1. 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

2. Tissue Stem Cells
Rare cells in mature tissues
Responsible for tissue renewal
Responsible for tissue repair

3. HUMAN CELL PRODUCTION NEED Routine, reliable, large scale
production of renewable functional
human cells for cell therapy medicine
and
drug development (e.g., hepatocytes)

4. 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!

5. 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.

6. Some Well Known Examples of the Cell Production Paradox…

7. Functional Transport Cells Stop Multiplying
Dividing
Tissue Stem Cells
Potten and Morris,
1988
“Streaming”
Zajicek, JTB,1986
Non-Dividing
Mature
Differentiated
Cells
Small Intestines:
Functional Transport Cells
Stop Multiplying
Replaced by Tissue Stem Cells
Potten et al., 2003

8. Similar Cell Multiplication Architecture:Similar Problem
Blood Cell Production
Similar Cell Multiplication Architecture:Similar Problem
Terminal
Arrest
&
Death
DIVIDING
TISSUE
STEM CELL
LIMITED DIVISION
MATURE CELLS

9. * Similar Process In Hepatocyte Production Portal Triad
Space of
Disse *
Limited Division
Mature Hepatocytes
(Under
normal
conditions)
Portal Triad
Tissue Stem Cell
Division
Death
Zajicek et al., 1985; Arber et al., 1988; Benedetti et al., 1988

10. * SOLUTION: 1) Mass produce tissue stem cells 2) Use to produce
mature cells
Portal
Triad
Space of
Disse *
Limited Division
Mature Hepatocytes
Portal Triad
Tissue Stem Cell
Division
Death
Zajicek et al., 1985; Arber et al., 1988; Benedetti et al., 1988

11. EXPANSION BARRIER IN CULTURE
TISSUE STEM CELL
EXPANSION BARRIER IN CULTURE
Tissue Stem Cell
Functional Cell
Asymmetric Self-Renewal
= Asymmetric Cell Kinetics

12. The critical expansion barrier in culture? ASYMMETRIC CELL KINETICS
time
Linear Cell Production Kinetics
ASYMMETRIC
CELL KINETICS

13. 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.

14. TECHNOLOGICAL SOLUTION: SUPPRESSION OF ASYMMETRIC CELL KINETICS
time
Exponential
Cell Kinetics!
SYMMETRIC
CELL KINETICS
(Ideally, produce
only tissue
stem cells)
“SACK” TECHNOLOGY

15. ASYMMETRIC CELL KINETICS
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.

16. CURRENT SACK PROGRESS SACK Clonal
Species Cell Type Effect Strains TSCs
Rat hepatocyte √ √ √
bile epithelial √ √ √
lung ? √ inc.
Mouse skeletal muscle √ √ inc.
hair follicle √ √ √
pancreas √ √ √
Human hepatic √ √ √
skin √ √ inc.
pancreas √ [√] √
kidney √ n/a n/a
hematopoietic √ √
(TSCs = confirmed tissue stem cells expanded; inc., incomplete studies; n/a, not applicable)

17. References Background Publications
Lee et al. (2003) Biotech. & Bioeng. 83,
– SACK expansion, rat liver
Paré and Sherley (2006) Curr. Topics Devel. Biol. 73,
– SACK principles
Huh et al. (2011) Sci. Rep. 1, 175; DOI: /srep00176
– Mouse hair follicle stem cells
Paré and Sherley (2013) J. Stem Cell Res. & Therapy 149.
DOI:104172/
– Human pancreatic stem cells
Patents
2010    Methods for Ex Vivo Propagation of Adult Hepatic Stem Cells -US
2010    Methods for Ex Vivo Propagation of Somatic Hair Follicle Stem Cells -US
2010    Hepatocyte Precursor Cell Lines -US
2013    Hepatocyte Precursor Cell Lines -US
Pending Patents
2014    Methods for Producing Human Pancreatic Islet Precursor Cells and Uses Thereof
-PCT/US2012/042644

18. THE SACK TECHNOLOGY
How does it work?

19. Biochemical Control of Asymmetric Cell Kinetics
IMPDH II gene
p53
AAAAAA
mRNA
IMP
IMPDH
The p53 cancer gene regulates
tissue stem cell kinetics state via
guanine ribonucleotide (rGNPs)
biosynthesis. IMPDH is the rate-
limiting enzyme for this pathway.
p53 expression reduces rGNPs,
promoting
asymmetric cell
kinetics by tissue
stem cells.
Liu et al., 1998, Mol. Biol.
Cell 9,
Rambhatla et al., 2001, J.
Biomed. & Biotech. 1,
XMP
GMP
Decreased
rGNPS
Symmetric Cell Kinetics
Asymmetric Cell Kinetics

20. Purine Salvage Control of Asymmetric Cell Kinetics
IMPDH II gene
p53
AAAAAA
mRNA
Hypoxanthine (Hx)
IMP
IMPDH
Hx, Xs, and Xn are natural, non-toxic
rGNP precursors that can be used to
expand rGNP pools and bypass p53
regulation of IMPDH to shift tissue
stem cells from asymmetric cell kinetics
to symmetric cell kinetics = “SACK
AGENTS”
Xanthosine (Xs),
Xanthine (Xn)
XMP
GMP
INCREASED
rGNPS
Symmetric Cell Kinetics
Asymmetric Cell Kinetics

21. Reversible Control of Asymmetric Cell Kinetics
IMPDH II gene
p53
AAAAAA
mRNA
IMP
IMPDH
When SACK agents are no longer
supplemented in the culture medium, expanded stem cells revert back to asymmetric cell kinetics with production
of mature cells, thereby achieving
renewable functional human cells.
…and reversible.
XMP
GMP
Decreased
rGNPS
Symmetric Cell Kinetics
Asymmetric Cell Kinetics

22. SACK Strategy for Renewable Production of Mature Human Cells
Suppress
Asymmetric
Cell Kinetics
Differentiation
Conditions
SACK removal
Asymmetric
Cell Kinetics
(initial
tissue cell
preparations)
Increased Symmetric
Stem Cell Kinetics
For
Mass Production
Of Tissue Stem Cells
- RENEWABLE -
Asymmetric
Cell Kinetics
With
Production
Of Mature Cells
In vivo or in vitro

23. SUMMARY AND CONCLUSIONS
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.
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)
SACK expanded tissue stem cells can be clonally derived
and stably genetically engineered.