Institute for Regenerative Medicine
Deriving Mesenchymal Stem Cells from Human
Amniotic Fluid – Potential for an Allogenei...
Wake Forest Institute for Regenerative Medicine
Wake Forest Institute for Regenerative
Medicine
• The Wake Forest Institut...
Wake Forest Institute for Regenerative Medicine
Wake Forest Institute for Regenerative
Medicine
Mission: Improve patient’s...
Wake Forest Institute for Regenerative Medicine
“FIRSTS” in Regenerative Medicine
Led a team of researchers that was the f...
Wake Forest Institute for Regenerative Medicine
ES Cells
Stem cells are present throughout
development and postnatal life
...
Wake Forest Institute for Regenerative Medicine
Cell sources before or at birth
Tissues & fluids support
the developing em...
Wake Forest Institute for Regenerative Medicine
Amniotic fluid sampling
Week 14-16
of gestation
Cell retrieval:
amniocente...
Amniotic Fluid Stem (AFS) Cell Technology
Selection of stem
cells (~ 1%)
Routine
culture
Genetic
testing
Therapeutic
appli...
Wake Forest Institute for Regenerative Medicine
Amniotic fluid-derived stem (AFS) cells
AFS cells
 Fresh AF or back-up
cy...
Wake Forest Institute for Regenerative Medicine
AFS cells maintain normal karyotype and
long telomeres
Telomere length
1. ...
Wake Forest Institute for Regenerative Medicine
Multilineage differentiation of verified hAFS
cell clone
1 2 3 4 5 6 7 8
O...
Wake Forest Institute for Regenerative Medicine
Marker profile of human AFS cells
Relativecellnumber
1 2 3 4
41 2 3
1 2 3 ...
Wake Forest Institute for Regenerative Medicine
Mesenchymal lineages from AFS cells
 Skeletal/cardiac
muscle
 Bone / car...
Wake Forest Institute for Regenerative Medicine
Properties of AFS cells (summary)
Readily isolated from amniotic fluid & ...
Wake Forest Institute for Regenerative Medicine
Wake Forest Institute for Regenerative Medicine
1. First paper to describe the presence of cells with a
hematopoietic pote...
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, Orland...
Figure 2. Human AFS cells inhibit lymphocyte activation in a dose dependent manner similar to
that of BM-MSCs.
Moorefield ...
Figure 3. AFS mediated immunosuppression does not require cell-cell contact.
Moorefield EC, McKee EE, Solchaga L, Orlando ...
Figure 4. Soluble factors released from AFS cells and BM-MSCs in response to activation.
Moorefield EC, McKee EE, Solchaga...
Wake Forest Institute for Regenerative Medicine
Bone differentiation of AFS cells
Mineralized calcium
In culture
Implantat...
Wake Forest Institute for Regenerative Medicine
Project 3: manufacturing process of AFS cells for
clinical study in subjec...
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,
i...
Wake Forest Institute for Regenerative Medicine
Key Questions
• Clinical utility of mesenchymal SC from
amniotic fluid vs ...
Wake Forest Institute for Regenerative Medicine
Where we stand
New stem cell-based products are reaching
the clinic
Grea...
Wake Forest Institute for Regenerative Medicine
Wake Forest Institute for
Regenerative Medicine
Special thanks to Dr. Shay...
This work was made possible, in part, by grants from the following institutions:
NIH: NIDDK
NIH: HLI
Department of Defense...
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Deriving Mesenchymal Stem Cells from Human Amniotic Fluid – Potential for an Allogeneic Cellular Therapy Product

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

  1. 1. 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
  2. 2. 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.
  3. 3. 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.
  4. 4. 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)
  5. 5. 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
  6. 6. 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
  7. 7. 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
  8. 8. Amniotic Fluid Stem (AFS) Cell Technology Selection of stem cells (~ 1%) Routine culture Genetic testing Therapeutic applications Amniocentesis Differentiation
  9. 9. 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.
  10. 10. 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
  11. 11. 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
  12. 12. Wake Forest Institute for Regenerative Medicine 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)
  13. 13. Wake Forest Institute for Regenerative Medicine Mesenchymal lineages from AFS cells  Skeletal/cardiac muscle  Bone / cartilage  Adipose UndifferentiatedDifferentiated Mineralization
  14. 14. 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
  15. 15. Wake Forest Institute for Regenerative Medicine
  16. 16. 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
  17. 17. Wake Forest Institute for Regenerative Medicine
  18. 18. 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
  19. 19. 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
  20. 20. 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
  21. 21. 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
  22. 22. 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 + scaffoldScaffold alone
  23. 23. 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
  24. 24. Wake Forest Institute for Regenerative Medicine A. Peister and R. Guldberg Bone tissue engineering In vitro In vivo
  25. 25. 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
  26. 26. 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
  27. 27. 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
  28. 28. Wake Forest Institute for Regenerative Medicine Wake Forest Institute for Regenerative Medicine Special thanks to Dr. Shay Soker for Slides
  29. 29. 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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