Isolation, characterization, and mesodermic differentiation of stem cells from adipose tissue of camel (Camelus dromedarius)
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- Mohammadi-Sangcheshmeh, A., Shafiee, A., Seyedjafari, E. et al. In Vitro Cell.Dev.Biol.-Animal (2013) 49: 147. doi:10.1007/s11626-012-9578-9
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Adipose-derived stem cells are an attractive alternative as a source of stem cells that can easily be extracted from adipose tissue. Isolation, characterization, and multi-lineage differentiation of adipose-derived stem cells have been described for human and a number of other species. Here we aimed to isolate and characterize camel adipose-derived stromal cell frequency and growth characteristics and assess their adipogenic, osteogenic, and chondrogenic differentiation potential. Samples were obtained from five adult dromedary camels. Fat from abdominal deposits were obtained from each camel and adipose-derived stem cells were isolated by enzymatic digestion as previously reported elsewhere for adipose tissue. Cultures were kept until confluency and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteogenic, and chondrogenic potential. The morphology of resultant camel adipose-derived stem cells appeared to be spindle-shaped fibroblastic morphology, and these cells retained their biological properties during in vitro expansion with no sign of abnormality in karyotype. Under inductive conditions, primary adipose-derived stem cells maintained their lineage differentiation potential into adipogenic, osteogenic, and chondrogenic lineages during subsequent passages. Our observation showed that like human lipoaspirate, camel adipose tissue also contain multi-potent cells and may represent an important stem cell source both for veterinary cell therapy and preclinical studies as well.
KeywordsAdipose tissueStem cellAdipogenicOsteogenicChondrogenicCamelus dromedarius
Potential application of stem cells is currently under intense investigation for treatment of a wide range of animal diseases particularly those of cell-based orthopedic therapies (Braun et al. 2010). Mesenchymal stem cells (MSCs), which are pluripotent cells with the potential for differentiating into variety of cell types, have become a promising source for novel cell therapeutic applications (Pittenger et al. 1999; Nadri and Soleimani 2007). Under appropriate conditions, these cells can be induced to differentiate into neurons, myoblasts, cardiomyocytes, adipocytes, chondrocytes, and osteoblasts (Jiang et al. 2002). Due to their easy isolation, expansion, and broad differentiation potential, MSCs emerged and have been widely studied in regenerative medicine and tissue engineering research for more than a decade (Baksh et al. 2004; Li et al. 2005; Seyedjafari et al. 2010).
MSCs can be obtained in relatively large numbers from a variety of tissues and organs. Previously described isolations for such tissues include bone marrow, deciduous teeth, skeletal muscle, cord blood as well as various fetal tissues (Pittenger et al. 1999; Kisiel et al. 2012). Likewise, it is now well established that adipose tissue represents an abundant, practical, and appealing source of multi-potent cells with the capacity of self-renewal and multi-lineage differentiation in vitro. In comparison to other sources, adipose-derived stem cells (ASCs) are easy to harvest from the subcutaneous fat stores through minimally invasive procedures (Qu et al. 2007; Braun et al. 2010; Meligy et al. 2012). They are also more readily available from both fetuses and adult research animals in large quantities. Accordingly, numerous evidences showed that the capacity of ASCs for differentiation into specific cell types was more efficient than those of other sources (Toupadakis et al. 2010; Shafiee et al. 2011b; Al-Nbaheen et al. 2012).
Much effort has been devoted to the investigation of ASCs as potential progenitor cells over the past decades, which has been prompted by their applications in cell therapy and tissue engineering. Previous research on ASCs has shown that they are capable of maintaining their proliferative potential up to 8–10 passages without any detectable impairment of their self-renewal capacity (Gruber et al. 2012). However, not only differences in differentiation potential have been observed among ASCs obtained from various species (Arrigoni et al. 2009) but also diversity existed in depots of adipose tissue within the same organism (Requicha et al. 2012). Of importance, studies by others and our previous studies have demonstrated that MSCs isolated from adipose tissue of mouse, rat, and rabbit can differentiate into adipocytes, chondrocytes, and osteocytes (Miura et al. 2006; Yoshimura et al. 2007; Taha and Hedayati 2010; Ahmadbeigi et al. 2011a, b). The data obtained from canine and equine have provided further information regarding the isolation, growth, and differentiation of fat cell progenitors in large animal models (Braun et al. 2010; Kisiel et al. 2012).
As per literature survey, so far there is no report for isolation and characterization of ASCs and their differentiation potential into mesenchymal lineage in species of camel. Therefore, establishment of a standard protocol for isolation, characterization of ACSs, and their differentiation into the regenerating tissues has many advantages for cell replacement therapy and tissue engineering in camel. These advantages are even more important in dromedary camels (Camelus dromedarius), where bone fractures are a real problem and more prone to accrue (Al-Sobayil 2010). Therefore, in this study we aimed to isolate and characterize the ASC population from camel adipose tissue, and to investigate the influence of the conventional in vitro cell culture environment on the proliferation ability and multi-lineage differentiation potential of the cells after serial passages.
Materials and Methods
All chemical reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.
Isolation and proliferation of stem cell in vitro
The experimental protocol was approved by the ethical committee at Stem Cell Technology Research Center, Tehran, Iran. For sampling of fat tissue, the abdominal cavity of the slaughtered camels was opened along the midline, and the abdominal adipose tissues were harvested from each animal. Adipose tissue was obtained from abdominal deposits of five adult dromedary camels. Camel ASCs were isolated by enzymatic digestion as previously performed in our laboratory (Shafiee et al. 2011b). Briefly, adipose tissues were washed extensively by phosphate buffer saline, dissected into 1-mm2 pieces followed by treating with 0.2% collagenase type I in Dulbecco’s modified Eagle’s medium (DMEM). The cell suspension was centrifuged at 350×g for 15 min, and the supernatant was discarded. Cell pellet was suspended in DMEM (low glucose) with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin 100 U/ml, streptomycin 0.1 mg/ml) at 37°C atmosphere of 5% CO2. After 24 h, non-adherent cells were discarded by changing the culture medium. The remaining cells were allowed to grow under normal conditions. The colonies were identified by their morphology almost 5 ± 1.2 d after culture. Thereafter, the cells were trypsinized and subsequently re-plated on a new dish. Eighteen to twenty (with average of 14.5 ± 1.2) days after culture when the cells become confluent, they were considered as passage 0 and propagated under culture conditions with careful monitoring and medium change. The cells were then divided into two aliquots with the first passaged into fresh culture flask upon reaching confluence and the other frozen (in 90% FBS and 10% DMSO) and stored in liquid nitrogen for later use. By using trypan blue staining, the survival rate of all thawed ASC lots was verified to be >85% before use in the study. To study the variability of cryopreserved progenitor cells, all ASCs thawed cells were subjected to the trypan blue exclusion test and examined for their proliferative capacity. Cells from passage 2 to passage 3 were used for all experiments.
Multi-lineage differentiation potential of ASCs
Induction of adipogenic differentiation was accomplished as previously described (Gheisari et al. 2012). Briefly, the cells were seeded at a density of 2 × 104 cells/cm2 into eight-chamber slides and cultured in basal media (DMEM with 10% FBS) supplemented with 0.5 mM hydrocortisone, 60 mM indomethacine, and 0.5 mM isobutylmethylxanthine. To assess adipogenic capacity, lipid accumulation was identified in differentiated cells with in situ Oil Red O (ORO) staining 21 d after induction. An undifferentiated batch of cells allocated to the control groups without differentiating supplements.
For osteogenic differentiation, the adherent cells were cultured in osteogenesis induction medium containing basal medium (DMEM + 10% FBS) supplemented with 10 mM β-glycerophosphate, 0.1 μM dexamethasone, and 50 μg/ml ascorbic acid 2-phosphate. This was followed by detection of early mineralization using Alizarin Red S 21 d after culture. Control group consisted of cells cultured in media without any differentiating supplements.
Chondrogenic differentiation of ASCs was induced by a 21-d culture in micropellet, as described elsewhere (Shafiee et al. 2011a). Briefly, MSCs (2.5 × 105 cells) were pelleted by centrifugation in 15-ml conic tubes and cultured in DMEM supplemented with 0.1 μM dexamethasone, 50 μg/ml ascorbic acid 2-phosphate, 1% insulin–transferrin–sodium selenite supplement (Gibco, Grand Island, NY), and 10 ng/ml TGF-β (Peprotech, Rocky Hill, NJ).
Alkaline phosphatase (ALP) activity
For ALP activity assay, total protein of cells was extracted using 200 μl radioimmune precipitation (RIPA) lysis buffer. ALP activity was detected with an ALP assay kit (Parsazmun, Tehran, Iran) using p-nitrophenyl phosphate (p-NPP) as substrate. The amount of ALP in the cells was normalized against total protein content.
The amount of deposited Ca2+ on stem cells following osteogenic induction was measured using Cresolphthalein Complex one method with a calcium content assay kit (Parsazmun). The extraction of Ca2+ was performed using 0.6 M HCl.
For karyotype analysis, cells were collected from primary culture and every two passages up to passage 8. The collected cells were treated with 0.15 μg/ml colcemid (Gibco) for 2 h followed by exposure to 0.075 M KCl at 37°C for 16 min. Thereafter, the cells were fixed in ice-cold 3:1 methanol/glacial acetic acid and were then dropped onto pre-cleaned chilled slides. Chromosomes were visualized using standard G-band staining technique. At least 20 metaphase spreads were screened and five banded karyotypes were evaluated for chromosomal rearrangements.
At least three replications were performed for each experiment. Values were expressed as mean ± SD. One-way analysis of variance was used to evaluate all data sets. A P value of less than 0.05 was considered statistically significant.
Characterization of ASCs
Multi-lineage differentiation potential of ASCs
Notable changes in organization of the cells within the culture have been observed after a 3-wk exposure of ASCs to osteogenic medium. The altered organization was accompanied with the changes in morphological appearance including elongation of the cells. This morphological alteration was more apparent after the second wk as compared with the control group. After 8 to 10 d of induction, ASCs began to deposit calcium on their matrix and formed the mineralized nodular aggregates which resulted in a greatly enhanced formation of bone nodules. The capacity of the ASCs to differentiate towards an osteogenic lineage and to deposit calcium was easily demonstrated with alizarin red staining (Fig. 3B). Calcium deposition was not observed in cells from the control group.
The differentiation toward the chondrocyte has been confirmed by accumulation of glycosaminoglycans (GAGs) in chondrocytes using Alcian blue 8G staining (Fig. 3C). Non-induced ASCs that has been cultured under normal medium condition without chondrogenic media did not form a well-structured pellet and scattered during the sectioning process.
Adipose tissue represents a plentiful reservoir of cells with multi-lineage potential of differentiation into a variety cell types, including adipocytes, chondrocytes, and osteoblasts. Compared to their umbilical cord blood counterpart, adipose tissue not only constitutes an abundant and accessible source of cells but also can be managed readily for in vitro manipulation. Attempts to subculture and differentiation of the adipose precursor cells have been of particular attention over the last decade, mainly due to their simple isolation procedures and relevant stem cell numbers that can be extracted (Furuya et al. 2012; Kisiel et al. 2012; Minteer et al. 2012).
Similar to our previous study in mice and rabbit, MSCs were successfully isolated and expanded from adipose tissues of camel with relative ease, simplicity, and speed (Ahmadbeigi et al. 2011a). The isolated cells demonstrated a spindle-shaped or elongated fibroblast-like morphology. Adherent cells from camel adipose tissue were found to have the ability to proliferate extensively in vitro and to maintain their morphological and growth characteristics upon culture.
In this study, the proliferation and expansion of camel ASCs (DT = 41.33 ± 1.52 h at passage 3) were relatively similar to those of MSCs isolated from human bone marrow and adipose tissue (Shafiee et al. 2011b). Compared to MSCs isolated from bone marrow of rodents such as rabbits and mice, camel ASCs exhibit a longer lifespan and higher potential of proliferation and expansion without any in vitro abnormality (Miura et al. 2006; Ahmadbeigi et al. 2011a; Ahmadbeigi et al. 2011b). Additional evidence complementing the above observation is provided recently by Vidal et al. (2012), who indicated that the stem cells derived from equine bone marrow have limited passage numbers of subculture in comparison with those derived from adipose tissue. These authors concluded that the adipose tissue and umbilical cord tissue may be preferable for tissue banking purposes than the BMSCs in equine.
The result of current study shows that cellular senescence occurs in camel ASCs after about eight population doublings. This includes morphological changes such as enhanced granularity of the cytoplasm followed by extensive vacuolization as well as decreased proliferation. Consistent with our finding, equine ASCs cultured up to more than fifth passage before signs of senescence (Braun et al. 2010). In a comparative study using small to large animal models, the number of pluripotent cells per milliliter of adipose tissue was variable and the yield of rabbit ASCs was lower than that in rat and pig. However, in that study, all ASCs populations showed both a stable doubling time during culture and marked clonogenic ability (Arrigoni et al. 2009).
Since self-renewal and multi-lineage differentiation potential are two major functional attributes of stem cells (Chan et al. 2011; Ahmad et al. 2012; Orbay et al. 2012), we attempted to evaluate the ability of ASCs for differentiation into three germ layer lineages and to investigate the stemness of isolated cells. Stem cells isolated from adipose tissue are logically prone to differentiate into adipocytes cells (Taha and Hedayati 2010; Shafiee et al. 2011b). The adipogenic differentiation pattern of camel ASCs was precisely as expected due to the great amount of adipogenic markers including large oil droplets throughout the cells after the period of adipogenic induction. As evident from literature, camel adipose tissues tended to have lower levels of cholesterol than beef and lamb adipose tissues (Abu-Tarboush and Dawood 1993). Therefore, as the technology of progenitor cell culture moves ahead, camel stem cells might be a suitable source for the large quantities of fat cells with favorable lipids profile as reflected by the decreased cholesterol and triglyceride levels.
Camel is an important multi-purpose popular local animal of Iran and more than 200,000 dromedary camels are freely living in the arid and semiarid deserts of this country (Razawi et al. 2009). The increasing popularity of camel in Iran and Middle East means veterinarians are more likely to deal with them as patients; therefore, dealing with camel fractures is quickly becoming part of the normal caseload. Accordingly, the literature on fractures in Old World Camelids is considerably greater than alpaca and llama, particularly in male dromedary camels due to fighting and racing injuries (Newman and Anderson 2009). Experimental and clinical evidences have been indicated that treatment of such fractures can be possible efficiently using cell therapy and tissue engineering approaches (Caplan 2005). Toward this, stem cells alone or differentiated into osteoblasts in combination with a matrix are transplanted in bone defects (Marcacci et al. 2007). In the present study, we observed that camel ASC can be efficiently transformed into osteoblasts with the ability of mineralization and producing bone-related enzymes such as ALP. As adipose tissue is an accessible tissue around the camel body, these cells can be easily isolated, propagated, and differentiated to osteoblasts followed by transplantation in camel bone defects. This autologous cell therapy excludes the risk of immunorejection based on allogeneic stem cell transplantation.
Contrary to adipogenic and osteogenic differentiation, cells need to be aggregated into cell pellet with a 3D arrangement for being induced to develop into chondrogenic differentiation. Our observation revealed that placing cell pellets in chondrogenic induction medium led to the formation of chondroblast-like cells. Production of ECM-like structures and components such as GAG is one of the hallmarks showing the commitment of stem cells into chondrogenic lineage. An increased accumulation of GAG was observed after 21 d of chondrogenic differentiation in cell pellets and showed the complete differentiation of camel ASCs to chondrocytes. This observation is consistent with earlier reports in which chondrogenic differentiation of ASCs has been successfully induced in several animal models (Braun et al. 2010; Taha and Hedayati 2010; Kisiel et al. 2012). In this light, it can be speculated that such mature cells are an ideal source for cell therapy and tissue engineering approaches.
Although information regarding expression and/or production markers to investigate the multi-lineage differentiation of stem cells are lacking in camel species, the phenotypic markers used in this study are well-established ones and are frequently utilized in authors’ laboratory for other species (Ahmadbeigi et al. 2011a, b).
Transformation of stem cells is one of the limitations and challenges in stem cell therapy approaches (Furlani et al. 2009; Rosland et al. 2009; Ahmadbeigi et al. 2011a, b). Herein, we performed karyotypic analysis after each passage to see the chromosome abnormalities. Cytogenetic analysis showed no abnormal karyotype before the senescence program is initiated. Our results confirmed that the camel ASC can be successfully expanded and passaged to reach sufficient amount of transferable cells which would be applicable for cell therapy and tissue engineering in camel species. Notably, this is the first time that karyotype of camel stem cell has been analyzed after multiple passages in culture.
In conclusion, the stem cells obtained from camel adipose tissue yields an adherent layer of fibroblast-like cells in the primary culture. After several passages, the adherent cells not only exhibited the same morphology and normal karyotype but also had a multi-lineage potential with the capacity to differentiate to adipogenic, osteogenic, and chondrogenic lineages. The general growth and culture properties of ACSs point to an important role for these cells in both veterinary medicine as well as preclinical studies.
This work was supported by a grant from the Stem Cell Technology Research Center, Tehran, Iran. The authors like to thank Miss Lida Langroudi for English editing the manuscript.