Publication of the Month

2007

December
Authors: Alejandro Nieponice, Lorenzo Soletti, Jianjun Guan, Bridget M. Deasy, Johnny Huard, William R. Wagner and David A. Vorp
Title: Development of a tissue-engineered vascular graft combining a biodegradable scaffold, muscle-derived stem cells and a rotational vacuum seeding technique.
Summary: There is a clinical need for a tissue-engineered vascular graft (TEVG), and combining stem cells with biodegradable tubular scaffolds appears to be a promising approach. The goal of this study was to characterize the incorporation of muscle-derived stem cells (MDSCs) within tubular poly(ester urethane) urea (PEUU) scaffolds in vitro to understand their interaction, and to evaluate the mechanical properties of the constructs for vascular applications. Porous PEUU scaffolds were seeded with MDSCs using our recently described rotational vacuum seeding device, and cultured inside a spinner flask for 3 or 7 days. Cell viability, number, distribution and phenotype were assessed along with the suture retention strength and uniaxial mechanical behavior of the TEVGs. The seeding device allowed rapid even distribution of cells within the scaffolds. After 3 days, the constructs appeared completely populated with cells that were spread within the polymer. Cells underwent a population doubling of 2.1-fold, with a population doubling time of 35 h. Stem cell antigen-1 (Sca-1) expression by the cells remained high after 7 days in culture (77±20% vs. 66±6% at day 0) while CD34 expression was reduced (19±12% vs. 61±10% at day 0) and myosin heavy chain expression was scarce (not quantified). The estimated burst strength of the TEVG constructs was 2127±900 mmHg and suture retention strength was 1.3±0.3 N. We conclude from this study that MDSCs can be rapidly seeded within porous biodegradable tubular scaffolds while maintaining cell viability and high proliferation rates and without losing stem cell phenotype for up to 7 days of in-vitro culture. The successful integration of these steps is thought necessary to provide rapid availability of TEVGs, which is essential for clinical translation.

November
Authors: Bo Zheng, Baohong Cao, Mihaela Crisan, Bin Sun, Guangheng Li, Alison Logar, Solomon Yap, Jonathan B Pollett, Lauren Drowley, Theresa Cassino, Burhan Gharaibeh, Bridget M Deasy, Johnny Huard & Bruno Péault
Title: Prospective Identification of Myogenic Endothelial Cells in Human Skeletal Muscle
Summary: This manuscript documents anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56+ myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56+ myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro, and may have potential for the treatment of human muscle disease.
Source: Nature Biotechnology 25, 1025 - 1034 (2007)

October
Authors: Qi Mi; Beatrice Rivie`; Gilles Clermont; David L. Steed; Yoram Vodovotz
Title: Agent-based model of inflammation and wound healing: insights into diabetic foot ulcer pathology and the role of transforming growth factor-b1
Summary: Inflammation and wound healing are inextricably linked and complex processes, and are deranged in the setting of chronic, nonhealing diabetic foot ulcers (DFU). An ideal therapy for DFU should both suppress excessive inflammation while enhancing healing. We reasoned that biological simulation would clarify mechanisms and help refine therapeutic approaches to DFU. We developed an agent-based model (ABM) capable of reproducing qualitatively much of the literature data on skin wound healing, including changes in relevant cell populations (macrophages, neutrophils, fibroblasts) and their key effector cytokines (tumor necrosis factor-a [TNF], interleukin [IL]-1b, IL-10, and transforming growth factor [TGF]-b1). In this simulation, a normal healing response results in tissue damage that first increases (due to wound-induced inflammation) and then decreases as the collagen levels increase. Studies by others suggest that diabetes and DFU are characterized by elevated TNF and reduced TGF-b1, although which of these changes is a cause and which one is an effect is unclear. Accordingly, we simulated the genesis of DFU in two ways, either by (1) increasing the rate of TNF production fourfold or (2) by decreasing the rate of TGF-b1 production 67% based on prior literature. Both manipulations resulted in increased inflammation (elevated neutrophils, TNF, and tissue damage) and delayed healing (reduced TGF-b1 and collagen). Our ABM reproduced the therapeutic effect of platelet-derived growth factor/platelet releasate treatment as well as DFU debridement. We next simulated the expected effect of administering (1) a neutralizing anti-TNF antibody, (2) an agent that would increase the activation of endogenous latent TGF-b1, or (3) latent TGF-b1 (which has a longer half-life than active TGF-b1), and found that these therapies would have similar effects regardless of the initial assumption of the derangement that underlies DFU (elevated TNF vs. reduced TGF-b1). In silico methods may elucidate mechanisms of and suggest therapies for aberrant skin healing.
Source: Wound Repair and Regeneration (2007) 15 671-682

September
Authors: Zheng B, Caro B, Crisan M, Sun B Li G, Logar A, Yap S, Pollett JB, Drowley L, Cassino T, Gharaibeh B, Deasy BM, Huard J, Peault B
Title: Prospective identification of myogenic endothelial cells in human skeletal muscle.
Summary: Myoendothelial cells taken from the blood vessels are much more efficient at forming muscle than other sources of stem cells known as satellite and endothelial cells. A thousand myoendothelial cells transplanted into the injured skeletal muscle of immunodeficient mice produced, on average, 89 muscle fibers, compared with nine and five muscle fibers for endothelial and satellite cells, respectively. Myoendothelial cells also showed no propensity to form tumors, a concern with other stem cell therapies.
Source: Nature Biotechnology. 2007 Sep;25(9): 1025-34

August
Authors: Kim GG, Donnenberg VS, Donnenberg AD, Gooding W, Whiteside TL
Title: A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h (51)Cr-release assay.
Summary: Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.
Source: J Immunol Methods. 2007 Aug 31;325(1-2):51-66

July
Author(s) Péault B, Rudnicki M, Torrente Y, Cossu G, Tremblay JP, Partridge T, Gussoni E, Kunkel LM, Huard J.
Title Stem and progenitor cells in skeletal muscle development, maintenance, and therapy
Summary Satellite cells are dormant progenitors located at the periphery of skeletal myofibers that can be triggered to proliferate for both self-renewal and differentiation into myogenic cells. In addition to anatomic location, satellite cells are typified by markers such as M-cadherin, Pax7, Myf5, and neural cell adhesion molecule-1. The Pax3 and Pax7 transcription factors play essential roles in the early specification, migration, and myogenic differentiation of satellite cells. In addition to muscle-committed satellite cells, multi-lineage stem cells encountered in embryonic, as well as adult, tissues exhibit myogenic potential in experimental conditions. These multi-lineage stem cells include side-population cells, muscle-derived stem cells (MDSCs), and mesoangioblasts. Although the ontogenic derivation, identity, and localization of these non-conventional myogenic cells remain elusive, recent results suggest their ultimate origin in blood vessel walls. Indeed, purified pericytes and endothelium-related cells demonstrate high myogenic potential in culture and in vivo. Allogeneic myoblasts transplanted into Duchenne muscular dystrophy (DMD) patients have been, in early trials, largely inefficient owing to immune rejection, rapid death, and limited intramuscular migration--all obstacles that are now being alleviated, at least in part, by more efficient immunosuppression and escalated cell doses. As an alternative to myoblast transplantation, stem cells such as mesoangioblasts and CD133+ progenitors administered through blood circulation have recently shown great potential to regenerate dystrophic muscle.
Source Mol Ther. 2007 May;15(5):867-77

June
Author(s) Fujimoto KL, Tobita K, Merryman WD, Guan J, Momoi N, Stolz DB, Sacks MS, Keller BB, Wagner WR
Title An elastic, biodegradable cardiac patch induces contractile smooth muscle and improves cardiac remodeling and function in subacute myocardial infarction
Summary Our objective in this study was to apply an elastic, biodegradable polyester urethane urea (PEUU) cardiac patch onto subacute infarcts and to examine the resulting cardiac ventricular remodeling and performance. BACKGROUND: Myocardial infarction induces loss of contractile mass and scar formation resulting in adverse left ventricular (LV) remodeling and subsequent severe dysfunction. METHODS: Lewis rats underwent proximal left coronary ligation. Two weeks after coronary ligation, a 6-mm diameter microporous PEUU patch was implanted directly on the infarcted LV wall surface (PEUU patch group, n = 14). Sham surgery was performed as an infarction control (n = 12). The LV contractile function, regional myocardial wall compliance, and tissue histology were assessed 8 weeks after patch implantation.

RESULTS: The end-diastolic LV cavity area (EDA) did not change, and the fractional area change (FAC) increased in the PEUU patch group (p < 0.05 vs. week 0), while EDA increased and FAC decreased in the infarction control group (p < 0.05). The PEUU patch was largely resorbed 8 weeks after implantation and the LV wall was thicker than infarction control (p < 0.05 vs. control group). Abundant smooth muscle bundles with mature contractile phenotype were found in the infarcted myocardium of the PEUU group. The myocardial compliance of the PEUU group was distributed between normal myocardium and infarction control (p < 0.001). CONCLUSIONS: Implantation of a novel biodegradable PEUU patch onto a subacute myocardial infarction promoted contractile phenotype smooth muscle tissue formation and improved cardiac remodeling and contractile function at the chronic stage. Our findings suggest a new therapeutic option against post-infarct cardiac failure.

Source J Am Coll Cardiol. 2007 Jun 12;49(23):2292-300

May
Author(s) Christopher R Shepard and Alan Wells
Title Demethylation of the E-cadherin promoter driven by hepatocytes allows for cell fate-determining signals in invasive breast cancer cells
Summary Epithelial-cadherin’s (E-cadherin) transcriptional silencing in most advanced tumors, due to promoter methylation, enables tumor cells to disseminate from the primary mass. However, E-cadherin-positive metastatic carcinoma foci do originate from mainly E-cadherin-negative primaries. It is unknown if this is due to dissemination of the minority of E-cadherin-positive cells or re-expression of E-cadherin during metastasis. Here, we demonstrate that co-culture of hepatocytes with invasive breast cancer cells lacking E-cadherin triggers an epigenetic reversion in the breast cancer cells resulting in demethylation of the E-cadherin promoter and subsequent expression on the protein level. We show a similar time-course for epigenetic reversion of primary human breast cancer explants co-cultured with hepatocytes. Further, we show that the E-cadherin ligation between breast cancer cells and hepatocytes is functional and activates the canonical MAPK pathway and Akt pathway in these cancer cells. Our epigenetic-reversion hypothesis for E-cadherin represents not only a paradigm shift in the current thinking that absence of E-cadherin is a fundamental issue, but would also reveal new strategies to combat the initial stages of metastatic disease in breast cancer patients.
Source FASEB J. 21: 128.4

April
Author(s) Peault B, Rudnicki M, Torrente Y, Cossu G, Tremblay JP, Partridge T, Gussoni E, Kunkel LM, Huard J.
Title Stem and Progenitor Cells in Skeletal Muscle Development, Maintenance, and Therapy
Summary Satellite cells are dormant progenitors located at the periphery of skeletal myofibers that can be triggered to proliferate for both self-renewal and differentiation into myogenic cells. In addition to anatomic location, satellite cells are typified by markers such as M-cadherin, Pax7, Myf5, and neural cell adhesion molecule-1. The Pax3 and Pax7 transcription factors play essential roles in the early specification, migration, and myogenic differentiation of satellite cells. In addition to muscle-committed satellite cells, multi-lineage stem cells encountered in embryonic, as well as adult, tissues exhibit myogenic potential in experimental conditions. These multi-lineage stem cells include side-population cells, muscle-derived stem cells (MDSCs), and mesoangioblasts. Although the ontogenic derivation, identity, and localization of these non-conventional myogenic cells remain elusive, recent results suggest their ultimate origin in blood vessel walls. Indeed, purified pericytes and endothelium-related cells demonstrate high myogenic potential in culture and in vivo. Allogeneic myoblasts transplanted into Duchenne muscular dystrophy (DMD) patients have been, in early trials, largely inefficient owing to immune rejection, rapid death, and limited intramuscular migration-all obstacles that are now being alleviated, at least in part, by more efficient immunosuppression and escalated cell doses. As an alternative to myoblast transplantation, stem cells such as mesoangioblasts and CD133+ progenitors administered through blood circulation have recently shown great potential to regenerate dystrophic muscle.
Source Molecular Therapy (2007) 15 5, 867-877

March
Author(s) Thomas W. Gilbert, Michael S. Sacks, Jonathan S. Grashow, Savio L.-Y. Woo, Stephen F. Badylak, Michael B. Chancellor
Title Fiber Kinematics of Small Intestinal Submucosa Under Biaxial and Uniaxial Stretch
Summary Improving our understanding of the design requirements of biologically derived collagenous scaffolds is necessary for their effective use in tissue reconstruction. In the present study, the collagen fiber kinematics of small intestinal submucosa (SIS) was quantified using small angle light scattering (SALS) while the specimen was subjected to prescribed uniaxial or biaxial strain paths. A modified biaxial stretching device based on Billiar and Sacks (J. Biomech., 30, pp. 753–7, 1997) was used, with a real-time analysis of the fiber kinematics made possible due to the natural translucency of SIS. Results indicated that the angular distribution of collagen fibers in specimens subjected to 10% equibiaxial strain was not significantly different from the initial unloaded condition, regardless of the loading path _p=0.31_. Both 10% strip biaxial stretch and uniaxial stretches of greater than 5% in the preferred fiber direction led to an increase in the collagen fiber alignment along the same direction, while 10% strip biaxial stretch in the cross preferred fiber direction led to a broadening of the distribution. While an affine deformation model accurately predicted the experimental findings for a biaxial strain state, uniaxial stretch paths were not accurately predicted. Nonaffine structural models will be necessary to fully predict the fiber kinematics under large uniaxial strains in SIS.
Source J. Biomech. Eng. 128, 890 (2006)

February
Author(s) Manuela Tavian, Bo Zheng, Estelle Oberlin, Mihaela Crisan, Bin Sun, Johnny Huard, and Bruno Peault
Title The Vascular Wall as a Source of Stem Cells
Summary We have characterized the emerging hematopoietic system in the human embryo and fetus. Two embryonic organs, the yolk sac and aorta, support the primary emergence of hematopoietic stem cells (HSCs), but only the latter contributes lymphomyeloid stem cells for definitive, adult-type hematopoiesis. A common feature of intra- and extraembryonic hematopoiesis is that in both locations hematopoietic cells emerge in close vicinity to vascular endothelial cells. We have provided evidence that a population of angiohematopoietic mesodermal stem cells, marked by the expression of flk-1 and the novel BB9/ACE antigen, migrate from the paraaortic splanchnopleura into the ventral part of the aorta, where they give rise to hemogenic endothelial cells and, in turn, hematopoietic cells. HSCs also appear to develop from endothelium in the embryonic liver and fetal bone marrow, albeit at a much lower frequency. This would imply that the organism does not function during its whole life on a stock of hematopoietic stem cells established in the early embryo, as is usually accepted. We next examined whether the vessel wall can contribute stem cells for other cell lineages, primarily in the model of adult skeletal muscle regeneration. By immunohistochemistry and flow cytometry, we documented the existence in skeletal muscle, besides genuine endothelial and myogenic cells, of a subset of satellite cells that coexpress endothelial cell markers. This suggested the existence of a continuum of differentiation from vascular cells to endothelial cells that was confirmed in long-term culture. The regenerating capacity of these cells expressing both myogenic and endothelial markers is being investigated in skeletal and cardiac muscle, and the results are being compared with those generated by satellite cells. Altogether, these results point to a generalized progenitor potential of a subset of endothelial, or endothelium-like, cells in blood vessel walls, in pre- and postnatal life.
Source Ann. N.Y. Acad. Sci. 1044: 41–50 (2005). © 2005 New York Academy of Sciences. doi: 10.1196/annals.1349.006

January
Author(s) Xue Wang, Yong Wang, Hong Pyo Kim, Kiichi Nakahira, Stefan W. Ryter, and Augustine M. K. Choi
Title Carbon Monoxide Protects against Hyperoxia-induced Endothelial Cell Apoptosis by Inhibiting Reactive Oxygen Species Formation
Summary Hyperoxia causes cell injury and death associated with reactive oxygen species formation and inflammatory responses. Recent studies show that hyperoxia-induced cell death involves apoptosis, necrosis, or mixed phenotypes depending on cell type, although the underlying mechanisms remain unclear. Using murine lung endothelial cells, we found that hyperoxia caused cell death by apoptosis involving both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) pathways. Hyperoxia-dependent activation of the extrinsic apoptosis pathway and formation of the death-inducing signaling complex required NADPH oxidase-dependent reactive oxygen species production, because this process was attenuated by chemical inhibition, as well as by genetic deletion of the p47phox subunit, of the oxidase. Overexpression of heme oxygenase-1 prevented hyperoxia-induced cell death and cytochrome c release. Likewise, carbon monoxide, at low concentrations, markedly inhibited hyperoxia-induced endothelial cell death by inhibiting cytochrome c release and caspase-9/3 activation. Carbon monoxide, by attenuating hyperoxia-induced reactive oxygen species production, inhibited extrinsic apoptosis signaling initiated by death-inducing signal complex trafficking from the Golgi apparatus to the plasma membrane and downstream activation of caspase-8. We also found that carbon monoxide inhibited the hyperoxia-induced activation of Bcl-2-related proteins involved in both intrinsic and extrinsic apoptotic signaling. Carbon monoxide inhibited the activation of Bid and the expression and mitochondrial translocation of Bax, whereas promoted Bcl-XL/Bax interaction and increased Bad phosphorylation. We also show that carbon monoxide promoted an interaction of heme oxygenase-1 with Bax. These results define novel mechanisms underlying the antiapoptotic effects of carbon monoxide during hyperoxic stress.
Source J. Biol. Chem., Jan 2007; 282: 1718 - 1726

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