“Potenziale impiego di cellule staminali di

derivazione amniotica nella medicina

rigenerativa nella specie equina”

Reproduction Unit

Large Animal Hospital

Veterinary Medicine Faculty, Lodi

Dr. Anna Lange Consiglio, PhD

Stem cells for new frontiers: tissue engineering

Why not simply transplant donated organs?

In human medicine, requests greatly exceed supply

In veterinary medicine

• no donors;

• ethic problems if abandoned pets are used;

• large animals remain excluded.

Sources of stem cells

Adult stem cells • are localized within the tissues

but in a limited number

• are difficult to isolate and to proliferate in vitro

• differentiate only in a few types of tissue

BUT

• are immunocompatible with the donor

Embryonic stem cells • are cultivated in vitro and

capable of unlimited proliferation

• differentiate into all tissues of the body

BUT

• are isolated from the embryo leading to its destruction

• arouse intense ethical debate

• are not immunocompatible

• are tumorigenic

Differences

Main sources of adult MSCs

Important therapeutic application in human and veterinary regenerative medicine

Bone marrow

Fat tissue

Therapeutic application with adult MSCs

A field of research from over 60 years.

Why so few clinical advances?

HUMAN MEDICINE

Indication References

Myocardial infarction Al-Khaldi et al., 2003

Imperfecta

osteogenesis

Horwitz et al., 2001

Defect long bone Quarto et al., 2001

Metachromatic

leukodystrophy and

Hurler syndrome

Koc et al., 2002

Idiopathic aplastic

anemia

Fouillard et al., 2003

VETERINARY MEDICINE

Indication References

Myocardial infarction Orlic et al., 2001; Saito et al.,

2002

Muscular dystrophy Gussoni et al., 1999

Pulmonary fibrosis Ortiz et al., 2003

Spinal fusion Mushler et al., 2003

Segmental bone defects Bruther and kurt 1998,

Craniotomy defect Krebsbach et al., 1998

Tendon injury Yiung et al., 1998

Meniscus Murphy et al., 2003

Characteristics of adult MSCs

Donor’s age

In vitro passage number (6-10)

Bone marrow

Invasive procedure Low density

Fat tissue Invasive procedure Low density

potential • proliferation • differenziation

decrease

Vidal et al, Stem Cells Development, 2011

Regenerative medicine

Amniotic liquid (In ‘t Anker et al., 2003)

Umbilical cord (Sarugaser et al., 2005)

Umbilical cord blood (Secco et al., 2008)

Amniotic membrane (Alviano et al., 2007)

Fetal adnexa in human medicine

SOURCE SPECIES

UMBILICAL CORD (Wharton Jelly)

- Pig (Carlin et al., 2006) - Horse (Hoyonowski et al., 2007; Cremonesi et

al., 2008; Passeri et al., 2009; Toupadakis et al., 2009; Corradetti et al., 2010, 2011; Martino et al., 2011)

- Dog (Filioli Uranio et al., 2011)

UMBILICAL CORD BLOOD

- Horse (Guest, 2008; Reed e Johnson, 2008) - Bovine (Raoufi et al., 2010) - Dog (Seo et al., 2009)

AMNIOS - Horse (Lange Consiglio et al., 2010, 2012) - Dog (Filioli Uranio et al., 2011) - Bovine (Corradetti et al., 2013)

AMNIOTIC LIQUID -Sheep (Mauro et al., 2010) - Horse (Park et al., 2010; Lovati et al., 2011) - Dog (Filioli Uranio et al., 2011)

Fetal adnexa in veterinary medicine

Extra-fetal tissues

• Tissues discarded at birth (no ethics)

• No impact on the health of mother and child

Alternative sources of MSCs

blastocoelic cavity

epiblast

hypoblast

trophectoderm

primitive yolk sac

hypoblast

Chorion amniotic fold

amniotic cavity

amniotic layers: epithelial and mesenchymal

extra-embrionic

coelom

epiblast

trophectoderm

endoderm

extra-embrionic mesoderm

Embryological origin of amnion

• AMNION is continuos with epiblast

• Pluripotency?

What are the factors necessary in stem cells therapy?

To collect a large number of cells inexpensively and

non invasively To have cells with high target of proliferation and differentiation to repair organ damages To have cells with

characteristics of homing To have cells without

immunogenic properties and

with immunomodulatory properties

To collect a large number of cells inexpensively and non invasively

Amnion Allantois

Enzymatic digestion (25 gr)

100*106 AMCs 250*106 AECs

Lange-Consiglio et al., 2011 J Tissue Eng and Reg Med Lange-Consiglio et al., 2013 Equine Veterinary Jourmal

25 ml di BM = 8-25*106 MCs (1 CFU ogni 600 cellule)

(1 CFU ogni 240 cellule)

After 14 days mesenchymal amniotic-derived cells show higher proliferative capacity (3 replicates)

To have cells with high target of proliferation

Lange-Consiglio et al., 2011 J tissue Eng and Reg Med Lange-Consiglio et al., 2013 Equine Veterinary Jourmal

To have cells with high target of differentiation

Induced Control

Lange-Consiglio et al., 2011 J tissue Eng and Reg Med

Speed of differentiation

Nodules observed

• 3a week in mesenchymal amniotic cells

• 5a week in bone marrow cells

Lange-Consiglio et al., 2013 Equine Veterinary Jourmal

To have cells with characteristics of homing to the site of injury or disease

10 μl of Candida albicans antigen After 48h, 1*108 AMCs labeled by PKH26 were administrated

by the intravenous or by endobronchial route

Punch biopsies were taken on 48 h, 1 week and 2 weeks after the administration of AMCs cells

To have cells with characteristics of homing to the site of injury or

disease

• by intravenous route, cells were present after 48h • by endobronchial route amniotic cells arrived in site

after 7-14 days unpublished data

To have cells with immunogenic and immunomodulatory properties

Pregnancy is a unique event in which a genetically and

immunologically foreign fetus survives to full term whitout

rejection by the mother’s immune system

Immunogenic properties

MHC I

MHC II

AMCs

P1 P5

Lange Consiglio et al. TERM, 2011

Immunomodulatory properties It is necessary a more complete understanding of the

mechanisms that equine MSCs use to modulate the immune system

but in equine no studies

have analyzed the ability of MSCs derived

from amnion to alter lymphocyte proliferation

and to produce immunomodulatory mediators

There are many investigation in different species

AMCs co-cultured with PBMC or Lymphocytes

non activated or activated

by PHA

Assay of proliferation of T-

cells and PBMC with [3H]-thymidine

Lymphocyte T or PBMC

Mediator secretion assay

(ELISA)

AMCs plated in dish or transwell

and irradiated with γ rays

AMCs co-cultured with unstimulated PBMC

In the absence of PBMC stimulation:

• AMCs did not induce

proliferation responses in PBMC

• This indicate that these cells do not induce allogeneic responses

Unpublished data

Inhibition of PBMC proliferation both through cell-cell contact and in a transwell

system

The inhibitory potential of AMCs was maintained for up to three AMCs culture passages, even though at this passage the inhibition was lower in the transwell setup

AMCs co-cultured with stimulated PBMC

Unpublished data

inflammation

Lymphocyte

IL-12

IFN-γ

IFN-β

TNF-α

Pro-inflammatory factors

Anti-inflammatory factors

lymphocyte

lymphocyte

lymphocy

te

lymphocyte

lymphocyte

lymphocyte

These data are compatible with data in other species

MSCs

Pro-inflammatory factors

Anti-inflammatory factors

Control of inflammatory process

These data provide a critical starting point for beginning to dissect out how

Regenerative medicine

equine AMCs respond to and alter the

inflammatory niche the horse may best serve as a large animal model for human diseases

Human regenerative medicine

Veterinary regenerative medicine

To date, the majority of research in adult-derived equine MSC therapy is focused on orthopedic injuries and the regeneration of bone, cartilage and tendon

The SDFT in the horse is a tendon that has many similarities to the human Achilles tendon in both its structure and matrix composition

Horse as large animal model for tendinophaties

• Energy-Storing Tendons

• Essential for efficiency of high-speed locomotion,

• Large size of both species

• Collagene (tipo I) • GAG (decorina e

aggreacan) • tenociti

Tendinophaties

• Matrix degradation by METALLOPROTEINASE

• TENOCYTES (caspase and apoptosis)

• CHONDROGENIC DIFFERENTIATION: fibrocartilaginous scar tissue after injury (> collagen III, IX, X, GAG, aggrecan,)

• Sport (types and durations of physical stress )

• Age

• Acute or chronic disease • Extrinsic (trauma) and intrinsic

(overstrain) factors

Hypertermia

Hypoxia

> ROS

• Preclinical studies show that cell therapies have the potential to regenerate rather than repair tendon tissue, a process termed tenogenesis.

• A number of different cell lines, with varying degrees of differentiation, have being evaluated including stem cells, tendon-derived cells and dermal fibroblasts

• Determine the ideal cell source, the number of cells to administer, or the optimal bioscaffold for clinical use

• Stem cells or growth factors?

Regenerative Medicine

Our Study

• autologous BM-MSCs (44 horses)

• eterologous AMCS (48 horses)

TRANSPLANTATION IN TENDINOPATHIES

Regenerative Medicine • Putative progenitor cell niches in tendon (Lovati et al., 2011)

(microdamage)

• Exogenous stem cells (BM-MSCs) 25-50% re-enjuries

BM-MCs: intralesional tendon treatment

2 SL 1 PSD 1 DDFT 8 SDFT

5 SDFT

2 SL 3 SDFT

Total number of treated horses: 44

Transplantation 16-35 days after injury

Lange-Consiglio et al., Cytotherapy 2013 in press

AMCs: intralesional tendon treatment

4 SDFT

Total number of treated horses: 48

Transplantation 6-15 days after injury Lange-Consiglio et al., Cytotherapy 2013 in press

Discipline Numbers of horses re-injury

Percentage of re-injury (%)

Show jumping 3/12 25.00

Dressage 4/9 44.44

Eventing 1/5 20.00

Trotter 0/5 0.00

Flat racing thoroughbreds 1/8 12.50

Treatment with

BM-MCs

Discipline Numbers of horses re-injury

Percentage of re-injury (%)

Show jumping 0/22 0.00

Dressage 2/14 14.26

Eventing 0/4 0.00

Trotter 0/5 0.00

Flat racing thoroughbreds 0/2 0.00

Treatment with AMCs

23.08%

4.26%

Followed-up was monitored for 2 years after returning to full work

Lange-Consiglio et al., Cytotherapy 2013 in press

25

50

150

days

Treatment with BM-MCs Treatment with

AMCs

After 60 days

NO SIGNIFICANT ADVERSE EFFECTS AFTER AMCs TREATMENT

Animals resume their activities between 4 and 5 months after treatment Animals resume their activities between

4 and 12 months after treatment Lange-Consiglio et al., Cytotherapy 2013 in press

CONCLUSIONS

AMCs: joint treatment

• 1 Cruciate ligament arthroscopy assistment

•2 Subcondral bone cysts medial femoral condyle ecoguided implant in GA

• 1 Elbow subcondral bone cyst Trans cortical drilling, cyst debridment

followed by injection of AMCS

AMSC: joint treatment

• I Cruciate Ligament arthroscopy assisted

• 2 subcondral bone cysts medial femoral condyle

• ecoguided implant in GA

• 1 elbow subcondral bone cyst

• trans cortical drilling, cyst debridment followed by injection

AMSC: joint treatment

• I Cruciate Ligament arthroscopy assisted

• 2 subcondral bone cysts medial femoral condyle

• ecoguided implant in GA

• 1 elbow subcondral bone cyst

• trans cortical drilling, cyst debridment followed by injection

AMSC: joint treatment

• I Cruciate Ligament arthroscopy assisted

• 2 subcondral bone cysts medial femoral condyle

• ecoguided implant in GA

• 1 elbow subcondral bone cyst

• trans cortical drilling, cyst debridment followed by injection

AMSC: joint treatment

• I Cruciate Ligament arthroscopy assisted

• 2 subcondral bone cysts medial femoral condyle

• ecoguided implant in GA

• 1 elbow subcondral bone cyst

• trans cortical drilling, cyst debridment followed by injection

AMSC: joint treatment

• I Cruciate Ligament arthroscopy assisted

• 2 subcondral bone cysts medial femoral condyle

• ecoguided implant in GA

• 1 elbow subcondral bone cyst

• trans cortical drilling, cyst debridment followed by injection

AMSC: joint treatment

• I Cruciate Ligament arthroscopy assisted

• 2 subcondral bone cysts medial femoral condyle

• ecoguided implant in GA

• 1 elbow subcondral bone cyst

• trans cortical drilling, cyst debridment followed by injection

AMSC: joint treatment

• I Cruciate Ligament arthroscopy assisted

• 2 subcondral bone cysts medial femoral condyle

• ecoguided implant in GA

• 1 elbow subcondral bone cyst

• trans cortical drilling, cyst debridment followed by injection

Regeneration mechanism?

•Differentiation

•Cell fusion with already-differentiated cell

•Stimulate differentiation of tissue cells

•Angiogenis (new blood supply)

•Anti-apoptosis (stop cell death)

•Anti-inflammatory (inhibit degeneration)

•Anti-fibrotic (inhibit scarring)

•Supply growth factor

Horse as large animal model for chronic respiratory diseases

•Heaves or recurrent airway obstruction (RAO) is a common disease of horses similar to asthma (10-20% of adult horses)

HEAVES SHARES MANY FEATURES OF HUMAN ASTHMA

•lower airway inflammation

•non-completely reversible airflow obstruction

•bronchial hyperresponsiveness (Lowell, 1964; Robinson et al., 1995)

•increased respiratory efforts at rest, coughing and exercise intolerance

There is a general belief that asthma is a condition unique to humans

Similarities and differences between heaves of horse and other animal models of asthma (Leclere et al., 2011)

Similarities between horse and human

• Well-developed bronchial circulation (McLaughlin RF et al., 1961; McLaughlin RF Jr., 1990; Magno M., 1990)

• Poorly developed lobulation and respiratory

bronchioles

• Thick pleura

• Terminal bronchioles

• Bronchial artery-pulmonary shunts (McLaughlin RF et al., 1961)

Heaves and neutrophils Heaves is characterized by a neutrophils recruitment into

airway secretions after spontaneous antigenic

stimulation (50-60-90%)

There is also evidence that neutrophils are the predominant cells in the airway secretions of patients

with severe asthma

Interesting study of Lavoie et al. (2001) tested the neutrophilia in heaves asthma-like

Method

• Bronchoalveolar lavage fluid (BALF) using a fibre-optic endoscopy

• Total cell count by hemacytometer

• Differential cell count on cytospin slide stained with Giemsa

• In situ hybridization for equine IL-4, IL-5 , IFN-γ mRNA

Horses with heaves Control horses with no history or clinical sign of respiratory diseases

Results • Neutrophils predominated in

BALF of horses with heaves

• Lymphocytes and macrophages were the predominant cells in BALF from control

• The number of cells expressing IL-5 and IL-4 mRNA was

significantly greater in BALF cells of horses with heaves

• This cytokine profile (Th2-type) is associated with neutrophilia but not with eosinophilia

Like human asthma

Heaves is an allergic condition similar to that of human asthma

ALTER LUNG FUNCTION

contribute to development of non-reversible airway obstruction both in heaves and severe asthma

induce many structural changes such as • epithelial detachment and

regeneration • goblet cell hyperplasia • bronchial smooth muscles

hyperplasia

are capable of releasing mediators such as • elastase • free oxygen radicals • leukotriene B4

Treatment

Not known cure for heaves:

If not combined with organic dust avoidance, after drug administration the control of pulmonary neutrophilia remains modest

Organic dust avoidance Bronchodilatators and

corticosteroids

Adverse effects of corticosteroids

Administered sistematically

On the immune system, they cause:

• Transient peripheral neutrophilia and lymphopenia

• Alter the lymphocyte subpopulations

• Decrease the antibody response to vaccination

Administered by inhalation

• They have little systemic effects in horses with heaves

Innovative therapies are needed

• Potential ability of these endogenous cells is clearly limited when the injury is severe enough to cause irreversible respiratory failure

The neutrophilia determines structural changes sometimes not

reversible

The existence of endogenous adult stem/progenitor cells has been reported in various

key regions of the lung (40 TYPES OF CELLS)

(Ginagrieco et al., 2002; Kim et al., 2005)

(Hong et al., 2001)

(Hong et al., 2004)

Amniotic-derived cells and our hypothesis

• Probably they protect from rejection, potentially permitting their use in allotransplantation, and control inflammatory process

• AMCs may have beneficial effects in their own right in the therapy of heaves

HOMING IMMUNOMODULATORY PROPERTIES

IMMUNO-PRIVILEGED

Therapeutic potential of mesenchymal stem cells and their paracrine factors in acute lung injury

Several other paracrine factors may be important in reducing lung injury and enhancing repair (Matthay et al., 2010)

To test the effect of AMCs therapy

1. Labeled AMCs cells with PKH26

2. Evaluated the presence and permanence of AMCs cells in the horse BALF and biopsies after bronchial instillation

3. Evaluated the effect of AMCs on mouse lung inflammation induced by an endotoxin

1. PKH26 staining

Extremely stable fluorescence allows in vivo cell tracking studies particularly when labeled cells are to be followed for periods longer than a few weeks

Stable incorporation of fluorescence into lipid regions of the cell membrane

Unpublished data

Presence of AMCs

• BAL of the right accessory lung lobe performed in horses of both groups after 48 hours, 1, 2 and 4 weeks

• Cytospin for differential cell count

• Multiple endobronchial biopsies at the end of the BAL performed at 4th week

Group 1: animals treated with 20 ml PBS into the right accessory lung lobe (CTR)

Group 2: animals treated with 1*108 labeled AMCs into 20

ml PBS solution

2. Presence of fluorescent AMCs in BALF

1 week 30%

2 weeks 10%

4 weeks 5%

Unpublished data

2. Differential count (neutrophils)

• LIGHT NEUTROPHILIA DUE TO STABLES and BALF • IN TREATED HORSE IS CONTRASTED BY AMCs Unpublished data

2. Presence of fluorescent AMCs in endobronchial biopsy

4 weeks 10-15% of AMCs

Unpublished data

• Lesion induced by LPS that is an E. coli endotoxin

• LPS instilled into the distal airspaces of one lung lobe resulted in

acute pulmonary edema

increase in lung vascular permeability

elevated levels of most biologically active cytokines and chemokines found in pulmonary edema fluid

(Gupta et al., 2007; Mei et al., 2007; Lee et al., 2009)

T

This study is in progress

3. Evaluation of AMCs on lung inflammation

Summary

• We hypothesized that AMCs treatment shift the balance from pro-inflammatory to anti-inflammatory cytokines for their immunomodulatoy effect (rationale of the therapeutic strategy)

• Effect by direct contact or by production of soluble factors? Probably both effect as well as in our immunomodulatory experiment

Pro-inflammator factors

Anti-inflammator factors

Thanks to : • Prof Fausto Cremonesi, Reproduction Unit, University of Milan

• Dr Aurora Meucci, Reproduction Unit, University of Milan

• Dr Bruna Corradetti and prof Davide Bizzaro, Universitiy of Ancona for molecular biology

• Prof. Sheila Laverty and Prof. Jean Pierre Lavoie, Université de Montréal, Québec, Canada

• Prof . Francesco Ferrucci and Dott.ssa Enrica Zucca, Internal Medicine Unit, University of Milan for in vivo study

• Prof. Silvana Arrighi, University of Milan, for histology

• Dr Ornella Parolini, Centro Ricerca Menni Brescia Hospital, for immunomodulation study

• Dr Stefano Tassan, private practitioner, for in vivo study

• Many graduate and undergraduate students

Thank for your attention!