Biomarker Discovery
and Development
Colon Cancer
(CC) Biomarkers
Supporting
Analysis Data
I.
Isolation,
culturing,
and demonstration
of stem
cell
characteristics
of cells
isolated
from
adult
human
gi tissues.
A. Stem
Cell Marker Expression of Cultured Stem
Cells.
B.
Demonstration of Cultured Stem Cells' Retention of Their
Self-Renewal Capability During Culturing.
C.
Demonstration of Cultured Stem Cells’ Pluripotency During Culturing.
The research performed at
AlfaGene
is greatly increasing our understanding of both adult stem cells and
cancer stem cells, identifying biosignature/biomarker and functional
changes in CRC, and providing novel basic information regarding many
cellular processes, including their role in both normal intestinal
epithelial cell biology and the pathogenesis of CRC.
Identified potential biomarkers will provide both therapeutic
targets and therapeutic treatment efficacy markers for the treatment
of CRC and other cancers in other tissues.
Furthermore, the cell lines
and differential expression data produced will provide an incredibly
important and long sought after resource for both academic and
pharmaceutical industry groups.
I. Isolation,
culturing,
and demonstration
of stem
cell
characteristics
of cells
isolated
from
adult
human
gi tissues.
AlfaGene
is able to, not only, isolate and culture, adult stem cells from GI
tissues, but also, derive differentiated epithelial cells from these
cultured stem cells. Stem
cells from the esophagus, stomach, duodenum, jejunum, ileum, cecum,
ascending colon, transverse colon, sigmoid, and rectum of single
adult human were isolated and cultured.
The classification of these cells as stem cells was verified
via demonstration of three aspects of their nature:
expression of known stem cell markers (I.A.),
self-renewal (I.B.), and
pluripotency (I.C.).
A. Stem
Cell Marker Expression of Cultured Stem
Cells.
RT-PCR, Western, and IH analysis demonstrated that the isolated and
cultured cells after three passages were positive for stem cell
makers Nanog, LIN28, Oct4, and SOX2, as well as, the suspected
intestinal stem cell markers Lgr5 and Bmi-1 (Figure 1).
B.
Demonstration of Cultured Stem Cells' Retention of Their
Self-Renewal Capability During Culturing
A small intestine stem cell line cultured for 20 passages,
undergoing approximately 40 cellular divisions, still continued to
express stem cell markers
(Figure 2).
C.
Demonstration of Cultured Stem Cells’ Pluripotency During Culturing.
The human adult stem cell lines established by AlfaGene continue,
during culturing, to
differentiate into the four epithelial cell lines
present in the intestinal tract: enterocyte, Goblet, enteroendocrine,
and Paneth.
Therefore, we examined the mRNA expression of molecular markers that
are specific for each of these four epithelial lineages in our newly
developed cell system. The markers used to identify columnar
epithelial cells include intestinal alkaline phosphatase (ALP1) and
sucrase isomaltase (SI). The markers used to identify Goblet cells
include mucin-2 (MUC2) and trefoil factor 3 (TFF3). Mucin-2, a
secreted gel-forming mucin, is the major gel-forming mucin secreted
by Goblet cells of the small and large intestines and is the main
structural component of the mucus gel, whereas, intestinal trefoil
factor 3 is a nonmucin protein and a product of fully differentiated
goblet cells. Chromogranin A (CHGA) is utilized as a marker to
identify enteroendocrine chromaffin cells. The markers used to
identify Paneth cells include lysozyme (LYZ) and defensin (DEFA5).
The data shown below, clearly demonstrate that the isolated cells
are truly pluripotent and have the ability to become, at a minimum,
any of these four types of epithelial cells found lining the luminal
wall of the small intestine.
Demonstration of cultured stem cells’ pluripotency by their ability,
over time in culture, to differentiate into the epithelial cell
types present in the intestinal epithelium
in vivo.
