Deriving Mesenchymal Stem Cells from Human Amniotic Fluid – Potential for an Allogeneic Cellular Therapy Product

Institute for Regenerative Medicine
Deriving Mesenchymal Stem Cells from Human
Amniotic Fluid – Potential for an Allogeneic Cellular
Therapy Product
Julie G. Allickson, PhD, MS, MT(ASCP), Director, Translational Research

Wake Forest Institute for Regenerative Medicine
Wake Forest Institute for Regenerative
Medicine
• The Wake Forest Institute for
Regenerative Medicine (WFIRM) is a
leader in translating scientific
discovery into clinical therapies.
• The interdisciplinary team is working
to engineer more than 30 different
replacement tissues and organs.

Wake Forest Institute for Regenerative
Medicine
Mission: Improve patient’s lives by developing
regenerative medicine therapies
and support technologies
Institute Director: Dr. Anthony Atala
Team: more than 300 faculty and staff
World’s First Laboratory-Engineered Organ: Institute researchers were
the first in the world to engineer an organ in the lab that was
successfully implanted into patients.

“FIRSTS” in Regenerative Medicine
Led a team of researchers that was the first in the world to successfully
engineer urine tubes (urethras) in the laboratory and implant them in patients.
(2011: reported long-term results; 2004: first implantation)
First team in the world to engineer functional experimental solid organs
(miniature livers and penile erectile tissue) using a strategy of recycling donor
organs, with potential applications to other organs, including the kidney and
pancreas. (2010)
Selected to co-lead the Armed Forces Institute of Regenerative Medicine, an
$85 million, federally funded project to apply the science of regenerative medicine
to battlefield injuries. (2008)
Identified and characterized a new class of stem cells derived from amniotic
fluid and placenta, which show promise for the treatment of many diseases.
These amnion stem cells have been proven to differentiate into many tissue
types, including blood vessel, bone, liver and muscle. (2007)
First team in the world to create a laboratory-grown organ -- engineered
bladder tissue that was successfully implanted in patients. (2006: reported
long-term results; 1998: first implantation.)
Founder of the Regenerative Medicine Foundation, a non-profit created to
enable the advancement of new treatments and therapies based on regenerative
medicine, and ultimately, to realize the goals of personalized medicine. (2005)
First team in the world to create a functional solid organ experimentally, a
miniature kidney that secretes urine. (2003) World’s First Laboratory-Engineered
Organ Institute researchers were the first in the world to engineer an organ in
the lab that was successfully implanted into patients.
First team in the world to engineer functional blood vessels that were
implanted pre-clinically and survived long term. (2001)

ES Cells
Stem cells are present throughout
development and postnatal life
Fertilized egg 3 days 5-7 days 6 weeks
‘Adult’ Stem Cells
18 weeks

Cell sources before or at birth
Tissues & fluids support
the developing embryo and
fetus during pregnancy
Available for non-invasive
sampling or recovery at
term
Samples:
Amniotic fluid
Chorionic villi
Placenta
Umbilical cord

Amniotic fluid sampling
Week 14-16
of gestation
Cell retrieval:
amniocentesis is easy
and currently already
used for prenatal
diagnosis

Amniotic Fluid Stem (AFS) Cell Technology
Selection of stem
cells (~ 1%)
Routine
culture
Genetic
testing
Therapeutic
applications
Amniocentesis
Differentiation

Amniotic fluid-derived stem (AFS) cells
AFS cells
 Fresh AF or back-up
cytogenetics lab culture
 Select c-Kitpos (CD117) cells
 Establish clonal and cell lines
De Coppi, P. et al. (2007). Isolation of amniotic stem cell lines with potential for therapy. Nat
Biotechnol.

AFS cells maintain normal karyotype and
long telomeres
Telomere length
1. Control – short
2. Control – long
3. AFS ~20
doublings
4. AFS ~250
doublings
DNA Content
Normal diploid DNA
content
Normal cell cycle
checkpoints
Karyotype
Normal G-banding
pattern
Y chromosome proves
fetal origin

Multilineage differentiation of verified hAFS
cell clone
1 2 3 4 5 6 7 8
Osteogenic (3)
U D
mrf4
desmin
myoD
Myogenic (4)
U D
pparγ2
LP
Adipogenic (5)
U D
VCAM
CD31
Endothelial (6)
U D
albumin
Hepatic (7)
U D
nestin
Neurogenic (8)
U D
osteocalcin
AP
runx2
Proviral junction
DNA fragment

Marker profile of human AFS cells
Relativecellnumber
1 2 3 4
41 2 3
1 2 3 4
Oct3/4
41 2 3
41 2 3
Log fluorescence intensity
SSEA-3 SSEA-4
1 2 3 4
Tra-1-81
41 2 3
Tra-1-60
41 2 3
CD29
41 2 3
CD44 CD73
41 2 3
CD90
41 2 3
CD105
41 2 3
CD45
41 2 3
CD34 CD133
41 2 3
HLA-
ABC
Negative:
SSEA-1, SSEA-3,
Tra-1-81, Tra-1-60
[some weak +]
CD4, CD34, CD45,
CD133
HLA-DR (MHC
Class II)

Mesenchymal lineages from AFS cells
 Skeletal/cardiac
muscle
 Bone / cartilage
 Adipose
UndifferentiatedDifferentiated
Mineralization

Properties of AFS cells (summary)
Readily isolated from amniotic fluid & cytogenetics lab
cultures by immunoselection for c-Kit (CD117)
Clonal or cell lines obtained routinely
Extensive culture without apparent senescence
Some lines > 250 population doublings
Doubling time ca 36 hrs
Normal karyotype, long telomeres
Non-tumorigenic in SCID/beige mice

1. First paper to describe the presence of cells with a
hematopoietic potential in murine and human AF.
2. Cells expressing surface markers and genes typically associated
with hematopoietic potential and were able to differentiate all
along the hematopoietic pathway.
3. Hematopoietic differentiation results obtained with murine
AFKL cells were similar to those seen with c-Kit+Lin- cells from
the site of fetal hematopoiesis .
4. Under appropriate differentiation conditions, murine and
human KL cells were able to generate all the blood lineages (ie,
myeloid and erythroid colonies), as well as mixed CFU-GEMM
and B, NK, and T lymphocytes.
Summary

Figure 1. The effect of IFN-γ on the immunophenotype of AFS cells and BM-MSCs.
Moorefield EC, McKee EE, Solchaga L, Orlando G, et al. (2011) Cloned, CD117 Selected Human Amniotic Fluid Stem Cells Are Capable of
Modulating the Immune Response. PLoS ONE 6(10): e26535. doi:10.1371/journal.pone.0026535
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026535

Figure 2. Human AFS cells inhibit lymphocyte activation in a dose dependent manner similar to
that of BM-MSCs.

Figure 3. AFS mediated immunosuppression does not require cell-cell contact.

Figure 4. Soluble factors released from AFS cells and BM-MSCs in response to activation.

Bone differentiation of AFS cells
Mineralized calcium
In culture
Implantation of inkjet-printed construct
(8 wks)
µCT scan (18 weeks)
AFS cells + scaffoldScaffold alone

Project 3: manufacturing process of AFS cells for
clinical study in subjects with diabetes
Project 2: Assess AFS cell-mediated control of
blood sugar in mice and non human primates
with diabetes
Development of Amniotic Fluid Stem Cell
Therapy for Individuals With Type 1 Diabetes
Project 1: In vitro differentiation of AFS cells to beta cells
23

A. Peister and R. Guldberg
Bone tissue engineering
In vitro
In vivo

Chromogenic in situ hybridization of
injected amniotic fluid stem cells,
integration of stem cells into the cultured
developing kidneys
L. Perin, S. Giuliani, D. Jin, S. Sedrakyan, G. Carraro, R. Habibian, D. Warburton, A. Atala and R. E. De Filippo
Cell Proliferation Vol. 40, 6 Pages: 936-948 2007
Structural differentiation of amniotic
fluid stem cells within developing
embryonic kidneys demonstrating
integration of stem cells
Injection of hAFS cells into neonatal
mouse kidney

Key Questions
• Clinical utility of mesenchymal SC from
amniotic fluid vs adult (e.g., bone marrow,
adipose tissue).
• Developmental origin(s) of broadly
multipotent / pluripotent cells found in
amniotic fluid and Full differentiation
potential of stem cells from birth-related
sources vs “adult” and ES cells
• Best banking / production strategies for
regenerative medicine

Where we stand
New stem cell-based products are reaching
the clinic
Great hopes for the future
BUT
Development is still at an early stage, POC
moving to Definitive studies
Safety must be paramount
There will be strength in unity
Critical thinking
Open minds
Understand the biology

Wake Forest Institute for
Regenerative Medicine
Special thanks to Dr. Shay Soker for Slides

This work was made possible, in part, by grants from the following institutions:
NIH: NIDDK
NIH: HLI
Department of Defense (AFIRM, OTRP)
Department of Energy
National Kidney Foundation
Muscular Dystrophy Association
The Crown Foundation
The Frase Foundation
The Nakos Foundation
JDRF
Musculoskeletal Transplant Foundation
Tengion, Inc
Plureon
Stovall, Inc
AugmentRx

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