cellule staminali di derivazione amniotica nella medicina ... · derivazione amniotica nella...
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“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.
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
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
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
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. Differential count (neutrophils)
• LIGHT NEUTROPHILIA DUE TO STABLES and BALF • IN TREATED HORSE IS CONTRASTED BY 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