|
Background:
RSPO1 is the critical ingredient used in the maintenance and proliferation of mouse and human organoid progenitor stem cells.
To date, positive results have been reported with stem cells of gastric, colonic, intestinal, pancreatic and liver lineage by
thought leaders in the industry such as Dr. Hans Clevers (Hubrecht Institute) and Dr. Calvin Kuo (Stanford University).
These cells can be passaged indefinitely and can produce unlimited amount of RSPO1 protein, allowing laboratories conducting
research with organoids to save time and money. The RSPO1 cells are authenticated and tested to be free from mouse or human
pathogenic agents. Greater than 90% of drugs entering clinical trials fail. Often compounds that look promising after in vitro
screening fail after in vivo analysis. The unreliability of in vitro assays to predict in vivo outcomes is a major contributor
to the increasing expense of the discovery process. It is estimated that the average cost to bring a drug to market is now
five billion dollars (Forbes, 2014). Thought leaders postulate that 3D organoid culture models more closely emulate in
vivo conditions, an advance over 2-D culture for cancer research, drug screening, toxicology studies, regenerative medicine
and host/disease pathology research. By introducing the first commercially available HA-R-Spondin-Fc293T cell line to the
market, we are making a positive step toward the adoption of 3D culture in drug screening and other cell based research applications.
Roof plate-specific Spondin-1 (R-Spondin 1 or RSPO1), also known as CRISTIN3, is a 27 kDa secreted activator protein that
belongs to the R-Spondin family. R-Spondins positively regulate Wnt/beta-catenin signaling, most likely by acting as a ligand
for LGR4-6 receptors and an inhibitor for ZNRF3. R-Spondin-1 induces proliferation of intestinal crypt epithelial cells,
increases intestinal epithelial healing, and supports intestinal epithelial stem cell renewal [1-5].
The 293T cell line is stably transfected to express murine Rspo1 with an N-terminal HA epitope tag and fused to a C-terminal
murine IgG2a Fc fragment. This cell line is used to produce either purified Rspo1 or Rspo1 conditioned media. The murine
Rspo1 protein has been used extensively in
organoid culture
to maintain Lgr5+ stem cells, and the FC and HA tags make it easy to purify or characterize.
Figure 1. Production of R-Spondin1 for organoid culture - The 293T cell line
is stably transfected to express murine Rspo1 with an N-terminal HA epitope tag and fused to a C-terminal murine IgG2a
Fc fragment. A) The HA-R-Spondin1-Fc 293T cell line is cultured with zeocin to select for stably transfected cells.
B) Production of HA-R-Spondin1-Fc is characterized using Western Blot for R-Spondin1 protein (arrow).
C) HA-R-Spondin1-Fc induces activation of Wnt/β-catenin response when evaluated using the Top-Flash Luciferase assay.
References:
-
Ootani, A., et al. 2009
Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche,Nat Med, 15(6): p.701-706
-
Barker, N., et al. 2010
Lgr5+ve Stem Cells Drive Self-Renewal in the Stomach and Build Long-Lived Gastric Units In Vitro. Cell Stem Cell, 6(1): p.25-36
-
Sato, T., et al., (2009)
Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche,Nature, 459(7244): p.262-265
-
Sato, T. and H. Clevers, (2013)
Growing Self-Organizing Mini-Guts from a Single Intestinal Stem Cell: Mechanism and Applications, Science, 340(6137): p.1190-1194
-
Jung, P., et al, (2011)
Isolation and in vitro expansion of human colonic stem cells, Nat Med, 17(10): p.1225-7
|