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

Exploring the Impact of COVID on Global Telecommunication Networks and ICT Solutions

The emergence of COVID-19 and its spread all over the world caused a significant increase in network traffic. Therefore, for more secure and consistent communications, it is crucial to investigate the factors that affect network traffic. In this study, the review of COVID-19 consequences on global telecommunication networks with an emphasis on network traffic is presented. The impact of lockdown on digital telecommunication networks in various countries around the globe is investigated. The rapid expansion of the virus forced countries to set up lockdown measures, and this caused people to stay at home; therefore, network traffic increased significantly from March 2020 to the middle of April, and then it slightly changed to be more stabilized until the middle of May 2020. Such increased network traffic has affected many aspects, such as mobile networks, roaming factors, and economic situations. In this research, supporting programs to protect network connectivity are studied around the globe. In a situation where people are mostly working remotely, security is a challenging issue that should be taken into careful consideration. This study provides a broad understanding of how COVID-19 affected digital communications and how governments responded to unprecedented crises

An expansive human regulatory lexicon encoded in transcription factor footprints.

Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis-regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein-DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency

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

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

Freshly sorted EC injected intramuscularly also form new vessels and integrate with existing vasculature vs. control cells.

<p>A, Tissues sections from mice injected intramuscularly and examined 14 days post injection reveals freshly sorted PKH stained EC also have angiogenic capability. Although freshly sorted EC did not migrate as far as cultured EC several PKH⁺/vWF⁺ vessels highlighted by anti-vWF staining with Alexa 647 (magenta) were visible beyond the point of injection. B, Contrary to the capability of freshly sorted and cultured EC to form new vessels and integrate with existing ones, control cells sorted as Sca-1⁻, CD31⁻ and CD45⁻ injected under the same conditions did not exhibit any angiogenic potential. Scale bars = 50 µm.</p

EC injected intramuscularly migrate, form new vessels and integrate with existing vasculature.

<p>Top, a montage of a region proximal to the injection site shows the needle track in the TA muscle. Bottom, a montage of a region approximately 80 µm distal to the injection site shows extensive migration beyond 1 mm from the needle track (white line). Montage represents an overlay of the PKH26 red fluorescence over bright field microscopy. Formation of new vessels and incorporation into existing vasculature is highlighted by anti-vWF staining in Alexa 647 (magenta). Green arrows point to areas where PKH26⁺ cells are making connections with or integrated into endogenous vessels. Scale bars = 50 µm.</p

Flow-cytometry characterization of sorted EC.

<p>A, Stepwise selection for Sca-1⁺, CD31⁺, CD34^dim and CD45⁻endothelial cells (EC) begins by first gating (pink box, left dot plot) on small and low granulated cells within a SSC-A and FSC-A dot plot. CD45⁺ cells are then identified and gated out by comparing the sample (green peak) with the unstained control (red peak) to reveal a Sca-1⁺ and CD31⁺ population of muscle EC within the CD45⁻population (pink bar, middle histogram). The Sca-1⁺ (vertical)/CD31⁺ (horizontal) endothelial population (pink oval, right dot plot) which is dim for CD34 can then be gated for further analysis or cell sorting. B, Endothelial CD31 FITC expression correlates with cell size as FSC-A comparison indicates larger cells (blue) express more CD31. C, 45% of EC are positive for ac-LDL rhodamine uptake (red) within the entire CD45⁻ population (green) portrayed in Sca-1 APC (vertical) and CD31 FITC (horizontal). D, The endothelial cells defined as Sca-1⁺, CD31⁺ and CD34^dimare homogeneously negative for CD45 PerCP, positive for Sca-1 APC and CD31 FITC, and dim for CD34 PE Cy7 (green peaks) vs. unstained controls (red peaks). Analysis of separate aliquots from the same muscle preparations revealed EC lack Syndecan 4 PE, AC133 FITC, NCAM PE and CXCR4 PE (green peaks EC samples, red peaks unstained control).</p