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progenitor cells

progenitor cells

Methods xxx (2015) xxx–xxx Contents lists available at ScienceDirect Methods journal homepage: www.elsevier.com/locate/ymeth Vascular smooth muscle...

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Methods xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Methods journal homepage: www.elsevier.com/locate/ymeth

Vascular smooth muscle cell differentiation from human stem/ progenitor cells Sarah K. Steinbach a,b, Mansoor Husain a,b,c,d,e,f,g,h,⇑ a

McEwen Centre for Regenerative Medicine, Toronto General Research Institute, 101 College St., Toronto, Ontario M5G-1L7, Canada Division of Experimental Therapeutics, Toronto General Research Institute, 101 College St., Toronto, Ontario M5G-1L7, Canada c Departments of Medicine, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S-1A8, Canada d Departments of Physiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S-1A8, Canada e Departments of Laboratory Medicine & Pathobiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S-1A8, Canada f Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S-1A8, Canada g Ted Rogers Centre for Heart Research, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S-1A8, Canada h Peter Munk Cardiac Centre, University Health Network, 200 Elizabeth St., Toronto, Ontario M5G-2C4, Canada b

a r t i c l e

i n f o

Article history: Received 17 July 2015 Received in revised form 3 December 2015 Accepted 7 December 2015 Available online xxxx Keywords: Vascular smooth muscle cell Embryonic stem cells Induced pluripotent stem cells Lateral plate mesoderm Neural crest Serum-free Coronary

a b s t r a c t 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 platederived VSMCs including ‘coronary-like’ 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. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction 1.1. Overview

Abbreviations: VSMCs, vascular smooth muscle cells; VEGF, vascular endothelial growth factor; PAH, pulmonary arterial hypertension; TGF-b, transforming growth factor beta; IL-1b, interleukin 1 beta; MMPs, matrix metalloproteinases; TIMPs, tissue inhibitor of metalloproteinases; hPSCs, human pluripotent stem cells; SKP, skin-derived precursor; ESC, embryonic stem cell; iPSC, induced pluripotent stem cell; BMP, bone morphogenetic protein; T, Brachyury; KDR, kinase insert domain receptor; Flk1, fetal liver kinase 1; VEGFR2, vascular endothelial growth factor receptor 2; PDGFRa, platelet derived growth factor receptor alpha; DKK1, Dickkopf homolog 1; EBs, embryoid bodies; IMDM, Iscove’s Modified Dulbecco’s Medium; MTG, monothioglycerol; bFGF, basic fibroblast growth factor; ASMA, alpha smooth muscle actin; MRTFA/B, myocardin related transcription factor A/B; SRF, serum response factor; qRT-PCR, quantitative real-time polymerase chain reaction; hASMC, human aortic smooth muscle cell; hBSMC, human bladder smooth muscle cell; FITC, fluorescein isothiocyanate. ⇑ Corresponding author at: Division of Experimental Therapeutics, Toronto General Research Institute, 101 College St., Toronto, Ontario M5G-1L7, Canada. E-mail address: [email protected] (M. Husain).

Vascular smooth muscle cells (VSMCs) are a type of smooth muscle that controls the diameter of medium and large blood vessels. VSMCs contract or relax to control blood pressure and act to distribute blood to areas where tissue oxygenation and nutrients are needed. From a regenerative medicine perspective, VSMCs are a therapeutically relevant cell type shown to enhance angiogenesis and wound healing, and improve heart function after myocardial infarction [1–5]. A normal vasculature is essential to maintaining tissue homeostasis and to providing the necessary oxygenation and nutrients to cells of the human body. When tissue homeostasis is disrupted, such as occurs with impaired vascular function, various complications arise. Cardiac and peripheral vascular diseases, often caused by atherosclerosis, cause a great deal of morbidity and mortality in the Western world. Current treatment strategies include stents or tissue grafts to restore blood flow to affected tissues. However,

http://dx.doi.org/10.1016/j.ymeth.2015.12.004 1046-2023/Ó 2015 Elsevier Inc. All rights reserved.

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the use of grafts can be insufficient for the recovery of blood flow and to restore functional integrity of the affected tissue. This may be due to a multitude of factors, including cell death, the development of graft disease and incomplete functional integration. We posit that it may be possible to mitigate these issues by finding appropriate progenitor cell types that might serve as the ideal starting material for regenerative applications. The ideal progenitor cell type for regenerative therapies must meet certain criteria. It must be able to expand to limitless numbers, be disease free, resistant to developing disease, be immune tolerant/compatible and must integrate functionally into their milieu. To date, the ‘‘holy grail” of progenitor cells has not been found, however studies are currently underway to deal with these challenges by engineering or modifying progenitor cell sources. In designing new cellular therapies to treat the multitude of diseases for which they may represent a cure, novel strategies need to be employed. For example, in designing progenitor cells to expand to high numbers, one might accelerate or deregulate the cell cycle with the unintended risk of cancer. Because of this, progenitors need to be designed such that they can be geared to enter the cell cycle in vitro, but kept in check in vivo. Similarly, in designing vascular grafts that promote angiogenesis, one might inadvertently promote the blood supply of a malignant tumor. For example, protein therapies such as VEGF have been shown to promote the survival of vascular grafts, but have also been shown to promote angiogenesis in cancer [6]. Genetic modification of various progenitor cell populations has also been attempted. In the treatment of pulmonary arterial hypertension (PAH), endothelial progenitor cells have been introduced with angiopoietin-1, VEGF, adrenomedullin, calcitonin gene-related peptide and endothelial nitric oxide synthase [7]. All of these manipulations have improved the pathophysiology of PAH, however, genetic manipulation can be costly, introduces the toxicity risk of viral and/or non-viral transfection vectors and can be difficult to scale [7]. Autologous adult stem cells present the least risk, however they also present the least potential for trans-differentiation and regeneration. By contrast, pluripotent stem cells have the greatest potential for differentiation and regeneration, but pose the greatest risk in terms of neoplasia. In the case of VSMCs, their relevant progenitors are derived from many different lineages as will be described below. These differences in lineage have been shown to have differential effects on function. It is currently unknown if matching the lineage of VSMC to the graft site would improve integration and therefore functional recovery. 1.2. Embryonic origins of VSMCs Using fate mapping approaches and chimeras, 8 independent embryonic sources of VSMCs have been identified [8]. These include the secondary heart field, the neural crest, the proepicardium, the somitic mesoderm, splanchnic mesoderm, mesothelium, mesoangioblasts and various adult stem/progenitor cells [8]. In particular, VSMCs from the aortic root are derived from the secondary heart field [9,10], those of the aortic arch are derived from the neural crest [11], while the pro-epicardium gives rise to VSMCs of the coronary arteries [12,13]. Somitic mesoderm gives rise to VSMCs in the descending thoracic aorta [14], and VSMCs of the abdominal aorta are derived from the splanchnic mesoderm [14]. Various adult stem/progenitor cells are found heterogeneously throughout the vasculature and their origins may be diverse [15–20]. VMSCs of different embryonic origins have been observed to respond differentially to different growth conditions such as serum and TGF-b. For example, neuro-ectoderm-sourced VSMCs proliferate in serum-free conditions, whereas mesoderm-sourced VSMCs require serum for proliferation [21]. Moreover, on the addition of TGF-b1, cell proliferation was increased in neural crest-derived

VSMCs, in contrast to mesoderm-derived VSMCs which were either unresponsive or growth inhibited [22]. Also, c-Myb expression was induced on the addition of TGF-b1 in neural crest-derived VSMCs, but not in mesoderm-derived VSMCs [22]. VSMCs isolated from different parts of the aorta, and thus of different embryonic origin, display differential responses to IL-1b, an atherogenic cytokine [23]. Gene expression levels, and corresponding protein levels of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) vary in response to IL-1b treatment depending on the VSMC lineage being treated [24,25]. This may have significant implications, as MMPs play a critical role in vascular remodeling, aneurysm formation and atherogenesis [24]. This is based on the ability of MMPs to degrade vessel wall connective tissue, which precedes the development of many vascular diseases. These differential effects in VSMC function appear relevant to atherosclerosis. Aortic homograft transplantation studies of animals fed a high fat diet, where the atherosclerosis prone abdominal aorta (of splanchnic origin) has been transplanted to the atherosclerotic resistant thoracic aorta (of somitic origin) have demonstrated that atherosclerosis susceptibility is dependent on intrinsic differences within the vessel wall [26,27]. This supports the premise of lineage-dependent VSMC diversity. Similar studies transplanting the abdominal aorta into the pulmonary circulation (of secondary heart field origin) have yielded similar results in response to an atherogenic diet [28]. This phenomenon can be translated to humans whereby it has been shown that different vascular segments display their own unique atherogenic response [29]. Moreover, aortic dissections occur preferentially at the interfaces where VSMCs of distinct embryological origins meet [8]. Together, these studies support the notion that intrinsic VSMC differences may be attributed to their lineage heterogeneity. 1.3. Pluripotent stem cells If we accept that the key to defining optimal functional integration may be lineage specification, we must ask how we might best achieve origin-dependent lineage restriction. Should this be through pluripotent stem cells (PSCs) or through adult progenitors such as the skin-derived precursor (SKP, see below) [30]? Since PSCs represent the cellular source with greatest regenerative and differentiation potential, we have focused this review on embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Developing different differentiation protocols using PSCs provides investigators with the prospect of developing cell-based therapies for many genetic and non-genetic human diseases, to perform drug screens, to study the earliest stages of human development and to model disease in vitro. The cardiovascular lineage in particular has been an area of concentrated interest as cardiovascular disease is a leading cause of death, congenital heart abnormalities are relatively common, and unanticipated cardiac toxicity prevents many new drugs from reaching clinical application. Using in vivo model systems, scientists have been able to identify the key regulatory pathways that control the establishment of several embryonic lineages in the embryo. At the present time, the most successful differentiation strategies recapitulate key regulatory pathways in vitro. The pluripotent nature of ESCs and iPSCs makes them attractive candidate sources for developing different VSMCs of distinct sources/origins and therefore differential function and disease susceptibility. Protocols to differentiate pluripotent stem cells into somite, lateral plate and neural crestderived VSMCs have been devised and have produced VSMC with discrete functional/biological differences [25]. In the case of iPSCs, the power of this technology lies in its potential to generate a limitless number of genetically modifiable and personalized PSCderived VSMC obtained from autologous patient tissue. This provides a strong platform for disease modeling and for generating

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vascular cells/grafts for repairing damaged vasculature and treating ischemic vascular disease. 1.4. Skin-derived precursors (SKPs) SKPs are Sox2+ progenitor cells which reside in the dermal papilla and sheath of hair follicles [31]. They generate ectodermal and mesenchymal derivatives such as neurons, Schwann cells, adipocytes, chondrocytes and VSMCs [30,32–35]. SKPs from the whisker pads of mice are derived from the neural crest, whereas SKPs from dorsal skin are derived from the somitic mesoderm [36]. SKPs can be cultured from human foreskin and have been shown to differentiate almost exclusively into VSMC with the addition of TGFb1 or -b3 [30]. These VSMC are functional and can integrate into the vasculature in vivo. Indeed, neural crest stem cells derived directly from the neural tube differentiate into VSMC under similar conditions [37]. Recently, cerebrovascular pathophysiology has been modeled in neural crest-derived VSMCs underscoring the importance of lineage specificity in modeling disease [38]. 1.5. Aim This methods paper is designed to aid the investigator in differentiating, in a directed serum-free manner, human PSCs or SKPs into lineage-specific VSMC. However, the same framework or roadmap can be applied to differentiate and characterize different stem/progenitor cell populations (SKPs) [30]. For example, differentiation of SKPs from the face or foreskin would be a neural crest-derived protocol. Other mesenchymal progenitors such as bone marrow or adipose tissue derived progenitor cells have been similarly characterized [39,40]. 2. Cardiac-directed differentiation in defined serum-free medium 2.1. Overview To direct the differentiation of human PSCs into the cardiac lineage, a staged protocol was designed to recapitulate and activate the key developmental processes of cardiac specification in vitro [41–43]. Studies of different model organisms have shown that the Activin/Nodal/TGF-b, Wnt, and BMP pathways all play critical roles in the establishment of the cardiovascular lineages. This includes endothelial cells, VSMCs and cardiomyocytes. In summary, the first step is to induce a primitive streak-like population, which results in the expression of Brachyury (T) expressing mesoderm, followed by the induction and specification of cardiac mesoderm and ending with the expansion of the cardiovascular lineages [44]. In the past, KDR+ (also known as Flk1 or VEGFR2 in the mouse) has been used to monitor the establishment of cardiac mesoderm. However, this is problematic as KDR+ is expressed on several mesodermal segments [43]. Using PDGFRa+ in addition to KDR+ more accurately identifies cardiac mesoderm. This comes from in vivo studies which demonstrated that Flk1+ is coexpressed with PDGFRa+ in cardiac mesoderm at E7.5 and is found on cardiac progenitor cells in the cardiac crescent of Nkx2.5 null embryos [45]. A distal-proximal Nodal gradient must first be formed to induce different primitive streak-like populations [46–51]. Low levels of Nodal signaling promote posterior mesoderm or hematopoietic development, while increasing Nodal levels induces more anterior mesoderm which includes the cardiac and somitic mesoderm, and definitive endoderm. This can be strikingly recapitulated in vitro where it is observed that the KDR+ PDGFRa+ (K+P+) population requires higher concentrations of Activin/Nodal than the single

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K+P population which is more hematopoietic. Indeed, the K+P population is thought to represent the primitive and definitive hemangioblast [52]. This differentiation method induces VSMCs from hPSCs through an embryoid body (EB)/cardiac mesoderm intermediate. Induction of the cardiac lineages can be divided into 3 steps (Fig. 1A). 1. Induction of the primitive streak. 2. Induction and specification of cardiac mesoderm, through the initiation of the K+P+ progenitor population. 3. Expansion of the cardiac lineages. Materials STEMPRO 34 (Invitrogen #10639-011) L-Ascorbic Acid (Sigma #A-4544) Monothioglycerol (MTG) (Sigma #M-6145) Transferrin (Roche #10 652 262) hBMP4 (R&D Systems #314-BP) hbFGF (R&D Systems #233-FB) hVEGF (R&D Systems #293-VE) Activin A (R&D Systems #338-AC/CF) Dickkopf homolog 1 (DKK) (R&D Systems #5439-DK/CF) Growth Factor Reduced Matrigel (BD #356231) 2.1.1. Induction of the primitive streak To induce the primitive streak, a combination of Activin A and BMP4 is used [43,44,53]. This results in the transient expression of the early mesoderm marker T (Brachyury). For days 0–1, 0.5 ng/ml of BMP4 is added to the PSC culture. This is the aggregation step where EBs are formed. Resuspend trypsinized hPSCs in aggregation medium [STEMPRO 34, 1% glutamine, 150 lg/ml transferrin, 50 lg/ml ascorbic acid, 3 ll/ml MTG (from a 26 ll/2 ml stock), 0.5 ng/ml BMP4 and 10 lM ROCK inhibitor]. Incubate the cell suspension overnight with agitation at a speed of 70 rpm in a tissue culture incubator at 37 °C in hypoxic conditions (5% CO2/5% O2/90% N2). The optimal concentration of cells for aggregation is 2.5  105–5  105 cells/ml and this step is performed in 6 well ultra low attachment plates (Corning). Once the aggregation step is complete (after approximately 24 h), move to the primitive streak induction step. To induce the primitive streak on days 1–3, a mixture of BMP4 and Activin A is used. Filter the EBs through a 100 lm filter into a 50 ml canonical tube and allow the EBs to settle for 15–20 min in an incubator at 37 °C in hypoxic conditions. Aspirate the aggregation medium and gently resuspend the EBs in induction medium 1 [STEMPRO 34, 1% glutamine, 150 lg/ml transferrin, 50 lg/ml ascorbic acid, 3 ll/ml MTG (from a 26 ll/2 ml stock), 10 ng/ml BMP4, 5 ng/ml bFGF and 3 ng/ml Activin A]. Culture the cells in 6 well ultra low attachment plates in hypoxic conditions until day 3–4. The induction time can vary and should be determined for each hPSC line. This cocktail of cytokines results in the induction of the K+P+ population. 2.1.2. Induction of K+P+ progenitor cells In order to specify cardiac mesoderm, Dickkopf homolog 1 (DKK1) is added to the EBs. VEGF is included to promote the expansion of the K+P+ population. Thus, from days 3 to 6, 150 ng/ml of DKK1 and 10 ng/ml of VEGF are added to the primitive streaklike population. Harvest the EBs and wash in IMDM to remove the inductive cytokines. Centrifuge the EBs at 200g for 5 min and aspirate supernatant. Resuspend the EB pellet in 2 ml of induction media 2 and replate into new 6 well ultra low attachment plates. The components of induction media 2 are STEMPRO 34, 1% glutamine, 150 lg/ml transferrin, 50 lg/ml ascorbic acid, 3 ll/ml

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A Formation of primitive streak-like population

Induction & specification of cardiac mesoderm

Maintenance and expansion of the cardiovascular lineages Monolayer P6

PSC growth Aggregation Stage1 0 -2

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BMP4 Feeder Feeder dependent depletion Normoxia

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B Collagenase XI

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7 days Fig. 1. (A) Schematic representation of the protocol used for human ESC differentiation toward the VSMC lineage (modified from [53]). (B) Schematic representation of the differentiation protocol for SKPs into VSMCs.

MTG (from a 26 ll/2 ml stock), 10 ng/ml VEGF and 150 ng/ml DKK1. Cells are cultured in a 37 °C incubator maintained in 5% CO2/5% O2/90% N2 (anoxic conditions) [43,44,53].

2.1.3. Expansion of the cardiac lineages To expand the cardiac lineages, VEGF is again added to the cultures from days 6 to 12. Harvest and replate the EBs under hypoxic conditions in induction media 3 [STEMPRO 34, 1% glutamine, 150 lg/ml transferrin, 50 lg/ml ascorbic acid, 3 ll/ml MTG (from a 26 ll/2 ml stock), 5 ng/ml VEGF]. After day 12, 5 ng/ml of bFGF is added to support the continued expansion of the cardiovascular lineages and cells can be transferred to 5% CO2/air. The cultures are maintained with VEGF and bFGF until day 28, after which cells are differentiated into VSMCs [53]. Interestingly, different human ESC or iPSC lines may display differential responses to the stimulation of these key regulatory signaling pathways. Thus, it is suggested that one perform a pilot experiment whereby different concentrations of Activin A and BMP4 are titrated to achieve the maximal differentiation into the K+P+ cardiac progenitor population. Between 0 and 10 ng/ml of Activin A should be titrated and between 1 and 30 ng/ml of BMP4 should be titrated to induce the primitive streak-like population.

2.3. Switching from growth in EBs to a monolayer EBs are dissociated with collagenase type II (0.8 mg/ml) containing 1 lg/ml DNase and TripLE. This dissociates the EBs into single cells. Pipette EBs into 15 ml tube and allow 20 min for EBs to settle to the bottom of the tube and aspirate the medium. Add 0.8 mg/ml collagenase type II in HBSS into the 15 ml tube (2 ml/tube) and tilt mix in a 37 °C incubator for 1–2 h. Add 10 lM of ROCK inhibitor to improve survival of cells. Examine the cells every hour to observe cloudiness of collagenase II. Add 1 lg/ml DNase to avoid clumping. Spin cells at 1000 rpm for 3 min and add 2 ml of TripLE (Gibco). Resuspend the cells and incubate in a 37 °C water bath for 2 min. Add complete medium to stop the reaction [STEMPRO 34, 1% glutamine, 150 lg/ml transferrin, 50 lg/ml ascorbic acid, 3 ll/ml MTG (from a 26 ll/2 ml stock)]. Spin at 1000 rpm for 3 min and resuspend in 5 ml of complete medium. Take 10 ll for cell counting. Cells can then be used for FACS analysis or for culture in a monolayer. For cell culture on a monolayer, cells are grown on 0.65% matrigel coated dishes on 48 well, 24 well or 6 well dishes. The media that should be used is high glucose DMEM supplemented with VEGF (10 ng/ml) and bFGF (5 ng/ml). To enrich for VSMCs, cultures were passaged with trypsin EDTA on plates coated with growth factor reduced matrigel 3 or more times at a cell density of 1  105 cells/ml. This removes endothelial cells and cardiomyocytes from the culture [53].

2.2. Differentiation of cardiac mesoderm containing EBs into VSMC

3. Differentiation of SKPs into VSMC in serum-free conditions

Once the cardiovascular lineage has been specified and expanded, the process of VSMC differentiation occurs in two steps: (1) maintenance of VSMCs, and (2) selection of VSMCs excluding endothelial cells and cardiomyocytes through passaging [53].

To obtain VSMC that are neuroectoderm sourced, or derived from the neural crest, the skin represents a source of SKPs. Skin in the facial region is thought to be neural crest derived and so are its resident progenitor cells. Human skin samples can be easily

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attained from children undergoing circumcision. These foreskin samples contain an abundant number of SKPs. We have also been able to culture SKPs from sternum skin, leg skin and neck skin using this protocol. 3.1. Isolation of human SKPs from foreskin or other anatomical locations Materials: DMEM with Glutamax (Invitrogen #10567-014) F-12 with Glutamax (Invitrogen, #31765-035) HBSS (Invitrogen #14175-095) B27 Supplement (Invitrogen, #17504-044) Fungizone (Invitrogen, #15290-018) Penicillin/Streptomycin (Lonza #17-602E) Collagenase Type XI (Sigma #C7657) FGF2 (PeproTech #100-18B) EGF (BD #CACB354052) Cell Strainer 70 lm (Falcon # CA21008-952) DNase I (Sigma # D5025) Liberase Blendzyme I (Roche #05 401 089 001) TGF-b1 (PeproTech #100-21C) TGF-b3 (R&D Systems #243-B3-002) Tissue (if large) is cut into 1 cm2 pieces using a blade [54]. The cut skin is then incubated in 553 ll of 9 mg/ml Liberase Blendzyme (Roche) in 25 ml HBSS for 24–48 h at 4 °C, or when the tissue becomes white and fluffy. The next day, peel off the epidermis which should come off easily with a pair of tweezers under a dissecting microscope. Place all of the HBSS Blendzyme solution back into the canonical tube and incubate at 37 °C for 20–40 min. Add 400 ll DNase I (2 mg/ml), mixing by inversion, and then let the sample settle for 1 min. Add 1 ml of FBS and allow the mixture to settle on ice for 2–5 min. Add 2 ml of fresh DMEM: F-12 3:1 to the mixture and begin to dissociate the cells mechanically using a 10 ml pipette. After vigorously mashing the tissue against the bottom of the tube with the pipette, remove the supernatant into a fresh tube. Add another 2 ml and repeat the process until the media is no longer cloudy with cells. Filter the supernatant through a 70 lm cell strainer. Centrifuge the filtered supernatant at 300g for 6 min and resuspend the pellet in 1 ml of DMEM:F-12 3:1. Count the cells with a haemocytometer using trypan blue to exclude dead cells. Plate cells at a density of 100,000– 150,000 cells/ml in SKP proliferation media [DMEM:F12 3:1, 1% penicillin/streptomycin, 1 lg/ml Fungizone, 2% B27, 40 ng/ml FGF2, 20 ng/ml EGF]. After approximately 2 weeks, floating colonies, or spheres, should be observed in the tissue culture flask [54]. To passage/dissociate the spheres, centrifuge to pellet the cells and keep the conditioned media. Filter the conditioned medium through a 0.22 lm filter and set aside for passaging of the SKPs. Resuspend the pellet of SKPs in 1 ml of Collagenase XI (1 mg/ml). Incubate at 37 °C for 30–40 min. Using a P1000 pipette, gently triturate the spheres (50 times) to dissociate the spheres into single cells. Add 20 ml of media to dilute the collagenase and centrifuge the mixture at 300 g for 6 min. Decant the supernatant and culture the cells in 50% conditioned medium supplemented with SKP proliferation medium growth factors. 3.2. Differentiation of human SKPs into VSMC To differentiate SKPs into VSMC, SKPs should be dissociated into single cells as was previously described for passaging SKPs (Fig. 1B). In order to effectively differentiate human SKPs into VSMC one must plate the cells at a low density (5000 cells/cm2)

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[30,55]. Prepare DMEM:F-12 3:1 and add in 2 ng/ml TGF-b1 or b3 to the medium. Add the cells to the medium and incubate at 37 °C in 5% CO2/air for 4–7 days. After the cells are initially plated, they appear small and compact. However, significant morphological changes occur during the 7 days of differentiation with TGF-b (Fig. 2). It is curious that many adult stem/progenitor cell populations require TGF-b for differentiation. For example, adipose tissuederived and bone marrow-derived mesenchymal stem cells, as well as epicardial progenitor cells all require TGF-b for VSMC differentiation [39,40,56]. This suggests that despite the diverse lineages of adult stem/progenitor cells, similar pathways are required to initiate VSMC differentiation. However, despite the conservation in the signaling pathways driving differentiation, intrinsic differences in the origins of these progenitor cell populations likely yield different VSMC populations with differential function. 4. Characterization of human PSC and SKP-derived VSMC 4.1. Phenotyping 4.1.1. Markers There are several markers that can be used to identify a VSMC and they should be used in combination. These include a smooth muscle actin (ASMA), Calponin, Smoothelin and SM22a (Transgelin) which are cytoskeletal proteins involved in cell contraction. There are two different kinds of smooth muscle: vascular and visceral. The vascular-specific SMC is of greater lineage heterogeneity than the visceral SMC. A perfect example of lineage diversity of VSMCs in the vasculature is the aorta which has been previously described in this article. The origins of visceral smooth muscle are ill defined and are thought to derive from the mesoderm, although the precise mesodermal compartments are unknown [57]. To distinguish between visceral and vascular SMC, one can look at the gene expression of Smoothelin-A and Smoothelin-B. Smoothelin-B is vascular-specific, whereas Smoothelin-A is visceral-specific [58]. Smoothelin-A is a truncated isoform of Smoothelin-B, and thus primers can be designed that are specific to Smoothelin B [58]. Primers designed to target Smoothelin A will detect both Smoothelin A and Smoothelin B transcripts [30,53,59]. Telokin and SMc actin are visceral markers that one can also determine the abundance of visceral vs. vascular SMCs [60,61]. The process of VSMC-dependent gene transcription has been relatively well characterized. Thus, one can also look at the transcription factors involved in differentiating SMCs such as myocardin, MRTFA, MRTFB and SRF [30,62–64]. Furthermore, to determine if your VSMC is proliferative or embryonic, expression levels of SMemb should be quantified and compared to differentiated and embryonic VSMC. SMemb has been shown to be upregulated in embryonic VSMC and in phenotypically modulated VSMC such as those found in the neointima after vascular injury [65,66]. 4.1.2. Quantitative real time-PCR (qRT-PCR) To quantify transcript levels using RT-PCR, total RNA should be isolated with Trizol or another RNA isolation kit. In our hands, 2 lg of RNA (from 100,000 to 500,000 cells) produced good qRT-PCR results. To reverse transcribe the RNA, Superscript III (Invitrogen) is used. To remove any traces of genomic DNA, RNA was treated with DNase I (Fermentas) and spiked with enhanced green fluorescent protein (eGFP) RNA (10 ng) to monitor consistency between cDNA synthesis reactions. Random hexamers (Invitrogen), dNTPs (Fermentas), and RNase Inhibitor (New England Biolabs) are also added to the reaction mixture. Final primer concentrations used were 0.2 lmol/l, and an annealing temperature of 63 °C was used

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ASMA

ASMA

50 μm

50 μm

Fig. 2. Dissociated SKPs undergo significant morphological changes with the addition of TGF-b1. Left panel are dissociated SKPs 1 day after plating. Right panel are dissociated SKPs after 7 days of culture in 2 ng/ml TGF-b1. ASMA: a-smooth muscle actin. Blue is nuclear Hoechst stain. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

during the amplification cycle for all primers. Human SKP-derived VSMCs were compared to human VSMC lines and shown to express levels of VSMC markers similar to the positive controls [30]. Human PSC-derived VSMCs were also positive for these markers by qRT-PCR [53]. 4.1.3. Immunocytochemistry For immunocytochemistry, antibodies are readily available for Smoothelin (Santa Cruz, sc-28562), ASMA (Sigma–Aldrich, A2547), Calponin (Abcam, ab46794) and SM22a (ProteinTech, 10493-1-AP). These markers should be used in conjunction with each other to determine if your PSC-derived or adult progenitorderived VSMC are indeed bonafide. Human SKP-derived VSMCs were positive for ASMA, Calponin and SM22a indicating that these cells were indeed VSMCs (Fig. 3). When phenotyping VSMC, one should always compare human PSC-derived VSMC to primary VSMC cultures. Frozen down stocks of human aortic SMC (hASMC) and human bladder SMC (hBSMC) are commercially available. We have purchased human primary cultures of hASMC from Cascada and hBSMC from Lonza. Choose any other cell type for negative controls like human keratinocytes or epithelial cell cultures which should give you a negative result with the aforementioned antibodies.

that they are functional in response to different agonist treatments. Calcium imaging establishes cell contraction and calcium responses in response to agonist treatment. VSMCs cultured in a monolayer are incubated with 5 lM of the membrane-permeable Ca2+-sensitive dye Fluo-4 AM or Fluo-5F AM (Life Technologies). Stock solutions of these Ca2+-sensitive dyes were made at 5 mM, aliquoted and frozen at 80 °C. To disperse the non-polar AM ester in aqueous media, an equal volume of 20% (w/v) Pluronic F-127 in DMSO (Life Technologies) should be added to the Fluo-4 or Fluo-5F AM working solution. We incubated the cells with the dye for 45 min at 37 °C in 5% CO2/air, however the time should be optimized for your particular cell type. The optimal amount of time would be when the cells begin to fluoresce. After the incubation period, cells are subsequently washed with fresh Tyrode solution consisting of 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose pH 7.4. All Ca2+ imaging can be performed with Tyrode solution in an environmental chamber at 37 °C and 5% CO2/air using an Olympus FV1000 confocal microscope or other imaging modality. Images should be obtained at a resolution of 512  512 pixels and recorded continually until the experiment is completed. Laser excitation is at 488 nm and emission is recorded at 516 nm. Using calcium imaging, we were able to deduce that hPSC-derived VSMC described in this protocol are like human coronary VSMC [53].

4.2. Functional assays 4.2.1. Calcium imaging It is one thing to establish that your hPSC-derived VSMC express markers distinguishing them as VSMC, but another to determine

50μm

ASMA

50μm

4.2.2. In vivo integration assay Another way to assess whether your hPSC-derived VSMCs are functional is to determine whether they can functionally integrate into the vasculature in vivo. We have used two approaches for visu-

Calponin

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SM22α

Fig. 3. Human foreskin-derived SKPs differentiated with TGF-b1are positive for VSMC markers ASMA, Calponin and SM22a. Human foreskin-derived SKPs were differentiated with TGF-b1 for 7 d, fixed, stained and probed with the aforementioned antibodies. These cells stained positive for ASMA, Calponin and SM22a indicating that they are VSMCs. ASMA: a-smooth muscle actin. Blue is nuclear Hoechst dye. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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alizing the integration of differentiated cells into the vasculature: (1) en face imaging of matrigel plugs subcutaneously injected into the animal [67]; or a (2) dorsal skin-fold window chamber model [68]. For en face imaging of matrigel plugs, the following should be performed. Differentiated PSC-derived VSMC are resuspended at 1.5  106 cells/ml in 1.5 ml high glucose DMEM with 2% FBS and 500 ng/ml bFGF2 (BD). This 1.5 ml cell suspension is then diluted 1:1 with growth factor-reduced Matrigel (BD) and kept on ice. NOD-SCID mice are anesthetized with ketamine and xylazine (1:2). The cell/Matrigel suspensions (300 ll) are then injected subcutaneously in the mouse abdomen with 27-gauge needles. After 2 –3 weeks, the mice are anesthetized with isoflurane, and the Matrigel plug exposed for intravital microscopy. To simultaneously observe blood flow within vessels, fluorescein isothiocyanate (FITC)-conjugated dextran (1 mg/ml concentration) at 3 mg/kg body weight can be injected via the tail vein. A GFP expressing human PSC line can be easily attained to perform these integration assays. However, different Cell Tracker dyes (Life Technologies) have been successfully used to trace injected cells and monitor their integration [30]. For visualization of integration with the window chamber model, a skilled surgical expert and window chamber mount is required. Here, 4- to 6-week-old female athymic nude mice were used for these experiments. PSC-derived VSMC can be resuspended in 50% Matrigel and injected directly into the retractor muscle of the mouse dorsal skin fold. The benefit of using this method is that fluorescence imaging can be performed weekly over 4 weeks. Tail vein injections of FITC-conjugated dextran are used to visualize blood flow. Both hPSC- and SKP-derived VSMCs integrated into the vasculature further indicating that these cells were functional [30,53].

5. Conclusion In this article we discuss the methods for generating lateral plate mesoderm-derived, coronary-like VSMCs from PSCs and neural crest-derived VSMC from SKPs. Various VSMC beds are derived from the lateral plate: the coronary arteries, the aortic root and the abdominal aorta. Moreover, there may be adult VSMC progenitors that may reside in the local vasculature which may be involved in steady state renewal of VSMCs that are lateral plate derived. As is observed with the ESC and iPSC differentiation scheme outlined in this article, PSCs faithfully recapitulate the early stages of embryonic development. Therefore, this differentiation scheme may be used to elucidate the key steps and pathways in generating lateral plate mesoderm-derived VSMCs. In addition, this method may be very useful for personalized medicine and regenerative approaches. VSMCs derived from iPSCs may be used to construct bioengineered blood vessels for coronary and peripheral artery bypass or hemodialysis grafts. They may also be used to model hereditary familial syndromes that affect the VSMC such as Marfan’s or Loeys-Dietz Syndrome or even systemic conditions such as progeria [69]. The frame work for this article can be applied to characterizing other VSMC populations derived from alternate adult stem/progenitor cell sources. Other sources of adult VSMC progenitors are fat, bone marrow, vessels, umbilical cord, placenta and skin. Within this framework, it may be useful to compare the VSMC types from each different source and compare them with primary VSMCs derived from different vascular beds to get a more comprehensive view of which cells are more mature or more similar to that in mature tissues. Moreover, the lineage-specific differences inherent in different VSMC derived from different embryonic origins may be elucidated and used to treat disorders of the vasculature tailored to their cell of origin.

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Acknowledgements This research was funded by Canadian Institutes of Health Research (MOP#136850), an Acceleration Award from the McEwen Centre for Regenerative Medicine, and salary support awards to MH from the Heart & Stroke Foundation of Canada (CI7495) and SKS from the Heart & Stroke Richard Lewar Centre of Excellence, University of Toronto.

References [1] R.K. Li et al., Smooth muscle cell transplantation into myocardial scar tissue improves heart function, J. Mol. Cell. Cardiol. 31 (3) (1999) 513–522. [2] S. Davani et al., Mesenchymal progenitor cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a rat cellular cardiomyoplasty model, Circulation 108 (Suppl. 1) (2003) II253–II258. [3] Y.S. Yoon et al., Clonally expanded novel multipotent stem cells from human bone marrow regenerate myocardium after myocardial infarction, J. Clin. Invest. 115 (2) (2005) 326–338. [4] S. Gojo et al., In vivo cardiovasculogenesis by direct injection of isolated adult mesenchymal stem cells, Exp. Cell Res. 288 (1) (2003) 51–59. [5] K.S. Midwood, L.V. Williams, J.E. Schwarzbauer, Tissue repair and the dynamics of the extracellular matrix, Int. J. Biochem. Cell Biol. 36 (6) (2004) 1031–1037. [6] P. Carmeliet, VEGF as a key mediator of angiogenesis in cancer, Oncology 69 (Suppl. 3) (2005) 4–10. [7] W.S. Foster, C.M. Suen, D.J. Stewart, Regenerative cell and tissue-based therapies for pulmonary arterial hypertension, Can. J. Cardiol. 30 (11) (2014) 1350–1360. [8] M.W. Majesky, Developmental basis of vascular smooth muscle diversity, Arterioscler. Thromb. Vasc. Biol. 27 (6) (2007) 1248–1258. [9] K.L. Waldo et al., Secondary heart field contributes myocardium and smooth muscle to the arterial pole of the developing heart, Dev. Biol. 281 (1) (2005) 78–90. [10] J. Maeda et al., Tbx1 is regulated by forkhead proteins in the secondary heart field, Dev. Dyn. 235 (3) (2006) 701–710. [11] X. Jiang et al., Fate of the mammalian cardiac neural crest, Development 127 (8) (2000) 1607–1616. [12] T. Mikawa, D.A. Fischman, Retroviral analysis of cardiac morphogenesis: discontinuous formation of coronary vessels, Proc. Natl. Acad. Sci. USA 89 (20) (1992) 9504–9508. [13] T. Mikawa, R.G. Gourdie, Pericardial mesoderm generates a population of coronary smooth muscle cells migrating into the heart along with ingrowth of the epicardial organ, Dev. Biol. 174 (2) (1996) 221–232. [14] P. Wasteson et al., Developmental origin of smooth muscle cells in the descending aorta in mice, Development 135 (10) (2008) 1823–1832. [15] J. Yamashita et al., Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors, Nature 408 (6808) (2000) 92–96. [16] J. Sainz et al., Isolation of ‘‘side population” progenitor cells from healthy arteries of adult mice, Arterioscler. Thromb. Vasc. Biol. 26 (2) (2006) 281–286. [17] K.A. Jackson et al., Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells, J. Clin. Invest. 107 (11) (2001) 1395–1402. [18] Y. Hu et al., Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice, J. Clin. Invest. 113 (9) (2004) 1258–1265. [19] K.M. Howson et al., The postnatal rat aorta contains pericyte progenitor cells that form spheroidal colonies in suspension culture, Am. J. Physiol. Cell Physiol. 289 (6) (2005) C1396–C1407. [20] J.N. Passman et al., A sonic hedgehog signaling domain in the arterial adventitia supports resident Sca1+ smooth muscle progenitor cells, Proc. Natl. Acad. Sci. USA 105 (27) (2008) 9349–9354. [21] S. Topouzis, M.W. Majesky, Smooth muscle lineage diversity in the chick embryo, Dev. Biol. 178 (2) (1996) 430–445. [22] P.F. Gadson Jr. et al., Differential response of mesoderm- and neural crestderived smooth muscle to TGF-beta1: regulation of c-myb and alpha1 (I) procollagen genes, Exp. Cell Res. 230 (2) (1997) 169–180. [23] K. Isoda et al., Deficiency of interleukin-1 receptor antagonist promotes neointimal formation after injury, Circulation 108 (5) (2003) 516–518. [24] Z.S. Galis, J.J. Khatri, Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly, Circ. Res. 90 (3) (2002) 251– 262. [25] C. Cheung et al., Generation of human vascular smooth muscle subtypes provides insight into embryological origin-dependent disease susceptibility, Nat. Biotechnol. 30 (2) (2012) 165–173. [26] H. Haimovici, N. Maier, Fate of aortic homografts in canine atherosclerosis. 3. Study of fresh abdominal and thoracic aortic implants into thoracic aorta: role of tissue susceptibility in atherogenesis, Arch. Surg. 89 (1964) 961–969. [27] H. Haimovici, N. Maier, Experimental canine atherosclerosis in autogenous abdominal aortic grafts implanted into the jugular vein, Atherosclerosis 13 (3) (1971) 375–384. [28] W.C. Woyda, E.M. Berkas, D.J. Ferguson, The atherosclerosis of aortic and pulmonary artery exchange autografts, Surg. Forum 11 (1960) 174–176.

Please cite this article in press as: S.K. Steinbach, M. Husain, Methods (2015), http://dx.doi.org/10.1016/j.ymeth.2015.12.004

8

S.K. Steinbach, M. Husain / Methods xxx (2015) xxx–xxx

[29] M.E. DeBakey, D.H. Glaeser, Patterns of atherosclerosis: effect of risk factors on recurrence and survival-analysis of 11,890 cases with more than 25-year follow-up, Am. J. Cardiol. 85 (9) (2000) 1045–1053. [30] S.K. Steinbach et al., Directed differentiation of skin-derived precursors into functional vascular smooth muscle cells, Arterioscler. Thromb. Vasc. Biol. 31 (12) (2011) 2938–2948. [31] J. Biernaskie et al., SKPs derive from hair follicle precursors and exhibit properties of adult dermal stem cells, Cell Stem Cell 5 (6) (2009) 610–623. [32] K.J. Fernandes et al., A dermal niche for multipotent adult skin-derived precursor cells, Nat. Cell Biol. 6 (11) (2004) 1082–1093. [33] K.J. Fernandes, J.G. Toma, F.D. Miller, Multipotent skin-derived precursors: adult neural crest-related precursors with therapeutic potential, Philos. Trans. R. Soc. Lond. B Biol. Sci. 363 (1489) (2008) 185–198. [34] J.G. Toma et al., Isolation of multipotent adult stem cells from the dermis of mammalian skin, Nat. Cell Biol. 3 (9) (2001) 778–784. [35] J.G. Toma et al., Isolation and characterization of multipotent skin-derived precursors from human skin, Stem Cells 23 (6) (2005) 727–737. [36] H. Jinno et al., Convergent genesis of an adult neural crest-like dermal stem cell from distinct developmental origins, Stem Cells 28 (11) (2010) 2027–2040. [37] N.M. Shah, A.K. Groves, D.J. Anderson, Alternative neural crest cell fates are instructively promoted by TGFbeta superfamily members, Cell 85 (3) (1996) 331–343. [38] C. Cheung et al., Modeling cerebrovascular pathophysiology in amyloid-beta metabolism using neural-crest-derived smooth muscle cells, Cell Rep. 9 (1) (2014) 391–401. [39] E.S. Jeon et al., Sphingosylphosphorylcholine induces differentiation of human mesenchymal stem cells into smooth-muscle-like cells through a TGF-betadependent mechanism, J. Cell Sci. 119 (Pt 23) (2006) 4994–5005. [40] Y. Narita et al., Effects of transforming growth factor-beta 1 and ascorbic acid on differentiation of human bone-marrow-derived mesenchymal stem cells into smooth muscle cell lineage, Cell Tissue Res. 333 (3) (2008) 449–459. [41] S.J. Kattman, E.D. Adler, G.M. Keller, Specification of multipotential cardiovascular progenitor cells during embryonic stem cell differentiation and embryonic development, Trends Cardiovasc. Med. 17 (7) (2007) 240–246. [42] S.J. Kattman, T.L. Huber, G.M. Keller, Multipotent flk-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages, Dev. Cell 11 (5) (2006) 723–732. [43] S.J. Kattman et al., Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines, Cell Stem Cell 8 (2) (2011) 228–240. [44] L. Yang et al., Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population, Nature 453 (7194) (2008) 524–528. [45] O.W. Prall et al., An Nk2-5/Bmp2/Smad1 negative feedback loop controls heart progenitor specification and proliferation, Cell 128 (5) (2007) 947–959. [46] F.L. Conlon et al., A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse, Development 120 (7) (1994) 1919–1928. [47] P. Gadue et al., Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells, Proc. Natl. Acad. Sci. USA 103 (45) (2006) 16806–16811. [48] K. Gritsman, W.S. Talbot, A.F. Schier, Nodal signaling patterns the organizer, Development 127 (5) (2000) 921–932. [49] L.A. Lowe, S. Yamada, M.R. Kuehn, Genetic dissection of nodal function in patterning the mouse embryo, Development 128 (10) (2001) 1831–1843.

[50] C.C. Lu, E.J. Robertson, Multiple roles for Nodal in the epiblast of the mouse embryo in the establishment of anterior-posterior patterning, Dev. Biol. 273 (1) (2004) 149–159. [51] B. Thisse, C.V. Wright, C. Thisse, Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo, Nature 403 (6768) (2000) 425–428. [52] S. Irion et al., Temporal specification of blood progenitors from mouse embryonic stem cells and induced pluripotent stem cells, Development 137 (17) (2010) 2829–2839. [53] O. El-Mounayri et al., Serum-free differentiation of functional human coronary-like vascular smooth muscle cells from embryonic stem cells, Cardiovasc. Res. 98 (1) (2013) 125–135. [54] J.A. Biernaskie et al., Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny, Nat. Protoc. 1 (6) (2006) 2803–2812. [55] R.Y. Tsai, R.D. McKay, Cell contact regulates fate choice by cortical stem cells, J. Neurosci. 20 (10) (2000) 3725–3735. [56] A.D. Witty et al., Generation of the epicardial lineage from human pluripotent stem cells, Nat. Biotechnol. 32 (10) (2014) 1026–1035. [57] H. Kuriyama et al., Physiological features of visceral smooth muscle cells, with special reference to receptors and ion channels, Physiol. Rev. 78 (3) (1998) 811–920. [58] J. Kramer et al., Identification and characterization of novel smoothelin isoforms in vascular smooth muscle, J. Vasc. Res. 38 (2) (2001) 120–132. [59] J. Kramer et al., A novel isoform of the smooth muscle cell differentiation marker smoothelin, J. Mol. Med. (Berl) 77 (2) (1999) 294–298. [60] A.M. Hoggatt, G.M. Simon, B.P. Herring, Cell-specific regulatory modules control expression of genes in vascular and visceral smooth muscle tissues, Circ. Res. 91 (12) (2002) 1151–1159. [61] A.M. Kovacs, W.E. Zimmer, Molecular cloning and expression of the chicken smooth muscle gamma-actin mRNA, Cell Motil. Cytoskeleton 24 (1) (1993) 67–81. [62] K.L. Du et al., Myocardin is a critical serum response factor cofactor in the transcriptional program regulating smooth muscle cell differentiation, Mol. Cell. Biol. 23 (7) (2003) 2425–2437. [63] J. Li et al., Myocardin-related transcription factor B is required in cardiac neural crest for smooth muscle differentiation and cardiovascular development, Proc. Natl. Acad. Sci. USA 102 (25) (2005) 8916–8921. [64] D. Wang et al., Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor, Cell 105 (7) (2001) 851– 862. [65] M. Aikawa et al., Human smooth muscle myosin heavy chain isoforms as molecular markers for vascular development and atherosclerosis, Circ. Res. 73 (6) (1993) 1000–1012. [66] M. Kuro-o et al., CDNA cloning of a myosin heavy chain isoform in embryonic smooth muscle and its expression during vascular development and in arteriosclerosis, J. Biol. Chem. 266 (6) (1991) 3768–3773. [67] M.J. Frontini et al., Fibroblast growth factor 9 delivery during angiogenesis produces durable, vasoresponsive microvessels wrapped by smooth muscle cells, Nat. Biotechnol. 29 (5) (2011) 421–427. [68] M.C. Skala et al., Longitudinal optical imaging of tumor metabolism and hemodynamics, J. Biomed. Opt. 15 (1) (2010) 011112. [69] G.H. Liu et al., Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome, Nature 472 (7342) (2011) 221–225.

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