Antonio UccelliNeuroimmunology UnitDept. Of NeurosciencesUniversity of Genoa, Italy

Workshop on Transplant and Cellular Therapy for Autoimmune

DiseasesCIBMTR, April 19-20 2013,

Milwaukee, WI

HSC InfusionChemotherapy

MSC

Induction of immune tolerance

Neuroprotection

Induction of oligodendrogenesis

Induction of neurogenesis

Transdifferentiation and neuroprotection

Immunomodulation

NPC

HSC

HSC Mobilization

Eradication of autoimmunity

Reset of the immune system

Immune reconstitution

Uccelli and Mancardi, Curr Opin Neurol 2010

Stem Cells Portfolio for the treatment of MS

The hematopoietic stem cell niche is the site where HSCs home in the BM in close contact with osteoblasts, endothelial and stromal cells, a rare cell population of non-heamatopoietic origin(representing < 0.01% of total BM cells) named mesenchymal stem cells (MSC)

Uccelli A, et al, Nat Rev Immunol 2008

Why should we use MSC for the treatment of brain repair?

Neural differentiation: prolonged (30 days) and specific

nestin

MAP2 GABA-A

GFAP

In vitro neuronal (trans)differentiation of MSC:is this biologically relevant in vivo?

Anghileri et al, 2008

Mesenchymal stem cells protect CNS neurons against glutamate excitotoxicity

Voulgari-Kokota et al Exp Neurol 2012

Mesenchymal stem cells instruct oligodendrogenic fate decision on adult neural stem cells.

Rivera et al., Stem Cells, 2006

Control MSC-CM

GalC

GFAP MBP

MSC promote in vivo neurogenesis

• MSCs promote proliferation, migration, and survival of mouse NSCs• Implantation of human MSCs increased synthesis of trophic factors

Munoz et al, PNAS 2006

MSC inhibit T cell proliferation arresting cell division

Benvenuto et al, Stem Cells 2007

MSC inhibit B cell functions

Corcione et al, Blood 2005

B cells B cells +MSC 1:1

B cells B cells +MSC 1:1

B cells B cells +MSC 1:1

***

cpm

0

20000

40000

60000

80000 B cells

B cells + MSC 1:1

B cells + MSC 1:2

MSC

IgM IgG IgA

ng/m

l

0

50

100

150

200

250

300

350

IgM IgG IgA

B cells

B cells + MSC 1:1 **

***

MSC inhibit B cell proliferation

MSC inhibit immunoglobulins production

MSC inhibit B cells differentiation to antibody secreting cells

In collaboration with V. Pistoia

CD11c CD86

A

100 101 102 103 104FL1-H

100 101 102 103 104FL2-H

100 101 102 103 104FL1-H

100 101 102 103 104FL2-H

100 101 102 103 104FL3-H

0

50

0

50

0

50

0

50

CD80 CD40

MHC cl.I MHC cl.II

CCR7 CD49d 1

78% vs 51% 72% vs 59 % 

41% vs 20% 

68% vs 32%  89% vs 75%

45% vs 16% 

55% vs 22% 48% vs 19%

Control DC

MSC treated DC

MSC impair LPS-induced maturation of DC

Chiesa et al, PNAS 2011

A DC + CD4+ TMSC exposed DC +

CD4+ T

Cel

l num

ber

500

0

500

0

50

0

CFSE

CD

69

CFSE

DC + CD8+ TMSC exposed DC

+ CD8+ T

48h

B

CFSE

69885 16035

67755 57210

4500 2610C

ell n

umbe

r

48h

72h

DC + CD8+ TMSC exposed DC

+ CD8+ T400

400

0

0

MSC impair DC antigen presentation to DO11.10 CD4+ T cells and cross-presentation to OT.1 CD8+ T cells

20800 2226

78110 2428

2MOVA

MOVA

0.2MOVA

Chiesa et al, PNAS 2011

The complex interplay between MSCs and the immune system

Uccelli et al, Nat Rev Immunol 2008

Do all these results translate into relevant in vivo biological effects

in experimental models of neurological diseases?

0

1000

2000

3000

4000

5000

6000

7000

8000

T ce

lls p

rolif

erat

ion

(CPM

)

Mog 1 M Mog 3 M Mog 10 M

Inhibition of Ag-specific T and B cell response

MSCs ameliorate EAE inducing in vivo tolerance to myelin antigens inside secondary lymphoid organs (Zappia et al 2005; Gerdoni et al 2007)

Zappia et al, Blood 2005

50 m

T cell

MSC

A B

Demyelination

T cells

Macrophages

B cells

Axonal loss

EO4 A

DC

B

GFAP NeuN

GFP

Neuroprotection without transdifferentiation

Inhibition of T cell encephalitogenic potential

Amelioration of EAE

Lymph nodes engraftment

Is MSCs therapeutic effect based also on long term engraftment

and cell replacement?

MSC are effective across MHC constraints“are they immunoprivileged?”

• MSC T cell inhibition overcome inter and intraspecies barriers

• Syngeneic, allogeneic and xenogeneic MSC can inhibit EAE

Zappia and Casazza, unpublished resultsRafei et al, Mol Ther 2010

Allogeneic marrow stromal cells are immune rejected by MHC class I– and class II–mismatched recipient mice

Eliopoulos et al, Blood 2005

MSCs are killed by cells of the innate immunity

Spaggiari et al, Blood 2006

Prigione et al, Stem Cells 2009

NK cells

γδ cells

Bone Marrow-Derived Mesenchymal Stem Cells after Infusion principally distribute to the lungs

Gao et al, 2001 Barbash et al, 2003 Lee et al, 2009

0

0.5

1

1.5

2

2.5

3

1° 3° 5° 7° 9° 11° 13° 15° 17° 19° 21° 23° 25° 27° 29° 31° 33° 35° 37° 39° 41° 43° 45°

ctrl iv

ctrl ic

msc iv

msc ic

*P < 0.05: **P < 0.01

Identification of the optimal route of administration

ctrl iv msc iv ctrl ic msc ic0

10

20

30

40

50

60

Dem

yelin

atio

n ar

eas

(%)

* *

10X

MSC I.C.MSC I.V.

10X10X

10X

CTRL I:V: CTRL I.C.

Morando et al., Stem Cell Res & Ther 2012

MAP2 MSC 40x

MAP2 CTRL 40x

Demyelination

T cells

Macrophages

B cells

Axonal loss

MSCs do not transdifferentiate but

protect neurons in vivo

EO4 A

DC

B

GFAP

NeuN

GFP

Gerdoni et al, Ann Neurol 2007

Conditioned growth medium from human

MSCs biases the development of

neurosphere derived cells toward

oligodendrocytes and neurons, and

promotes functional recovery in EAE

Bai et al Nat Neurosci 2012

MSC reduce the EAE-dependent oxidative stress in the CNS

Lanza et al, JNC 2009

GFAP

IB4

MSCs reduces astrogliosis and microglia activation in EAE

SPINAL CORD CTR 40X SPINAL CORD MSC 40X

Casazza and Principato unpublished results

Perry, V. H. et al. (2010) Nat. Rev. Neurol.

Microglia dynamically changes phenotype and functions depending on environmental cues

Microglial plays a key role in CNS homeostasis

Neumann H et al. Brain 2009;132:288-295

iNOS

HOX1

TNF

PARP

N9 +LPS +LPS+MSCN9 +LPS +LPS+MSC

N9 +LPS +LPS+MSC

IL1β

N9 +LPS +LPS+MSC

Metallothioneins * p<0.001

N9 +LPS +LPS+MSC

N9 +LPS +LPS+MSC

* p<0.05 * p<0.01 * p<0.001

* p<0.001* p<0.001

MSC inhibit microglia production of proinflammatory molecules and upregulation of stress associated proteins following LPS activation

Giunti et al, Stem Cells 2012

MSC reverse and enhance the LPS-mediated decrease of molecules associated with neuroprotection

IGF1

CX3CR1 CD200RNURR1

N9 +LPS +LPS+MSC

* p<0.05

N9 +LPS +LPS+MSC

* p<0.05

N9 +LPS +LPS+MSCN9 +LPS +LPS+MSC

* p<0.05

EP2 * p<0.05

N9 +LPS +LPS+MSC

* p<0.05

Giunti et al, Stem Cells 2012

MSC induce functional changes on microglia

N9 +LPS +LPS+MSC

Intracellular Ca2+ * p<0.001

N9 +LPS +LPS+MSC

Phagocytosis * p<0.001 TREM2 * p<0.001

N9 +LPS +LPS+MSC

N9 +LPS +LPS+MSC

Giunti et al, Stem Cells 2012

The disruption of CX3CL1-CX3CR1 signaling induces neurotoxicity in mouse models of neurodegeneration (Cardona et al, Nat Neurosci, 2006)

CX3CL1 expressed by neurons modulates the activity of CX3CR1-positive microglia

MSC express and produce fractalkine under inflammatory conditions

MSC +IFN +IFN+LPS

+LPS MSC +IFN +IFN+LPS

+LPS

CX3CL1 production * p<0,05 * p<0,01CX3CL1 expression

Giunti et al, Stem Cells 2012

CX3CL1 silencing reverts MSC effect on the microglia expression of inflammatory and neuroprotective genes

IL1β

* p<0.01** p<0.01

* p<0.05** p<0.05NURR1CX3CR1

* p<0.01** p<0.01

* p<0.001** p<0.05TNF

N9 +LPS +LPS+MSC

+LPS+MSCCX3CL1si

N9 +LPS +LPS+MSC

+LPS+MSCCX3CL1si

N9 +LPS +LPS+MSC

+LPS+MSCCX3CL1si

N9 +LPS +LPS+MSC

+LPS+MSCCX3CL1si

N9 +LPS +LPS+MSC

+LPS+MSCCX3CL1si

EP2 * p<0.01** p<0.01

Giunti et al, Stem Cells 2012

Silencing of fractalkine in reverts functional changes exerted by MSCs on microglia

N9 +LPS +LPS+MSC +LPS+MSC+ aCX3CL1

Phagocytosis * p<0.001** p<0.001

N9 +LPS +LPS+MSC

+LPS+MSC+aCX3CL1

Intracellular Ca2+ * p<0.001** p<0.001

N9 +LPS +LPS+MSC

+LPS+MSC+aCX3CL1

TREM2 * p<0.001** p<0.001

N9 +LPS +LPS+MSC

+LPS+MSCsiCX3CL1

Giunti et al, Stem Cells 2012

CONCLUSIONS

• MSC – microglia interactions lead to the inhibition of the production of proinflammatory molecules and enhances molecules associated to neuroprotection.

• MSC promote microglia’s functions involved in CNS homeostasis• CX3CL1 produced by MSC plays a major role in the induction of a

functional switch of microglia from a detrimental one to a beneficial one further pointing out a major role for molecules released by MSC upon interaction with the inflammatory environment.

Uccelli et al, Mol Med 2012

MSCs improve survival and motor functions in SOD1/G93A(+) mice through inhibition of glutamate driven excitotoxicity

Mesenchymal stem cells improves experimental epilepsy rescuing hippocampal neurons from apoptosis

Voulgari-Kokota et al, Exp Neurol 2012

Systemic administration of MSC increases neuronal survival in global cerebral ischemia

Perasso et al 2010

T

B

+

+

-

-

-

dem

yelin

ated

axo

n

TNF-

IFN-

NO

MSC inhibit the autoimmune attack to the CNS and promote neural cell survival

Adapted from Uccelli et al Trends Immunol 2007

NeuronsNeural precursor cells

Oligodendrocytes

Astrocytes

MicrogliaROI

++

Neuron

• Transdifferentiation potential ✗• Engraftment/cell contact mechanisms ✗• Direct inhibition of the effector functions of cells of the

immune system ✔• By-stander effect through the release of:

– Anti-apoptotic and anti-oxidant molecules ✔– Trophic factors ✔– Factors inducing the mobilization and induction of

proliferation of local precursors (committed) cells ✔• Non-cell autonomous mechanisms mediated by:

– Dendritic cells – Monocytes/macrophages– microglia

MoA of MSC for brain repair: take home messeage

"Stemness" Does Not Explain the Repair of Many Tissues by

Mesenchymal Stem/Multipotent Stromal Cells (MSCs)

There has recently been an explosion of interest in adult stem/progenitor cells that have the potential to repair

tissues, with over 3,000 citations to publications (PubMed) and numerous announcements of clinical trials in which the cells are used to treat individuals

with a broad range of diseases. At the same time, the data present “a paradox” as the cells originally

attracting attention because of their stem-cell like properties, frequently repair injured tissues without

much evidence of either engraftment or differentiation.Darwin Prockop 2007

The studies on MSC have been supported by grants of the Italian Foundation for Multiple Sclerosis (FISM), Istituto Superiore di Sanità (ISS) “National Program on Stem Cells”, by the Ministero della Salute, Ricerca Finalizzata, by the Ministero dell’Università e della Ricerca, Progetti PRIN, by the

Fondazione CARIGE, by the Fondazione CARIPLO and the Regione LIGURIA

• Neuroimmunology Unit – University of Genoa– F. Benvenuto – S. Casazza– D. Giunti– L. Lovato– F. Ivaldi– S. Morando– B. Parodi– L. Serpero– T. Vigo– A. Uccelli

• Dept. Of Neurosciences,- University of Genoa– GL. Mancardi

• ISPE San Raffaele Hospital• G. Martino

• Department of Biology University of Genoa• L. Vergani

• Dept. Of Nuclear Medicine University of Genoa– G. Sambuceti

Institute of Anatomy University of BariD. Virgintino

Dept of biomedical sciences - University of TeramoM. Maccarrone

• Hematology – S. Martino Hospital, Genoa– F. Gualandi

• University of California San Francisco– J. Oksenberg– S. Baranzini

• Gaslini Insitute, Genoa– F. Frassoni– S. Chiesa– G. Candiano – E. Traggiai– L. Moretta– V. Pistoia

• Dept. of Biochemistry – University of Genoa– A. De Flora

• Dept. Neuropharmacology, University Genoa– GB. Bonanno

– CNR Genoa– Cesare Usai

– Tor Vergata University – Rome– D. Centonze– A. Urbani

– University of Verona – Italy– B. Bonetti

– Ospedali Riuniti Bergamo – Italy– M. Introna

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