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CHARACTERIZATION OF MULTIPOTENT EQUINE ADIPOSE TISSUE-DERIVED
PROGENITOR CELLS. CLINICAL CASE REPORTS OF ALLOGENEIC CELL
THERAPY IN HORSES
Poster · April 2009
DOI: 10.13140/RG.2.2.29290.00969
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Lisley I. Mambelli
Instituto Butantan
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Enrico Santos
CELLTROVET
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André L V Zoppa
University of São Paulo
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CHARACTERIZATION OF MULTIPOTENT EQUINE ADIPOSE TISSUE-DERIVED
PROGENITOR CELLS. CLINICAL CASE REPORTS OF ALLOGENEIC CELL
THERAPY IN HORSES.
LI. Mambelli1,2; E.J.C Santos, PhD*1; Lizier, N.F3; Maranduba, C.M.C.3; P.J.R. Frazão4; M.B. Chaparro4; A.L.V. Zoppa, PhD4. 1. Celltrovet Veterinary Activities Ltda. (CELLTROVET), Sao Paulo, Brazil, 2. Department of anatomy, School of Veterinary Medicine and Animal Science,
University of Sao Paulo, Sao Paulo, Brazil. 3. Laboratory of Genetic, Butantan Institute, São Paulo, Brazil; 4.Department of Surgery, School of Veterinary
Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil.
E-mail: [email protected]
Introduction. In horses, stem cell therapies are a promising tool to the treatment of many injuries, which are common consequences of athletic endeavor, resulting in high morbidity and often
compromising the performance. Previously, we reported the isolation and differentiation of equine adipose tissue-derived progenitor cells (eAT-PC) into mesodermal derivates and also showed the
potential of these cells to maintain their stemness even after crypreservation. The aim of this study was further characterization of eAT-PC differentiation potential and application of allogenic eAT-
PC for the treatment of tendonitis in horses.
Methods. eAT-PC were maintained under conditions previously described (Mambelli et al., 2009). Differentiation towards smooth and skeletal muscles and also neuronal cells was performed after
thawing following routine protocols, slightly modified. Mouse anti-human antibodies: anti-myosin, anti-α-actinin, anti-MyoD1, anti-beta-tubulin-III; as well as rabbit anti-human anti-nestin and
anti-glial fibrilary acidic protein (GFAP) were used after cell fixation in 4% paraformaldehyde. The expression of cell specific proteins was analyzed under confocal microscopy. Twelve animals
with tendonitis received 107 of eAT-PC into the injured tissue under local anesthetic and ultrasonographic control. After one month, ultrasonographic control was performed again. All procedures
were approved by horse owners under signature of a veterinary service contract.
Results. After the induction of miogenic differentiation, the cells presented first sings of morphological changes similar to muscle cells, at day 10. Myosin, α-actinin and MyoD1 antibodies
showed positive immunostaining with progenitor cells confirming muscles cells differentiation. Neuronal differentiation was evidenced by morphological changes, which lead to outgrowth
formation and nucleous dislocation. Prior differentiation into neuronal lineages, eAT-PC already presented strong nestin positive immunolabelling. After differention neuron-like cells derived from
eAT-PC reacted positively with such markers as beta-III-tubulin and GFAP. Functional test are being provided. Respective controls used in both studies did not present any specific labeling with
the same antibodies. Since our study was based on clinical cases, the animals were heterogenous for age, weight and sex, but all of them were athletic horses. One month after eAT-PC application
into the lesion, the formation of healthy tissue has been observed. All treated horses showed a functional recovery and were able to return to their normal activity, without lesion recurred.
Conclusion. Extending our previous findings, we showed that eAT-PC posess all characteristics of mutipotent adult stem cells. Besides differentiation into bone, cartilage and adipose cells
(Mambelli et al., 2009) additionally, they were able to produce smooth, skeletal muscles and neuron-like cells. Their application in horses provided functional recovery of damaged tendons and
treated animals were capable to return to their normal activity. Our findings classify eAT-PC isolated and cultured in vitro, as a promising tool for cell-therapy, which maintain their potential even
after cryopreservation. Further studies are needed in order to understand the mechanism of their action on damaged tissues recuperation.
Figure 1. Isolation of equine adipose tissue-derived progenitor cells (A-E). (B)
Biopsy of horse fat. (C) The pieces of fat were washed and digested by
collagenase type IV. (D) Tissue explants. (E) Fibroblast-like cells isolated from
equine adipose tissue explant.
A
B
C
D
E
Isolation
eAT-PC Morphology and Cell Doubling
A
B
Figure 4. Morphology of eAT-PC at different passages, P2 (A) and P20 (B). The cells proliferative potencial was evaluated by cell doubling
method before and after cryopreservation (C).
C
Figure 9. Osteogenic Differentiation before (A–C, G) and after (D–F, H) cryopreservation. (A) Initial
mineralization of extracellular matrix observed (without any type of staining) after 11 days of
differentiation. (B) Strong mineralization observed after 21 days. (C) The cells cultured (21 days) in
control osteogenic medium did not present any signs of mineralization. Von Kossa staining revealed
calcified extracellular matrix in experimental culture (D) at day 11 and (E) at day 21. (F) Control
culture (21 days) remained von Kossa negative (G, H). Overview of osteogenic differentiation observed
at day 21 in two experimental (upper) and two control (down) wells before (G) and after (H)
cryopreservation. Positive immunostaining (green) was observed after induction of osteogenic
differentiation (11 days) with antiosteocalcin LF-32 (I) and anti-bone sialoprotein (K) antibodies (both
after cryopreservation). Control undifferentiated eAT-PC did not react with both antibodies (J, L).
Osteogenic Differentiation
A B
C D
Adipogenic Differentiation
Figure 6. Adipogenic Differentiation (A,C-D). Non-
stained experimental culture (A) and stained by Oil Red O
(B) control culture. Experimental culture stained by Oil
Red O, four (C) and seven (D) days after induction of
adipogenic differentiaton.
A B
C D
E F
Chondrogenic Differentiation
Figure 8. Three-Dimensional Chondrogenic
Differentiation (A-C, E). (A-B): Histological study of
differentiated eAT-MSCs stained by Toluidine blue.
Immunohistochemistry by confocal microscopy
analysis with specific antibodies against Aggrecan (C)
and Collagen type II (E). As expected, the controls not
reacted with the same markers (D and F). Nuclei
stained by DAPI in blue.
Figure 7. Miogenic Differentiation (A-C). Non-stained
experimental culture (A). Immunocitochemistry by confocal
microscopy analysis with specific antibodies against Miosin (B)
and MyoD1 (C). Nuclei stained by DAPI in blue.
Miogenic Differentiation
A
B C
Neural Differentiation
A B
C D
Figure 10. Neural Differentiation (A-D). Non-stained experimental culture (A). Experimental culture
stained by hematoxylin ⁄ eosin (B). Immunocitochemistry by confocal microscopy analysis with specific
antibodies against β-tubulin III (C) and GFAP (D). Nuclei stained by DAPI in blue.
Before application of eAT-PC
One month after application of eAT-PC
A
B
Figure 11. Ultrasonographic control (A-B). (A) Lesion
ultrasonographic control before application of eAT-PC. (B) Lesion
ultrasonographic control one month after application of eAT-PC,
showing a healthy tissue formation.
Ultrasonographic Control
GFP
A B
C
Figure 5. eAT-PC transductioned with GFP (A-B). (C)
eAT-PC transductioned with GFP were found in mouse
skin.
200 bp
Figure 2. RT-PCR. Expression analysis of
horse associated genes as mesenchymal
(CMSC; 191bp) and embryonic (CESCG1;
157bp) markers.
Figure 3. Karyotype analysis of eAT-PC
stained with Giemsa. Normal karyotype
(2n=64 chromosomes)
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