Materials and Methods

Animals and UCB collection—Umbilical-cord blood samples were collected from mares housed at 2 Thoroughbred breeding facilities and 1 Quarter Horse farm in California and the Center for Equine Health at the University of California-Davis. Consent for participation was obtained from owners and farm managers for mares housed at the private facilities. In brief, immediately after parturition and prior to breaking of the umbilical cord, the umbilical vein of each mare was isolated and manually disinfected by cleansing the region 3 times with chlorhexidine surgical scrub solution,^a followed by 4 × 4-cm gauze pads^b soaked in 70% isopropyl alcohol.^c Then, personnel collected as much UCB as possible via venipuncture (2.5-cm, 16-gauge needle) into a 250-mL bag containing citrate-phosphate–dextrose-adenine anticoagulant.^d Most samples were placed in a 4° or 22°C shipping container^e equipped with a continuous temperature–recording device^f for overnight shipping.

UBC-sample processing—When samples arrived at the Veterinary Blood Bank of the William R. Pritchard Veterinary Medical Teaching Hospital, University of California-Davis, the shipping containers were opened, the temperature data were recorded, and a 5-mL aliquot of UCB was removed for microbiological analysis, CBC, and determination of cell viability. Microbiological analyses were performed at the microbiology laboratory of the teaching hospital according to routine microbiological methods (aerobic bacterial culture, anaerobic bacterial culture, and fungal culture). The CBCs were performed by personnel at the hematology laboratory of the teaching hospital by use of an automated hematology analyzer.^g Cell viability was assessed microscopically with a hemacytometer and 0.4% trypan blue^h stain, which was applied in accordance with the manufacturer's instructions.

For processing, hetastarchⁱ (20% by volume) was added to the collection bag. Samples were processed with a closed automated system^j that reduces blood volume and the number of RBCs and enriches nucleated cells from UCB samples, following manufacturer's instructions. A final volume of 21 mL/sample was obtained. After processing, half of the sample was processed fresh, and the remainder was cryopreserved.

Isolation and culture of MSCs—For culture, all UCB samples were diluted 1:1 with Dulbecco PBS solution,^h layered over 10 mL of sterile lymphocyte-isolation medium,^k and centrifuged at 500 × g for 20 minutes. Mononuclear cells were plated in DMEM (low glucose concentration)^h supplemented with 10% defined fetal bovine serum,^l 10% equine serum,^l and 1% penicillin-streptomycin,^h hereafter referred to as maintenance medium. Cells were passaged at 70% confluence by incubating for ≤ 5 minutes with 0.05% trypsin-EDTA.^h Cell culturing was continued until 1 × 10⁷ MSCs (cell dose) were obtained. For the first 24 UCB samples received by the laboratory, mononuclear cells were collected and plated at cell densities ranging from 7.5 × 10⁵ cells/cm² to 5.0 × 10⁶ cells/cm² in standard tissue culture plastic flasks,^m and samples were incubated at 37°C with 5% CO₂ and 21% O₂. After 1 × 10⁷ MSCs were reached, the first 6 highly proliferative MSC samples were maintained in culture for long-term passage until the cells underwent senescence. Senescent cells were defined as cells that increased in size relative to other cells, altered in morphology from spindle shaped to polygonal, and ceased to proliferate.^15,28 Culture of nucleated cells from UCB in standard tissue plastic flasks^m and normoxic conditions (21% O₂), as described previously, was performed for the first 24 samples received.

Because hypoxic conditions reportedly increase proliferation of human and rat MSCs,^19,26,27 the next 5 UCB samples received by the laboratory were used to compare MSC growth kinetics in normoxic conditions with growth kinetics in hypoxic conditions. Each UCB sample was divided in 2. Half of the initial UCB-derived nucleated cells were plated on standard tissue culture plastic plates and placed in an incubator set to standard normoxic conditions (21% O₂), and the other half of the nucleated cells were placed under reduced oxygen tension (hypoxic conditions [5% O₂]) that was induced by use of incubators flushed with humidified gas mixtures of N₂ and CO₂ to adjust incubator conditions to 5% O₂, 5% CO₂, and 90% N₂.

After basic growth kinetics were defined, the next 9 UCB samples received by the laboratory were used to evaluate MSC isolation and proliferation by use of fibronectin-coated plates.ⁿ Each sample was divided in 2. Half of the initial UCB-derived nucleated cells were plated in standard tissue culture plastic plates,^m and the other half were plated on fibronectin-coated, 6-well tissue culture platesⁿ at identical densities (1 × 10⁶ cells/cm²). Cell colonies were counted at the time of first passage. Afterward, MSCs were plated on standard tissue culture plastic plates at 1 × 10⁶ cells/cm². Because use of the fibronectin-coated plates yielded an improvement in the isolation rate and monolayer expansion of MSCs, this technique was adopted as standard culture protocol for the remaining 13 UCB samples, and nucleated cells recovered from those samples were plated at 1 × 10⁶ cells/cm² in fibronectin-coated plates.

Evaluation of effects of cryopreservation—Immediately after UCB sample processing, half of most samples were cryopreserved. In brief, volume-reduced and RBC-depleted UCB samples (21-mL final volume) were stored at 4°C for a minimum of 20 minutes. Five milliliters of dimethyl sulfoxide–dextran^o (55% dimethyl sulfoxide [wt/vol], 5% dextran [wt/vol; molecular weight, 40 kDa]) was slowly added through a 0.2-μm filter (final freezing volume, 26 mL; range, 1 × 10⁸ cells/mL to 1 × 10⁹ cells/mL). Storage bags were heat sealed and placed in a small metal canister.^p Samples were frozen in an automated, robotic controlled-rate liquid nitrogen cryopreservation system,^q in accordance with the manufacturer's instructions.

Thawing and processing after cryopreservation—Six processed, volume-depleted, nucleated cell–enriched UCB samples that were cryopreserved prior to MSC isolation were later thawed to evaluate postthaw cell recovery, cell viability, and sample sterility. Thawing was performed as described elsewhere,²⁹ with adaptations for equine UCB. Briefly, UCB samples were placed in vapor-phase liquid N₂ for 15 minutes, then quickly immersed in a 37°C water bath. Thawing solution (150 mL) consisting of 2.5% (wt/vol) bovine albumin^r and 5% (wt/vol) dextran^s in saline (0.9% NaCl) solution was slowly added to the UCB. Samples were centrifuged at 400 × g and 10°C, and cells were resuspended in 10 mL of MSC maintenance medium.

In addition, MSCs that were isolated and expanded from 5 fresh UCB samples were cryopreserved, then thawed to evaluate cell recovery and cell viability of expanded MSCs after thawing. Thawing was performed as described for UCB samples.

In vitro differentiation of MSCs—Mesenchymal stem cells obtained from 8 UCB samples were induced to differentiate in osteogenic, adipogenic, and chondrogenic conditions. For osteoblast differentiation, 1 × 10⁴ MSCs (from passages 4 to 7) were plated in each well of a 24-well plate in DMEM (low glucose concentration) supplemented with 5% defined fetal bovine serum,^l 5% equine serum,^l and 1% penicillin-streptomycin.^h After 3 days of culture, osteoinductive medium containing ascorbic acid 2-phosphate^t (25 μg/mL), β-glycerolphosphate^u (5mM), dexamethasone^o (100nM), and rhBMP6^v (200 ng/mL) was added.³⁰ The medium was replaced every 3 days with fresh osteoinductive medium without rhBMP6 because bone morphogenic proteins act at an early stage of osteoblast differentiation and therefore continuous treatment is likely not necessary.^30,31 After 8 days of induction, the cells were harvested for detection of alkaline phosphatase and staining with alizarin red S.

Cells were stained for alkaline phosphatase by use of an alkaline phosphatase cytochemical staining kit^w in accordance with the manufacturer's instructions. Briefly, UCB MSCs were rinsed 3 times in PBS solution and fixed in citrate-acetone-formaldehyde solution. The MSCs were rinsed in ultrapure water and stained with substrate solution for 15 minutes at room temperature (approx 22°C). The cells were subsequently rinsed 3 times in deionized water and examined by means of photomicroscopy.

At day 12 to 16 of induction, the UCB MSCs were washed in 1X PBS solution and fixed with 50% ethanol for 3 minutes. The ethanol solution was removed, and the cells were stained with 1% alizarin red S^t for 5 minutes. Each well was rinsed 3 or 4 times with PBS solution, then rinsed once in deionized water to reduce background staining.

For evaluation of differentiation in adipogenic conditions, 2 × 10⁴ UCB MSCs (passage 4 to 7) were cultured for 2 days in maintenance medium. The medium was subsequently replaced with adipose base medium containing DMEM (high glucose concentration),^h 15% rabbit serum,^10,32,h and 1% penicillin-streptomycin.^h For the first 3 days of adipoinduction, the medium was supplemented with bovine insulin^t (10 μg/mL), isobutyl methylxanthine^t (0.5mM), dexamethasone^o (1μM), and troglitazone^x (5 μg/mL). The troglitazone was added because, in our experience, adipogenesis is increased with troglitazone supplementation, compared with adipogenesis with insulin alone. After 3 days, the isobutyl methylxanthine supplement was removed from the medium. After 3 additional days, the dexamethasone supplement was removed from the medium.³³ After 18 days (4 cycles of induction), the cells were washed twice in PBS solution, fixed in 2% paraformaldehyde for 30 minutes, rinsed twice in PBS solution, rinsed with 60% isopropanol, and stained with oil red O solution^t (0.3% in 60% isopropanol) for 30 minutes. The oil red O solution was removed, and cells were rinsed in 60% isopropanol, then PBS solution.

For evaluation of chondrogenic differentiation, UCB MSCs (5 × 10⁵; passage 4 to 7) were pelleted for 5 minutes at 500 × g. The supernatant was removed and replaced with medium containing DMEM (high glucose concentration)^h and 1% insulin, transferrin, and sodium selenite supplement^a with dexamethasone^o (1μM), ascorbic acid 2-phosphate^t (25 μg/mL), L-proline^t (40 μg/mL), and sodium pyruvate^t (100 μg/mL). For induction, the medium was additionally supplemented with recombinant human transforming growth factor-β3^v (10 ng/mL) and rhBMP6^v (10 ng/mL). Cell cultures were incubated in conical tubes at 37°C and 5% CO₂. The medium was changed every 3 days until cells were harvested. On day 21, the medium was aspirated, and the micromasses were washed 3 times with PBS solution; fixed in 2% paraformaldehyde for 2 hours; rinsed 3 times; stored in 70% ethanol until embedded in paraffin, sectioned, and stained with Alcian blue^y or Masson trichrome stain C^t; and examined by means of photomicroscopy.

Immunocytochemical analysis—Antibodies were used to verify the presence of cytoskeletal proteins characteristic of cells of mesenchymal origin (vimentin) and verify the absence of staining for other differentiated cell lineages including endothelial cells (factor VIII_ra or Von Willebrand factor), epithelial cells (pan cytokeratin), leukocytes (CD18), and bone (osteocalcin). Cells were also stained for smooth muscle actin and osteonectin, which are typically highly expressed in cells of smooth muscle and bone, respectively, and variably expressed in MSCs and other cell lineages.^34–36

Low- and high-passage MSCs from 3 UCB samples were harvested by use of 0.25% trypsin-EDTA.^h Cells were centrifuged at 113 × g for 5 minutes onto glass slides in a cytofuge.^z Immunocytochemistry was performed with a routine streptavidin-biotin detection system as described elsewhere,³⁷ with few modifications. Cells were stained for vimentin^aa (mAb clone 384; 1:100 dilution), smooth muscle actin^bb (mAb clone 1A4; 1:200 dilution), osteonectin^bb (mAb clone OST1; 1:300 dilution), CD18^cc (mAb clone 2G1; 1:10 dilution), factor VIII_ra^aa (rabbit polyclonal; 1:2,000 dilution), osteocalcin^bb (prediluted mAb clone OC1), and pan cytokeratin^dd (mAb clone Lu-5; 1:100 dilution). A frozen section of equine tissue of various types was used as a positive control sample; the section contained the specific antigen and was run in parallel with the MSC samples. Omission of the primary antibody and substitution of normal goat serum were used as a negative control sample.

Statistical analysis—Data are reported as mean ± SE. The Student paired t test was used to evaluate differences between paired data (eg, MSC expansion in hypoxic vs normoxic culture conditions). The unpaired t test was used to evaluate differences between unpaired data (eg, preprocessing and postprocessing cell counts). A basic statistical software package^ee was used for both types of analyses. Univariate and multivariate linear regression analyses were performed to examine associations between various horse, sample shipment, cell-processing, and cell-culture factors and MSC-isolation success, by use of commercial software.^ff A value of P < 0.05 was considered significant for all analyses.

Results

Animals—Seventy-nine horses were enrolled in the study during the 2008 foaling season (January 28 to May 28, 2008). Eight of these horses were subsequently removed for the following reasons: a UCB sample was not successfully collected (n = 5 horses; 6%), foaling was not observed (2; 3%), and sample volume was too small (1; 1%). Samples of UCB were collected without complication from 71 (90%) horses. Mares ranged from 4 to 20 years of age (mean, 9.8 years).

UCB sample processing—Forty-seven (66%) samples were shipped to the laboratory in shipping containers at 4°C, 16 (23%) samples were delivered by hand, and 8 (11%) samples were shipped in shipping containers at 22°C. The interval between UCB sample collection and cell culture ranged from 7 to 159 hours (mean, 56 hours). Microbiological testing was performed at the time of sample receipt and again after processing. Results of all fungal and anaerobic bacterial cultures were negative. Aerobic bacteria were isolated from 5 (7%) samples, which were initially received by the laboratory; the samples were not contaminated during processing. Two of the 5 UCB samples were shipped at 22°C; another 2 were hand delivered, maintained at room temperature, and not processed for 90 hours after foaling. The remaining sample was delivered in a temperature-controlled shipping container; however, the temperature probe failed because of operator error. As such, the temperature of the container was not known. None of the 5 samples were discarded because of bacterial contamination. Cells from 3 samples were cultured in medium containing antimicrobials without incident; all 3 samples yielded MSCs and proliferated to 1 × 10⁷ cells. One MSC sample was used for in vitro differentiation experiments.

The volumes of UCB samples ranged from 82 to 400 mL (mean, 216 mL). Automated processing of the samples significantly (P < 0.001) increased the concentration of nucleated cells (mean ± SE concentration before processing, 3.9 ± 0.16 × 10³ cells/μL; concentration after processing, 22.8 ± 1.3 × 10³ cells/μL) and significantly (P < 0.001) reduced the RBC concentration (hct; concentration before processing, 30.1 ± 0.7 × 10³ cells/μL; concentration after processing, 11.5 ± 0.2 × 10³ cells/μL). The percentage recovery of nucleated cells from UCB samples averaged 87.0 ± 1.6%; the percentage recovery for mononuclear cells was 95.4 ± 4.3%. Viability of nucleated cells before and after automated processing was determined in a subset of 32 samples, which represented the entire range of transport times. Cell viability before processing averaged 94.5 ± 1.5%, and that after processing was 92.3 ± 2.3%; the difference between means was not significant (P = 0.57).

Isolation of MSCs—Cells from each UCB sample were cultured until 1 × 10⁷ MSCs were obtained. Of the 71 cord blood samples received, 51 were plated for MSC culture. Overall, moderate to high numbers of MSCs were expanded in 41 of the 51 (80%) samples. These samples reached 1 × 10⁷ MSCs between the second and seventh passages, with most reaching cell dose by the fourth passage. Mesenchymal stem cells were not isolated or failed to expand to > 1 × 10⁶ cells in 10 (20%) samples (Table 1). Samples that were highly successful at proliferation (28/51 [55%]) reached 4 × 10⁶ MSCs in a mean of 21 days and 1 × 10⁷ MSCs in a mean of 31 days. Samples that were moderately successful at proliferation (13/51 [25%]) reached 4 × 10⁶ MSCs in a mean of 41 days. The MSCs recovered were elongate, spindle shaped, and morphologically similar to those described for horses¹⁰ and humans.²⁸ Age of mare did not influence success of MSC isolation from UCB.

Several variables were assessed for association with success or failure of MSC isolation and monolayer expansion. No association was detected between mare age, number of prior parturitions, hours before automatic cell processing, nucleated cell plating density, or UCB volume and the success of MSC isolation and monolayer expansion. The nucleated cell count after processing significantly (P < 0.001) influenced the probability of success or failure of MSC growth. The mean nucleated cell count for highly and moderately successful samples was 25.7 × 10³ cells/μL, whereas the mean nucleated cell count for samples that failed to yield MSC growth was 14.1 × 10³ cells/μL. The significance of this finding was confirmed by linear regression analysis in which nucleated cell count was evaluated for correlations with final cell number (r = 0.2; P < 0.001), days to yield 1 × 10⁷ cells (r = 0.26; P < 0.001), and days to yield 4 × 10⁶ cells (r = 0.22; P < 0.001).

Mesenchymal stem cells were successfully isolated from 27 of 28 (96%) UCB samples with a nucleated cell count > 21 × 10³ cells/μL. Because nucleated cell count could have been related to volume of the UCB sample, multivariate regression analysis of factors associated with success or failure of MSC isolation was performed to include the variables UCB sample volume and nucleated cell count. Results of that analysis were less significant than results for the analysis that included only nucleated cell count as a predictor variable.

The first 6 UCB MSC samples identified as highly proliferative were cultured until senescence to assess the long-term proliferation potential of UCB-derived MSCs. Morphologic features of senescence were compatible with those described for human UCB- and bone marrow–derived MSCs and included flat cells with blunt ends or polygonal, jagged cells that ceased to grow.²⁸ The 6 highly proliferative clones of equine UCB-derived MSCs did not undergo senescence until a mean of 21 passages (cultured for mean of 132 days).

Hypoxic culture conditions—Initial MSC colonies were cultured from all 5 UCB samples in normoxic and hypoxic conditions. All samples cultured at 5% O₂ proliferated rapidly to the desired cell number (cell dose; 1 × 10⁷ MSCs), whereas only 2 samples cultured at 21% O₂ grew to a comparable number of MSCs. Cells cultured in hypoxic conditions took fewer days to reach cell dose (35 vs 66 days); however, the difference was not significant (P = 0.10). Culture of UCB nucleated cells in 5% O₂ increased the probability of successful isolation of high numbers of MSCs, compared with culture of cells in 21% O₂.

Fibronectin substrate—Nucleated cells from 9 UCB samples that were initially cultured in fibronectin-coated plates reached a significantly (P = 0.016) higher number of MSCs than did samples cultured in standard culture plastic plates. Cells in fibronectin-coated plates expanded at a rate 2.6 times as great as that of cells in standard plastic culture plates (8.2 × 10⁵ cells/d vs 3.1 × 10⁵ cells/d, respectively). Seven nucleated cell samples isolated on fibronectin reached MSC cell dose in a mean of 22 days. Mesenchymal stem cells were not isolated from the other 2 samples. Initial numbers of MSC colonies ranged from 1 to 20. However, the number of colonies detected after the first passage was not associated with successful isolation. Overall, UCB-derived nucleated cells plated on fibronectin expanded more rapidly than did cells plated on plastic, with a resultant increase in successful MSC isolation rate and monolayer expansion.

In vitro differentiation—Osteogenic differentiation was induced in 8 of 8 UCB MSC samples as evidenced by increased activity of alkaline phosphatase and robust mineralization defined by alizarin red S stain, compared with results in control samples (Figure 1). Induction of adipogenic differentiation in 6 of 6 UCB MSC samples was characterized by minimal apparent accumulation of small lipid vacuoles after oil red O staining. However, mature adipocytes, characterized by a teardrop morphology, never fully developed. The addition of rabbit serum to the adipogenic medium in and of itself resulted in a small amount of adipogenic differentiation. After chondrogenic differentiation, 1 of 1 UCB MSC sample yielded a large micromass pellet that was not evident in the senescent control UCB-derived cells that had been similarly induced to differentiate. Alcian blue and Masson trichrome staining of the cell pellet revealed an organized, condensed structure with chondrocyte lacunae in 1 sample. Additional differentiation experiments were performed with UCB-derived MSCs that had been frozen and thawed prior to differentiation. No differences were detected in the ability of frozen-thawed MSCs to differentiate into cartilage, bone, or fat (data not shown).

Representative photomicrographs depicting the osteogenic (first column), adipogenic (second column), and chondrogenic (third column) differentiation potential of MSCs isolated from equine UCB samples and cultured in control (top row) and induction (bottom row) media. A—Adherent, spindle-shaped, nondifferentiated MSCs incubated in osteogenic control medium. Alizarin red S stain; bar = 200 μm. B—Adherent, spindle-shaped, nondifferentiated MSCs incubated in adipogenic control medium. Oil red O stain; bar = 200 μm. C—Senescent MSCs cultured in defined chondrogenesis medium formed a small, necrotic pellet and failed to form an organized, condensed structure with chondrocyte lacunae. Masson trichrome stain; bar = 100 μm. D—Robust mineralization is evident in MSCs cultured in defined osteogeneic differentiation medium. Alizarin red S stain; bar = 200 μm. E—Accumulation of lipid-rich small vacuoles is evident in MSCs cultured in defined adipogenic medium. Oil red O stain; bar = 200 μm. F—Proliferative MSCs cultured in defined chondrogenesis medium formed an organized large pellet that consisted of a condensed structure with chondrocyte lacunae. Masson trichrome stain; bar = 100 μm.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1526

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Representative photomicrographs depicting the osteogenic (first column), adipogenic (second column), and chondrogenic (third column) differentiation potential of MSCs isolated from equine UCB samples and cultured in control (top row) and induction (bottom row) media. A—Adherent, spindle-shaped, nondifferentiated MSCs incubated in osteogenic control medium. Alizarin red S stain; bar = 200 μm. B—Adherent, spindle-shaped, nondifferentiated MSCs incubated in adipogenic control medium. Oil red O stain; bar = 200 μm. C—Senescent MSCs cultured in defined chondrogenesis medium formed a small, necrotic pellet and failed to form an organized, condensed structure with chondrocyte lacunae. Masson trichrome stain; bar = 100 μm. D—Robust mineralization is evident in MSCs cultured in defined osteogeneic differentiation medium. Alizarin red S stain; bar = 200 μm. E—Accumulation of lipid-rich small vacuoles is evident in MSCs cultured in defined adipogenic medium. Oil red O stain; bar = 200 μm. F—Proliferative MSCs cultured in defined chondrogenesis medium formed an organized large pellet that consisted of a condensed structure with chondrocyte lacunae. Masson trichrome stain; bar = 100 μm.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1526

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Representative photomicrographs depicting the osteogenic (first column), adipogenic (second column), and chondrogenic (third column) differentiation potential of MSCs isolated from equine UCB samples and cultured in control (top row) and induction (bottom row) media. A—Adherent, spindle-shaped, nondifferentiated MSCs incubated in osteogenic control medium. Alizarin red S stain; bar = 200 μm. B—Adherent, spindle-shaped, nondifferentiated MSCs incubated in adipogenic control medium. Oil red O stain; bar = 200 μm. C—Senescent MSCs cultured in defined chondrogenesis medium formed a small, necrotic pellet and failed to form an organized, condensed structure with chondrocyte lacunae. Masson trichrome stain; bar = 100 μm. D—Robust mineralization is evident in MSCs cultured in defined osteogeneic differentiation medium. Alizarin red S stain; bar = 200 μm. E—Accumulation of lipid-rich small vacuoles is evident in MSCs cultured in defined adipogenic medium. Oil red O stain; bar = 200 μm. F—Proliferative MSCs cultured in defined chondrogenesis medium formed an organized large pellet that consisted of a condensed structure with chondrocyte lacunae. Masson trichrome stain; bar = 100 μm.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1526

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Isolation and expansion of MSCs from cryopreserved UCB samples—Six processed UCB samples were frozen for 9 to 70 days prior to thawing. All thawed samples were microbiologically sterile, and nucleated cell viability was 93.8 ± 2.8%. Mesenchymal stem cells were recovered from all 6 samples, and the mean interval to first passage was 13.3 ± 2.2 days (range, 9 to 24 days). The MSCs in 4 of the samples were expanded, and cell dose was achieved in 34 ± 2.4 days. This interval was not significantly (P = 0.24) different from that for the initial monolayer expansion of MSCs from fresh UBC samples (Table 1). The remaining 2 samples had slower monolayer expansion kinetics and never reached cell dose.

Isolation and expansion of cryopreserved MSCs—Mesenchymal stem cells isolated from UCB samples were frozen for 17 to 106 days at passage numbers ranging from 2 to 7. After samples were thawed, MSC viability was 84.0 ± 3.8% (Table 1). As was evident with cryopreserved UCB nucleated cells, initial MSC colonies were obtained from all 5 samples. Successful monolayer expansion of 1 × 10⁶ frozen-thawed MSCs to 1 × 10⁷ MSCs was accomplished in 4 of 5 samples. Frozen-thawed MSCs expanded from 1 × 10⁶ frozen MSCs to 1 × 10⁷ MSCs in significantly (P = 0.03) fewer days (26 days) than their paired fresh counterparts (37).

Immunocytochemical phenotype of equine UCB-derived MSCs—All equine UCB-derived MSCs were uniformly lacking CD18, pan cytokeratin, and factor VIII_ra (Figure 2; Table 2). Early-passage, highly proliferative MSCs had negative immunocytochemical results for all markers except a few cells (< 20%) that expressed vimentin and smooth muscle actin. Conversely, MSCs that were poorly proliferative or senescent (including paired cells that were immunophenotyped at early passage when they were highly proliferative and immunophenotyped at senescence) had a more differentiated pattern, with > 80% of the cells expressing vimentin, > 50% of the cells expressing actin and osteonectin, and a few cells expressing osteocalcin.

Representative photomicrographs of MSCs isolated from equine UCB and immunocytochemically stained to detect CD18 (top row), vimentin (second row), actin (third row), and osteonectin (fourth row), comparing early-passage, highly proliferative cells (left column) and late-passage, senescent cells (right column). Proliferative (A) and senescent (B) MSCs did not express CD18, which is a panleukocyte marker. Most MSCs in early passage (C) did not express vimentin, which is a mesenchymal-cell cytoskeletal protein; however, the onset of senescence (D) yielded strong vimentin expression, indicating a phenotype change. Most MSCs in early passage (E) did not have smooth muscle actin, which is a cytoskeletal protein; however, senescent MSCs (F) had an increase in actin expression. Mesenchymal stem cells in early passage (G) were uniformly lacking osteonectin, which is a calcium-binding glycoprotein in osteoblasts, osteocytes, and other cells; however, senescent MSCs (H) had an increase in osteonectin expression. Bars = 10 μm.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1526

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Representative photomicrographs of MSCs isolated from equine UCB and immunocytochemically stained to detect CD18 (top row), vimentin (second row), actin (third row), and osteonectin (fourth row), comparing early-passage, highly proliferative cells (left column) and late-passage, senescent cells (right column). Proliferative (A) and senescent (B) MSCs did not express CD18, which is a panleukocyte marker. Most MSCs in early passage (C) did not express vimentin, which is a mesenchymal-cell cytoskeletal protein; however, the onset of senescence (D) yielded strong vimentin expression, indicating a phenotype change. Most MSCs in early passage (E) did not have smooth muscle actin, which is a cytoskeletal protein; however, senescent MSCs (F) had an increase in actin expression. Mesenchymal stem cells in early passage (G) were uniformly lacking osteonectin, which is a calcium-binding glycoprotein in osteoblasts, osteocytes, and other cells; however, senescent MSCs (H) had an increase in osteonectin expression. Bars = 10 μm.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1526

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Representative photomicrographs of MSCs isolated from equine UCB and immunocytochemically stained to detect CD18 (top row), vimentin (second row), actin (third row), and osteonectin (fourth row), comparing early-passage, highly proliferative cells (left column) and late-passage, senescent cells (right column). Proliferative (A) and senescent (B) MSCs did not express CD18, which is a panleukocyte marker. Most MSCs in early passage (C) did not express vimentin, which is a mesenchymal-cell cytoskeletal protein; however, the onset of senescence (D) yielded strong vimentin expression, indicating a phenotype change. Most MSCs in early passage (E) did not have smooth muscle actin, which is a cytoskeletal protein; however, senescent MSCs (F) had an increase in actin expression. Mesenchymal stem cells in early passage (G) were uniformly lacking osteonectin, which is a calcium-binding glycoprotein in osteoblasts, osteocytes, and other cells; however, senescent MSCs (H) had an increase in osteonectin expression. Bars = 10 μm.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1526

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Discussion

Equine UCB is a viable, accessible source of multipotent MSCs. Compared with other adult-derived tissue sources of MSCs, UCB has several potential advantages as a source of MSCs. A high volume of UCB can be readily, ethically, and easily collected. Mesenchymal stem cells derived from UCB are also useful because MSC number or differentiation potential does not decline with horse age.^16,17 Human UCB-derived MSCs appear to have a higher proliferative potential¹⁵ and increased plasticity,^10,12,34,38 compared with other adultderived MSCs. However, human UCB samples also appear to contain lower numbers of MSCs than do bone marrow and fat,¹⁵ and the reliability of obtaining MSCs from each UCB sample is debatable. In the study reported here, we successfully isolated and expanded MSCs from approximately 80% of equine UCB samples, which is a higher rate than is reported elsewhere¹⁰ for horses. Compared with the study of that report, our study included a larger number of samples with higher initial sample volume. Our study also differed technically in that it involved storage and transport of samples at 4°C, automated UCB processing, and alternative MSC isolation techniques including the use of fibronectin, hypoxic conditions, and culture medium that contained equine serum. Similar to findings for human UCB,¹⁵ the data in the study reported here suggested that the number of MSCs in UCB may be low. However, our data also suggested that clonally derived MSCs could proliferate for > 20 passages.

High numbers of viable nucleated cells can be routinely recovered from equine UCB samples when temperature-controlled shipping and automated processing are used. In our study, nucleated cell number in volume-reduced and RBC-depleted UCB was critical in predicting successful MSC isolation. These data concurred with findings of other researchers who found that a high mean nucleated cell number was associated with efficient isolation of MSCs from human UCB.⁹ The high nucleated cell count of equine UCB may be partially responsible for the increased success of MSC isolation from equine versus human UCB (successful 30% to 63% of the time).^9,12,15

Neither initial UCB volume nor short interval from UCB sample collection to processing was associated with an increase in successful isolation of MSCs in our studies. In fact, it appeared that UCB samples with a long interval to processing yielded a higher MSC recovery rate than did samples with a short interval to processing, although the difference in intervals was not significant. This finding contrasts with findings for human UCB, which suggest that an interval < 15 hours from sample collection to isolation of MSCs and a high initial net volume of UCB are important for successful MSC isolation.⁹

Shipment of equine UCB samples at 4°C permitted long transit times with minimal risk of bacterial overgrowth; however, even samples with a minuscule amount of bacterial contaminants were successfully cultured for MSC isolation. With long intervals from collection to processing of UCB samples, there may be a preferential loss of terminally differentiated granulocytes and monocytes from UCB (essentially a form of lineage depletion). Lineage depletion facilitates MSC isolation from human UCB samples, and the inclusion of cells, including granulocytes, lymphocytes, and monocytes, may also hinder efficient MSC isolation from equine UCB when particular conditions such as hypoxia are not used.^28,39 Neither UCB sample volume nor the number of MSC colonies initially isolated was associated with eventual successful monolayer expansion of MSCs. Highly proliferative MSC clones were obtained from UCB samples from which 1 cell colony was initially isolated and were not obtained from samples from which greater numbers (up to 20) of colonies were initially isolated. This observation suggested that differences in MSC expansion kinetics likely relate to the different proliferation potentials of the initiating CFUs.

Hypoxic conditioning of human MSCs results in enhanced cell proliferation and cell migration in vitro, likely because of the activation of signaling pathways that control cell-cycle rates and induction of genes that increase their migratory phenotypes.^19,20 Hypoxic preconditioning of MSCs prior to in vivo transplantation has in part been justified by the knowledge that MSCs are derived from and must migrate to and adapt to sites of low oxygen tension in vivo. Our research, although limited in scope, suggested that mild hypoxia similarly increased the rate of equine MSC proliferation and overall culture success. The effect of mild hypoxia may be attributable in part to the induction of signaling pathways but also to a decrease in survival of contaminating leukocytes in UCB. Ongoing research by our laboratory group has suggested that hypoxic culture conditions may also be used to salvage MSCs from UCB samples that otherwise may not have expanded successfully. Given that the use of hypoxic incubators adds expense and some technical challenges to routine tissue culture, additional research is needed to define the mechanisms by which hypoxia acts to increase equine MSC proliferation and to substantiate that hypoxic preconditioning results in tangible in vivo improvement of tissue regeneration.

Similar to findings in another study,¹⁰ equine UCB-derived MSCs in the study reported here were capable of in vitro differentiation toward osteogenic, chondrogenic, and adipogenic cells when appropriate induction media were used (dexamethasone for osteogenesis and rabbit serum for adipogenesis). Nonetheless, as reported for equine bone marrow,² equine UCB,³⁹ and human UCB-derived MSCs,⁹ equine UCB-derived MSCs have limited adipogenesis. The method of adipogenic induction used in our study was similar to that used by other investigators for human UCB-derived MSCs, and those investigators also found lipid-rich vacuole accumulation with a lack of adipocyte generation.⁹ Inefficient adipogenesis may be attributable to species differences in adipogenic potential of MSCs or different requirements in differentiation protocols for equine cells. It may also be related to the ontogenetic age of the MSCs because an increase in adipogenesis is correlated with an increase in donor age.⁴⁰ Adipocytes are lacking in fetal bone marrow but are clearly present in adult bone marrow and adipose tissue. Overall, our findings concurred with those of other research¹⁰ and suggested that the MSCs derived from equine UCB are multipotent.

Immunophenotyping is used extensively to characterize human stem cells derived from bone marrow, adipose, and placental tissues. Despite this, many researchers have moved away from extensive phenotyping because no universal marker exists to identify MSCs.⁴¹ Many of the antibodies used routinely for phenotyping human and rodent cells are either not available for or do not cross-react in other animal species, including horses.⁴² We elected to phenotype our cells with a limited panel of well-characterized antibodies with known cross-reactivity to equine cell epitopes. The onset of senescence in the study reported here was associated with an immunophenotypic change in MSCs as evidenced primarily by strong, positive staining for the mesenchymal-cell cytoskeletal protein vimentin. This finding opposes that of another study²⁸ in which human UCB-derived MSCs were stained with vimentin. In that study, highly proliferative MSCs were stained strongly for vimentin. In our study, early-passage, highly proliferative, spindle-shaped equine UCB-derived MSCs did not stain for vimentin. However, vimentin was expressed in these same cells at the onset of senescence as well as in cells that were never highly proliferative. In addition, cells undergoing senescence were variably positive for other cytoskeletal protein markers characteristic of lineage-committed cells, including smooth muscle actin, osteonectin, and osteocalcin.

In humans, bone marrow- and UCB-derived MSCs also have variable expression of α smooth muscle actin and osteonectin^43,44 as well as phenotypic variation with passage.²⁸ Results of the present study suggested that long-term passage was associated with phenotypic change and a shift toward lineage commitment. The onset of cell senescence and altered morphologic features of cells appeared to be associated with the loss of multipotentiality and the expression of markers of differentiation. Long-term passage kinetics and the interval to onset of senescence have not been reported for equine adipose tissue–or bone marrow–derived MSCs; however, our data concur with findings in human UCB, which suggested that some MSC clones derived from UCB may have a higher proliferation rate than for other adult-derived MSCs.¹⁵

Other reports^10,39 of the isolation of MSCs from equine UCB samples allude to problems with cryopreservation of UCB-derived nucleated cells or expanded MSCs for later thawing and monolayer expansion. In the present study, we used a commercially available UCB processor that significantly enriched the nucleated cell fraction and reduced blood volume and RBC number in equine UCB. Processed samples were subsequently cultured fresh, and results were compared with those of aliquots that had been immediately frozen in an automated controlled-rate freezer and thawed in accordance with protocols approved for human UCB banking. Similar to results of another study⁴⁵ in which human UCB samples were used, our results suggested that equine UCB-derived MSCs could readily be obtained from cryopreserved, enriched nucleated cells when appropriately handled. Our findings were also similar to those in a report² of successful cryopreservation, thawing, and monolayer expansion of equine bone marrow–derived MSCs for up to 6 months with no significant alterations in cell viability, morphology, proliferation, or differentiation. Successful cryopreservation of UCB-derived nucleated cells and UCB-derived MSCs is an important first step in the development of cellular treatments and tissue banks. Equine MSCs may be useful in models of injury in large animals, and our findings may be directly relevant in the development of regenerative cell-based treatments for human orthopedic and traumatic injuries.