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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
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