Chapter 1. Introduction and Background
1.3 CD133
1.3.5 Expression of CD133 in tissues
Human CD133 messenger RNA (mRNA) transcripts are detected in many cell lines and in most tissues apart from mature peripheral blood leukocytes171. However, the human AC133 epitope, used initially to discover CD133, is restricted to stem- and progenitor cells that include endothelial progenitor cells193, hematopoietic stem cells165, foetal brain stem cells194, embryonic epithelium167,168, prostatic epithelial stem cells195, myogenic cells196 and are found in certain cancers such as retinoblastoma166,197, teratocarcinoma166 and leukaemia198. This discrepancy is again confirmed in mice where the murine counterpart of AC133, 13A4, detects approximately the same epitope in mouse CD133. Using this antibody, CD133 was detected in tubular epithelium of the kidney and in the ependymal epithelium in the brain167,199 whereas CD133 mRNA is more widespread as is evident from the analysis of β-galactosidase (LacZ) reporter gene expression under the control of CD133 promoter in mutant mice94,171. This difference in mRNA and protein detection of mammalian CD133 could be explained by tissue-specific glycosylation, as AC133 antibody recognizes only a specific form of glycosylated CD133165. In contrast, it is known that 13A4 recognises both unglycosylated as well as glycosylated CD133167. Moreover, AC133 and 13A4 antibody may selectively bind to a unique alternatively spliced isoform of CD133. Together, these findings explain the earlier observations of CD133 expression restricted to stem and progenitor cells.
Haematopoietic stem cells
Perhaps one of the best-documented areas of CD133 expression has been in haematopoietic stem cells. Haematopoietic stem cells are the progenitors to all different cell types from the lymphoid lineages (NK-cells, B-cells, T-cells) to the
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myeloid lineages (macrophages and monocytes, megakaryocytes/platelets, neutrophils, basophils, eosinophils, dendritic cells, erythrocytes)200.
CD133 is known to mark a pluripotent population of haematopoietic stem cells derived from different sources such as human bone marrow, foetal liver and peripheral blood201. Yin et al. have shown that the receptor is downregulated to undetectable levels as the haematopoietic stem cell matures165.
Approximately 0.52% of bone marrow and 0.16% of cord blood mononuclear cells express CD133202. CD133 is expressed on 83% of cord blood CD34+ cells and up to 70% in bone marrow CD34+ cells165. Analysis showed that CD133 is undetectable on committed precursors, such as red blood cells, granulocytes and pre-B-cells, and that populations of CD133+ cells consist mainly of long-term culture initiating cells, the most primitive human haematopoietic cells which can be assayed in vitro203. CD133+ cells appear to be precursors to CD34+ cells as Gallacher showed that CD133+ cells were the only subset of a CD34-CD38- lineage population from human umbilical blood that could generate CD34+ cells in vitro with a 400 fold increase of engraftment capability in NOD/SCID (Non- obese diabetic/severe combined immunodeficiency) mice than that observed in the CD133- subset202.This was partially confirmed by Summers’ work which produced CD34+ cells from CD133+ CD34- cells in culture204. As the cells differentiate, they first begin to express markers such as CD38 and finally express differentiation markers such as CD13 and CD33.
In vivo applications of CD133+ stem cells in tissue regeneration have already been shown to be beneficial. Umbilical cord blood or bone marrow-derived CD133+ cells have been applied to myocardial tissue regeneration after infarction. Two reports showed that CD133+ cells improved myocardium recovery through
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an increase in angiogenesis and prevention of scar thinning and diastolic dilation205,206.
Other stem cells
Within human tissue, CD133 expression is widespread and not solely restricted to haematopoietic stem or progenitor cells. CD133+ cells isolated from foetal liver, umbilical cord blood, bone marrow and mobilized blood were capable of in vitro differentiation to neuronal cells and astrocytes, oligodendrocytes and glial cells207. CD133+ cells from peripheral blood markedly improved angiogenesis, astrogliosis, axon growth and functional recovery in a mouse spinal cord injury model compared to CD133- cells208.
Similarly, CD133+ cells isolated from the human foetal brain or skin tissues were capable of forming neurospheres in vitro and to differentiate into neurons and glia194. Moreover, when human CD133+ neurosphere cells were transplanted into neonatal NOD-SCID mice, they proliferated, migrated and differentiated into fully integrated neurons and glial cells209.
Grafting of integrin α2β1hi/CD133+ prostate basal cells in immunodeficient mice are capable of forming fully differentiated acini structures with evidence of both secretory and squamous cells present whereas the CD133- population leads to the formation of fibromuscular stroma with a complete absence of functional glands195.
CD133+ cells from the stroma of human cornea have the capacity to proliferate in vitro; colonies derived from CD133+ cells could be differentiated into fibroblastic cells, indicating that CD133+ cells represent stem cell of the corneal stroma210.
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Initial reports of CD133 expression in the kidney showed that its presence is restricted to the interstitium and that they lack the haematopoietic stem cell markers CD34 and CD45 but do express Paired Box 2 (Pax2) indicating their renal origin211. Interestingly, these cells are capable of differentiating into endothelial and epithelial cells expressing markers of both distal and proximal epithelium211.
Furthermore, stem cell therapy involving CD133+ cells derived from bone marrow have been used in clinical trials for patients recovering from cardiac arrest212, hepatic cirrhosis213 and chronic total occlusion and ischemia214. As of February 2017, 17 clinical trials are ongoing using CD133+ cells in regenerative medicine215. It is clear from the highlighted cases that CD133+ cells possess tremendous benefits for tissue engineering but more research is required to understand the role of CD133 in these processes.
CD133 in differentiated cells
Despite the focus of most research on CD133 in stem and progenitor cells, there is considerable evidence of CD133 expression in terminally differentiated cells of glandular organs such as the salivary glands, pancreas, mammary glands and liver as well as in kidneys216–218. This comes as no surprise as CD133 mRNA transcripts, unlike the AC133 epitope, have been observed in Caco-2 cells that underwent enterocytic differentiation219. The lack of CD133 protein detection using the AC133 antibody is partly due to its dependence on the glycosylation status and could be circumvented with the rabbit antiserum αhE2 targeting amino acid residues Gly240-Ser388. Using this antiserum, CD133 has been detected in the parietal layer of Bowman’s capsule of nephrons present in the juxtamedullary region of the cortex similar to 13A4 epitope detection in the adult mouse
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kidney167,216. Immunoreactivity of αhE2 but not AC133 has also been detected on the apical side of lactiferous ductal epithelia in the mammary glands corresponding to high CD133 mRNA transcripts216.
Immervoll and Corbeil both demonstrated for the first time that the human AC133 epitope, previously thought to be inversely correlated with differentiation, can also be detected in differentiated cells, namely at the differentiated apical/endoluminal face of ductal pancreatic epithelia with increasing expression towards the acini where CD133 staining was more cytoplasmic as opposed to the usual membrane detection. Although these cells express the pancreatic differentiation marker Carbonic anhydrase II it is believed that these cells positive for the AC133 epitope could be in fact dedifferentiated or transdifferentiated cells. However, given that approximately 30% of the ductal cells are positive for AC133, it is clear that this epitope is not a marker for stem and progenitor cells only.