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Adipose-derived stem cells (ADSCs) are multipotent, and can be differentiated into many cell types in vitro. In this study, tissues from pigs were chosen to identify and characterize ADSCs. Primary ADSCs were sub-cultured to passage 28. The surface markers of ADSCs: CD29, CD71, CD73, CD90, and CD166 were detected by reverse-transcription polymerase chain reaction assays and the markers CD29, CD44, CD105, and vimentin were detected by immunofluorescence. Growth curves and the capacity of clone-forming were performed to test the proliferation of ADSCs. Karyotype analysis showed that ADSCs cultured in vitro were genetically stable. To assess the differentiation capacity of the ADSCs, cells were induced to differentiate into osteoblasts, adipocytes, epithelial cells, neural cells, and hepatocyte-like cells. The results suggest that ADSCs from pigs showed similar biological characteristics with those separated from other species, and their multi-lineage differentiation shows potential as an application for cellular therapy in an animal model.
Les cellules souches dérivées du tissu adipeux (CSDAs) sont pluripotentes, et peuvent se différencier en plusieurs types de cellules in vitro. Dans la présente étude, des tissus d’origine porcine ont été choisis afin d’identifier et de caractériser les CSDAs. Des CSDAs primaires ont été souscultivées jusqu’au passage 28. Les marqueurs de surface des CSDAs suivants : CD29, CD71, CD73, CD90, et CD166 ont été détectés par réaction d’amplification en chaîne par la polymérase utilisant la transcriptase réverse et les marqueurs CD29, CD44, CD105, et vimentine ont été détectés par immunofluorescence. Des courbes de croissance et la capacité à former des clones ont été effectuées afin de tester la prolifération des CSDAs. L’analyse du caryotype a montré que les CSDAs cultivées in vitro sont génétiquement stables. Afin d’évaluer la capacité de différentiation des CSDAs, des cellules ont été induites à se différencier en ostéoblastes, adipocytes, cellules épithéliales, cellules neurales, et des cellules apparentées aux hépatocytes. Les résultats suggèrent que les CSDAs porcines ont montré des caractéristiques biologiques similaires à celles de d’autres espèces, et que leur différentiation en plusieurs lignées montre un potentiel pour une application de thérapie cellulaire dans un modèle animal.
(Traduit par Docteur Serge Messier)
Adipose tissue is an abundant, less expensive, and safe bodily tissue to study. It can be isolated in large quantities by minimally invasive liposuction. Immature pluripotent cells are important targets for tissue engineering, regenerative medicine, and gene therapy (1). Adipose-derived stem cells (ADSCs) are the richest sources of multipotent cells and can be easily cultivated and expanded. These cells pose less psychological and physical impact to the donor than bone marrow cells (BMCs). The phenotype and expansive capacity of ADSCs are stable, without spontaneous re-differentiation and shift of cellular markers after dozens of passages (2). They have a high capacity to differentiate and self-renew within and across lineage barriers (3). Adipose-derived stem cells can be differentiated into osteogenic, adipogenic, myogenic, chondrogenic, and neurogenic lineages (3–5). Adipose-derived stem cells also have low immunogenicity, which offers great promise for use in regenerative medicine (6–8). Other research shows ADSCs represent a practical, abundant, and appealing source for cell replacement of donor tissue (9).
Currently, the shortage of available donors and graft rejection hamper widespread transplantation. Stem cell therapy is a current research topic and holds great promise for disease treatment. Adipose-derived stem cells can grow rapidly in vitro. They are available for harvesting in large numbers using a less invasive operation. In this research, we describe the isolation and culture procedures of pig ADSCs, and demonstrate that ADSCs are able to differentiate into many cells types.
Animal experiments were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee at Chinese Academy of Agricultural Sciences. Adipose tissues were harvested from the visceral (omental region) of pigs under aseptic conditions. Briefly, the extracellular matrix was dissociated with 0.1% (m/v) type I collagenase (Sigma) and the harvested pellet was re-suspended in complete medium (10). At 70% to 80% confluence, the cells were passaged with 0.125% trypsin. Generally, after 3 to 4 passages, the cells were homogenous.
Cells were incubated in the following antibodies: i) mouse anti-pig CD29 (1:100; Abcam); ii) rat anti-pig CD44 (1:100; Abcam); iii) mouse anti-pig CD105 (1:100; Abcam); and iv) mouse anti-pig Vimentin (1:100; Abcam). The cells were then incubated by FITC-conjugated goat anti-mouse immunoglobulin and goat anti-rat immunoglobulin (bioss) and examined using a Nikon TE-2000-E confocal microscope.
RNA was extracted from the cells from passages 4, 14, 20, and 26 using Trizolreagent (Invitrogen). We used the TaKaRa RNA PCR Kit (AMV) Ver. 3.0 for reverse-transcription polymerase chain reaction (RT-PCR). The cDNAs were amplified by Emerald Amp Max PCR (TaKaRa). The specific primers are listed in Table I. The PCR products were visualized by 2% agarose gel electrophoresis.
To assess growth dynamics, ADSCs at different passages were seeded in triplicate in 24-well plates (1 × 104 cells/well) (11). The population doubling time (PDT) was calculated as follows:
Where: t0 = start time of culture, t = termination time of culture, N0 = initial number of cells in culture, and Nt = the final number of cells in culture.
Cells from passages 4, 12, and 27 were seeded in 24-well microplates at a density of 1 × 104 cells/well, and numbers of colony-forming units (CFU) were counted to calculate the colony-forming rate, which is formulated as CFU numbers/starting cell number per 24-well × 100%.
The karyotype of P8 cells was analyzed. Cells were subjected to hypotonic treatment and fixed, and the chromosome numbers were counted from 100 spreads under an oil immersion objective upon Giemsa staining.
Adipose-derived stem cells from passage 16 were plated onto 60-mm dishes. When the cells confluenced to 70%, the medium was changed to adipocyte-inducing medium. Cells from the control group were fed with DMEM medium. After 10 d, the induced cells were fixed with 4% (m/v) paraformaldehyde and stained with Oil-Red O for 30 min to visualize lipid droplets. The adipogenic specific genes LPL and PPARγ were detected by RT-PCR.
The ADSCs from passage 18 were plated onto 60-mm dishes for osteogenic differentiation. The cells were incubated with osteogenic-inducing medium when they confluenced to 80%. Cells from the control group were fed with DMEM medium. The cells were fixed with 4% paraformaldehyde (m/v) at 10 and 22 d after induction. These cells were also stained with alizarin to visualize the mineralized matrix. The osteogenic-specific genes OPN, COL1, RUNX2 were detected by RT-PCR.
To induce hepatogenic differentiation of ADSCs, passage 13 cells were incubated on a 60-mm dish. When the cells confluenced to 50%, the medium was changed to hepatic-inducing medium. After 15 d, the cells were fixed with 4% paraformaldehyde (m/v) and stained with periodic acid Schiff to visualize the glycogen deposits. The hepatogenic specific genes AFP and ALB were detected by RT-PCR.
The ADSCs from passage 8 were incubated on a 6-well plate. After the cells confluenced to 50%, the medium was changed to L-DMEM medium containing 10% FBS (v/v), 1% B27, 1% glutamine, 1% streptomycin, 10 ng/mL bFGF, and 15 ng/mL EGF. After 10 d, the cells were conformed with immunofluorescent assays of epithelial markers PCNA and CK18. The epitherial specific genes CK18 and E-Carderin were detected by RT-PCR.
The ADSCs from passage 9 were plated onto a 6-well plate. After the cells confluenced to 70%, the medium was changed to a neurogenic-induction medium. The induction was divided into 2 steps: i) the cells were incubated with the neurogenic induction medium-1 for 7 d; ii) the cells were then incubated with the neurogenic induction medium-2 for 7 d. The cells were conformed with immunofluorescent assays of neural markers MAP2, GFAP, and β-tubulin. The neural-specific genes NF and map2 were detected by RT-PCR.
Primary cells isolated from adipose tissue adhered to plates and began to elongate after 48 h (Figure 1A-a). After about 3 d, the cells exhibited a fibroblast-like morphology (Figure 1A-b). Cells expanded rapidly and confluenced to 90% 10 d later (Figure 1A-c). Cells were cultured up to passage 28 with most cells showing signs of senescence, such as vacuolization and karyopyknosis (Figure 1A-d).
Specific surface antigen markers of ADSCs were detected by immunofluorescence and RT-PCR. Results of immunofluorescence staining demonstrated that the ADSCs were CD29, CD44, CD105, and Vimentin positive (Figure 2). The RT-PCR indicated that the ADSCs expressed CD29, CD71, CD73, CD105, and CD166 but didn’t express endothelial marker CD31 (Figure 1C).
Growth and proliferation of ADSCs were similar at P4, P14, P20, and P26 according to the growth curves analysis (Figure 3). After a latency phase of 1 to 2 d, cell growth entered the logarithmic phase, and reached the plateau phase at approximately day 8. The PDT was 28, 31, 32, and 36 h for P4, P14, P20, and P26, respectively.
Colony formation was observed by microscopy after 6 d. Colony-forming levels were 37 ± 0.2%, 30.6 ± 0.1%, and 24 ± 0.3% for passages 4, 12, and 27, respectively, demonstrating the capacity of cultured ADSCs for self-renewal (Figure 4).
The diploid chromosome number of pig ADSCs was 2n = 38, consisting a pair of sex chromosomes per gender. There was no normal diploid chromosome ploidy missing or broken (Figure 5).
The adipogenic potential was evaluated by differentiation of post-confluent ADSCs. The obvious lipid droplet appeared after 10 d of induction, and it was in the cytoplasm. The lipid droplet was stained strongly with Oil O Red through fatty acids, and presented as bright red inclusions (Figure 5a). The expression of adipocyte specific markers LPL and PPARγ was tested by RT-PCR for 10-day differentiated cells. The undifferentiated cells were tested as controls. The results are shown in Figure 5b.
Morphological changes were used as evidence for osteogenic differentiation during the induction. At the end of the 22-day induction period, calcium crystals appeared in the cytoplasm and the differentiation was confirmed by alizarin staining for calcium (Figure 5c). The expression of osteoblast specific markers OPN, COL1, and RUNX2 was tested by RT-PCR for the 10-day and 22-day differentiated cells. The undifferentiated cells were tested as controls. The results are shown in Figure 5d.
Morphological changes in cultured ADSCs at passage 13 were observed. Along with the differentiation, the cells at passage 13 lost their fibroblastic morphology gradually and became a flatter and broader in shape; at the 15th day of induction, these cells formed a polygonal shape. The ability of glycogen-storage at differentiated cells was formed using PAS staining (Figure 5e) AFB, a mature functional hepatocyte-specific marker and AFP, a marker of immature hepatocytes, were tested by RT-PCR. Both differentiated cells expressed the AFB and ALP, but the undifferentiated cells were not expressed (Figure 5f).
During the induction, many morphological changes appeared in the cultured cells. The cells at passage 6 lost their fibroblastic morphology gradually and became flatter and broader in shape; at the 13th day of differentiation, the cells appeared cobblestone in shape. Induction of 13-day cells was further confirmed by immunofluorescence stain for CK18 (Figure 5g). CK18 is a specific antibody for epithelial cells. Specific surface markers were evaluated by RT-PCR and the undifferentiated cells were used as controls (Figure 5i).
The ADSCs were pre-induced for 7 d, after which spindle-shaped cells began to contract with an irregular form. Following induction, cell bodies further contracted and became round, triangular, or cone-shaped with multipolar processes. The processes continued to grow with many branches forming and cone-like terminal expansions were observed. A number of cells demonstrated very long processes, which appeared similar to the long axon of neurons. Immunofluorescence staining results showed that the MAP2, GFAP, and β-tubulin markers of neural cells were expressed in the differentiated cells (Figure 5j). The RT-PCR showed that the induced group cells were positive for NF and map2.
In this study, ADSCs were successfully isolated from the visceral omental region of pigs. The tissues were washed with PBS 8 times and digested through 1% I type collagenase for 1 h. The cells were cultured in a medium that contained 10% FBS, 1% B27, 1% glutamine, 1% streptomycin, and 10 ng/mL bFGF. Because bFGF can promote mitosis and stimulate anabolism, it is important to the growth of ADSCs and B27 is a secondary growth factor that provides nutrients for ADSCs. The medium can guarantee ADSCs in the best state of growth. There were few cells that had adhered 48 h after inoculation. The cells first confluence to 80% was 10 d after inoculation. During the first 10 d, the medium was changed every 2 d. Later, cells were passaged stably over 2 d on average. At the 26th passage of ADSCs, the characteristics of aging appeared and the times of passage were prolonged by 4 d. This was maybe due to the cells themselves, several times the enzyme digestion, or the medium. Further research is required to discover the specific reasons for this.
There were no specific surface markers used to identify and screen for MSCs. So RT-PCR, immunofluorescent staining, and flow cytometry were used to test for the surface markers of ADSCs. The results showed ADSCs highly expressed CD29, CD44, CD71, CD73, CD90, CD166, and vimentin, weekly expressed CD105, and were unexpressed CD31.
We explored the ability of clone formation for ADSCs at passages 4, 12, and 26. Results showed that ADSCs have a strong ability of clone formation in a good growth state. In order to detect whether there was any variation in the proliferation of ADSCs, we conducted karyotype analysis. Results demonstrated there was no chromosome missing or broken in the ADSCs.
In order to study the pluripotency of ADSCs, we differentiated the cells into ectoderm, mesoderm, and endoderm. We differentiated the ADSCs into epithelial and neurogenic cells for ectoderm. The results of cell differentiation were verified by cell morphology, RT-PCR, and immunofluorescence stain. For epithelial cell morphology, the control groups were typical fibroblast-like shape and the induced group cells changed into a cobblestone-like shape. For RT-PCR, the control groups didn’t express epithelial-cell specific markers CK18 and E-cadherin, while the induced group cells highly expressed CK18 and E-cadherin. For immunofluorescence staining, the induced cells were positive to the antibody CK18, which is a marker for epithelial cells. For the neurogenic cells, the control group cells still retained a fibroblast-like shape, while the induced group cells changed into a typical neuron like morphology and long axons were observed. Results of immunofluorescence staining showed the induced group cells were positive for GFAP, MAP2, and β-tubulin. The cells were then differentiated into adipocytes and osteoblasts for mesoderm. And we detected the results by cell morphology, staining and RT-PCR. For adipogenic differentiation, the induced cells appeared as obvious fat drops in the cytoplasm, expressed the adipocytes specific markers LPL and PPARγ, and the fat drops stained red with Oil-Red O. For osteogenic differentiation, the induced cells had an oval-like shape with many aggregations. Along with the increase of aggregations, calcium nodules were emerging in the cytoplasm. The induced cells were stained with alizarin, and with Oil-Red O and expressed the osteoblast specific markers OPN, COL1, and RUNX2. For hepatic differentiation, the induced cells changed from a fibroblast-like shape to a polygonal or round shape. And RT-PCR results showed the induced cells expressed mRNA for ALB and AFP, while these were not expressed in control groups. We evaluated the efficiency of differentiation by PAS staining and the induced cells were dyed purple in the cytoplasm. Results showed that ADSCs have the ability of crossing layer differentiation. Though ADSCs can differentiate into 3 layers, the function of induced cells was not tested; therefore, more research is needed in this capacity.
Adipose tissue is an abundant source of mesenchymal stem cells, which have shown promise in the field of regenerative medicine. Various clinical trials have shown the regenerative capability of adipose-derived stem cells in subspecialties of medical fields such as plastic, orthopedic, oral maxillofacial, and cardiac surgeries. Breast reconstruction and augmentation trials have been reported by Yoshimura et al (11–12). Adipose-derived stem cells were first used to stimulate craniofacial bone repair in calvarial defects (13). These stem cells have been used to heal chronic fistulas in Crohn’s disease (14–15).
Myocardial infarction is a life-threatening medical emergency, which is the greatest cause of death in developing countries. Until now, myocardial infarction is mainly dealt with through drugs and surgery, which can have many side effects on patients. Adipose tissue taken from around the heart has shown that ADSCs can differentiate into myocardial cells in vitro. Stem cell therapy is an effective way to cure many diseases, so it is significant if ADSCs derived from visceral (omental region) could be differentiated into myocardial cells and be used to induce cells to cure myocardial infarction.
In conclusion, ADSCs were isolated from visceral omental region of the pig, and the self-renewal ability and differential potential were evaluated in vitro. The present study illustrates the potential application of adipose tissue as an adult stem cell source for regenerative therapies.