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
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.
Wake Forest Institute for Regenerative Medicine
“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)
Wake Forest Institute for Regenerative Medicine
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
Wake Forest Institute for Regenerative Medicine
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
Wake Forest Institute for Regenerative Medicine
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
Wake Forest Institute for Regenerative Medicine
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.
Wake Forest Institute for Regenerative Medicine
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
Wake Forest Institute for Regenerative Medicine
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
Wake Forest Institute for Regenerative Medicine
Marker profile of human AFS cells R
elat
ive
cell
num
ber
1 2 3 4
4 1 2 3
1 2 3 4
Oct3/4
4 1 2 3
4 1 2 3
Log fluorescence intensity
SSEA-3 SSEA-4
1 2 3 4
Tra-1-81
4 1 2 3
Tra-1-60
4 1 2 3
CD29
4 1 2 3
CD44 CD73
4 1 2 3
CD90
4 1 2 3
CD105
4 1 2 3
CD45
4 1 2 3
CD34 CD133
4 1 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)
Wake Forest Institute for Regenerative Medicine
Mesenchymal lineages from AFS cells Skeletal/cardiac
muscle
Bone / cartilage
Adipose Undifferentiated Differentiated
Mineralization
Wake Forest Institute for Regenerative Medicine
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
Wake Forest Institute for Regenerative Medicine
Wake Forest Institute for Regenerative Medicine
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
Wake Forest Institute for Regenerative Medicine
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.
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 3. AFS mediated immunosuppression does not require cell-cell contact.
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 4. Soluble factors released from AFS cells and BM-MSCs in response to activation.
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
Wake Forest Institute for Regenerative Medicine
Bone differentiation of AFS cells Mineralized calcium
In culture
Implantation of inkjet-printed construct (8 wks) µCT scan (18 weeks)
AFS cells + scaffold Scaffold alone
Wake Forest Institute for Regenerative Medicine
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
Wake Forest Institute for Regenerative Medicine
A. Peister and R. Guldberg
Bone tissue engineering
In vitro
In vivo
Wake Forest Institute for Regenerative Medicine
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
Slide
Wake Forest Institute for Regenerative Medicine
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
Wake Forest Institute 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
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