis. In this work, aligned and random polyaniline/polyetersulfone (PANI/PES) nanofibrous scaffolds doped by Camphor-10-sulfonic acid (β) (CPSA) were fabricated via electrospinning and used to conduct electrical impulses in a unidirectional and multidirectional fashion, respectively. A bioreactor was subsequently engineered to apply electrical impulses to cells cultured on PANI/PES scaffolds. We established cardiovascular disease-specific induced pluripotent stem cells (CVD-iPSCs) from the fibroblasts of patients undergoing cardiothoracic surgeries. The CVD-iPSCs were seeded onto the scaffolds, cultured in cardiomyocyte-inducing factors, and exposed to electrical impulses for 1 h/day, over a 15-day time period in the bioreactor. The application of the unidirectional electrical stimulation to the cells significantly increased the number of cardiac Troponin T (cTnT+) cells in comparison to multidirectional electrical stimulation using random fibrous scaffolds. This was confirmed by real-time polymerase chain reaction for cardiac-related transcription factors (NKX2.5, GATA4, and NPPA) and a cardiac-specific structural gene (TNNT2). Here we report for the first time that applying electrical pulses in a unidirectional manner mimicking the unidirectional wave of electrical stimulation in the heart, could increase the derivation of cardiomyocytes from CVD-iPSCs.
Circulation, Nov 23, 2010
Circulation, Nov 26, 2013
Methods, 2015
Transplantation of vascular smooth muscle cells (VSMCs) is a promising cellular therapy to promot... more Transplantation of vascular smooth muscle cells (VSMCs) is a promising cellular therapy to promote angiogenesis and wound healing. However, VSMCs are derived from diverse embryonic sources which may influence their role in the development of vascular disease and in its therapeutic modulation. Despite progress in understanding the mechanisms of VSMC differentiation, there remains a shortage of robust methods for generating lineage-specific VSMCs from pluripotent and adult stem/progenitor cells in serum-free conditions. Here we describe a method for differentiating pluripotent stem cells, such as embryonic and induced pluripotent stem cells, as well as skin-derived precursors, into lateral plate-derived VSMCs including &amp;amp;amp;#39;coronary-like&amp;amp;amp;#39; VSMCs and neural crest-derived VSMC, respectively. We believe this approach will have broad applications in modeling origin-specific disease vulnerability and in developing personalized cell-based vascular grafts for regenerative medicine.
Circulation, Nov 26, 2013
Canadian Journal of Cardiology, 2015
The Journal of Cell Biology, 2009
Fwd shuttles Rab11 to the cleavage furrow by both kinase-dependent and -independent mechanisms.
Circulation Research, 2011
Cell cycle progression in vascular smooth muscle cells (VSMCs) is a therapeutic target for resten... more Cell cycle progression in vascular smooth muscle cells (VSMCs) is a therapeutic target for restenosis. Objective: Having discovered that calmodulin (CaM)-dependent cyclin E/CDK2 activity underlies Ca 2؉-sensitive G 1-to-S phase transitions in VSMCs, we sought to explore the physiological importance of the CaM-cyclin E interaction. Methods and Results: A peptide based on the CaM binding sequence (CBS) of cyclin E was designed to interfere with CaM-cyclin E binding. Compared with control peptides, CBS blocked activating Thr160 phosphorylation of CDK2, decreased basal cyclin E/CDK2 activity, and eliminated Ca 2؉-sensitive cyclin E/CDK2 activity in nuclear extracts from mouse VSMCs. Nucleofection with CBS, or treatment with CBS conjugated to the HIV-1 TAT protein transduction domain to improve bioavailability, inhibited G 1-to-S cell cycle progression in a dose-dependent manner. These effects were not observed with control peptides. TAT-CBS inhibited 3 Hthymidine incorporation in primary human aortic SMCs (HA-SMCs) in vitro, manifested greater transduction into HA-SMCs compared with endothelial cells in vitro, and limited decreased SM22␣ expression, neointima formation, and medial thickening without affecting collagen deposition or reendothelialization in a mouse model of carotid artery injury in vivo. The antiproliferative effects of CBS remained evident in mouse embryonic fibroblasts derived from wild-type mice but not cyclin E1/E2 double knockout mice. Conclusions: A synthetic peptide designed to disrupt CaM-cyclin E binding inhibits Ca 2؉ /CaM-dependent CDK2 activity, cell cycle progression, and proliferation in VSMCs and limits arterial remodeling following injury. Importantly, this effect appears to be cyclin E-dependent and may form the basis of a potentially novel therapeutic approach for restenosis. (Circ Res. 2011;108:1053-1062.) Key Words: vascular smooth muscle cell Ⅲ cell cycle Ⅲ restenosis Ⅲ calmodulin Ⅲ cyclin E/CDK2 V ascular smooth muscle cells (VSMCs) normally proliferate at very low rates in the media of adult arteries, remaining in the growth arrested (G 0) phase of the cell cycle. A shift in the balance between growth stimulatory and inhibitory factors can lead to cell cycle reentry and transformation from contractile and quiescent to proliferative and synthetic phenotypes. Thus activated, VSMCs can remodel the artery by altering the extracellular matrix, replicating in the media, and migrating to the intima to undergo further cycles of proliferation. Indeed, unregulated proliferation of VSMCs is a principal mechanism underlying the pathogenesis of common vascular diseases, such as atherosclerosis and restenosis. 1,2 Decades of work have implicated ionic calcium (Ca 2ϩ) as a regulator of eukaryotic cell cycle progression. 3 In VSMCs, we previously made 3 related discoveries regarding Ca 2ϩ-mediated cell cycle regulation: (1) a coordinated increase in the free intracellular Ca 2ϩ concentration is required for G 1-to-S phase cell cycle transition 4,5 ; (2) this occurs through cell cycleassociated expression and activation of specific Ca 2ϩ pumps and channels 5-7 ; and (3) is at least partly mediated by Ca 2ϩ /calmodulin (CaM)-dependent cyclin E/CDK2 activity. 8 Our findings suggested that Ca 2ϩ sensitivity of the G 1-to-S phase cell cycle transition requires the direct binding of the major Ca 2ϩ signal transducer CaM to cyclin E, through a specific and conserved CaM-binding motif in cyclin E. The functional importance of this motif was accentuated by the observation that a cyclin E mutant lacking this motif was unable to produce Ca 2ϩ /CaM-stimulated activity of CDK2. 8
Cardiovascular Research, 2013
Time for primary review: 32 days Aims Despite the diverse developmental origins of vascular smoot... more Time for primary review: 32 days Aims Despite the diverse developmental origins of vascular smooth muscle cells (VSMCs), recent attempts to generate VSMCs from human embryonic stem cells (hESCs) differentiated along various lineages did not yield distinct cell phenotypes. The aim of this study was to derive and characterize functional coronary-like VSMCs from hESCs using serum-free cardiac-directed differentiation. Methods and results Embryoid bodies (EBs) from three pluripotent stem cell lines subjected to cardiac-directed differentiation in defined media were characterized over 30 days for VSMC-specific gene expression by qRT-PCR, immunofluorescence microscopy and fluorescence-activated cell sorting (FACS). EBs composed of cardiomyocytes, endothelial cells (ECs), fibroblasts, and VSMCs underwent FACS on d28 to reveal that the VSMCs form a distinct subpopulation, which migrate with ECs in an in vitro angiogenesis assay. To enrich for VSMCs, d28 EBs were dissociated and cultured as monolayers. Over several passages, mRNA and protein levels of cardiomyocyte, endothelial, and fibroblast markers were abolished, whereas those of mature VSMCs were unchanged. Vascular endothelial growth factor and basic fibroblast growth factor were critical for the separation of the cardiac and VSMC lineages in EBs, and for the enrichment of functional VSMCs in monolayer cultures. Calcium cycling and cell shortening responses to vasoconstrictors in hESC-derived VSMCs in vitro were indistinguishable from primary human coronary artery SMCs, and distinct from bladder and aorta SMCs. VSMCs identically derived from green fluorescent protein-expressing hESCs integrated in and contributed to new vessel formation in vivo. Conclusion The ability to generate hESC-derived functional human coronary-like VSMCs in serum-free conditions has implications for disease modelling, drug screening, and regenerative therapies.
Canadian Journal of Cardiology, 2013
Canadian Journal of Cardiology, 2012