PLACENTA UMANA da materiale di scarto biologico.. verso il loro utilizzo per la Terapia Rigenerativa

(1)

PLACENTA UMANA

da materiale di scarto biologico..

verso il loro utilizzo per la

Terapia Rigenerativa

Ornella Parolini

Centro di Ricerca E.Menni

(2)

Amnion Smooth chorion False Fetal surface of placenta 1 False knot of placenta Umbilical cord

(3)

Why an interest in human

placenta?

• Identify stem cells for cell therapy

approaches:

– Stem cell potential – Stem cell potential

– No transplant rejection

• Placenta may combine these two essential

features on the basis of:

– Embryological origin

(4)

(5)

Fetal membranes Uterus wall

FETAL MATERNAL TOLERANCE:

Pregnancy is a unique event

in which a genetically and

immunologically

foreign fetus

survives to full term

Umbilical cord trophoblast

survives to full term

without rejection by

the mother's immune

(6)

From Placenta:….

• FRAGMENTS of tissue:

–

Amniotic membrane

–

Cord fragments

–

Cord fragments

• CELLS:

–

from amniotic membrane, chorionic membrane,

cord, Wharton Jelly,

cord blood

• Media derived from culture of placenta

derived cells

(7)

Amnion

(8)

Basal Membrane

Reflected fetal membranes

Amnion Chorion Amnion Epithelium Compact Layer Cellular Layer Spongy Layer

Amniotic Epithelial Cell Amniotic Mesenchymal Cell

400 x EE Amnion

Amnion Chorion

(9)

Adherence to plastic

Formation of fibroblast colony-forming units

A specific pattern of surface antigen expression

Minimal criteria for defining hAMSC

Negative (<2%) CD 45

CD 34

(At passages 2-4) Positive (>95%) CD 90

CD 73

Differentiation potential toward one or more lineages, including osteogenic, adipogenic, chondrogenic and vascular/endothelial

Fetal origin

Parolini O. Stem Cells 2008 CD 34

CD 14 HLA-DR

CD 73 CD 105

Other mesenchymal and hematopoietic markers: CD44+, CD29+,HLA-A,B,C+, CD13+,

(10)

Differentiation potential of hAMSC and hCMSC

Osteogenic Chondrogenic Adipogenic

Soncini M et al. TERM 2007

Osteogenic Chondrogenic Adipogenic

(11)

IMMUNOMODULATORY FEATURES OF

AMNIOTIC DERIVED CELLS

(12)

0 5000 10000 15000 20000 R R+S* R+A* cp m *** A ** R: responder (hPBMNC)

S*: stimulator (hPBMNC irradiated cells) A: Amnion (irradiated cells)

pPBMNC: porcine PBMNC

0 5000 10000 15000

a Pig+hPBMC* Pig+hA* Pig+hC* (CD45-Gly-) Pig * * * * cp m ** pPBMC pPBMC + hPBMC* pPBMC + hA* pPBMC + hC(CD45-GlyA-)* ** B Transplantation, 2004

(13)

1. AMSC inhibit lymphocyte proliferation

PBMC + CD3/CD28 PBMC + CD3/CD28 + AMSC PBMC+ allo PBMC* PBMC+ allo PBMC* + AMSC

Transient Inhibition

• Magatti M, et al. Stem Cells 2008

0 20 40 60 80 100 120 % p ro li fe ra ti o n % p ro li fe ra ti on R + S1* R + S1* + AMC Re + S1* Re + S2* Transient Inhibition

(14)

GM-CSF + IL-4 [3-5 days]

monocytes

In vitro differentiation of monocyte-derived dendritic cells

(DC):

LPS [2 days] immature-DC (iDC) mature-DC (mDC) m a tu ra tio n

(15)

AMSC inhibit monocyte differentiation toward dendritic cells and affect ability of DC to induce T cell proliferation

monocyte CD1a CD14 mDC CD1a CD14 [AMSC-mDC*] CD1a CD14 14

• Magatti M, et al. CellTransplantation2009

0 20000 40000 60000 80000 100000 120000 140000 500 2000 10000 *** ** ** 0 14 12 10 8 6 4 2 ▲ monocytes ○ iDC □ [iDC-AMSC] ● mDC ■ [mDC-AMSC]

(16)

• Amniotic derived cells don’t induce T cell proliferation, except when used at a low concentration

• Amniotic membrane MSC inhibit

lymphocyte proliferation

AM C

Immunological characteristics

• The lymphocyte inhibition is mediated by soluble factor(s)

• The inhibition is reversible

• Amniotic membrane MSC inhibit the differentiation from monocytes to

dendritic cells

T cells

(17)

In Vivo studies:

AM derivatives

(cells, membranes, conditioned

medium..)

transplantation

Mouse model Pulmonary fibrosis in a

Neonatal mice and neonatal swines

Mouse model Pulmonary fibrosis in a (bleomycin-induced lung fibrosis)

Rat model Myocardial ischemia (Coronary artery ligation)

Rat animal model Liver fibrosis (Bile duct ligation)

(18)

ALLO-TRANSPLANTATION AMNION CELLS

B l e o myc i n f i b ro s i s i n d u c e d i n a m o u s e m o d e l

C57BL/6 mice BLEO C57BL/6 mice BLEO 34 XENO-TRANSPLANTATION Balb/c GFP mice C57BL/6 mice BLEO C57BL/6 mice BLEO C57BL/6 mice BLEO BALB/c mice

From HUMAN PLACENTA

(19)

Type II Pneumocytes Type I Pneumocytes Capillaries Fibroblasts Pulmonary alveolar structure Bleomycin intratracheal injection

Bleomycin is a chemotherapeutic agent that produces alveolar

epithelial cell damage through DNA strand breakage by

Reactive Oxygen Radicals.

Damaged cells secrete growth factors and chemokines which stimulate inflammatory cell recruitment (⇨⇨⇨⇨ thickening of

interalveolar spaces)

Inflammatory cells produce pro-fibrotic cytokines (TGF-ββββ, IL-13) which stimulate fibroblast migration,

proliferation and fibroblast activation to myofibroblasts (αααα-SMA positive cells)

Myofibroblasts are the major responsible of extracellular matrix deposition leading to

formation of patchy pulmonary fibrosis (⇨⇨⇨⇨ alveolar occlusion)

Collagen deposition B LE O Bronchial Cells Alveolar Macrophages Pneumocytes Fibroblasts

(20)

Bronchi

Vessels

*

_*

*

Alveolar

Spaces

*

_*

*

interstitium

(21)

Bronchi

*

Alveolar

Spaces

interstitium

Vessels

*

(22)

Bronchi

Vessels

(23)

Semiquantitative Modified Hagood System for Fibrosis Determination (Hagood et al., 2005) Fibrosis Extent 1: 0-25% 2: 26-50% 3: 51-75% 4: 76%-100% Alveolar Obliteration (1-2-3)* Fibroblast proliferation (1-2-3)* Collagen Deposition (1-2-3)* X + + = Fibrosis Score 400X 400X 400X 400X 400X * 1=Mild 2=Moderate 3=Severe

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day 14 * * F ib ro s is s e v e ri ty (s c o re u n it s ) F ib ro s is e x te n t (% o f a re a in v o lv e d ) 2 3 4 5 25 50 75 100 n=19 n=8 n=7 ** Allo-transplantation 1

“Bleo” “Bleo+Cells” “Cells”

IP route IT route IP route IT route * F ib ro s is s e v e ri ty (s c o re u n it s ) 0 1 0 25 n=3 n=3

(25)

day 14 * 2 3 4 5 50 75 100 F ib ro s is s e v e ri ty (s c o re u n it s ) F ib ro s is e x te n t (% o f a re a in v o lv e d ) n=19 n=8 n=7 ** Xeno-transplantation 1

“Bleo” “Bleo+Cells” “Cells”

IP route IT route IP route IT route 0 1 2 0 25 F ib ro s is s e v e ri ty (s c o re u n it s ) (% o f a re a in v o lv e d ) n=4 n=3

(26)

a1 a2 Allo-transplantation Xeno-transplantation In tr a -t ra ch ea l d el iv er y In tr a -p er it o n ea l d el iv er y a3 a4

(27)

Amniotic membrane-derived cell transplantation significantly

reduced bleomycin-induced lung fibrosis

Which disease animal models

have we studied?

Pulmonary fibrosis

Amniotic membrane application

(Cargnoni A. et al.; Cell Transplantation, 2009)

Myocardial ischemia

Liver fibrosis

Amniotic membrane application significantly improved cardiac functions in ischemic rat hearts for at least 2 months post-injury

Amniotic membrane application significantly reduced liver fibrosis induced in rats by BDL

(Cargnoni A. et al.; Cell Transplantation 2009)

(28)

•

In all disease models studied, placenta-derived cells and amniotic membrane exerted anti-fibrotic effects

•

No appreciable cell engraftment was observed in any of the target organs of cell transplantation /amniotic

General considerations

from our data:

of the target organs of cell transplantation /amniotic membrane application

•

Soluble factors, rather than cells per sé, may represent the true “actors”

(29)

Bleomycin

Bleomycin +

Could conditioned medium (CM) derived from amniotic cell culture replace placenta-derived cell therapy or amniotic membrane application? Bleomycin + Control Medium Bleomycin + AM-Conditioned Medium

(30)

(31)

(32)

In vivo studies demonstrate mainly the

ability of amniotic cells/amniotic membrane

REGENERATION versus REPAIR

ability of amniotic cells/amniotic membrane

to modify the environment and exert paracrine

effects that improve local surrounding tissue,

favouring repair by the host cells

(33)

IPLASS aims to become a worldwide networking platform to promote Research on all aspects concerning knowledge, experimentation

and clinical use of placenta-derived cells

www.iplass.net

and clinical use of placenta-derived cells

Committee Members:

Ornella Parolini (ITALY) President

Cesar Borlongan (USA) Vice President

Marco Evangelista (Australia) Secretary

Heinz Redl (Austria) Stephen Strom (USA)

Bing Liu (China) Sankar Venkatachalam (India)

Steffen Zeisberger (Switzerland) Marta Magatti (Italy)

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