Direct Isolation, Culture and Transplant of Mouse Skeletal Muscle Derived Endothelial Cells with Angiogenic Potential

Background: Although diseases associated with microvascular endothelial dysfunction are among the most prevalent illnesses to date, currently no method exists to isolate pure endothelial cells (EC) from skeletal muscle for in vivo or in vitro study. Methodology: By utilizing multicolor fluorescent-activated cell sorting (FACS), we have isolated a distinct population of Sca-1 +, CD31 +, CD34 dim and CD45 2 cells from skeletal muscles of C57BL6 mice. Characterization of this population revealed these cells are functional EC that can be expanded several times in culture without losing their phenotype or capabilities to uptake acetylated low-density lipoprotein (ac-LDL), produce nitric oxide (NO) and form vascular tubes. When transplanted subcutaneously or intramuscularly into the tibialis anterior muscle, EC formed microvessels and integrated with existing vasculature. Conclusion: This method, which is highly reproducible, can be used to study the biology and role of EC in diseases such as peripheral vascular disease. In addition this method allows us to isolate large quantities of skeletal muscle derived EC with potential for therapeutic angiogenic applications

Isolation and Characterization of Neural Crest-Derived Stem Cells from Dental Pulp of Neonatal Mice

Dental pulp stem cells (DPSCs) are shown to reside within the tooth and play an important role in dentin regeneration. DPSCs were first isolated and characterized from human teeth and most studies have focused on using this adult stem cell for clinical applications. However, mouse DPSCs have not been well characterized and their origin(s) have not yet been elucidated. Herein we examined if murine DPSCs are neural crest derived and determined their in vitro and in vivo capacity. DPSCs from neonatal murine tooth pulp expressed embryonic stem cell and neural crest related genes, but lacked expression of mesodermal genes. Cells isolated from the Wnt1-Cre/R26R-LacZ model, a reporter of neural crest-derived tissues, indicated that DPSCs were Wnt1-marked and therefore of neural crest origin. Clonal DPSCs showed multi-differentiation in neural crest lineage for odontoblasts, chondrocytes, adipocytes, neurons, and smooth muscles. Following in vivo subcutaneous transplantation with hydroxyapatite/tricalcium phosphate, based on tissue/cell morphology and specific antibody staining, the clones differentiated into odontoblast-like cells and produced dentin-like structure. Conversely, bone marrow stromal cells (BMSCs) gave rise to osteoblast-like cells and generated bone-like structure. Interestingly, the capillary distribution in the DPSC transplants showed close proximity to odontoblasts whereas in the BMSC transplants bone condensations were distant to capillaries resembling dentinogenesis in the former vs. osteogenesis in the latter. Thus we demonstrate the existence of neural crest-derived DPSCs with differentiation capacity into cranial mesenchymal tissues and other neural crest-derived tissues. In turn, DPSCs hold promise as a source for regenerating cranial mesenchyme and other neural crest derived tissues

Neurotrophin-3 is a novel angiogenic factor capable of therapeutic neovascularization in a mouse model of limb ischemia

OBJECTIVE: To investigate the novel hypothesis that neurotrophin-3 (NT-3), an established neurotrophic factor that participates in embryonic heart development, promotes blood vessel growth. METHODS AND RESULTS: We evaluated the proangiogenic capacity of recombinant NT-3 in vitro and of NT-3 gene transfer in vivo (rat mesenteric angiogenesis assay and mouse normoperfused adductor muscle). Then, we studied whether either transgenic or endogenous NT-3 mediates postischemic neovascularization in a mouse model of limb ischemia. In vitro, NT-3 stimulated endothelial cell survival, proliferation, migration, and network formation on the basement membrane matrix Matrigel. In the mesenteric assay, NT-3 increased the number and size of functional vessels, including vessels covered with mural cells. Consistently, NT-3 overexpression increased muscular capillary and arteriolar densities in either the absence or the presence of ischemia and improved postischemic blood flow recovery in mouse hind limbs. NT-3–induced microvascular responses were accompanied by tropomyosin receptor kinase C (an NT-3 high-affinity receptor) phosphorylation and involved the phosphatidylinositol 3-kinase–Akt kinase–endothelial nitric oxide synthase pathway. Finally, endogenous NT-3 was shown to be essential in native postischemic neovascularization, as demonstrated by using a soluble tropomyosin receptor kinase C receptor domain that neutralizes NT-3. CONCLUSION: Our results provide the first insight into the proangiogenic capacity of NT-3 and propose NT-3 as a novel potential agent for the treatment of ischemic disease

Absence of CD34 on Murine Skeletal Muscle Satellite Cells Marks a Reversible State of Activation during Acute Injury

Background: Skeletal muscle satellite cells are myogenic progenitors that reside on myofiber surface beneath the basal lamina. In recent years satellite cells have been identified and isolated based on their expression of CD34, a sialomucin surface receptor traditionally used as a marker of hematopoietic stem cells. Interestingly, a minority of satellite cells lacking CD34 has been described. Methodology/Principal Findings: In order to elucidate the relationship between CD34+ and CD34- satellite cells we utilized fluorescence-activated cell sorting (FACS) to isolate each population for molecular analysis, culture and transplantation studies. Here we show that unless used in combination with a7 integrin, CD34 alone is inadequate for purifying satellite cells. Furthermore, the absence of CD34 marks a reversible state of activation dependent on muscle injury. Conclusions/Significance: Following acute injury CD34- cells become the major myogenic population whereas the percentage of CD34+ cells remains constant. In turn activated CD34- cells can reverse their activation to maintain the pool of CD34+ reserve cells. Such activation switching and maintenance of reserve pool suggests the satellite cell compartment is tightly regulated during muscle regeneration

The Cellular and Molecular Axis of Muscle Regeneration

Thesis (Ph.D.)--University of Washington, 2014Skeletal muscle has significant regenerative capacity, which is impaired with muscular dystrophy and aging. Muscle function and repair requires the involvement of several cellular compartments and molecular interactions. With disease cellular responses are influenced by the alteration of signaling pathways that are involved in the normal process of muscle regeneration. Disruptions in regenerative signaling coincide with the activation of pathways responsible for tissue pathology. Therefore, the cellular and molecular axis of muscle regeneration follows a strict program that when interrupted by disease, cannot sustain repair and results in muscle degeneration. The cellular compartments of the skeletal muscle respond as a collective to repair damage. Each cellular population is influenced by distinct pathways and cellular interactions. In the absence of disease, the process of regeneration is mediated by satellite cells, endothelial cells and collagen producing cells to respond to injury and regenerate muscle fibers, vessels, and reconstitute damaged connective tissue respectively. With disease, signaling pathways that influence cellular responses to injury are altered. As described in this dissertation, we discovered Sphingosine-1-Phosphate (S1P) and Platelet Derived Growth Factor Receptor-α (PDGFRα) are two signaling pathways with opposing effects in muscular dystrophy. With muscular dystrophy, the accumulation of fibrosis perturbs the regenerative response. Therefore, we hypothesis that alterations in the muscle's repair processes contribute to pathogenesis; pro-regenerative pathways (such as S1P) diminish as pro-fibrotic pathways (such as PDGFRα) remain active. Understanding the cellular crosstalk and both processes of degeneration and regeneration are crucial for the development of therapies that can reduce muscle pathology and promote repair. Herein, we explore the axis of molecular signaling and cellular responses that influence muscle regeneration during injury and wasting. Such a holistic approach is necessary for continuing our advance in treating muscle wasting diseases. Our main findings support our hypothesis that regeneration and degeneration are intimately linked. Two cell populations affected by muscular dystrophy (endothelial cells and satellite cells) are diverse resident cells involved in S1P signaling of the muscle. In contrast, collagen producing cells are activated by PDGFRα to promote fibrosis and perturb regeneration. In summary, our findings, support the development of combinatory therapies that target specific pathways, such as S1P and PDGFRα, to promote regenerative signaling while negating the effects of degenerative signaling on muscle repair. Despite the potential of such cellular and molecular strategies, significant barriers exit in the culture and politics of science that I will discuss in the closing commentary. Here I will describe the current scientific crisis, which in my own opinion extends not only from a decline in funding, but abuse and misuse of resources has rampant for years. In addition the structure of scientific training and funding has compounded this crisis, as more PhD's continue to be trained despite the recognizable and ongoing decline in scientific employment. Therefore, in the past two decades, the field of biological science has adapted a policy analogous to our government's stance on global warming; ignore the immanent catastrophe. This will not come in the form of rising oceans or temperatures, but stagnation of discoveries and treatment for diseases. This calamity can be averted if we, as a community, divert from politics and personal gain, but instead focus our efforts on conducting meaningful science with the public's best interests in mind

FACS sorted EC can be culture-expanded and preserve endothelial functions in culture.

<p>A, Growth rate comparison for muscle EC lines derived individually from four 25 MO C57BL6 males, cultured in DMEM with 10% FCS and 10 ng/ml VEGF for 60 days. B, Vascular bundle formation occurs rapidly within 18 hrs of cells being seeded on pure matrigel vs. controls without matrigel. Photographs were taken at 1.5 hour intervals and after 4.5 hours left overnight in culture. C, Top panel shows endothelial specific NO production. Left, muscle EC can produce NO as detected by green fluorescence emission of DAF-2T. Middle, NO production was inhibited by L-NAME. Right, muscle EC stained with rabbit anti-eNOS followed by anti-rabbit Alexa 647 show strong staining in endothelial caveolae. Bottom panel shows other functional assays as well as maintenance of vWF expression. Left, cultured muscle EC uptake ac-LDL- rhodamine (red fluorescence). Middle, skeletal muscle derived EC in culture retain vWF expression for more than 80 days as portrayed by anti-vWF with Alexa 594. Right, 99.9% of freshly FACS-sorted muscle EC stained positive for vWF immunoperoxidase with DAB (brown). Scale bar = 50 µm.</p

EC of the skeletal muscle form microvessels in matrigel injected subcutaneously over the dorsum.

<p>A–B, Tissue sections of the matrigel plug recovered from the dorsal skin of C57BL6 mice analyzed 14 days post injection reveal microvessel formation. Staining for anti-CD31 and anti-vWF followed with a secondary Alexa 647 antibody highlight mature vessels formed within the matrigel by injected cells (PKH26⁺). C, Conjugated FITC anti-SMA staining indicates the recruitment of endogenous smooth muscle cells in the formation of microvessels. Scale bar = 50 µm.</p

Muscle EC abundance between gender, muscle goups and age.

<p>A, Gender comparison of the percentage (y-axis) of EC within the CD45⁻ population between 2 MO mice (n = 3). * designates p<0.05. B, The percentage of EC within the CD45⁻ population among different skeletal muscles. C, Age comparison of the percentage of EC within the CD45⁻ population between 1, 12 and 24 MO mice (n = 3). * designates p<0.05 between 1 vs. 12 MO mice. ** designates p<0.05 between 12 vs. 25 MO mice. P values calculated by student t-test.</p

EC injected subcutaneously in matrigel migrate into the muscle.

<p>Top, a montage of the matrigel plug shows PKH26⁺ microvessels in the matrigel plug over the TA muscle. Bottom, A montage of an area approximately 80 µm distal shows migration of PKH26⁺ EC. EC invaded the TA from the matrigel plug and formed new microvessels along their migration path and also integrated into existing vessel. Staining with anti-vWF in Alexa 647 (magenta) highlights EC angiogenic capability within the matrigel plug and muscle. Scale bars = 50 µm.</p