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.