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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: lisley@celltrovet.com.br

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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