Stem Cells Derived from Amniotic Fluid and Placenta Anthony Atala

Stem Cells Derived from Amniotic Fluid and Placenta

Anthony Atala, MD Wake Forest Institute for Regenerative Medicine Wake Forest University School of Medicine 1 Winston Salem, NC

Amniotic fluid and placenta

• Tissues & fluids support developing embryo / fetus • Non-invasive sampling or recovery at term

– Chorionic villi / Placenta

– Amniotic fluid

• Amniocentesis (14 weeks to term) and chorionic villus sampling (12 weeks to term) are widely accepted methods for prenatal diagnosis, and are known to contain cells from all 3 germ layers • Amniotic fluid and placental tissue: Possible source for alternate stem cells?

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Amniotic fluid and placental derived stem cells

• Fresh cells or back-up cytogenetics lab culture • Select c-Kitpos (CD117pos) cells • Establish clonal lines - no feeder layers

AFS cells 4

400 human amniotic fluid and placental samples (10 - 40 wks)

Stem cells were isolated and differentiated to:

Bone Fat Muscle Endothelium Liver

Clonality confirmed by Southern blot showing retroviral insertion fingerprint

Parent Offspring Differentiated cells (3 germ layers)======>

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Preservation of telomere length

SSEA-4 OCT4

Amniotic / Placental Stem Cells

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CD105 CD73 CD146 CD34 CD45

CD44 CD29 CD90 SSEA4 HLA-ABC

AFS cells show higher similarity to ES and iPS cells than bone marrow MSC Expression profile analysis of “stemness genes” in embryonic, iPS, BM-MSC and AFS cells

Isolated cells after 250 population doublings

Normal Karyotype Normal G banding

Do not for teratomas when injected in vivo 12

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AFS costimulatory molecule expression

100 100

80 80

60 60 CD80 MHC II 40 40 % ofMax % ofMax

20 20

0 0 100 101 102 103 100 101 102 103 FL1-H FL1-H 100 100

80 80

60 60 CD86 MHC I 40 % ofMax 40 % ofMax

20 20

0 0 0 1 2 3 100 101 102 103 10 10 10 10 FL1-H FL1-H 13AFS cells show little to no expression of MHCII and co-stimulatory molecules

hAFS cells inhibit T-cell activation in vitro

- + 1:16 1:8 1:4

• Enzyme-linked immunosorbent spot (EliSpot) T ++++ PHA++++ • Procedure: AFS - 1:16 1:8 1:4 – Coat wells with anti-IFN-γ – Add T cells, activator (PHA) and AFS cells to wells – Incubate 24 hours – Remove cells by washing – Develop 14• Each spot is an activated T cell clone producing IFN-γ

The cells are able to form embryoid bodies

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16Valli et al., Oncogene, 2010

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Two possible uses for amniotic fluid or placental derived stem cells:

1. Immunomodulation

• The cells could be used in a non-autologous manner for immunomodulation, in a similar manner to current bone marrow mesenchymal stem cells • AFS cells preserve their telomere length, therefore an advantage is that the same cells can be used off the shelf for many years, thus avoiding batch to batch variability (as opposed to bone marrow, where there is a limited expansion potential and different batches need to be obtained over time) • The AFS cells are clonal, therefore the same outcome can be expected in terms of the effect of the cells each time

2. Regenerative medicine application of amniotic fluid stem cells

Amniocentesis Routine Genetic culture testing

Selection of stem cells (~ 1%)

Differentiation Ectoderm Mesoderm Therapeutic applications Endoderm

• The cells can be used in an autologous manner, thus avoiding rejection, or a bank of 100,000 unique specimens could supply approximately 90% 20 of the US population with a perfect genetic match for transplantation

Fat differentiation

8 Days 16 Days Control 16 Days

Light

Oil-Red-O

8d - 16d 8d 16d - + + Lipoprotein lipase

pparγ2

β2-microglobulin 21

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Bone differentiation of human AFS cells

Mineralization

Diff.

Undiff.

4 days 8 days 16 days 24 days 32 days Alkaline Phosphatase Stain Differentiated Undifferentiated

cells 400 6 8d-16d- 24d 32d 8d+ 16d+ 24d+32d+ 300 mRNA - - AP 200 cbfa 1

100 Diff. Osteocalcin Undiff. 0 4 days 8 days 16 days 24 days 32 days β2 microglobulin 22nMolp-Nitrophenol/min/10 Alkaline Phosphatase Secretion

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Cartilage differentiation

Alizarin Red staining of AFSC osteogenic cultures 0.35

0.30

0.25

0.20

0.15 Absorption 0.10

0.05

0.00 0 1 234 Weeks in culture

Control TGF-β3TGF-β3 + dex TGF-β1 + dex 25Kolambkaret al., J Mol Histol 2007

Cardiac differentiation

Human Undifferentiated Heart AFS -RT

MEF 2C

MEF 2D DAPI Connexin 43

N-Cadherin

Merge SOX2 H-Cadherin

GAPDH

26Guan et al., J. Tiss Eng & Regen Med, 2011

A Cardiac Troponin I Cardiac Troponin T sox2 25 180 1400 160 1200 20 140 1000 120 15 100 800 80 600 10 60 400 40 5 200

20 Relative quantification Relative quantification quantification Relative 0 0 Relative Quantification 0 Undiff.Undiff 5 days diff. 10 days diff. Undiff.Undiff. 55 Days days Diff.diff. 10 Daysdays diff.Diff. Undiff.undiff 5 5 days diff.diff 10 days diffdiff.

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Co-cultured neonatal rat cardiomyocytes (NRCs) with hAFS cells formed both mechanical and electrical connections

B C A

CONNEXIN43 DAPI PKH26

D E

• Connexin 43 and N-Cadherin distribution is mainly cytoplasmic and peri-nuclear CONNEXIN43 DAPI ANTI-NUMA Human Nuclei before co-culture, and membrane bound FG after co-culture

28 N-Cadherin DAPI PKH26

0 hour

B1 B2

A2 A3

6 hours B3 B4

C 50% No TPA

hHLA-ABC TPA A4 A5 40%

30% 12 hours 20% Percentage Percentage 10%

0% Calcein 0612 Time (Hours) • PKH-26 labeled hAFS cells become calcein positive when cultured with calcein preloaded NRCs, and this is partially blocked by TPA 29 (involves gap junction connexin 43)

Endothelial differentiation

Phase KDR/flk1

8d- 8d+ CD31

VCAM

Factor P1H12 VIII

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Liver differentiation

Control Day45 HNF4α c-met

MDR

Albumin α-fetoprotein

Immunoblot: hepatocyte proteins

Morphology (insert shows higher mag.)

31 Albumin mRNA Urea secretion

Lung differentiation

• Using microinjection into cultured mouse embryonic lungs, hAFSC can integrate into the epithelium and express the early human differentiation marker thyroid transcription factor 1 (TTF1)

32 Carraro et al., Stem Cells, 2008

• hAFSC express endogenous PDPN (green); In adult nude mice, following hyperoxia injury, tail vein-injected hAFSC localize in the distal lung and express both TTF1 and the type II pneumocyte marker surfactant protein C 33

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• Specific damage of Clara cells through naphthalene injury produced integration and differentiation of hAFSC at the bronchioalveolar and bronchial positions 34 with expression of the specific Clara cell 10-kDa protein

hAFSC did not show fusion

• Naphthalene injured lung was analyzed by triple staining forty days after hAFSC intratracheal administration; Merging of picture A and B show a hAFSC detected by Y chromosome that expresses CC10 but is negative for X chromosome; FISH for Y and X chromosome on cytospin sample shows two Y positive hAFSC that do not have X chromosome signal 35

Kidney

• Structural differentiation of amniotic fluid stem cells within developing embryonic kidneys demonstrating integration of stem cells

• Chromogenic in situ hybridization of injected amniotic fluid stem cells shows integration of stem cells into the kidneys 36 L. Perin, et al., Cell Proliferation, 2007

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AFSC for renal therapy

• AFSCs can survive and replicate in vivo when directly injected into the kidney (ATN) • AFSCs integrate into the tubules and differentiate into tubule-like cells as well as glomeruli precursors • AFSCs injected into both kidneys after glycerol damage, ameliorate the creatinine and BUN levels, decrease tubular epithelial cells apoptosis and increase cell proliferation

38De Filippo et al., PLOS One, 2010

39De Filippo et al., PLOS One, 2010

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Necrotizing Enterocolitis (NEC)

• Most common neonatal surgical emergency • 1-7% NICU admissions • Mortality: 20 - 40%

NEC model

NEC NEC+AFS cells Feeding

Birth Hypoxia

Lipopolysaccharide

24h Phosphate buffered saline

2x106 AFS cells 48h

72h

96h 41 DeCoppi et al., 2010

72 hrs post-injection

GFP/dapi 42

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PCR analysis

Heart Brain Spleen

+ Bone marrow Bowel Kidneys

Lungs 43 Liver

EGFP amplification on genomic DNA

Brain

Bone marrow

Lungs

Heart

Kidneys

Spleen

Liver

Intestine

020406080100 Percentage of positive organs 44

Intestinal macroscopic assessment

Breastfed

NEC

NEC + AFS cells

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Intestinal microscopic assessment

Breastfed

NEC

46 NEC+AFS cells

Human and murine amniotic fluid stem cells display hematopoietic activity

• Cells differentiated into all cell lineages including myeloid, erythroid and leukoid cells • The bone marrow depleted rodents showed repopulation of bone marrow into all three lineages after injection of the stem cells • Potential of cells to replenish bone marrow banks without additional donors 47

Clinical utility of amniotic fluid and placental stem cells

• The stem cells, derived from amniotic fluid and placental tissue, can be obtained during pregnancy or at the time of birth from discards • The cells are neither embryonic nor adult stem cells, but have properties of both • This system avoids the teratoma, tumor potential, and rejection concerns surrounding the use of other stem cells • The stem cells can be rapidly expanded to large quantities sufficient for clinical translation, thus avoiding the limitations of adult stem cells • The stem cells could be stored at the time of birth for future “self” use, or could be banked in large quantities, thus avoiding rejection

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Amniotic/ Marrow/ Embryonic IPS Tissue Specific Placenta Cord

Growth +++ +++ +++ + ++ Potential

Tumor +++ +++ +++ Free

Rejection +++ +++ +++ +++ Free

Lineage +++ +++ ++ + + Potential

DeCoppi et al., 2007

We would like to Acknowledge Dr. Paolo DeCoppi who worked with our team, was the first author of the initial paper, and is now is our collaborator 50 at Great Ormond Street Hospital and University College London

• Some of the work in this presentation was made possible, in part, by grants from the following institutions: –NIH –DOD – The Frase Foundation – Juvenile Diabetes Research Foundation – Diabetes Research Institute – Christopher Mosely Foundation – Errett Fisher Foundation

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