Isolation, proliferation, cytogenetic, and molecular characterization and in vitro differentiation potency of canine stem cells from foetal adnexa: A comparative study of amniotic fluid, amnion, and umbilical cord matrix

Cell Morphology, Population Doubling Time, and Cell Viability

In all three cell types, the morphology of proliferating cells typically appeared fibroblast-like (Fig. 1A–C for AF-MSCs, AM-MSCs, and UCM-MSCs, respectively), and clusters of rapidly expanding cells were observed. In Figure 1D, a cluster of AF-MSCs is shown.

The population doubling time (DT) was measured, calculated, and drawn as a graph (Fig. 2A–C). In all three cell lines, the proliferation rate was reduced with passages. In particular, a significant increase in DT was found in AF-MSCs as compared to passage 1 (P1) versus P3/P4 (P < 0.05) and P1 versus P6/P7/P8 (P < 0.001). At P1, 1.12 ± 0.04 days were necessary to double the cell number, whereas at P8, DT was 1.50 ± 0.05 days (Fig. 2A). In AM-MSCs, 1.37 ± 0.05 days were necessary at P1 to double cell number, whereas at P6, DT was 1.48 ± 0.04 days (P < 0.01), and from P7, the significance level increased to P < 0.001 (1.67 ± 0.13 and 1.66 ± 0.1 for P7 and P8, respectively; Fig. 2B). In UCM-MSCs, DT significantly increased in P2/P4 versus P1 (P < 0.01) and P3/P5 versus P1 (P < 0.05). The significance level increased to P < 0.001 in P6/P7/P8 versus P1 (Fig. 2C). At P1, 1.14 ± 0.04 days were necessary to double cell number, whereas at P8, DT was 2.42 ± 0.72 days.

In addition, cell viability was measured. In particular, cell viability in AF-MSCs and AM-MSCs did not change with the passages (percentage of viable cells: mean values = 95.51 ± 2.81 and 91.97 ± 2.34, respectively; Fig. 3A,B). In UCM-MSCs, cell viability remained at approximately constant levels up to P6, and significantly decreased from P7 (P < 0.05; Fig. 3C).

Stem Cell and Hematopoietic Markers mRNA Expression

The expression of stem cell markers was evaluated in all three cell lines at P1 and P2 (Fig. 4). In P1, AF-MSCs expressed Oct-4, CD44, DLA-DRA1, and DLA-79. In P2, only CD44 was expressed. CD184 and CD29 expressions were not observed in this cell line. For AM-MSCs, cells in P1 expressed Oct-4, CD44, CD184, and CD29. In P2, these cells expressed Oct-4 and CD184, but they did not express CD44 and CD29. For UCM-MSCs, P1 cells expressed Oct-4, CD44, CD184, and CD29, as in the P1 of AM-MSCs. In P2, CD44 and CD184 were expressed; Oct-4 and CD29 were not observed. The housekeeping gene HPRT1 was expressed in all cell lines and passages. Expression of the embryonic stem cell marker Nanog, the hematopoietic markers CD34 and CD45, and the major histocompatibility complex (MHC) genes DLA-DQA1 and DLA-88 were never found in any cell lines or passages. Canine subcutaneous fibroblasts, used as negative controls, did not express stemness or MHC markers. Dog leucocytes, used as positive controls for hematopoietic and MHC markers, expressed hematopoietic and MHC markers but they did not express embryonic or mesenchymal stemness markers. In order to confirm the identity of Oct-4 in all three cell lines, sequence analysis of the RT-PCR-amplified fragments has been performed (Ceinge Biotecnologie Avanzate—Nucleic Acid Sequencing Services—University of Naples, Naples, Italy) and amplifications products show identity to canine Oct-4 (NCBI Nucleotide Database: XM_538830.1).

Karyotype and Telomerase Activity

It is known that the karyotype of stem cells can undergo changes during passaging, and show rates of chromosomal abnormalities (Buzzard et al., 2004); for this reason, we performed karyotype analysis on all our samples. All observed metaphases of AF-MSCs (n = 15), AM-MSCs (n = 23), and UCM-MSCs (n = 13) were normal in chromosomal number and structure, thus confirming the genomic stability of the cells in culture (Fig. 5). Telomerase activity and telomere maintenance are associated with the immortality of cancer cells, germ-line cells, MSCs, and embryonic stem (ES) cells (Shay and Wright, 1996; Shay et al., 1996; Hiyama and Hiyama, 2007). Accordingly, a telomerase activity detection assay was used to distinguish immortal cells from differentiated somatic cells. We performed a Telomere Repeat Amplification Protocol (TRAP) assay (TRAPeze® Telomerase Detection Kit; Chemicon International Inc., Temecula, CA) on AF-MSCs, AM-MSCs, and UCM-MSCs, and analyzed telomerase expression and activities in comparison with canine subcutaneous tissue, canine immortalized fibroblasts, canine newly isolated fibroblasts and canine tumor cells (Materials and Methods section). The TRAP assay is a two enzyme-based system in which the extracted and to-be-tested telomerase adds telomeric repeats to a substrate first, and then Taq polymerase amplifies it. All of our samples showed high telomerase activity except for the subucutaneous tissue (Fig. 6). Since telomerase is a heat-sensitive enzyme, every sample was tested for heat sensitivity as a control. All the heat-treated test extracts confirmed the inactivation of enzyme except for the one corresponding to the canine breast adenoma ZMTH3 (smear detected), even after increasing the length of heat-inactivation two-fold and three-fold (Fig. 6). The extra 36-bp band representing the internal control (standard internal control; S-IC) was visible in all assays except for AF and AM, probably due to the excessively high telomerase activity in these samples.

In Vitro Differentiation

When induced to neuronal differentiation, cells of the three cell lines were positive for nestin expression (Fig. 7A). These cells showed the presence of Nissl bodies and a neuronal-like morphology (Fig. 7B) as compared to the fibroblast-like appearance observed in cells cultured in basal conditions (Fig. 1).

In cells induced to differentiate in an adipogenic medium, leptin expression (Fig. 7C) and the presence of lipids droplets (Fig. 7D) were evident after 25 days of adipogenic differentiation in all three cell lines. Adiponectin expression was found only in AF-MSCs (Fig. 7C).

In cells induced to differentiate in an osteogenic medium for 14 days, semi-quantitative RT-PCR analysis revealed over-expression of the osteogenic gene osteonectin. In AF-MSCs, AM-MSCs, and UCM-MSCs, expression of this gene was found to be 1.2-, 1.2-, and 1.3-fold higher, respectively, in differentiated as compared to undifferentiated cells. Expression levels of osteocalcin between differentiated cells and controls did not change in any of the three cell lines. Osteopontin was expressed only in AF-MSCs (Fig. 7E). Calcium mineralization was also found in all three cell lines, as revealed by Von Kossa staining (Fig. 7F).

After 14 days of in vitro culture in a chondrogenic medium, all cell lines were stained with Alcian blue, showing the typical metachromasia of cartilage, thus chondrogenic differentiation was demonstrated by the mucopolysaccharide-rich extracellular matrix (Fig. 7G).

Cell Isolation and Culture

Samples were recovered after elective ovariohysterectomy in four bitches at 25–40 days of gestational age. AF, AM, and UCM of canine fetuses were used for the isolation of mononuclear cells. Pieces of the AM and UCM were placed in two different Petri plates with sterile and pre-warmed phosphate buffered saline (PBS; P4417, Sigma, Milan, Italy) with antibiotics (penicillin 100 U/mL, streptomycin 100 µg/mL solution, P0781; Sigma). After three flushes with PBS, pieces were placed in two new Petri plates. Pieces where cultured at the temperature of 38.5°C with 5% CO₂ in culture medium: Dulbecco Modified Eagles Medium (DMEM; D5546; Sigma), fetal bovine serum (FBS; 10%; F3018, Sigma), L-glutamine (2 mM; G7513; Sigma), penicillin (100 U/mL) streptomycin (100 µg/mL), and amphotericin (0.25 µg/mL) solution (A5955, Sigma), and epidermal growth factor (EGF; 10 ng/mL; E4127, Sigma). After 5 days, pieces were removed and cells were cultured until their isolation. The collected AF was delivered in Falcon tubes and spun to separate cells from the liquid fraction. The recovered cells were re-suspended in culture medium, as previously described for AM and UCM, and placed into T25 cell culture flasks. Two cell lines were evaluated for each tissue type (AF, AM, and UCM).

Cell Expansion

Plated cells were monitored during the first expansion period (passage 0 = P0), and after 48–72 hr, the medium was changed to remove non-adherent cells. Approximately 10 days later, when well-developed colonies of fibroblast-like cells appeared, cultures were washed with PBS, harvested with 0.05% trypsin–0.02% EDTA (ECM0920D; Euroclone, Milan, Italy), and re-plated in six-well plates (3516; Corning, New York, NY). Cells were counted for each passage, with Burker's chamber by dilution (1:1) in trypan blue (T8154; Sigma), starting to P1. Cell viability was expressed as a percentage of unstained cells. Since then, cells were maintained for some passages to perform growth and proliferation studies.

The three types of cell lines were seeded in six-well plates at a density of 1,000 cells/cm² and the doubling-time analysis was performed. Culture passages were performed every 4 days and cell counts were performed for each culture passage. Obtained values are used to allow the calculation of doubling time (DT). Population DT was calculated using the formula DT = CT/CD where CT (culture time) is the time between passage “n” and passage “n + 1,” and CD (cell doubling) = ln(nf/ni)/ln2 where “nf” is the harvested cell number and “ni” the seeded cell number (Lei et al., 2007). On the days of the first and second counts, aliquots of the cells were cryopreserved for each cell line for molecular characterization; aliquots of fresh samples from the third passage were used to perform in vitro differentiation, and aliquots of fresh samples of the fourth passage were used for karyotype analysis and the TRAP assay.

Karyotyping

Karyotype analysis of AF-MSCs, AM-MSCs, and UCM-MSCs at P4 was performed according to the standard procedures of Q banding. Briefly, the cell line was processed with colcemid (0.1 µg/mL in culture medium; D1925; Sigma) for 4 hr followed by three washes in a fixative (3 methanol:1 acetic acid). Chromosomes were banded by immersion in quinacrine mustard (Q2876; Sigma), fully washed with tap water, and mounted in 1× PBS. Metaphases were acquired by CCD camera using a Leica fluorescence microscope. Chromosomes were counted, and the banding pattern was compared to standard Canis familiaris karyotype according to Breen et al. (1999).

Telomere Repeat Amplification Protocol Assay

The TRAP assay is based on an improved version (Kim and Wu, 1997) of the original method described by Kim et al. (1994), and assesses the telomerase activity on samples by measuring the capacity of the extracted enzyme to carry out the amplification of the target telomeric repeat (TS) available in the commercial kit. This assay works as a two-enzyme system, utilizing telomerase activity and the polymerase chain reaction (PCR). During the first step of the reaction, telomerase adds a number of telomeric repeats (GGTTAG) onto the 3′ end of a substrate oligonucleotide (TS). In the second step, the extended products are amplified by PCR using the TS and RP (reverse) primers, generating a ladder or smear of products with six base increments starting at 50 nucleotides. Further, since telomerase is a heat-sensitive enzyme, a negative control can be obtained for every sample after heat inactivation of the sample itself.

A 36-bp amplification product represents a standard internal control (S-IC) for the experiment and should always be visible in all the samples (+ and −). Many cell/tissue extracts contain inhibitors of Taq polymerase and can give potentially false-negative results. To distinguish this from other problems, the TRAPeze® Primer Mix contains the internal control oligonucleotides K1 and TSK1 that, together with TS, produce a 36-bp band (S-IC) in every lane. The S-IC band also serves as a control for amplification efficiency in each reaction, and can be used for quantitative analysis of the reaction products. Cell extracts of AF-MSCs, AM-MSCs, and UCM-MSCs at P4 were assayed for telomerase activity following the instruction of the TRAPeze® telomerase detection kit (Chemicon International Inc.). Cell extracts were prepared by lysing around 1 × 10⁶ cells with 1× CHAPS lysis buffer. For each sample, 10 µl of cell extract were heat treated by incubating at 85°C for 10 min prior to the TRAP assay to inactivate telomerase. ZMTH3 cell line, canine immortalized fibroblasts, canine newly isolated fibroblasts and canine subcutaneous tissue were used as controls. Cell extracts were used for PCR in a mix composed as follows: cell extract (2 µl), 10× TRAP buffer (5 µl), 50× dNTP mix (1 µl), TS primer (1 µl), TRAP primer mix (1 µl), Taq polymerase (2 U, 0.4 µl), and PCR-Grade Water (39.6 µl) for a final volume of 50 µl. Reaction products were analyzed using 12.5% polyacrylamide gel.

Neurogenic Differentiation

Cells at P3 of the three cell lines were plated in six-well plates. Twenty-four hours prior to neuronal induction, culture media were replaced with pre-induction media consisting of DMEM, 20% FBS and 1 mM β-mercaptoethanol (M7522, Sigma; Seo et al., 2009). For neuronal differentiation, the pre-induction medium was removed and the cells were washed with PBS and transferred to neuronal induction media composed of DMEM, 20% FBS, 2% dimethyl sulfoxide (D5879, Sigma), and 200 µM butylated hydroxyanisole (B1253, Sigma; Woodbury et al., 2000). Cells were cultured for 5 days and, after differentiation, Nissl staining was performed as reported by Hou et al. (2003) and Lu et al. (2010). Molecular tests were performed for nestin and glial fibrillary acidic protein (GFAP).

Adipogenic Differentiation

For adipogenic differentiation, cells at P3 of the three cell lines were cultured for 25 days in a differentiation medium composed as follows: DMEM, 10% FBS, 2 mM L-glutamine, 1× penicillin (100 U/mL), streptomycin (100 µg/mL), and amphotericin (0.25 µg/mL) solution, 1 µM dexamethasone (D2915, Sigma), 200 mM indomethacin (I7378, Sigma), and 10 µg/mL insulin (I6634; Sigma). The medium was changed twice weekly. Differentiation was evaluated by Red Oil-O staining (0.1% in 60% isopropanol; Bossolasco et al., 2006; Hoynowski et al., 2007) to visualize lipid droplets, and by molecular tests for adiponectin and leptin expression.

Osteogenic Differentiation

For osteogenic differentiation, cells at P3 of the three cell lines were cultured for 14 days in a differentiation medium composed of DMEM, 10% FBS, 1× penicillin (100 U/mL), streptomycin (100 µg/mL), and amphotericin (0.25 µg/mL) solution, 0.1 µM dexamethasone, 0.25 mM L-ascorbic acid 2-phosphate (A8960, Sigma), and 10 mM β-glycerophosphate (50020, Sigma). The medium was changed twice weekly. Differentiation was evaluated by Von Kossa staining (Ilancheran et al., 2007; Mauro et al., 2010) with some modifications (1% silver nitrate and 5% sodium thiosulfate) to detect calcium deposits, and by molecular tests for osteopontin, osteocalcin and osteonectin.

Chondrogenic Differentiation

Chondrogenic differentiation was assessed in a monolayer culture by incubating cells at P3 for 14 days in DMEM containing 100 nM dexamethasone, 50 µg/mL L-ascorbic acid 2-phosphate, 1 mM sodium pyruvate, 40 µg/mL proline, ITS (insulin 5 µg/mL, transferrin 5 µg/mL, selenious acid 5 ng/mL; I3146, Sigma), and 5 ng/mL TGF-β1 (100-36E, Peprovet, DBA, Milan, Italy). Differentiation was demonstrated by staining with Alcian blue (pH 2.5; Wang et al., 2004; Ghosh et al., 2010).

Reverse Transcription–Polymerase Chain Reaction (RT-PCR) Analysis

Total mRNA was isolated from AF-MSCs, AM-MSCs, and UCM-MSCs using NucleoSpin RNA II (FC140955L; Macherey-Nagel, Düren, Germany), and converted into cDNA using an Enhanced Avian HS RT-PCR kit (HSRT100-1KT, Sigma). The cDNA was amplified by using the PCR mix reaction: cDNA (100 ng), PCR buffer (1×; 5 µL), dNTP mix (10 mM each; 1 µL), PCR primers (mixture of 50 µM each primer; 1 µL), Hot Master Taq DNA Polymerase (1U; 2200320; 5Prime, Milan, Italy) and nuclease-free water for a final volume of 50 µl. The expression of the following set of genes was evaluated for molecular characterization before in vitro differentiation: canine Nanog homebox, canine Pou class 5 homebox 1 (POU5F1 alias Oct-4), canine CD44, canine Chemokine (C-X-C motif) receptor 4 (CXCR4 alias CD184), canine 1D Isoform of Integrin 1β (CD29), canine CD34, canine CD45, canine MHC class II DLA-DRA1 and DLA-DQA1and canine MHC class I DLA-79 and DLA-88. To test cell differentiation, the following markers were used: canine nestin and canine glial fibrillary acidic protein (GFAP) for neurogenic differentiation; canine adiponectin (ADIPOQ) and canine leptin for adipogenic differentiation; canine bone gamma-carboxyglutamate protein osteocalcin, canine osteopontin and canine osteonectin for osteogenic differentiation. Canine hypoxanthine phosphoribosyltransferase 1 (HPRT1) was used as an internal control for RNA extraction, RT procedure, and PCR. All oligonucleotide primers spanned a least one intron. Table 1 shows primer sequences, and the expected length of corresponding amplified fragments. The RNA templates of all analyzed genes were amplified for 35 cycles of 94°C (45 sec, denaturation), 59°C (45 sec, annealing), 72°C (40 sec, elongation), followed by one cycle at 72°C for 5 min, except CD45 and DLA-88, which were amplified with a 57°C annealing temperature. Negative controls (PCR without template and RNA without RT) were performed in each experiment (data not shown). Subcutaneous fibroblasts were used as negative controls for embryonic, mesenchymal, hematopoietic, and MHC markers. Dog leucocytes were used as positive controls for hematopoietic and MHC markers expression. PCR products were visualized with ethidium bromide on a 2% agarose gel.

Semi-Quantitative RT-PCR Analysis

Semi-quantitative RT-PCR analysis was performed on AF-MSCs, AM-MSCs, and UCM-MSCs before and after osteogenic differentiation considering that, in these cell lines, osteogenic markers osteonectin and osteocalcin were found to be expressed in differentiated and undifferentiated cells. To check the exponential phase amplification of these two genes, we conducted a test with 120-µL final-volume PCR mixture (200 ng of cDNA, 1 U of HotMaster taq polymerase Eppendorf, 0.25 mM dNTPs, 1 mM each primer), taking 10 µl from the tube starting from cycle 27 up to 35. The same procedure was followed for the house-keeping HPRT1 gene. PCR amplification was conducted up to the optimal cycle identified in the previous step for both the targets and house-keeping gene. The obtained amplification products, together with a 100-bp DNA ladder, were run on 2% agarose gel stained with ethidium bromide and images were digitally captured with a CCD camera (Gel Doc 2000 Gel Documentation System; BioRad, Milan, Italy). Densitometric analysis was performed using the QuantityOne software (BioRad), and values were expressed as relative absorbance units. Messenger-RNA expression was normalized for each gene against the amount of HPRT1 mRNA. Three replicates were performed.

Statistical Analysis

The Student's t-test was used to evaluate the statistical significance of the results. Values with P < 0.05 were considered as statistically different.