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

http://images.sciencedaily.com/2012/07/120703133741-large.jpg

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.



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 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)


Mesenchymal lineages from AFS cells Skeletal/cardiac

muscle

Bone / cartilage

Adipose Undifferentiated Differentiated

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

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 + scaffold Scaffold 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

Slide


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



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