Stem Cell-derived Smooth Muscle Cells Research Articles

LRP1 Repression by SNAIL Results in ECM Remodeling in Genetic Risk for Vascular Diseases.

Genome-wide association studies implicate common genetic variations in the LRP1 (low-density lipoprotein receptor-related protein 1) locus at risk for multiple vascular diseases and traits. However, the underlying biological mechanisms are unknown. Fine mapping analyses included Bayesian colocalization to identify the most likely causal variant. Human induced pluripotent stem cells were genome-edited using CRISPR-Cas9 to delete or modify candidate enhancer regions and generate LRP1 knockout cell lines. Cells were differentiated into smooth muscle cells through a mesodermal lineage. Transcription regulation was assessed using luciferase reporter assay, transcription factor knockdown, and chromatin immunoprecipitation. Phenotype changes in cells were conducted using cellular assays, bulk RNA sequencing, and mass spectrometry. Multitrait colocalization analyses pointed at rs11172113 as the most likely causal variant in LRP1 for fibromuscular dysplasia, migraine, pulse pressure, and pulmonary function trait. We found the rs11172113-T allele to associate with higher LRP1 expression. Genomic deletion in induced pluripotent stem cell-derived smooth muscle cells supported rs11172113 to locate in an enhancer region regulating LRP1 expression. We found transcription factors MECP2 (methyl CpG binding protein 2) and SNAIL to repress LRP1 expression through an allele-specific mechanism, involving SNAIL interaction with disease risk allele. LRP1 knockout decreased induced pluripotent stem cell-derived smooth muscle cell proliferation and migration. Differentially expressed genes were enriched for collagen-containing extracellular matrix, connective tissue development, and lung development. LRP1 knockout and deletion of rs11172113 enhancer showed potentiated canonical TGF-β (transforming growth factor beta) signaling through enhanced phosphorylation of SMAD2/3. Analyses of the protein content of decellularized extracts indicated partial extracellular matrix remodeling involving enhanced secretion of CYR61, a known LRP1 ligand involved in vascular integrity and TIMP3, implicated in extracellular matrix maintenance and also known to interact with LRP1. Our findings support allele-specific LRP1 gene repression by the endothelial-to-mesenchymal transition regulator SNAIL. We propose decreased LRP1 expression in smooth muscle cells to remodel the extracellular matrix enhanced by TGF-β as a potential mechanism of this pleiotropic locus for vascular diseases.

Endothelial Cells Increase Mesenchymal Stem Cell Differentiation in Scaffold-Free 3D Vascular Tissue.

In this study, we present a versatile, scaffold-free approach to create ring-shaped engineered vascular tissue segments using human mesenchymal stem cell-derived smooth muscle cells (hMSC-SMCs) and endothelial cells (ECs). We hypothesized that incorporation of ECs would increase hMSC-SMC differentiation without compromising tissue ring strength or fusion to form tissue tubes. Undifferentiated hMSCs and ECs were co-seeded into custom ring-shaped agarose wells using four different concentrations of ECs: 0%, 10%, 20%, and 30%. Co-seeded EC and hMSC rings were cultured in SMC differentiation medium for a total of 22 days. Tissue rings were then harvested for histology, Western blotting, wire myography, and uniaxial tensile testing to examine their structural and functional properties. Differentiated hMSC tissue rings comprising 20% and 30% ECs exhibited significantly greater SMC contractile protein expression, endothelin-1 (ET-1)-meditated contraction, and force at failure compared with the 0% EC rings. On average, the 0%, 10%, 20%, and 30% EC rings exhibited a contractile force of 0.745 ± 0.117, 0.830 ± 0.358, 1.31 ± 0.353, and 1.67 ± 0.351 mN (mean ± standard deviation [SD]) in response to ET-1, respectively. Additionally, the mean maximum force at failure for the 0%, 10%, 20%, and 30% EC rings was 88.5 ± 36. , 121 ± 59.1, 147 ± 43.1, and 206 ± 0.8 mN (mean ± SD), respectively. Based on these results, 30% EC rings were fused together to form tissue-engineered blood vessels (TEBVs) and compared with 0% EC TEBV controls. The addition of 30% ECs in TEBVs did not affect ring fusion but did result in significantly greater SMC protein expression (calponin and smoothelin). In summary, co-seeding hMSCs with ECs to form tissue rings resulted in greater contraction, strength, and hMSC-SMC differentiation compared with hMSCs alone and indicates a method to create a functional 3D human vascular cell coculture model.

Abstract 3115: Mechanics And Genomic Profiles Reveal Aging And Developmental Lineage Heterogeneities In Human Induced Pluripotent Stem Cell-derived Smooth Muscle Cells.

Effective stem cell therapies and disease modeling depend on understanding epigenetic factors of aging. Accurately modeling age-dependent phenotypes such as aortic, and smooth muscle cell stiffening, requires cell models that reflect donor age. Improved knowledge of how donor aging influences stem cell-derived smooth muscle cells is essential for precise disease modeling and creating targeted therapeutics. We hypothesize that traits of vascular aging are retained in human induced pluripotent stem cells (iPSCs) and manifest in differentiated smooth muscle cells (SMCs). We aim to compare iPSC stiffness and gene expression between young (under 40, n=5 lines) and old (over 60, n=4 lines) donors, and examine age-dependent differences in their iPSC-SMCs. We measured cell stiffness using atomic force microscopy (AFM), and analyzed bulk and single-cell RNA gene expression in iPSCs and iPSC-SMCs of neuroectoderm (NE) or paraxial mesoderm (PM) lineages, respectively. AFM revealed no significant age-related differences in iPSC colony stiffness. Bulk RNA expression showed high similarity (>94% Pearson correlation) between young and old donor iPSCs, with principal component analysis indicating low variability and no clear age separation. Single-cell stiffness measurements found old NE-SMCs significantly stiffer than young ones (p=0.032), with no marked difference in PM-SMCs between ages. Old and young NE-SMCs were stiffer than their respective PM-SMC counterparts (old, p=0.006; young, p=0.007). UMAP analysis of single-cell RNA expression displayed distinct groupings for old and young NE-SMCs, but not for PM-SMCs. Cluster analysis revealed three clusters predominantly of old NE-SMCs and one of young NE-SMCs. Our study reveals age-related stiffening in NE lineage iPSC-SMCs, but not in PM lineage or iPSCs, indicating that iPSCs preserve certain donor age-related traits that manifest as lineage-specific phenotypes in SMCs post-differentiation. RNA sequencing showed similar expression patterns in young and old iPSCs, but age-specific differences in NE-SMCs. The integration of AFM and RNA expression data sheds light on the emergence of age-dependent phenotypes in differentiated, sensitive cell types, which are not present in immature cells.

Esophageal wound healing by aligned smooth muscle cell-laden nanofibrous patch.

The esophagus exhibits peristalsis via contraction of circularly and longitudinally aligned smooth muscles, and esophageal replacement is required if there is a critical-sized wound. In this study, we proposed to reconstruct esophageal tissues using cell electrospinning (CE), an advanced technique for encapsulating living cells into fibers that allows control of the direction of fiber deposition. After treatment with transforming growth factor-β, mesenchymal stem cell-derived smooth muscle cells (SMCs) were utilized for cell electrospinning or three-dimensional bioprinting to compare the effects of aligned micropatterns on cell morphology. CE resulted in SMCs with uniaxially arranged and elongated cell morphology with upregulated expression levels of SMC-specific markers, including connexin 43, smooth muscle protein 22 alpha (SM22α), desmin, and smoothelin. When SMC-laden nanofibrous patches were transplanted into a rat esophageal defect model, the SMC patch promoted regeneration of esophageal wounds with an increased number of newly formed blood vessels and enhanced the SMC-specific markers of SM22α and vimentin. Taken together, CE with its advantages, such as guidance of highly elongated, aligned cell morphology and accelerated SMC differentiation, can be an efficient strategy to reconstruct smooth muscle tissues and treat esophageal perforation.

Induced pluripotent stem cell-derived smooth muscle cells to study cardiovascular calcification.

Cardiovascular calcification is the lead predictor of cardiovascular events and the top cause of morbidity and mortality worldwide. To date, only invasive surgical options are available to treat cardiovascular calcification despite the growing understanding of underlying pathological mechanisms. Key players in vascular calcification are vascular smooth muscle cells (SMCs), which transform into calcifying SMCs and secrete mineralizing extracellular vesicles that form microcalcifications, subsequently increasing plaque instability and consequential plaque rupture. There is an increasing, practical need for a large scale and inexhaustible source of functional SMCs. Here we describe an induced pluripotent stem cell (iPSC)-derived model of SMCs by differentiating iPSCs toward SMCs to study the pathogenesis of vascular calcification. Specifically, we characterize the proteome during iPSC differentiation to better understand the cellular dynamics during this process. First, we differentiated human iPSCs toward an induced-SMC (iSMC) phenotype in a 10-day protocol. The success of iSMC differentiation was demonstrated through morphological analysis, immunofluorescent staining, flow cytometry, and proteomics characterization. Proteomics was performed throughout the entire differentiation time course to provide a robust, well-defined starting and ending cell population. Proteomics data verified iPSC differentiation to iSMCs, and functional enrichment of proteins on different days showed the key pathways changing during iSMC development. Proteomics comparison with primary human SMCs showed a high correlation with iSMCs. After iSMC differentiation, we initiated calcification in the iSMCs by culturing the cells in osteogenic media for 17 days. Calcification was verified using Alizarin Red S staining and proteomics data analysis. This study presents an inexhaustible source of functional vascular SMCs and calcifying vascular SMCs to create an in vitro model of vascular calcification in osteogenic conditions, with high potential for future applications in cardiovascular calcification research.

Generation and Characterization of Human Mesenchymal Stem Cell-Derived Smooth Muscle Cells

Cardiovascular diseases are the leading cause of death worldwide. A completely autologous treatment can be achieved by using elastogenic mesenchymal stem cell (MSC)-derived smooth muscle cells (SMC) at the affected tissue site of vascular diseases such as abdominal aortic aneurysms (AAA). Thus, our work focused on evaluating the efficacy of (a) the combination of various growth factors, (b) different time periods and (c) different MSC lines to determine the treatment combination that generated SMCs that exhibited the greatest elastogenicity among the tested groups using Western blotting and flow cytometry. Additionally, total RNA sequencing was used to confirm that post-differentiation cells were upregulating SMC-specific gene markers. Results indicated that MSCs cultured for four days in PDGF + TGFβ1 (PT)-infused differentiation medium showed significant increases in SMC markers and decreases in MSC markers compared to MSCs cultured without differentiation factors. RNA Seq analysis confirmed the presence of vascular smooth muscle formation in MSCs differentiated in PT medium over a seven-day period. Overall, our results indicated that origin, growth factor treatment and culture period played a major role in influencing MSC differentiation to SMCs.

Abstract P174: Nuclear Smooth Muscle α-actin Is Critical For Smooth Muscle Cell Differentiation And To Prevent Cerebrovascular Disease

Missense mutations in ACTA2, encoding α-smooth muscle actin (α-SMA), predispose to thoracic aortic aneurysms. A subset of these ACTA2 mutations also predispose to childhood onset cerebrovascular disease characterized by occlusion of the distal internal carotid arteries and small vessel disease. In our studies to identify how specific ACTA2 mutations predispose to cerebrovascular disease, we confirmed that α-SMA translocates to the nucleus in wildtype (WT) smooth muscle cells (SMCs), is enriched in the nucleus over β-actin with differentiation of SMCs, associates with the INO80 chromatin remodeling complex, and selectively binds to the promoters of SMC contractile genes. Expression of individual mutant α-SMAs in 293 cells determined that missense mutations associated with cerebrovascular disease inhibit this nuclear translocation. To further examine the effects of reduced nuclear α-SMA with ACTA2 mutations, we used two model systems: pluripotent stem cell-derived SMCs from patients with ACTA2-associated cerebrovascular disease (ACTA2 R179C) as well as SMCs explanted from a knockin mouse model (Acta2SMC-R179C/+). Both mouse and human cells harboring the R179C mutation proliferate and migrate more than WT SMCs, are less differentiated than WT SMCs, and have increased expression of pluripotency-associated genes. Although the mutation is heterozygous, these cells show a dominant negative impact on nuclear α-SMA function through dramatically reduced levels of α-SMA in the nucleus, in the INO80 chromatin remodeling complex, and on the promoters of SMC-specific genes. Finally, forced nuclear localization of α-SMA in WT SMCs increases levels of SMC contractile proteins. Taken together, we have identified a novel role for α-SMA in driving differentiation of SMCs, and our data supports that defects in this nuclear role drive cellular phenotypes consistent with the cerebrovascular disease seen in patients with ACTA2 R179 mutations.

Induced pluripotent stem cell-derived smooth muscle cells increase angiogenesis and accelerate diabetic wound healing.

Aim:To assess the potential of human induced pluripotent stem cell-derived smooth muscle cells (hiPSC-SMC) to accelerate diabetic wound healing.Methods:hiPSC-SMC were embedded in 3D collagen scaffolds and cultured in vitro for 72 h; scaffolds were then applied to diabetic, nude mouse, splinted back wounds to assess in vivo healing. Cultured medium after scaffold incubation was collected and analyzed for expression of pro-angiogenic cytokines.Results:hiPSC-SMC secrete increased concentration of pro-angiogenic cytokines, compared with murine adipose derived stem cells. Delivery of hiPSC-SMC-containing collagen scaffolds accelerates diabetic wound healing and is associated with an increased number of total and M2 type macrophages.Conclusion:hiPSC-SMC promote angiogenesis and accelerate diabetic wound healing, making them a promising new candidate for treatment of diabetic wounds.

IPSC-Derived Endothelial Cells Affect Vascular Function in a Tissue-Engineered Blood Vessel Model of Hutchinson-Gilford Progeria Syndrome.

SummaryHutchinson-Gilford progeria syndrome (HGPS) is a rare disorder caused by a point mutation in the Lamin A gene that produces the protein progerin. Progerin toxicity leads to accelerated aging and death from cardiovascular disease. To elucidate the effects of progerin on endothelial cells, we prepared tissue-engineered blood vessels (viTEBVs) using induced pluripotent stem cell-derived smooth muscle cells (viSMCs) and endothelial cells (viECs) from HGPS patients. HGPS viECs aligned with flow but exhibited reduced flow-responsive gene expression and altered NOS3 levels. Relative to viTEBVs with healthy cells, HGPS viTEBVs showed reduced function and exhibited markers of cardiovascular disease associated with endothelium. HGPS viTEBVs exhibited a reduction in both vasoconstriction and vasodilation. Preparing viTEBVs with HGPS viECs and healthy viSMCs only reduced vasodilation. Furthermore, HGPS viECs produced VCAM1 and E-selectin protein in TEBVs with healthy or HGPS viSMCs. In summary, the viTEBV model has identified a role of the endothelium in HGPS.

Pro-elastogenic effects of mesenchymal stem cell derived smooth muscle cells in a 3D collagenous milieu

Intrinsically poor auto-regenerative repair of proteolytically-disrupted elastic matrix structures by resident SMCs in the wall of abdominal aortic aneurysms (AAAs) prevents growth arrest and regression of these wall expansions. Supporting their possible future use in a regenerative cell therapy for AAAs, in a prior study, we showed that bone marrow mesenchymal stem cell-derived Smooth Muscle Cells (BM-SMCs) secrete biological factors that have significant pro-elastogenic and anti-proteolytic effects on aneurysmal rat aortic SMCs (EaRASMCs) in non-contact co-cultures. We also identified one stable BM-SMC phenotype (cBM-SMC) generated by differentiating BM-MSCs on a 2D fibronectin substrate in the presence of PDGF (Platelet Derived Growth Factor) and TGF-β1 (Transforming Growth Factor-β1) that exhibited superior elastogenicity and pro-elastogenic/anti-proteolytic properties. In this study, we further investigated the ability of these cBM-SMCs to maintain these superior elastogenic properties in a 3D collagenous milieu alone and in co-culture with EaRASMC to evaluate their potential as an alternative cell source for cell therapy in AAA. Some of our key observations were higher contractility and greater amount of structurally intact elastin production in both standalone culture of cBM-SMCs as well as co-culture of cBM-SMCs with EaRASMCs as shown by VVG (Verhoeff-Van Gieson) staining and Pontamine Sky Blue labeling and lower MMP-9 protein expression in standalone culture in 3D collagenous environment. Our overall result indicates that cBM-SMCs possess the ability to provide elastogenic impetus in a 3D collagenous AAA milieu which is otherwise not conducive to elastogenesis. Therefore our study strongly suggest the utility of cBM-SMCs as a potential cell source for cell therapy to augment elastic matrix neo-assembly and fiber formation and attenuate proteolysis in a collagenous milieu that is evocative of the de-elasticized aneurysmal wall. Statement of SignificanceAbdominal aortic aneurysm (AAA) or ballooning of the aorta is one of the leading causes of cardiovascular disease (CVD) related death caused by significantly increased proteolytic activity in the aortic wall. Reversing pathophysiology of this condition is challenging due to intrinsically poor regeneration of elastin by aortic smooth muscle cells. Current management of AAA is limited to passive monitoring of the disease until it becomes large enough to receive surgical intervention and no drug based therapy currently exists. Cell based therapy can be a potential alternative treatment in this scenario because it provides elastogenic impetus to the aneurysmal SMCs, compensates for the dead SMCs and serves as a robust source of elastin while being delivered with minimal invasiveness. Hence this work will have significant impact in the field of tissue engineering and regenerative medicine.

Abstract 650: Stem Cell-derived SMCs Are Useful for Vascular Tissue Engineering

Objectives: Tissue engineered vascular grafts with long-term patency are in great need in the clinics, for which smooth muscle cells (SMCs) are essential. Recently several laboratories have established methods to obtain SMCs from embryonic stem cells or iPS cells. However, the mechanisms of stem cell differentiation are not fully understood. And also it is hard to obtain a large number of SMCs with higher purity. To solve these issues, we take the advantage of mesenchymal stem cells (MSCs) from human umbilical cord that were cultivated and differentiated in vitro. Methods: MSCs from human umbilical cord were cultivated and differentiated to vascular lineages. Signal pathways involved in stem cell differentiation were studied using Western blot, siRNA, PCR, immunoprecipation and transfection. For vascular tissue engineering, stem cell-derived SMCs were applied to decelularized scaffold to create the vessels in a Bioreactor. Results: In response to TGFβ1 stimulation, MSCs were abundantly expressing SMC markers including αSMA, SM22, calponin and SMMHC at the gene and protein levels. Functionally, differentiated SMCs displayed a contraction ability in vitro and formed vessels in matrigel plug assay SCID mice. Micro-RNA (miR) array analysis showed the upregulation of miR-503 and the downregulation of miR-222-5p at early time points after TGFβ1 treatment, which was confirmed by TaqMan microRNA assay. Mechanistically, miR-503 was demonstrated to promote SMC differentiation through directly targeting Smad7, a negative regulator of TGFβ1 Smad-related signaling pathways. The expression of miR-503 was Smad4-dependent and Smad4 was enriched at the promoter region of miR-503. In addition, miR-222-5p inhibited SMC differentiation through targeting ROCK2 and αSMA 3’-UTR. Finally, SMCs differentiated from stem cells exhibited the ability to migrate into decellularized mouse aorta and gave rise to vascular graft with smooth muscle layer. Conclusions: We established a method to produce a large number of human SMCs from MSCs, which demonstrated the potential of utilizing the cells to generate vascular grafts. And we provide the mechanistic data demonstrating the impact of miRNA-503/222-5p on TGFβ1-mediated signaling in SMC differentiation from stem cells.

Maintaining Elastogenicity of Mesenchymal Stem Cell-Derived Smooth Muscle Cells in Two-Dimensional Culture.

Abdominal aortic aneurysms (AAAs) are localized expansions of the abdominal aorta that grow slowly to rupture. AAA growth is driven by irreversible elastic matrix breakdown in the aorta wall by chronically upregulated matrix metalloproteases (MMPs). Since adult vascular smooth muscle cells (SMCs) poorly regenerate elastic matrix, we previously explored utility of bone marrow mesenchymal stem cells and SMCs derived therefrom (BM-SMCs) for this purpose. One specific differentiated phenotype (cBM-SMCs) generated on a fibronectin substrate in presence of exogenous transforming growth factor-β and platelet-derived growth factor exhibited superior elastogenicity versus other phenotypes, and usefully provided proelastogenic and antiproteolytic stimuli to aneurysmal SMCs. Since in vivo cell therapy demands large cell inoculates, these derived SMCs must be propagated in vitro while maintaining their superior elastogenic, proelastogenic, and antiproteolytic characteristics. In this work, we thus investigated the culture conditions that must be provided to this propagation phase, which ensure that the differentiated SMCs maintain their phenotype and matrix regenerative benefits. Our results indicate that our BM-SMCs retain their phenotype in long-term culture even in the absence of differentiation growth factors and fibronectin substrate, but these conditions must be continued to be provided during postdifferentiation propagation if they are to maintain their superior elastic matrix deposition, crosslinking, and fiber formation properties. Our study, however, showed that cells propagated under these conditions exhibit higher expression of MMP-2, but favorably, no expression of elastolytic MMP-9. Hence, the study outcomes provide crucial guidelines to maintain phenotypic stability of cBM-SMCs during their propagation in two-dimensional culture before their delivery to the AAA wall for therapy.

Abstract 143: Enrichment of Dimethylation of Lysine 4 on Histone 3 and Resident Vascular Stem Cell Transition to Vascular Smooth Muscle Cells

Data support a role for stem cell-derived vascular smooth muscle cells (SMC) in arteriosclerosis. Epigenetics play a critical role in SMC differentiation where histone proteins associated with the myosin heavy chain (Myh11) promoter are post-translationally modified by dimethylation of lysine 4 on histone 3 (H3K4me2). Studies report that ‘de-differentiated’ SMC do not exhibit loss of H3K4me2 at the Myh11 promoter even when mRNA levels decrease thereby allowing this modification to be used to track differentiated SMC. Our aim was to determine the level of H3K4me2 and H3K27me3 methylation of differentiated SMCs and compare to resident vascular stem cells and stem cell-derived SMC. Murine adventitial Sca1 + stem cells (APCs), rat medial Sox10 + multipotent vascular stem cells (MVSCs) and CD44 + bone-marrow derived mesenchymal stem cells (MSCs) were all examined for methylation of lysine 4 (H3K4me2) and lysine 27 (H3K27me3) on histone 3 associated with the Myh11 promoter, before and after SMC differentiation and compared to fresh aortic differentiated SMC and sub-cultured de-differentiated SMC in vitro by Chromatin Immunoprecipitation (ChIP) assay. Cells were also examined for Myh11 expression, stemness (telomerase activity) and multipotentcy. Differentiated rat and murine SMC were enriched for H3K4Me2 at the Myh11 promoter, compared to H3K27me3. Phenotypically de-differentiated sub-cutured rat and murine SMC were enriched for H3K4Me2 when compared to H3K27me3, but to a much lesser extent when compared to differentiated aortic SMC. In contrast, resident APCs and MVSCs, and bone-marrow derived MSCs, were all enriched for H3K27me3, concomitant with significant telomerase activity and multipotent differentiation capacity. The levels of enrichment of H3K27me3 dropped significantly after SMC differentiation with TGF-βeta1 concomitant with a significant enrichment of H3K4me2 to levels that mimicked the level of enrichment in de-differentiated SMC when compared to aortic differentiated SMC. De-differentiated SMC exhibit reduced enrichment of H3K4me2 at the Myh11 promoter region when compared to differentiated aortic SMCs, but mimic the level of enrichment of H3K4me2 observed following resident vascular stem cell differentiation to SMCs.

Abstract 573: Vibrational Spectroscopy Discriminates Differentiated Vascular Smooth Muscle Cells From Mesenchymal Stem Cells and Their Vascular Progeny

Background: The source of intimal vascular smooth muscle cells (SMCs) following vascular remodelling has been controversial, with either de-differentiated SMCs and/or stem cell-derived SMCs playing a putative role. Fourier transform Infrared (FTIR) and Raman spectroscopy are complementary forms of vibrational spectroscopy which provide an excellent platform for extracting important biochemical data in a label-free manner to discriminate cell types. Aim: Determine whether native differentiated SMCs can be distinguished from mesenchymal stem cells (MSCs) and MSC-derived SMCs using vibrational spectroscopy. Methods: Freshly isolated rat aortic differentiated SMCs (up to passage 4), CD44+ bone marrow derived mesenchymal stem cells (MSCs), MSC-derived smooth muscle cells (mdSMCs - after TGF-β1 treatment for 14 d) and osteoblasts (mdOSTs - after osteogenic inductive stimulation for 21 d) were grown and fixed on calcium fluoride slides before their respective spectra were recorded by Raman and FTIR Spectroscopy. Multivariate statistical algorithms, including Principal Components Analysis (PCA) and Linear Discriminant Analysis (LDA), were applied to the spectra in order to classify the cell types based on their biochemical variation. Results: The recorded spectra for each cell type revealed significant visible differences between the cells across all recordings. The PCA score plot discriminated the cells based on their unique characteristics. A combination of PCA-LDA were applied for classification, and a leave one out cross validation resulted in sensitivities and specificities that were >95%. Conclusion: Vibrational spectroscopy discriminates differentiated SMCs from MSC and their vascular progeny and may be useful for identifying these cells as early biomarkers of disease.

Magnetically Responsive Bone Marrow Mesenchymal Stem Cell-Derived Smooth Muscle Cells Maintain Their Benefits to Augmenting Elastic Matrix Neoassembly

Abdominal aortic aneurysms (AAA) represent abnormal aortal expansions that result from chronic proteolytic breakdown of elastin and collagen fibers by matrix metalloproteases. Poor elastogenesis by adult vascular smooth muscle cells (SMCs) limits regenerative repair of elastic fibers, critical for AAA growth arrest. Toward overcoming these limitations, we recently demonstrated significant elastogenesis by bone marrow mesenchymal stem cell-derived SMCs (BM-SMCs) and their proelastogenesis and antiproteolytic effects on rat aneurysmal SMCs (EaRASMCs). We currently investigate the effects of super paramagnetic iron oxide nanoparticle (SPION) labeling of BM-SMCs, necessary to magnetically guide them to the AAA wall, on their functional benefits. Our results indicate that SPION-labeling is noncytotoxic and does not adversely impact the phenotype and elastogenesis by BM-SMCs. In addition, SPION-BM-SMCs showed no changes in the ability of the BM-SMCs to stimulate elastin regeneration and attenuate proteolytic activity by EaRASMCs. Together, our results are promising toward the utility of SPIONs for magnetic targeting of BM-SMCs for in situ AAA regenerative repair.

Abstract 617: Phenotype as a Basis for Selection of BM-MSC-Derived SMCs for Regenerative Repair of Abdominal Aortic Aneurysms

Abdominal aortic aneurysms (AAA) are a leading cause of death in the elderly with over 150,000 new diagnoses annually in the US. They involve bulging of the abdominal aorta due to chronic proteolytic degradation of the wall structural extracellular matrix (ECM), mainly elastin and collagen. There are currently no established medical management approaches for treating small AAAs and early surgical intervention provides no benefit. AAA growth arrest and regression are also prevented by inherently poor auto regenerative repair of elastic matrix by adult vascular smooth muscle cells (SMCs). We have identified bone marrow mesenchymal stem cell-derived SMCs (BM-SMCs) as superior cell types for regenerative cell therapy due to their high elastogenicity and pro-elastogenic and anti-proteolytic effects on AAA SMCs. In this study we investigated if and how phenotypic coordinates of BM-SMCs on a contractile to synthetic phenotypic continuum impact their above functional benefits. BM-SMC subpopulations were generated under 2% & 10% v/v FBS conditions in presence of transforming growth factor beta-1 (TGFβ), with or without platelet derived growth factor (PDGF-BB) under low & high glucose conditions. Phenotypic coordinates of the BM-SMCs were established by analysis of SMC marker (alpha SM actin (αSMA), SM22, caldesmon, smoothelin, MHC) expression with RT-PCR, western blotting, and immunofluorescence (IF) staining. Elastogenicities of the cell subpopulations were compared by analysis of tropoelastin and matrix elastin synthesis (Fastin assay), crosslinking (ELISA for desmosine), IF, and TEM; contractile properties were compared via a carbachol assay and whole cell patch-clamp for intracellular Ca 2+ /K + activity. Overall, our results indicate that 10% FBS + TGF with/without PDGF express higher contractile markers and higher elastic matrix yield when compared to controls (aortic SMCs and MSCs) and other test cases. Together with ongoing studies that are evaluating pro-elastogenic and anti-proteolytic effects of our generated BM-SMCs on AAA SMCs in non-contact coculture, we believe that the results are promising towards identifying superior elastogenic cell sources for a regenerative cell therapy for AAAs, which we are validating in a rat AAA model.

Pro-elastogenic effects of bone marrow mesenchymal stem cell-derived smooth muscle cells on cultured aneurysmal smooth muscle cells.

Abdominal aortic aneurysms (AAAs) involve slow proteolysis and loss of structural matrix components (collagen and elastin), which lead to wall thinning, weakening and ultimate rupture. At this time, no established non-surgical therapy is available to slow or arrest AAA growth. Inhibiting matrix metalloproteases (MMPs; e.g. MMP2 and -9) overexpressed within AAAs is insufficient to arrest AAA growth, since resident smooth muscle cells (SMCs) are poorly elastogenic and cannot overcome elastolysis to reinstate a healthy elastic matrix. Towards overcoming this limitation, this first study sought to determine the utility of rat bone marrow mesenchymal stem cell (BM-MSC)-derived SMCs to stimulate elastin and elastic matrix synthesis and assembly by aneurysmal SMCs (EaRASMCs). BM-MSCs were successfully differentiated into cells of an SMC lineage (SMLCs). Our study indicates that BM-MSC-derived SMLCs secrete trophic factors, contained in conditioned medium (CM) from their cultures, that, when exposed to EaRASMC cultures in real time, stimulate elastin precursor and matrix deposition and crosslinking by these elastogenically deficient cells, with added benefits in terms of attenuating MMPs, specifically MMP9. The results thus lend support to a proposed cell therapy for AAAs, based on the use of BM-MSC-derived SMLCs. Although we observed no particular improvement in elastic fibre formation, no attenuation of MMP2 activity and increase in amounts of active MMP2 enzyme, we believe that this study justifies follow-up studies to improve upon these outcomes. Future studies will explore the effects of concentrated CM collected from long-term SMLC cultures on EaRASMCs and also investigate the elastogenic output of SMLCs themselves. Copyright © 2014 John Wiley & Sons, Ltd.

Microbioreactors to manipulate oxygen tension and shear stress in the microenvironment of vascular stem and progenitor cells

The dynamics of dissolved oxygen (DO) and shear stress in the vasculature microenvironment play a major role in determining the fate of stem cells in adults and during early embryonic development. In this study, we present a microbioreactor (MBR) that provides independent control over oxygen tension and shear stress in cultures of stem and progenitor cell types. We first describe the design principles and use a model-driven approach for the optimization of the MBR geometry and operating conditions prior to its fabrication and assembly. We then demonstrate the utilization of the MBR for culturing adult human endothelial progenitors, human umbilical vein endothelial cells, and human embryonic stem cell-derived smooth muscle cells under different DO and shear stress levels.

Integrated Membrane Protein Analysis of Mature and Embryonic Stem Cell-derived Smooth Muscle Cells Using a Novel Combination of CyDye/Biotin Labeling

Cultivated vascular smooth muscle cells (SMCs) were surface-labeled with CyDyes followed by biotinylation. After enrichment on avidin columns, proteins were separated on large format gradient gels by SDS-PAGE. A comparison between CyDye-tagged and non-tagged gel bands revealed a substantial increase of protein identifications from membrane, membrane-associated, and extracellular matrix proteins with a corresponding reduction in co-purified intracellular proteins. Notably the majority of identified proteins were involved in cellular adhesion processes. To demonstrate the quantitative potential of this platform, we performed a comparison between mature and embryonic stem cell-derived smooth muscle cells (esSMCs) and identified the membrane proteins E-cadherin, integrin alpha6, and CD98 (4F2) to be significantly up-regulated in esSMCs suggesting that SMCs derived from embryonic stem cells maintain characteristics of their embryonic stem cell origin. This was subsequently confirmed by RT-PCR: despite expressing a panel of smooth muscle markers (calponin, Sm22, and aortic smooth muscle actin), esSMCs remained positive for markers of stem cell pluripotency (Oct4, Nanog, and Rex1). In summary, we describe a novel strategy for the profiling of cell membrane proteins. The procedure combines DIGE technology with biotin/avidin labeling to discriminate membrane and membrane-associated proteins from intracellular contaminants by fluorescence tagging and permits semiquantitative differential expression analysis of membrane proteins.

Proteomic analysis reveals higher demand for antioxidant protection in embryonic stem cell-derived smooth muscle cells

Embryonic stem (ES) cells can differentiate into vascular smooth muscle cells (SMCs), but differences in protein composition, function and behaviour between stem cell-derived and mature SMCs remain to be characterized. Using differential in gel electrophoresis (DIGE) and MS, we identified 146 proteins that differed between ES cell-derived SMCs (esSMCs) and aortic SMCs, including proteins involved in DNA maintenance (higher in esSMCs), cytoskeletal proteins and calcium-binding proteins (higher in aortic SMCs). Notably, esSMCs showed decreased expression of mitochondrial, but a compensatory increase of cytosolic antioxidants. Subsequent experiments revealed that mitochondrial-derived reactive oxygen species (ROS) were markedly increased in esSMCs. Despite a three-fold rise in glutathione (GSH) reductase activity, esSMCs had lower levels of reduced GSH, and depletion of GSH by diethyl maleate or inhibition of GSH reductase by carmustine (BCNU) resulted in more pronounced cell death compared to aortic SMCs, while addition of antioxidants improved the viability of esSMCs. We present the first proteomic analysis of esSMCs demonstrating that stem cell-derived SMCs are more sensitive to oxidative stress due to increased generation of mitochondrial-derived ROS and require additional antioxidant protection for survival.