Human sinusoidal subendothelial cells regulate homing and invasion of circulating metastatic prostate cancer cells to bone marrow

: Subendothelial cells (pericytes) are the clonogenic, multipotent and self-renewing skeletal stem cells (SSCs) found in bone marrow (BM) stroma. They express genes maintaining hematopoietic stem cell (HMC) niche identity and, transplanted in immunocompromised mice, organize the hematopoietic microenvironment (HME) generating humanized bone/BM ossicles. To create a mouse model of hematogenous metastasis of human prostate cancer (PC) cells to human bone/BM, we injected PC cells in the blood circulatory system of Severe Combined Immunodeficiency (SCID)/beige mice bearing heterotopic ossicles. Results indicate that PC cells could efficiently home to mice-implanted extraskeletal BM ossicles, but were not able to colonize mice skeletal segments. In humanized bone/BM ossicles, early foci of PC cells occupied a perisinusoidal position, in close contact with perivascular stromal cells. These findings demonstrate the importance of the SSC compartment in recreating a suitable environment to metastatic PC cells. Our data support the hypothesis that BM SSCs committed to a pericyte fate can specify for homing niches of PC cells, suggesting an involvement of specific interactions with subendothelial stromal cells in extravasation of circulating metastatic PC cells to BM

Is chronic exposure to raw water a possible risk factor for amyotrophic lateral sclerosis? A pilot case-control study

Background: The etiopathogenesis of amyotrophic lateral sclerosis (ALS) is still largely unknown. Methods: We performed a case-control study (33 cases and 35 controls) in Umbria, Italy. We investigated associations between common lifestyle, clinical factors, as well as environmental exposures potentially implicated with ALS onset. Face-to-face interviews were carried out. All cases were recruited and diagnosed according to El Escorial criteria. Case-control comparisons were made for educational and residential status, occupational exposures, and clinical and lifestyle factors prior to cases’ dates of diagnosis. Results: Our results showed an increased risk of ALS for subjects chronically exposed to raw water use (odds ratio (OR) = 6.55, 95% confidence interval (CI): 2.24–19.12). Garden activities showed a tight association with ALS as well, very likely as a consequence of chronic raw water exposure. Indeed, we could exclude an impact for pesticides, as no significant differences were observed in pesticide exposure in the two groups interviewed. However, cases were more often exposed to fertilizers. After adjustment for age, sex, and heavy physical activities, exposure to raw water was still associated with increased ALS risk (OR = 4.74, 95% CI: 1.33–16.85). Discussion: These findings suggest an association between ALS and exposure to raw water, which should be further investigated for the presence of chemicals interfering with nervous system functionality

Clonogenic, myogenic progenitors expressing MCAM/CD146 are incorporated as adventitial reticular cells in the microvascular compartment of human post-natal skeletal muscle

Recent observation identifies subendothelial (mural) cells expressing MCAM, a specific system of clonogenic, self-renewing, osteoprogenitors (a.k.a, "mesenchymal stem cells") in the microvascular compartment of post-natal human bone marrow (BM). In this study, we used MCAM/CD146, as a marker to localize, isolate and assay subendothelial clonogenic cells from the microvasculature of postnatal human skeletal muscle. We show here that these cells share with their BM counterpart, anatomic position (subendothelial/adventitial) and ex vivo clonogenicity (CFU-Fs). When assayed under the stringent conditions, these cells display a high spontaneous myogenic potential (independent of co-culture with myoblasts or of in vivo fusion with local myoblasts), which is otherwise only attained in cultures of satellite cells. These muscle-derived mural cells activated a myogenic program in culture. Cultured CD146+ cells expressed the myogenic factors (Pax7, Pax3 and Myf5), NCAM/CD56, desmin as well as proteins characteristic of more advanced myogenic differentiation, such as myosin heavy chain. In vivo, these cells spontaneously generate myotubes and myofibrils. These data identify the anatomy and phenotype of a novel class of committed myogenic progenitor in human post-natal skeletal muscle of subendothelial cells associated with the abluminal surface of microvascular compartment distinct from satellite cells

CD146⁺ mural cells are spontaneously myogenic <i>in vivo</i>.

<p>To assess myogenic potential of human muscle-derived CD146⁺ cell populations we used a stringent <i>in vivo</i> myogenesis assay. A) In a heterotopic transplantation assay, CD146⁺ muscle cells were suspended in growth factor-reduced Matrigel^TM and injected into the epifascial space of the back of SCID/beige mice, and harvested 3 weeks later. Plugs harvested demonstrated that: B) No myogenic differentiation was observed with human dermal-derived fibroblasts, while remaining viable in the plug; C) <i>In vivo</i> activation of a myogenic program in CD146⁺ muscle cells. Human CD146⁺ muscle cells revealed an extensive formation not only of myotubes expressing human-specific myogenic markers, desmin, MyHC, CD56 (counterstained with hematoxylin), spectrin, dystrophin, myoglobin, but even of striated myofibers (<i>black arrows</i>) and non-conventional muscle structures (syncytia myoglobes, <i>red arrows</i>), as demonstrated by H&E staning. Scale bar = 80 μm, 75 μm, 50 μm, 40 μm, 25 μm, 10 μm, 5 μm.</p

Muscle CFU-F-derived cells are spontaneously myogenic in culture.

<p>A) Expression by FACS of CD146 and B-C) by fluorescent immunocytochemistry generation of myofibers in cultures of CD146-sorted muscle CFU-Fs, unsorted muscle CFU-Fs, muscle explant cultures and skin fibroblasts. Only rare myotubes are observed in cultures of muscle explant cultures. Data are expressed as percentage double positive cells MyHC/Dapi (<i>***P</i> < 0.001). Data are shown as mean ± standard deviation (SD). Scale bar = 120 μm. D) Sorted and cultured CD146⁺ human muscle-derived cells progressively turn on expression of myogenic markers PAX7, PAX3, Myf5, CD56, Desmin and MyHC, as verified by fluorescent immunocytochemistry. Scale bar = 120 μm, 60 μm. E) Terminal differentiation into myotubes could be achieved by replating CD146⁺ muscle-derived clonogenic cells at very high density with high serum. Multinucleated myotubes are obvious after Giemsa staining and expressing MyHC. F) Karyotype of human skeletal muscle cells generated by pooling colonies of M-CFU-Fs, after 10 population doublings, showed an euploid number of chromosomes.</p

Cells expressing CD146 in the vascular walls of human adult skeletal muscle.

<p><b>Identification of M-CFU-Fs as subendothelial CD146⁺ cells.</b> A) Immunolocalization of CD146 in human adult skeletal muscle. Immunoreactivity is restricted to microvascular walls. <i>endo</i>, endothelial cell nucleus. <i>arrow</i>, subendoethelial cell nucleus. Scale bar = 10 μm. B) Localization of CD146 and CD34 in muscle sections. Detail of a capillary, adjacent to a myofiber. Overview demonstrating that endothelial cells [(endo) in B] express CD34 but not CD146. Subendothelial pericytes [(peri) in B] express CD146. Scale bar = 10 μm. C) In muscle cells, expression of CD146, CD56, ALP and CD34 by FACS fluorescent. Expression of CD146 and CD56 is mutually exclusive in distinct cell subsets, with no co-expression. Co-expression of CD34 and ALP along with CD146, in human muscle cell suspensions before culture. CD146 is expressed in certain subsets of endothelial cells and ALP^<b>+</b> cells. D) Isotype control and FACS analysis of collagenase-generated cell suspensions of muscle, demonstrating an ~1% fraction of CD146-expressing cells. Freshly isolated muscle CD146⁺ cells were plated at clonal density (1.6 cells/cm²). At clonal density, muscle CD146⁺ but not muscle CD146^- cells formed discrete fibroblastic colonies (arrows point to colonies, Giemsa stain). CFE assay of unsorted total cells, FACS-sorted CD146^<b>+</b> cells and CD146^- cell subsets. All muscle CFU-Fs are found in the CD146-expressing subset of muscle cells. Representative cultures are shown. Discrete fibroblastic colonies formed by plating collagenase-released muscle cells at clonal density (1.6 cells/cm²). E) Cell morphology in single colonies generated by CD146^<b>+</b> muscle-derived clonogenic cells at 14 days (scale bars, 200 μm) in culture, in 20% serum, and on plastic. Developing colony photographed 9 days after plating (a). Fully developed colony photographed at day 14 after plating (b). Both demonstrating the characteristic whorled pattern and fibroblast-like cell morphology. F) FACS analysis of a multi-clonal strain obtained by combining multiple primary colonies. Note high expression of multiple markers of BM-derived CFU-Fs (and “mesenchymal stem cells”), CD44, CD90, CD105, CD49a, ALP, and high/bright expression of CD146. Endothelial (CD34, CD133) and hematopoietic markers (CD45) are negative. G) Proliferation curve of muscle-derived CD146⁺ multi-clonal cell strain.</p

Human muscle cell strains generated by explants cultures.

<p>A) <i>In vitro</i> FACS characterization of human muscle cell strains generated by explants cultures. B) The % of CD146⁺ cells in the cultures declined as a function of increasing plating density, reaching ~65% in <i>in vitro</i> characterization of human muscle a primary culture generated by seeding cells at a definitively non-clonal density (1.6x10⁴ cells/cm²). The lowest % (~43%) was observed in cell strains generated by explants. A substantial proportion (34–57%) of cells in non-clonal cultures (plated at 1.6x10⁴ cells/cm² or explant cultures) fail to express CD146⁺. C) Conventional <i>in vitro</i> myogenic differentiation assays were assessed by plating muscle-derived CFU-Fs CD146⁺ cells onto Matrigel^TM coated dishes, with DMEM/2% Horse Serum. After 7 days, extensive formation of myotubes expressing specific myogenic markers were observed. No myogenic differentiation was observed with non-muscle fibroblasts derived cell strain. MyHC, Myosin Heavy chain, Scale bar = 120 μm.</p

<i>In vivo</i> generation of satellite cells, microvascular cells and contribution to regenerating myofibers in a model of muscle damage by transplanted CD146⁺ cells.

<p>Muscle-derived CD146⁺ cells were injected intramuscular into cardiotoxin-injured muscle <i>tibialis anterior</i> of SCID/beige mice, injured 1 day earlier by an intramuscular injection of CTX. A) After 4 weeks transplanted human muscle-derived cells, identified by expression of human Lamin A/C (red), were distributed to the interstitium (arrow), but also to the surface of myofibers (MF; green) in a satellite cell-like position (arrowhead), and to walls of small blood vessels (bv, double arrows). Scale bar = 110 μm, 100 μm, 50 μm, 40 μm. B) Cells in a satellite cell-like position express human CD56 (red). Scale bar = 150 μm, 70 μm, 40 μm. C) Cells blood vessel walls (bv) express human CD146 (red, arrowhead). The inset shows immunoreactivity of CD146 was restricted to microvascular walls of the interstitial tissue. D) Clusters of myofibers expressing human dystrophin 3 and spectrin (red) were generated by muscle CD146⁺ cells by in vivo transplantation into SCID/beige/CTX-treated mice (DAPI: nuclear stain). Scale bar = 350 μm. Human dermal fibroblasts were used as negative control. Scale bar = 280 μm.</p