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Transcript
SUPPRESSION OF ASYMMETRIC
CELL KINETICS (“SACK”)
A general method for selective and
conditional expansion of tissue stem cells
from ASYMMETREX
Contact
James L. Sherley, M.D., Ph.D. Director
[email protected]
Tissue Stem Cells
1. Rare cells in mature tissues
2. Responsible for tissue renewal
3. Responsible for tissue repair
HUMAN CELL PRODUCTION NEED
Routine, reliable, large scale
production of renewable functional
human cells for cell therapy medicine
and
drug development (e.g., hepatocytes)
HUMAN CELL PRODUCTION NEED
Routine, reliable, large scale
production of renewable functional
human cells?
But “renewable functional human cells”
is a tissue cell physiology oxymoron!
The Cell Production Paradox
Generally,
mature functional human cells
have limited multiplication capacity,
i.e., they are unable to divide
for their own self-renewal.
Some Well Known Examples
of the
Cell Production Paradox…
Small Intestines:
Functional Transport Cells
Stop Multiplying
Replaced by Tissue Stem Cells
Potten and Morris,
1988
Non-Dividing
Mature
Differentiated
Cells
“Streaming”
Zajicek, JTB,1986
Potten et al., 2003
Dividing
Tissue Stem Cells
Blood Cell Production
Similar Cell Multiplication Architecture:Similar Problem
Terminal
Arrest
&
DIVIDING
TISSUE
STEM CELL
Death
LIMITED DIVISION
MATURE CELLS
Similar Process
In Hepatocyte
Production
Space of
Disse
Limited Division
Mature Hepatocytes
(Under
normal
conditions)
Portal
Triad
Portal Triad
Tissue Stem Cell
Division
*
Death
Zajicek et al., 1985; Arber et al., 1988; Benedetti et al., 1988
SOLUTION:
1) Mass produce
tissue stem cells
2) Use to produce
mature cells
Portal Triad
Tissue Stem Cell
Division
Space of
Disse
Limited Division
Mature Hepatocytes
Portal
Triad
*
Death
Zajicek et al., 1985; Arber et al., 1988; Benedetti et al., 1988
TISSUE STEM CELL
EXPANSION BARRIER IN CULTURE
Tissue Stem Cell
Functional Cell
Asymmetric Self-Renewal
= Asymmetric Cell Kinetics
The critical expansion barrier
in culture?
ASYMMETRIC CELL KINETICS
time
Linear Cell Production Kinetics
ASYMMETRIC
CELL KINETICS
The critical ASC expansion barrier
in culture:
ASYMMETRIC CELL KINETICS
time
ASYMMETRIC
CELL KINETICS
With time,
stem cells are
lost due to dilution by
the non-dividing differentiating cells they produce.
TECHNOLOGICAL SOLUTION:
SUPPRESSION OF
ASYMMETRIC CELL KINETICS
time
Exponential
Cell Kinetics!
SYMMETRIC
CELL KINETICS
(Ideally, produce
only tissue
stem cells)
“SACK” TECHNOLOGY
and…REVERSIBLE
SUPPRESSION OF
ASYMMETRIC CELL KINETICS
time
ASYMMETRIC
CELL KINETICS
Kinetically “suppressed”
expanded tissue stem cells
retain the ability to revert back
to asymmetric production of mature cells in vivo or in vitro.
CURRENT SACK PROGRESS
Species
Rat
Mouse
Human
SACK Clonal
Cell Type
Effect Strains TSCs
hepatocyte
√
√
√
bile epithelial
√
√
√
lung
?
√
inc.
skeletal muscle
√
√
inc.
hair follicle
√
√
√
pancreas
√
√
√
hepatic
√
√
√
skin
√
√
inc.
pancreas
√
[√]
√
kidney
√
n/a
n/a
hematopoietic
√
√
(TSCs = confirmed tissue stem cells expanded; inc., incomplete studies; n/a, not applicable)
References
Background Publications
Lee et al. (2003) Biotech. & Bioeng. 83, 760-771
– SACK expansion, rat liver
Paré and Sherley (2006) Curr. Topics Devel. Biol. 73, 141-171
– SACK principles
Huh et al. (2011) Sci. Rep. 1, 175; DOI: 10.1038/srep00176
– Mouse hair follicle stem cells
Paré and Sherley (2013) J. Stem Cell Res. & Therapy 149.
DOI:104172/2157-7633.1000149
– Human pancreatic stem cells
Patents
2010
2010
2010
2013
Methods for Ex Vivo Propagation of Adult Hepatic Stem Cells -US 7824912
Methods for Ex Vivo Propagation of Somatic Hair Follicle Stem Cells -US 7655465
Hepatocyte Precursor Cell Lines -US 7645610
Hepatocyte Precursor Cell Lines -US 8404481
Pending Patents
2014
Methods for Producing Human Pancreatic Islet Precursor Cells and Uses Thereof
-PCT/US2012/042644
THE SACK TECHNOLOGY
How does it work?
Biochemical Control of Asymmetric Cell Kinetics
IMPDH II gene
AAAAAA mRNA
p53
The p53 cancer gene regulates
tissue stem cell kinetics state via
guanine ribonucleotide (rGNPs)
biosynthesis. IMPDH is the ratelimiting enzyme for this pathway.
p53 expression reduces rGNPs,
promoting
asymmetric cell
kinetics by tissue
stem cells.
IMP
IMPDH
Symmetric Cell Kinetics
Liu et al., 1998, Mol. Biol.
Cell 9, 15-28.
Rambhatla et al., 2001, J.
Biomed. & Biotech. 1, 28-37.
XMP
GMP
Decreased
rGNPS
Asymmetric Cell Kinetics
Purine Salvage Control of Asymmetric Cell Kinetics
IMPDH II gene
AAAAAA mRNA
p53
IMP
Hypoxanthine (Hx)
Hx, Xs, and Xn are natural, non-toxic
rGNP precursors that can be used to
expand rGNP pools and bypass p53
XMP regulation of IMPDH to shift tissue
stem cells from asymmetric cell kinetics
to symmetric cell kinetics = “SACK
GMP
AGENTS”
IMPDH
Xanthosine (Xs),
Xanthine (Xn)
INCREASED
rGNPS
Symmetric Cell Kinetics
Asymmetric Cell Kinetics
Reversible Control of Asymmetric Cell Kinetics
IMPDH II gene
AAAAAA mRNA
p53
IMP
When SACK agents are no longer
supplemented in the culture medium,
expanded stem cells revert back to
XMP asymmetric cell kinetics with production
of mature cells, thereby achieving
renewable functional human cells.
GMP
IMPDH
…and reversible.
Decreased
rGNPS
Symmetric Cell Kinetics
Asymmetric Cell Kinetics
SACK Strategy for Renewable
Production of Mature Human Cells
Suppress
Asymmetric
Cell Kinetics
Asymmetric
Cell Kinetics
(initial
tissue cell
preparations)
Differentiation
Conditions
SACK removal
Increased Symmetric
Asymmetric
Stem Cell Kinetics
Cell Kinetics
For
With
Mass Production
Production
Of Tissue Stem Cells
Of Mature Cells
- RENEWABLE In vivo or in vitro
SUMMARY AND CONCLUSIONS
I.Asymmetrex’s patented SACK Technology provides a general
method for expanding human tissue stem cells that can be
developed as a renewable source of cells for cell therapy, drug
development, and cell research.
II.The SACK Technology can be used to produce renewable
tissue stem cell strains from many different species (e.g., rat,
mouse, human) and tissue types (e.g., liver, lung, kidney,
hematopoietic, skin, pancreas, hair follicle)
III.SACK expanded tissue stem cells can be clonally derived
and stably genetically engineered.