Cell Biology International (2009) 33, 100-105 - Simona Passeri and others - Isolation and expansion of equine umbilical cord-derived matrix cells (EUCMCs)

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Cell Biology International (2009) 33, 100–105 (Printed in Great Britain)

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Isolation and expansion of equine umbilical cord-derived matrix cells (EUCMCs)

Simona Passeri^a, Francesca Nocchi^b, Roberta Lamanna^b, Simone Lapi^b, Vincenzo Miragliotta^a^*, Elisabetta Giannessi^a, Francesca Abramo^c, Maria Rita Stornelli^a, Micheletino Matarazzo^d, Daniele Plenteda^d, Patrizia Urciuoli^b, Fabrizio Scatena^b and Alessandra Coli^a

^aDepartment of Veterinary Anatomy, Biochemistry and Physiology, University of Pisa, Pisa, Italy
^bCell Biology and Tissue Regeneration Laboratory-Immunohaematology 2 Unit – Azienda Ospedaliera Universitaria Pisana, Pisa, Italy
^cDepartment of Animal Pathology, University of Pisa, Pisa, Italy
^dCentro Militare Veterinario of Grosseto, Grosseto, Italy

Abstract

Stem cells from extra-embryonic sources can be obtained by non-invasive procedures. We have standardized a method for the expansion of equine umbilical cord-derived matrix cells (EUCMCs) for potential therapy.

EUCMCs were isolated from the umbilical cord of five mares immediately after delivery. For expansion, cells were grown in α-MEM and MSCBM. Moreover, to measure the effect of growth factor supplementation, epidermal growth factor (EGF) was added to α-MEM.

α-MEM and MSCBM media performed similarly in terms of population doubling and CFU number value. EGF supplementation of α-MEM determined a significant increase of the population doubling value. EGF supplementation did not affect the adipogenic and chondrogenic differentiation while bone nodule sizes an increased with the osteogenic protocol.

Both α-MEM and MSCBM can be used to cultivate EUCMCs. α-MEM supplemented with EGF might represent an advantage for EUCMCs expansion. The results could be useful in choosing the culture medium since α-MEM is more cost-effective than MSCBM.

Keywords: Umbilical cord-derived matrix cells, Umbilical cord, Stem cells, Equine, Epidermal growth factor.

^*Corresponding author. Department of Veterinary Anatomy, Biochemistry and Physiology, University of Pisa, viale delle Piagge 2, Pisa 56124, Italy. Tel.: +39 050 221 6859; fax: +39 050 221 6868.

1 Introduction

Stem cells represent the most promising population for cell therapy and have gained considerable attention during the last few years. Recent studies have been focused on making more feasible their isolation and expansion under well standardized cell culture conditions, eventually directing their proliferation with growth factors in order to transplant them into patients for clinical gains.

As humans, horses suffer from skin and musculoskeletal diseases which represent an enormous source of wastage for the equine industry; attempts to resolve these diseases might therefore contribute to the development of novel therapies that can be useful also for the human analog disorders.

Presently, three types of stem cells have been described: embryonic stem cells, found in the inner cell mass of the early embryo, extra-embryonic stem cells isolated from extra-embryonic tissues (amnios, placenta, umbilical cord matrix) and adult stem cells (Igura et al., 2004; Zhang et al., 2006). Among adult stem cells, mesenchymal stem cells (MSCs) are reported to be able to self renew and to differentiate into cells of connective tissue lineages, including bone, fat, cartilage and muscle (Barry and Murphy, 2004; Lee and Hui, 2006). Recently, horse MSCs have been isolated from bone marrow (Koerner et al., 2006; Vidal et al., 2006; Arnhold et al., 2007; Kisiday et al., 2008), adipose tissue (Vidal et al., 2007; Kisiday et al., 2008), peripheral blood (Koerner et al., 2006) and umbilical cord blood (Koch et al., 2007; Reed and Johnson, 2008). Horse extra-embryonic stem cells have also been isolated from umbilical cord matrix (Hoynowski et al., 2007) for their potential suitability in clinical application.

The horse extra-embryonic tissues represent a source of stem cells and in this direction, Hoynowski et al. (2007) isolated and characterized a stem cell population from the equine umbilical cord matrix. They studied the expression of a number of markers that are associated with an embryonic phenotype (Oct-4, SSEA-4 c-Kit) or to an adult phenotype (CD54, CD90, CD105, CD146) by FACS analysis. Moreover they reported the in vitro differentiation ability of these cells toward the adipogenic, chondrogenic and osteogenic lineages. Taken together, all the above mentioned findings demonstrate that equine umbilical cord matrix cells (EUCMCs) have functional features similar to MSCs. The reported expression of Oct-4, SSEA-4 and c-Kit witnesses a more primitive phenotype that can make them stand between embryonic and adult stem cells. A major issue, in stem cell therapy, is the establishment of a non-invasive withdrawal of tissues together with an abundant source of cells and a low level of contamination. The reported findings in horses (Hoynowsky et al., 2007) and humans (Wang et al., 2004; Fong et al., 2007; Secco et al., 2008; Qiao et al., 2008) indicate umbilical cord matrix as a rich source of EUCMCs whose collection is obviously non-invasive. On the other hand the partum canal and the delivery environment represent a non-sterile condition, particularly if we refer to the equine species.

Epidermal growth factor (EGF) is already known to intervene in modulating the growth and repair of various tissues. After binding to its receptor (EGFR) it activates an intracellular pathway able to promote migration, adhesion, proliferation, and survival in various cell types (Lembach, 1976; Wells, 1999; Roux and Blenis, 2004). Both EGFR expression and EGF responsiveness have been reported in marrow-derived MSC (Gronthos and Simmons, 1995; Satomura et al., 1998). Fan et al. (2008) recently reported that EGF is able to induce EGFR signaling and to promote MSC proliferation and migration without any “side-effects” on pluripotency (Tamama et al., 2006).

Here we report a method to collect, isolate, expand EUCMCs comparing different antibiotic concentration (to control contamination), different culture media and the suitability of epidermal growth factor (EGF) during the expansion phase. A standardized method to collect and expand EUCMCs will allow the feasibility of a large number sample storage that would be used when needed in the grown foal (autologously) or in unrelated horses (heterologously).

2 Materials and methods

2.1 Umbilical cord sampling

Umbilical cords were obtained by five 10–13years old standard bred mares. All umbilical cord (UC) harvesting procedures were performed under guidelines determined by the Local Ethical Committee. UCs (portions of 30cm from the point of rupture) were collected from five mares soon after delivery, put into RPMI-1640 medium (Invitrogen, CA, USA) supplemented with 2% penicillin/streptomycin (P/S) (Cambrex, NJ, USA) and 1% amphotericine-b (Invitrogen), stored at 4°C. Subsequently, each UC was cut in pieces (4–6cm in length), divided into three samples (A, B, C) that were used as follows: sample A was immediately processed for isolation of EUCMCs as detailed below; sample B was immersed in RPMI 2% P/S+2% amphotericine-b, stored at 4°C for 24h and then processed; sample C was immersed in RPMI 5% P/S+2% amphotericine-b, stored at 4°C for 24h and then processed. After bacterial and fungal contamination was assessed by cytological analysis of the culture medium, only sample C was considered suitable for further experiments.

2.2 Isolation and expansion of EUCMCs

UC samples were washed by flushing a phosphate-buffered saline (PBS) (Euroclone, MI, Italy) solution with a syringe into the cord vessels, thus removing blood traces. Each vessel was carefully stripped away and the surrounding mucous connective tissue (MCT) removed with a scalpel blade, minced in 1mm³ pieces and immersed in 10mL of a collagenase solution (1mg/mL) for 30min at 37°C. The suspension was then filtered through a 100μm filter (Millipore, Billerica, MA, USA).

Nucleated cells were counted in an hemocytometer by staining with 0.4% Trypan blue (Sigma, St. Louis, MO, USA), centrifuged at 500×g for 10min, and the pellets were resuspended in alpha modified minimum essential medium (α-MEM) (Cambrex, NJ, USA) supplemented with 20% fetal calf serum (FCS) (Eurobio, France), 100U/mL penicillin, 100μg/mL streptomycin, 2mM l-glutamine (Euroclone, MI, Italy). Cells were plated at 10⁵cells/cm² in 25cm² flasks (Sarstedt, Nümbrecht, Germany). After 24h the non-adherent cells were removed by washing with PBS and fresh medium was added twice a week for about 14days or until adherent cell reached 90% confluence (passage 0, P0). Cells were then harvested (P1) for further expansion using trypsin 0.25% solution and 1mM EDTA (Euroclone, Milan, Italy) for 5min at 37°C, replated at 5000cells/cm², grown to near confluence and harvested with the same protocol. At the end of each passage the cells were counted by a hemocytometer; living cells were identified by Trypan blue exclusion.

2.3 Selection of culture medium

From passage 1, cells were grown in α-MEM and mesenchymal stem cells basal medium (MSCBM, Cambrex). α-MEM was always supplemented with 20%-FCS, penicillin 100U/ml, streptomycin 0.1mg/ml, l-glutamine 2mM. Media were replaced every 3days.

To evaluate the effect of growth factor supplementation, epidermal growth factor (EGF, Sigma, St. Louis, MO, USA) was added to α-MEM to a concentration of 10ng/mL.

EUCMCs were plated at 5000cells/cm² in a 25cm² tissue culture flask with either α-MEM or MSCBM or α-MEM+EGF to determine growth kinetics. Cells at 90% confluence were trypsinized, counted with a hemocytometer and re-plated as mentioned above.

2.4 Colony forming unit-fibroblast (CFU-F) assay

To evaluate the EUCMC number in the primary culture, nucleated cells isolated from UC were plated at 10⁵cells/cm² in six well plates and incubated for 8days. After incubation the cells were rinsed twice with PBS. Colonies (CFU-F) were then stained with Crystal Violet (0.5%) (Diagnostic International Distribution, Milan, Italy) in methanol (Sigma, St. Louis, MO, USA) at room temperature for 10min, rinsed twice with PBS and visually counted using a phase contrast microscope (Leica, Germany).

The frequency of EUCMC in horse UC was estimated by dividing the total number of nucleated cells plated at P0 with the number of CFU-F counted in the primary culture.

2.5 Population doubling and fold increase evaluation

Population doubling (PD) was calculated according to the following formula: (logNh−logN0)/log2 where Nh is the cell number at the end of passage and N0 the initial cell number.

Population doubling at the end of primary culture was calculated by comparing the number of cells at the end of P0 with the estimated number of EUCMCs at the beginning of the primary culture. Cumulative population doubling (CPD) was calculated by adding the population doubling value at P0 to the sum of the population doubling values obtained for each passage.

Fold increase was calculated by dividing the number of harvested cells at 90% confluence by the number of plated cells for each passage.

2.6 Differentiation protocols

To ascertain the differentiation ability of EUCMCs, P3 cells already grown in α-MEM were plated at 5000cells/cm² in four well chamber slides (Sigma, St. Louis, MO, USA) and re-incubated in α-MEM for 10days. The three differentiative protocols were performed as follows.

2.6.1 Osteogenesis

Cultures were incubated in α-MEM supplemented with 20% FCS, 100U/mL penicillin, 100μg/mL streptomycin, 2mM l-glutamine, 20mM β-glycerol phosphate (Sigma, St. Louis, MO, USA), 100nM dexamethasone (Sigma, St. Louis, MO, USA) and 250μM ascorbate 2-phosphate (Sigma, St. Louis, MO, USA) for 3weeks. Cells were fixed with a 10% buffered formalin solution (Sigma, St. Louis, MO, USA) for 20min at room temperature (RT) and stained with Alizarin Red (Sigma, St. Louis, MO, USA) pH 4.1 for 20min at RT.

2.6.2 Adipogenesis

Cultures were incubated in α-MEM that was supplemented with 20% FCS, 100U/mL penicillin, 100μg/mL streptomycin, 12mM l-glutamine, 5μg/mL insulin (Lilly), 50μM indomethacin (Sigma), 1μM dexamethasone (Sigma, St. Louis, MO, USA) and 0.5μM 3-isobutyl-1-methylxanthine (IBMX, Sigma, St. Louis, MO, USA) for 2weeks. Cells were fixed with 10% formalin for 20min at RT and stained with 0.5% Oil Red O (Sigma, St. Louis, MO, USA) in methanol (Sigma) for 20min at RT.

2.6.3 Chondrogenesis

Cultures were incubated for 3weeks in chondrocyte basal medium (CBM, Cambrex Bio Science, Walkersville, MD, USA). Cells were fixed with 10% formalin for 20min at RT and stained with Alcian Blue solution (Sigma, St. Louis, MO, USA) pH 2.5for 20min at RT. Cell nuclei were counterstained with Weigert's iron hematoxylin.

To test the effect of EGF supplementation on differentiation ability cells already grown in α-MEM+EGF were plated at 5000cells/cm² in four well chamber slides and re-incubated in α-MEM+EGF for 10days. Subsequently, adipogenic, osteogenic and chondrogenic differentiations were attempted by using the same protocols mentioned above and adding EGF (10ng/ml) to the media.

2.7 Statistical analysis

Values are reported as mean±SD. All statistical analyses were performed using Graph-Pad Prism software (GraphPad, San Diego, CA, USA). The population doubling for each passage and each medium was compared using ANOVA and T test. The correlation between CFU-F at P0 and CPD was determined by regression analysis. Differences were considered statistically significant at p<0.05.

3 Results

3.1 Umbilical cord sampling

While fungal contamination was never observed, all cultures obtained from samples A showed a bacterial contamination. Sixty percent of UCs incubated in RPMI supplemented with 2% P/S+2% amphotericine-b (samples B) showed a bacterial contamination, while those in RPMI supplemented with 5% P/S+2% amphotericine-b (samples C) did not show any contamination.

3.2 Isolation and culture of EUCMC

The average number of equine UC cells collected was 1.6±0.6×10⁷ (n=5). The CFU number calculated at P0 (i.e. primary culture) was 136.7±39.4 and the frequency was 1:118.

There was a positive correlation between number of mononucleated cells obtained from each umbilical cord and the total number of CFUs obtained at P0 (r²=0.96; p<0.01).

The average number of equine UC mesenchymal cells (EUCMC) obtained at the end of primary culture was 4.1±1.8×10⁶ (n=5), corresponding to 13.7 PD. EUCMC have been cultured up to 12 passages and the CPD was 36.5±3.4.

3.3 Selection of culture medium

The PD values obtained from each passage, for α-MEM and MSCBM, were not statistically different.

EGF supplementation of α-MEM determined a significant increase of the PD value (p=0.007) for each passage. The CPD value was 55±7 after 19 passages were performed till cells reached senescence.

Fold increase calculated for cells cultured with α-MEM+EGF resulted to be statistically higher (p<0.01) than α-MEM cultured cells (Fig. 1).

Fig. 1

Histogram showing the fold increase values obtained in 12 passages by using either α-MEM or α-MEM+EGF. α-MEM=white columns; α-MEM+EGF=black columns; asterisks indicate statistically significant differences. The mean value of the passage duration was 70.3±42.9h for α-MEM and 53.4±25.1h for α-MEM+EGF.

No significant differences in fold increase were detected between α-MEM and MSCBM cultured cells.

3.4 Morphological observations

Both large and occasionally multi-nucleated cells and small, spindle-shaped, mononucleated cells were present in the primary culture. This heterogeneity could no longer be found by the second passage as the smaller spindle-shaped fibroblastoid cells appeared to predominate and to proliferate even after numerous passages. Individual spindle-shaped cells appeared after 3–4days of primary culture, while colonies were observed as early as 5days post seeding and the first subculture was done 7days after initial seeding. The EUCMCs also showed a stellate shape that did not change for all passages (Fig. 2).

Fig. 2

Photomicrograph showing the three morphological types of P0 EUCMCs: large cells, spindle-shaped cells and (insert) stellate cells.

3.5 Differentiation

Differentiation of EUCMCs into adipocytic, osteoblastic and chondrocytic lineages was observed.

After adipogenic induction, the cell morphology changed from the elongated confluent fibroblastic cells to more oval-shaped cells, which showed a distinct ring of red coarse vacuoles around the cell periphery after Oil Red O staining. These vacuoles appeared to develop by day 2 and became more numerous and larger with time (Fig. 3A).

Fig. 3

Photomicrographs showing the differentiation ability of EUCMCs. (A) Adipogenic differentiation detected by Oil Red O staining; (B) osteogenic differentiation detected by Alizarin Red S staining; (C) chondrogenic differentiation detected by Alcian Blue staining. Scale bars are equal to 100μm.

Osteogenic differentiation induced cell cultures to change their morphology from adherent monolayer of swirling spindle-shaped cells, which was still apparent in the control cultures, to multilayered cell clusters surrounded by a matrix-like substance positive to the Alizarin Red S stain. Cultures showed rapid mineralization and nodule formation. A weak reactivity to Alizarin Red staining was visible in the control cultures. The colonies forming bone nodules were characterized by an accumulation of overcrowd fibroblast-like cells in direct contact with one another. The cells bordering the nodules were of a fibroblastic morphology, while those were visible toward the center were more polygonal (Fig. 3B).

Chondrogenic differentiation of EUCMC was identified by marked deposition of glycosaminoglycans in the matrix, observable after Alcian blue staining (Fig. 3C).

EGF supplemented to the differentiation media did not affect the adipogenic and chondrogenic differentiation while an increase in bone nodule size was observed with the osteogenic protocol.

4 Discussion

In our experience, bacterial/fungal contamination is one of the main problems usually encountered in UC sampling. This is obviously due to the non-sterile environment where the procedure is usually performed and to the possible contact with fecal material and the mare perineal area. To decrease bacterial/fungal contamination three sampling protocols have been tested and the best result was obtained with an over-night immersion in RPMI 5% P/S+2% amphotericin-B.

To optimize in vitro cell growth, two different culture media have been tested. MSCBM, a commercial ready to use culture medium already supplemented with growth factors able to support stem cell proliferation but very expensive, and α-MEM, commonly used to expand MSC (Javazon et al., 2001; Fukuchi et al., 2004; Smith et al., 2004). The positive correlation found between the number of mononucleated cells (1.6±0.6×10⁷) and the number of generated CFU (136.7±39.4) is in agreement with previously reported findings (Da Silva Meirelles and Nardi, 2003), and represent key information in order to optimize the isolation of these cells.

The CPD value (36.5±3.4) found in the isolated EUCMCs does not concord with what already was observed by Sarugaser et al. (2005), that reported a PD value around 50 obtained from cells derived from human UC and with the observations of Mitchell et al. (2003) who reported a PD value of 80 in the swine species. A possible explanation of these differences might depend on the different species studied and on different culture media used. In our study cell proliferation was not influenced by the culture medium (α-MEM vs MSCBM). In fact, both media performed similarly in terms of CFU number, population doubling and fold increase values. These findings could be useful in choosing the optimal culture medium since α-MEM results more cost-effective than MSCBM.

On the other hand, α-MEM supplemented with EGF dramatically increased the cumulative population doubling value (55±7) as well as the number of passages before cells became senescent; the obtained fold increase was significantly higher in respect to unsupplemented α-MEM. These findings are corroborated by the reported effects of EGF on MSCs isolated from other species; thus witnessing that EUCMC express the EGF receptor that, as reported in humans (Krampera et al., 2005) mice and pigs (Tamama et al., 2006), when bound with its ligand stimulates cell migration, proliferation and survival.

When cultured in specific culture media, EUCMCs differentiated into the adipogenic, osteogenic and chondrogenic lineages. This capability assesses the typical properties of MSCs in that they can differentiate into lineages of mesenchymal origin, as already described (Bruder et al., 1998; Pittenger et al., 1999; Muraglia et al., 2000).

The effect of EGF supplementation has also been tested on the differentiation ability of EUCMCs. Adipogenic and chondrogenic differentiation was not affected by EGF supplementation. In contrast, the osteogenic differentiation proceeded to the formation of larger bone nodules. Therefore, as reported also by Tamama et al. (2006), EGF does not inhibit MSC differentiation. The increased size of bone nodules might depend on the effect that EGF has on cell proliferation; it is interesting that Sarugaser et al. (2005) reported that MSCs exhibited spontaneous bone nodules formation even in non-osteogenic culture conditions. Therefore, EGF being a strong inducer of mitosis, cells are quickly forced to the favorable differentiative lineage with the consequent formation of bigger nodules.

In conclusion, in the study presented herein we propose a suitable sampling procedure with an appropriate use of antibiotic/antimycotic supplement in order to avoid undesirable contaminations that would lead to the wastage of cell cultures. In addition we propose EGF as an advantageous supplement to allow an optimal isolation and expansion of the EUCMCs.

Acknowledgements

The work was partially supported by the Italian Education University and Research Ministry (60% funds).

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Received 11 June 2008/18 September 2008; accepted 13 October 2008

doi:10.1016/j.cellbi.2008.10.012

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