Ryan St Clair

CD133 expression is not restricted to stem cells, and both CD133+ and CD133– metastatic colon cancer cells initiate tumors

Colon cancer stem cells are believed to originate from a rare population of putative CD133+ intestinal stem cells. Recent publications suggest that a small subset of colon cancer cells expresses CD133, and that only these CD133+ cancer cells are capable of tumor initiation. However, the precise contribution of CD133+ tumor-initiating cells in mediating colon cancer metastasis remains unknown. Therefore, to temporally and spatially track the expression of CD133 in adult mice and during tumorigenesis, we generated a knockin lacZ reporter mouse (CD133lacZ/+), in which the expression of lacZ is driven by the endogenous CD133 promoters. Using this model and immunostaining, we discovered that CD133 expression in colon is not restricted to stem cells; on the contrary, CD133 is ubiquitously expressed on differentiated colonic epithelium in both adult mice and humans. Using Il10-/-CD133lacZ mice, in which chronic inflammation in colon leads to adenocarcinomas, we demonstrated that CD133 is expressed on a full gamut of colonic tumor cells, which express epithelial cell adhesion molecule (EpCAM). Similarly, CD133 is widely expressed by human primary colon cancer epithelial cells, whereas the CD133- population is composed mostly of stromal and inflammatory cells. Conversely, CD133 expression does not identify the entire population of epithelial and tumor-initiating cells in human metastatic colon cancer. Indeed, both CD133+ and CD133- metastatic tumor subpopulations formed colonospheres in in vitro cultures and were capable of long-term tumorigenesis in a NOD/SCID serial xenotransplantation model. Moreover, metastatic CD133- cells form more aggressive tumors and express typical phenotypic markers of cancer-initiating cells, including CD44 (CD44+CD24-), whereas the CD133+ fraction is composed of CD44lowCD24+ cells. Collectively, our data suggest that CD133 expression is not restricted to intestinal stem or cancer-initiating cells, and during the metastatic transition, CD133+ tumor cells might give rise to the more aggressive CD133(- )subset, which is also capable of tumor initiation in NOD/SCID mice.

Cytokine Preconditioning Promotes Codifferentiation of Human Fetal Liver CD133+ Stem Cells Into Angiomyogenic Tissue

Background—CD133 (AC133) is a surface antigen that defines a broad population of stem cells, including myogenic and endothelial progenitors. CD133+ cells are rare in adult tissues, and the factors that support their differentiation into mature angiomyogenic cells are not known. These hurdles have hampered the use of CD133+ cells for therapeutic purposes. Because human fetal liver is a rich source of CD133+ cells, we sought to identify the growth factors that promote codifferentiation of these cells into angiogenic and myogenic cells. Methods and Results—Human fetal liver CD133+ and CD133− cell subpopulations were cultured with 5′-azacytidine or vascular endothelial growth factor (VEGF165) and/or brain-derived nerve growth factor (BDNF). CD133+ but not CD133− cells from human fetal liver codifferentiated into spindle-shaped cells, as well as flat adherent multinucleated cells capable of spontaneous contractions in culture. The resulting spindle-shaped cells were confirmed to be endothelial cells by immunohistochemistry analysis for von Willebrand factor and by acetylated LDL uptake. Multinucleated cells were characterized as striated muscles by electron microscopy and immunohistochemistry analysis for myosin heavy chain. Presence of VEGF165 and BDNF significantly enhanced angiomyogenesis in vitro. Inoculation of cells derived from CD133+ cells, but not CD133− cells, into the ear pinna of NOD/SCID mice resulted in the formation of cardiomyocytes, as identified by immunostaining with cardiac troponin-T antibody. These cells generated electrical action potentials, detectable by ECG tracing. Conclusions—CD133 defines a population of human fetal liver cells capable of differentiating into both angiogenic and myogenic cells. Preconditioning of these CD133+ cells with VEGF165 and BDNF enhances the angiomyogenesis. CD133+ fetal liver cells ultimately may be used for therapeutic angiomyogenesis.

Morphology and molecular organization of the adult neuromuscular junction of Drosophila

While the larval neuromuscular junction (NMJ) of Drosophila has emerged as a model system to study synaptic function and development, little attention has been given to the study of the adult NMJ. Here we report an immunocytochemical and morphological characterization of an adult NMJ preparation of the prothorax. All muscles examined were innervated by small, uniform type II terminals (0.5–1.5 μm), a subset of which contained octopamine. Terminals classified as type I varied in their morphology across different muscles, ranging from strings or clusters of boutons (0.8–5.5 μm) to an elongate terminal (80–100 μm long) with few branches and contiguous swellings (3–15 μm) along its length. Analysis of the molecular composition of the NMJs during the first 5 days after eclosion revealed four major findings: 1) type I boutons increase in size during early adulthood; 2) Fasciclin II‐immunoreactivity is not detectable at type I terminals, while DLG‐immunoreactivity is observed at the synapse; 3) a Shaker‐GFP fusion protein that localizes to all type I boutons in the larva is differentially localized at adult prothoracic NMJs; and 4) while all type I terminals contain glutamate, the glutamate receptor subunits, DGluRIIA and DGluRIIB, are expressed and clustered in only a subset of muscles. These findings suggest that maturation of the adult NMJ occurs during early adulthood and that muscle‐specific properties may play a role in organizing synaptic components in the adult. Furthermore, these results demonstrate that there are major differences in the molecular organization of the adult and larval NMJs. J. Comp. Neurol. 468:596–613, 2004.

Corneal Nerve Structure and Function After Long-Term Wear of Fluid-Filled Scleral Lens

Purpose: The aim of this study was to determine whether long-term wear of a fluid-filled scleral lens alters basal tear production, corneal sensation, corneal nerve density, and corneal nerve morphology in 2 disease categories. Methods: Patients recruited from the Prosthetic Replacement of the Ocular Surface Ecosystem (PROSE) treatment program at the Weill Cornell Medical College were categorized into 2 groups: distorted corneas (DC) or ocular surface disease (OSD). We measured tear production, central corneal sensation, subbasal nerve density and tortuosity, and stromal nerve thickness before and after long-term wear of the prosthetic device used in PROSE treatment, defined as at least 60 days of wear for a minimum of 8 hours a day. Results: Twenty patients were included in the study. After long-term wear of the prosthetic device, tear production decreased in patients with DC (21.2 ± 8.5 to 10.4 ± 4.6 mm; P < 0.0001) but did not change in patients with OSD (7.5 ± 5.2 to 8.7 ± 7.2 mm; P = 0.71). Corneal sensation increased in the DC group (45.6 ± 9.2 to 55.0 ± 5.6 mm; P < 0.05). There was no significant change in sensation in patients with OSD (45.0 ± 8.7 to 49.1 ± 14.8 mm; P = 0.37). Subbasal nerve density, subbasal nerve tortuosity, and stromal nerve thickness remained unchanged in both DC and OSD groups after long-term wear (P > 0.05). Conclusions: Patients with DC had significantly reduced basal tear production and increased corneal sensation after long-term wear of the scleral lens, but patients with OSD did not show any changes in tear production or corneal sensation.