DOI: 10.1002/vms3.74
Original Article
Bone marrow-derived multipotent mesenchymal
stromal cells from horses after euthanasia
Carmen Schro€ck* , Carina Eydt†, Florian Geburek‡, Lena Kaiser*, Felicitas Pa€bst§,¶,
Janina Burk§,¶, Christiane Pfarrer† and Carsten Staszyk*
*Institute for Veterinary Anatomy, -Histology and -Embryology, Justus-Liebig-University, Giessen, Germany, †Institute of Anatomy, University of Veterinary
Medicine Hannover, Hannover, Germany, ‡Clinic for Horses, Justus-Liebig-University, Giessen, Germany, §Translational Centre for Regenerative Medicine
(TRM), University of Leipzig, Leipzig, Germany and ¶Faculty of Veterinary Medicine, Large Animal Clinic for Surgery, University of Leipzig, Leipzig,
Germany
Abstract
Allogeneic equine multipotent mesenchymal stromal cells (eMSCs) have been proposed for use in regenerative
therapies in veterinary medicine. A source of allogeneic eMSCs might be the bone marrow from euthanized
horses. The purpose of this study was to compare in vitro characteristics of equine bone marrow derived eMSC
(eBM-MSCs) from euthanized horses (eut-MSCs) and from narcotized horses (nar-MSCs). Eut-MSCs and nar-
MSCs showed typical eMSC marker profiles (positive: CD44, CD90; negative: CD11a/CD18 and MHCII) and
possessed tri-lineage differentiation characteristics. Although CD105 and MHCI expression varied, no differ-
ences were detected between eut-MSCs and nar-MSCs. Proliferation characteristics did not differ between eut-
MSCs and nar-MSCs, but age dependent decrease in proliferation and increase in MHCI expression was
detected. These results suggest the possible use of eut-MSCs for therapeutic applications and production of
commercial available eBM-MSC products.
Keywords: equine stem cells, flow cytometry, horse, proliferation assay, regenerative therapy.
Correspondence: Carsten Staszyk, Institute for Veterinary Anatomy, -Histology and -Embryology, Justus-Liebig-University,
Giessen, Germany.
E-mail: carsten.staszyk@vetmed.uni-giessen.de
approaches autologous sternal bone marrow is har-
Introduction
vested in order to isolate and expand eBM-MSCs.
Equine bone marrow-derived multipotent mesenchy- Although the application of autologous MSCs avoids
mal stromal cells, obtained from live horses, are the the risk of immunorejection of the applied cells,
focus of multiple experimental and clinical studies there are several problems associated with obtaining
evaluating their characteristics with regard to their autologous eBM-MSCs. First, the MSCs may be
use in regenerative therapies (Arnhold et al. 2007; altered by disease status and treatment with pharma-
Brehm 2008; Cortes et al. 2013; Renzi et al. 2013; ceutical products. Second, the harvesting procedure
Smith et al. 2013). As far as the authors are aware, bears the risk of thoracic and cardiac punctures
however, there have been no experimental studies (Jacobs et al. 1983; Durando et al. 2006). Third, the
reported which use eBM-MSCs obtained from eutha- production of an injectable autologous eBM-MSC
nized horses. It is widely accepted that multipotent product is a time consuming procedure that takes up
mesenchymal stromal cells (MSCs) selected for ther- to five weeks (Brems & Jebe 2008). These disadvan-
apeutic use express MSC-typical cell surface markers tages might be avoided by the use of allogeneic
such as CD44, CD90 and CD105, and provide exten- MSC-products obtained from euthanized or slaugh-
sive proliferative capacity in vitro (Iacono et al. tered horses. Thus, the alternative use of allogeneic
2012b; Spaas et al. 2013). For most therapeutic MSCs has been proposed for regenerative therapies
© 2017 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd. 239
Veterinary Medicine and Science (2017), 3, pp. 239–251
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which
permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no
modifications or adaptations are made.
240 C. Schro€ck et al.
in people (Alison & Caplan 2009; Heng et al. 2009)
as well as in horses (Watts et al. 2011; Iacono et al. Methods
2012a; Lange-Consiglio et al. 2013). The experimen-
Animals
tal use of allogeneic equine MSCs for treatment of
tendon lesions and other orthopaedic disorders has Animal subjects included in this study were 9 warm-
delivered promising results without significant blood horses, owned by the Equine Clinic of the
adverse effects (Carrade & Borjesson 2013; Lange- University of Veterinary Medicine Hannover, which
Consiglio et al. 2013). For the in vitro experiments, were first narcotized and later euthanized at the
the use of MSCs from euthanized horses would fol- Equine Clinic of the University of Veterinary Medi-
low the concept of replacement, reduction and cine Hannover for reasons unrelated to this study.
refinement in animal experiments. The aim of this The horses were divided into two groups: In five
study was to compare in vitro characteristics of eBM- horses bone marrow aspirates were gained before
MSCs obtained from euthanized and live horses par- euthanasia, in five horses bone marrow aspirates
ticularly focussing on immunophenotype. According were gained after euthanasia; one horse belonged to
to the suggestion of the ISCT (International Society both groups.
for Cellular Therapy) for the immunophenotyping of Horses that had undergone previous bone marrow
human mesenchymal stromal cells, a panel of posi- (BM) aspiration were excluded from this study. Par-
tive (CD44, CD90, CD105, MHCI) and negative ticular attention was paid to collect BM from donors
(CD11a/CD18, MHCII) commercially available stem from a wide range in age.
cell markers was assessed which had previously been
shown to cross react with equine cells. (Dominici
Isolation and expansion of BM-MSCs from live
et al. 2006; Burk et al. 2013). Instead of a commonly
horses
used doubling time assay, which takes an exponential
growing of the cell population for granted, a newly Bone marrow was aspirated from the sterna of five
designed proliferation assay was used to describe the horses under general anesthesia (No. 2, 4, 6, 8, 9b
proliferation curve, based on the logistical function according to Table 1) using a 13 G bone marrow
that includes lag-, log-, and stationary phase. Addi- biopsy needle (Angiotech, Wyomissing, USA).
tionally, the new proliferation assay enables discrimi- Horses were sedated with xylazine (Xylazin 2%
,
nation between fast-proliferating and slow- CP-Pharma GmbH, Burgdorf, Germany) and gen-
proliferating subpopulations within a given cell eral anesthesia was induced by ketamine (Narketan

population, possibly leading to a selection of fast 100 mg/mL, Vetoquinol GmbH, Ravensburg, Ger-
proliferating cells in the future. many) and midazolam (Midazolam
 5 mg/mL, B.
Table 1. Data of bone marrow donors.
Horse Bone marrow Euthanized/ Sex Breed Age Disease
aspirate narcotized
1 1 Eut Gelding Warmblood 28 years Colic
2 2 Nar Gelding Warmblood 28 years Infirmity
3 3 Eut Mare Haflinger 23 years Colic
4 4 Nar Mare Warmblood 22 years Lameness
5 5 Eut Gelding Warmblood 12 years Colic
6 6 Nar Mare Warmblood 16 years Lameness
7 7 Eut Gelding Haflinger 7 years Lameness
8 8 Nar Mare Warmblood 3 years Unknown
9a 9 Eut Mare Warmblood 2 weeks premature Sepsis
9b 10 Nar Mare Warmblood 2 weeks premature Sepsis
© 2017 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.
Veterinary Medicine and Science (2017), 3, pp. 239–251
MSC from euthanised horses 241
Braun Melsungen AG, Melsungen, Germany) using
Isolation and expansion of BM-MSCs from
standard drug dosages. The horses were placed in
euthanized horses
dorsal recumbency and general anesthesia was main-
tained by isoflurane (Isofluran CP
, CP-Pharma Five horses (No. 1, 3, 5, 7, 9a, Table 1) were eutha-
GmbH, Burgdorf, Germany) in clean oxygen. nized for reasons unrelated to this study. The horses
Horse No. 9a/b (Table 1) was sedated and narco- were sedated with xylazine (Proxylaz
, Bela-Pharm
tized as later described for euthanized horses. One GmbH & Co. KG, Vechta, Germany) and narcotized
sample of bone marrow was harvested under general using ketamine (Ketamin, 10%
, Bela-Pharm
anesthesia (BM aspirate No. 9, Table 1) and then a GmbH & Co. KG, Vechta, Germany) and diazepam
second one within 30 min after euthanasia (BM aspi- (Diazepam 10 mg
, Rotexmedica, Trittau, Ger-
rate No. 10, Table 1). many). Subsequently, these horses received T61

Five millilitre bone marrow aspirates were (Intervet, Unterschleißheim, Germany) for euthana-
obtained from sternebra 4 or 5 using ultrasound guid- sia. Standard drug dosages were used for all drugs.
ance according to Eydt et al. (2014) and carried to Within 30 min after euthanasia, 2 9 5 mL BM were
the lab within 15 min inside a styrofoam container. aspirated and processed as described for the live
The bone marrow samples were centrifuged at 103 horses.
rcf for 15 min and the generated cell pellets resus-
pended in 10 mL proliferation medium (pmol/L,
Flow cytometry analysis for immunophenotyping
Mensing et al. 2011). The suspension was filtered
through a 70 lm filter and was carefully layered over Two positive markers (CD90 and CD105) and one
14 ml Easycoll
 (1.086 g/mL, Biochrom AG, Berlin, negative marker (MHCII) were selected according
Germany). After density gradient centrifugation at to the recommendation of the ISCT for identifying
400 rcf for 35 min (without brake) the mononuclear human MSCs [19]. Additionally, the presence of
cell population (MNCs) was aspirated and cells were CD44, MHCI (positive markers) and absence of
washed two times in PBS (DPBS
 1x, Life Technolo- CD11a/CD18 (negative marker) was determined.
gies GmbH, Darmstadt, Germany). All cells Cells were incubated with 10% horse serum
harvested from 5 mL BM were seeded as passage 0 (donor horse serum, heat inactivated, PAA Labo-
into two 25 cm² cell culture flasks, containing ratories GmbH, Pasching, Austria) in PBS for
5 mL pmol/L. Medium was changed 4–24 h after 15 min at room temperature and washed using
seeding, and subsequently every 2–3 days. When 70– washing buffer (WB) containing 98.5% PBS, 1%
80% confluence was reached, after 7–22 days, cells BSA (PAA Laboratories GmbH, Pasching, Aus-
were detached using trypsin (0.05% Trypsin-EDTA, tria), 0.01% NaN3 (Merck-Schuchardt, Hohen-
Life Technologies GmbH, Darmstadt, Germany) brunn, Germany) and 0.5% goat serum (Institute
and half of the cells were cryopreserved for later use, for Veterinary Anatomy, Histology and Embryol-
the other half was passaged. In the first passage, cells ogy, Giessen, Germany) to block non-specific anti-
from each aspirate were seeded in two 75 cm² cell body binding. Subsequently, cells were pelleted in
culture flasks (1 9 105 to 3 9 105 cells) containing aliquots containing 2 9 105 cells on a 96-well plate
13 mL pmol/L and medium was changed every with V-bottom and incubated with primary anti-
2–3 days until 70–80% confluence was reached and bodies (Table 2). Cells were then washed twice
cells were further passaged or cryopreserved. The with WB and stained with appropriate secondary
same procedure was repeated in the passage 2 and 3. antibodies (Table 2). Then, cells were washed
Cells in passage 1, 2 or 3 reached confluence after twice, resuspended in PBS and at least 1 9 105
6–15 days. Approximately 4 9 106 cells were har- vital cells were analysed by flow cytometry (Accuri
vested from two 75 cm² flasks and used for flow C6
, BD Bisoscience, Heidelberg, Germany) using
cytometry analysis. Flow cytometry and proliferation Accuri C6 software (BD Bisoscience, Heidelberg,
assays were performed with cells from passages 1–3. Germany). Unlabelled cells, secondary antibody
© 2017 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.
Veterinary Medicine and Science (2017), 3, pp. 239–251
242 C. Schro€ck et al.
© 2017 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.
Veterinary Medicine and Science (2017), 3, pp. 239–251
Table 2. List of antibodies used.
Primary antibody Secondary antibody
Name Isotype Reactivity Clone Dilution Product number Fluorescence Isotype Reactivity Dilution Product
number
CD44 Rat IgG2b,k Mouse IM7 1:400 (1.25lg/mL) BD 553131 APC Goat Ig Rat IgG 1:600 (0.33lg/ mL) BD 551019
CD90 Mouse IgG1,k Human 5E10 1:400 (1.25lg/ mL ) BD 555593 PE Goat Ig Mouse IgG1, 1:800 (0.25lg/ mL) BD 550589
CD105 Mouse IgG1 Human SN6 1:500 (2lg/ mL) AbD Serotec IgG2a, IgG2b,
MCA 1557T IgG3, IgM,
MHCI Mouse IgG2a Horse CVS22 1:200 (5lg/ mL) AbD Serotec IgA
MCA1086GA
MHCII Mouse IgG1 Horse CVS20 1:200 (5lg/ mL) AbD Serotec
MCA1085GA
CD11a/ Mouse IgG1 Horse CVS9 1:200 (5lg/ mL) AbD Serotec
CD18 MCA1081GA
Isotype Corresponding antibodies Dilution Product number Fluorescence Isotype Reactivity Dilution Product
controle number
Rat IgG2b,k CD44 1:800 (1.25lg/ mL) Invitrogen APC Goat Ig Rat IgG 1:600 (0.33lg/ mL) BD 551019
02-9288
Mouse IgG1 CD105, MHCII, CD11a/CD18 1:200 (5lg/ mL) Invitrogen PE Goat Ig Mouse IgG1, 1:800 (0.25lg/ mL) BD 550589
02-6100 IgG2a, IgG2b,
Mouse IgG2a MHCI 1:200 (5lg/ mL) Invitrogen IgG3, IgM, IgA
02-6200
MSC from euthanised horses 243
only labelled cells and isotype controls were used 1.0 ml PBS. Afterwards, cell suspensions were
as control samples. Two different secondary anti- divided into 2 9 0.5 mL. One portion was thor-
bodies without spectral overlap were used for mul- oughly mixed with 0.5 mL eFluor 670 (1:200). The
ticolour analyses (Table 2). For negative markers other portion (control) was mixed with 0.5 mL
(MHCII, CD11a/CD18) positive controls were per- PBS. Both portions were incubated for 10 min at
formed on eBM derived MNCs, which include 37°C in the dark. Staining was stopped by adding
hematopoietic stem cells (data not shown). 2.5 mL cold pmol/L and by incubation on ice for
5 min (in the dark). Subsequently, the cells were
washed three times with pmol/L. Cells were
Gating strategy
counted by flow cytometry and seeded on 6-well
Dead cells were marked and excluded from the anal- plate at a density of 1 9 10³ cells/cm². During a
ysed gate, using the viability dye 7-AAD (BD Biso- period of 10 days, cells were collected successively
science, Heidelberg, Germany) in accordance with at six time points (including lag-, log- and station-
the manufacturers0 instruction. ary-phase) and were analysed by flow cytometry.
Obtained data were analysed by logistic regression
using the statistical software program BMDP6D
Modified Proliferation assay
(BMDP/Dynamic, Release 8.1 [Dixon, 1993]). The
The proliferation dye eFluor 670 (eBioscience, following parameters were determined and used
Frankfurt, Germany) is equally distributed to daugh- for comparison among the different donors (Fig. 1):
ter cells during mitosis. The manufacturer’s staining
protocol for suspension cells was adapted to the spe-
cial needs of adherent equine MSCs. 1. G: boundary value, the maximum cell number
To synchronize the cell cycle, 3.5 9 106 eBM- attainable from 9.6 cm² culture dish
MSCs, suspended in DMEM (DMEM 1x, 4.5 g/L 2. G1: time to reach the half boundary value
D-Glucose, Life Technologies GmbH, Darmstadt, 3. G2: maximal proliferation speed of the cell
Germany), were placed on a soft shaker at 8°C. population
After 12 h, cells were washed twice with pre- 4. G3: time to reach 1.5 9 105 cells
warmed (37°C) PBS and were resuspended in 5. G4: cell number after 150 h of cultivation time
Fig. 1. Example of a proliferation curve of eBM-MSCs. Logistical regression displaying the boundary value, the maximum cell number attainable
from 9.6 cm² culture dish (G), the time to reach half boundary value (G1), the maximal proliferation speed of the cell population (G2), the time
to reach 1.5 9 105 cells (G3) and the cell number after 150 h of cultivation time (G4).
© 2017 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.
Veterinary Medicine and Science (2017), 3, pp. 239–251
244 C. Schro€ck et al.
The calculated values (G–G4) were statistically
Adipogenic differentiation
analysed using a paired-sample t-test (eut- and nar-
MSCs from similarly aged donors were paired), a 2 9 104 cells/cm² were seeded on a 24-well plate sup-
covariance analysis and correlation diagrams, in plied with 0.5 mL pmol/L. Medium was changed
order to analyse differences between the assigned every 2–3 days until 70% confluence. Afterwards,
groups of MSCs from narcotized horses and MSCs adipogenic differentiation medium was added. Nega-
from euthanized horses and to determine the impact tive controls were formed by supplying pmol/L
of the donor age. A p-value below 0.05 was regarded instead of the differentiation medium. At day 14, the
as statistically significant. presence of fat droplets was demonstrated using Oil
After 3 days the number of cell divisions under- red O staining.
gone by each cell was calculated by measuring the
decrease in fluorescence intensity in relation to the
Results
initial fluorescence intensity. This information was
used to determine the number of cells from different Viable, plastic adherent and proliferating cells were
cell generations within a population. The assessment obtained from all 10 bone marrow aspirates.
of the generation number was limited to the 6th cell
generation as the fluorescence intensity of eFluor
Immunophenotypic characterisation
670 decreases after that time. All experiments were
performed in triplicate. All eBM-MSCs from narcotized horses and from
euthanized horses presented high percentages (>90%)
of CD44 and CD90 positive cells (Figs. 2, 3). The per-
Differentiation assays
centages of other positive markers of MSCs (MHCI,
To confirm tri-lineage differentiation capacity CD105) varied widely between MSC populations.
according to the ISCT (Dominici et al. 2006), osteo- MHCI was present in 91.4%  7.1 of eut-MSCs and
genic, chondrogenic and adipogenic differentiation in 76.7%  19.1 of nar-MSCs respectively. CD105
assays were performed using MSCs obtained from was expressed in 33.9%  27.2 of eut-MSCs and in
the oldest and the youngest horses as examples of 45.7%  20.9 of nar-MSCs respectively. None of the
the assigned groups (No. 1, 2, 9a and 9b; Table 1). detected differences were statistically significant.
The expression of the MSC negative marker
(CD11a/CD18) and of MHCII was consistently low
Osteogenic differentiation/chondrogenic
(<2.2%) in all nar-MSCs and eut-MSCs.
differentiation
A covariance analysis showed a significant
3 9 105 cells were placed into a 15 mL tube within correlation (P = 0.016) between the age of the donor
0.5 mL medium for osteogenic or chondrogenic dif- horse and the expression of MHCI. The percentage
ferentiation and centrifuged for 1 min with 100 rcf. of MHCI expressing cells increased per year of age
Pellets were cultured under standard conditions, 5% by 1%.
CO2 at 37°C. Negative controls were supplied with
pmol/L instead of differentiation media. Medium was
Proliferation characteristics
changed every 2–3 days. At day 14, the pellets were
fixed in 10% buffered formalin, and processed rou- According to the calculated cell divisions, cells were
tinely for histology. Osteogenic differentiation was assigned to 4 proliferation types (Figure 4):
demonstrated by Von-Kossa staining showing miner-
alization (Arnhold et al. 2007; Cortes et al. 2013). 1. Type I: Slow-proliferating cells (generation 1 and
Chondrogenic differentiation was demonstrated by 2)
Alcian blue staining showing acidic polysaccharides 2. Type II: Moderate-proliferating cells (generations
(Colleoni et al. 2009; Cortes et al. 2013). 3–5)
© 2017 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.
Veterinary Medicine and Science (2017), 3, pp. 239–251
MSC from euthanised horses 245
Fig. 2. Flow cytometry analysis. Histograms display fluorescence intensity on x-axis and cell counts on y-axis. Nar-eBM-MSC and eut-eBM-MSC
highly expressed CD44 and CD90. CD105 and MHC I expression varied in wide ranges. CD11a/CD18 and MHCII were expressed by very few
cells. Left peaks represent isotype control stainings, right peaks represent antibody staining with corresponding percentage of positive cells
(percentages shown as mean of measured doublets).
© 2017 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.
Veterinary Medicine and Science (2017), 3, pp. 239–251
246 C. Schro€ck et al.
Fig. 3. Marker expression of eBM-MSCs. Box plots of the distribution of percentages of eBM-MSCs from euthanized and narcotized horses,
which are positive for the marker expressions. Boxes represent the lower and upper quartiles, lines inside the boxes are medians, whiskers rep-
resent minimum and maximum values and circles represent mean values. Eut-MSCs and nar-MSCs show no significant differences (P > 0.05).
Fig. 4. Proliferation speed of eBM-MSCs. Percentage of slow- (Type I, red), moderate- (Type II, green), fast- (Type III, purple) and non- (Type
IV, blue) proliferating cells in expanded cultures from bone marrow aspirates 1–10. Means of triplets displayed.
3. Type III: Fast-proliferating cells (generations >5) contained less than 2.1% fast-proliferating cells. All
4. Type IV: Non-proliferating cells (generation 0) nar-MSCs (except horse No. 4) contained more than
2.4% fast-proliferating cells (Figure 4).
From 30 cell cultures (triplets from each culture Correlation diagrams demonstrated that the maxi-
obtained, that is, 15 nar-MSCs, 15 eut-MSCs), 23 (12 mum proliferation speed (i.e. parameter G2)
nar-MSC and 11 eut-MSC cultures) contained a decreased with increasing age of the donor. No sta-
heterogeneous mixture of all defined proliferation tistically significant differences between nar-MSCs
types, and the remaining 3 nar-MSC and 4 eut-MSC and eut-MSCs were detected.
cultures contained no fast-proliferating cells. In 10
nar-MSCs and 11 eut-MSCs, the majority of cells
Tri-lineage differentiation
were assigned to the moderate proliferating cell type.
In 5 nar-MSCs and in 4 eut-MSCs the majority of All investigated cells showed their tri-lineage differ-
cells were slow-proliferating cells. All eut-MSCs entiation potential and differentiated into
© 2017 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.
Veterinary Medicine and Science (2017), 3, pp. 239–251
MSC from euthanised horses 247
Fig. 5. Results of tri-lineage differentiation assays of eBM-MSCs. Von Kossa staining following osteogenic differentiation (a) and corresponding
negative control (b). Alcian blue staining following chondrogenic differentiation (c) and corresponding negative control (d). Oil red O staining fol-
lowing adipogenic differentiation (e) and corresponding negative control (f). Scale bar = 50 lm.
adipogenic, chondrogenic and osteogenic directions (Brems & Jebe 2008; Bourzac et al. 2010), however,
(Fig. 5). All controls did not differentiate. there are previous studies, that obtained BM-MSCs
also from narcotized horses (Frisbie et al. 2009).
Although the risks of this method are minimal
Discussion
(Kasashima et al. 2011; Eydt et al. 2014), the horses
The potential use of allogeneic equine MSCs for are exposed to stress and pain. Another disadvantage
regenerative therapies could help to overcome disad- is the long post-harvesting time required to produce
vantages associated with the harvest of autologous an injectable autologous cell product (Brems & Jebe
bone marrow. The references show, that autologous 2008). The disadvantages of the autologous sampling
bone marrow is usually taken from sedated horses method could possibly be avoided if MSCs from one
© 2017 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.
Veterinary Medicine and Science (2017), 3, pp. 239–251
248 C. Schro€ck et al.
healthy horse, cryopreserved in many samples, could
Immunophenotyping
be used for therapy in many patients. Additionally
using MSCs from euthanized horses may increase The MSC markers used in this study (positive mark-
their availability for research. ers CD44, CD90, MHCI, CD105, negative markers
Recently, bovine BM-MSCs were successfully har- CD11a/CD18, MHCII) were selected in accordance
vested from abattoir-derived bovine fetuses (Cortes with several studies characterizing equine MSC from
et al. 2013). Vital, proliferating cells, expressing a different sources (Burk et al. 2013). Although the
typical set of MSC markers were obtained and multi- utilized antibodies for CD44 and CD90 were pro-
lineage differentiation was demonstrated. As shown duced against non-equine species, they possess a high
in our data, vital and proliferating cells are also level of cross reactivity and were previously recog-
obtainable from euthanized horses. Mesenchymal nized as reliable markers for equine MSC (Arnhold
stromal cells harvest and expansion was successful et al. 2007; Mensing et al. 2011; Burk et al. 2013). To
for at least 30 min after euthanasia, however, the ensure reliable immunophenotyping, we used equine
maximum time frame for successful MSC harvest specific antibodies against MHCI, CD11a/CD18 and
from dead animals remains to be determined, as nei- MHCII. As expected, the vast majority of plastic
ther our study nor the study presented by Cortes adherent and expanding cell cultures highly
et al. (2013) were designed to investigate this intrigu- expressed CD44, CD90 and MHCI, but did not show
ing parameter. In our preliminary experiments, vital CD11a/CD18 and MHCII reactivity. Although the
eBM-MSCs were successfully obtained even five cross reactivity of anti-human CD105 was confirmed
hours after euthanasia (data not shown). by the manufacturer, the expression of CD105 was
Unfortunately it was not possible to gain paired highly variable. Therefore, CD105 did not serve as a
samples from each horse before and after euthanasia, reliable marker, as previously reported for equine
as was done for horse No. 9. Therefore, we tried to peripheral blood MSCs (Spaas et al. 2013).
get samples from horses in a wide range of age in According to the established marker panel, no sig-
both groups to get an overview about the characteris- nificant differences between nar-MSCs and eut-MSCs
tics of the cells from horses of different ages. were present. Thus, cells with the ability to express
Previous studies suggested the use of fetal abat- typical stem cell markers are obtainable from eutha-
toir-derived bovine BM-MSCs for regenerative ther- nized horses for at least 30 min after euthanasia. This
apies because the cells show typical characteristics of result supports a hypothesis that emphasizes the need
bovine MSCs from live animals (Cortes et al. 2013). for a hypoxic environment in the stem cell niche. It
However, there is no information about possible dif- has been proposed that due to a hypoxic environment
ferences between MSCs obtained from abattoir- prevents cellular processes that promote cell differenti-
derived fetuses and those from live animals. ation and therefore conserve the undifferentiated sta-
Although the harvest of BM-MSCs from eutha- tus of the stem cell (Ivanovic 2009). It is hypothesized
nized horses provides a new opportunity to collect that lack of adverse reactions for allogeneic treatments
large quantities of cells, potential differences between is due to the absence of MHCII and the immunosup-
BM-MSCs obtained from euthanized horses and live pressive effect of MSCs in general (Heng et al. 2009;
horses might restrict the speculative application. Rafei et al. 2009; Roemeling- Rhijn et al. 2013; Li et al.
Therefore, we performed a comparative evaluation of 2014). Nevertheless, the range of different conditions
BM-MSC characteristics that were recognized as cru- the horses had before nar- or eut-MSCs were har-
cial parameters for the potential use of MSCs in vested, did not affect the expression of the markers.
regenerative therapies. Special attention was paid to
use a reliable panel of equine MSC markers to investi-
Proliferation capacity
gate selected criteria relevant for allogeneic applica-
tions and to provide detailed information about the An essential aspect for evaluating MSCs in vitro is
particular proliferation capacity of the cells. their capacity to proliferate (Dominici et al. 2006).
© 2017 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.
Veterinary Medicine and Science (2017), 3, pp. 239–251
MSC from euthanised horses 249
Moreover, it is assumed that the in vitro proliferative demonstrated telomere shortening in human BM-
capacity is positively correlated with the regenerative MSC with increasing donor age, limiting the regener-
capacity of MSCs (Gilbert & Blau 2011). Most ative capacity of these cells (Baxter et al. 2004).
assessments of the proliferative capacity are con-
ducted by calculating the cell doubling time (DT)
Conclusion
based on the number of seeded cells and the number
of cells after a defined time in the culture (Vidal To the best of the authors0 knowledge, the harvest of
et al. 2007; Iacono et al. 2012b; Cortes et al. 2013). vital and proliferative eBM-MSCs from euthanized
This procedure provides valid results for a phase of horses has not been previously described. In this
exponential cell proliferation. However, prolifera- study, eBM-MSCs obtained from euthanized horses
tion of cell populations in vitro is rather character- and from live horses did not differ in terms of expres-
ized by a non-exponential, s-shaped proliferation sion of MSC markers or proliferative capacities. We
curve. Thus, the mere doubling time provides only suggest that eut-MSCs are suitable at least for scien-
limited information. tific studies based on in vitro techniques. This would
In order to detect cells displaying different prolif- meet the obligation for replacement, reduction and
eration capacities within a cell population we con- refinement in animal experiments. Moreover, the
ducted a modified proliferation assay utilizing a results obtained here suggest a potential for use of
proliferation dye and flow cytometry. As previously eut-MSC as commercially available products in allo-
investigated, the data corresponded to the typical s- geneic MSC therapies. This assumption has been
shaped proliferation curve of expanding cells in vitro supported by several clinical studies that successfully
and therefore allowed detailed analysis of prolifera- utilized allogeneic equine MSCs derived from fetal
tion characteristics. The identification of different tissues (Watts et al. 2011; Iacono et al. 2012a; Lange-
cell generations elucidated a heterogeneous mixture Consiglio et al. 2013) and from BM of adult donor
of subpopulations with different proliferative capaci- horses (Guest et al. 2008). Moreover, allogeneic
ties. This result was unexpected as other investigated MSCs were already successfully applied in clinical
equine MSC cultures were characterized by very human studies (Alison & Caplan 2009; Chang et al.
homogeneous cell content (Arnhold et al. 2007). 2014; Guest et al. 2008).
However, homogeneity of MSCs in these reports was This study is limited by the number of donor
determined according to their fibroblast-like mor- horses and their individual differences in disease, age
phological characteristics. Furthermore, the applied and genetics. Although these differences might have
proliferation assays were not able to detect differ- effects on the characteristics of the harvested MSCs,
ences in proliferation characteristics among cells no significant differences according to the used mar-
within a single culture. Pre-selection of fast-prolifer- ker panel were detected. These results justify further
ating cells could improve the quality of cell products investigations to confirm the presented data and to
for research and therapy. However, the data evaluate the possible negative effects of euthanasia
obtained here do not provide cellular markers suit- on the target cells, with special consideration for the
able for the predictive selection of those cells. technique and chemicals used for that purpose. The
Although statistical analyses did not reveal any use of MSCs from slaughtered instead of euthanized
proliferation differences between eut-MSCs and nar- horses could be considered to avoid influences from
MSCs, there was a negative correlation between the diseases or drugs on the obtained cells.
age of the donor horse and the maximum prolifera-
tion speed. This might be of importance for selecting
Acknowledgements
appropriate donors. Besides an accelerated in vitro
expansion speed, eBM-MSCs collected from younger The authors wish to thank Prof. Dr. Kerstin Fey,
horses might possess advantageous regenerative Dr. Katja Roscher and Kim Theuerkauf from the
capacities. This assumption is based on studies that Clinic for Horses of the Justus Liebig University,
© 2017 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.
Veterinary Medicine and Science (2017), 3, pp. 239–251
250 C. Schro€ck et al.
Gießen, Germany for the support in bone marrow cells. American Journal of Veterinary Research 231,
acquisition and Mrs Sherwood-Brock for proof read- 1095–1105.
Baxter M.A., Wynn R.F., Jowitt S.N., Wraith J.E., Fairbairn
ing the manuscript.
L.J. & Bellantuono I. (2004) Study of telomere length
reveals rapid aging of human marrow stromal cells fol-
Source of funding lowing in vitro expansion. Stem Cells 22, 675–682.
Bourzac C., Smith L.C., Vincent P., Beauchamp G.,
This work was supported by a grant from the Ger- Lavoie J. & Laverty S. (2010) Isolation of equine bone
man Federal Ministry for Economic Affairs and marrow-derived mesenchymal stem cells: a comparison
between three protocols. Equine Veterinary Journal 42,
Energy, 372 AiF Project GmbH (Grant #
519–527.
KF2994201AJ2). Brehm W.. (2008) Equine mesenchymal stem cells for the
treatment of tendinous lesions in the horse. IVIS, 10th
International Congress of World Equine Veterinary
Conflict of Interest Association. 2008.
Brems R., Jebe E.(2008). Comparison of treatments with
None of the authors have any financial or non-finan-
autolog; cultured stem cells from adipose tissue or bone
cial competing interest which could inappropriately
marrow. IVIS, 10th International Congress of World
influence or bias the content of the paper. Equine Veterinary Association.
Burk J., Badylak S.F., Kelly J. & Brehm W. (2013) Equine
cellular therapy-from stall to bench to bedside? Cytome-
Ethical Statement try Part A 83, 103–113.
Carrade D.D. & Borjesson D.L. (2013) Immunomodula-
Nine warmblood horses were euthanized for reasons
tion by mesenchymal stem cells in veterinary species.
other than for this study in the Equine Clinic of the Comparative Medicine 63, 207–217.
University of Veterinary Medicine Hannover. This Chang Y.S., Ahn S.Y., Yoo H.S., Sung S.I., Choi S.J., Oh
study was approved by the Ethics Committee of the W.I. & Park W.S. (2014) Mesenchymal stem cells for
University of Veterinary Medicine, Hannover, Foun- bronchopulmonary dysplasia: phase 1 dose-escalation
clinical trial. Journal of Pediatrics 164, 966–972.
dation, Germany and by the responsible German
Colleoni S., Bottani E., Tessaro I., Mari G., Merlo B.,
federal state authority (Lower Saxony State Office
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for Consumer Protection and Food Safety, 362 33.9- entiation of equine mesenchymal stem cells: effect of
42502-04-11/0572). donor, source, amount of tissue and supplementation
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Contributions
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differentiation of bone marrow mesenchymal stem cells
and design. CSc, LK, FG and CP contributed to col-
from abattoir-derived bovine fetuses. BMC Veterinary
lection and compilation of data. CSc, FP, JB and CSt Research 9, 133.
analysed and interpreted the data. CSc and CSt Dixon W.J. (chief editor), (1993). BMDP Statistical Soft-
wrote the paper. All authors revised and edited the ware Manual, Volume 1 and 2, University of California
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Dominici M., Le Blanc K., Mueller I., Slaper-Cortenbach
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