The stem cell marker CD133 (Prominin-1) is expressed in various human glandular epithelia

Abstract

Human prominin-1 (CD133) is expressed by various stem and progenitor cells originating from diverse sources. In addition to stem cells, its mouse ortholog is expressed in a broad range of adult epithelial cells, where it is selectively concentrated in their apical domain. The lack of detection of prominin-1 in adult human epithelia might be explained, at least in part, by the specificity of the widely used AC133 antibody, which recognizes an epitope that seems dependent on glycosylation. Here we decided to re-examine its expression in adult human tissues, particularly in glandular epithelia, using a novel monoclonal antibody (80B258) generated against the human prominin-1 polypeptide. In examined tissues, we observed 80B258 immunoreactivity at the apical or apicolateral membranes of polarized cells. For instance, we found expression in secretory serous and mucous cells as well as intercalated ducts of the large salivary and lacrimal glands. In sweat glands including the gland of Moll, 80B258 immunoreactivity was found in the secretory (eccrine and apocrine glands) and duct (eccrine glands) portion. In the liver, 80B258 immunoreactivity was identified in the canals of Hering, bile ductules, and small interlobular bile ducts. In the uterus, we detected 80B258 immunoreactivity in endometrial and cervical glands. Together these data show that the overall expression of human prominin-1 is beyond the rare primitive cells, and it seems to be a general marker of apical or apicolateral membrane of glandular epithelia. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Figure 1

Generation and characterization of the monoclonal antibody 80B258 directed against human prominin-1 polypeptide. (A) Schematic representation of the glutathione S-transferase (GST)-αhE2 fusion protein containing part of the first extracellular loop of prominin-1 (residues Gly240 to Ser388) used as antigen to generate the MAb 80B258. Black boxes indicate transmembrane domains of prominin-1, whereas the signal peptide is in gray. The forks indicate the potential N-glycosylation sites. (B) 80B258 MAb recognizes the recombinant human prominin-1. Lysates of Chinese hamster ovary (CHO) cells transiently transfected with either human prominin-1 (CD133) or, as a control, vector DNA alone (MOCK) were incubated in absence (–) or presence (F) of peptide-N-glycosidase F (PNGase F) and analyzed by immunoblotting with either 80B258 or AC133 MAb. Arrow, endo-H-insensitive 120-kDa form of recombinant human prominin-1; asterisk, endo-H-sensitive 105-kDa form of recombinant human prominin-1; arrowhead, 92-kDa product after N-de glycosylation. (C) 80B258 MAb recognizes the authentic human prominin-1. Lysates of human hematopoietic stem and progenitor cells were analyzed by immunoblotting with either AC133 or 80B258 MAb or αhE2 antiserum. Arrow, 120-kDa form of human prominin-1. Right panels (Lanes 4 and 5) are a longer exposure of the same blots shown in left panels (Lanes 1 and 2, respectively). The position of prestained apparent molecular mass markers (in kDa) is indicated on the left (B,C).

Figure 2

Localization of prominin-1 in human salivary glands. Paraffin-embedded sections of submandibular (A–C), sublingual (D,E), and parotid (F) glands were immunolabeled with 80B258 MAb and counterstained with either light green (A–E) or hematoxylin-eosin (F). To unmask the 80B258 epitope, samples were either heated in the microwave (A,B,B′) or incubated with SDS solution (C–F) before labeling. (A–F) The 80B258 immunoreactivity was observed in the apical and the top part of lateral membranes of epithelial cells (black and white arrowheads, respectively) located in the serous acini (black dotted line) and serous demilunes (orange dotted line). The restricted lateral membranes of cells forming intercellular canaliculi were positive (yellow arrowheads). The apical localization of 80B258 immunoreactivity was observed in cells lining mucous tubuli (B,D,E, blue dotted line). The luminal surface of the intercalated ducts (black line) in submandibular and parotid glands (C,F1, blue arrowheads) and intralobular duct (black line) in sublingual gland (D,E, blue arrowheads) showed 80B258 immunoreactivity, whereas striated ducts were negative (A,F, red dotted line). The insets in A, C, and F demarcate regions shown at higher magnification in A1, C1, and F1, respectively. (B′) Negative control, i.e., without primary antibody. Bar = 50 μm.

Figure 3

Localization of prominin-1 in human sweat glands of axilla. Paraffin-embedded sections containing eccrine (A–E), apocrine (F–I), and apoeccrine-like (J) sweat glands were immunolabeled with 80B258 MAb and counterstained with either light green (all panels except B) or hematoxylin-eosin (B). For unmasking the 80B258 epitope, samples were either heated in the microwave (all panels except C) or incubated with SDS solution (C) before labeling. (A–E) The 80B258 immunoreactivity was detected at the luminal surface of cells (black arrowheads) located in the secretory portion (black dotted line) of eccrine sweat glands. The restricted lateral membranes of cells forming intercellular canaliculi (yellow arrowheads), the apical surface of the superficial duct cell layer (blue arrowheads), and secretion (white asterisks) found in the lumen of the duct portion (red dotted line) were positive. (F–I) The 80B258 immunoreactivity was detected at the luminal surface of cells (black arrowheads) and secretion (white asterisks) found in the lumen of the secretory portion of apocrine sweat glands. Note that 80B258 immunoreactivity was associated with tubular structures that are just before or under an extrusion phase of secretion (blue dotted line) and postsecretory or resting state (black dotted line). Some tubules were negative (H, white arrow). The inset in I demarcates a region displayed at higher magnification in I1. In cells in the process of secretion, i.e., those harboring an apical dome-like shape, 80B258 immunoreactivity was confined to the top part of the apical domain (red arrows). (A′,F′) Negative control, i.e., without primary antibody. Black asterisks indicate the absence of labeling in secreted materials (F′). (J,J1,J2) In apoeccrine-like sweat glands, 80B258 immunoreactivity (black arrowheads) was restricted to the cells exhibiting a straight apical plasma membrane (blue dotted line), whereas those with an apical dome-like shape (red dotted line) were negative. The insets in J demarcate two regions shown at higher magnification in J1 and J2. Cells surrounding intercellular canaliculi were positive (yellow arrowheads). Bar = 50 μm.

Figure 4

Localization of prominin-1 in the human lacrimal gland and the gland of Moll of eyelid. Paraffin-embedded sections containing the accessory lacrimal gland of Wolfring (A–D), the gland of Moll (E), and conjunctival epithelium (F) were immunolabeled with 80B258 MAb and counterstained with light green. For unmasking the 80B258 epitope, samples were either heated in the microwave (A,B) or incubated with SDS solution (C–F) before labeling. (A–D) The 80B258 immunoreactivity was observed at the apical and the top part of lateral membranes of serous secretory cells (black and white arrowheads, respectively), and at the widened endings of intercellular canaliculi (yellow arrowheads) of the lacrimal gland. The luminal surface of the intercalated ducts was labeled (blue arrowheads). (C′) Negative control, i.e., without primary antibody. (E,F) The 80B258 immunoreactivity was observed at the apical membrane (black arrowheads) of either columnar cells lining secretory tubule of the apocrine gland of Moll (E) or epithelial cells located at the superficial layer of conjunctiva (F). The inset in E demarcates a region displayed at higher magnification in E1. Bar = 50 μm.

Figure 5

Localization of prominin-1 in human normal endometrial and cervical glands. Paraffin-embedded sections of materials derived from bioptic samples were immunolabeled with 80B258 MAb and counterstained with light green. For unmasking the 80B258 epitope, samples were incubated with SDS solution before labeling. (A–C) The 80B258 immunoreactivity was observed at the luminal surface (black arrowheads) of epithelial cells lining endometrial glands that are located in pars spongiosa of the endometrium (A). Insets in B demarcate two regions shown at higher magnification in B1 and B2. Numerous motile cilia (blue arrowheads) and other smaller membrane structures (red arrowhead) were labeled. A tangential section shows the strong labeling of motile cilia (C, blue arrowheads). (D) 80B258 immunoreactivity was observed at the luminal surface of epithelial cells (black arrowheads) lining cervical glands outlined by a black dotted line. The insets in D and D1 demarcate regions shown at higher magnification in D1 and D2, respectively. (A′,B′,D1′) Negative control, i.e., without primary antibody. White arrowhead indicates immuno-negative cilia (B′). Bar = 50 μm.

Figure 6

Localization of prominin-1 in the human liver. Paraffin-embedded sections were immunolabeled with 80B258 MAb and counterstained with light green. For unmasking the 80B258 epitope, samples were either heated in the microwave (A,B) or incubated with SDS solution (C–H) before labeling. (A–F) 80B258 immunoreactivity was detected at the apical membrane of cells present in the canals of Hering (A,B, white arrowheads), intraportal ductules (A–E, blue arrowheads), and small interlobular bile ducts (F, blue arrowhead). (G,H) No 80B258 immunoreactivity was detected in larger bile ducts. (C′) Negative control, i.e., without primary antibody. h, hepatocytes; PS, portal space, outlined by a red dotted line. Bar = 50 μm.

Figure 7

Localization of prominin-1 in the human pancreas. Paraffin-embedded sections were immunolabeled with 80B258 MAb and counterstained with light green (A,B) or hematoxylin-eosin (C,D). For unmasking the 80B258 epitope, samples were incubated with SDS solution before labeling. (A–D) 80B258 immunoreactivity was detected at the luminal surface of cells located in intercalated ducts (blue arrowheads) and the apical membrane of centroacinar cells (black arrowheads) of the secretory portion outlined by a dotted line. (A′) Negative control, i.e., without primary antibody. Bar = 50 μm.